Search

Yang Wang,1,* Zheng Wang,1,* Xiao Wu,1,* Song Zhu,2,* Qiru Guo,1 Zhong Jin,1 Zixian Chen,3 Delai Zhang,2 Wangming Hu,1 Huan Xu,1 Liangqin Shi,1 Lan Yang,1 Yong Wang1 1College of Basic Medicine, Chengdu University of Traditional Chinese Medicine, Chengdu, People’s Republic of China; 2Hospital of Chengdu University of Traditional Chinese Medicine, Chengdu, People’s Republic of China; 3School of Ethnic Medicine, Chengdu University of Traditional Chinese Medicine, Chengdu, People’s Republic of China*These authors contributed equally to this workCorrespondence: Yong Wang, College of Basic Medicine, Chengdu University of Traditional Chinese Medicine, Chengdu, Sichuan, People’s Republic of China, Tel +86 18280350393, Email yongwang1008@hotmail.com; wangyong@cdutcm.edu.cnPurpose: Dysfunction of endothelium is associated with multiple pathological vascular diseases. However, how to regulate reendothelialization after vascular injury is not well defined. This study aims to determine whether and how Paeonol controls reendothelialization following artery injury.Methods: The endothelium of murine carotid artery was denuded by catheter guide wires injury. H&E staining and IF staining were performed to determine whether Paeonol is critical for reendothelialization. BRDU Incorporation Assay, Boyden Chamber Migration Assay, Tube Formation Assay, and Spheroid Sprouting Assay were used to investigate whether Paeonol is involved in regulating proliferation and migration of endothelial cells. The underlying mechanism of how Paeonol regulates reendothelialization was determined by Molecular docking simulation and CO-IP Assay.Results: Paeonol treatment significantly inhibits neointima formation in carotid artery ligation model by promoting proliferation and migration of endothelial cells. Mechanistically, Paeonol enhances c-Myc expression, consequently interacts with VEGFR2 results in activating VEGF signaling pathway, and eventually promotes reendothelialization after vascular injury.Conclusion: Our data demonstrated that Paeonol plays a critical role in regulating vascular reendothelialization, which may be therapeutically used for treatment of pathological vascular diseases.Graphical Abstract: Keywords: Paeonol, c-Myc, VEGFR2, vascular injury, reendothelialization

Background The mechanisms underlying lesions of dopaminergic (DA) neurons, an essential pathology of Parkinson’s disease (PD), are largely unknown, although oxidative stress is recognized as a key factor. We have previously shown that the pro-oxidative aldehyde acrolein is a critical factor in PD pathology, and that acrolein scavenger hydralazine can reduce the elevated acrolein, mitigate DA neuron death, and alleviate motor deficits in a 6-hydroxydopamine (6-OHDA) rat model. As such, we hypothesize that a structurally distinct acrolein scavenger, dimercaprol (DP), can also offer neuroprotection and behavioral benefits. Methods DP was used to lower the elevated levels of acrolein in the basal ganglia of 6-OHDA rats. The acrolein levels and related pathologies were measured by immunohistochemistry. Locomotor and behavioral effects of 6-OHDA injections and DP treatment were examined using the open field test and rotarod test. Pain was assessed using mechanical allodynia, cold hypersensitivity, and plantar tests. Finally, the effects of DP were assessed in vitro on SK-N-SH dopaminergic cells exposed to acrolein. Results DP reduced acrolein and reversed the upregulation of pain-sensing transient receptor potential ankyrin 1 (TRPA1) channels in the substantia nigra, striatum, and cortex. DP also mitigated both motor and sensory deficits typical of PD. In addition, DP lowered acrolein and protected DA-like cells in vitro. Acrolein’s ability to upregulate TRPA1 was also verified in vitro using cell lines. Conclusions These results further elucidated the acrolein-mediated pathogenesis and reinforced the critical role of acrolein in PD while providing strong arguments for anti-acrolein treatments as a novel and feasible strategy to combat neurodegeneration in PD. Considering the extensive involvement of acrolein in various nervous system illnesses and beyond, anti-acrolein strategies may have wide applications and broad impacts on human health.

