Investigation On Hydrogen Sulfide Regulated Vascular Smooth Muscle Cells Autophagy And Its Antagonistic Role In Aortic Dissection

Aortic dissection(AD)is a kind of vascular disease,which is highly fatal.Degeneration of the medial wall of the aorta is the pathological part to AD formation.It is a process of massive depletion of vascular smooth muscle cells(VSMCs)and degradation of the extracellular matrix.Hydrogen sulfide(H₂S)is currently considered as a third gas signaling molecule.Many studies have shown that H₂S plays an important role in the regulation of many chronic diseases,especially in cardiovascular diseases,such as by inhibiting cardiovascular remodeling,vasodilating,and scavenging oxygen free radicals to improve the endogenous antioxidant defense ability and inhibit inflammation.Recently,it has been found that H₂S content in the vascular tissue homogenized in AD patients is lower than that in the control group.H₂S can up-regulate autophagy through AMPK/mTOR signaling pathway,and play a cardiovascular protective role by inhibiting endoplasmic reticulum stress(ERS)through IRE-?-XBP-1 signaling pathway.However,the role and mechanism of CSE/H₂S system in AD have not been reported.The aim of this study was to investigate the effect of H₂S up-regulated VSMCs autophagy on the occurrence of AD.Therefore,it is expected to clarify the mechanism of AD and provide a new strategy for the prevention and treatment of AD.Part1:CSE/H₂S system and autophagy is down-regulated in aortic dissectionAims:To study the change of CSE/H₂S system and autophagy level in AD.Methods:Collect sample:1.Patients admitted to the First Affiliated Hospital and the Second Affiliated Hospital of University of South China from March 2017 to February 2019 with AD and artificial vascular replacement(AD group with aortic sample and serum sample);2.Body donors in the kidney transplantation department(Control group with aortic sample);3.Normal physical examination subjects were enrolled(Control group with serum sample).HE staining and Masson staining were used to observe the pathomorphologic changes of aorta.The content of H₂S in serum was detected by colorimetry.The secretion of inflammatory factors was detected by ELISA.The expressions of CSE,P62,LC3,MMP2/9 and IRE-?were detected by immunohistochemistry,western blot and immunofluorescence,respectively.Results:There was no significant difference in age,history of diabetes and smoking rate between AD group and body donors control group,AD group andnormal physical examination control group.Compared with body donors control group,arterial wall fractures,it has free cavity,muscle fibers and collagen fibers arrangement is irregular,the CSE expression decreases(31.2%,P<0.05),autophagy activity(P62expression levels rises to 4.4 times,LC3II/I ratio decreases 58.6%,P<0.05)decreases,the expression of TIMP2/MMP-2/9 level decreases(26.8%and 44.9%,P<0.05)and p-IRE-?expression level rises(2.2 times,P<0.05).Compared with normal physical examination control group,the serum H₂S level decreases(26.3%,P<0.05),proinflammatory factors CRP,D2D,IL 6 and TNF alpha levels increase(38.8times,34.2 times,4.5 times and 2.5times,P<0.05),the suppression of inflammation factor(IL-10)levels drops(42.5%,P<0.05).Conclusions:At AD,CSE/H₂S system was down-regulated,autophagy activity and the suppression of inflammation factor were decreased,proinflammatory factors and MMP levels were increased,and ERS response was aggravated.Part2:The effect of hydrogen sulfide on AD formation and its mechanismAims:To investigate the effect of H₂S on AD formation in rats and its mechanismMethods:BAPN was used to replicate rat AD model,and SD rats were divided into four groups:control group(without BAPN),BAPN group,BAPN+Na HS group and BAPN+Na HS+CQ group.At 4 weeks,the animals were sacrificed,the general conditions and the incidence of AD in the four groups were compared,and the pathomorphological changes of aorta were observed by HE staining and Masson staining.The content of H₂S in serum was detected by colorimetry.The expressions of CSE,P62,LC3,MMP-2/9 and IRE-?were detected by immunohistochemistry,western blot and immunofluorescence respectively,and the secretion of inflammatory factors was detected by ELISA.Results:Compared with the Control group,the daily diet amount and weight amount of rats in BAPN group,BAPN+Na HS group and BAPN+Na HS+CQ group was less,but the difference was not statistically significant.All rats in the Control group were normal and AD did not occur.The incidence of AD in BAPN group was 83.3%.Compared with BAPN group,the incidence of AD in BAPN+Na HS group was 35.7%,which was significantly lower,and the difference was statistically significant(P<0.05).Compared with BAPN group,the incidence of AD in BAPN+Na HS+CQ group was 84.6%,which was higher,but the difference was't statistically significant.Compared with Control group,the BAPN group rat aortic wall have obvious false lumen,elastic fibers arranged disorder,vascular wall thickened obviously,the content of H₂S decreased(28.0%,P<0.05),autophagy activity(P62 expression level rises to 3.2 times,LC3?