Study On The Mechanism Of IMD Protecting HUVECs Damage Induced By Amiodarone Via Wnt/β-catenin Signal Pathway

Objective:1.To reveal the mechanism of HUVECs damage induced by amiodarone;2.To explore the protective effect and mechanism of IMD on HUVECs damage induced by amiodarone.Methods:1.HUVECs,cultured in vitro,was treated with different concentrations of amiodarone,and CCK8 detected cell viability.According to the results of CCK8,the best drug concentration was selected for follow-up experiments.HUVECs was treated with the best drug concentration for 6h 12 h,24 h,36 h,48 h and CCK8 detected cell viability.According to the results of CCK8,the best action time was selected for follow-up experiments.Western Blot detected the expression of apoptosis-related proteins.2.HUVECs was divided into four groups: control group,AM group,IMD group and IWR group.Cell viability was detected by CCK8,cell migration was detected by Wound healing assays,mitochondrial membrane potential was detected by JC-1 method,and the expression of apoptotic proteins(Bcl-2,Bax and Casepase3)and Wnt/ β-catenin signaling pathway related proteins(p-β-catenin,β-catenin)were detected by Western Blot.The expressions of apoptosis and inflammation related genes(apoptosis related genes:Bcl-2,Bax and Casepase3;inflammation related genes: IL-1β,IL-6,TNF-α and IL-10)were detected by PCR,and the production of ROS,the release rate of LDH and the activity of SOD were detected by kit.Results:1.Amiodarone induced apoptosis,inflammatory response and oxidative stress injury in HUVECs.Amiodarone decreased the activity of HUVECs and increased the expression of apoptosis-related proteins in a concentration-and time-dependent manner,and could induce inflammatory response and oxidative stress injury of HUVECs.The CCK8 results showed that the HUVECs activity decreased with the increase of amiodarone concentration and treatment time;the Western Blot results suggested that the higher the amiodarone concentration and the longer the treatment time,the more obvious the protein expression level of anti-apoptosis gene(Bcl-2)decreased,and the more significant the protein expression level of pro-apoptosis genes(Bax and Casepase3)increased;the results of Real-time PCR suggested that amiodarone upregulated the expression of HUVECs pro-inflammatory gene(IL-1β,IL-6,TNF-α)mRNA and down-regulated the expression of anti-inflammatory gene(IL-10)mRNA;the results of ROS,LDH and SOD showed that amiodarone increased the production of HUVECs ROS and the release of LDH,and decreased the activity of HUVECs antioxidant enzyme SOD.2.IMD exerted its protective effect on amiodarone-induced HUVECs damage by activating Wnt/β-catenin signal pathway.IMD pretreatment improved the decrease of HUVECs activity,migration ability and mitochondrial membrane potential induced by amiodarone,decreased the rate of apoptosis,and reduced the inflammatory reaction and oxidative stress injury of HUVECs.However,the use of IWR-1-endo(a Wnt/β-catenin signal pathway blocker)weakens the protective effect of IMD to some extent.CCK8 results showed that IMD pretreatment improved the decrease of HUVECs activity induced by amiodarone;Western-blot results showed that amiodarone blocked Wnt/β-catenin signal pathway in a concentration-dependent manner,IMD activated Wnt/β-catenin signal pathway,cell scratch test showed that IMD pretreatment improved the decrease of HUVECs migration induced by amiodarone,and IWR-1-endo pretreatment weakened the effect of IMD to some extent.The results of mitochondrial membrane potential detection showed that amiodarone caused the decrease of mitochondrial membrane potential,IMD pretreatment had a protective effect on the decrease of mitochondrial membrane potential,and IWR-1-endo pretreatment weakened the effect of IMD to some extent.The results of Western-blot and Real-time PCR showed that compared with AM group,the protein and mRNA expression of anti-apoptosis gene(Bcl-2)were up-regulated,while the protein and mRNA expression of pro-apoptosis genes(Bax and Casepase3)were down-regulated in IMD group.Compared with IMD group,the protein and mRNA expression of anti-apoptosis gene in IWR group were down-regulated,while the protein and mRNA expression of pro-apoptosis gene were up-regulated.The results of Real-time PCR showed that compared with AM group,the expression of pro-inflammatory gene(IL-1β,IL-6,TNF-α)mRNA was down-regulated and the expression of anti-inflammatory gene(IL-10)mRNA was up-regulated in IMD group.Compared with IMD group,the expression of pro-inflammatory gene mRNA was up-regulated and anti-inflammatory gene mRNA was down-regulated in IWR group.The detection results of ROS,LDH and SOD showed that IMD pretreatment reduced the production of ROS,decreased the release rate of LDH and activated the activity of SOD.Compared with IMD group,IWR group showed the opposite trend.Conclusion:1.Amiodarone induced apoptosis,inflammation and oxidative stress injury of HUVECs.2.IMD had a protective effect on HUVECs damage induced by amiodarone,which may be achieved by activating Wnt/β-catenin signal pathway.