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Spontaneous recovery of ischemic stroke is very limited and often results in the loss of motor and sensory function. Till now, rehabilitative training is the most widely accepted therapy to improve long-term outcome. However, its effectiveness is often suboptimal, largely due to a sharp decline of neuroplasticity in adults. In this study, we hypothesized that a combination of proprioceptive stimulation and rehabilitative training will promote neuroplasticity and functional recovery post injury. To test this hypothesis, we first established a photothrombotic stroke model that lesions the hindlimb sensorimotor cortex. Next, we demonstrated that injecting Cre-dependent AAV-retro viruses into the dorsal column of PV-Cre mice achieves specific and efficient targeting of proprioceptors. With chemogenetics, this method enables chronic activation of proprioceptors. We then assessed effects of combinatorial treatment on motor and sensory functional recovery. Our results showed that pairing proprioceptive stimulation with rehabilitative training significantly promoted skilled motor, but not tactile sensory functional recovery. This further led to significant improvement when compared to rehabilitation training or proprioceptor stimulation alone. Mechanistically, combinatorial treatment promoted cortical layer V neuronal mTOR activity and sprouting of corticospinal axon into the area where proprioceptive afferents terminate in the denervated side of the spinal cord. Serving as a proof of principle, our study thus provided novel insights into the application of combining proprioceptive stimulation and rehabilitative training to improve functional recovery of ischemic stroke and other traumatic brain or spinal cord injuries., (Copyright © 2021 Elsevier Inc. All rights reserved.)

There has been a concern that sodium-glucose cotransporter 2 (SGLT2) inhibitors could reduce skeletal muscle mass and function. Here, we examine the effect of canagliflozin (CANA), an SGLT2 inhibitor, on slow and fast muscles from nondiabetic C57BL/6J mice. In this study, mice were fed with or without CANA under ad libitum feeding, and then evaluated for metabolic valuables as well as slow and fast muscle mass and function. We also examined the effect of CANA on gene expressions and metabolites in slow and fast muscles. During SGLT2 inhibition, fast muscle function is increased, as accompanied by increased food intake, whereas slow muscle function is unaffected, although slow and fast muscle mass is maintained. When the amount of food in CANA-treated mice is adjusted to that in vehicle-treated mice, fast muscle mass and function are reduced, but slow muscle was unaffected during SGLT2 inhibition. In metabolome analysis, glycolytic metabolites and ATP are increased in fast muscle, whereas glycolytic metabolites are reduced but ATP is maintained in slow muscle during SGLT2 inhibition. Amino acids and free fatty acids are increased in slow muscle, but unchanged in fast muscle during SGLT2 inhibition. The metabolic effects on slow and fast muscles are exaggerated when food intake is restricted. This study demonstrates the differential effects of an SGLT2 inhibitor on slow and fast muscles independent of impaired glucose metabolism, thereby providing new insights into how they should be used in patients with diabetes, who are at a high risk of sarcopenia., (© 2022 The Author(s).)

Aortic dissection and rupture are triggered by decreased vascular wall strength and/or increased mechanical loads. We investigated the role of mTOR signaling in aortopathy using a well-described model of angiotensin II-induced dissection, aneurysm, or rupture of the suprarenal abdominal aorta in Apoe-deficient mice. Although not widely appreciated, nonlethal hemorrhagic lesions present as pseudoaneurysms without significant dissection in this model. Angiotensin II-induced aortic tears result in free rupture, contained rupture with subadventitial hematoma (forming pseudoaneurysms), dilatation, or healing, while the media invariably thickens regardless of mural tears. Medial thickening results from smooth muscle cell hypertrophy and extracellular matrix accumulation, including matricellular proteins. Angiotensin II activates mTOR signaling in vascular wall cells, and inhibition of mTOR signaling by rapamycin prevents aortic rupture but promotes dissection. Decreased aortic rupture correlates with decreased inflammation and metalloproteinase expression, whereas extensive dissection correlates with induction of matricellular proteins that modulate adhesion of vascular cells. Thus, mTOR activation in vascular wall cells determines whether aortic tears progress to dissection or rupture. Previous mechanistic studies of aortic aneurysm and dissection by angiotensin II in Apoe-deficient mice should be reinterpreted as clinically relevant to pseudoaneurysms, and mTOR inhibition for aortic disease should be explored with caution.

Clinical utilization of doxorubicin (DOX), which is a commonly used chemotherapeutic, is restricted due to toxic effects on various tissues. Using hesperetin (HST), an antioxidant used in Chinese traditional medicine protects testis against DOX-induced toxicity although the molecular mechanisms are not well-known. The study was aimed to examine the possible role of the mechanistic target of rapamycin kinase (mTOR) and dynamin 1-like dynamin-related protein 1 (DRP1) in the therapeutic effects of HST on the DOX-induced testicular toxicity. Rats were divided into Control, DOX, DOX + HST, and HST groups (n = 7). Single-dose DOX (15 mg/kg) was administered intraperitoneally and HST (50 mg/kg) was administered by oral gavage every other day for 28 days. Total antioxidant status (TAS), histopathological evaluations, immunohistochemistry, and gene expression level detection analyses were performed. Histopathologically, DOX-induced testicular damage was ameliorated by HST treatment. DOX reduced testicular TAS levels and increased oxidative stress markers, 8-Hydroxy-deoxyguanosine (8-OHdG), and 4-Hydroxynonenal (4-HNE). Also, upregulated mTOR and DRP1 expressions with DOX exposure were decreased after HST treatment in the testis (p < 0.05). On the other hand, DOX-administration downregulated miR-150-5p and miR-181b-2-3p miRNAs, targeting mTOR and mRNA levels of beclin 1 (BECN1) and autophagy-related 5 (ATG5), autophagic markers. Furthermore, these levels were nearly similar to control testis samples in the DOX + HST group (p < 0.05). The study demonstrated that HST may have a therapeutic effect on DOX-induced testicular toxicity by removing reactive oxygen species (ROS) and by modulating the mTOR and DRP1 expressions, which have a critical role in regulating the balance of generation/elimination of ROS., (Copyright © 2021. Published by Elsevier Inc.)

In temporal lobe epilepsy (TLE), abnormal axon guidance and synapse formation lead to sprouting of mossy fibers in the hippocampus, which is one of the most consistent pathological findings in patients and animal models with TLE. Glypican 4 (Gpc4) belongs to the heparan sulfate proteoglycan family, which play an important role in axon guidance and excitatory synapse formation. However, the role of Gpc4 in the development of mossy fibers sprouting (MFS) and its underlying mechanism remain unknown. Using a pilocarpine-induced mice model of epilepsy, we showed that Gpc4 expression was significantly increased in the stratum granulosum of the dentate gyrus at 1 week after status epilepticus (SE). Using Gpc4 overexpression or Gpc4 shRNA lentivirus to regulate the Gpc4 level in the dentate gyrus, increased or decreased levels of netrin-1, SynI, PSD-95, and Timm score were observed in the dentate gyrus, indicating a crucial role of Gpc4 in modulating the development of functional MFS. The observed effects of Gpc4 on MFS were significantly antagonized when mice were treated with L-leucine or rapamycin, an agonist or antagonist of the mammalian target of rapamycin (mTOR) signal, respectively, demonstrating that mTOR pathway is an essential requirement for Gpc4-regulated MFS. Additionally, the attenuated spontaneous recurrent seizures (SRSs) were observed during chronic stage of the disease by suppressing the Gpc4 expression after SE. Altogether, our findings demonstrate a novel control of neuronal Gpc4 on the development of MFS through the mTOR pathway after pilocarpine-induced SE. Our results also strongly suggest that Gpc4 may serve as a promising target for antiepileptic studies., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: Highest incidence of oral cancer is reported in India with reduced survival rate in the advanced stages due to lack of effective biomarkers. Therefore, it is essential to develop novel biomarkers for the better management of this disease. In the current study, TNFAIP8/TIPE protein family comprising of four proteins is explored for its role in oral cancer., Methods: IHC analysis of oral cancer TMA and Western blot analysis of tobacco treated oral cancer cells were performed to determine the differential expression of TIPE proteins in oral cancer. Further, CRISPR/Cas9-mediated gene editing was done to generate TIPE proteins' knockouts and MTT, colony formation, wound healing, cell cycle and Western blot analysis were performed to determine the effect of gene knockouts on various cancer hallmarks and the associated molecular targets of TIPE proteins., Results and Discussion: IHC results revealed that expression of TIPE, TIPE2 and TIPE3 were upregulated and TIPE1 was downregulated in oral cancer tissues compared to normal tissues. Similar results were observed upon treating oral cancer cells with tobacco carcinogens. Furthermore, knockout of TIPE or TIPE2 or TIPE3 significantly reduced the survival, proliferation, colony formation and migration of oral cancer cells whereas knockout of TIPE1 had an opposite effect. Further, TIPE, TIPE2 and TIPE3 knockout-mediated inhibition of proliferation was associated with inhibition of cell cycle progression at S or G2/M phases, and downregulation of proteins involved in cancer progression. We found that TIPE, TIPE1 and TIPE2 proteins regulate oral cancer progression through modulation of Akt/mTOR signaling cascade, whereas TIPE3 acts through an Akt-independent mTOR/STAT3 pathway., Conclusion: Collectively, the TIPE proteins were proved to play significant roles in the progression of oral cancer thus warranting research and clinic attention for their therapeutic and prognostic values and raising the importance of specific targeting of TIPE proteins in cancer treatment., (Copyright © 2021. Published by Elsevier Inc.)

