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Abstract: Quercetin is known for its antihypertensive effects. However, its role on hypertensive renal injury has not been fully elucidated. In this study, hematoxylin and eosin staining, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining, and Annexin V staining were used to assess the pathological changes and cell apoptosis in the renal tissues of angiotensin II (Ang II)-infused mice and Ang II-stimulated renal tubular epithelial cell line (NRK-52E). A variety of technologies, including network pharmacology, RNA-sequencing, immunohistochemistry, and Western blotting, were performed to investigate its underlying mechanisms. Network pharmacology analysis identified multiple potential candidate targets (including TP53, Bcl-2, and Bax) and enriched signaling pathways (including apoptosis and p53 signaling pathway). Quercetin treatment significantly alleviated the pathological changes in renal tissues of Ang II-infused mice and reversed 464 differentially expressed transcripts, as well as enriched several signaling pathways, including those related apoptosis and p53 pathway. Furthermore, quercetin treatment significantly inhibited the cell apoptosis in renal tissues of Ang II-infused mice and Ang II-stimulated NRK-52E cells. In addition, quercetin treatment inhibited the upregulation of p53, Bax, cleaved-caspase-9, and cleaved-caspase-3 protein expression and the downregulation of Bcl-2 protein expression in both renal tissue of Ang II-infused mice and Ang II-stimulated NRK-52E cells. Moreover, the molecular docking results indicated a potential binding interaction between quercetin and TP53. Quercetin treatment significantly attenuated hypertensive renal injury and cell apoptosis in renal tissues of Ang II-infused mice and Ang II-stimulated NRK-52E cells and by targeting p53 may be one of the potential underlying mechanisms., Competing Interests: The authors reports no conflicts of interest., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Objective: To explore the regulatory effect of Pien Tze Huang (PZH) on targeting partner of NOB1 (PNO1) and it's down-stream mediators in colorectal cancer (CRC) cells., Methods: Quantitative polymerase chain reaction was performed to determine mRNA levels of PNO1, TP53, and CDKN1A. Western blotting was performed to determine protein levels of PNO1, p53, and p21. HCT-8 cells were transduced with a lentivirus over-expressing PNO1. Colony formation assay was used to detect cell survival in PNO1 overexpression of HCT-8 cells after PZH treatment. Cell-cycle distribution, cell viability and cell apoptosis were performed to identify the effect of PNO1 overexpression on cell proliferation and apoptosis of HCT-8 cells after PZH treatment. Xenograft BALB/c nude mice bearing HCT116 cells transduced with sh-PNO1 or sh-Ctrl lentivirus were evaluated. Western blot assay was performed to detect PNO1, p53, p21 and PCNA expression in tumor sections. Terminal deoxynucleotidyl transferase dUTP nick end labling (TUNEL) assay was used to determine the apoptotic cells in tissues., Results: PZH treatment decreased cell viability, down-regulated PNO1 expression, and up-regulated p53 and p21 expressions in HCT-8 cells (P<0.05). PNO1 overexpression attenuated the effects of PZH treatment, including the expression of p53 and p21, cell growth, cell viability, cell cycle arrest and cell apoptosis in vitro (P<0.05). PNO1 knockdown eliminated the effects of PZH treatment on tumor growth, inhibiting cell proliferation inhibition and apoptosis induction in vivo (P<0.05). Similarly, PNO1 knockdown attenuated the effects of PZH treatment on the down-regulation of PNO1 and up-regulation of p53 and p21 in vivo (P<0.05)., Conclusion: The mechanism by which PZH induces its CRC anti-proliferative effect is at least in part by regulating the expression of PNO1 and its downstream targets p53 and p21., (© 2024. The Chinese Journal of Integrated Traditional and Western Medicine Press and Springer-Verlag GmbH Germany, part of Springer Nature.)

As a main extraction compound from Scutellaria baicalensis Georgi, Baicalin exhibits various biological activities. However, the underlying mechanism of Baicalin on hypertension-induced heart injury remains unclear. In vivo, mice were infused with angiotensin II (Ang II; 500 ng/kg/min) or saline using osmotic pumps, followed by intragastrically administrated with Baicalin (5 mg/kg/day) for 4 weeks. In vitro, H9C2 cells were stimulated with Ang II (1 μM) and treated with Baicalin (12.5, 25 and 50 μM). Baicalin treatment significantly attenuated the decrease in left ventricular ejection fraction and left ventricular fractional shortening, increase in left ventricular mass, left ventricular systolic volume and left ventricular diastolic volume of Ang II infused mice. Moreover, Baicalin treatment reversed 314 differentially expressed transcripts in the cardiac tissues of Ang II infused mice, and enriched multiple enriched signalling pathways (including apoptosis, autophagy, AMPK/mTOR signalling pathway). Consistently, Baicalin treatment significantly alleviated Ang II-induced cell apoptosis in vivo and in vitro. Baicalin treatment reversed the up-regulation of Bax, cleaved-caspase 3, cleaved-caspase 9, and the down-regulation of Bcl-2. Meanwhile, Baicalin treatment alleviated Ang II-induced increase of autophagosomes, restored autophagic flux, and down-regulated LC3II, Beclin 1, as well as up-regulated SQSTM1/p62 expression. Furthermore, autophagy inhibitor 3-methyladenine treatment alleviated the increase of autophagosomes and the up-regulation of Beclin 1, LC3II, Bax, cleaved-caspase 3, cleaved-caspase 9, down-regulation of SQSTM1/p62 and Bcl-2 expression after Ang II treated, which similar to co-treatment with Baicalin. Baicalin treatment reduced the ratio of p-AMPK/AMPK, while increased the ratio of p-mTOR/mTOR. Baicalin alleviated Ang II-induced cardiomyocyte apoptosis and autophagy, which might be related to the inhibition of the AMPK/mTOR pathway., (© 2024 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Esophageal cancer (EC) is the sixth leading cause of cancer‑related mortality worldwide, with the incidence gradually increasing each year. Therefore, further clarifying the mechanism underlying the development of EC may be beneficial for identifying novel biomarkers and targets for its treatment. The present study aimed to determine the functional roles of glioblastoma‑amplified sequence (GBAS), a newly identified gene that has been reported to play crucial roles in multiple types of cancer, including in the malignant behavior of EC cells, such as cell viability, colony formation, cell apoptosis and cell cycle progression. The results of the present study revealed that, in vitro , the knockdown of GBAS significantly suppressed cell viability and colony formation in TE‑1 and KYSE‑150 cell lines, using a Celigo cell count analysis and colony formation assay respectively, whereas the apoptotic rate of EC cells was significantly increased by the knockdown of GBAS using Annexin V APC staining. Furthermore, following GBAS knockdown, the cell cycle progression of TE‑1 and KYSE‑150 cells was arrested in the G1 phase using PI staining. In conclusion, the findings of the present study suggested that GBAS may serve a role in EC by regulating cell viability, apoptosis and cell cycle progression.

Ethnopharmacological Relevance: Kuanxiong aerosol (KXA) is a common clinical drug based on Fangxiang Wentong (FXWT) therapy in the treatment of angina pectoris. However, the pharmacological mechanism of KXA in the prevention and treatment of myocardial injury (MI) is not clear., Aim of the Study: The purpose of this study was to explore the protective effect of KXA on isoproterenol (ISO)-induced MI in rats., Materials and Methods: The study included male Wistar Kyoto rats (age: 6 weeks). The rats were randomly divided into the following 5 groups (n = 6 per group): control group, ISO group, isosorbide mononitrate (ISMN) group (5 mg/kg), KXA-L group (0.1 mL/kg), and KXA-H group (0.3 mL/kg). The rats in the last three groups were given intragastric administration for 14 days, and rats in control group and ISO group were given the same amount of normal saline daily. ISO (120 mg/kg) was used to induce MI on the 13th and 14th days. We assessed electrocardiograms (ECGs), myocardial specific enzymes, histopathological changes, and apoptosis., Results: We found that KXA reduced the increase in the ST-segment amplitude (elevation or depression) and the levels of myocardial marker enzymes induced by ISO in MI rats, improved the pathological changes in myocardial tissue, and reduced cardiomyocyte apoptosis. At the same time, KXA significantly inhibited the up-regulation of caspase-3 and Bax expression and down-regulation of Bcl-2 expression induced by ISO. RNA sequencing showed that 90 up-regulated genes induced by ISO were down-regulated after KXA treatment, whereas 27 down-regulated genes induced by ISO were up-regulated after KXA treatment. In addition, KEGG pathway enrichment analysis showed that the mitogen-activated protein kinase (MAPK) signaling pathway may be an important target of KXA in the treatment of ISO-induced MI in rats. The results of RNA sequencing verified by Western blot analysis showed that KXA significantly inhibited the activation of the ISO-induced MAPK pathway., Conclusions: KXA improves cardiac function in MI rats by inhibiting apoptosis mediated by the MAPK signaling pathway., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: FMRP is a selective mRNA-binding protein that regulates protein synthesis at synapses, and its loss may lead to the impairment of trace fear memory. Previously, we found that FMRP levels in the hippocampus of rats with post-traumatic stress disorder (PTSD) were decreased. However, the mechanism underlying these changes remains unclear., Methods: Forty-eight male Sprague-Dawley rats were randomly divided into four groups. The experimental groups were treated with the single-prolonged stress (SPS) procedure and injected with a lentivirus-mediated inhibitor of miR-142-5p. Behavior test as well as morphology and molecular biology experiments were performed to detect the effect of miR-142 downregulation on PTSD, which was further verified by in vitro experiments., Results: We found that silence of miRNA-142 (miR-142), an upstream regulator of FMRP, could alleviate PTSD-like behaviors of rats exposed to the SPS paradigm. MiR-142 silence not only decreased the levels of proinflammatory mediators, such as interleukin-1β, interleukin-6, and tumor necrosis factor-α, but also increased the expressive levels of synaptic proteins including PSD95 and synapsin I in the hippocampus, which was one of the key brain regions associated with PTSD. We further detected that miR-142 silence also downregulated the transportation of nuclear factor kappa-B (NF-κB) into the nuclei of neurons and might further affect the morphology of neurons., Conclusions: The results revealed miR-142 downregulation could alleviate PTSD-like behaviors through attenuating neuroinflammation in the hippocampus of SPS rats by binding to FMRP.

