Protective Effect And Mechanism Of Polysaccharides From Citrus Aurantium On HepG2 Injury Induced By INH/RFP

Objective:To determine the content of Sagittaria sagittifolia polysaccharide,based on the determination of Sagittaria sagittifolin polysaccharide(SSP)on the non-toxic dose range,we study the protective effect of SSP in INH+RFP-induced hepatotoxicity in in HepG2 cells,and explore the mechanisms underlying this effect via regulating Nrf2/ARE signaling pathway,CYP450s,inhibition of hepatocyte apoptosis.This study can be used to provide the experimental basis for the development of SSP as liver protection products.Methods:1 H2SO4-Phenol method was used to measure the content of SSP.The pure SSP was obtained through rough extraction,deproteinization,and pure.The standard curve was established using Glucose standard solution,the absorbance was determined via H2SO4-Phenol method at 490nm wavelength.The repeatability test,precision test,stability test,and recovery rate experiment were used to verifying the accuracy of the experiment.2 The protection effect of SSP against INH/RFP-induced hepatotoxicity in HepG2 cellsHepG2 cells were divided into seven groups:Normal group,SSP protection group(0.125,0.25,0.5,1,2 mg/mL SSP).The MTT method was used to determine the SSP non-toxic dose range.HepG2 cells were divided into eight groups:Normal group,Model group(0.1 mg/mL INH+0.2 mg/mL RFP),SSP protection group(0.1mg/mL INH+0.2 mg/mL RFP+0.125,0.25,0.5,1,2 mg/mL SSP),cell cell survival and the ALT,AST,and LDH activity in cell supernatant were determined by MTT and Reitman-Frankel method in 12,24,and 48h,then the the best dose and time effect of SSP was confirmed.3 The protective mechanisms of SSP against INH/RFP-induced hepatotoxicity in HepG2 cells3.1 SSP protect against INH/RFP-induced hepatotoxicity via activating Nrf2/ARE signaling pathwayHepG2 cells were divided into three groups:Normal group,Model group(0.1mg/mL INH+0.2 mg/mL RFP),SSP protection group(best dose),the mRNA expression of Nrf2、Keap1、HO-1、GCLC were determined by RT-PCR method.Then the mRNA expression of Nrf2 was interfered by si-RNA,HepG2 cells were divided into four groups:Model group,SSP protection group,Model interfere group,SSP protection interfere group,the mRNA expression of Nrf2 was determined by RT-PCR method.Then the content of Nrf2、Keap1、HO-1、GCLC were determined by elisa method.After the best dose of SSP was confirmed,HepG2 cells were divided into three groups:Normal group,Model group(0.1mg/mL INH+0.2 mg/mL RFP),SSP protection group(best dose),the protein expression of Nrf2、Keap1、HO-1.GCLC were determined by Western-Blot method.HepG2 cells were divided into seven groups:Normal group,Model group(0.1mg/mL INH+0.2 mg/mL RFP),SSP protection group(0.1mg/mL INH+0.2 mg/mL RFP+0.125,0.25,0.5,1,2 mg/mL SSP),the content of MDA,SOD,and CAT were determined by ultraviolet spectrophotometer.3.2 SSP protect against INH/RFP-induced hepatotoxicity via regulating CYP450SHepG2 cells were divided into three groups:Normal group,Model group(0.1mg/mL INH+0.2 mg/mL RFP),SSP protection group(best dose),the mRNA expression of CYP2E1 and CYP3A4 were determined by RT-PCR method.Then the content of CYP2E1 and CYP3A4 were determined by elisa method.After the best dose of SSP was confirmed,HepG2 cells were divided into three groups:Normal group,Model group(0.1mg/mL INH+0.2 mg/mL RFP),SSP protection group(best dose),the protein expression of CYP2E1 and CYP3A4 were determined by Western-Blot method.3.3 SSP protect against INH/RFP-induced hepatotoxicity via inhibiting hepatocyte apoptosisHepG2 cells were divided into three groups:Normal group,Model group(0.1mg/mL INH+0.2 mg/mL RFP),SSP protection group(best dose),the mRNA expression of Bcl-2 and Bax were determined by RT-PCR method.Then the content of Bcl-2,Bax,Caspase3 and Caspase9 were determined by elisa method.After the best dose of SSP was confirmed,HepG2 cells were divided into three groups:Normal group,Model group(0.1mg/mL INH+0.2 mg/mL RFP),SSP protection group(best dose),the protein expression of Bcl-2 and Bax were determined by Western-Blot method.Results:1 H2SO4-Phenol method was used to measure the content of SSP.The average content of SSP was 34.31%,There was a good linear relationship between 20～100μg,r2=0.9928,average recovery was 104%,and RSD was 4.72(n=5),the colour of the test solution is stable within 2 h.2 The protection effect of SSP against INH/RFP-induced hepatotoxicity in HepG2 cellsThe cell survival had no difference between the SSP protection group and normal group(P>0.05),that means the non-toxic dose range of SSP were 0.125,0.25,0.5,1,2 mg/mL.Compared with the normal group,the cell survival was significantly declined and ALT,AST,and LDH activity increased in model group(P<0.05).Compared with the