Disuse syndrome indicates psychosomatic hypofunction caused by excess rest and motionless and muscle atrophy is termed disuse muscle atrophy. Disuse muscle atrophy-induced muscle weakness and hypoactivity further induces muscle atrophy, leading to a vicious cycle, and this is considered a factor causing secondary sarcopenia and subsequently frailty. Since frailty finally leads to a bedridden state requiring nursing, in facing a super-aging society, intervention for a risk factor of frailty, disuse muscle atrophy, is important. However, the main treatment of disuse muscle atrophy is physical therapy and there are fewer effective preventive and therapeutic drugs. The objective of this study was to search for Kampo medicine with a disuse muscle atrophy-improving effect. Ninjin'yoeito is classified as a qi-blood sohozai (dual supplement) in Chinese herbal medicine, and it has an action supplementing the spleen related to muscle. In addition, improvement of muscle mass and muscle weakness by ninjin'yoeito in a clinical study has been reported. In this study, the effect of ninjin'yoeito on disuse muscle atrophy was investigated. A disuse muscle atrophy model was prepared using male ICR mice. After surgery applying a ring for tail suspension, a 1-week recovery period was set. Ninjin'yoeito was administered by mixing it in the diet for 1 week after the recovery period, followed by tail suspension for 14 days. Ninjin'yoeito administration was continued until autopsy including the hindlimb suspension period. The mice were euthanized and autopsied immediately after completion of tail suspension, and the hindlimb muscles were collected. The food and water intakes during the hindlimb unloaded period, wet weight of the collected muscle, and muscle synthesis and muscle degradation-related factors in blood and muscle were evaluated. Ingestion of ninjin'yoeito inhibited tail suspension-induced reduction of the soleus muscle wet weight. In addition, an increase in the blood level of a muscle synthesis-related factor, IGF-1, and promotion of phosphorylation of mTOR and 4E-BP1 in the soleus muscle were observed. It was suggested that ninjin'yoeito has a disuse muscle atrophy-improving action. Promotion of the muscle synthesis pathway was considered the action mechanism of this., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Context: Berberine (BBR) is used to treat diarrhoea and gastroenteritis in the clinic. It was found to have anticolon cancer effects., Objective: To study the anticolon cancer mechanism of BBR by connectivity map (CMAP) analysis., Materials and Methods: CMAP based mechanistic prediction was conducted by comparing gene expression profiles of 10 μM BBR treated MCF-7 cells with that of clinical drugs such as helveticoside, ianatoside C, pyrvinium, gossypol and trifluoperazine. The treatment time was 12 h and two biological replications were performed. The DMSO-treated cells were selected as a control. The interaction between 100 μM BBR and target protein was measured by cellular thermal shift assay. The protein expression of 1-9 μM BBR treated SW480 cells were measured by WB assay. Apoptosis, cell cycle arrest, mitochondrial membrane potential (MMP) of 1-9 μM BBR treated SW480 cells were measured by flow cytometry and Hoechst 33342 staining methods., Results: CMAP analysis found 14 Hsp90, HDAC, PI3K or mTOR protein inhibitors have similar functions with BBR. The experiments showed that BBR inhibited SW480 cells proliferation with IC 50 of 3.436 μM, induced apoptosis, autophage, MMP depolarization and arrested G1 phase of cell cycle at 1.0 μM. BBR dose-dependently up-regulated PTEN, while inhibited Notch1, PI3K, Akt and mTOR proteins at 1.0-9.0 μM ( p  < 0.05). BBR also acted synergistically with Hsp90 and HDAC inhibitor (0.01 μM) in SW480 cells at 0.5 and 1.0 μM., Discussion and Conclusions: The integrative gene expression-based chemical genomic method using CMAP analysis may be applicable for mechanistic studies of other multi-targets drugs.

Diabetic nephropathy (DN) is one of the most serious and major renal complications of diabetes. Previously, Six-transmembrane Protein of Prostate 2 (STAMP2) was reported to contribute to nutritional stress. The purpose of this study is to investigate whether overexpression of STAMP2 attenuates diabetic renal injuries in DN rats. We induced the DN rat model by high-fat diet and low-dose streptozotocin and evaluated the metabolite and urine albumin/creatinine. Recombinant adeno-associated virus vectors were injected for overexpression of STAMP2. Pathophysiologic and ultrastructure features of DN by histochemical stain and transmission electron microscope, autophagy-related proteins and signaling pathway by western blotting were assessed. We found the expression of STAMP2 was decreased and autophagy was blunted in DN rat kidneys. Overexpressing STAMP2 significantly ameliorated metabolic disturbance, insulin resistance, and specifically restoring diabetic renal injury. Furthermore, overexpressing STAMP2 improved the autophagy deficiency in DN rats, as revealed by changes in the expressions of Beclin1, p62, and LC3. Furthermore, STAMP2 overexpressing promoted autophagy by inhibiting the mTOR and activating the AMPK/SIRT1 signaling pathway. Our results suggested that STAMP2 overexpression attenuated renal injuries via upregulating autophagy in DN rats. STAMP2 overexpressing promoted autophagy may been involved with inhibition of the mTOR/ULK1 and activation of the AMPK/SIRT1 signaling pathway., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

BACKGROUNDThe aberrant activation of the PI3K/mTOR signaling circuitry is one of the most frequently dysregulated signaling events in head and neck squamous cell carcinoma (HNSCC). Here, we conducted a single-arm, open-label phase IIa clinical trial in individuals with oral premalignant lesions (OPLs) to explore the potential of metformin to target PI3K/mTOR signaling for HNSCC prevention.METHODSIndividuals with OPLs, but who were otherwise healthy and without diabetes, underwent pretreatment and posttreatment clinical exam and biopsy. Participants received metformin for 12 weeks (week 1, 500 mg; week 2, 1000 mg; weeks 3-12, 2000 mg daily). Pretreatment and posttreatment biopsies, saliva, and blood were obtained for biomarker analysis, including IHC assessment of mTOR signaling and exome sequencing.RESULTSTwenty-three participants were evaluable for response. The clinical response rate (defined as a ≥50% reduction in lesion size) was 17%. Although lower than the proposed threshold for favorable clinical response, the histological response rate (improvement in histological grade) was 60%, including 17% complete responses and 43% partial responses. Logistic regression analysis revealed that when compared with never smokers, current and former smokers had statistically significantly increased histological responses (P = 0.016). Remarkably, a significant correlation existed between decreased mTOR activity (pS6 IHC staining) in the basal epithelial layers of OPLs and the histological (P = 0.04) and clinical (P = 0.01) responses.CONCLUSIONTo our knowledge this is the first phase II trial of metformin in individuals with OPLs, providing evidence that metformin administration results in encouraging histological responses and mTOR pathway modulation, thus supporting its further investigation as a chemopreventive agent.TRIAL REGISTRATIONNCT02581137FUNDINGNIH contract HHSN261201200031I, grants R01DE026644 and R01DE026870.

Neuroinflammation has emerged as a key contributor in the pathogenesis of Alzheimer's disease (AD). Mammalian target of rapamycin (mTOR) is a key regulator of metabolism, cell growth and protein synthesis. And an elevated mTOR activity has been detected in AD-affected brain areas. Previous studies have suggested that all-trans retinoic acid (atRA) and rapamycin (RAPA), an mTOR inhibitor, protect lipopolysaccharide (LPS)-induced neuronal inflammation through inhibiting nuclear import of NFκB. The aim of this study was to test the effects of atRA on mTOR expression. Here we discovered that mTOR and p-mTOR expression are elevated in LPS-treated mice or primary rat neurons, while atRA blocks the mTOR gene upregulation via a SIRT1-dependent mechanism. The results of this study demonstrated that atRA may protect LPS-induced neuronal inflammation through suppressing mTOR signaling., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Autophagy, which mediates the delivery of cytoplasmic substrates to the lysosome for degradation, is essential for maintaining proper cell homeostasis in physiology, ageing, and disease. There is increasing evidence that autophagy is defective in neurodegenerative disorders, including motor neurons affected in amyotrophic lateral sclerosis (ALS). Restoring impaired autophagy in motor neurons may therefore represent a rational approach for ALS. Here, we demonstrate autophagy impairment in spinal cords of mice expressing mutant TDP-43 Q331K or co-expressing TDP-43 WTxQ331K transgenes. The clinically approved anti-hypertensive drug rilmenidine was used to stimulate mTOR-independent autophagy in double transgenic TDP-43 WTxQ331K mice to alleviate impaired autophagy. Although rilmenidine treatment induced robust autophagy in spinal cords, this exacerbated the phenotype of TDP-43 WTxQ331K mice, shown by truncated lifespan, accelerated motor neuron loss, and pronounced nuclear TDP-43 clearance. Importantly, rilmenidine significantly promoted mitophagy in spinal cords TDP-43 WTxQ331K mice, evidenced by reduced mitochondrial markers and load in spinal motor neurons. These results suggest that autophagy induction accelerates the phenotype of this TDP-43 mouse model of ALS, most likely through excessive mitochondrial clearance in motor neurons. These findings also emphasise the importance of balancing autophagy stimulation with the potential negative consequences of hyperactive mitophagy in ALS and other neurodegenerative diseases., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Increasing evidence reveals that the Rho GTPase-activating protein is a crucial negative regulator of Rho family GTPase involved in tumorigenesis. The Rho GTPase-activating protein 25 (ARHGAP25) has been shown to specifically inactivate the Rho family GTPase Rac1, which plays an important role in pancreatic adenocarcinoma (PAAD) progression. Therefore, here we aimed to clarify the expression and functional role of ARHGAP25 in PAAD. The ARHGAP25 expression was lower in PAAD tissues than that in normal pancreatic tissues based on bioinformatics analysis and immunohistochemistry staining. Overexpression of ARHGAP25 inhibited cell growth of AsPC-1 human pancreatic cancer cells in vitro, while opposite results were observed in BxPC-3 human pancreatic cancer cells with ARHGAP25 knockdown. Consistently, in vivo tumorigenicity assays also confirmed that ARHGAP25 overexpression suppressed tumor growth. Mechanically, overexpression of ARHGAP25 inactivated AKT/mTOR signaling pathway by regulating Rac1/PAK1 signaling, which was in line with the results from the Gene set enrichment analysis on The Cancer Genome Atlas dataset. Furthermore, we found that ARHGAP25 reduced HIF-1α-mediated glycolysis in PAAD cells. Treatment with PF-04691502, a dual PI3K/mTOR inhibitor, hampered the increased cell growth and glycolysis due to ARHGAP25 knockdown in PAAD cells. Altogether, these results conclude that ARHGAP25 acts as a tumor suppressor by inhibiting the AKT/mTOR signaling pathway, which might provide a therapeutic target for PAAD., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Background: AdipoR2, which belongs to the seven-transmembrane-domain receptor family, has been shown to play an important role in the development of human tumours, but the underlying mechanisms are poorly understood. In this study, we found that AdipoR2 expression correlates with glioma grade. In addition, we also investigated the mechanisms behind the antiproliferative effects of AdipoR2 in U251 cells (a human glioma cell line) using colony formation and WST-8 growth assays., Methods: The U251 cell line was cultured in vitro. Western blotting was used to detect the expression of relevant proteins. Quantitative RT-PCR was used to detect AdipoR1 and AdipoR2 expression. Flow cytometry was used to detect cell cycle assay results. The gene expression profiles of glioma samples from the CGGA database were analysed by MATLAB and GSEA software., Results: The AMPK/mTOR pathway plays a central role in the regulation of cell proliferation, differentiation and migration and may promote tumorigenesis. Therefore, we can control cancer progression by modulating the AMPK/mTOR pathway. However, there is no information on the relationship between AdipoR and AMPK/mTOR in central nervous system tumours such as GBM. In this study. We found 648 upregulated genes and 436 downregulated genes correlated with AdipoR2 expression in 158 glioma samples. GSEA suggested that AdipoR2 is a cell cycle-associated gene. The results of the flow cytometry analysis indicated that AdipoR2 induced G0/G1 cell cycle arrest in U251 cells. Furthermore, we identified the AMPK/mTOR signalling axis to be involved in AdipoR2-induced cell cycle arrest., Conclusions: Our results suggest that AdipoR2 may represent a novel endogenous negative regulator of GBM cell proliferation. These findings also suggest that AdipoR2 may be a promising therapeutic target in GBM patients.