Dysregulation of neuronal development may cause neurodevelopmental disorders. However, how to regulate embryonic neuronal development and whether this regulation can be medical interrupted are largely unknown. This study aimed to investigate whether and how andrographolide (ANP) regulates embryonic neuronal development. The pregnant mice at embryonic day 10.5 (E10.5) were administrated with ANP, and the embryonic brains were harvested at E17.5 or E18.5. Immunofluorescence (IF), Immunohistochemistry (IHC) performed to determine whether ANP is critical in regulating neuronal development. Real-time quantitative PCR, western blotting, cell counting kit-8 assay, Flow Cytometry assay, Boyden Chamber Migration assay carried out to evaluate whether ANP regulates neuronal proliferation and migration. Protein-protein interaction, CO-immunoprecipitation and IF staining carried out to evaluate whether ANP regulates the interaction between PFKFB3, NeuN and TBR1. Knockdown or overexpression of PFKFB3 by adenovirus infection were used to determine whether ANP inhibits neuronal development through PFKFB3 mediated glycolytic pathway. Our data indicated that ANP inhibited the maturation of embryonic neurons characterized by suppressing neuronal proliferation and migration. ANP regulated the interaction between PFKFB3, NeuN, and TBR1. Knockdown of PFKFB3 aggravated ANP mediated inhibition of neuronal proliferation and migration, while overexpression of PFKFB3 attenuated ANP mediated neuronal developmental suppression. In summary, ANP suppressed the expression of PFKFB3, and interrupted the interaction between TRB1 and NeuN, resulting in suppressing neuronal proliferation, migration and maturation and eventually inhibiting murine embryonic neuronal development.

Despite decades of research into the pathology mechanisms of Parkinson's disease (PD), disease-modifying therapy of PD is scarce. Thus, searching for new drugs or more effective neurosurgical treatments has elicited much interest. Clioquinol (CQ) has been shown to have therapeutic benefits in rodent models of neurodegenerative disorders. However, it's neuroprotective role and mechanisms in PD primate models and PD patients, especially in the advanced stages, are not fully understood. Furthermore, issues such as spontaneous recovery of motor function and high symptom variability in different monkeys after the same toxic protocol, has not been resolved before the present study. In this study, we designed a chronic and long-term progressive protocol to generate a stabilized PD monkey model showed with classic motor and non-motor deficits, followed by treatment analysis of CQ. We found that CQ could remarkably improve the motor and non-motor deficits, which were based on the reduction of iron content and ROS level in the SN and further improvement in pathology. Meanwhile, we also showed that ferroptosis was probably involved in the pathogenesis of PD. In addition, the study shows a positive effect of CQ on AKT/mTOR survival pathway and a blocking effect on p53 medicated cell death in vivo and in vitro.

Iron homeostasis is critical for maintaining normal brain physiological functions, and its mis-regulation can cause neurotoxicity and play a part in the development of many neurodegenerative disorders. The high incidence of iron deficiency makes iron supplementation a trend, and ferric citrate is a commonly used iron supplement. In this study, we found that the chronic oral administration of ferric citrate (2.5 mg/day and 10 mg/day) for 16 weeks selectively induced iron accumulation in the corpus striatum (CPu), substantia nigra (SN) and hippocampus, which typically caused parkinsonism phenotypes in middle-aged mice. Histopathological analysis showed that apoptosis- and oxidative stress-mediated neurodegeneration and dopaminergic neuronal loss occurred in the brain, and behavioral tests showed that defects in the locomotor and cognitive functions of these mice developed. Our research provides a new perspective for ferric citrate as a food additive or in clinical applications and suggests a new potential approach to develop animal models for Parkinson’s disease (PD).

Ferric citrate (FC) has been used as an iron fortifier and nutritional supplement, which is reported to induce colitis in rats, however the underlying mechanism remains to be elucidated. We performed a 16-week study of FC in male healthy C57BL/6 mice (nine-month-old) with oral administration of Ctr (0.9 % NaCl), 1.25 % FC (71 mg/kg/bw), 2.5 % FC (143 mg/kg/bw) and 5 % FC (286 mg/kg/bw). FC-exposure resulted in colon iron accumulation, histological alteration and reduce antioxidant enzyme activities, such as glutathione (GSH), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and total antioxidant capacity (T-AOC), together with enhanced lipid peroxidation level, including malondialdehyde (MDA) level and 4-Hydroxynonenal (4-HNE) protein expression. Exposure to FC was associated with upregulated levels of the interleukin (IL)- 6, IL-1β, IL-18, IL-8 and tumor necrosis factor α (TNF-α), while down-regulated levels of IL-4 and IL-10. Exposure to FC was positively associated with the mRNA and protein expressions of cysteine-aspartic proteases (Caspase)- 9, Caspase-3, Bcl-2-associated X protein (Bax), while negatively associated with B-cell lymphoma 2 (Bcl2) in mitochondrial apoptosis signaling pathway. FC-exposure changed the diversity and composition of gut microbes. Additionally, the serum lipopolysaccharide (LPS) contents increased in FC-exposed groups when compared with the control group, while the expression of colonic tight junction proteins (TJPs), such as Claudin-1 and Occludin were decreased. These findings indicate that the colonic mucosal injury induced by FC-exposure are associated with oxidative stress generation, inflammation response and cell apoptosis, as well as the changes in gut microbes diversity and composition.

