The Effect Of Cinobufotalin On H₂₂ Hepatoma Cell Induce Immune Cell To Synthesis And Release β-end In Intro

Objective In this study, we established the co-culture model of splenic lymphocyteand H22hepatoma carcinoma cell in mice in vitro. We observed that whether Cinobufotalincan promote the release of β-END in supernate and the expression of CRF、IL-1and thegene expression of POMC and CTSL. To explore the proliferation of Cinobufotalin affectimmune cell in alone and in the co-culture model. To detect the chemotaxis ofCinobufotalin influence immune cell, in order to provide theory for the mechanism ofCinobufotalin treated cancer pain based on the pathway of β-END-μ-POMC.Methods (1) We prepared splenic lymphocyte and H22hepatoma carcinoma ascite cellfrom mice, take this two cells to co-culture according to a certain concentration ratio.(2)The content of β-END in supernate were measured by ELISA in order to select the optimaltime and the optimal concentration ratio of the index mensuration. ELISA method detectedCinobufotalin influence the content of cytokine in supernate.(3) MTT assay andCytometry were used to test the cell proliferation.(4) Fluorescence quantitative PCR wasused to determine the gene expression of POMC and CTSL.(5) The chemotaxis wasdetected by Transwell chemotaxis assay.Results (1) We established the co-culture model of splenic lymphocyte and H22hepatoma carcinoma cell in vitro successfully.(2) Acording to the content thatCinobufotalin promote the release of β-END in supernate, we choose the optimal time was24h and the optimal concentration ratio of splenic lymphocyte and H22hepatomacarcinoma cell was10:1. Moreover, the different concentration Cinobufotalin all improvedthe content of β-END in supernate significantly.(3) Cinobufotalin can obviously improvedthe proliferation of immune cell in alone and in the co-culture model. Compared withblank control group, the both has significant differences at24h and48h. Cinobufotalin caninhibited H22hepatoma carcinoma cell in alone and in the co-culture model.(4) The geneexpression of POMC and CTSL in Cinobufotalin group, which showed a significantdifference compared with control group.(5) Transwell assay demonstrated that thechemotaxis of immune cell was improved in Cinobufotalin group.Conclusion In extracorporeal co-culture model, Cinobufotalin increased the contentof β-END and CRF in supernate, and improved the gene expression of POMC and CTSL.Cinobufotalin could inhibitting the H22hepatoma carcinoma cell while promoting theproliferation of immune cell. At the same time, Cinobufotalin could induce the chemotaxis of immune cell. Speculate that Cinobufotalin may play a local analgesic effect based on thepathway of β-END-μ-POMC by promoting the proliferation of immune cell andrecruitment and increasing the synthesis and release of β-END.