The current study aimed to investigate linagliptin for its potential role in the prevention of liver fibrosis progression. Balb-C mice were randomly allocated into five groups (10 each): ( i ) control; ( ii ) mice were injected intraperitoneally with 50 μL carbon tetrachloride (CCl 4 ) in corn oil in a dose of 0.6 μL/g three times per week for four weeks; ( iii ) linagliptin was administered orally in a daily dose of 10 mg/kg simultaneously with CCl 4 ; ( iv ) silymarin was administered orally in a daily dose of 200 mg/kg concomitantly with CCl 4 ; and ( v ) only linagliptin was administered. Hepatic injury was manifested in the CCl 4 group by elevation of biochemical parameters (alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP)), and hepatic fibrosis was evident histopathologically by increased METAVIR score and immunostaining expression of alpha-smooth muscle actin (α-SMA), as well as increased liver tissue oxidative stress parameters, transforming growth factor-β1 (TGF-β1), and mammalian target of rapamycin (mTOR). Linagliptin was able to stop the progression of liver fibrosis, evident histopathologically with reduced METAVIR score and α-SMA expression. The possible mechanism may be via suppression of oxidative stress, TGF-β1, and mTOR, which was associated with improvement of serum biochemical parameters ALT and AST. In conclusion, linagliptin might help to protect the liver against persistent injury-related consequences.

Various genetic and epigenetic mechanisms have been suggested to play roles as the underlying pathophysiology of Multiple Sclerosis (MS). Changes in different parts of the mTOR signaling pathway are among the potential suggested mechanisms based on the specific roles of this pathway in CNS. MTOR, RPS6KB1, and EIFEBP1 genes are among important genes in the mTOR pathway, responsible for the proper function of acting proteins in this signaling pathway. This study aimed to investigate the relative expression levels of these genes in the blood samples of relapsing-remitting MS (RRMS) patients compared to healthy controls. In this case-control study blood samples were collected from 30 newly diagnosed RRMS patients and 30 age and sex-matched healthy controls. mRNA level of MTOR, RPS6KB1, and EIFEBP1 genes were assessed using Real-Time PCR. The expression of MTOR, RPS6KB1, and EIF4EBP1 genes was up regulated in MS patients compared to healthy controls (p < 0.001 for all mentioned genes). Considering gender differences, expression of the mentioned genes was increased among female patients (all P < 0.001). However, no statistically significant changes were observed among male patients. Based on the receiver operating characteristic, MTOR gene had the highest diagnostic value followed by EIF4EBP1 and RPS6KB1 genes in differentiating RRMS patients from controls. In conclusion, we found the simultaneous upregulation of MTOR, RPS6KB1, and EIF4EBP1 genes among RRMS patients. MTOR showed to have the highest diagnostic value compared to other 2 genes in differentiating RRMS patients. Further studies evaluating the importance of these findings from pharmacological and prognostic perspectives are necessary.

Although aging in the liver contributes to the development of chronic liver diseases such as NAFLD and insulin resistance, little is known about the molecular and metabolic details of aging in hepatic cells. To examine these issues, we used sequential oxidative stress with hydrogen peroxide to induce premature senescence in AML12 hepatic cells. The senescent cells exhibited molecular and metabolic signatures, increased SA-βGal and γH2A.X staining, and elevated senescence and pro-inflammatory gene expression that resembled livers from aged mice. Metabolic phenotyping showed fuel switching towards glycolysis and mitochondrial glutamine oxidation as well as impaired energy production. The senescent AML12 cells also had increased mTOR signaling and decreased autophagy which likely contributed to the fuel switching from β-oxidation that occurred in normal AML12 cells. Additionally, senescence-associated secretory phenotype (SASP) proteins from conditioned media of senescent cells sensitized normal AML12 cells to palmitate-induced toxicity, a known pathological effect of hepatic aging. In summary, we have generated senescent AML12 cells which displayed the molecular hallmarks of aging and also exhibited the aberrant metabolic phenotype, mitochondrial function, and cell signaling that occur in the aged liver.

Ethnophamacological Relevance: The Chinese herbal prescription JieZe-1 (JZ-1) is based on the modification of Yihuang Tang, which was first described in Fu Qingzhu Nvke by the famous Qing Dynasty doctor Shan Fu as a treatment for leukorrheal diseases. As an in-hospital preparation, JZ-1 has been used in Tongji Hospital for many years to treat various infectious diseases of the lower female genital tract, including cervicitis, vaginitis, genital herpes and condyloma acuminatum. Our previous studies have shown that JZ-1 has curative effects on Candida albicans, Trichomonas vaginalis and Ureaplasma urealyticum infections., Aim of the Study: Genital herpes is among the most common sexually transmitted diseases (STDs) worldwide and is mainly caused by herpes simplex virus type-2 (HSV-2). Current therapies can relieve symptoms in patients but do not cure or prevent the spread of the virus. This study was designed to investigate the effect of JZ-1 on HSV-2 infection and its mechanism, which is based on autophagy induction, to provide new ideas and a basis for the study of antiviral drugs., Materials and Methods: Evaluation of the antiviral activity of JZ-1 was conducted by MTT assay and western blotting. Then, Western blot and immunofluorescence analyses, observations through transmission electron microscopy and experiments with the recombinant lentivirus vector mRFP-GFP-LC3B were used to monitor autophagic flux in VK2/E6E7 cells. To explore the mechanism by which JZ-1 regulates autophagy, western blotting and real-time quantitative PCR (qRT-PCR) were used to determine the expression of phosphoinositide 3-kinase (PI3K)/Akt/mTOR pathway proteins and to detect changes in critical molecules in the pathway after the application of a PI3K inhibitor. Additionally, the mRNA expression levels of inflammatory cytokines, namely, IL-6, IFN-α, IFN-β and TNF-α, were measured with qRT-PCR., Results: HSV-2 infection inhibited autophagy in the VK2/E6E7 cells. Further study revealed that the activation of the PI3K/Akt/mTOR pathway induced by HSV-2 infection may result in the blocked autophagic flux and inhibited autophagosome and autolysosome formation. JZ-1 exhibited significant antiviral activity in the VK2/E6E7 cells, which showed increased cell vitality and reduced viral protein expression, namely, earliest virus-specific infected cell polypeptides 5 (ICP5) and glycoprotein D (gD). We found that JZ-1 treatment inhibited the upregulation of the PI3K/Akt/mTOR pathway proteins and promoted autophagy to combat HSV-2 infection, while PI3K inhibitor pretreatment prevented the enhanced autophagy induced by JZ-1. Moreover, JZ-1 attenuated the increase in inflammatory cytokines that had been induced HSV-2 infection., Conclusion: Our results showed that JZ-1 protects against HSV-2 infection, and this beneficial effect may be mediated by inducing autophagy via inhibition of the PI3K/Akt/mTOR signaling axis., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Dysregulation of the circadian timing system (CTS) frequently appears during colorectal cancer (CRC) progression. In order to better understand the role of the circadian clock in CRC progression, this study evaluated in vitro how knockdown of a core circadian protein BMAL1 (BMAL1-KD) influenced the behavior of two primary human CRC cell lines (HCT116 and SW480) and a metastatic CRC cell line (SW620).Unexpectedly, BMAL1-KD induced CRC cell-type specific responses rather than the same phenomenon throughout. First, BMAL1-KD increased AKT/mTOR activation in each CRC cell line, but to different extents. Second, BMAL1-KD-induced P53 activation varied with cell context. In a wild type P53 background, HCT116 BMAL1-KD cells quickly underwent apoptosis after shBMAL1 lentivirus transduction, while surviving cells showed less P53 but increased AKT/mTOR activation, which ultimately caused higher proliferation. In the presence of a partially functional mutant P53, SW480 BMAL1-KD cells showed moderate P53 and mTOR activation simultaneously with cell senescence. With a moderate increased AKT but unchanged mutant P53 activation, SW620 BMAL1-KD cells grew faster.Thus, under different CRC cellular pathological contexts, BMAL1 knockdown induced relatively equal effects on AKT/mTOR activation but different effects on P53 activation, which finally triggered different CRC cell fates.

