AP39 Inhibits High Homocysteine-induced Vascular Senescence By Promoting Mitochondrial Remodeling Through FUNDC1

Objective:The aim of this study was to investigate whether AP39ameliorates vascular senescence caused by hyperhomocysteine and to investigate the role of FUNDC1-mediated mitochondrial remodeling in this process.Methods:（1）To determine the ameliorative effect of AP39 on endothelial cell senescence,human umbilical vein endothelial cells were treated with different concentrations（250μM,500μM,750μM,1 m M,2m M）of HCY for 24 hours to determine the optimal concentration,which was verified by Western Blot for senescence-associated proteins（P53,P21）.The optimal drug concentration was then explored by pretreatment with different concentrations of AP39（1n M,10n M,30n M,100n M,300n M）and validated by Western Blot for senescence-associated proteins（P53,P21）.Inhibition of H₂S was followed by Western Blot to observe changes in the expression of mitochondrial remodeling proteins and senescence proteins.And FUNDC1 protein expression changes were screened in the process.SA-β-Gal stain was used to further determine whether AP39 indeed ameliorated HCY-induced senescence in human umbilical vein endothelial cells.（2）To clarify the role of FUNDC1 in the process of mitochondrial remodeling and endothelial cell senescence,FUNDC1 gene expression was silenced using si-RNA followed by Western Blot to detect mitochondrial remodeling proteins（DRP1,p-DPR1,MFN1,MFN2,PGC-1α）and senescence-related proteins（P53,P21,IL-6）to clarify the Effect of FUNDC1 on mitochondrial remodeling and senescence phenotype of human umbilical vein endothelial cells（3）To clarify whether AP39 improves endothelial cell senescence by promoting mitochondrial remodeling through FUNDC1,si-FUNDC1 was used to silence FUNDC1 followed by pretreatment of cells with AP39 to observe the improvement of HCY-induced endothelial cell senescence and mitochondrial remodeling,using Western Blot to detect senescence proteins and mitochondrial remodeling proteins,SA-β-Gal staining was used to observe changes in endothelial cell senescence,and JC-1 probe was used to observe changes in mitochondrial membrane potential.To identify whether the mechanism of AP39 improving HCY-induced human umbilical vein endothelial cells is by promoting mitochondrial remodeling through FUNDC1.（4）To further clarify in animal tissues whether AP39 can promote mitochondrial remodeling via FUNDC1 and thus improve vascular senescence,50 male 8-week-old SD rats were fed in an SPF（Specific Pathogen Free）environment,weighing approximately 220 g,and randomly divided into 5 groups:Control group,HCY group（high homocysteine-induced vascular senescence model group,fed with drinking water containing10 g/L of L-methionine for 10 weeks）,HCY+AP39 group（rats in the HCY group were given a continuous intraperitoneal injection of AP39（100 n M/kg/d）4 weeks prior to execution）,HCY+AP39+PAG group（rats in the HCY+AP39 group were given a continuous intraperitoneal injection of PAG（endogenous hydrogen sulfide generating enzyme CSE inhibitor,30 mg/Kg）4 weeks prior to execution）,and AP39 control group（Control group rats were intervened with continuous intraperitoneal injection of AP39（100n M/kg/d）prior to execution）.After the rats were executed,β-galactosidase staining was used to observe the staining of neck vascular tissue in each group of SD rats,Western Blot was used to observe the expression changes of senescence-related proteins,mitochondrial remodeling proteins,FUNDC1,CSE and other proteins.RT-q PCR was used to detect the expression changes of senescence-related genes（IL-6,P21）,FUNDC1gene and mitochondrial remodeling gene（MFN1）.HE staining was used to observe the changes of vascular morphology in each group of SD rats,Masson staining was used to observe the collagen fiber deposition in the vascular tissues of the neck of SD rats in each group.Transmission electron microscopy was used to observe the morphological changes and quantitative changes of mitochondria in the vascular tissue of the neck of SD rats in each group.Results:（1）In vitro experiments:Compared with Control group,Western Blot showed the highest expression of senescence-related proteins such as P53 and P21 when the HCY concentration was 1 m M（P<0.05）.Based on this dose,Western Blot showed a significant decrease in the expression of FUNDC1 and CSE proteins in HCY group（P<0.05）,SA-β-Gal staining showed a significant increase in fluorescence intensity（P<0.05）,Western Blot showed a significant decrease in the expression of mitochondrial remodeling proteins like p-DRP1,MFN1,MFN2,PGC-1α（P<0.05）,and JC-1 probe showed a significant decrease in mitochondrial membrane potential（P<0.05）.In vivo experiment:Western Blot showed significantly higher expression of aging-related proteins such as P53,P21 and IL-6 in vascular tissues of SD rats in HCY group compared with those in Control group（P<0.05）.RT-q PCR showed a significant increase in the expression of IL-6 and P21 genes（P<0.05）and a decrease in the expression of FUNDC1 and the mitochondrial remodeling gene MFN1（P<0.05）.