Wnt/?-catenin Pathway Mediated The Heat Exposure-Induced Inhibition Of Proliferation And Differentiation Of Porcine Intestinal Epithelial Cells And Expansion Of Stem Cells

High temperature,as one of the most important stressors of livestock production in summer,can cause the atrophy of intestinal mucosa and damage the intestinal epithelial barrier function.The imbalance of intestinal epithelial homeostasis under continuous exposure to the high-temperature conditions may be related to the disturbance of intestinal epithelial renewal process which derives from intestinal stem cells(ISCs)and is regulated by the Wnt/?-catenin signaling pathway in the niche.To investigate the mechanism of heat exposure(41?)on the renewal process of intestinal epithelium in piglets,MTT and cell counting assays were performed to detect the proliferation of IPEC-J2(porcine jejunal epithelial cell)in the experiment,then cell apoptosis,cell differentiation,cell barrier and Wnt/?-catenin signaling pathway were detected by Western blotting,cell immunofluorescence,flow cytometry and transwell at time point of proliferation difference.Furthermore,three dimensional(3-D)model of enteroid,cultured from crypts which are isolated from 5-day-old crossbred boars(Yorkshire × Landrace),were used to explore the effect of heat exposure on the expansion of intestinal stem cells(ISCs),and the changes of proliferation,apoptosis,differentiation,?-catenin and the tight junction proteins in enteroids were detected by RT-PCR,Wes and enteroid immunofluorescence.The study might provide a novel target for the repair of intestinal damage and a new strategy for manipulating gut health manipulation induced by high temperature.The results were as follows:(1)The results showed that the proliferation ability of IPEC-J2 cells were significantly inhibited(P<0.05)after exposed to heat(41?)at 48 h and 72 h and significantly decrease(P<0.05)the enteroid budding efficiency at 24 h.(2)Compared with the control group(37?),the heat exposure group(41?)markedly induced(P<0.05)IPEC-J2 cell apoptosis and increased the Caspase-3 expression level at 72 h,as well as in enteroids at 24 h(P<0.05).(3)Heat exposure decreased the terminal differentiation marker(KRT20),the ISCs marker(Lgr5 and Bmi1),the absorptive cell marker(SI)and the goblet cell marker (Muc2)at 72 h in IPEC-J2 cell and at 24 h in enteroids.(4)Compared with the control group(37?),the TEER values and permeability of fluorescein sodium salt(FS)were significantly reduced(P<0.05)and increased (P<0.05)respectively in heat exposure group(41?)at 72 h,and the tight junction protein(ZO-1,Occluding and Claudin-1)(P<0.05)expression in IPEC-J2 cell after exposure to heat at 72 h and in enteroids at 24 h.(5)After exposure to 41? for 72 h,the Wnt/?-catenin pathway was significantly down-regulated in IPEC-J2 cells.Specifically,the protein expression levels of Axin2 and Gsk3? were increased(P<0.05),and the ?-catenin,Cyclin D1 and c-Myc protein expression levels were decreased(P<0.05),as well as the ?-catenin(P<0.05)in enteroids at 24 h.Conclusion: Heat exposure inhibits cell proliferation and differentiation,induces apoptosis in IPEC-J2 cells and enteroids which appears to be mediated by the Wnt/?-catenin signaling pathway,and these effects result in barrier function damage.