Qingda granule (QDG), simplified from Qingxuan Jiangya Decoction, is a well-known traditional Chinese medicine formula that has been used for decades to treat hypertension. However, the cardioprotective effects of QDG on Ang II-induced hypertension remain unknown. This study aimed to investigate the effects of QDG on hypertension-induced cardiac hypertrophy and apoptosis, as well as explore its underlying mechanisms. Mice were infused with Ang II (500 ng/kg/min) or saline solution as control, then administered oral QDG (1.145 g/kg/day) or saline for two weeks. QDG treatment attenuated the elevation in blood pressure caused by Ang II, as well as the decreased left ventricle ejection fractions and fractional shortening. Moreover, QDG treatment significantly alleviated the Ang II-induced elevation of the ratio of heart weight to tibia length, as well as cardiac injury, hypertrophy, and apoptosis. In cultured H9C2 cells stimulated with Ang II, QDG partially reversed the increase in cell surface area and number of apoptotic cells, up-regulation of hypertrophy markers ANP and BNP, and activation of caspases-9 and -3. QDG also partially reversed Ang II-induced accumulation of reactive oxygen species (ROS), depolarization of the mitochondrial membrane, release of cytochrome C, up-regulation of Bax, and decrease in levels of p-PI3K, p-AKT, and Bcl-2. These results suggest that QDG can significantly attenuate Ang II-induced hypertension, cardiac hypertrophy and apoptosis, and it may exert these effects in part by suppressing ROS production and activating the PI3K/AKT signaling pathway., (Copyright © 2020 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Huoxin Pill (HXP), a Traditional Chinese Medicine, is used widely to treat patients with coronary heart disease and angina pectoris in China. However, the underlying protective mechanism of HXP on cardiac apoptosis and fibrosis has never been evaluated. Therefore, the aim of this study was to investigate the role of HXP in a myocardial infarction (MI) mouse model. The mice were randomly divided into 3 groups and subjected to surgical ligation of the left anterior descending (LAD) coronary artery or sham surgery (n = 6 for each group) and treated with HXP (50 mg/kg/day) or saline by gavage for 2 weeks. At 2 weeks post MI, we found that HXP significantly enhanced myocardial function and attenuated the increase of heart weight index (HWI) and pathological changes in MI mice. RNA-sequencing and KEGG pathway analyses identified 660 differentially expressed genes and multiple enriched signaling pathways including p53 and TGF-β. In support of these findings, HXP attenuated cardiac apoptosis and decreased p53 and Bax protein expression, while increasing Bcl-2 protein expression in cardiac tissues of MI mice. Furthermore, HXP treatment inhibited cardiac fibrosis and significantly down-regulated TGF-β1 protein expression and Smad2/3 phosphorylation in cardiac tissues. In summary, HXP can improve cardiac function in mice after MI by attenuating cardiac apoptosis and fibrosis partly via supression of the p53/Bax/Bcl-2 and TGF-β1/Smad2/3 pathways., (Copyright © 2020 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Salidroside possesses excellent anti-tumor activity in many types of malignant tumor. In present study, we focused on the effects of salidroside on hepatocellular carcinoma (HCC). The viability of human HCC cells was assayed using MTT. Apoptosis in the cells and tissues samples were detected by Annexin V/PI or TUNEL staining assays. The levels of apoptosis and endoplasmic reticulum (ER) stress related proteins were measured by western blotting analysis. We found salidroside significantly suppressed cell viability and promoted apoptosis in HCC cells. Salidroside could activate intrinsic and extrinsic apoptotic pathways, by increasing activities of caspase-3, caspase-8 and caspase-9, up-regulating levels of Bax, Cytochrome c and decreasing level of Bcl-2 in HepG2 cells. Moreover, it was found salidroside induced ER stress and increased expression of p-PERK, eIF2a, p-eIF2a, ATF-6 and CHOP in HepG2 cells. Interestingly, knockdown of CHOP impaired salidroside induced inhibitory effects on HepG2 cells, suggesting the important role of ER stress in cytotoxic effect of salidroside. Finally, we have confirmed salidroside induced ER stress and inhibited development of HepG2 in an xenograft mouse model. In conclusion, our data suggest salidroside inhibits viability and induces apoptosis of HCC both in vitro and vivo, and this effect is partially mediated by activation of ER stress., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Aims: Central diabetes insipidus (CDI), a typical complication caused by pituitary stalk injury, often occurs after surgery, trauma, or tumor compression around hypothalamic structures such as the pituitary stalk and optic chiasma. CDI is linked to decreased arginine vasopressin (AVP) neurons in the hypothalamic supraoptic nucleus and paraventricular nucleus, along with a deficit in circulating AVP and oxytocin. However, little has been elucidated about the changes in AVP neurons in CDI. Hence, our study was designed to understand the role of several pathophysiologic changes such as endoplasmic reticulum (ER) stress and apoptosis of AVP neurons in CDI., Methods: In a novel pituitary stalk electric lesion (PEL) model to mimic CDI, immunofluorescence and immunoblotting were used to understand the underlying regulatory mechanisms., Results: We reported that in CDI condition, generated by PEL, ER stress induced apoptosis of AVP neurons via activation of the PI3K/Akt and ERK pathways. Furthermore, application of N-acetylcysteine protected hypothalamic AVP neurons from ER stress-induced apoptosis through blocking the PI3K/Akt and ERK pathways., Conclusion: Our findings showed that AVP neurons underwent apoptosis induced by ER stress, and ER stress might play a vital role in CDI condition through the PI3K/Akt and ERK pathways., (© 2018 The Authors. CNS Neuroscience & Therapeutics Published by John Wiley & Sons Ltd.)

Objective: To investigate the effects of ethanol extract of Patrinia scabiosaefolia (EEPS) on chemo-resistance of colorectal cancer cells (CRC) and explore the possible molecular mechanisms., Methods: 5-fluorouracil (5-FU)-resistant human colorectal carcinoma cell line (HCT-8/5-FU) and its parental cells HCT-8 were treated with EEPS (0, 0.25, 0.50, 1 or 2 mg/mL), or 5-FU (0, 100, 200, 400, 800 or 1600 μmol/L). The 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay was performed to evaluate the cell viability. Cell density was observed by phase-contrast microscope, cell counting and colony formation assay were used to determine the cell proliferation of HCT-8/5-FU cells treated with 0, 0.5, 1 or 2 mg/mL EEPS. Cell apoptosis was determined by Hoechst staining. Western-blot was performed to detect the phosphorylation of AKT as well as the protein expression level of B-cell CLL/lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax)., Results: Compared with HCT-8 cells, MTT assay results indicated that HCT-8/5-FU cells were resistant to 5-FU treatment (P<0.05), and sensitive to EEPS treatment (P>0.05). Moreover, compared with untreated HCT-8/5-FU cells, 1 and 2 mg/mL of EEPS treatment significantly reduced cell density, cell number, inhibited cell survival (P<0.05), and induced apoptosis in HCT-8/5-FU cells. Furthermore, 1 and 2 mg/mL of EEPS significantly decreased the phosphorylation level of p-AKT and Bcl-2 protein expression, and increased the expression of Bax protein (P<0.05)., Conclusion: EEPS is a promising therapeutic agent that may overcome chemo-resistance in cancer cells, likely through suppression of the AKT pathway and promotion of cancer cell apoptosis.

