| bjective: To investigate the protective effect and its relatedness of ethanol extract-induced L-02 cell oxidative injury through the Keap1-Nrf2-ARE signaling pathway based on ethanol-induced oxidative damage model of human hepatocytes(L-02)in vitro.mechanism.Methods:1.Preparation of drug-containing serum of Pange Oral Liquid: Forty male Sprague Dawley rats weighing 175-215 g were randomly divided into 2 groups with 20 rats in each group as blank control group(with drug-containing serum group,etc.)The volume of physiological saline),Qi Ge oral solution group(8.85 g crude drug /100 g body weight / day),daily morning and evening after intragastric administration once a day,5 consecutive days after abdominal aortic blood collection preparation of drug-containing serum.2.The effect of Xiaoge Oral Liquid containing serum on the vitality of normal L-02 cells: Different volume ratios(1.25%,2.5%,5%,10%)were added to L-02 cells.Serum,the effect of drug-containing serum on the viability of L-02 cells was examined.3.L-02 cell oxidative damage model: The final concentration of ethanol was 0.5%,1%,1.5%,2%,2.5%,3%,5% to interfere with L-02 cells 6h,12 h,24h modeling,respectively.The cell viability was detected by CCK8 method.The activity of AST and LDH in the cell supernatant was detected by an automatic biochemical analyzer.The content of malondialdehyde(MDA)in L-02 cells was measured by thiobarbituric acid method.A microplate reader was used to measure cellular reactive oxygen species(ROS)levels and combined CCK-8 results to determine the final ethanol concentration and time of the model.4.Screening of the optimum concentration of drug-containing serum in Fuge Oral Liquid: To determine the concentration of 2.5% ethanol and different volume ratios(1.25%,2.5%,5%,and 10%)of Puzheng Oral Liquid(ie divided into ZGG1.25%+YC2.5% group,ZGG2.5%+YC2.5% group,ZGG5%+YC2.5%group,ZGG10%+YC2.5% group)L-02 cells were co-cultured At 24 h,cell viability was detected by CCK8 method.The activity of AST and LDH enzymes in cell culture supernatant was measured by an automatic biochemical analyzer.The content of malondialdehyde(MDA)in L-02 cells was determined by thiobarbituric acid method.The level of reactive oxygen species(ROS)in the cells was measured using a fluorescence microplate reader to verify the antioxidation effect of different volume ratios of Geju oral liquid containing serum,and the optimal concentration was selected for subsequent experiments.5.The protective effect of Xiaoge Oral Liquid serum on ethanol-induced oxidative damage of L-02 cells: The experiment was divided into 3 groups:normal control group(10% normal rat serum culture)and model group(optimal ethanol effect.Concentration and time)and drug-containing sera(optimum concentration of drug-containing sera of Zhi Ge oral solution + optimal ethanol concentration and time),and cells were collected after 24 hours of treatment with L-02 cells,and they were measured by thiobarbituric acid method.The activity of superoxide dismutase(SOD)in the cells was observed by immunofluorescence staining for the nuclear transfer of Nrf2 in the Keap1-Nrf2-ARE pathway of L-02 cells using real-time fluorescent quantitative polynucleotide chain reaction(q PCR).Detection of diphasic enzymes glutamylcysteine ??ligase subunit(GCLC),glutathione S-transferase P1subunit(GSTP1),glutathione S-transferase(GSTT1)in the Keap1-Nrf2-ARE pathway),oxidase(NQO1),heme oxygenase(HO1)m RNA expression levels,and from this pathway to discuss Ge oral liquid serum on ethanol-induced L-02 cell oxidative damage protection and mechanism.Results:1.The survival rate of the cells was not significantly different after treatment with different volume ratios of quercetin oral drug-containing serum for 24 h in normal L-02 cells(P>0.05).2.After different concentrations of ethanol intervened in L-02 cells for 6h,12 h and 24 h,the cell survival rate gradually decreased with the increase of ethanol concentration and the prolongation