Hydrogen Sulfide Inhibits H₂O₂-induced Senescence Of Vascular Smooth Muscle Cells Through Activation Of The Klotho/FoxO1 Pathway

Background and objective: Cardiovascular disease has risen to the highest mortality rate of disease of the world.With the advancement of science and technology and the improvement of medical level,research and prevention of cardiovascular disease has also been greatly developed.In cardiovascular disease research,more and more attention is paid to the risk factor-senescence.Vascular smooth muscle cell senescence is the basis of vascular senescence.It is important to explore the mechanism of vascular smooth muscle cell senescence in the prevention of vascular senescence and the prevention of cardiovascular and other system-related diseases.Hydrogen sulfide?H2S?has been thought to be a metabolic waste in the body and has now been shown to be an important biologically active gas signaling molecule in the body.Studies have shown that H₂S has the effect of delaying cell senescence, the mechanism remains to be further improved.Klotho gene is an "anti-aging" gene.The current academic community generally believe that the secretion of Klotho protein is an anti-aging hormone associated with human coronary artery disease,by inhibiting FoxO1 phosphorylation to enhance FoxO1 activity,and then play anti-Effect.It has been reported that H₂S can regulate the expression of Klotho in neuronal cells.The aim of this study was to explore the role of Klotho /FoxO1 pathway in H2S-induced anti-hydrogen peroxide-induced smooth muscle cell senescence.Method and results: 1.the vascular smooth muscle cells were treated with H₂O₂ concentration?0,25,50,100,150?mol/L?for 1 hour,the complete medium was replaced and cultured in a carbon dioxide incubator?37 ?,5% CO₂?for 24 hours.The optimal H₂O₂ concentration was determined by western blotting and ?-galactosidase staining.The results of ?-galactosidase staining showed that the number of positive cells in the control group?0 ?mol / L H₂O₂ group was less than 5%?.The number of green dye positive cells in 25?mol / L H₂O₂ group was still about 10%.50?mol / L H₂O₂ group,the number of positive cells is also less,about 13%.The number of positive cells in the 100?mol/L H₂O₂ group was significantly increased,about 65%,which was about 11 times of the control group?P <0.01?.Compared with the control group,the expression of P16,P21 and P53 protein in the 100?mol/L H₂O₂ group was significantly up-regulated by 288.8%,200.8% and 175.4%?P <0.01?,respectively.The results showed that 100?mol/L was the best H₂O₂ concentration induced by vascular smooth muscle cell senescence.2.Na HS pretreated vascular smooth muscle cells for 30 minutes,the concentrations were: 0,25,50,100,200?mol/L.After treatment with 100?mol/L H₂O₂ for 1 hour,the complete medium was replaced and incubated in a carbon dioxide incubator?37 ° C,5% CO₂?for 24 hours.The related proteins were then detected by Western blotting and stained with ?-galactosidase.?-galactosidase staining results showed that the control group?0?mol/L Na HS group?positive cells,about 63%.The number of positive cells in the 25 ?mol/L Na HS group was about 47%lower than that in the control group.The number of green-stained cells in the 50?mol/L Na HS group was significantly reduced by about 25%.The number of positive cells in the 100?mol/L Na HS group was significantly decreased,and the number of positive cells in the200?mol/L Na HS group was also decreased by about 12%?P <0.01?.Compared with the control group,the expression of P16,P21 and P53 protein in the 100?mol/L Na HS group decreased by 143.6%,166.4% and319.6%?P <0.01?,respectively.The expression of P16,P21 and P53 protein in the 200?mol/L Na HS group decreased by 