Apoptosis and receptor-interacting protein kinase 1/3(RIPK1/3)-mediated necroptosis contribute to the cerebral ischemia/reperfusion (I/R) injury. Emricasan is an inhibitor of caspases in clinical trials for liver diseases while ponatinib could be a potential inhibitor for RIPK1/3. This study aims to investigate the effect of emricasan and/or ponatinib on cerebral I/R injury and the underlying mechanisms. Firstly, we evaluated the status of apoptosis and necroposis in a rat model of cerebral I/R under different conditions, which showed noticeable apoptosis and necroptosis under condition of 2-h ischemia and 24-h reperfusion; next, the preventive or therapeutic effect of emricasan or ponatinib on cerebral I/R injury was tested. Administration of emricasan or ponatinib either before or after ischemia could decrease the neurological deficit score and infarct volume; finally, the combined therapeutic effect of emricasan with ponatinib on I/R injury was examined. Combined application of emricasan and ponatinib could further decrease the I/R injury compared to single application. Emricasan decreased the activities of capase-8/-3 in the I/R-treated brain but not the protein levels of necroptosis-relevant proteins: RIPK1, RIPK3, and mixed lineage kinase domain-like (MLKL), whereas ponatinib suppressed the expressions of these proteins but not the activities of capase-8/-3. Combination of emricasan with ponatinib could suppress both capase-8/-3 and necroptosis-relevant proteins. Based on these observations, we conclude that combination of emricasan with ponatinib could synergistically reduce I/R injury in rat brain through simultaneous prevention of apoptosis and necroptosis. Our findings might lay a basis on extension of the clinical indications for emricasan and ponatinib in treating ischemic stroke.

BACKGROUND Previous studies suggested that inhibition of apoptosis prevents the dysfunction of ischemia-reperfusion injury. In the pathogenesis of ischemia-reperfusion injury, JNK/ERK1/2 and p38 play an essential role in regulation of cell apoptosis. Electroacupuncture (EA), a form of acupuncture, has demonstrated superiority in preventing ischemia-reperfusion injury, but the underlying mechanism is unclear. In the present study, we explored the effects of electroacupuncture at Shenting (GV24) and Baihui (GV20) acupoints on focal cerebral ischemia-reperfusion (MCAO) rats, and explored whether JNK/ERK1/2- and p38-mediated cell apoptosis are involved. MATERIAL AND METHODS The rats were divided into a sham operation control group, an ischemia group, and an electroacupuncture group with acupuncture applied for 10 days (30 min per day). TTC staining was used to calculate the ischemic brain volume. TUNEL staining and transmission electron microscopy were used to detect cell apoptosis. Western blot analysis and Bio-Plex were used to detect JNK, p38, ERK1/2, Bcl-2, and Bax protein expression. RESULTS We found that electroacupuncture at day 10 significantly reduced cerebral infarction. In addition, electroacupuncture suppressed activation of JNK and p38, while enhancing the activation of ERK1/2 in the peri-ischemic regions. Consequently, the effect of electroacupuncture on these pathways resulted in the inhibition of apoptosis, which was demonstrated by TUNEL and transmission electron microscopy. We found that electroacupuncture upregulated the anti-apoptotic Bcl-2/Bax ratio in peri-ischemic regions. CONCLUSIONS Our findings suggest that inhibition of cell apoptosis via regulating multiple signaling pathways might be a mechanism whereby electroacupuncture has a positive therapeutic effect on post-stroke impairment.

Ethnopharmacological Relevance: Pien-Tze-Huang (PZH) is a famous formula of traditional Chinese medicine used to treating stroke. However, the protective effect of PZH and its mechanisms in acute ischemic stroke remain to be explored., Aim of the Study: To investigate the protective effect of PZH on neuronal apoptosis in acute cerebral ischemic injury rats and explore its underlying mechanisms., Materials and Methods: The effects of PZH were studied in acute ischemic stroke rats induced by transient middle cerebral artery occlusion, and the mitochondria-mediated apoptotic proteins including cytochrome C (Cyt C), Bax, Bcl-xl, P53, caspase-3, and caspase-9 as well as AKT and glycogen synthase kinase-3 beta (GSK-3β) were assessed., Results: Four days of PZH treatment (180 mg/kg) could significantly reduce cerebral infarct volume, improve neurological deficit, attenuate inflammatory response, and inhibit neuronal apoptosis in acute ischemic stroke rats. Moreover, PZH treatment significantly decreased cytosolic Cyt C, Bax, P53, cleaved caspase-3, and cleaved caspase-9 levels, but elevated mitochondrial Cyt C and Bcl-xl levels. PZH treatment also increased phosphorylation of AKT and GSK-3β., Conclusion: PZH potently protects the brain from cerebral ischemia/reperfusion injury in vivo, and inhibiting mitochondria-mediated neuronal apoptosis as well as attenuating inflammatory responses may be involved in this effect. This study provides experimental basis of PZH in treating acute cerebral ischemic stroke, which would provide some novel insights for its prevention and treatment of ischemic stroke., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Hedyotis diffusa Willd (HDW) is a major component frequently used in Traditional Chinese Medicine for the clinical treatment of colorectal cancer (CRC) and its associated drug resistance. However, the underlying mechanism of HDW circumventing drug resistance of cancer cells remains to be elucidated. Cancer cell resistance to apoptosis and activation of the phosphatidylinositol‑3‑kinase (PI3K)/protein kinase B (AKT) signaling pathway have been implicated as major factors in the acquired resistance to chemotherapeutic anti‑cancer drugs. The present study investigated the effect and mechanisms of action of ethanol extract of Hedyotis diffusa Willd (EEHDW) on the proliferation and apoptosis of CRC 5‑fluorouracil (5‑FU) resistant (HCT‑8/5‑FU) cells. CRC HCT‑8/5‑FU cell viability following treatment with EEHDW was determined using MTT and colony formation assay. In addition, Annexin V/propidium iodide staining with flow cytometry analysis and 4',6‑diamidino‑2‑phenylindole staining were used to determine the apoptosis of drug‑resistant cancer cells following treatment with EEHDW. The mRNA and protein expression levels of B cell leukemia/lymphoma (Bcl‑2), Bcl‑2 associated X (Bax), cyclin dependent kinase 4 (CDK4), cyclin D1 and p21 were evaluated using reverse transcription‑polymerase chain reaction and western blot analysis, respectively. Furthermore, activation of the PI3K/AKT signaling pathway and expression of phosphatase and tensin homolog (PTEN), PI3K, AKT and phosphorylated (p)‑AKT were determined. EEHDW significantly reduced cell viability, inhibited cell colony formation and promoted apoptosis of HCT‑8/5‑FU cells. Furthermore, EEHDW significantly downregulated the expression of Bcl‑2, cyclin D1 and CDK4 and upregulated the expression of Bax and p21. In addition, EEHDW inhibited the activation of the PI3K/AKT pathway by increasing expression of PTEN and suppressing the expression of PI3K and p‑AKT. The present study provided the first direct evidence that EEHDW may overcome drug‑resistance in human CRC cells by inhibiting PI3K/AKT signaling pathway and provides a basis for the improved therapeutic use of HDW in the clinical treatment of cancer.

Autophagy may have protective effects in renal ischemia-reperfusion (I/R) injury, although the underlying mechanisms remain unclear. Augmenter of liver regeneration (ALR), a widely distributed multifunctional protein that is originally identified as a hepatic growth factor, may participate in the process of autophagy. To investigate the role of ALR in autophagy, ALR expression is knocked-down in human kidney 2 (HK-2) cells with short hairpin RNA lentivirals. Then, the level of autophagy is measured in the shRNA/ALR group and the shRNA/control group in an in vitro model of ischemia-reperfusion (I/R). The results indicate that the level of autophagy in two groups increase, accompanied by increased reactive oxygen species production, especially in the shRNA/ALR group. The AMPK/mTOR signaling pathway is hyperactive in the shRNA/ALR group. Inhibition of autophagy with the AMPK inhibitor compound C induce apoptosis, especially in the shRNA/ALR group. These findings collectively indicate that ALR negatively regulates the autophagy process through an association with the AMPK/mTOR signaling pathway. Autophagy inhibit apoptosis and play a protective role under conditions of oxidative stress.

Qingxuan Jiangya Decoction (QXJYD), a traditional Chinese medicine formula prescribed by academician Ke-ji Chen, has been used in China to clinically treat hypertension for decades of years. However, the molecular mechanisms of its action remain largely unknown. In this study, we examined the therapeutic efficacy of QXJYD against elevated systolic blood pressure in the spontaneously hypertensive rat (SHR) model, and investigated the underlying molecular mechanisms. We found that oral administration of QXJYD significantly reduced the elevation of systolic blood pressure in SHR but had no effect on body weight change. Additionally, QXJYD treatment significantly decreased the media thickness and ratio of media thickness/lumen diameter in the carotid arteries of SHR. Moreover, QXJYD remarkably promoted apoptosis of vascular smooth muscle cells and reduced the expression of anti-apoptotic B-cell leukemia/lymphoma 2. Furthermore, QXJYD significantly decreased the plasma Angiotensin II level in SHR. Collectively, our findings suggest that reversing vascular remodeling via inducing VSMC apoptosis could be one of the mechanisms whereby QXJYD treats hypertension.