Bufonis (VB), an animal drug called Chansu in China, is the product of the secretion of Bufo gargarizans Cantor or B. melanostictus Schneider. As a traditional Chinese medicine (TCM) for a long time, it has been widely used in the treatment of heart failure, ulcer, pain, and various cancers. Cinobufaginn (CNB), the cardiotonic steroid or bufalene lactone extracted from VB, has the effects of detoxification, detumescence, and analgesia.The present study aimed to define the effects of CNB on non-small-cell lung cancer (NSCLC) and identify the potential molecular mechanisms.A549 cells were treated with cinobufagin and cell viability, apoptosis, migration, and invasion were then evaluated using Cell Counting Kit-8 (CCK8) assays, flow cytometry, and Transwell assays, respectively. Moreover, the levels of proliferating cell nuclear antigen (PCNA), cytokeratin8 (CK8), poly ADP-ribose polymerase (PARP), Caspase3, Caspase8, B-cell lymphoma/lewkmia-2(Bcl-2), Bcl2-Associated X(Bax), forkhead box O1 (FOXO1), and euchromatic histone-lysine N-methyltransferase2 (G9a, EHMT2) in A549 cells were evaluated using qRT-PCR and/or Western blot analysis (WB), Co-IP, immunofluorescence, and immunohistochemistry. An in vivo imaging system, TUNEL, Immunofluorescence, and immunohistochemistry were also used to detect proliferating cell nuclear antigen(PCNA), Ki67, E-Cadherin(E-Cad), FOXO1, and G9a in mouse xenograft model experiments.CNB suppressed cell proliferation, migration, and invasion but promoted apoptosis in A549 cells in a dose- and time-dependent manner, while cinobufagin had no cytotoxic effect on BEAS-2B cells. In vivo, cinobufagin inhibited the proliferation, migration, and invasion of A549 cells and promoted their apoptosis. The occurrence of the above phenomena was accompanied by an increase in FOXO1 expression and a decrease in G9a expression. In A549 cells, CNB did not reverse the changes in the proliferation, migration, invasion, and apoptosis of A549 cells after FOXO1 was successfully silenced.Our study provides the first evidence that cinobufagin suppresses the malignant biological behaviours of NSCLC cells in vivo and in vitro and suggests that mechanistically, this effect may be achieved by inhibiting the expression of the histone methyltransferase G9a and activating the tumour suppressor gene FOXO1. Taken together, our findings provide important insights into the molecular mechanism underlying cinobufagin's anticancer activity, and suggest that cinobufagin could be a candidate for targeted cancer therapy.

Polyphyllin has been reported to exhibit anticancer effects against various types of cancer via the proapoptotic signaling pathway. The aim of the present study was to investigate the role of the endoplasmic reticulum stress and death receptor signaling pathways in PPI-induced apoptosis of human hepatocellular carcinoma HepG2 cells. Analysis demonstrated that PPI could significantly inhibit the proliferation and induce apoptosis of HepG2 cells in a dose- and time-dependent manner. Investigation into the molecular mechanism of PPI indicated that PPI notably mediated ER stress activation via IRE-1 overexpression and activation of the caspase-12 to protect HepG2 cells against apoptosis. In addition, PPI markedly induced the expression of death receptors signaling pathways-associated factors, including tumor necrosis factor (TNF) receptor 1/TNF-αand FAS/FASL. Additionally, suppression of the death receptor signaling pathways with a caspase-8 inhibitor, Z-IETD-FMK, revealed an increase in the death rate and apoptotic rate of HepG2 cells. Collectively, the findings of the present study suggested that the ER stress and death receptor signaling pathways were associated with PPI-induced HepG2 cell apoptosis; however, endoplasmic reticulum stress may serve a protective role in this process. The combination of PPI and Z-IETD-FMK may activate necroptosis, which enhances apoptosis. Therefore, the results of the present study may improve understanding regarding the roles of signaling pathways in PPI regulated apoptosis and contribute to the development of novel therapies for the treatment of HCC.

