Curcumin Antagonizes Vascular Endothelial Cell Pyroptosis Induced By Trimethylamine Oxide And Its Mechanism

Objective: Trimethylamine oxide(TMAO),an intestinal flora product,which can induce atherosclerosis(AS).therefore,MAO has become a potential target for the prevention and treatment of AS.By controlling diet,reducing intestinal flora of TMAO production,and inhibiting TMAO The generation of somatic compounds and the conversion of TMA to TMAO are new ways to regulate the AS-induced effects of TMAO.We found earlier that TMAO can cause vascular endothelial cell pyroptosis.This article aims to investigate whether curcumin with good anti-AS effect can antagonize TMAO-induced vascular endothelial cell pyroptosis and explore its mechanism.Methods: Human umbilical vein vascular endothelial cells(HUVEC)were cultured in vitro and passaged.The toxicity range of curcumin was measured by MTT method,and the appropriate concentration of curcumin was obtained.Then the concentration effect(0 ?M,20 ?M,40 ?M,60 ?M)and time effect(0h,6h,12 h,24h)of curcumin on TMAO-induced HUVEC-related death related molecules were studied,and the optimal concentration and time were obtained.In order to study the role of mitochondrial gene UQCRC1 in TMAO-induced vascular endothelial cell scoring,according to the siRNA instructions,HUVEC cell lentivirus was transfected with siRNA of mitochondrial gene UQCRC1.The sequence of UQCRC1 siRNA was siRNA: 5,-GCAGAACTGTAGTCTGGAA-3.Western blot detection of protein expression level,real-time fluorescence PCR detection of mRNA,BCA method to determine the protein content of samples,spectrophotometric detection of ATP level,DCFH-DA detection of ROS,bioinformatics online website analysis of UQCRC1 gene promoter sequence CG island.The statistical analysis of all data was expressed as mean ± standard deviation(± SD),and the data was analyzed and plotted with Graphpad Prism 5.0.1.A 95% confidence interval was selected,and P <0.05 was considered significant.RESULTS:(1)HUVEC cells were treated with curcumin at 0 ?M,20 ?M,40 ?M,60 ?M,80 ?M,and 100 ?M,and their OD values were measured.It can be seen that compared with 0 ?M,20 ?M,40 ?M,60 ?M curcumin has no significant difference compared with 0 ?M,and the cell viability of 80 ?M and 100 ?M curcumin is significantly reduced.Therefore,the concentration of curcumin in subsequent experiments was 60 ?M or less.(2)When the cells were cultured to about 70%,two hours before adding TMAO,curcumin(0,20,40,60 ?M)with different concentrations was added to continue culture.Western blot was used to detect the expression of coking death-related protein.The results showed that compared with the control group,TMAO significantly up-regulated the protein expression levels of Caspase-1,NLRP3,and IL-1?.After adding curcumin,TMAO up-regulated the Caspase-1,NLRP3,and IL-related molecules.The expression of-1? was significantly inhibited,and it had a concentration effect.With the increase of curcumin concentration,the expression level of scorch-related protein in curcumin decreased,and it showed a certain concentration dependence.But for Caspase-1,NLRP3,IL-1?,the concentration dependence is not exactly the same.Among them,the inhibitory effect of IL-1? with 40 ?M curcumin is the best.The levels of IL-1 ? in the 60 ?M curcumin group and the 60 ?M group are higher than those in the 40 ?M curcumin group.The measurement of Caspase-1 by the effect of curcumin is similar to IL-1?,but not as good The measurement results of IL-1? are as obvious as those of Caspase-1.The measurement results of NLRP3 have obvious concentration effects.The curcumin of 20?M,40?M,and 60?M has a greater tendency to decrease as its concentration increases.There is no significant difference,which may be related to the too small range of concentration changes.(3)The endothelial cells were treated with curcumin at 40 ?M for different time(0h,6h,12 h and 24h),and the expression of coke death related protein was detected by Western blot.The results showed that compared with the control group,TMAO significantly increased the protein expression levels of endothelial cell death related proteins Caspase-1,NLRP3,and IL-1?.After adding curcumin,it can be seen that curcumin down-regulated endothelial cell death in a time-dependent manner.Protein expression levels of related proteins Caspase-1,NLRP3,IL-1?.Among them,the expression levels of these three molecules were not significantly different from the TMAO group at 6h,and there were significant differences between these three molecules and the TMAO group at 12 h and 24 h,but the decrease was the most significant at 24 h and had statistical significance.(4)ROS detection kit was used to detect ROS levels in blank group,TMAO group and curcumin + TMAO group.The experimental results show that the fluorescence intensity of the TMAO group is significantly higher than that of the control group,and the fluorescence intensity is reduced compared with the TMAO group after adding curcumin.This result indicates that TMAO can promote the production of HUVECROS,and curcumin can partially reverse the induction of ROS by TMAO.(5)ATP detection kit was used to detect ATP production in blank group,TMAO group and curcumin + TMAO group.The results showed that TMAO significantly down-regulated ATP levels in HUVEC cells,while 40 ?M curcumin could antagonize the effect of TMAO.Compared with the TMAO group,the difference was significant.However,curcumin does not completely reverse TMAO's reducing effect on ATP,it can only partially reverse it.Using RNA interference to silence UQCRC1 gene and inhibit UQCRC1 expression.First,three UQCRC1 siRNAs were synthesized,and after transfection with HUVEC,the transfection efficiency was measured.The results showed that the silencing effect of these three UQCRC1 siRNAs was satisfactory.Compared with the control group,the UQCRC1 expression levels were significantly suppressed.After UQCRC1 was silenced,the expression levels of Caspase-1,NLRP3,and IL-1? increased significantly,and the expression levels of Caspase-1,NLRP3,and IL-1? significantly decreased after the addition of curcumin.Even lower than the control group.(9)After the interference of UQCRC1,the ROS level increased significantly,and when curcumin was added,the ROS production dropped significantly.The situation with ATP is the same as with ROS.(10)Through bioinformatics analysis,a CpG island was found on the UQCUC1 gene promoter.After treatment with 600 ?M TMAO for 24 hours,the protein expression level of TET2 decreased significantly,but the treatment was supplemented with 40 ?M curcumin to intervene,and the expression of TET2 was partially restore.This result indicates that the expression of DNA methylolase TET2 is affected by TMAO and curcumin,and the effects of the two are opposite,that is,TMAO down-regulates TET2 expression,and curcumin may up-regulates TET2 expression.CONCLUSIONS: Curcumin inhibits TMAO-induced pyroptosis and mitochondrial dysfunction via up-regulating the expression of UQCRC1 in HUVEC.