Acquired resistance to epidermal growth factor inhibitors has been reported to be associated with cross‑resistance to radiation. Paris Saponins (PSs) exert a wide range of pharmacological activities, including cell apoptosis induction, multidrug resistance inhibition, angiogenesis inhibition and tumor cell migration by modulating various signaling pathways. The present study aimed to investigate the radiosensitization effects of PSII, PSVI and PSVII in a gefitinib‑resistant PC‑9‑ZD lung adenocarcinoma cell line, and the possible mechanism underlying their function. A clonogenic assay was performed to determine the effects of PS radiosensitization on the PC‑9‑ZD cell line. The cell cycle was analyzed by flow cytometry, and cell apoptosis was analyzed with Annexin V/propidium iodide and Hoechst staining. Protein expression levels were detected by western blotting. The results of the present study revealed a significant increase in PC‑9‑ZD cell line radiosensitivity following treatment with PSs. PSs induced G2/M cell cycle phase arrest and apoptosis of the irradiated PC‑9‑ZD cells. Notably, the expression levels of B cell lymphoma 2 (Bcl‑2) were downregulated, and those of caspase‑3, Bcl‑2‑associated X protein (Bax) and p21/Waf1/Cip1 were upregulated following treatment with PSs. The present results demonstrated that PSs induced radiosensitivity in gefitinib‑resistant cells by inducing G2/M phase arrest and by enhancing the apoptotic response via the modulation of caspase‑3, Bax, Bcl‑2 and p21/Waf1/Cip1 expression.

Palmitate has been implicated in the induction of cardiomyocyte apoptosis via reducing the activity of 5' AMP-activated protein kinase (AMPK). We sought to evaluate whether high-density lipoproteins (HDLs), known for their cardioprotective features and their potential to increase AMPK activity, can reduce palmitate-induced cardiomyocyte apoptosis and whether this effect is AMPK-dependent. Therefore, cardiomyocytes were isolated from adult Wistar rat hearts via perfusion on a Langendorff-apparatus and cultured in free fatty acid-free BSA control medium or 0.5 mM palmitate medium in the presence or absence of HDL (5 μg protein/ml) with or without 0.1 μM of the AMPK-inhibitor compound S for the analysis of Annexin V/propidium, genes involved in apoptosis and fatty acid oxidation, and cardiomyocyte contractility. We found that HDLs decreased palmitate-induced cardiomyocyte apoptosis as indicated by a reduction in Annexin V-positive cardiomyocytes and an increase in Bcl-2 versus Bax ratio. Concomitantly, HDLs increased the palmitate-impaired expression of genes involved in fatty acid oxidation. Furthermore, HDLs improved the palmitate-impaired cardiomyocyte contractility. All effects were mediated in an AMPK-dependent manner, concluding that HDLs reduce palmitate-induced cardiomyocyte apoptosis, resulting in improved cardiomyocyte contractility through a mechanism involving AMPK., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The widely-used Chinese medicinal herb Hypericum japonicum, also known as Hypericum japonicum Thunb or Tianjihuang, displays potent anti‑carcinogenic effects against liver cancer. However, the molecular mechanism underlying the therapeutic effects of Hypericum japonicum remains to be elucidated. The present study investigated the in vivo efficacy of ethyl acetate extract of Hypericum japonicum (EAEHJ) against tumor growth in an H22 cell‑bearing liver cancer mouse model. Treatment with EAEHJ significantly reduced tumor weight, but had no effect on murine body weight. The results of the present study also showed that EAEHJ induced H22 cell apoptosis in vivo. In addition, the anti‑carcinogenic effects of EAEHJ were investigated in vitro. The results of the present study demonstrate that both phospholipid asymmetry in the plasma membrane and mitochondrial membrane potential were deregulated in HepG2 human hepatoma cells, following treatment with EAEHJ. Treatment with EAEHJ also increased the ratio of pro‑apoptotic B‑cell lymphoma 2 (Bcl‑2)‑associated X protein (Bax) to anti‑apoptotic Bcl‑2, and activated the caspase‑9 signaling pathway. These results suggest that EAEHJ is able to trigger the apoptosis of liver cancer cells via the mitochondria-dependent pathway.

The signal transducer and activator of transcription 3 (STAT3) pathway is one of the main growth factor‑mediated signal transduction pathways and is closely associated with the occurrence and development of benign prostatic hyperplasia (BPH). Qianliening capsules (QC) have significant therapeutic effects on BPH; however, the precise mechanism underlying its anti‑BPH activity remains to be elucidated. To further elucidate the molecular mechanism of the therapeutic effect of QC on BPH, the present study used epidermal growth factor (EGF), which has a role in the pathogenesis of BPH, to stimulate the growth of human prostate WPMY‑1 cells and activate the STAT3 pathway in the WPMY‑1 cells. The cell viability was determined using an MTT assay and the cell morphology was observed by phase‑contrast microscopy. Fluorescence activated cell sorting analysis with Annexin‑V/propidium iodide (PI) staining and PI staining were performed to examine cell apoptosis and the cell cycle. The activation of caspase‑9 and ‑3 were evaluated by colorimetric assay. STAT3 phosphorylation and transcriptional activity were detected by western blot analysis and the luciferase gene reporter, respectively. The mRNA and protein expression levels of B‑cell lymhoma 2 (Bcl‑2), Bcl‑2‑associated X protein (Bax), cyclin D1, cyclin‑dependent kinase 4 (CDK4) and p21 were measured by reverse transcription quantitative polymerase chain reaction and western blot analysis, respectively. In the present study, QC was found to significantly and dose‑dependently inhibit the EGF‑stimulated growth of WPMY‑1 cells, as evidenced by QC‑induced cell -morphological changes and a reduction in cell viability. In addition, QC treatment markedly induced the activation of caspase‑9 and ‑3. QC treatment also inhibited the EGF‑mediated increase of STAT3 phosphorylation levels and transcriptional activity in WPMY‑19 cells, accompanied by downregulation of the expression of Bcl‑2, cyclin D1 and CDK4 and upregulation of the expression of Bax and p21. These results suggested that QC effectively inhibited the proliferation and promoted the apoptosis of human prostate cells via modulation of the STAT3 signaling pathway and its target genes, which is likely to be one of the mechanisms underlying its activity in BPH treatment.

Lin28 plays important roles in the development, maintenance of pluripotency and progression of various types of cancers. Lin28 represses the biogenesis of let-7 microRNAs and is implicated in both development and tumorigenesis. Oncogenic regulation of let-7 microRNAs has been demonstrated in several human malignancies, yet their correlation with Lin28 has not yet been studied in gastric cancer. Therefore, in the present study, we explored the possible mechanisms involved in the effects by Lin28 on the proliferation, migration, cell cycle arrest and apoptosis in gastric cancer cells via alteration of let-7 miRNA. The expression levels of Lin28 and let-7 were detected by real-time PCR in gastric cancer cell lines in vitro. Lin28 was overexpressed in the BGC-823 cells via lentiviral transfection, and let-7 expression was assessed. Cell proliferation and migration capabilities were investigated by MTT and Transwell assays, while cell cycle distribution and the apoptosis rate were detected using flow cytometry. The expression of Lin28 was moderately expressed in the GES cells while underexpressed in the BGC-823, SGC-7901 and HGC-27 cells. Let-7a miRNA was highly expressed in the GES, BGC-823, SGC-7901 and HGC-27 cells. Overexpression of Lin28 was inversely correlated with the downregulated expression of let-7a, and markedly suppressed the proliferation, migration, cell cycle progression and induced apoptosis in the BGC-823 cells. These findings demonstrated that overexpression of Lin28 can suppress the biological behavior of gastric cancer in vitro, and let-7 miRNA may play an important role in the process. We suggest that Lin28 may be a candidate predictor or an anticancer therapeutic target for gastric cancer patients.

Background: We hypothesized that preconditioning (PC) with a short exposure to isoflurane (ISO) would reduce neurodegeneration induced by prolonged exposure to ISO in neonatal rats, as previously shown in neuronal cell culture., Methods: We randomly divided 7-day-old Sprague-Dawley rats into 3 groups: control, 1.5% ISO, and PC + 1.5% ISO. The control group was exposed to carrier gas (30% oxygen balanced in nitrogen) for 30 minutes and then to carrier gas again for 6 hours the following day. The 1.5% ISO group was exposed to carrier gas for 30 minutes and then to 1.5% ISO for 6 hours the following day. The PC + 1.5% ISO group was preconditioned with a 30-minute 1.5% ISO exposure and then exposed to 1.5% ISO for 6 hours the following day. Blood and brain samples were collected 2 hours after the exposures for determination of neurodegenerative biomarkers, including caspase-3, S100β, caspase-12, and an autophagy biomarker Beclin-1., Results: Prolonged exposure to ISO significantly increased cleaved caspase-3 expression in the cerebral cortex of 7-day-old rats compared with the group preconditioned with ISO and the controls using Western blot assays. However, significant differences were not detected for other markers of neuronal injury., Conclusions: The ISO-mediated increase in cleaved caspase-3 in the postnatal day 7 rat brain is ameliorated by PC with a brief anesthetic exposure, and differences were not detected in other markers of neuronal injury.