Background: We observed the effects of resveratrol on the expression of molecules involved in the mTOR signaling pathway in pathological scar fibroblasts, including PI3K, Akt and mTOR., Methods: We detected the expression of PI3K, Akt and mTOR in pathological scar and normal skin fibroblasts through immunofluorescence. After being treated with different concentrations of resveratrol, the expression of PI3K, Akt and mTOR mRNA and protein were detected by RT-PCR and Western Blot respectively., Results: Results showed that the expression of PI3K, Akt and mTOR were significantly enhanced in pathological scar fibroblasts and mainly expressed in the nucleus, with no expression in normal skin fibroblasts. Results from RT-PCR and Western Blot tests demonstrated that after Res intervention with different concentrations for pathological scar fibroblasts, the relative expression quantity of PI3K mRNA and protein decreased and showed a dose dependent relationship. Compared to the control group, the differences were statistically significant (P<0.05). However, decrease in the expression of PI3K mRNA and protein was not obvious and there were no significant differences in comparison to the control group (P>0.05)., Conclusions: The mechanism of resveratrol in the inhibition of the proliferation of pathological scar fibroblasts may be related to its down-regulation in the expression of Akt and mTOR, which are the key molecules of mTOR signaling pathway.

Background: Mammalian target of rapamycin (mTOR) inhibitors have been shown to have excellent effects in the management of kaposiform hemangioendothelioma (KHE); however, the mechanism of action is unclear. This study identified the expressions of mTOR pathway-related proteins in different vascular tumors to provide insight into the pathogenesis of KHE., Methods: We retrospectively reviewed the pathologic specimens of 30 patients (KHE, 15; tufted angioma [TA], 5; infantile hemangioma [IH], 5; and lymphatic malformation [LM], 5). The immunohistochemical expression of mTOR-related proteins tuberous sclerosis complex 2 (TSC2), phosphatase and tensin homologue (PTEN), phosphorylated eukaryotic translation initiation factor 4E binding protein 1 (p-4EBP1), phosphorylated mTOR (p-mTOR), and phosphorylated ribosomal protein S6 kinase B1 (p-P70S6K) were analyzed using Image-Pro Plus software. KHE had the following pattern of expression in the spindle vascular endothelial cells: TSC2 (-); PTEN (-); p-4EBP1 (+); p-mTOR (+); and p-P70S6K (+)., Results: All 3 patients treated with sirolimus had good responses. The TA results were similar to KHE with no significant differences (p-4EBP1: p = 0.0687; p-mTOR: p = 0.0832). The expressions of TSC2, PTEN, p-4EBP1, p-mTOR, and p-P70S6K were negative or weakly positive in IH with a statistically significant difference compared to KHE (p-4EBP1: p < 0.001; p-mTOR: p < 0.001; p-P70S6K: p < 0.001). LM had no significant differences when compared to KHE., Conclusions: The absence of TSC2 and PTEN caused abnormal activation of the mTOR signaling pathway and may be involved in the pathogenesis of KHE. The expression of mTOR-related proteins in TA and LM was similar to KHE, unlike IH. The KHE pattern of expression [PTEN (-), TSC2 (-), p-mTOR (+), p-P70S6K (+), and p-4EBP1 (+)] suggested that sirolimus may be a good therapeutic choice., (© 2020 S. Karger AG, Basel.)

Objective: To investigate the effect of metformin (MET) on enhancing the sensitivity of human pancreatic cancer cells to gemcitabine (GEM) by regulating the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway., Materials and Methods: The GEM-resistant human pancreatic cancer PANC-1/GEM cell line was established, and the proliferation ability of PANC-1 and PANC-1/GEM cell lines was detected using the Cell Counting Kit-8 (CCK-8), which was then detected by flow cytometry after they were labeled by Ki67. Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) and Western blotting were adopted to detect the difference in the mTOR expression between PANC-1 and PANC-1/GEM cell lines. The proliferation ability of PANC-1/GEM/MET and PANC-1/GEM cell lines was determined using CCK-8 after drug-resistant cell lines were treated with 20 mmol/L MET combined with 0.4 μmol/L GEM or 0.4 μmol/L GEM alone for 48 h. Colony formation assay was applied to detect the proliferation ability of cells. The difference in the expression of mTOR/PI3K/Akt between PANC-1/GEM/MET and PANC-1/GEM cell lines was tested via qRT-PCR and Western blotting, respectively., Results: Compared with PANC-1 cells, PANC-1/GEM cells had significantly enhanced proliferation ability (p<0.01). Flow cytometry results showed that the proliferation ability of PANC-1/GEM cells was notably enhanced (p<0.01). The expression level and phosphorylation level of mTOR in drug-resistant cell lines were increased (p<0.01). After the drug-resistant cell lines were treated with 20 mmol/L MET for 48 h, the proliferation ability of PANC-1/GEM/MET cells was evidently decreased compared with that of PANC-1/GEM cells (p<0.01). The messenger ribonucleic acid (mRNA) and protein expression levels of mTOR/PI3K/Akt were markedly down-regulated (p<0.01)., Conclusions: MET can regulate the PI3K/Akt/mTOR signaling pathway to enhance the sensitivity of human pancreatic cancer cells to GEM.

Reactive oxygen species (ROS) act through multiple pathways to induce apoptosis of retinal capillary pericytes, which is an early marker and the primary cause of the progression of diabetic retinopathy. However, the specific molecular mechanisms behind ROS-induced retinal capillary pericyte loss in diabetic retinopathy remains elusive. In this study, we investigated the molecular regulation and effects of DJ-1/PARK7 on oxidative stress and injury of rat retinal pericytes (RRPs). To perform the research, RRPs were isolated from rat retina and cultured in medium with for 2 days: control group (5.6 mM glucose), high glucose group (30 mM glucose), hypertonic group (5.6 mM glucose + 24.4 mM mannitol). We found decreased expression of DJ-1 and increased apoptosis of RRPs in high glucose group. To further study the role of DJ-1, four groups were divided as follows: normal control group (5.6 mM glucose), high glucose (30 mM glucose), empty vector control group (pcDNA3.1,30 mM glucose), DJ-1 overexpression group (pcDNA3.1-myc-DJ-1,30 mM glucose). DJ-1, P53, p-P53, cleaved caspase-3, manganese superoxide dismutase (MnSOD), catalase (CAT) and PI3K/Akt/mTOR signaling pathway in each group was detected by Western Blot. RRPs apoptosis was detected by Terminal-deoxynucleoitidyl Transferase mediated Nick End Labeling (TUNEL) and 4'6- diamidino-2-phenylindole (DAPI). Mitochondrial function was detected by jc-1 and fluorescent probes DCFH-DA was used to determine reactive oxygen species (ROS). We found that high glucose (30 mM) lasting two days can induce significant apoptosis of RRPs, increase ROS production and expressions of p-p53 and active caspase-3, impair mitochondrial function, decrease the activities of MnSOD and CAT, and decrease expression of DJ-1, p-AKT and p-mTOR. In contrast, DJ-1/PARK7 overexpression significantly increases expression of DJ-1, p-AKT and p-mTOR, increases expression and activities of MnSOD and CAT, improves mitochondrial function, decreases expression of apoptotic gene protein p-p53 and active caspase-3, reduces ROS production and reduces the apoptotic rate of RRPs induced by high glucose. These results suggest that DJ-1 may play a role in protecting RRPs from high glucose induced-oxidative injury. DJ-1 might improve mitochondrial function, inhibit ROS production and enhance antioxidant capacity to reduce apoptosis of retinal pericytes through the PI3K/AKT/mTOR signaling pathway which may be related to early pathogenesis of diabetic retinopathy., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Although having promising anti-myeloma properties, the pan-histone deacetylase inhibitor (HDACi) panobinostat lacks therapeutic activity as a single agent. The aim of the current study was to elucidate the mechanisms underlying multiple myeloma (MM) resistance to panobinostat monotherapy and to define strategies to overcome it. Sensitivity of MM cell lines and primary CD138+ cells from MM patients to panobinostat correlated with reduced expression of the chemokine receptor CXCR4, whereas overexpression of CXCR4 in MM cell lines increased their resistance to panobinostat. Decreased sensitivity to HDACi was associated with reversible G0/G1 cell growth arrest while response was characterized by apoptotic cell death. Analysis of intra-cellular signaling mediators revealed the pro-survival mTOR pathway to be regulated by CXCR4 overexpression. Combining panobinostat with mTOR inhibitor everolimus abrogated the resistance to HDACi and induced synergistic cell death. The combination of panobinostat/everolimus resulted in sustained DNA damage and irreversible suppression of proliferation accompanied by robust apoptosis. Gene expression analysis revealed distinct genetic profiles of single versus combined agent exposure. Whereas panobinostat increased the expression of the cell cycle inhibitor p21, co-treatment with everolimus abrogated the increase in p21 and synergistically downregulated the expression of DNA repair genes and mitotic checkpoint regulators. Importantly, the combination of panobinostat with everolimus effectively targeted CXCR4-expressing resistant MM cells in vivo in the BM niche. In summary, our results uncover the mechanism responsible for the strong synergistic anti-MM activity of dual HDAC and mTOR inhibition and provide the rationale for a novel potential therapeutic approach to treat MM., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: The unfolded protein response plays versatile roles in physiology and pathophysiology. Its connection to cell growth, however, remains elusive. Here, we sought to define the role of unfolded protein response in the regulation of cardiomyocyte growth in the heart., Methods: We used both gain- and loss-of-function approaches to genetically manipulate XBP1s (spliced X-box binding protein 1), the most conserved signaling branch of the unfolded protein response, in the heart. In addition, primary cardiomyocyte culture was used to address the role of XBP1s in cell growth in a cell-autonomous manner., Results: We found that XBP1s expression is reduced in both human and rodent cardiac tissues under heart failure. Furthermore, deficiency of XBP1s leads to decompensation and exacerbation of heart failure progression under pressure overload. On the other hand, cardiac-restricted overexpression of XBP1s prevents the development of cardiac dysfunction. Mechanistically, we found that XBP1s stimulates adaptive cardiac growth through activation of the mechanistic target of rapamycin signaling, which is mediated via FKBP11 (FK506-binding protein 11), a novel transcriptional target of XBP1s. Moreover, silencing of FKBP11 significantly diminishes XBP1s-induced mechanistic target of rapamycin activation and adaptive cell growth., Conclusions: Our results reveal a critical role of the XBP1s-FKBP11-mechanistic target of rapamycin axis in coupling of the unfolded protein response and cardiac cell growth regulation.

