Hippo-YAP signaling pathway regulates autophagy of human periodontal ligament cells under cyclic tensile stress / 华西口腔医学杂志

OBJECTIVES@#This work aimed to investigate the molecular mechanism of cyclic tensile stress (CTS) stimulating autophagy in human periodontal ligament cells (hPDLCs).@*METHODS@#hPDLCs were isolated and cultured from normal periodontal tissues. hPDLCs were loaded with tensile stress by force four-point bending extender to simulate the autophagy of hPDLCs induced by orthodontic force du-ring orthodontic tooth movement. XMU-MP-1 was used to inhibit the Hippo signaling pathway to explore the role of the Hippo-YAP signaling pathway in activating hPDLC autophagy by tensile stress. The expression levels of autophagy-related genes (Beclin-1, LC3, and p62) in hPDLCs were detected by real-time quantitative polymerase chain reaction. Western blot was used to detect the expression levels of autophagy-related proteins (Beclin-1, LC3-Ⅱ/LC3-Ⅰ, and p62) and Hippo-YAP pathway proteins (active-YAP and p-YAP) in hPDLCs. Immunofluorescence was used to locate autophagy-related proteins (LC3-Ⅱand p62) and Hippo-YAP pathway proteins (active-YAP) of hPDLCs.@*RESULTS@#CTS-activated autophagy in hPDLCs and expression of autophagy-related proteins initially increased and then decreased; it began to increase at 30 min, peaked at 3 h, and decreased (P<0.05). CTS increased the expression of active-YAP protein and decreased the expression of p-YAP protein (P<0.05). When XMU-MP-1 inhibited the Hippo-YAP signaling pathway (P<0.05), active-YAP protein was promoted to enter the nucleus and autophagy expression was enhanced (P<0.05).@*CONCLUSIONS@#The Hippo-YAP signaling pathway is involved in the regulation of autophagy activation in hPDLCs under CTS.

Tanshinone IIA inhibits hypoxia/reoxygenation-induced cardiomyocyte apoptosis and autophagy by regulating ABCE1 / 中华危重病急救医学

OBJECTIVE@#To investigate the effects of tanshinone IIA on apoptosis and autophagy induced by hypoxia/reoxygenation in H9C2 cardiomyocytes and its mechanism.@*METHODS@#H9C2 cardiomyocytes in logarithmic growth phase were divided into control group, hypoxia/reoxygenation model group and tanshinone IIA low-dose, medium-dose and high-dose groups (50, 100, 200 mg/L tanshinone IIA were treated after hypoxia/reoxygenation respectively). The dose with good therapeutic effect was selected for follow-up study. The cells were divided into control group, hypoxia/reoxygenation model group, tanshinone IIA+pcDNA3.1-NC group and tanshinone IIA+pcDNA3.1-ABCE1 group. The cells were transfected with the overexpressed plasmids pcDNA3.1-ABCE1 and pcDNA3.1-NC and then treated accordingly. Cell counting kit-8 (CCK-8) was used to detect H9C2 cell activity in each group. The apoptosis rate of cardiomyocytes was detected by flow cytometry. The ATP-binding cassette transporter E1 (ABCE1), apoptosis-related proteins Bcl-2 and Bax, caspase-3, autophagy-related proteins Beclin-1, microtubule-associated protein 1 light chain 3 (LC3II/I) and p62 mRNA expression level of H9C2 cells in each group were detected by real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-qPCR). The protein expression levels of the above indexes in H9C2 cells were detected by Western blotting.@*RESULTS@#(1) Cell activity and ABCE1 expression: tanshinone IIA inhibited the activity of H9C2 cells induced by hypoxia/reoxygenation, and the effect was significant at medium-dose [(0.95±0.05)% vs. (0.37±0.10)%, P < 0.01], mRNA and protein expression of ABCE1 were significantly reduced [ABCE1 mRNA (2-ΔΔCt): 2.02±0.13 vs. 3.74±0.17, ABCE1 protein (ABCE1/GAPDH): 0.46±0.04 vs. 0.68±0.07, both P < 0.05]. (2) Expression of apoptosis-related proteins: medium-dose of tanshinone IIA inhibited the apoptosis of H9C2 cells induced by hypoxia/reoxygenation [apoptosis rate: (28.26±2.52)% vs. (45.27±3.07)%, P < 0.05]. Compared with the hypoxia/reoxygenation model group, medium-dose of tanshinone IIA significantly down-regulated the protein expression of Bax and caspase-3 in H9C2 cells induced by hypoxia/reoxygenation, and significantly up-regulated the protein expression of Bcl-2 [Bax (Bax/GAPDH): 0.28±0.03 vs. 0.47±0.03, caspase-3 (caspase-3/GAPDH): 0.31±0.02 vs. 0.44±0.03, Bcl-2 (Bcl-2/GAPDH): 0.53±0.02 vs. 0.37±0.05, all P < 0.05]. (3) Expression of autophagy-related proteins: compared with the control group, the positive rate of LC3 in the hypoxia/reoxygenation model group was significantly increased, while the positive rate of LC3 in the medium-dose of tanshinone IIA group was significantly decreased [(20.67±3.09)% vs. (42.67±3.86)%, P < 0.01]. Compared with hypoxia/reoxygenation model group, medium-dose of tanshinone IIA significantly down-regulated Beclin-1, LC3II/I and p62 protein expressions [Beclin-1 (Beclin-1/GAPDH): 0.27±0.05 vs. 0.47±0.03, LC3II/I ratio: 0.24±0.05 vs. 0.47±0.04, p62 (p62/GAPDH): 0.21±0.03 vs. 0.48±0.02, all P < 0.05]. (4) Expression of apoptosis and autophagy related proteins after transfection with overexpressed ABCE1 plasmid: compared with tanshinone IIA+pcDNA3.1-NC group, the protein expression levels of Bax, caspase-3, Beclin-1, LC3II/I and p62 in tanshinone IIA+pcDNA3.1-ABCE1 group were significantly up-regulated, while the protein expression level of Bcl-2 was significantly down-regulated.@*CONCLUSIONS@#100 mg/L tanshinone IIA could inhibit autophagy and apoptosis of cardiomyocytes by regulating the expression level of ABCE1. So, it protects H9C2 cardiomyocytes injury induced by hypoxia/reoxygenation.