Dexmedetomidine, a highly selective α2-adrenergic agonist, has been reported to attenuate isoflurane-induced cognitive impairment and neuroapoptosis. However, the underlying molecular mechanisms remain poorly understood. The aim of this study was to investigate whether mitogen-activated protein kinase (MAPK) pathway was involved in dexmedetomidine-induced neuroprotection against isoflurane effects. Seven-day-old (P7) neonatal Sprague-Dawley rats were pretreated with various concentrations of dexmedetomidine, and then exposed to 0.75% isoflurane or air for 6h. Terminal deoxyribonucleotide transferase-mediated dUTP nick end labeling (TUNEL) was used to detect neuronal apoptosis in their hippocampus. Activated caspase-3, extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun NH2-terminal kinases (JNK), p38, phospho-ERK1/2, phospho-JNK and phospho-p38 proteins were detected by Western blotting in the hippocampus at the end of exposure. Also, P7 rats were pretreated with 75 μg/kg dexmedetomidine alone, or given the ERK inhibitor U0126 before dexmedetomidine pretreatment, or pretreated with the p38 MAPK inhibitor SB203580 or JNK inhibitor SP600125 alone, and then exposed to 0.75% isoflurane for 6h. Isoflurane induced significant neuroapoptosis, increased the protein expression of phospho-JNK, phospho-c-Jun, phospho-p38 and phospho-nuclear factor-κB (NF-κB), decreased the level of phospho-ERK1/2 protein and reduced the ratio of Bcl-2/Bax in the hippocampus. Dexmedetomidine pretreatment inhibited isoflurane-induced neuroapoptosis and restored proteins expression of MAPK pathways and the Bcl-2/Bax ratio after isoflurane exposure. Moreover, SB203580 and SP600125 also partly attenuated the isoflurane-induced protein changes. However, U0126 did not reverse dexmedetomidine-induced neuroprotection. Our results indicate that the JNK and p38 pathways, not the ERK pathway are involved in dexmedetomidine-induced neuroprotection against isoflurane effects., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Polyphyllins, a major component of Rhizoma paridis, have been extensively used in non-small cell lung cancer (NSCLC). The aim of the present study was to evaluate the effects of Paris saponin I (PSI) on a panel of gefitinib-resistant NSCLC cell lines and its inhibition of tumor growth in a nude mouse model. The MTT assay was used to assess growth inhibition. The cell cycle was analyzed using flow cytometry and apoptosis was assessed using Annexin V/propidium iodide staining. The morphology of the apoptotic cells was determined by transmission electron microscopy. The protein expression levels of B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax) and caspase-3 were detected using western blot analysis. In addition, the glucose metabolism in tumor-bearing mice was evaluated using 18F-fludeoxyglucose (FDG) micro-positron emission tomography imaging. The PSI-induced growth inhibition rate was observed to significantly increase in a time- and dose-dependent manner. Furthermore, PSI induced significant G2/M-phase arrest and apoptosis. The expression levels of Bcl-2 decreased, while those of Bax and caspase-3 increased following PSI treatment. 18F-FDG-uptake in the PSI treatment groups was significantly decreased compared with that in the control group in vivo. In conclusion, PSI is a potent antitumor agent that acts by inhibiting the proliferation of gefitinib-resistant cells, and has potential as a candidate for a natural drug for gefitinib-resistant therapy. PSI-induced apoptosis, which occurred via multiple pathways, including G2/M-phase arrest and upregulation of the Bax/Bcl-2 ratio and caspase-3 expression, ultimately led to cell death and tumor inhibition.

Passive movement has been found to improve evidently ischemic stroke patients' impaired capacity of learning and memory, but the optimal time window of initiating the therapy and the underlying mechanism are not fully understood. In this study, the effect of passive movement at different time windows on learning and memory of rats with cerebral infarction was detected. The results showed that the expression of caspase-3 and escape latency in the passive movement group were all considerably lower than those in the model group (P < 0.05), while the expression of Bcl-2 mRNA was significantly higher than those in the model group (P < 0.05). Moreover, we found that there were most significant changes of escape latency and expressions of Bcl-2 mRNA and caspase-3 when the therapy started at 24 h after focal cerebral infarction. These results suggest that passive movement is able to contribute to the recovery of learning and memory of rats with cerebral infarction, which is partially mediated by inhibiting neuron cell apoptosis, and the optimal therapeutic time is at 24 h after cerebral infarction.

Objective: To evaluate the effect of Bear Bile Powder(, BBP) on the growth and apoptosis of HepG2 human hepatocellular carcinoma cells, and investigate the possible molecular mechanisms mediating its anti-cancer activity., Methods: HepG2 cells were treated with 0.4-1.0 mg/mL of BBP for 24, 48 and 72 h. The viability of HePG2 cells was determined by MTT assay. Cellular morphology was observed via phase-contrast microscopy. Fluorescence-activated cell sorting analysis with Annexin-V/propidium idodide and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazol-carbocyanine iodide (JC-1) staining was performed to determine cell apoptosis and the loss of mitochondrial membrane potential, respectively. Activation of caspase-9 and -3 was evaluated by a colorimetric assay., Results: The treatment with 0.4-1 mg/mL of BBP for 24, 48, or 72 h respectively reduced cell viability significantly by 7%-60%, 20%-90% or 25%-98%, compared with the untreated control cells (P<0.01). In addition, BBP treatment induced morphological changes in HepG2 cells. Furthermore, after treated with 0, 0.4, 0.6, 0.8 and 1.0 mg/mL of BBP, apoptosis cells (including early and late apoptotic cells) were 18.0%±1.3%, 34.9%±2.2%, 33.9%±2.8%, 37.4%±2.8% and 46.0%±2.5%, respectively (P<0.05); and the percentage of cells with reduced JC-1 red fluorescence were 6.6%±0.8%, 8.5%±0.8%, 13.5%±1.6%, 17.6%±2.3% and 46.7%±3.6%, respectively (P<0.01). Finally, BBP treatment significantly and dose-dependently induced activation of both caspase-9 and caspase-3 in HepG2 cells (P<0.05)., Conclusions: BBP could inhibit the growth of HepG2 hepatocellular cancer cells through mitochondrion-mediated apoptosis, which may, in part, explain its anti-cancer activity. BBP may be a potential novel therapeutic agent for the treatment of hepatocellular carcinoma.

Scutellaria barbata D. Don (SB) has long been used as a major component in numerous Chinese medical formulas to clinically treat various types of cancer. Previously, we reported that the extracts of SB were able to suppress colon cancer growth in vivo and in vitro, possibly by inducing cancer cell apoptosis and inhibiting cell proliferation and tumor angiogenesis. However, the anticancer mechanisms of its bioactive ingredients remain largely unclear. In the present study, using three human colon cancer cell lines SW620, HT-29 and HCT-8, the antitumor effect of different solvent fractions of SB were evaluated and the potential underlying molecular mechanisms were investigated. Using an 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, it was revealed that the chloroform fraction of SB (ECSB) exhibited the most potent inhibitory effect on the growth of all three colon cancer cell lines and SW620 cells exhibited the most sensitive response to ECSB treatment (IC50=65 µg/ml). In addition, by performing fluorescence-activated cell sorting, transmission electron microscopy and colony formation assays, it was observed that ECSB significantly induced apoptosis and inhibited proliferation in SW620 cells in a dose-dependent manner. Furthermore, ECSB treatment resulted in the upregulation of the pro-apoptotic Bax/Bcl-2 ratio and a decrease in the expression of the pro-proliferative cyclin D1 and cyclin-dependent kinase 4. The results from the present study may provide a scientific foundation for the development of novel anticancer agents from the bioactive ingredients in the ECSB.

We evaluated the neuroprotective effect of electro-acupuncture (EA) on cerebral ischemia-reperfusion (IR) injury and deeply investigated the relationship between this neuroprotective effect and PI3K/Akt pathway. Rats underwent focal cerebral IR injured by suture method and received the in vivo therapeutic efficacy of EA at points of Zusanli(ST36) and Quchi(LI11) after the operation. We found that the EA treatment significantly (p<0.05) improved neurological deficit and cerebral infarction. Furthermore, EA profoundly activated PI3K/Akt signaling resulted in the inhibition of cerebral cell apoptosis in the ischemic penumbra. Simultaneously EA increased the expression of PI3K, p-Akt, p-Bad and Bcl-2 at the protein level and the expression of Bcl-2 at the mRNA level. On the contrary, EA inhibited the Bax and cleaved Caspase-3-positive expression. The selective PI3K inhibitor LY294002 compromised EA-induced neuroprotective effects and reduced the elevation of p-Akt, p-Bad and Bcl-2 levels. Our data suggested that the PI3K/Akt pathway played a critical role in mediating the neuroprotective effects of EA treatment at points of Zusanli and Quchi after the ischemic stroke., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