β-galactosidase staining showed significantly higher positive staining rate in vascular tissues of the neck,HE staining showed discontinuity of intima,obvious protrusion of lesion and disordered arrangement of smooth muscle of middle membrane,Masson staining showed a significant increase in collagen fiber deposition in the neck vascular tissue（P<0.05）,and transmission electron microscopy showed a significant decrease in the number of mitochondria and a significant decrease in mitochondrial remodeling.The results suggest that HCY causes vascular senescence,vascular injury and pathological remodeling,and inhibits mitochondrial remodeling,and this process is associated with FUNDC1 and CSE gene expression.（2）In vitro experiments:Compared with HCY group,Western Blot showed the lowest expression of senescence-related proteins such as P53and P21 when the concentration of AP39 was 100 n M（P<0.05）.On the basis of this dose,Western Blot showed a significant decrease in the expression of FUNDC1 and CSE proteins in the HCY+AP39 group（P<0.05）,SA-β-Gal staining showed a significant decrease in fluorescence intensity（P<0.05）,Western Blot showed a significant increase in the expressions of mitochondrial remodeling proteins like p-DRP1,MFN1,MFN2,PGC-1α（P<0.05）,and JC-1 probe showed a significant recovery in mitochondrial membrane potential（P<0.05）.In vivo experiments:Western Blot showed that the expressions of aging-related proteins such as P53,P21 and IL-6 was significantly lower in the vascular tissue of SD rats in HCY+AP39 group compared with those of SD rats in HCY group（P<0.05）.RT-q PCR showed a significant decrease in the expression of IL-6 and P21 genes（P<0.05）and an increase in the expression of FUNDC1 and the MFN1（P<0.05）.β-galactosidase staining showed that the positive rate of staining in the vascular tissue of the neck was significantly lower.HE staining showed that continuity and integrity of intima were improved,and smooth muscle alignment was improved.Masson staining showed a significant decrease in the amount of collagen fiber deposition in the neck vascular tissue（P<0.05）.And transmission electron microscopy showed a significant increase in the number of mitochondria and a significant increase in the degree of mitochondrial remodeling.The results indicate that AP39improved HCY-induced vascular senescence,vascular injury and pathological vascular remodeling,and promoted mitochondrial remodeling,and this process was associated with FUNDC1 and CSE gene expression.（3）In vitro experiments:Western Blot showed increased expression of P21 protein（P<0.05）and decreased expression of mitochondrial MFN2 protein（P<0.05）in endothelial cells of HCY+AP39+PAG group compared with HCY+AP39 group.In vivo experiments:Western Blot showed that senescence proteins,such as P53,P21,IL-6,were elevated again in the vascular tissue（P<0.05）.RT-q PCR showed that IL-6 and P21gene expression was again increased（P<0.05）,while FUNDC1 and MFN1 were again decreased（P<0.05）.β-galactosidase staining showed that the positive staining rate in the neck vascular tissue was significantly increased again,HE staining showed that there was a decrease in endothelial integrity and continuity,and uniformity of smooth muscle arrangement decreased obviously.Masson staining showed that the neck Masson staining showed a significant decrease in collagen fiber deposition in the neck vascular tissue（P<0.05）.And transmission electron microscopy showed a significant decrease in the number of mitochondria again and a significant decrease in the degree of mitochondrial remodeling.The results indicate that the process of improving vascular aging,vascular injury,pathological vascular remodeling,and promoting mitochondrial remodeling by AP39 could be blocked by the process of inhibiting endogenous H₂S production,and the drug effect of AP39 was related to promoting endogenous H₂S production（4）In vitro experiments:Compared with Control group,in the endothelial cells of si-FUNDC1 group,Western Blot showed that the expressions of senescence-related proteins like P53,P21 and IL-6 were significantly increased（P<0.05）,while the expressions of mitochondrial remodeling proteins like p-DRP1,MFN1,MFN2 and PGC-1αwere significantly decreased（P<0.05）.The results suggest that inhibition of FUNDC1 gene expression can lead to endothelial cell senescence and inhibit mitochondrial remodeling.（5）In vitro experiments:Compared with HCY+AP39 group,in the HCY+AP39+si-FUNDC1 group endothelial cells,Western Blot showed a recovery in the expression of senescence-related proteins such as P53,P21 and IL-6（P<0.05）,SA-β-Gal staining showed a significant increase in fluorescence intensity（P<0.05）,Western Blot showed that the expression of mitochondrial remodeling proteins such as p-DRP1,MFN1,MFN2 and PGC-1αdecreased significantly（P<0.05）,and JC-1 probe showed that the mitochondrial membrane potential decreased significantly（P<0.05）.The results suggest that inhibition of FUNDC1gene expression can reverse the effects of AP39 in improving endothelial cell senescence and promoting mitochondrial remodelingConclusion:AP39 promotes mitochondrial remodeling through FUNDC1 to effectively improve high homocysteine-induced vascular senescence,which offers new therapeutic prospects for vascular senescence research.