197.5%,198.4% and395.1%,respectively?P <0.01?.The results showed that H₂S had the effect of antagonizing H₂O₂-induced senescence of vascular smooth muscle cells.And in the subsequent experiments will be applied100?mol/L Na HS anti-H₂O₂-induced vascular smooth muscle cell senescence.3.The cells were randomly divided into 3 groups: control group,H₂O₂ group and Na HS + H₂O₂ group.And then Western blotting was used to detect the related proteins.Western blotting showed that the expression of Klotho protein in the H₂O₂ group decreased by 10.6%?P<0.01?compared with the control group.Compared with H₂O₂ group,the expression of Klotho protein in Na HS + H₂O₂ group increased by 43.1%?P <0.01?.On the other hand,studies have shown that the activity of FoxO1 by post-translational modification?phosphorylation and deacetylation?,FoxO1 phosphorylation can promote FoxO1 from nuclear translocation to cytoplasm,reduce FoxO1 activity.Therefore,we also observed the expression of phosphorylated FoxO1 protein in each experimental group.Compared with the control group,the expression of p-FoxO1 protein in H₂O₂ group increased by 7.4%?P <0.01?.Compared with H₂O₂ group,the expression of p-FoxO1 protein in Na HS + H₂O₂ group was decreased by 30.2%?P <0.01?.The results showed that H₂S up-regulated the expression of Klotho protein and down-regulated the expression of phosphorylated FoxO1 in the process of H₂S anti-vascular smooth muscle cell senescence.4.The cells were randomly divided into 4groups: control group,H₂O₂ group,H₂O₂+Na HS group, H₂O₂+Na HS+FoxO1 si RNA group.?-galactosidase staining: H₂O₂ group compared with the control group,the positive cell rate increased by272.7%?P <0.01?.H₂O₂+Na HS group compared with H₂O₂ group,the positive cell rate decreased by 65.5%?P <0.01?.H₂O₂+Na HS+FoxO1 si RNA group compared with H₂O₂+Na HS group,the positive cell rate increased by 166.7%?P <0.01?.Western blotting showed that the expression of P16,P21 and P53 protein was increased by 51.1%,76.6%and 25.1%?P <0.01?in H₂O₂ group compared with the control group.Compared with H₂O₂ group,the expression of P16,P21 and P53 protein decreased by 41.2%,45.8% and 52.2%,respectively?P <0.01?.Compared with H₂O₂+Na HS group,the expression of P16,P21 and P53 protein in H₂O₂+Na HS+FoxO1 si RNA group increased by 45.1%,48.9% and49.2%,respectively?P<0.01?.The results showed that FoxO1 was a target of H₂S anti-vascular smooth muscle cell senescence.Silencing FoxO1 attenuates H₂S anti-induced vascular smooth muscle cell senescence.5.the cells were randomly divided into 4 groups: control group,H₂O₂ group,H₂O₂+Na HS group,H₂O₂+Na HS+Klotho si RNA?-galactosidase staining: H₂O₂ group compared with the control group,the positive cell rate increased by 166.7%?P <0.01?.H₂O₂+Na HS group compared with H₂O₂ group,the positive cell rate decreased by 75.1%?P<0.01?.H₂O₂+Na HS + Klotho si RNA group compared with H₂O₂+Na HS group,the positive cell rate increased by 310.2%?P <0.01?.Western blotting showed that the expression of P16,P21 and P53 was increased by33.8%,82.4% and 45.5%?P <0.01?in H₂O₂ group compared with the control group.Compared with H₂O₂ group,the expression of P16,P21 and P53 protein in H₂O₂+Na HS group decreased by 39.4%,43.5% and 25.2%,respectively?P<0.01?.The expression of P16,P21 and P53 protein in H₂O₂+Na HS+Klotho si RNA group was significantly increased by 30.5%,57.1% and 50.7%?P <0.01?compared with H₂O₂+Na HS group.Experimental results show that silencing Klotho will weaken H₂S anti-induced vascular smooth muscle cell senescence.Conclusion: H₂S can inhibit the senescence of vascular smooth muscle cells induced by H₂O₂.The mechanism is related to upregulation of Klotho expression,reduction of FoxO1 phosphorylation levels,and enhancement of FoxO1 activity.