Effects Of Intestinal Smooth Muscle-derived GDNF On Intestinal Epithelial Tight Junctions And Smooth Muscle Phenotype Transformation

Objective Inflammatory bowel disease（IBD）is a non-specific inflammatory disease of the gut that seriously affects the quality of life and economic burden of humans.Glial cells derived neurotrophic factor（GDNF）can not only maintain the integrity of the enteric nervous system,but also promote the maturation of intestinal epithelial cells and regulate the intestinal immune response.Recent studies have confirmed that intestinal smooth muscle cells（ISMC）are the main source of GDNF in the process of inflammation,and the hypertrophy and hyperplasia and the synthesis of collagen fibers of ISMC are important reasons for intestinal stenosis formation under chronic inflammation.The role of ISMC-derived GDNF in IBD has not been investigated.In vitro culture of primary ISMC has disadvantages such as timeconsuming,labor-intensive,and limited number of passages,which limit their studies in vitro.In this study,by establishing an immortalized rat ISMC line,we investigated the effect of ISMC-derived GDNF on epithelial tight junctions and smooth muscle phenotype transformation.Methods Firstly,primary cultured neonatal rat ISMC were transfected with a lentiviral vector containing the simian virus 40 large T antigen（Sv40lt）gene,and immortalized ISMC were obtained after antibiotic screening and single clone selection.The levels of Sv40 lt DNA and m RNA in immortalized cells,the expression of smooth muscle markers α-SMA and desmin in different passage,and the proliferation ability of high passage and the response to carbachol was detected.The luciferase gene（luc2）was transfected into the immortalized ISMC by lentivirus and Sleeping Beauty transposition system,and the fluorescence signal in mice after subcutaneous injection of the immortalized ISMC was dynamically detected by in vivo imaging of small animals.Next,the secretion of GDNF was determined when ISMC was treated with different concentrations of TNF-α,IL-1β and IFN-γ respectively.Further,the Gdnf gene was overexpressed in the above-mentioned immortalized ISMC by genetic engineering technology,and the culture supernatants of GDNF overexpressed and control cells were used to culture the intestinal epithelial cell line IEC-6,and the proliferation and transepithelial electrical resistivity（TEER）and the expression of tight junction proteins ZO-1,E-cadherin and occludin were detected.The intestinal epithelial cell tight junction damage model induced by TNF-α was established,and the effects of ISMC-derived GDNF on IEC-6 TEER and tight junction protein ZO-1 and occludin were detect,and in order to verify whether the above changes were caused by GDNF,vandetanib,the inhibitor of GDNF receptor Ret,was added and the above indicators were re-examined.The proliferation,migration and synthesis abilities and the expression of SMMHC,α-SMA,SM22 and calponin were detected in ISMC after GDNF overexpression,and the changes of above indicators were re-examined after vandetanib was added.The changes of p-AKT and p-ERK1/2 in ISMC after GDNF overexpression were detected,and the changes of p-AKT and p-ERK1/2 were re-examined after vandetanib was added.Results In this experiment,an immortalized rat ISMC line was successfully established.The morphology of the immortalized ISMC is similar to that of primary ISMC,and it can express the smooth muscle markers α-SMA and desmin.The expression of Sv40 lt gene can be detected in the immortalized ISMC,and it has been stably passaged to more than 50 generations.There is no significant difference in the expression of α-SMA and desmin between different passage.Cells that have been passaged for many times still have strong proliferation ability and response to carbachol.In vivo imaging of small animals confirmed that the immortalized ISMC had no tumorigenic properties.Different concentrations of TNF-α,IL-1β and IFN-γ act on ISMC,only TNF-α can promote the secretion of GDNF from ISMC,and TNF-αpromotes the secretion of GDNF from ISMC in a time and concentration-dependent manner.ISMC-derived GDNF promotes the proliferation of IEC-6 and increases the expressions of TEER and ZO-1,E-cadherin and occludin;ISMC-derived GDNF can partially restore the TNF-α-induced reduction of TEER,ZO-1,and occludin levels,while vandetanib can restore TEER to baseline levels;after overexpression of GDNF,ISMC proliferation and migration abilities are enhanced,and the expressions of collagen I and osteopontin were increased,while the expression levels of contractile proteins SMMHC,α-SMA,SM22 and calponin were decreased,while vandetanib reduced the proliferation and migration of GDNF-overexpressing ISMC,while SMMHC,α-SMA,SM22 and calponin expression level increased.After overexpression of GDNF,the levels of p-AKT and p-ERK in ISMC cells increased,and vandetanib could reduce the levels of p-AKT and p-ERK in ISMC cells overexpressed GDNF.Conclusion TNF-α promotes the secretion of GDNF in ISMC,and ISMCderived GDNF promotes the formation of intestinal epithelial tight junctions and regulates smooth muscle phenotype transformation.