Preliminary Study On The Anti Myocardial Ischemia-Reperfusion Activity And Mechanism Of The Total Flavonoids And Its Components From Anchusa Italica Retz.in Vitro

Objective: To study the active components in the total flavonoids of Anchusa italica Retz.in amyocardial inducing myocardial ischemia-reperfusion injury(MIRI).To explore whether it can inhibit I/R injury by suppressing the activated NF-κB signal pathway,regulating autophagy level,reducing cell pyroptosis,inhibiting cardiomyocyte apoptosis,and improving mitochondrial function.Methods:(1)The pretreatment concentration and time of rutin,rosmarinic acid,narcoside,kaempferol 3-O-rutoside and total flavonoids were studied by establish of hypoxia/reoxygenation(H/R)model in vitro.CCK-8 method was used to detect the survival rate of primary cardiac myocytes,enzyme-linked immunoassay was used to detect the lactate dehydrogenase(LDH)levels in cell culture supernatant,malondialdehyde(MDA)content and superoxide dismutase(SOD)activity in cells.(2)To establish the H/R model in vitro,rutin,rosmarinic acid,narcoside,kaempferol3-o-rutoside,total flavonoid and puerarin as the positive drug were used for pretreatment.The detection indexes were the proportion of cardiomyocyte apoptosis,the expression of mitochondrial membrane potential(Δψm),the key protein of NF-κB signaling pathway and apoptosis,autophagy and pyroptosis related protein.Results:(1)The concentration and time of pretreatment were 50μg/m L rutin intervention for 3 hour,25μg/m L rosmarinic acid intervention for 3 hours,25μg/m L Narcissus intervention for 3 hours,100μg/m L kaempferol-3-O-rutoside intervention for 3 hours and 50μg/m L total flavonoids intervention for 1 hour.Compared with hypoxia/reoxygenation group,each group could reduce the leakage of LDH(P<0.01),the content of MDA in cells(P<0.01),SOD activity significantly improved and the difference was statistically significant(P<0.01).Among them,the total flavonoids has the most significant repairing effect on the damage caused by H/R treatment.(2)After H/R,the apoptosis index of cardiomyocytes in the administration group was significantly lower than that in the hypoxia/reoxygenation group,and the mitochondrial membrane potential was increased(P<0.01).At the same time,the activation of the key protein of NF-κB signaling pathway was inhibited,the proportion of Bax/Bcl-2 representing apoptosis was significantly lower than that of the model group(P<0.01),the expression of beclin-1 protein was significantly lower and the expression of p62 was up-regulated(P<0.01),the NLRP3,IL-1β and GSDMD protein in the treatment group were significantly lower than those in the model group(P<0.01).Conclusion: The total flavonoids and four monomers of Anchusa italica Retz.can improve the activity of cardiac myocytes,resist the over oxidation of cardiac myocytes and enhance the activity of SOD.This protective effect is that the drug intervention before H/R can inhibit the over autophagy of cardiac myocytes by inhibiting the activation of NF-κB signal pathway,relieve pyroptosis,reduce the apoptosis of cardiac myocytes and reduce the damage of cardiomyocytes caused by H/R.