Oxidative stress induces autophagy to inhibit the proliferation and apoptosis of human bone marrow mesenchymal stem cells (hBMSCs) / 细胞与分子免疫学杂志

Objective To investigate the effect of H2O2-induced oxidative stress on autophagy and apoptosis of human bone marrow mesenchymal stem cells (hBMSCs). Methods hBMSCs were isolated and cultured. The cells were divided into control group, 3-MA group, H2O2 group, H2O2 combined with 3-MA group. DCFH-DA staining was used to analyze the level of reactive oxygen species (ROS). hBMSCs were treated with 0, 50, 100, 200, 400 μmol/L H2O2, and then the cell viability was detected by CCK-8 assay. The level of autophagy was detected by monodansylcadaverine (MDC) staining and LysoTracker Red staining. The cell apoptosis was detected by flow cytometry. Western blotting was used to detect the expression of beclin 1, mTOR, phosphorylated mTOR (p-mTOR), cleaved caspase-3(c-caspase-3) and caspase-3 proteins. Results Compared with the control group and 3-MA group, ROS level and autophagosomes were increased and the proliferation and apoptosis were decreased in H2O2 group. The protein expression of beclin 1, mTOR, c-caspase-3 was up-regulated, while the p-mTOR was down-regulated. Compared with the 3-MA group, the H2O2 combined with 3-MA group also had an increased ROS level and autophagosomes, but not with significantly increased apoptosis rate; The protein expression of beclin 1, mTOR, c-caspase-3 was up-regulated, and the p-mTOR was down-regulated. Conclusion H2O2 can induce hMSCs to trigger oxidative stress response. It enhances the autophagy and inhibits the proliferation and apoptosis of hBMSCs.