Nasopharyngeal carcinoma (NPC) has a unique and complex etiology, which is not completely understood. The aim of this study is to investigate the expression patterns of phosphatidylinositol 3-kinase (PI3K), protein kinase B (PKB), and mammalian target of rapamycin (mTOR) proteins in patients with NPC and their relationship with NPC progression and prognosis. Between January 2008 and March 2010, PI3K, PKB, and mTOR protein expressions were detected using immunohistochemistry among 119 patients with NPC and 30 healthy people. A 5-year follow-up was conducted for all patients. Correlations of PI3K, PKB, and mTOR proteins with the clinicopathological features and prognosis of NPC were evaluated using Spearman's rank correlation coefficient and Kaplan-Meier curve. Cox's regression analysis was performed to analyze the risk factors for the prognosis of NPC. First, PI3K, PKB, and mTOR were highly expressed in patients with NPC. The expressions of PI3K, PKB, and mTOR proteins were associated with T stage, N stage, clinical stage, relapse, and distant metastasis. Meanwhile, PI3K is positively correlated with PKB and PKB is positively correlated with mTOR in NPC. Higher PI3K, PKB, and mTOR protein expressions were related to a shorter survival time and a lower survival rate in NPC. Cox regression analysis revealed that age, T stage, N stage, PI3K, PKB, and mTOR were independent risk factors for NPC patient survival. Altogether, our data suggest that overexpression of PI3K, PKB, and mTOR proteins is an important indicator of poor survival in NPC. In addition, inhibition of PI3K-PKB-mTOR signaling may also contribute to the development of new therapeutic strategies for NPC., (© 2018 Wiley Periodicals, Inc.)

The purpose of this study was to determine the impact of acute ethanol administration on the major signal transduction pathways in skeletal muscle responsible for regulating the protein synthetic and degradative response to refeeding. Adult male C57Bl/6 mice were fasted overnight; mice were then either refed normal rodent chow for 30 min or a separate group of mice remained food deprived (i.e., fasted). Thereafter, mice were administered either 3 g/kg ethanol or saline. Gastrocnemius/plantaris was collected 1 h later and analyzed. Acute ethanol decreased basal and prevented the refeeding-induced increase in muscle protein synthesis. While ethanol prevented a nutrient-stimulated increase in S6K1 phosphorylation, it did not alter the increase in 4E-BP1 phosphorylation. Downstream of S6K1, ethanol also attenuated the refeeding-induced increase in S6 and eIF4B phosphorylation, as well as the decrease in eEF2 phosphorylation. Although ethanol decreased ERK and p90 RSK phosphorylation, activation of this signaling pathway was not altered by refeeding in either control or ethanol-treated mice. Related to protein degradation, in vitro-determined proteasome activity and the content of total ubiquitinated proteins were not altered by ethanol and/or refeeding. Control mice appeared to exhibit a refeeding-induced decrease in autophagy as suggested by the increased FoxO3 and ULK1 phosphorylation and total p62 protein as well as decreased LC3B-II; however, ethanol blunted these refeeding-induced changes. These data suggest that ethanol can acutely prevent the normally observed mTOR-dependent increase in protein synthesis and reduction in autophagy in response to nutrient stimulation, but does not appear to acutely alter proteasome activity.

Objectives: NMDA antagonists and nitric oxide synthase (NOS) inhibitors induce antidepressant-like effects and may represent treatment options for depression. The behavioural effects of NMDA antagonists seem to depend on Tyrosine kinase B receptor (TrkB) activation by BDNF and on mechanistic target of rapamycin (mTOR), in the medial prefrontal cortex (mPFC). However, it is unknown whether similar mechanisms are involved in the behavioural effects of NOS inhibitors. Therefore, this work aimed at determining the role of TrkB and mTOR signalling in the prelimbic area of the ventral mPFC (vmPFC-PL) in the antidepressant-like effect of NOS inhibitors., Methods: Pharmacological treatment with LY235959 or ketamine (NMDA antagonists), NPA or 7-NI (NOS inhibitors), BDNF, K252a (Trk antagonist) and rapamycin (mTOR inhibitor) injected systemically or into vmPFC-PL followed by behavioural assessment., Results: We found that bilateral injection of BDNF into the vmPFC-PL induced an antidepressant-like effect, which was blocked by pretreatment with K252a and rapamycin. Microinjection of LY 235959 into the vmPFC-PL induced antidepressant-like effect that was suppressed by local rapamycin but not by K252a pretreatment. Microinjection of NPA induced an antidepressant-like effect insensitive to both K252a and rapamycin. Similarly, the antidepressant-like effects of a systemic injection of ketamine or 7-NI were not affected by blockade of mTOR or Trk receptors in the vmPFC-PL., Conclusion: Our data support the hypothesis that NMDA blockade induces an antidepressant-like effect that requires mTOR but not Trk signalling into the vmPFC-PL. The antidepressant-like effect induced by local NOS inhibition is independent on both Trk and mTOR signalling in the vmPFC-PL.

Gene expression studies revealed a large degree of variability in gene expression patterns particularly in tissues even in genetically identical individuals. It helps to reveal the components majorly fluctuating during the disease condition. With the advent of gene expression studies many microarray studies have been conducted in prostate cancer, but the results have varied across different studies. To better understand the genetic and biological regulatory mechanisms of prostate cancer, we conducted a meta-analysis of three major pathways i.e. androgen receptor (AR), mechanistic target of rapamycin (mTOR) and Mitogen-Activated Protein Kinase (MAPK) on prostate cancer. Meta-analysis has been performed for the gene expression data for the human species that are exposed to prostate cancer. Twelve datasets comprising AR, mTOR, and MAPK pathways were taken for analysis, out of which thirteen potential biomarkers were identified through meta-analysis. These findings were compiled based upon the quantitative data analysis by using different tools. Also, various interconnections were found amongst the pathways in study. Our study suggests that the microarray analysis of the gene expression data and their pathway level connections allows detection of the potential predictors that can prove to be putative therapeutic targets with biological and functional significance in progression of prostate cancer.

Background: We performed profiling of the immune microenvironment of castration-resistant (CRPC) and castration-sensitive (CSPC) prostate cancer (PC) in order to identify novel targets for immunotherapy., Methods: PD-L1 and CD3/CD8 immunohistochemistry, PD-L1/2 fluorescent in situ hybridization, tumor mutation burden, microsatellite instability, and RNA-seq of 395 immune-related genes were performed in 19 CRPC and CSPC. Targeted genomic sequencing and fusion analysis were performed in 17 of these specimens., Results: CD276, PVR, and NECTIN2 were highly expressed in PC. Comparison of CRPC versus CSPC and primary versus metastatic tissue revealed the differential expression of immunostimulatory, immunosuppressive, and epithelial-to-mesenchymal transition (EMT)-related genes. Unsupervised clustering of differentially expressed genes yielded two final clusters best segregated by CRPC and CSPC status., Conclusion: CD276 and the alternative checkpoint inhibition PVR/NECTIN2/CD226/TIGIT pathway emerged as relevant to PC checkpoint inhibition target development., (© 2019 Wiley Periodicals, Inc.)

Previous studies have proposed that caloric restriction (CR) regulates many cell functions and prolongs the lifespan of an organism. Our previous studies proposed that CR also prevents follicular activation and preserves the ovarian reserve in mice by activating SIRT1. To test if SIRT1 preserves the ovarian reserve and prolongs the ovarian longevity, we generated SIRT1 knock-in mice that can overexpress SIRT1 in oocytes of the mouse. Ovaries of the mice at ages 35 days and 15 months were collected, and the follicular development and follicular reserve were examined. The vaginal opening and onset of estrus of transgenic female mice (both the homozygous and heterozygous for SIRT1 overexpression) were later than that of wild-type mice. Both the homozygous and heterozygous SIRT1-overexpressing mice had a larger and stronger reproductive capacity than wild-type mice. Moreover, 35-day-old and 15-month-old homozygous and heterozygous SIRT1-overexpressing mice also had a higher mean number and percentage of healthy follicles, fewer atretic follicles than wild-type mice, and the mean number and percentage of primordial follicles in both the homozygous and heterozygous SIRT1-overexpressing mice were higher than wild-type mice at the same age. However, the phenotypes of heterozygous and homozygous transgenic mice came no difference. Immunohistochemistry showed increased expression of SIRT1 and FOXO3a, and decreased expression of mTOR in both the homozygous and heterozygous SIRT1-overexpressing mice compared with wild-type mice. Thus, oocyte-specific SIRT1-overexpressing mice continuously activate FOXO3a and suppress mTOR and have a larger reproductive capacity, larger follicle reserve and longer ovarian lifespan., (Copyright © 2018. Published by Elsevier B.V.)

