Investigation On The Antagonistic Effect Of H₂S Against Endothelial-to-Mesenchymal Transition Induced By Abnormal Shear Stress And Its Role In Atherogenesis

Experimental background:Atherosclerosis?As?is a pathological change that occurs in large and medium-sized arteries.It is an important pathophysiological basis of cardiovascular disease.Several factors are involved in the process of the development of As,with abnormal shear stress in As.The formation of lesions plays an important role.It was found that the proliferating mesenchymal cells in As plaque not only originate from the media of the blood vessels,but also a large part of the endothelial cells from the intima were transformed by the endothelium.Endothelial-to-mesenchymal transition?EndMT?is a special type of epithelial mesenchymal transition,which is the process by which endothelial cells are transformed into mesenchymal cells under a variety of factors.During this process,endothelial cells gradually Loss of its morphology and function,or the phenotypic characteristics of mesenchymal cells including proliferation,migration,and collagen synthesis.In recent years,it has been found that the abnormal shear stress-induced endothelial interstitial transformation plays an important role in vascular diseases such as As.Hydrogen sulfide?H₂S?is a new type of gas signaling molecule with vascular protection function.However,The effect and mechanism of endothelial interstitial transformation under abnormal shear stress is not clear.Purpose:By establishing an abnormal shear stress model,the effect of exogenous hydrogen sulfide on ApoE^-/-mice was observed,and its mechanism of inhibiting endothelial interstitial transformation and atherosclerosis was preliminarily explored.Method:Forty-five ApoE^-/-mice were randomly divided into 3 groups?n=15?:PBS control group,Na HS group,and PPG group.NaHS group mice were injected intraperitoneally.The dose of exogenous donor NaHS of H₂S was 56?mol/kg/day.PPG group mice received intraperitoneal injection of CSE inhibitor PPG at a dose of 37.5 mg/kg/day,Intraperitoneal injection of equal doses of PBS in the control group.After adaptive feeding for 1 week,abnormal shear was established after anesthesia.The shear stress model was fasted?approximately 12 hours?before the ligation operation.After injection,they were injected with PBS,NaHS,and PPG,respectively,fed with high-fat and high-cholesterol diet,and sacrificed after 2 weeks.Blood samples were obtained by eyeball sampling,anticoagulation and centrifugation,serum H₂S concentration wasmeasuredbymodifiedmethylenebluemethod and spectrophotometer.The left common carotid artery was taken and frozen sections were performed.HE and oil red O staining was used to observe the changes of As plaque.The expression of endothelial cell markers?CD31,VE-cadherin?and interstitial cell markers??-SMA,FAP?,N-cadherin?in the left common carotid artery was detected by immunofluorescence.The left common carotid artery of ApoE^-/-mice was taken and the expression of TGF?1 and pSmad2/3 was detected by immunofluorescence.The human umbilical vein endothelial cells?HUVECs?induced by TGF?1 successfully undergone the process of endothelial interstitial transformation.The expression of pSmad2/3protein in three groups of cells was detected by Western blot.Experimental results:1.There was no significant change in blood lipid and body weight in each group of mice.2.The level of plasma H2 S in NaHS group was increased about 26.3%compared with the control group?P<0.05?,while the plasma H2 S level in the PPG group was decreased about 45.20%compared?P<0.01?.The expression of CSE was detected in three groups of mice.The expression of CSE in the sodium hydrosulfide group was significantly increased in the control group,while the expression of CSE was significantly decreased in the PPG group compared with the other two groups.3.The comparison of the right and left common carotid arteries in each group showed that the area of the left carotid artery relative to the right side was significantly increased?with no lesion on the right side?,indicating that abnormal shear stress can promote the development of As lesions.The mouse common carotid oil red 0 showed that compared with the PBS and PPG groups,the plaque area of the carotid As in the NaHS group was reduced approximately37.58%?P<0.05?and 61.32%?P<0.05?,respectively,compared with the Contrl group and the PPG group.4.Immunofluorescence of frozen sections of common carotid arteries showed that in the NaHS group,the co-expression of CD31 with?-SMA and FAP?was significantly reduced compared with the other two groups.The expression of VE-cadherin was relatively increased,while the expression of mesenchymal marker N-cadherin was significantly decreased,and the degree of endothelial interstitial transformation in NaHS mice was significantly reduced.5.Immunofluorescence were used to detect the expression of TGF?1 and pSMAD2/3.The expression of TGF?1 and pSMAD2/3 in NaHS group was significantly reduced compared with other two groups.6.HUVECs were incubated for 48 h with 10 ng/ml TGF?1,100?mol/L NaHS+10ng/ml TGF?1,and normal cultured cells were used as controls.Western blot was used to detect the expression of CD31,?SMA,FAP?,VE-Cadherin,N-Cadherin and pSmad2/3 in three groups of cells.Western blotting showed that compared with the control group,the expression of CD31 and VE-Cadherin in the TGF?1 group was decreased,while the expression of?SMA,FAP?,N-Cadherin,and pSmad2/3 was significantly increased.Compared with TGF?1 group,the expression of CD31 and VE-Cadherin in TGF?1+NaHS group increased,but the expression of?SMA,FAP?and N-Cadherin decreased,and the expression of pSmad2/3 decreased?P<0.05?.Conclusion:Hydrogen sulfide can reduce the transformation of endothelium caused by abnormal shear stress and reduce the occurrence of atherosclerosis.Its mechanism is related to the down-regulation of TGF?1/Smad expression.