| Part one ?-lipoic acid reverses the decrease of H2S levels induced by diabetes or high glucose conditionAims:We aimed to identify the decrease of serum hydrogen sulfide?H2S?levels induced by type 2 diabetes mellitus?T2DM?and the ameliorative effect of?-lipoic acid on H2S levels.Methods:In the clinical trail,101 Chinese patients with T2DM and 20age-and sex-matched Chinese subjects without diabetes were recruited in the studytoobserveserumH2Slevels.AllpatientswithT2DM were hospitalized in the first endocrinology department,Third hospital of Hebei Medical University from June 2016 to December 2016.The participants in control group were recruited from healthy volunteers.Additionally,Sprague-Dawley?SD?rats with T2DM induced by high-glucose-high-fat diet and low-dose streptozotocin?27.5 mg/kg?and cultured vascular smooth muscle cells?VSMCs?incubated in high glucose condition were also used to detect the changes of H2S levels.Meanwhile,?-lipoic acid was used in patients/rats with T2DM and cultured VSMCs under high glucose condition to detect the effects on H2S levels and CSE?cystathionine?-1yase?expression.Propargylglycine?PPG?,as an endogenous inhibitor of H2S,was used to identify the effects of?-lipoic acid on H2S levels further.Results:Serum H2S levels significantly decreased in Chinese patients with T2DM,and it was in accordance with the results in diabetic rats.Moreover,H2S levels in the homogenate and the medium of cultured VSMCs under high glucose condition were also decreased.?-lipoic acid reversed these changes in vivo and in vitro.And the up-regulatory effects of?-lipoic acid were weakened by PPG.In addition,diabetes or high glucose condition induced a decrease of CSE expression in VSMCs,which was reversed by?-lipoic acid.Conclusion:Diabetes or high glucoses condition induces a decrease of H2S level.?-lipoic acid could up-regulate the H2S levels and endogenous H2S production in VSMCs via elevating the CSE expression.Part two ?-lipoic acid regulates autophagy and improves the diabetic vascular smooth muscle function by elevating H2S levelAims:Our experiment was to reveal whether?-lipoic acid could regulate autophagy and could improve the diabetic vascular smooth muscle function by elevating H2S level.Methods:VSMCs were isolated from the thoracic and abdominal aorta of male SD rats and cultured in this study.The cells between passages 5 and 8were used in this experiment.Serum-starved VSMCs were stimulated with 30mmol/L glucose.In some groups,VSMCs were primarily cultured in HG for12 h,and then,were co-treated with 100?mol/L sodium hydrosulfide?NaHS,an exogenous donor of H2S?or 500?mol/L?-lipoic acid for 36 h.Meanwhile,rapamycin,an autophagy activator,was also used to incubate VSMCs.It was to identify whether H2S/ALA could protect VSMCs via regulating autophagy.Aditionally,the cells were pre-treated with PPG for 30 min before NaHS or?-lipoic acid treatment to analysis whether ALA regulated autophagy by elevating H2S levels.In vivo,SD rats with T2DM were induced by high-glucose-high-fat diet and low-dose streptozotocin?27.5 mg/kg?.The rats were randomly divided into five groups:control group,diabetes mellitus?DM?group,?-lipoic acid group,PPG group,PPG+?-lipoic acid group.Hematoxylin and Eeosin staining was used to observe the pathologic changes of aortas.Vascular function examination was performed for the endothelium-dependent relaxation,endothelium-independent relaxation,and angiotensin II-induced contraction.Electron microscope examinations with a transmission electron microscope were used to detect autophagic vacuoles.Western-blot analysis was used to access protein levels of autophagy-related protein.Results:In cultured VSMCs under high glucose condition,Beclin-1 and LC3BII/LC3BI expression was markedly elevated,while the cell viability and P62 expression was down-regulated.These effects were partly restored by NaHS or?-lipoic acid.The protective effects of NaHS or?-lipoic acid were attenuated by rapamycin or PPG.In rats with T2DM,?-lipoic acid protected the vascular function and reduced autophagy relative index.PPG could weaken the effects of?-lipoic acid on VSMCs of diabetic rats.Conclusion:?-lipoic acid could down-regulate autophagy and could improve the diabetic vascular smooth muscle function by elevating H2S levels.Part three ?-lipoic acid inhibits autophagy via AMPK/mTOR signal pathway in the vascular smooth muscle cells under high glucose by elevating H2S levelAims:Our study was to elucidate wether?-lipoic acid inhibits AMP-activated protein kinase?AMPK?/mammalian target of rapamycin?mTOR?signal pathway,downregulates the autophagy level and improves cell viability in vascular smooth muscle cells in high glucose conditions by elevating H2S levels.Methods:VSMCs were isolated from SD rats and cultured in this study.Serum-starved VSMCs were stimulated with 30 mmol/L glucose.In some groups,VSMCs were primarily cultured in HG for 12 h,and then,were co-treated with 100?mol/L NaHS or 500?mol/L?-lipoic acid for 36 h.Cells werepre-treatedwithPPG,5-amino-1-?-d-ribofuranosyl-imidazole-4-carboxamide?AICAR,an AMPK activator?,compound C?an AMPK blocker?,or not for 30 min before NaHS or?-lipoic acid treatment according to the requirements.Cell viability was measured with CCK-8 assay.SD rats were randomly divided into five groups:control group,DM group,?-lipoic acid group,PPG group,?-lipoic acid+PPG group.Western-blot analysis was used to access protein levels of AMPK,p-AMPK,mTOR,p-mTOR,and autophagy related protein in cultured VSMCs and in the arteries of the SD rats.Results:AMPK/mTOR signal pathway was activated by high glucose and decreased by NaHS or?-lipoic acid in VSMCs.The down-regulatory effects of NaHS or?-lipoic acid on AMPK/mTOR pathway were attenuated by AICAR followed by an increase of autophagy activity.Compound C enhanced the effects of NaHS or?-lipoic acid on AMPK/mTOR pathway and autophagy.P-AMPK expression was increased and p-mTOR expression was decreased in?-lipoic acid+PPG group compared with the?-lipoic acid group.In rats with T2DM,?-lipoic acid could also reduce AMPK/mTOR activation induced by diabetes,and the regulation of?-lipoic acid was inhibited by PPG.Conclusion:?-lipoic acid inhibits AMPK/mTOR signal pathway followed by a decrease of autophagy activity in the VSMCs under high glucose by elevating H2S levels.Conclusions:1.Diabetes or high glucoses condition induces a decrease of H2S levels.?-lipoic acid could up-regulates H2S levels.2.?-lipoic acid down-regulates autophagy and improves the diabetic vascular smooth muscle function by elevating H2S levels.3.?-lipoic acid modulates autophagy via AMPK/mTOR signal pathway in the VSMCs under high glucose condition by elevating H2S levels. |
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