The Effect Of Realgar On MAPK/Nrf2/HO-1 Signaling Pathway Of Rat Primary Hepatocytes And The Protective Effect Of Artificial Calculus Bovis

Objective:Realgar contains a small amount of arsenic trioxide（As₂O₃,i As^Ⅲ,or arsenic）which is easily absorbed by the gastrointestinal tract and is the toxic component of realgar.However,due to the influence of traditional medication habits（non-toxic side effects of traditional Chinese medicine）,incidents of drug-induced arsenic poisoning caused by the unscientific（long-term,excessive,and non-standard）use of monogargar or its compound preparations have been reported.Its clinical manifestations are systemic multiple system damage,mainly digestive system and skin damage.Studies have shown that oxidative stress and oxidative damage are one of the main mechanisms of arsenic toxicity.Nrf2,as an antioxidant,plays an important role in resisting oxidative stress.At the same time,MAPK signaling pathway is involved in the activation of Nrf2.After Nrf2is activated,downstream antioxidant gene expression is initiated,including NQO1,GST,SOD,HO-1,andγ-GCS to further exert antioxidant effects.Calculus bovis was first recorded in Shennong’s Materia Medica.Clinical studies have shown that artificial calculus can reduce liver damage and protect the liver.At the same time,modern research shows that artificial calculus bovis is a natural antioxidant and has obvious antagonistic effects on oxidative stress injury induced by various toxicants.Therefore,in this study,in vitro cell culture experiments were used to investigate the changes of MAPK and Nrf2/HO-1 pathways in rat liver cell injury induced by realgar,elucidate the mechanism of realgar damage on hepatocyte toxicity,and find early changes in its toxicity and sensitive organisms.This study has important theoretical and practical significance for early effective measures to prevent and control the occurrence of chronic arsenic poisoning,guide the rational use of medicines and improve the quality of the population.At the same time,the mechanism of compatibility and detoxification of artificial calculus to realgar was analyzed to provide an experimental basis and theoretical basis for the research on the prevention of liver toxicity of realgar and traditional Chinese medicine compatibility and detoxification theory.Methods:Isolation,culture and identification of rat primary hepatocytes,and establishment of a model of primary hepatocytes culture in vitro.MTT method was used to detect the hepatocytes viability of different levels of realgar,different concentrations of artificial calculus bovis,and artificial calculus bovis exposed to realgar after pretreatment.The appropriate exposure dose of realgar and artificial calculus bovis was selected,and its effect on cell morphology was observed under a microscope.GPX enzyme activity detection kit was used to detect the effects of realgar extracts with different concentrations on GPX enzyme activity in cells.Western Blot（WB）was used to detect the effects of realgar extracts of different concentrations on the expressi on levels of MAPK（p38,JNK）,Nrf2,HO-1,Bcl-2,and Bax proteins in rat primary hepatocytes.Western Blot（WB）was used to detect changes in the expression levels of Nrf2 and HO-1protein in hepatocytes after hepatocytes were pretreated with artificial calculus bovis extract at different concentrations for 1 h and then exposed to realgar extract.The data obtained from the experiments are expressed as mean±standard deviation,GraphPad Prism5 software was used for one-way ANOVA and two-way ANOVA to test the significance of differences between groups.P<0.05 was considered statistically significant.Results:1.Realgar extract at 1.25-2.5 mg/mL（w/v）（as arsenic concentration:5.06-10.13μg/mL（w/v））will cause morphological changes and decrease in cell viability of rat primary liver cells and showed a dose-response relationship.The difference was statistically significant compared with the control group（P<0.05）,and at 1.75 mg/mL（w/v）cell viability is close to 50%;2.Artificial calculus bovis extract at 0.10-0.25mg/mL（w/v）,there was no significant difference in cell viability compared with the control group（P>0.05）;3.Artificial calculus bovis extract at 0.175 and 0.25 mg/m L（w/v）after pretreatment for 1 h and then exposed to 1.75 mg/mL（w/v）realgar extract,the hepatocytes viability is significantly higher than 1.75 mg/mL（w/v）realgar extract rat primary hepatocytes treated alone（P<0.05）;4.Realgar extract at 1.25-1.75 mg/mL（w/v）can cause significant GSH-Px enzyme activity in rat primary hepatocytes reduce（P<0.05）;5.Compared with the control group,realgar extract at 1.25-1.75 mg/mL（w/v）can significantly increase the expression of JNK and p38 protein in rat primary hepatocytes（P<0.05）,while Nrf2,HO-1 protein expression levels were significantly higher than those in the control group（P<0.05）;Compared with the control group,when the realgar extract was 1.75 mg/mL（w/v）the expression level of the anti-apoptotic protein Bcl-2 decreased（P>0.05）,and the expression level of the apoptotic protein Bax increased significantly.（P<0.05）;6.The artificial calculus bovis extract was pretreated at 0.175 and 0.25 mg/mL（w/v）for 1 h and then exposed to 1.75 mg/mL（w/v）realgar extract,and compare with 1.75 mg/m L（w/v）realgar extrac,the expressiton of Nrf2 and HO-1 proteins in hepatocytes was significantly reduced,and the difference was statistically significant（P<0.05）Conclusion:1.Realgar can cause the rat’s primary liver cell vitality and antioxidative capacity to decrease significantly,resulting in oxidative damage.Its mechanism is related to the activation of JNK,p38,Nrf2 and HO-1.2.Artificial calculus bovis protects liver cells from realgar.The mechanism is that artificial bezoar can inhibit the activation of realgar-induced Nrf2/HO-1 signaling pathway.