MicroRNAs (miRNAs) have been shown to be closely associated with cellular apoptosis, but their involvement in response to ethanol-induced gastric mucosal epithelial cell apoptosis remains largely unknown. The purpose of this study was to investigate the expression profile of apoptosis-associated miRNAs in ethanol-induced acute gastric mucosal injury and the mechanisms underlying injury. Gastric mucosal injury was induced in rats by oral administration of ethanol, and gastric tissues were collected for analysis of gastric ulcer index, apoptosis ratio, caspase-3 activity, and miRNAs expression. Cell cultures of human gastric mucosal epithelial cells (GES-1) were incubated with ethanol to induce apoptosis. Mimics or inhibitors of miRNAs or c-Jun N-terminal kinase (JNK) inhibitor were added to the cell culture medium. GES-1 cells were collected for analysis of apoptosis ratio, caspase-3 activity, miRNAs expression, and protein phosphorylation levels of JNK, p38 mitogen-activated protein kinase (p38MAPK), or extracellular signal-regulated kinase (ERK). In the animal experiments, gastric ulcer index, cellular apoptosis, and caspase-3 activity were significantly increased, accompanied by up-regulation of miR-145 and down-regulation of the microRNAs miR-17, miR-19a, miR-21, miR-181a, and miR-200c. In the human cell culture experiments, the anti-apoptotic effects of miR-19a and miR-21 or pro-apoptotic effect of miR-145 were confirmed by their corresponding mimics or inhibitor; the ethanol-induced GES-1 apoptosis as well as the changes in miRNAs expression were significantly attenuated in the presence of JNK inhibitor. These results demonstrated that miR-145, miR-19a, and miR-21 were the apoptosis-associated miRNAs in gastric mucosal epithelial cells. The regulation of expression of these 3 miRNAs in ethanol-induced GES-1 apoptosis involved the JNK pathway., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Disrupted apoptosis not only confers a survival advantage to cancer cells but also causes resistance to chemotherapies. Therefore, inducing cell apoptosis has become a promising strategy for anticancer treatment. Patrinia scabiosaefolia (PS) has long been used to clinically treat various types of malignancies including colorectal cancer (CRC). However, the precise mechanism of its tumoricidal activity remains largely unclear. Using a CRC mouse xenograft model and a human colon carcinoma cell line, HT-29, in the present study, we evaluated the antitumor activities of an ethanol extract of Patrinia scabiosaefolia (EEPS), and investigated the underlying molecular mechanisms. We found that EEPS inhibited CRC growth both in vivo and in vitro, without apparent adverse side-effects. Moreover, EEPS treatment promoted apoptosis in CRC tumor tissues and in HT-29 cells, suggesting that the inhibitory effect of EEPS on tumor growth was due to its pro-apoptotic activity. Furthermore, EEPS treatment inhibited the expression of anti-apoptotic Bcl-2 but enhanced pro-apoptotic Bax expression at both transcriptional and translational levels. Finally, EEPS induced the loss of mitochondrial membrane potential and activation of caspase-9 and -3 in HT-29 cells. Taken together, data in this study suggest that induction of cancer cell apoptosis via the mitochondrial-dependent pathway may be one of the mechanisms whereby PS exerts anticancer activity.

The Livistona chinensis seed has been used for centuries to clinically treat various types of cancer. However, the precise mechanism of its anticancer activity remains to be elucidated. In the present study, we evaluated the efficacy of the ethanol extract of Livistona chinensis seed (EELC) against tumor growth using a hepatocellular carcinoma (HCC) mouse xenograft model and an HCC cell line, HepG2, and investigated the molecular mechanisms mediating its biological activities. We found that EELC inhibited HCC growth both in vivo and in vitro, without apparent signs of toxicity. In addition, EELC treatment resulted in the induction of HCC cell apoptosis. Moreover, EELC-induced apoptosis was accompanied by the loss of mitochondrial membrane potential, activation of caspase-9 and caspase-3, and increase in the pro-apoptotic Bax/Bcl-2 ratio. Our findings suggest that promotion of mitochondrial-dependent apoptosis in cancer cells may be one of the mechanisms by which the Livistona chinensis seed is effective in cancer treatment.

Cognitive impairment is a serious mental deficit following stroke that severely affects the quality of life of stroke survivors. Nuclear factor‑κB (NF-κB)-mediated neuronal cell apoptosis is involved in the development of post-stroke cognitive impairment; therefore, it has become a promising target for the treatment of impaired cognition. Acupuncture at the Baihui (DU20) and Shenting (DU24) acupoints is commonly used in China to clinically treat post‑stroke cognitive impairment; however, the precise mechanism of its action is largely unknown. In the present study, we evaluated the therapeutic efficacy of electroacupuncture against post-stroke cognitive impairment and investigated the underlying molecular mechanisms using a rat model of focal cerebral ischemia-reperfusion (I/R) injury. Electroacupuncture at Baihui and Shenting was identified to significantly ameliorate neurological deficits and reduce cerebral infarct volume. Additionally, electroacupuncture improved learning and memory ability in cerebral I/R injured rats, demonstrating its therapeutic efficacy against post-stroke cognitive impairment. Furthermore, electroacupuncture significantly suppressed the I/R-induced activation of NF-κB signaling in ischemic cerebral tissues. The inhibitory effect of electroacupuncture on NF-κB activation led to the inhibition of cerebral cell apoptosis. Finally, electroacupuncture markedly downregulated the expression of pro-apoptotic Bax and Fas, two critical downstream target genes of the NF-κB pathway. Collectively, our findings suggest that inhibition of NF-κB‑mediated neuronal cell apoptosis may be one mechanism via which electroacupuncture at Baihui and Shenting exerts a therapeutic effect on post-stroke cognitive impairment.

The aim of this study was to evaluate the therapeutic efficacy of Qianliening capsule (QC) against benign prostatic hyperplasia (BPH) in vivo in a BPH rat model, as well as to investigate the effects of QC on prostatic cell apoptosis and the possible molecular mechanisms mediating its anti-BPH activity. Fifty male Sprague‑Dawley (SD) rats were randomly classified into five groups. The rats of the four groups were castrated and subcutaneously injected with testosterone propionate to generate BPH. One week after model establishment, BPH rats were orally administrated with various doses of QC daily for 28 days. The prostatic tissues from BPH rats were collected to evaluate prostatic index (PI). The histological changes of prostate were observed by hematoxylin and eosin staining. TUNEL analysis was performed to examine cell apoptosis. The mRNA expression of Bcl-2 and Bax in prostatic tissues was determined by reverse transcription‑polymerase chain reaction (RT-PCR). The protein expression of Bcl-2, Bax and cleaved caspase 3 were examined by immunohistochemistry. Administration with QC significantly decreased PI in a dose-dependent manner (P<0.05 or P<0.01) and improved prostatic hyperplasia in BPH rats. Additionally, QC treatment induced prostatic cell apoptosis in a dose-dependent manner. Moreover, QC promoted the cleavage of caspase 3, an indicator of apoptosis, in a dose-dependent manner. Furthermore, following QC treatment, the expression ratio of pro‑apoptotic Bax to anti‑apoptotic Bcl-2 in prostatic tissues was increased in a dose-dependent manner. As a result, QC was effective in the treatment of BPH in rats. Promoting apoptosis of prostatic cells may therefore be one of the mechanisms by which QC treats BPH.

The aim of this study was to evaluate the therapeutic efficacy of Rubus aleaefolius Poir total alkaloids (TARAP) against hepatocellular carcinoma growth in vivo and in vitro, and to investigate the possible molecular mechanisms mediating its biological activity. Nude mice were implanted with HepG2 human hepatocellular carcinoma cells and fed with vehicle (physiological saline) or 3 g/kg/d dose of TARAP, 5 days per week, for 21 days. The in vivo efficacy of TARAP against tumor growth was investigated by evaluating its effect on tumor volume and tumor weight in mice with HCC xenografts and its adverse effect was determined by measuring the body weight gain. The in vitro effect of TARAP on the viability of HepG2 cells was determined by MTT assay. HepG2 cell morphology was observed via phase-contrast microscopy. Apoptosis in tumor tissues or in HepG2 cells was analyzed by TUNEL assay or FACS analysis with Annexin V/PI, respectively. The loss of mitochondrial membrane potential in HepG2 cells was determined via JC-1 staining followed by FACS analysis. Activation of caspase-9 and -3 in HepG2 cells was examined by a colorimetric assay. The mRNA and protein expression of Bcl-2 and Bax in tumor tissues were measured by RT-PCR and immunohistochemistry. TARAP reduced tumor volume and tumor weight, but had no effect on the body weight gain in HCC mice. TARAP decreased the viability of HepG2 cells and induced cell morphological changes in vitro in a dose- and time-dependent manner. In addition, TARAP induced apoptosis both in tumor tissues and in HepG2 cells. Moreover, TARAP treatment resulted in the collapse of mitochondrial membrane potential in HepG2 cells, as well as the activation of caspase-9 and -3. Furthermore, administration of TARAP increased the pro-apoptotic Bax/Bcl-2 ratio in HCC mouse tumors, at both transcriptional and translational levels. TARAP inhibits hepatocellular carcinoma growth both in vivo and in vitro probably through the activation of mitochondrial-dependent apoptosis, which may, in part, explain its anticancer activity. These results suggest that total alkaloids in Rubus aleaefolius Poir may be a potential novel therapeutic agent for the treatment of hepatocellular carcinoma and other cancers.

