| Objective: To study the mechanism of Nrf2 target regulating mitochondrial quality Control system mediating the protective effect of essential oil from Stahlianthus involucratus rhizomes(EOSIR)on human umbilical vein endothelial cells(HUVECs)injury.Methods: HUVECs were induced by oxidized low-density lipoprotein(ox-LDL)to establish a cell injury model,the expression of Nrf2 factor was silenced by si RNA transfection.The cultured cells were divided into Control group,ox-LDL group,EOSIR group,si-Nrf2 group and si-NC group.The protein expression of Nrf2、HO-1、NQO1、P53、P21、Bax、Bcl-2 were detected by Western Blot;The m RNA expression of Nrf2、HO-1、NQO1 were detected by q RT-PCR;Griess method was used to detect the content of nitric oxide(NO);ELISA was used to analyze the content of human endothelin(ET-1)and human prostacyclin(PGI2);Cell senescence was observed by SA-β-gal staining;The cells were divided into Control group,ox-LDL group,EOSIR(0.01,0.05 and 0.1 μg/m L)group,and aspirin positive Control group in the mitochondrial quality test.Transmission electron microscopy was used to observe the mitochondrial morphology.the expression levels of MFN1,MFN2,Drp1,Opa1,TFAM,PGC-1α and LC3Ⅱ,P62 was detected by Western Blot.Nrf2 factor was further silenced to detect the expression level of mitochondria-related proteins;The content of ATP was detected by micromethod.Results:(1)Western Blot and q RT-PCR results showed that EOSIR inhibited the down-regulation of Nrf2,HO-1,NQO1 in ox-LDL induced HUVECs(P<0.01).After silencing Nrf2 factor,the regulation effect of EOSIR on Nrf2 and its downstream factors was also inhibited(P<0.05,P<0.01).(2)NO content was detected by Griess method,the ox-LDL group decreased significantly compared with the Control group,but increased after EOSIR intervention(P<0.01).After Nrf2 gene silencing,the effect of EOSIR induced content change was inhibited(P<0.01).(3)ELISA results showed that after EOSIR intervention,PGI2 content was significantly higher than that in the ox-LDL group,and ET-1 level was significantly lower than that in the ox-LDL group(P<0.01).After Nrf2 gene silenced,the improvement effect of EOSIR on the content of ET-1 and PGI2 was also inhibited(P<0.01).(4)The results of SA-β-gal staining showed that the positive rate of ox-LDL induced ox-LDL group was significantly higher than that of the Control group,and the positive rate was significantly decreased after the intervention of EOSIR.After the silencing of Nrf2 gene,the inhibition effect of EOSIR on cell aging staining was also weakened.(5)After EOSIR intervention,the protein level of P21,P53 and Bax were significantly decreased compared with the ox-LDL group,and the expression of Bcl-2 was significantly increased(P<0.05,P<0.01).After the silencing of Nrf2 gene,the regulation effect of EOSIR on the expression of cell aging and apoptosis proteins was also reversed(P<0.05,P<0.01).(6)Transmission electron microscopy observing the mitochondrial morphology,the number of autophagosomes in the EOSIR group was significantly reduced compared with the ox-LDL group,and the mitochondrial morphology returned to normal.(7)According to the results of Western Blot,the proteins level of MFN1,MFN2,Opa1,TFAM and PGC-1α in the EOSIR group were significantly increased compared with the ox-LDL group,and the level of Drp1 was significantly decreased.Meanwhile,the EOSIR intervention could significantly inhibit the up-regulation of autophagy related protein LC3Ⅱ by ox-LDL.And improve the P62 downgrade.After Nrf2 gene silencing,the regulation effect of EOSIR on the above mitochondria-related proteins was also decreased(P<0.05,P<0.01).(8)Compared with the ox-LDL group,the ATP content of EOSIR was significantly increased(P<0.01).After Nrf2 was silenced,the up-regulation effect of EOSIR on ATP content was also inhibited(P<0.05).Conclusion: The results suggest that EOSIR has a protective effect on ox-LDL-induced endothelial cell injury,which may be achieved through the activation of Nrf2 target to regulate mitochondrial quality Control system. |
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