Clinical diagnosis of Parkinson's disease (PD) is characterized by the classical features of tremor, bradykinesia and rigidity, which are present only when more than 70%-80% degeneration of dopaminergic (DA) neurons in the substantia nigra. The lack of means for early diagnosis of PD has elicited interest in searching for its risk factors, which, by now, are almost obtained at a single time point in PD process, and little developing risk factors, obtained from completely normal situation to the onset or even advanced stage of PD in individual person which could monitor the progress of PD, are present. Here we have detected some potential factors in the blood of MPTP induced PD monkeys along with the progress of the disease. All the PD monkeys showed mild PD symptoms since the 9th week and gradually reached a classic and stable parkinsonism stage at the 18th week. Our results have found that the expression of Parkin, USP30, MUL1, PINK1, and LRRK2 significantly increased at 1st, 3th, 3th, 5th, and 8th week respectively and remained high till the end; The expression of UCHL1 and TRIM24 significantly increased at the 1st and 18th week, respectively, then gradually decreased and significantly lower than normal value; DJ-1 showed significantly decreased since the 12th week, while SNCA showed no significantly changed excepted at the 5th week. And, the terminal results of whole blood were highly consistent with those of in SN. These results support that these genes change may as biomarkers to monitor the progress of PD, and may facilitate the development of biomarkers for early diagnosis.

Hypothalamus-pituitary-adrenal (HPA) axis, as the key moderator in energy metabolism, plays an important role in diabetes. The endogenous cannabinoid system (eCBs) involves in neuronal functions, and simultaneously cannabinoid receptors are almost expressed in all regions of the hypothalamus according to a spate of reports. However, few data investigate the changes of eCBs and HPA axis in type 2 diabetes. In this study, five diabetes mellitus rhesus monkeys, five prediabetes rhesus monkeys and five healthy rhesus monkeys were observed. In the present study, we detected cell swelling and necrosis extensively in the paraventricular nucleus (PVN) and neurohypophysis in prediabetes and overt diabetes monkeys. The adrenocorticotropic hormone in the pituitary gland, adrenocorticotropic hormone receptor, and 11β-hydroxysteroid dehydrogenase in the adrenal gland were all hyper-secreted and expressed from healthy to overt diabetes. Meanwhile, the cortisol concentration in the adrenal gland was increased along with the progress of diabetes. It could be concluded that hyperfunction of the HPA axis exists in the type 2 Diabetes pathogenesis. However, we also found a weakened expression and secretion of corticotrophin releasing hormone and glucocorticoids receptor in PVN. The expression of corticotropin releasing hormone receptor 1 in pituitary gland decreased in prediabetes monkeys, but increased in overt diabetes monkeys. The downregulation of cannabinoid receptor 1 and upregulation of monoglycerol lipase and fatty acid amide hydrolase in PVN was involved in the pathogenesis of type 2 diabetes. Collectively, we can conclude that changes in endocannabinoid hydrolase and cannabinoid receptor might indicate the effect of downregulation of eCBs. It can be assumed that hyper-function of the HPA axis from healthy to overt diabetes is due to the undermining inhibition of eCBs. However, the regulatory mechanism of eCBs targets on the HPA axis need to be further explored.

Oxidative stress has been implicated in a range of neurodegenerative diseases, in particular, Parkinson’s disease (PD). However, the pathogenic mechanism of its influence on PD progression remains unclear. In order to clarify this, PD models are generated by 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intramuscular injection in eight rhesus monkeys. During the of progress PD pathogenesis, the activities of some major antioxidant enzymes, such as serum superoxide dismutase (SOD), glutathione peroxidase (GPX), and glutathione-s-transferase (GST), were measured, which show continuously decrease. While, the GSH and MDA content, as well as the expression of gamma-glutamylcysteine synthetase (GCS) and glutathione-s-transferase pi (GST-pi) exhibit robust increase. Taken together, this study shows the dynamic changes of antioxidant enzymes and lipid peroxidation in blood of PD monkeys during its progress, which will be important for the diagnosis and treatment of PD.