Recent studies have shown that intratumoral heterogeneity is prevalent in esophageal squamous cell cancer (ESCC) based on DNA sequencing and chromosome analysis in multiple regions from the same tumor. This study aimed to investigate the expression of ZNF750, EP300, MTOR and KMT2D and their intratumoral heterogeneity (ITH) in patients with ESCC. A total of 106 cases, who underwent esophagectomy from 2008 to 2010, with two foci from each case, were tested by immunohistochemistry(IHC) as well as 12 cases were tested by RNAscope in this study.We found that 58/106 (54.72%), 66/106 (62.26%), 75/106 (70.75%%) of ESCC showed high expression of ZNF750, EP300, MTOR, respectively by IHC, and 8/12 (66.67%), 10/12 (83.33%), 4/12 (33.33%) and 6/12 (50%) showed high expression of ZNF750, EP300, MTOR and KMT2D, respectively by RNAscope. Multivariate analysis showed that MTOR expression was an independent infavorable prognostic factor of overall survival (OS) (HR = 1.921; P = 0.000). This study also found that 44/106(4151%), 37/106 (34.91%), 39/106(36.79%) of ESCC showed heterogeneous expression of ZNF750, EP300 and MTOR respectively by IHC, 8/12(66.67%), 8/12(66.67%), 4/12(33.33%), 4/12(33.33%) of ZNF750, EP300, MTOR and KMT2D respectively by RNAscope, IHC and RNAscope could successfully detect a high prevalence of ITH. In conclusion, findings of this study showed that ZNF750, EP300, MTOR and KMT2D heterogeneously expressed in ESCC. High expression of ZNF750 related to a better outcome, while EP300 and MTOR related to a poor prognosis., (Copyright © 2018. Published by Elsevier GmbH.)

The aim of this study is to investigate the protective effect of epigallocatechin-3-gallate (EGCG) on apoptosis and mTOR/AKT/GSK-3β pathway in substantia nigra neurons in 6-dopamine-induced Parkinson rats. A total of 30 healthy male SD rats were randomly divided into control group, the Parkinson model group, and Parkinson model+EGCG treatment group. The model and EGCG groups were injected into the right striatum with 6-OHDA to establish the Parkinson model, and the control group was injected with saline only. The EGCG group was intragastrically administered with EGCG 50 mg/kg daily for 4 weeks. The rats' turns, speed, and left forelimb usage; neuron apoptosis by TUNEL; and the α-synuclein protein expression in substantia nigra by immunohistochemical staining were studied. Western blotting was used to detect the relative protein (mTOR, AKT and GSK-3β) expressions. Compared with the model group, the EGCG group significantly reduced the rotation speed; increased the left forelimb usage (P<0.01); reduced the neuron apoptosis (P<0.01); decreased α-synuclein expression (P<0.01); and decreased the mTOR, AKT, and GSK-3β protein expressions (P<0.01). EGCG can reduce neuron cell apoptosis in substantia nigra neurons in 6-OHDA-induced Parkinson rats. The mechanism might be related to mTOR/AKT/GSK-3β activation.

Background: Obesity, insulin resistance, and type 2 diabetes are established risk factors for the development of Alzheimer's disease (AD). Given this connection, two drugs, metformin (MET) and resveratrol (RESV), are considered for the clearance of amyloid-β peptides through AMPK-mediated activation of autophagy. However, overactivation of AMPK observed in late-stage AD brains and relationships between AMPK and neurogenesis (through mTORC1 inhibition), questions treatment with these drugs., Objective: To examine if MET and/or RESV supplementation activates brain AMPK, regulates markers of autophagy, and affects markers of neuronal health/neurogenesis., Methods: 8-week-old male C57BL/6J mice were fed a low (N = 12; 10% kcal fat; LFD) or high fat diet (N = 40; 60% kcal fat; HFD) for 9 weeks to induce insulin resistance and obesity. HFD mice were then treated with/without MET (250 mg/kg/day), RESV (100 mg/kg/day), or COMBO (MET: 250 mg/kg/day, RESV: 100 mg/kg/day) for 5 weeks. Hippocampus and prefrontal cortex were extracted for western blotting analysis., Results: Cortex AMPK (T172) and raptor (S792, the regulatory subunit of mTORC1) phosphorylation were upregulated following RESV, COMBO treatments. mTOR (S2448) and ULK1 (S555) activation was seen following MET, COMBO and RESV, COMBO treatments, respectively, in the cortex and hippocampus. p62 content was decreased following RESV, COMBO, with LC3 content being increased following RESV treatment in the cortex. Brain derived neurotropic factor (BDNF) was significantly decreased following RESV, COMBO, and synaptophysin following all treatment in the cortex., Conclusion: These results demonstrate that while treatments upregulated markers of autophagy in the prefrontal cortex, reductions in neuronal health markers question the efficacy of AMPK as a therapy for AD.

Autosomal dominant polycystic kidney disease (ADPKD) is a common heritable human disease. Recently, the role of repressed autophagy in ADPKD has drawn increasing attention. Here, we investigate the mechanism underlying the effect of Saikosaponin-d (SSd), a sarcoplasmic/endoplasmic reticulum Ca 2+ ATPase pump (SERCA) inhibitor. We show that SSd suppresses proliferation in ADPKD cells by up-regulating autophagy. We found that treatment with SSd results in the accumulation of intracellular calcium, which in turn activates the CaMKKβ-AMPK signalling cascade, inhibits mTOR signalling and induces autophagy. Conversely, we also found that treatment with an autophagy inhibitor (3-methyladenine), AMPK inhibitor (Compound C), CaMKKβ inhibitor (STO-609) and intracellular calcium chelator (BAPTA/AM) could reduce autophagy puncta formation mediated by SSd. Our results demonstrated that SSd induces autophagy through the CaMKKβ-AMPK-mTOR signalling pathway in ADPKD cells, indicating that SSd might be a potential therapy for ADPKD and that SERCA might be a new target for ADPKD treatment.

Triple-negative breast cancer (TNBC) is a highly aggressive form of breast cancer. Aberrant expression of genes in mTOR pathway and their targeting miRNAs plays an important role in TNBC. The aim of this study was to determine the expression of mTOR and S6K1 and their targeting miRNAs in breast cancer cell lines and clinical samples. miRNAs targeting 3'-UTR of mTOR and S6K1 mRNAs were predicted using bioinformatic algorithms. MDA-MB-231, MCF-7, and MCF-10A as well as 20 TNBC samples were analyzed for gene and miRNA expression using quantitative real-time PCR (RT-qPCR). A receiver operating characteristic (ROC) curve analysis was performed for evaluation of candidate miRNAs as diagnostic biomarkers. miR-96 and miR-557 targeting mTOR and S6K1 mRNAs, respectively, were selected, and miR-3182 was selected as the miRNA targeting both genes. The miRNAs were down-regulated in cell lines, while their target mRNAs were up-regulated. Similar findings were observed in clinical samples. The ROC curve analysis revealed decline in expression of these miRNAs. We suggest that miR-96, miR-557, and miR-3182 can be used as inhibitory agents for mTOR and S6K1 in TNBC-targeted therapy.

TGFβ promotes podocyte hypertrophy and expression of matrix proteins in fibrotic kidney diseases such as diabetic nephropathy. Both mTORC1 and mTORC2 are hyperactive in response to TGFβ in various renal diseases. Deptor is a component of mTOR complexes and a constitutive inhibitor of their activities. We identified that deptor downregulation by TGFβ maintains hyperactive mTOR in podocytes. To unravel the mechanism, we found that TGFβ -initiated noncanonical signaling controls deptor inhibition. Pharmacological inhibitor of PI 3 kinase, Ly 294002 and pan Akt kinase inhibitor MK 2206 prevented the TGFβ induced downregulation of deptor, resulting in suppression of both mTORC1 and mTORC2 activities. However, specific isoform of Akt involved in this process is not known. We identified Akt2 as predominant isoform expressed in kidney cortex, glomeruli and podocytes. TGFβ time-dependently increased the activating phosphorylation of Akt2. Expression of dominant negative PI 3 kinase and its signaling inhibitor PTEN blocked Akt2 phosphorylation by TGFβ. Inhibition of Akt2 using a phospho-deficient mutant that inactivates its kinase activity, as well as siRNA against the kinase markedly diminished TGFβ -mediated deptor suppression, its association with mTOR and activation of mTORC1 and mTORC2. Importantly, inhibition of Akt2 blocked TGFβ -induced podocyte hypertrophy and expression of the matrix protein fibronectin. This inhibition was reversed by the downregulation of deptor. Interestingly, we detected increased phosphorylation of Akt2 concomitant with TGFβ expression in the kidneys of diabetic rats. Thus, our data identify previously unrecognized Akt2 kinase as a driver of TGFβ induced deptor downregulation and sustained mTORC1 and mTORC2 activation. Furthermore, we provide the first evidence that deptor downstream of Akt2 contributes to podocyte hypertrophy and matrix protein expression found in glomerulosclerosis in different renal diseases., Competing Interests: The authors have declared that no competing interests exist.

Dietary administration of orotic acid (OA), an intermediate in the pyrimidine biosynthetic pathway, is considered to provide a wide range of beneficial effects, including cardioprotection and exercise adaptation. Its mechanisms of action, when applied extracellularly, however, are barely understood. In this study, we evaluated potential effects of OA on skeletal muscle using an in vitro contraction model of electrically pulse-stimulated (EPS) C2C12 myotubes. By analyzing a subset of genes representing inflammatory, metabolic, and structural adaptation pathways, we could show that OA supplementation diminishes the EPS-provoked expression of inflammatory transcripts (interleukin 6, Il6; chemokine (C-X-C Motif) ligand 5, Cxcl5), and attenuated transcript levels of nuclear receptor subfamily 4 group A member 3 (Nr4A3), early growth response 1 (Egr1), activating transcription factor 3 (Atf3), and fast-oxidative MyHC-IIA isoform (Myh2). By contrast, OA had no suppressive effect on the pathogen-provoked inflammatory gene response in skeletal muscle cells, as demonstrated by stimulation of C2C12 myotubes with bacterial LPS. In addition, we observed a suppressive effect of OA on EPS-induced phosphorylation of AMP-activated protein kinase (AMPK), whereas EPS-triggered phosphorylation/activation of the mammalian target of rapamycin (mTOR) was not affected. Finally, we demonstrate that OA positively influences glycogen levels in EP-stimulated myotubes. Taken together, our results suggest that in skeletal muscle cells, OA modulates both the inflammatory and the metabolic reaction provoked by acute contraction. These results might have important clinical implications, specifically in cardiovascular and exercise medicine.