/I ratio decreases 48.9%,P<0.05)decreased,the expression of MMP-2/9 and p-IRE-?expression level rise(65.7%?52.7%and 3.41times,P<0.05),proinflammatory factor CRP,D2D,IL-6 and TNF-?levels increase(20.02 times,4.69 times,3.73 times and 4.21 times,P<0.05),the suppression of inflammation factor(IL-10)levels drops(59.0%,P<0.05).Compared with the BAPN group,rat aortic wall structure in the BAPN+Na HS group is relatively complete,elastic fiber arrangement is neat,aortic wall thickening isn't obviously,serum H₂S content(increases 1.94 times,P<0.05)and autophagy activity increases(P62 expression level decreases 33.33%,LC3?/I ratio increases1.47times,P<0.05),the expression of MMP-2/9 level(decreases 17.24%and 26.19%,P<0.05),p-IRE-?expression(decreases 35.14%,P<0.05),and proinflammatory factors(CRP,D2D,IL-6 and TNF-?levels decrease27.6%,28.7%,30.5%and 37.5%,P<0.05)drop,suppression of inflammation factor(IL-10)level increases(1.91 times,P<0.05).Compared with the BAPN group,in the BAPN+Na HS+CQ group,rat aortic wall thickening is more apparent,elastic fiber arrangement is more disorder,autophagy activity decrease(P62 expression level increases 1.4times,LC3?/I ratio decreases 47.8%,P<0.05),the expression of MMP-2/9 level(1.19 times and 1.26times,P<0.05),p-IRE-?expression(1.25 times,P<0.05)and proinflammatory factor CRP,D2D,IL-6 and TNF alpha levels(1.38 times,1.48 times,1.30 times,and 1.25times,P<0.05)increase,suppression of inflammation factor IL-10 level drops(18.36%,P<0.05).Conclusions:Autophagy is impaired when AD occurs in rats.Exogenous H₂S can reduce the incidence of AD,the mechanism is related to the up-regulation of autophagy,inhibition of inflammatory factor expression and endoplasmic reticulum stressPart3:H₂Sup-regulatesVSMCsautophagy through AMPK/mTOR/ATG5 pathwayAims:To investigate the effect of H₂S on VSMCs autophagy and observe the effect of AMPK/mTOR/ATG5 pathway on autophagy,inflammatory factor secretion level and ERS in VSMCs.Methods:VSMCs were isolated and cultured from the aorta of body donors control group and AD group.The morphology of VSMCs was observed under inverted microscope.The expression of?-actin in VSMCs was detected by immunofluorescence.AMPK si RNA,LV mTOR lentivirus and ATG5 si RNA were transfected respectively.The expression of AMPK,mTOR and ATG5 protein were observed by Western blot.The VSMCs were divided into control group(body donors control group aortic VSMCs),AD group(AD group aortic VSMCs),Na HS group(AD group aortic VSMCs),Na HS+AMPK si RNA group(AD group aortic VSMCs),Na HS+LV mTOR group(AD group aortic VSMCs),Na HS+ATG5 si RNA group(AD group aortic VSMCs)and Na HS+CQ group(AD group aortic VSMCs).The changes of autophagy(p62 and LC3)in VSMCs were detected by western blot,the levels of inflammatory factors(TNF-?,IL-6,IL-1?and IL-10)and ERS(p-IRE-?and GRP78)were detected by ELISA and western blot.Results:1.VSMCs isolated from human aortic tissue expressed?-actin.2.Compared with the control si RNA group,AMPK si RNA transfection significantly inhibited AMPK expression(68.10%,P<0.05).Compared with vector transfection group,LV mTOR lentivirus transfection significantly increased mTOR expression(increased to 2.36 times,P<0.05).Compared with control si RNA group,ATG5 si RNA transfection significantly inhibited ATG5 expression(decreased 63.53%,P<0.05).3.Compared with control group,the autophagy activity of VSMCs in AD group was lower(p62 expression increased to 2.43 times and LC3 II/I ratio decreased by 79.19%,P<0.05).Compared with AD group,autophagy activity of VSMCs in Na HS group was up-regulated(p62expression decreased by 57.39% and LC3 II/I ratio increased to 1.92times,P<0.05).Compared with Na HS group,the autophagy activity of VSMCs in Na HS+AMPK si RNA group,Na HS+LV mTOR group,Na HS+ATG5 group were significantly decreased(p62 expression increased to 1.60,1.52 and 1.61 times,P<0.05,LC3?/I ratio decreased by 35.95%,27.45%and 37.25%,respectively,P<0.05).4.Compared with control group,the levels of proinflammatory factors in VSMCs of AD group were increased(TNF-?,IL-6 and IL-1?were increased to 3.01,3.53 and 4.23 times,respectively,P<0.05),the level of IL-10 was decreased by 56.13%(P<0.05),the expression of p-IRE-?and GRP78was increased(increased to 3.80 and 3.86 times,respectively,P<0.05).Compared with AD group,the levels of proinflammatory factors in VSMCs of Na HS group were decreased(TNF-?,IL-6 and IL-1?were decreased by 47.84%,53.82%and 47.75%,respectively,P<0.05),the level of IL-10 was increased to 1.70 times(P<0.05),and the expression of p-IRE-?and GRP78 was decreased(50.37%and 46.91%,respectively,P<0.05).Compared with Na HS group,the level of proinflammatory cytokines of VSMCs in Na HS+AMPK si RNA group,Na HS+LV mTOR group,Na HS+ATG5 group and Na HS+CQ group was increased(TNF-?increased to 1.50 times,1.55 times,1.49 times and 2.38 times,P<0.05,IL-6 increased to 1.54 times,1.61 times,1.53 times and 2.72times,P<0.05,IL-1?increased to 1.42 times,1.40 times,1.51 times and2.30 times,P<0.05),and IL-10 level decreased(28.00%,30.67%,34.67%and 54.67%respectively,P<0.05).The expression of p-IRE-?and GRP78increased(p-IRE-?increased to 1.33 times,1.28 times,1.34 times and2.37 times,P<0.05,GRP78 expression increased to 1.40 times,1.44times,1.49 times and 2.42 times,respectively,P<0.05).Conclusions:Exogenous H₂S up-regulated the autophagy of VSMCs and inhibited the inflammatory response and ERS of VSMCs through AMPK/mTOR/ATG5 pathway.