Objective: To investigate the molecular mechanisms by which Qianliening Capsule (, QC) treats benign prostatic hyperplasia (BPH)., Methods: Human prostate stromal cell line WPMY-1 was treated with 0, 1, 3 and 5 mg/mL of QC for 24, 48 and 72 h, respectively, in the presence of 10 ng/mL basic fibroblast growth factor (bFGF). The viability of WPMY-1 cells was determined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell morphology was observed by phase-contrast microscopy. 4',6-diamidino-2-phenylindole (DAPI) staining and fluorescence activated cell sorting (FACS) analysis with Annexin-V/propidium iodide (PI) staining were performed to determine cell apoptosis. The loss of mitochondrial membrane potential was examined by FACS analysis with 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyarine iodide (JC-1) staining. Activation of caspase-3 and -9 was evaluated by colorimetric assay. The mRNA and protein expression levels of Bcl-2 and Bax were measured by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting, respectively., Results: Upon bFGF stimulation, the viability of WPMY-1 cells was increased to 122%-118% compared with the control cells (P <0.05). However, treatment with 1-5 mg/mL of QC for 24, 48 and 72 h decreased the viability of bFGF-stimulated cells to 80%-92%, 59%-82%, 36%-62% compared with the untreated cells (P <0.05). In addition, QC treatment reduced WPMY-1 cell density in a dose-dependent manner. Moreover, QC treatment dose-dependently induced the loss of plasma membrane asymmetry, the nuclear condensation and fragmentation, collapse of mitochondrial membrane potential, activation of caspase-9 and caspase-3, and increase of pro-apoptotic Bax/Bcl-2 ratio., Conclusion: Promoting mitochondrion-dependent apoptosis of prostate stromal cells might be one of the mechanisms by which QC treats BPH.

This study examined the effect of electro-acupuncture (EA) combined with transcranial magnetic stimulation (TMS) therapy at different time windows on learning and memory ability of rats with cerebral infarction and the underlying mechanism. Two hundred SD rats were randomly divided into four groups: normal group, sham-operated group, model group and EA+TMS group, and each group was then divided into five sub-groups in terms of the different time to start treatment post operation: 6, 12, 24, 48 and 72 h. Cerebral infarction models were established in the model and the EA+TMS groups by left middle cerebral artery occlusion/reperfusion (MCAO/R). After treatment for 14 d, the Morris water maze test was applied to examine the spatial learning and memory abilities of rats. In infarcted area, the expression of caspase-3 was immunohistochemically detected, and real-time fluorescent quantitative PCR was used to measure the expression of Bcl-2 mRNA. The results showed that in EA+TMS group compared with model group at the same treatment time windows, the escape latency was substantially shortened, the expression of caspase-3 was considerably decreased and the expression level of Bcl-2 mRNA significantly increased (P<0.05). In the EA+TMS sub-groups, the escape latency was shortest, the expression level of caspase-3 lowest, and the expression level of Bcl-2 mRNA highest at the treatment time window of 24 h. It was concluded that EA combined with TMS can promote neurological function of rats with cerebral infarction by increasing the expression level of Bcl-2 mRNA and decreasing the expression of caspase-3. The best time window is 24 h after perfusion treatment to ischemia.

Myeloperoxidase (MPO) is involved in myocardial ischemia-reperfusion (IR) injury and vascular peroxidase (VPO) is a newly identified isoform of MPO. This study was conducted to explore whether VPO is involved in IR-induced cardiac dysfunction and apoptosis. In a rat Langendorff model of myocardial IR, the cardiac function parameters (left ventricular pressure and the maximum derivatives of left ventricular pressure and coronary flow), creatine kinase (CK) activity, apoptosis, VPO1 activity were measured. In a cell (rat-heart-derived H9c2 cells) model of hypoxia-reoxygenation (HR), apoptosis, VPO activity, and VPO1 mRNA expression were examined. In isolated heart, IR caused a marked decrease in cardiac function and a significant increase in apoptosis, CK, and VPO activity. These effects were attenuated by pharmacologic inhibition of VPO. In vitro, pharmacologic inhibition of VPO activity or silencing of VPO1 expression significantly suppressed HR-induced cellular apoptosis. Our results suggest that increased VPO activity contributes to IR-induced cardiac dysfunction and inhibition of VPO activity may have the potential clinical value in protecting the myocardium against IR injury.

Signal transducer and activator of transcription 3 (STAT3) plays a critical role in cell survival and proliferation. Constitutive activation of STAT3 is strongly correlated with pathogenesis of various types of malignant tumors including colorectal cancer (CRC), and therefore is a major focus in the development of anti-cancer agents. Pien Tze Huang (PZH), a well-known traditional Chinese formula prescribed already in the Ming Dynasty, has been demonstrated to be clinically effective in the treatment of CRC. However, the precise mechanism of its anti-cancer activity remains largely unknown. In the present study we evaluated the efficacy of PZH against tumor growth in vivo in the CRC mouse xenograft model, and investigated the underlying molecular mechanisms. We found that administration of PZH reduced tumor volume and tumor weight but had no effect on body weight gain in CRC mice, demonstrating that PZH can inhibit colon cancer growth in vivo without apparent adverse effect. We also observed that PZH treatment inhibited the phosphorylation level of STAT3 in tumor tissues. Consequently, the inhibitory effect of PZH on STAT3 activation resulted in the up-regulation of Bax/Bcl-2 ratio as well as down-regulation of Cyclin D1 and CDK4 expression, leading to the induction of apoptosis as well as the inhibition of cell proliferation. These results suggest that promotion of cancer cell apoptosis and inhibition of proliferation via suppression of STAT3 pathway might be one of the mechanisms by which PZH treats colorectal cancer.

Millimeter wave (MW) is an electromagnetic wave with a wavelength between 1 and 10 mm and a frequency of 30-300 GHz that causes multiple biological effects and has been used as a major component in physiotherapies for the clinical treatment of various types of diseases including cancers. However, the precise molecular mechanism of the anticancer activity of millimeter wave remains to be elucidated. In the present study, we investigated the cellular effects of the MW in the U-2OS human osteosarcoma cell line. Our results showed that MW induced cell morphological changes and reduced cell viability in a dose- and time-dependent manner suggesting that MW inhibited the growth of U-2OS cells as demonstrated. Hoechst 33258 staining and Annexin V/propidium iodide double staining exhibited the typical nuclear features of apoptosis and increased the proportion of apoptotic Annexin V-positive cells in a dose-dependent manner, respectively. In addition, MW treatment caused loss of plasma membrane asymmetry, release of cytochrome c, collapse of mitochondrial membrane potential, activation of caspase-9 and -3, and increase of the ratio of pro-apoptotic Bax to anti-apoptotic Bcl-2. Taken together, the results indicate that the U-2OS cell growth inhibitory activity of MW was due to mitochondrial-mediated apoptosis, which may partly explain the anticancer activity of millimeter wave treatment.

Objective: To explore the effects and the molecular mechanism of puerariae radix flavones (PRF) on acute myeloid leukemia cell line Kasumi-1 cells in vitro., Methods: Kasumi-1 cells treated by PRF for 48 hours were observed with Wright's and Hoechst 33258 dying. The apoptotic cells were analyzed by flow cytometry with AnnexinV/PI staining. The expression levels of bcl-2, Bim and Caspase-3/-8/-9 protein were assayed by Western blot and the AML1-ETO fusion gene was detected by real-time polymerase chain reaction., Results: PRF could induce Kasumi-1 cells to apoptosis effectively. The proportion of apoptotic cells in 50, 200 and 500 µg/ml PRF treatment groups were (14.1 ± 0.8)%, (17.7 ± 1.3)% and (32.4 ± 1.4)%, respectively, and significantly higher than that of control \[(7.8 ± 0.7)%\]. The relative expression levels of the anti-apoptotic Bcl-2 protein were 0.85 ± 0.05, 0.62 ± 0.07 and 0.43 ± 0.05; the apoptotic Bim protein were 0.21 ± 0.06, 0.39 ± 0.04 and 0.75 ± 0.05; the caspase-3 and caspase-9 were 0.92 ± 0.04, 1.21 ± 0.07, 1.33 ± 0.04 and 0.35 ± 0.05, 0.53 ± 0.03, 0.69 ± 0.07, respectively. Compared to the blank control group, all these changes were significant (P < 0.01). Nevertheless, nearly no changes could be observed on the expression level of AML1-ETO fusion gene and caspase-8 protein., Conclusion: Apoptosis of Kasumi-1 cells induced by PRF might correlate to the down-regulation of Bcl-2 protein expression and the activation of caspase-3 and caspase-8 protein in the cells. It seemed that all these effects had no relationship with the AML1-ETO fusion gene.