Background: Successful implantation of embryos requires endometrial receptivity. Calcitonin is one of the factors influencing the implantation window. This study aimed to evaluate calcitonin effects on endometrial receptivity. To this end, the effects of calcitonin on the implantation window in the ovarian stimulation and the normal ovarian cycle were investigated by the morphological study of the endometrium as well as the expression of MSX.1, HB-EGF, and micro-RNA (miRNA) Let-7a; then the mechanisms of calcitonin effects were studied through the mammalian target of rapamycin (mTOR) and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathways., Materials and Methods: A total of 64 Bulb/c mice were divided into two groups: Normal ovarian cycle and ovarian stimulation. Each group consisted of four subgroups: Ctrl, CT, PP242, and CT + PP242. Calcitonin and PP242 were injected on the fourth day of pregnancy and 24 hr later all the mice were killed. Uterine tissue samples were used for morphological analysis and the endometrial epithelial and the stromal cells were isolated from myometrium for evaluation of gene and protein expression., Results: Ovarian stimulation increased the phosphorylation levels of mTOR and ERK1/2 and the expression of miRNA Let-7a. Calcitonin injection increased the expression of HB-EGF, Msx.1, and miRNA Let-7a in a normal ovarian cycle and in ovarian-stimulated mice. It also increased eukaryotic initiation factor 4E-binding protein 1 and ERK1/2 phosphorylation in normal ovarian cycles., Conclusion: Calcitonin improved the receptivity of the uterine endometrium by upregulation of the HB-EGF, Msx.1, and miRNA Let-7a likely through mTOR and ERK1/2 signaling pathway., (© 2018 Wiley Periodicals, Inc.)

Alcohol abuse causes brain damage and cognitive dysfunction. However, the underlying mechanisms remain elusive. Endoplasmic reticulum (ER) acts as machinery to ensure the proper folding of newly synthesized proteins. The perturbation of ER, i.e., ER stress, plays a pivotal role in some neurological disorders. Mammalian target of rapamycin (mTOR), a serine/threonine kinase, is involved in the regulation of ER stress. The current study sought to determine whether binge ethanol exposure induces ER stress in adult mouse brain and the role mTOR signaling during this process. Adult C57BL6 mice received binge ethanol exposure by daily gavage (5 g/kg, 25% ethanol w/v) for 1, 5 or 10 days. Binge ethanol exposure caused neurodegeneration and neuroinflammation after 5 days of exposure, and a concomitant increase of ER stress and inhibition of mTOR. However, ethanol exposure did not significantly alter spatial learning and memory, and spontaneous locomotor activity. Ethanol treatment induced ER stress and the death of cultured neuronal cells. Cotreatment with an ER stress inhibitor, sodium 4-phenylbutyrate (4-PBA) significantly diminished ethanol-induced ER stress and neuronal apoptosis, suggesting that ER stress contributes to ethanol-induced neurodegeneration. Furthermore, the blockage of mTOR activity by rapamycin increased ER stress in cultured neuronal cells; whereas the activation or inhibition of ER stress by tunicamycin or 4-PBA respectively had little effects on mTOR signaling. These results suggested that mTOR signaling is upstream of ER stress and may thereby mediate ethanol-induced ER stress., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Objectives: To investigate the mechanism of 2-methoxyestradiol (2-ME) in inhibiting hypoxia-induced collagen synthesis of fibroblasts in SSc., Methods: The expressions of hypoxia-inducible factor 1 alpha (HIF-1α) and connective tissue growth factor (CTGF) in skin specimens derived from SSc patients and healthy volunteers were examined by immunohistochemistry. HIF-1α was knocked down by lentiviral transduction, and SSc dermal fibroblasts cultured under normoxic (21% O2) or hypoxic (1% O2) condition were treated with PI3K inhibitor LY294002, rapamycin or 2-ME (25 μM). The protein levels of HIF-1α, CTGF, collagen I, p-Akt and p-mTOR were examined by western blotting or immunofluorescence. Apoptosis and cell cycle of fibroblasts were assessed by flow cytometry and by measuring caspase 3 activity, and cell proliferation was evaluated by Cell Counting Kit-8., Results: The expressions of HIF-1α and CTGF were increased in skins of SSc patients compared with healthy controls. Hypoxia up-regulated the protein levels of HIF-1α, CTGF and collagen I in SSc fibroblasts. In contrast, 2-ME inhibited PI3K/Akt/mTOR pathway and down-regulated protein levels of HIF-1α, CTGF and collagen I. Knockdown of HIF-1α reduced expressions of CTGF and collagen I, which were further down-regulated by 2-ME intervention. Moreover, 2-ME promoted the apoptosis and inhibited the proliferation of SSc fibroblasts by arresting the cell cycle at the G2/M phase., Conclusion: 2-ME reduced the production of CTGF and collagen I in SSc fibroblasts induced by hypoxia through PI3K/Akt/mTOR/HIF-1α signalling and inhibited the proliferation of fibroblasts. These findings suggested that 2-ME could be employed as a promising antifibrotic therapy for SSc.

Adiponectin (APN), an adipose tissue-excreted adipokine, plays protective roles in metabolic and cardiovascular diseases. In this study, the effects and mechanisms of APN on biological functions of rat vascular endothelial progenitor cells (VEPCs) were investigated in vitro. After administrating APN in rat VEPCs, the proliferation was measured by methyl thiazolyl tetrazolium (MTT) method, the apoptotic rate was test by Flow cytometry assay, mRNA expression of B-cell lymphoma-2 (Bcl-2) and vascular endothelial growth factor (VEGF) was determined by real-time reverse transcriptase polymerase chain reaction (RT-PCR), and protein expression of mechanistic target of rapamycin (mTOR), signal transducer and activator of transcription 3 (STAT3) and phospho-STAT3 (pSTAT3) was analyzed by Western blot. It was suggested that APN promoted the optical density (OD) value of VEPCs, enhanced mRNA expression of Bcl-2 and VEGF, and inhibited cell apoptotic rate. Furthermore, protein expression of pSTAT3 was also increased in the presence of APN. Moreover, APN changed-proliferation, apoptosis and VEGF expression of VEPCs were partially suppressed after blocking the mTOR-STAT3 signaling pathway by the mTOR inhibitor XL388. It was indicated that APN promoted biological functions of VEPCs through targeting the mTOR-STAT3 signaling pathway.

Background: This study aimed to investigate the interactions between silent information regulator 1 (SIRT1) and mammalian target of rapamycin (mTOR) in intraplaque angiogenesis and their potential mechanisms through in vivo and in vitro studies., Methods: An atherosclerosis model was established in 12 rabbits on a high-cholesterol diet. The rabbits were equally divided into 3 groups: a control group (high-lipid diet), RAP group (high-lipid diet supplemented with rapamycin) and RAP + NAM group (high-lipid diet supplemented with rapamycin and nicotinamide). At the end of 4 weeks, the area of plaques in the aorta was determined and the protein expression of CD31 and vascular endothelial growth factor (VEGF) was detected through hematoxylin and eosin staining and immunohistochemical staining, respectively. For in vitro study, a hypoxia model was established in human umbilical vein endothelial cells (HUVECs) by using the chemical method (CoCl 2 ). The MTT assay, scratch assay and tube formation assay were performed to evaluate the proliferation and angiogenesis abilities of HUVECs. Reverse transcription polymerase chain reaction was used to examine the mRNA levels of SIRT1, hypoxia-inducible factor-1α (HIF-1α), mTOR and p70 ribosomal S6 kinase (p70S6K). Western blotting was used to examine the protein levels of SIRT1, HIF-1α, mTOR, p-mTOR, p-raptor and p-p70S6K., Results: The results of the in vivo study indicated a significant inhibitory effect of rapamycin on plaque size and intraplaque angiogenesis (0.05 ± 0.02mm 2 versus 5.44 ± 0.50mm 2 , P < 0.05). This effect was attenuated by nicotinamide (0.76 ± 0.15mm 2 versus 0.05 ± 0.02mm 2 , P < 0.05). Compared with the RAP group, CD31- and VEGF-positive vessels were abundant in the RAP + NAM group. The RAP group showed lower expression of p-mTOR, p-p70S6K and HIF-1α than did the control group (P < 0.05), whereas the RAP + NAM group showed slightly higher expression of these factors than did the RAP group (P < 0.05). Furthermore, in vitro studies revealed that the inhibitory effect of rapamycin on the angiogenic ability of HUVECs and its significant inhibitory effects on the protein level of HIF-1α and the phosphorylation of proteins involved in the mTORC1 pathway, including mTOR, raptor and p70S6K (P < 0.05), were enhanced by cotreatment with SRT1720 and rapamycin (P < 0.05). In contrast to mTOR and SIRT1, the mRNA levels of p70S6K and HIF-1α were reduced by rapamycin (P < 0.05) and further reduced by cotreatment with SRT1720 and rapamycin., Conclusions: The study results indicate that SIRT1 might negatively regulate atherosclerotic angiogenesis via mTORC1 and HIF-1α signaling pathway and cointervention of SIRT1 and mTOR may serve as a crucial therapeutic strategy in cardiovascular medicine., (Copyright © 2018. Published by Elsevier Inc.)