Down-regulation of SIK2 expression alleviates myocardial ischemia-reperfusion injury in rats by inhibiting autophagy through the mTOR-ULK1 signaling pathway / 南方医科大学学报

OBJECTIVE@#To explore the role of salt-inducible kinase 2 (SIK2) in myocardial ischemia-reperfusion (IR) injury in rats.@*METHODS@#Fifteen male SD rats were randomized equally into sham operation group, myocardial IR model group, and SIK2 inhibitor group (in which the rats were treated with intravenous injection of 10 mg/kg bosutinib via the left femoral vein 24 h before modeling). Ultrasound was used to detect the cardiac function of the rats, and myocardial pathologies were observed with HE staining. Transmission electron microscopy was used to observe autophagy of myocardial cells, and Western blotting was performed to detect the contents of the autophagy-related proteins SIK2, LC3B, Beclin-1, p62 and the expressions of p-mTOR, mTOR, p-ULK1, and ULK1 in myocardial tissue.@*RESULTS@#Myocardial IR injury significantly increased the number of autophagosomes (P < 0.05) and the expression of SIK2 protein (P < 0.01) in the myocardial tissues. Treatment with bosutinib before modeling obviously lowered the expression of SIK2 protein (P < 0.01), alleviated myocardial pathologies, and reduced the number of autophagosomes (P < 0.05) in the myocardial tissue. The rats with myocardial IR injury showed obviously lowered LVEF and FS values (P < 0.001), which were significantly improved by bosutinib treatment (P < 0.05); no significant difference was detected in IVSDd or LVPWDd among the 3 groups (P > 0.05). Myocardial IR injury obviously increased the expressions of LC3-II/LC3-I and Beclin-1 proteins and lowered the expression of p62 protein (P < 0.01), and these changes were significantly rescued by bosutinib treatment (P < 0.05). The rat models of myocardial IR injury showed significantly increased expression of p-ULK1 (Ser757) (P < 0.01) and lowered expression of p-mTOR protein (P < 0.0001) in the myocardium, and these changes were obviously reversed by bosutinib (P < 0.01 or 0.05); there was no significant difference in mTOR and ULK1 expressions among the 3 groups (P > 0.05).@*CONCLUSION@#SIK2 may promote autophagy through the mTOR/ULK1 signaling pathway, and inhibiting SIK2 can reduce abnormal autophagy and alleviate myocardial IR injury in rats.

Effects of Zhongfeng capsule on autophagy related proteins expressions of brain tissue in rats with cerebral ischemia/reperfusion injury / 中国应用生理学杂志

Objective: To investigate the effects of Zhongfeng capsule on the autophagy-related proteins expression in rats with cerebral ischemia/reperfusion injury (CI/ RI), and to explore its neural protection mechanisms of the decoction. Methods: Rat middle cerebral artery ischemia/reperfusion injury model (ischemia for 2 h, reperfusion for 24 h) was prepared by the improved line plug method. Sixty male SD rats were randomly divided into sham operation group, model group, butylphthalide group(0.054 g/kg), Zhongfeng capsule high-dose groups (1.08 g/kg), Zhongfeng capsule middle-dose groups (0.54 g/kg), Zhongfeng capsule low-dose groups (0.27 g/kg), with 10 rats in each group. Rats were treated with Zhongfeng capsule by gavage once a day for 10 days. The rats were sacrificed and the brain tissue was obtained after the experiment in each group. Score neurological deficit was evaluated after 24 h of the last intervention in rat of each group. The pathological changes of brain tissue were observed by HE staining. The serum levels of estradiol (E2) and follicle stimulating hormone (FSH) were determined by ELISA. The expressions of key genes and proteins of PI3K/Akt/Beclin1 signaling pathway in brain tissue were detected by qRT-PCR and Western blot respectively. Results: Compared with the sham operation group, the body weight and protein expressions of p-PI3k and p-Akt in brain tissue of rats were decreased significantly in the model group, while the brain index, neurological deficit score, gene and protein expressions of Beclin1 and LC3 were increased markedly in the model group(P＜0.05 or P＜0.01). In the model group, nerve cells of brain tissue were loosely packed, interstitial edema, triangular in shape, nuclear pyknosis and dark-blue staining were observed. Compared with the model group, the body weight of rats was increased obviously, the neurological deficit score was decreased significantly and the pathological injury of brain tissue was alleviated evidently in high-dose of Zhongfeng capsule group (P＜0.05). The brain index, the gene and protein expressions of Beclin1 and LC3 were decreased apparently in Zhongfeng capsule treatment groups(P＜0.05 or P＜0.01), while the expressions of p-PI3k and p-Akt in brain tissue were increased evidently in Zhongfeng capsule treatment groups(P＜0.05 or P＜0.01). Conclusion: Zhongfeng capsule can inhibit autophagy and improve brain neurons lesion of CIRI rats, the mechanism may be related to regulate the expression of Beclin1 and LC3 in PI3K/Akt/Beclin1 signaling pathway.