Myeloperoxidase (MPO) is an important enzyme involved in the genesis and development of atherosclerosis. Vascular peroxidase 1 (VPO1) is a newly discovered member of the peroxidase family that is mainly expressed in vascular endothelial cells and smooth muscle cells and has structural characteristics and biological activity similar to those of MPO. Our specific aims were to explore the effects of VPO1 on endothelial cell apoptosis induced by oxidized low-density lipoprotein (ox-LDL) and the underlying mechanisms. The results showed that ox-LDL induced endothelial cell apoptosis and the expression of VPO1 in endothelial cells in a concentration- and time-dependent manner concomitant with increased intracellular reactive oxygen species (ROS) and hypochlorous acid (HOCl) generation, and up-regulated protein expression of the NADPH oxidase gp91(phox) subunit and phosphorylation of p38 MAPK. All these effects of ox-LDL were inhibited by VPO1 gene silencing and NADPH oxidase gp91(phox) subunit gene silencing or by pretreatment with the NADPH oxidase inhibitor apocynin or diphenyliodonium. The p38 MAPK inhibitor SB203580 or the caspase-3 inhibitor DEVD-CHO significantly inhibited ox-LDL-induced endothelial cell apoptosis, but had no effect on intracellular ROS and HOCl generation or the expression of NADPH oxidase gp91(phox) subunit or VPO1. Collectively, these findings suggest for the first time that VPO1 plays a critical role in ox-LDL-induced endothelial cell apoptosis and that there is a positive feedback loop between VPO1/HOCl and the now-accepted dogma that the NADPH oxidase/ROS/p38 MAPK/caspase-3 pathway is involved in ox-LDL-induced endothelial cell apoptosis., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Objective: To explore the inhibitory effect of calcitonin gene-related peptide (CGRP) on cardiomyocyte apoptosis and the underlying mechanism., Methods: In cultured rat cardiomyocytes, apoptosis was induced by the incubation of isoprenaline (10(-5) mol/L) for 48 h. CGRP (10(-8) or 10(-7) mol/L) was administrated for 1 h before incubating isoprenaline to evaluate its effect on cardiomyocyte apoptosis. At the end of the drug treatment, the rate of apoptotic cells and intracellular reactive oxygen species (ROS) were determined, and RNA was extracted to examine the expression of microRNA-1 and microRNA-133a., Results: Isoprenaline significantly increased the rate of apoptotic cells and intracellular ROS production concomitantly with the increased microRNA-1 expression and the decreased microRNA-133a expression, which were inhibited by pretreatment with CGRP. The effects of CGRP were reversed by CGRP receptor antagonist., Conclusion: CGRP can inhibit the isoprenaline-induced cardiomyocyte apoptosis. The beneficial effect of CGRP is related to regulating microRNA-1 and microRNA-133a expression through the prevention of isoprenaline-induced ROS production.

Objective: To investigate the cellular effects of Pien Tze Huang (PZH) in the HT-29 human colon carcinoma cell line., Methods: The viability of HT-29 cells was determined by MTT assay. A fluorescence-activated cell sorting (FACS) analysis with annexin-V/propidium iodide (PI) and JC-1 staining were performed to determine cell apoptosis and the loss of mitochondrial membrane potential, respectively. Activation of caspase 3 was evaluated by a colorimetric assay. The mRNA expression levels of Bcl-2 and Bax were measured by reverse transcription polymerase chain reaction (RT-PCR)., Results: PZH, in a dose- and time-dependent manner, reduced viability and induced apoptosis of HT-29 cells. Moreover, PZH treatment resulted in the collapse of the mitochondrial membrane potential, activation of caspase 3, and an increase in the Bax/Bcl-2 ratio., Conclusion: PZH inhibits the growth of HT-29 cells by inducing cancer cell apoptosis via regulation of the Bcl-2 family and activation of caspase 3, which may, in part, explain its anticancer activity.

Millimeter wave (MW) is an electromagnetic wave with a wavelength between 1 and 10 mm and a frequency of 30-300 GHz that causes multiple biological effects, both locally and globally. MW has been widely used in clinical medicine. Although our previous work demonstrated that MW is capable of inhibiting sodium nitroprussiate (SNP)-induced apoptosis in chondrocytes, the precise mechanism of the anti-apoptotic activity remains to be elucidated. The purpose of this study was to investigate the effects of MW in SNP-induced apoptotic chondrocytes. Sprague Dawley rat chondrocytes were isolated and cultured, and the cells were counted. Cell viability was evaluated using MTT assay. Cells were then treated with SNP and MW, and flow cytometry was used to detect apoptosis. Our results showed that MW treatment inhibited a SNP-induced mitochondrion-dependent pathway of apoptosis. MW treatment inhibited the loss of plasma membrane asymmetry (externalization of phosphatidylserine), collapse of mitochondrial membrane potential, and activation of caspase-9 and caspase-3. Taken together, the results indicate that MW inhibits the mitochondrion-dependent pathway of apoptosis in chondrocytes and this may, in part, explain its clinical effect in the treatment of osteoarthritis.

Objective: To study the inhibitory effect of Fuzheng Yiliu Granule (FYG) on hepatocellular cancer (HCC) and investigate the mechanism mediating its bioactivity., Methods: H22 tumor-bearing ICR mice were treated with FYG [3.6 g/(kg·d)] for 5 days. Tumor volume and tumor weight, percentages of CD3(+), CD4(+), CD8(+), and natural killer (NK) cells in peripheral blood, tumor apoptosis and serum levels of interleukin-2 (IL-2), and tumor necrosis factor-α (TNF-α) were evaluated. FYG-containing serum was prepared from SD rats treated for 7 days [high dose 3.6 g/(kg·d); middle dose 1.8 g/(kg·d); low dose 0.9 g/(kg·d)]. Cell cycle, cell viability, and apoptosis were evaluated after HepG2 cell line was cultured in FYG-containing serum for 48 h. The levels of IL-2 and TNF-α in FYG-containing serum were also determined., Results: FYG produced a potent antitumor effect (P<0.01) and induced marked apoptosis of the tumor tissue (P<0.05). Mice treated with FYG had higher percentages of CD3(+) and CD4(+) (P<0.05), and more NK cells (P<0.01) in the peripheral blood than those in the animals treated with normal saline. Mice receiving FYG had the highest serum levels of IL-2 and TNF-α (P<0.01). High-dose FYG-containing serum significantly decreased HepG2 cell viability, inhibited cell proliferation (P<0.05), and induced apoptosis (P<0.01). In addition, the levels of IL-2 and TNF-α of high-dose-containing serum were higher than the blank serum (P<0.01)., Conclusion: FYG could inhibit HCC growth by regulating immune function and inducing apoptosis of tumor cells in vivo and in vitro.

Bcl-2 homology domain-3 (BH3) peptides are potent cancer therapeutic reagents that target regulators of apoptotic cell death in cancer cells. However, their cytotoxic effects are affected by different expression levels of Bcl-2 family proteins. We recently found that the amphipathic tail-anchoring peptide (ATAP) from Bfl-1, a bifunctional Bcl-2 family member, produced strong pro-apoptotic activity by permeabilizing the mitochondrial outer membrane. Here, we test whether the activity of ATAP requires other cellular factors and whether ATAP has an advantage over the BH3 peptides in targeting cancer cells. Confocal microscopic imaging illustrates specific targeting of ATAP to mitochondria, whereas BH3 peptides show diffuse patterns of cytosolic distribution. Although the pro-apoptotic activities of BH3 peptides are largely inhibited by either overexpression of anti-apoptotic Bcl-2 or Bcl-xL or nullification of pro-apoptotic Bax and Bak in cells, the pro-apoptotic function of ATAP is not affected by these cellular factors. Reconstitution of synthetic ATAP into liposomal membranes results in release of fluorescent molecules of the size of cytochrome c from the liposomes, suggesting that the membrane permeabilizing activity of ATAP does not require additional protein factors. Because ATAP can target to the mitochondrial membrane and its pro-apoptotic activity does not depend on the content of Bcl-2 family proteins, it represents a promising candidate for anti-cancer drugs that can potentially overcome the intrinsic apoptosis-resistant nature of cancer cells.

Signal transducer and activator of transcription 3 (STAT3) plays an important role in tumor cell survival and proliferation and thus has become a major focus in the development of anti-cancer therapies. Patrinia scabiosaefolia has been used for the treatment of various types of cancer. However, the precise mechanism of the anti-cancer activity of Patrinia scabiosaefolia remains unclear. In this study, we evaluated the effect of the ethanol extract of Patrinia scabiosaefolia (EEPS) on proliferation and apoptosis in human multiple myeloma U266 cells that persistently express phosphorylated STAT3, and investigated the possible molecular mechanisms mediating its biological effects. We found that EEPS inhibited the phosphorylation of STAT3 in U266 cells. Consequently, the inhibitory effect of EEPS on STAT3 activation resulted in the suppression of cell proliferation and the induction of cell apoptosis. Moreover, EEPS treatment inhibited the expression of cyclin D1 (a promoter of cell proliferation) and Bcl-2 (an inhibitor of apoptosis), two important target genes of the STAT3 signaling pathway. Our findings for the first time demonstrate that Patrinia scabiosaefolia inhibits proliferation and promotes the apoptosis of cancer cells via inhibition of the STAT3 pathway, which may in part explain its anti-cancer activity.

Hedyotis Diffusa Willd has been used as a major component in several Chinese medicine formulations for the clinical treatment of colorectal cancer. However, the molecular mechanism of the anti-cancer activity of Hedyotis Diffusa Willd remains unclear. In the present study, we investigated the cellular effects of the ethanol extract of Hedyotis Diffusa Willd (EEHDW) in the HT-29 human colon carcinoma cell line. We found that EEHDW inhibited the growth of HT-29 cells demonstrating EEHDW-induced cell morphological changes and reduced cell viability in a dose- and time-dependent manner. Furthermore, we observed that EEHDW treatment resulted in DNA fragmentation, loss of plasma membrane asymmetry, collapse of mitochondrial membrane potential, activation of caspase-9 and caspase-3, and increase of the ratio of pro-apoptotic Bax to anti-apoptotic Bcl-2, suggesting that the HT-29 cell growth inhibitory activity of EEHDW was due to mitochondrion-mediated apoptosis, which may partly explain the anti-cancer activity of Hedyotis Diffusa Willd.