Aims: Muscle mass is regulated by the balance between the synthesis and degradation of muscle proteins. Loss of skeletal muscle mass is associated with an increased risk of developing metabolic diseases such as obesity and type 2 diabetes mellitus. The aim of this study was to clarify the effects of licorice flavonoid oil on muscle mass in KK-A y /Ta mice., Main Methods: Male genetically type II diabetic KK-A y /Ta mice received 0, 1, or 1.5 g/kg BW of licorice flavonoid oil by mouth once daily for 4 weeks. After 4 weeks, the femoral and soleus muscles were collected for western blotting for evaluation of the mTOR/p70 S6K, p38/FoxO3a, and Akt/FoxO3a signaling pathways., Key Findings: Ingestion of licorice flavonoid oil significantly enhanced femoral muscle mass without affecting body weight in KK-A y /Ta mice. Licorice flavonoid oil also decreased expression of MuRF1 and atrogin-1, which are both markers of muscle atrophy. The mechanisms by which licorice flavonoid oil enhances muscle mass include activation of mTOR and p70 S6K, and regulation of phosphorylation of FoxO3a., Significance: Ingestion of licorice flavonoids may help to prevent muscle atrophy., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Colorectal cancer (CRC) is one of the most frequent malignant tumors. Signaling by the PI3K/AKT pathway is crucial for CRC development and progression, including proliferation and migration. Celastrol has an anticancer effect, but its mechanism needs to be determined. Here, we showed that celastrol suppressed CRC cell proliferation and migration. Celastrol treatment also decreased the PI3K/AKT pathway components, and MMP3 and MMP7 expression levels. In addition, knockdown of AKT, not mTOR, inhibited MMP3 and MMP7 expression levels and AKT silencing promoted the celastrol-induced effects on CRC cell proliferation and migration. Taken together, these findings indicated that the celastrol-induced antitumor effects were mediated through MMP3 and MMP7 by the PI3K/AKT signaling pathway.

Impaired autophagic clearance of aggregated α-synuclein is considered as one of key mechanisms underlining Parkinson disease (PD). High-mobility group protein B1 (HMGB1) has recently been demonstrated to mediate persistent neuroinflammation and consequent progressive neurodegeneration by promoting multiple inflammatory and neurotoxic factors. In this study, we examined the influence of the overexpression of wild-type (WT) and mutant-type (MT, A53T and A30P) α-synuclein on the autophagy in neuroblastoma SH-SY5Y cells under starvation, and then investigated the regulation of endogenous HMGB1 on the α-synuclein degradation and on the starvation-induced autophagy in the α-synuclein-overexpressed SH-SY5Y cells. It was demonstrated that the overexpression of WT or MT α-synuclein significantly downregulated the starvation-induced conversion of LC3I to LC3II and autophagy protein (Atg) 5 expression, whereas markedly inhibited the starvation-downregulated mTOR in SH-SY5Y cells. On the other side, the lentivirus-mediated upregulation of endogenous HMGB1 promoted the degradation of WT or MT α-synuclein in SH-SY5Y cells autophagy-dependently via promoting Atg 5, but not mTOR, the Atg 5 knockdown downregulated the HMGB1-mediated promotion to α-synuclein degeneration. Thus, we concluded that α-synuclein inhibited the starvation-induced autophagy in neuroblastoma SH-SY5Y cells via inhibiting the mTOR/Atg 5 signaling. However, the endogenous HMGB1 promoted the autophagic degradation of α-synuclein via the Atg 5-dependent autophagy-initiation pathway, implying the protective role of endogenous HMGB1 in the neuroblastoma cells against the α-synuclein accumulation., (Copyright © 2018. Published by Elsevier Inc.)

Assisted-treadmill training, may be helpful in promoting muscle mass preservation after incomplete spinal cord injury (SCI). However, biological mechanism involved in this process is still not fully understood. This study investigated the effects of locomotor treadmill training on muscle trophism mediated by protein kinase B (Akt)/mammalian target of rapamycin (mTOR)/p70 ribosomal protein S6 kinase (p70S6K) in paraplegic rats. Adult female Wistar rats underwent an incomplete thoracic SCI induced by compression using an aneurysm clip. After 7 days, injured animals started a 3-week locomotor treadmill training with body weight-support and manual step help. Soleus trophism was measured by muscle weight and transverse myofiber cross-sectional area (CSA). An enzyme-linked immunosorbent assay (ELISA) and western blot analysis were used to detect brain-derived neurotrophic factor (BDNF), tropomyosin-related kinase B (TrkB), Akt, mTOR and p70S6K in paretic soleus. Trained animals did not show locomotor improved, but present an increase in muscle weight and myofiber CSA. Furthermore, the levels of Akt, p70S6K phosphorylation, mTOR and TrkB receptor were increased by training in soleus. In contrast, muscle BDNF levels were significantly reduced after training. The results suggest locomotor treadmill training partially reverts/prevents soleus muscle hypotrophy in rats with SCI. Furthermore, this study provided the first evidence that morphological muscle changes were caused by Akt/mTOR/p70S6K signaling pathway and TrkB up-regulation, which may increase the sensitivity of muscle, reducing autocrine signaling pathway demand of BDNF for cell growth.

Notoginsenoside R1 (NGR1) is a predominant phytoestrogen extracted from Panax notoginseng that has recently been reported to play important roles in the treatment of cardiac dysfunction, diabetic kidney disease, and acute liver failure. Studies have suggested that NGR1 may be a viable treatment of hypoxic-ischemic brain damage (HIBD) in neonates by reducing endoplasmic reticulum stress via estrogen receptors (ERs). However, whether NGR1 has other neuroprotective mechanisms or long-term neuroprotective effects is unclear. In this study, oxygen-glucose deprivation/reoxygenation (OGD/R) in primary cortical neurons and unilateral ligation of the common carotid artery (CCL) in 7-day-old postnatal Sprague Dawley (SD) rats followed by exposure to a hypoxic environment were used to mimic an HIBD episode. We assessed the efficacy of NGR1 by measuring neuronal damage with MTT assay and assessed brain injury by TTC staining and brain water content detection 24-48 h after OGD/HIE. Simultaneously, we measured the long-term neurophysiological effects using the beam walking test (5 weeks after HI) and Morris water maze test 5-6 weeks after HI. Expression of PI3K-Akt-mTOR/JNK (24 h after HI or OGD/R) proteins was detected by Western blotting after stimulation with HI, NGR1, LY294002 (PI3K inhibitor), 740Y-P (PI3K agonist), or ICI 182780(estrogen receptors inhibitor). The results indicated that NGR1 exerted neuroprotective effects by inhibiting neuronal apoptosis and promoting cell survival via the PI3K-Akt-mTOR/JNK signaling pathways by targeting ER in neonatal hypoxic-ischemic injury.

Background: Lymphangioleiomyomatosis (LAM) is a low-grade neoplasm characterized by the pulmonary infiltration of smooth muscle-like cells (LAM cells) and cystic destruction. Patients usually present with airway obstruction in pulmonary function tests (PFTs). Previous studies have shown correlations among histological parameters, lung function abnormalities and prognosis in LAM. We investigated the lung tissue expression of proteins related to the mTOR pathway, angiogenesis and enzymatic activity and its correlation with functional parameters in LAM patients., Methods: We analyzed morphological and functional parameters of thirty-three patients. Two groups of disease severity were identified according to FEV1 values. Lung tissue from open biopsies or lung transplants was immunostained for SMA, HMB-45, mTOR, VEGF-D, MMP-9 and D2-40. Density of cysts, density of nodules and protein expression were measured by image analysis and correlated with PFT parameters., Results: There was no difference in the expression of D2-40 between the more severe and the less severe groups. All other immunohistological parameters showed significantly higher values in the more severe group (p ≤ 0.002). The expression of VEGF-D, MMP-9 and mTOR in LAM cells was associated with the density of both cysts and nodules. The density of cysts and nodules as well as the expression of MMP-9 and VEGF-D were associated with the impairment of PFT parameters., Conclusions: Severe LAM represents an active phase of the disease with high expression of VEGF-D, mTOR, and MMP-9, as well as LAM cell infiltration. Our findings suggest that the tissue expression levels of VEGF-D and MMP-9 are important parameters associated with the loss of pulmonary function and could be considered as potential severity markers in open lung biopsies of LAM patients.

Shikonin, a natural naphthoquinone isolated from a traditional Chinese medicinal herb, which exerts anticancer effects in various cancers. However, the molecular mechanisms underlying the therapeutic effects of shikonin against endometrioid endometrial cancer (EEC) have not yet been fully elucidated. Herein, we investigated anticancer effects of shikonin on EEC cells and explored the underlying molecular mechanism. We observed that shikonin inhibits proliferation in human EEC cell lines in a dose-dependent manner. Moreover, shikonin-induced apoptosis was characterized by the up-regulation of the pro-apoptotic proteins cleaved-Caspase-3 and Bax, and the down-regulation of the anti-apoptotic protein Bcl-2. Microarray analyses demonstrated that shikonin induces many miRNAs' dysregulation, and miR-106b was one of the miRNAs being most significantly down-regulated. miR-106b was identified to exert procancer effect in various cancers, but in EEC remains unclear. We first confirmed that miR-106b is up-regulated in EEC tissues and cells, and knockdown of miR-106b suppresses proliferation and promotes apoptosis. Meanwhile, our results validated that the restored expression of miR-106b abrogates the antiproliferative and pro-apoptotic effects of shikonin. We also identified that miR-106b targets phosphatase and tensin homolog ( PTEN ), a tumor suppressor gene, which in turn modulates AKT/mTOR signaling pathway. Our findings indicated that shikonin inhibits proliferation and promotes apoptosis in human EEC cells by modulating the miR-106b /PTEN/AKT/mTOR signaling pathway, suggesting shikonin could act a potential therapeutic agent in the EEC treatment., (© 2018 The Author(s).)