of the intervention time,when the ethanol concentration was 2.5%,3%,5%,After 24 h intervention,the AST,LDH,and MDA and ROS content in the supernatant of the cells were significantly increased(P<0.05).Combined with the survival rate of the cells at different time points,the concentration of 2.5% ethanol was used to interfere with L-02 cells for 24 h.The ethanol-induced L-02 oxidative injury model was established for subsequent experiments.3.Different volume ratios(1.25%,2.5%,5%,10%)of Pu-Ge oral liquid containing serum and 2.5% ethanol(ie ZGG1.25%+YC2.5% group,ZGG2.5%+YC2.The survival rate of ZGG1.25%+YC2.5% group was higher than that of the normal group after the L-02 cells were incubated for 5%,ZGG5%+YC2.5% and ZGG10%+YC2.5%groups for 24 hours.There were no significant differences in ALT,LDH,and MDA and ROS contents in the cell supernatant(P>0.05).ZGG2.5%+YC2.5%,ZGG5%+YC2.5%,ZGG10%+YC2 The survival rate of.5% cells gradually decreased with the increase of the volume ratio of drug-containing serum of Qige Oral Liquid,but the survival rate was over 75%(P<0.05),and the AST,LDH in the cell supernatant of each group.The content of MDA and ROS in the cells increased gradually with the increase of the volume ratio of the drug-containing serum of Pange Oral Liquid(P<0.05).Compared with the model group,the survival rate of ZGG1.25%+YC2.5% group,ZGG2.5%+YC2.5% group,ZGG5%+YC2.5% group,and ZGG10%+YC2.5%group increased.P<0.05),ALT,LDH and MDA,ROS content in the cell supernatant of each group decreased(P<0.05).The final concentration of 1.25%Gegen oral solution serum was used as the optimal concentration.L-02 cells were co-incubated with 1.25% Liquor Oral Liquid and 2.5% ethanol for 24 hours for subsequent experiments.4.The protective effect of Puzheng Oral Liquid contained serum on ethanol-induced oxidative damage of L-02 cells:Compared with the normal control group,SOD enzyme activity in the model group was significantly reduced(P<0.05),MDA,ROS content increased significantly(P<0.05),there was no significant difference in SOD enzyme activity and MDA,ROS content between the drug-containing serum group(P<0.05);Compared with the model group,SOD enzyme activity in the drug-containing serum group was significantly higher(P<0.05).ROS content decreased significantly(P<0.05).Effect of Puqi Oral Liquid Containing Serum on Keap1-Nrf2-ARE Pathway of Ethanol-induced L-02 Cell Oxidative Injury:Compared with the normal control group,the expression level of Nrf2 nuclei in the model group increased slightly,and the second-phase enzyme GCLC The expression levels of GSTP1,GSTT1,NQO1,and HO1 m RNAs were significantly decreased(P<0.05).The expression levels of Nrf2 in the drug-containing serum group were significantly increased,and the expression levels of the second-phase enzymes GCLC,GSTP1,GSTT1,NQO1,and HO1 were significantly increased.Increased(P<0.05);compared with the model group,the expression level of Nrf2 nuclei in the drug-containing serum group was significantly increased,and the expression levels of the second-phase enzymes GCLC,GSTP1,GSTT1,NQO1,HO1 were significantly increased(P<0.05).Conclusions:1.The drug-containing serum of Puzheng Oral Liquid has no significant effect on the growth of normal L-02 cells;2.The effect of2.5% ethanol on L-02 cell viability for 24 h has little effect on L-02 cell viability,but it can induce L-02 cells.Oxidative damage;3.1.25% Fructus Puerariae Oral Liquid serum can significantly inhibit alcohol-induced L-02 cell AST,LDH leakage,reduce the content of MDA,ROS in cells,increase intracellular SOD enzyme activity,improve Keap1-Nrf2-ARE The expression of Nrf2 in the pathway and the expression of the diphasic enzyme in the Keap1-Nrf2-ARE signaling pathway indicated that Puzheng Oral Solution-containing serum could reduce the ethanol-induced oxidative stress injury,and its mechanism may be related to the activation of Keap1-Nrf2-ARE.Pathways related. |
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