The Significance Of Gpbar1 Signaling Pathway In AR42J Cell Injury Induced By Deoxycholic Acid And Intervention Of Qingyi Decoction Medicated Serum

Objectives:The changes of Gpbar1 protein expression induced by different concentrations of bile acids and the changes of its downstream signal pathway were compared and analyzed,and the effects of different doses of Qingyi decoction medicated serum on the expression of Gpbar1 protein were compared and analyzed.To explore the role of Gpbar1 protein and downstream pathway in acute pancreatitis and the intervention mechanism of Qingyi decoction medicated serum.Methods:Based on the previous research basis of our group,AR42J cells were induced with 0.8mmol/L and 0.4mmol/L DCA at different concentrations,and the models of acinar cell injury of severe pancreatitis with necrosis and mild pancreatitis with apoptosis were established respectively.Subsequently,QRT-PCR and Western-Blot were used to detect the effect of different concentrations of DCA on the expression of Gpbar1.The changes of intracellular cAMP content and calcium content were detected by flow cytometry.Then,the changes of Gpbar1 expression were detected by Western-Blot with different doses of Qingyi decoction medicated serum at different times.The changes of cAMP content were detected by Elisa.The changes of calcium concentration in fluo-3AM flow cytometry were also observed.Results:The expression levels of Gpbar1 protein,cAMP and Ca²⁺in DCA treated with different concentrations were significantly higher than those in negative control group?P<0.001?.And the high concentration DCA treatment group was obviously upregulated than the low concentration DCA treatment group?P<0.001?.In the 0.4mmol/L DCA induced cell injury model,the intervention group of Qingyi decoction medicated serum was compared with the model group.1.The expression of Gpbar1 protein in cells decreased with the increase of Qingyi decoction medicated serum dosage,but the decrease was not significant in the intervention of low dose Qingyi decoction medicated serum?P>0.05?.However,with the increase of action time of Qingyi decoction medicated serum,there was no significant change in protein expression?P>0.05?.2.The intracellular cAMP content decreased significantly with the increase of the dosage of Qingyi decoction medicated serum?P<0.05?.With the increase of action time of Qingyi decoction,medicated serum the content of intracellular cAMP also decreased,but when the intervention time of Qingyi decoction increased from 0.5h to 6h,the content of intracellular cAMP decreased no statistical significance?P>0.05?.cAMP decreased significantly at 12h?P<0.05?.3.The intracellular calcium content decreased significantly with the increase of Qingyi decoction dosage?P<0.05?.When the high dose Qingyi decoction was treated,there was no significant difference in the decrease of Ca²⁺between the high dose and the middle dose?P>0.05?.In comparison with low dose?P<0.05?,and Qingyi decoction only decreased the intracellular calcium content at 0.5 h and 12 h after intervention?P<0.05?.Other intervention time was not significantly compared with the decrease of Ca²⁺?P>0.05?.In the 0.8mmol/L DCA induced cell injury model,the intervention group of Qingyi decoction medicated serum was compared with the model group.1.The expression of cell Gpbar1 protein decreased with the increase of Qingyi decoction dose,the content of cell Gpbar1 protein decreased with the increase of Qingyi decoction dosage,but the low dose of Qingyi decoction did not decrease significantly?P>0.05?.The expression of Gpbar1 protein did not change significantly with the increase of action time of Qingyi decoction medicated serum?P>0.05?.2.The intracellular cAMP content decreased significantly with the increase of the dosage of Qingyi decoction?P<0.05?.There was no significant difference in the effect of high,medium and low dosage Qingyi decoction medicated serum on cAMP reduction?P>0.05?.With the increase of action time of Qingyi decoction medicated serum,the content of intracellular cAMP decreased,but there was no statistical significance?P>0.05?.3.The decrease of intracellular calcium ion in low dose Qingyi decoction medicated serum intervention group was not obvious?P>0.05?.Medium and high dose groups decreased significantly?P<0.05?.There was no significant difference in calcium content among groups with high,medium and low doses of Qingyi decoction medicated serum?P>0.05?.The effect of Qingyi decoction medicated serum on the decrease of Ca²⁺content was the same as that in the mild pancreatitis model,and the intracellular calcium content was significantly decreased only at 0.5h and 12h?P<0.05?.Other intervention time was not significant compared with the decrease of calcium ion?P>0.05?.Conclusion:1.When DCA induces AR42J cell injury,the expression of Gpbar1 protein is up-regulated,and the concentration of downstream cAMP and Ca²⁺also increases,and both increase with the increase of DCA concentration.2.Qingyi decoction medicated serum can reduce the expression of Gpbar1 and the concentration of downstream cAMP and Ca²⁺in DCA-induced AR42J cells,which may be one of the potential targets for the treatment of acute biliary pancreatitis.3.Within a certain dose range?10%-20%?,the intervention effect of Qingyi decoction medicated serum increased with increasing dose.4.In the DCA induced cell injury model,the expression of Gpbar1 protein and the concentration of cAMP did not change significantly with the increase of action time of Qingyi decoction medicated serum.