Protective Effect Of Sal B On Myocardial Ischemia Injury By The Medium Of Mitophagy-NLRP3 Inflammsome

Objectives:This study is undertaken to focus on the effect of Sal B on NLRP3 inflammsome and its possible pathways of regulation in rats model of myocardial ischemia injury and elucidate further the mechanism of Sal B against myocardial ischemia injury.Methods:The experimental study is divided into three chapters.Chapter 1.To establish ISO-induced myocardial ischemia injury model in SD rats.To study the protective effect of Sal B on ischemic myocardium by detecting Value of the T-wave in ECG,Myocardial injury serum biochemical indexes（CK,GOT and LDH）,Antioxidant serum biochemical indexes（SOD,MDA）,Anti-inflammatory serum biochemical indexes（IL-1β）,Myocardial tissue HE and TUNEL staining.To study Sal B on the correlation of NLRP3 expression by detecting the expression of NLRP3-related protein and mRNA in myocardium via immunohistochemistry,western blot and real-time PCR.Chapter 2.To establish H₂O₂-induced oxidative stress injury model and LPS + ATP combined induction inflammatory injury model in H₉C₂ cells.The survival rate of cells was detected by MTT assay.The effect of Sal B on myocardial injury was studied by measuring the oxidative and inflammatory factors in the supernatant of the cells.To study protective effects of Sal B on NLRP3 inflammsome in Cardiomyocytes injury by Western blot and Immunofluorescence.To further study effects of Sal B on pyroptosis by TUNEL and Calciem-AM staining.Chapter 3.Promote the activation of NLRP3 inflammsome in H₉C₂ cells by LPS+ATP combined induction.To study the possible mechanism of Sal B inhibiting NLRP3 inflammsome by detecting ROS,MMP,SIRT1,p-Foxo3a and mitophagy related proteins.The role of autophagy in the activation of NLRP3 inflammsome was further demonstrated by autophagy inhibitor 3-MA.Results:Part 1.Sal B can significantly reduce myocardial necrosis,inflammatory cell infiltration and reduce DNA damage,significantly reduce T-wave value of ECG（P<0.05 or P<0.01）.Compared with model group,CK values,GOT values and IL-1β values of rats in different dose groups were significantly lower,and MDA values and LDH values of rats in middle-and high-dose groups were significantly lower（P<0.05 or P<0.01）.However,T-SOD values of rats middle-and high-dose groups were significantly higher（P<0.05 or P<0.01）.The expression of NLRP3,ASC,caspase-1P20 and IL-1β protein and mRNA in the heart tissue of Sal B group was significantly decreased in a dose-dependent manner（P<0.05 or P<0.01）.Part 2.Sal B in H₉C₂ cells for 24 h,H₂O₂ in H₉C₂ cells for 2h,selecting the appropriate concentration of H₂O₂ model of 100μM,Sal B appropriate pretreatment concentration of 1,5,25μM.Compared with the H₂O₂ model group,the different doses of Sal B could improve the survival rate of cells and maintain the stability of cell morphology.Meanwhile,Sal B could significantly decrease the levels of LDH and MDA in the supernatant and increase the level of T-SOD in a dose-dependent manner（P<0.05 or P<0.01）.Western blot,IF and ELISA showed that Sal B could inhibit the activation of NLRP3 and secretion of IL-1β in oxidative and inflammatory injury.TUNEL and Calcein-AM staining showed that Sal B could protect the integrity of the cell membrane and reduce DNA damage,thereby inhibiting pyroptosis.Part 3.While LPS + ATP induced H₉C₂ cells,Sal B could promote the expression of SIRT1,p-Foxo3a and MnSOD（P<0.05 or P<0.01）,inhibit the increase of ROS and MMP,promote the expression of autophagy marker proteins LC3 Ⅱ,Beclin-1 and Parkin,and inhibit the expression of P62 and PINK1（P<0.05 or P<0.01）.In the case of autophagy inhibitor 3-MA,the effect of Sal B that promote mitophagy and inhibition of NLRP3 inflammsome activation was weakened.However,3-MA did not affect the promotion of Sal B on SIRT1 and p-Foxo3a.Conclusions:1.Sal B could inhibit the activation of NLRP3 inflammatory mediators in ischemic heart tissue,thereby reducing the inflammatory response to the purpose of protecting the heart.2.Sal B could protect the oxidative stress and inflammatory injury in H₉C₂ cells by inhibiting the activation of NLRP3 inflammsome.Sal B aslo could inhibit the secretion of inflammatory factors and pyroptosis while the activation of NLRP3 inflammsome.3.Sal B could inhibit the activation of NLRP3 inflammsome in H₉C₂ cells,its mechanism may be related with activation of SIRT1-FOXO3a-Parkin axis,improvement of myocardial mitophagy,reducing the accumulation of damaged mitochondria,thereby inhibiting the release of NLRP3 activation signal.