| Objective: The protective effect of the essential oil of rhizome from Ginger Notoginseng(EORGN)on human umbilical vein endothelial cells(HUVECs)damage induced by oxidized low-density lipoprotein(ox-LDL)was studied in this work,and the protective mechanism was explored by the Nrf2/ARE signaling pathway.Methods: The cell injury model was established with HUVECs induced by ox-LDL.The experiment was divided into six groups,which were blank control group,model group,low,middle and high doses of EORGN groups and aspirin(ASP)positive group.EORGN was extracted by steam distillation and the components of EORGN were analyzed by GC-MS.MTT assay was used to determine the optimal concentration and time of ox-LDL-induced HUVECs cell damage and analyze the effect of EORGN on the survival rate of HUVECs induced by ox-LDL.Hoechst 33342 staining experiment observed the effect of EORGN on ox-LDL-induced HUVECs cells changes of damage morphology.JC-1 staining experiment to observe the effect of EORGN on the changes of the mitochondrial membrane potential of HUVECs induced by ox-LDL.ELISA method to analyze the effect of EORGN on the contents of lactate dehydrogenase(LDH),and malondialdehyde(MDA)levels,and increased nitric oxide(NO),superoxide dismutase(SOD),catalase(CAT),and glutathione peroxidase(GSH-Px)in HUVECs induced by ox-LDL.Flow cytometry to detect the effect of EORGN on the reactive oxygen species(ROS)content and apoptosis of HUVECs induced by ox-LDL.RT-q PCR was used to detect the effect of EORGN on the expression level of Nrf2,nicotinamide adenine dinucleotide phosphate(NADPH)quinone oxidoreductase-1(NQO1),heme oxygenase-1(HO-1),Bcl-2 and Bax m RNA in HUVECs induced by ox-LDL.Western blot analysis was used to detect the effect of EORGN on the protein expression levels Nrf2,NQO1,HO-1,Bcl-2,Bax in HUVECs induced by ox-LDL.Results:(1)The yield of essential oil extracted by steam distillation was 3.78%.GC-MS preliminarily analyzed and identified fifty-four compounds in EORGN,accounting for 98.86%of the total essential oil.The highest content is camphene,accounting for22.38%,followed by α-pinene,accounting for 16.11%,the third is D-camphor,accounting for 9.74%,and the fourth is 3,6,7,8-tetrahydro-3,3,6,6-tetramethyl-as-indacen-1(2H)-one,accounting for8.41%.(2)MTT results showed that compared with the blank control group,EORGN had no toxicity to normal HUVECs cells in the concentration range of 0.01-60 μg/m L,but had obvious cytotoxicity when the concentration was more than 80 μg/m L(P<0.01).The survival rate of HUVECs induced by 200 μg/m L ox-LDL for 24 h was 54.32%(P<0.01),which was used as the best condition for modeling.Compared with the model group,EORGN significantly increased the survival rate of HUVECs induced by ox-LDL.(3)The results of Hoechst 33342 staining showed that compared with the blank control group,the model group had stronger blue fluorescence.The treatment with EORGN could significantly weaken the fluorescence intensity and improve the morphology of cell injury.(4)The results of JC-1 staining showed that compared with the blank control group,the injured cells in the model group showed increased green fluorescence intensity and decreased red fluorescence intensity.The treatment with EORGN could weaken the green fluorescence intensity and enhance the red fluorescence intensity.(5)The results of ELISA experiment showed that compared with the blank control group,the contents of LDH and MDA increased,and the contents of SOD,CAT,GSH-Px and NO decreased in the model group(P<0.05,P<0.01).The treatment with EORGN could decrease the contents of LDH,MDA and increase the contents of SOD,CAT,GSH-Px and NO(P<0.05,P<0.01).(6)The results of flow cytometry showed that compared with the blank control group,the content of ROS and the rate of apoptosis in the model group was significantly increased(P<0.01).The treatment with EORGN could significantly down-regulate the content of ROS and decrease the rate of apoptosis(P<0.05,P<0.01).(7)RT-q PCR analysis showed that ox-LDL could activate the expression of proapoptotic gene Bax m RNA,decreased the expression of anti-apoptosis gene Bcl-2 m RNA and decreased the expression of Nrf2,NQO1 and HO-1 m RNA(P<0.01).However,it was found that EORGN could significantly down-regulate the expression of Bax m RNA and up-regulated the expression of Nrf2,NQO1,HO-1 and Bcl-2 m RNA(P<0.05,P<0.01).(8)Western blot results showed that ox-LDL could activate the expression of pro-apoptotic protein Bax,decreased the expression level of anti-apoptosis protein Bcl-2,and decreased the expression level of Nrf2,NQO1 and HO-1 protein(P<0.01).However,it was found that EORGN could significantly down-regulate the expression level of Bax protein and up-regulated the protein expression levels of Nrf2,NQO1,HO-1 and Bcl-2(P<0.05,P<0.01).Conclusions: The essential oil from rhizome Ginger Notoginseng has a significant protective effect on HUVECs damage induced by ox-LDL,and can prevent and treat atherosclerosis by increasing the expression of antioxidant enzymes and anti-apoptosis.The mechanism was probably related to the activation Nrf2/ARE signaling pathway.Therefore,the essential oil from rhizome Ginger Notoginseng may have great potential in the treatment of atherosclerosis. |
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