Autophagy activation attenuates the neurotoxicity of local anesthetics by decreasing caspase-3 activity in rats / A ativação autofágica atenua a neurotoxicidade dos anestésicos locais ao diminuir a atividade da caspase‐ 3 em ratos

Abstract Background and objectives The mechanisms by which local anesthetics cause neurotoxicity are very complicated. Apoptosis and autophagy are highly coordinated mechanisms that maintain cellular homeostasis against stress. Studies have shown that autophagy activation serves as a protective mechanism in vitro. However, whether it also plays the same role in vivo is unclear. The aim of this study was to explore the role of autophagy in local anesthetic-induced neurotoxicity and to elucidate the mechanism of neurotoxicity in an intrathecally injected rat model. Methods Eighteen healthy adult male Sprague-Dawley rats were randomly divided into three groups. Before receiving an intrathecal injection of 1% bupivacaine, each rat received an intraperitoneal injection of vehicle or rapamycin (1 mg.kg-1) once a day for 3 days. The pathological changes were examined by Haematoxylin and Eosin (HE) staining. Apoptosis was analysed by TdT-mediated dUTP Nick-End Labelling (TUNEL) staining. Caspase-3, Beclin1 and LC3 expression was examined by Immunohistochemical (IHC) staining. Beclin1 and LC3 expression and the LC3-II/LC3-I ratio were detected by western blot analysis. Results After bupivacaine was injected intrathecally, pathological damage occurred in spinal cord neurons, and the levels of apoptosis and caspase-3 increased. Enhancement of autophagy with rapamycin markedly alleviated the pathological changes and decreased the levels of apoptosis and caspase-3 while increasing the expression of LC3 and Beclin1 and the ratio of LC3-II to LC3-I. Conclusions Enhancement of autophagy decreases caspase-3-dependent apoptosis and improves neuronal survivalin vivo. Activation of autophagy may be a potential therapeutic strategy for local anaesthetic-induced neurotoxicity.

Resumo Introdução e objetivos Os mecanismos de neurotoxicidade dos anestésicos locais são complexos. A apoptose e a autofagia são mecanismos altamente organizados que mantêm a homeostase celular durante o estresse. Estudos revelam que a ativação da autofagia atua como mecanismo de proteção in vitro. Não está claro se a autofagia também desempenha essa função in vivo. O objetivo deste estudo foi analisar o papel da autofagia na neurotoxicidade induzida por anestésico local e esclarecer o mecanismo dessa neurotoxicidade utilizando um modelo de injeção intratecal em ratos. Métodos Dezoito ratos Sprague‐Dawley machos adultos saudáveis foram divididos aleatoriamente em três grupos. Antes de receber a injeção intratecal de bupivacaína a 1%, cada rato recebeu injeção intraperitoneal de veículo ou rapamicina (1 mg.kg‐1) uma vez ao dia durante 3 dias. As alterações patológicas foram examinadas por coloração com Hematoxilina e Eosina (HE). A apoptose foi analisada por coloração com o método dUTP Nick‐End Labeling (TUNEL) mediado por TdT. A expressão de caspase‐3, Beclin1 e LC3 foram examinadas por coloração Imunohistoquímica (IHQ). A expressão de Beclin1 e LC3 e a razão LC3‐II/LC3‐I foram detectadas por análise de western blot. Resultados Após a injeção intratecal de bupivacaína, ocorreu lesão patológica nos neurônios da medula espinhal e os níveis de apoptose e caspase‐3 aumentaram. A ativação da autofagia causada pela rapamicina mitigou de forma expressiva as alterações patológicas e diminuiu os níveis de apoptose e caspase‐3, aumentando a expressão de LC3 e Beclin1 e a razão LC3‐II/LC3‐I. Conclusões O aumento da autofagia diminui a apoptose dependente da caspase‐3 e melhora a sobrevivência neuronal in vivo. A ativação da autofagia pode ser uma estratégia terapêutica potencial para a neurotoxicidade induzida por anestésicos locais.