The Effect Of LPS On The Proliferation And Apoptosis Of Enteric Glial Cells In Inflammatory Environment In Vitro

Objective:To investigate the effects of LPS on the proliferation and apoptosis of enteric glial cells in inflammatory environment,and to explore its mechanism.Methods:1.The expression of glial fibrillary acidic protein(GFAP)and calcium binding protein S-100? in enteric glial cells(EGCs)was detected by immunofluorescence staining.2.EGC was divided into blank control group,TNF-?+IFN-? group,LPS+TNF-?+IFN-? group,TNF-?+IFN-?+RET receptor inhibitor group and LPS+NF-?+IFN-?+RET receptor inhibitor group.MTT colorimetry assay and BrdU method were used to detect the proliferation of EGCs.TUNEL staining,Annexin V-FITC/PI double staining and Hoechst33342 staining were used to detect the apoptosis of EGCs.Caspase-3 activity kit were used to detect the Caspase-3 activity.The relative expression levels of RET,PI3 K,AKT and p-AKT were detected by Western blotting.Results:1.Immunofluorescence staining showed that GFAP and calcium binding protein S-100? could be expressed simultaneously in the cell lines used in this study.2.MTT results showed that different concentrations of LPS(100,200,400 ug/mL)inhibited the proliferation of EGCs(P < 0.05),and different concentrations of TNF-a(50,100,200 ng/mL)+IFN-?(100 ng/mL)significantly reduced the survival rate of EGCs(P < 0.05).MTT and BrdU results showed that compared with TNF-a(50 ng/mL)model group,LPS treatment significantly reduced cell proliferation(P < 0.05).At the same time,in TNF-a+IFN-? group and LPS+TNFa+IFN-? group,the proliferation of EGCs cells was further reduced after RET receptor inhibitor treatment(P < 0.05).3.The results of TUNEL staining and flow cytometry(Annexin V-FITC/PI double staining)showed that there was no significant change in apoptosis of EGCs between groups,and there was no significant difference between groups(P > 0.05).Morphological observation of Hoechst 33342 cell apoptosis showed that there was no obvious nuclear deviation and no significant change in the number of apoptotic bodies in each group.The results of Caspase-3 Activity Kit showed that there was no significant difference in relative activity of Caspase-3 among groups(P > 0.05).4.The results of Western blotting showed that the relative expression of RET,AKT and p-AKT proteins in EGCs of TNF-a+IFN-? group was down-regulated compared with the normal control group(P < 0.05).Compared with the model group of TNF-a+IFN-?,LPS treatment reduced the relative expression of RET,AKT and p-AKT proteins in EGCs(P<0.05).At the same time,the relative expression of RET,AKT and p-AKT proteins in EGCs treated with RET receptor inhibitors decreased further in TNF-a+IFN-? group and LPS+ TNF-a+IFN-? group(P <0.05).There was no significant difference in the expression of PI3 K in EGCs(P > 0.05).Conclusions:1.The cell lines used in this experiment were identified as rat enteric glial cell lines.2.LPS can inhibit the proliferation of EGCs in inflammatory environment,which may be related to the inhibition of RET and PI3K/AKT signaling pathway-related protein expression.LPS has no significant effect on the apoptosis of EGCs in inflammatory environment.