model group,the cell survival was significantly increased and ALT,AST,and LDH activity decreased in SSP protection group(P<0.05),moreover,it has no difference when the concentrations were 0.5mg/mL and 1,2 mg/mL(P>0.05),the ALT,AST,and LDH activity decreased most significantly when the reaction time was 24h.3 The protective mechanisms of SSP against INH/RFP-induced hepatotoxicity in HepG2 cells3.1 SSP protect against INH/RFP-induced hepatotoxicity via activating Nrf2/ARE signaling pathwayIn the RT-PCR experiment,compared with the normal group,the mRNA expression of Nrf2,HO-1,GCLC were increased,but the mRNA expression of Keap1 was decreased in model group(P<0.05).Compared with the model group,the mRNA expression of Nrf2,HO-1,GCLC were increased,but the mRNA expression of Keapl were decreased in SSP protection group(P<0.05).In the gene silencing experiment,compared with the model group,the mRNA expression of Nrf2 was increased in SSP protection group,but decreased in model interfere group.Compared with the model interfere group,the mRNA expression of Nrf2 was increased in the SSP protection interfere group(P<0.05).In the elisa experiment,compared with the normal group,the content of Nrf2,HO-1,GCLC were increased,but the content of Keapl was decreased in model group(P<0.05).Compared with the model group,the content of Nrf2,HO-1,GCLC were increased,but the content of Keapl were decreased in SSP protection group(P<0.05).The content of Nrf2 was highest when SSP was 0.5 mg/mL,the content of HO-1 and GCLC were highest when SSP was 1 mg/mL,the content of Keapl was lowest when SSP was 0.5 mg/mL.In the Western-Blot experiment,compared with the normal group,the protein expression of Nrf2,HO-1,GCLC were increased,but the protein expression of Keapl was decreased in model group(P<0.05).Compared with the model group,the protein expression of Nrf2,HO-1,GCLC were increased,but the protein expression of Keapl were decreased in SSP protection group(P<0.05).Compared with the normal group,the content of MDA increased,SOD,CAT,and GSH content were decreased significantly in model group(P<0.05).Compared with the model group,the content of MDA decreased,SOD,CAT,and GSH content were increased significantly in SSP protection group(P<0.05),and optimum concentration was 0.5mg/mL.3.2 SSP protect against INH/RFP-induced hepatotoxicity via regulating CYP450SIn the RT-PCR experiment,compared with the normal group,the mRNA expression of CYP2E1 and CYP3A4 were increased(P<0.05).Compared with the model group,the mRNA expression of CYP2E1 and CYP3A4 were decreased in SSP protection group(P<0.05).In the elisa experiment,compared with the normal group,the content of CYP2E1 and CYP3A4 were increased(P<0.05).Compared with the model group,the content of CYP2E1 and CYP3A4 were decreased in SSP protection group(P<0.05).The content of CYP2E1 and CYP3A4 were lowest when SSP was 1.5 mg/mL.In the Western-Blot experiment,compared with the normal group,the protein expression of CYP2E1 and CYP3A4 were increased(P<0.05).Compared with the model group,the protein expression of CYP2E1 and CYP3A4 were decreased in SSP protection group(P<0.05).3.3 SSP protect against INH/RFP-induced hepatotoxicity via inhibiting hepatocyte apoptosisIn the RT-PCR experiment,compared with the normal group,the mRNA expression of Bcl-2 and Bax were increased,but the mRNA expression of Bcl-2/Bax was decreased in model group(P<0.05).Compared with the model group,the mRNA expression of Bcl-2 and Bcl-2/Bax were increased,but the mRNA expression of Bax was decreased in SSP protection group(P<0.05).In the elisa experiment,compared with the normal group,the content of Bcl-2,Bax,Caspase3 and Caspase9 were increased(P<0.05).Compared with the model group,the content of Bcl-2 was increased,but the content of Bax,Caspase3 and Caspase9 were decreased in SSP protection group(P<0.05).The content of Bcl-2 was highest when SSP was 0.5 mg/mL,the content of Bax,Caspase3 and Caspase9 were lowest when SSP was 0.5 mg/mL.In the Western-Blot experiment,compared with the normal group,the protein expression of Bcl-2,and Bax were increased,but the protein expression of Bcl-2/Bax was decreased in model group(P<0.05).Compared with the model group,the protein expression of Bcl-2 and Bcl-2/Bax were increased,but the protein expression of Bax was decreased in SSP protection group(P<0.05).Conclusion:The average content of SSP was 34.31%via H2SO4-Phenol,and this method was sensitive,reproducilble.The SSP non-toxic dose range was 0.125,0.25,0.5,1,2 mg/mL.SSP has protective effect against INH/RFP-induced hepatotoxicity.And the mechanisms underlying this effect via the activation of Nrf2 pathway and its downstream antioxidant enzymes,the inhibition of CYP2E1 and CYP3A4 expression,the regulation of factor related apoptosis.