Qian Yang Yu Yin Granule Protects Against Hypertension-induced Renal Injury By Epigenetic Mechanism Mediated By Nicotinamide N-Methyltransferase(NNMT)

ObjectiveThe mechanism of renal injury in hypertension is complex,and the efficacy of existing treatment schemes is limited.Epigenetics is the potential pathogenesis of hypertensive kidney injury.Early clinical and experimental studies of Qian Yang Yu Yin Granules have confirmed the protective effect of QYYY on hypertensive kidney injury.The purpose of this study is to confirm that QYYY can intervene hypertensive kidney injury through epigenetic pathway in vivo and in vitro models.Methods1.In vivo basic experimentQYYY improved renal function of SHR through NNMT-mediated epigenetic pathway.In vitro experiment,rats were grouped into normal group,model group,QYYY low dose group(2.1 g/kg/d),QYYY high dose group(8.4 g/kg/d)and valsartan group.Angiotensin ?was used to model renal damage in hypertension for 2 weeks.After treatment with QYYY for 8 weeks,serum and urine creatinine and urinary microalbumin were detected as indicators of changes in kidney function.The expressions of NNMT,H3K4 methylation and cortactin acetylation were detected by western-blotting.The MNA,activity index of NNMT,was detected by Liquid chromatography with tandem mass spectrometry.2.In vitro basic experiment(1)QYYY inhibited the proliferation of HEK293T cells.The cell experiments were divided into normal group,model group,QYYY group and valsartan group.The effect of angiotensin ? on the proliferation of 293T cells at different time(6 h,12 h,24 h,48 h,72 h)and at different concentrations(10'4,10-5,10-6,10-7,10-8,10-9mmol/mL)was observed by MTT method.Then,the safe concentration of QYYY,including low dose concentration of 0.5mg/ml and high dose concentration of 10mg/ml,was used to observe the effect on proliferation of 293T cells induced by Ang ?.Edu fluorescence staining was used to verify the effect of QYYY on proliferation of 293T cells induced by Ang ?.The nuclei of Hoechst33342 staining were blue,and the dividing cells stained with Edu were green.The ratio of Edu/hoechst between groups was compared.The cell cycle flow method was used to detect the DNA content in each group and observe the blocking effect of QYYY on cell cycle.(2)Effects of QYYY on epigenetic factors.The levels of H3K4 methylation and cortaction acetylation were detected by western-blotting.DNA methylation levels were compared by the method of DNA ELISA and genome-wide methylation capture and sequencing.(3)Lentivirus transfection test was used to verify the relationship between NNMT and epigenetic factors.Lentivirus transfection raised NNMT gene expression,then MNA,inhibitors of NNMT,was used.After the steps above,H3K4 methylation,cortactin acetylation,SAH and SIRT1 were detected by western-blotting.(4)Effects of QYYY on NNMT related enzymes.After the effects of NNMT and epigenetic factors were confirmed,the effects of QYYY on protein expression and mRNA levels of NNMT related enzymes were observed.Results1.In vivo basic experiments.QYYY can improve renal function of SHR,reducing serum creatinine,urinary microalbumin,and promoting urinary creatinine excretion when compared with Ang ? group.Western-blot results showed that QYYY could decrease the expression of NNMT and ac-cortactin and increase the level of H3K4 trimethylation.The combination of liquid and mass suggested that QYYY could reduce MNA concentration.2.In vitro basic experiment.(1)MTT results showed that Ang ? 10-7mmol/mL used on 293T cells for 48 hours was effective in promoting cell proliferation(P<0.05),which was the optimal concentration in hypertensive renal injury cell model.The results also showed that 0.5 mg/mL and 10 mg/mL QYYY could inhibit cell proliferation induced by Ang ?.The Edu experiment confirmed that the low(0.5 mg/mL)and high(10 mg/mL)dose of QYYY could inhibit cell proliferation compared with the model group,and the proportion of Edu/hoechst in the low and high dose groups decreased to 42%and 39%.Cell cycle flow cytometry showed that QYYY could block cell proliferation in the G2/M phase.(2)Western blot results showed that the level of H3K4 trimethylation decreased and ac-cortactin increased in the Ang? group.QYYY could increase the level of H3K4 trimethylation and decrease the level of ac-cortactin.DNA ELISA indicated that the level of DNA methylation in the model group was higher than that in the control group.Genome-wide capture and sequencing text detected the average methylation differentiation level,differentiated genes,regions and chromosomes in the normal group,model group and QYYY group,and analyzed the possible related pathways.(3)Western blot results showed that after the up-regulation of NNMT expression by lentivirus transfection,the expression of H3K4me3 and SIRT1 were decreased and SAH and ac-cortactin were increased.After used with MNA,the trends of the protein above were in the opposite direction.(4)QYYY can reduce the protein and mRNA levels of NNMT,SAH,increase he protein and mRNA level of SIR?,and increase the mRNA level of SAM.ConclusionsIn vivo and vitro experiments,it have proved that QYYY can improve renal injury in hypertension through epigenetic pathway mediated by NNMT.