Effects Of Targeted Intervention Of NF-κB Pathway On Myocardial Remodeling Induced By Pressure Overload

Objective: Effect of targeted intervention of NF-κB-p65 on myocardial remodeling in rats with cardiac dysfunction induced by pressure overload.Methods:(1)Cell experiment: The myocardial injury model of H9C2 cells was induced by AngiotensinⅡ(AngⅡ).MTT assay was used to detect the cell activity to screen the optimal administration concentration.The cells were routinely cultured in a six-well plate,and the cells were divided.(1)Control group: Conventional high glucose medium.(2)Ang II group:AngⅡ(1μmol/L)treatment for 24 h.(3)Ang II + PDTC group: PDTC(100μmol/L)pretreatment for 1h,PBS washing 3 times,AngⅡ(1μmol/L)for 24 h.(4)PDTC group:PDTC(100μmol/L)treatment for 1h.Cells were collected and flow cytometry was used to detect apoptosis.Western blot was used to detect the expression of apoptosis-related proteins Bax and Bcl-2,cardiomyocyte injury-related protein atrial natriuretic peptide(ANP)and nuclear P65 in each group.(2)In animal experiment:6-week-old SD rats with relatively stable blood pressure were randomly divided.(1)Sham group: Close the abdominal cavity directly after laparotomy.(2)AAC group: Pressure overload cardiac insufficiency model was established by abdominal aortic coarctation(abdominal aortic coarctation,AAC).(3)AAC+e GFP group: Empty adeno-associated virus was injected 3days after abdominal aortic coarctation.(4)AAC+e GFP-p65 group: Three days after coarctation of abdominal aorta,silencing P65 adeno-associated virus was injected.After 8weeks,the weight and blood pressure were measured,the animals were killed,the left ventricular index was measured,and the heart and blood samples were taken;The expression of m RNA p65 was detected by q RT-PCR;The expression of total P65 in myocardial tissue was detected by immunohistochemistry;The expression of virus fluorescent marker protein GFP,Cleavedcaspase-3 and P65 in nucleus was detected by Western blot;Myocardial pathological changes were detected by Masson staining and HE staining;Myocardial apoptosis was detected by TUNEL staining.The contents of tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)in serum of rats were detected by ELISA.Results:(1)Cell experiment: The results of Western blot showed that compared with Control group,the expression of ANP increased,the expression of Bcl-2 decreased,the expression of apoptosis-related protein bax increased and the expression of P65 in nucleus increased in AngⅡ group(P<0.05).Compared with Ang Ⅱ group,the expression of myocardial injury-related protein ANP decreased,Bcl-2 expression increased and Bax expression decreased in AngⅡ+PDTC group(P<0.05).Compared with Control group,the expression of P65 in PDTC group decreased(P<0.05),but there was no significant change in other proteins.The results of flow cytometry showed that the percentage of apoptosis in AngⅡ group was higher than that in Control group,and that in AngⅡ+PDTC group was lower than that in Ang Ⅱ group(P<0.05).Compared with Control group,the percentage of apoptosis in PDTC group had no significant change(P>0.05).(2)Animal experiment: The results of q RT-PCR showed that p65 in AAC+e GFP-p65 group was lower than that in Sham group(P<0.05),while there was no significant change in AAC group and AAC+e GFP group(P>0.05).The results of hemodynamics and heart mass showed that compared with Sham group,systolic blood pressure(SBP)and diastolic blood pressure(DBP)in AAC group and AAC+e GFP group increased,while the maximum rising rate of left ventricular pressure(+dp/dtmax)and the maximum rate of decrease of left ventricular pressure(-dp/dtmax)decreased(P<0.05).Compared with AAC group,in AAC+e GFP-p65 group there was no difference in SBP and DBP(P>0.05),but +dp/dtmax and-dp/dtmax increased(P<0.05).Histopathological examination showed that compared with Sham group,in AAC group and AAC+e GFP group HE staining showed larger cross-sectional area of cells,TUNEL staining showed increased percentage of myocardial apoptosis,Masson staining showed increased collagen fibers around myocardium(P<0.05),and there was no significant change in P65 content by immunohistochemistry(P>0.05).Compared with AAC group,there was no difference in histopathological examination in AAC+e GFP group(P>0.05),while in AAC+e GFP-p65 group,the cross-sectional area of cells decreased by HE staining,the percentage of myocardial apoptosis by TUNEL staining and the number of pericardial collagen fibers by Masson staining decreased(P<0.05).The results of Western blot showed that the expression of P65 in the nucleus and Cleaved-caspase-3 protein in AAC group and AAC+e GFP group was higher than that of Sham group(P<0.05),and there was no difference between AAC+e GFP group and AAC group(P>0.05),but the expression of P65 in the nucleus and Cleaved-caspase-3 protein in AAC+e GFP-p65 group was lower than that of AAC group(P<0.05).Compared with Sham group,the expression of GFP protein in AAC+e GFP group and AAC+e GFP-p65 group was significantly higher(P<0.05).The results of ELISA detection showed that the contents of TNF-α and IL-6 in serum of rats in AAC group and AAC+e GFP group were higher than those in Sham group(P<0.05),and there was no difference between AAC+e GFP group and AAC group(P>0.05),while the content of TNF-α in serum of AAC+e GFP-p65 group was lower than that of AAC group(P<0.05),but there was no difference in IL-6 content(P>0.05).Conclusion: Targeting intervention NF-κB-p65 reduce myocardial remodeling induced by stress overload in rats with cardiac insufficiency.