Immunomodulatory Effects Of Shh/p53/NF-?B Signaling Pathways In AEC? Infected With BCG

In the process of infecting the body against Mycobacterium tuberculosis(MTB),alveolar epithelial cells(AEC)are the first "physical barrier" of alveolar resistance to infection.As one of the first target cells of MTB infection,AEC can recruit alveolar macrophage(AMs)into the alveoli by secreting cytokines,and plays an important regulatory role in the innate immunity against MTB infection.In recent years,studies show found that Hedgehog(Hh)signaling pathway has "cross-talk" relationship with p53 and NF-?B signaling pathways during the development of cancer,and regulates the body inflammatory response.This suggests that when MTB infects the host lung,the Shh,p53 and NF-?B signaling pathways in alveolar epithelial cells and alveolar macrophages may be activated and participate in the regulation of inflammatory cytokine secretion to protect against MTB infection!In this study,interference/overexpressing mouse alveolar epithelial cells TC-1 were infected with Bacillus Calmette-Guerin(BCG),and the supernatant was collected and added to macrophage RAW264.7 cells.To analyze the changes of p53 and NF-?B signaling pathway-related signaling molecules,the secretion of inflammatory cytokines in alveolar macrophages,a mouse BCG peritoneal infection model has been establish to observe the morphological changes of lung and spleen,the changes of Shh,p53 signaling pathway also tested,in order to reveal the immunoregulatory effects of Shh p53 and NF-?B signaling pathways in the regulation of immune-related cells against BCG infection in vitro and in vivo.We excuted the relevant tests and obtained the test results as follows:(1)From the cell level test,when the Shh gene was interfered in TC-1 cells,there was no significant change in Gli2,and the expression of PTCH protein was down-regulated;when the Shli gene was overexpressed in TC-1 cells,Gli2 and PTCH Protein expression is upregulated.After the Shh gene-interfering TC-1 cell supernatant was added to alveolar macrophage RAW264.7 cells,the expression of NF-?B protein was up-regulated in RAW264.7 cells;the supernatant of TC-1 cells overexpressing Shh gene CBP protein expression was down-regulated after addition to alveolar macrophage RAW264.7 cells.When using BCG infected Shh gene TC-1 cells,GIi2 and PTCH protein expression was up-regulated;when BCG infected Shh gene overexpressing TC-1 cells,PTCH expression was inhibited.The addition of Shh interference to the supernatant of TC-1 after BCG infection increased the expression of p53,CBP,MDM2,TLR4 and inhibited the expression of NF-?B after BCG infection;Shh overexpresses TC-1 after BCG infection addition of RAW264.7 cells to the supernatant promoted the expression of MDM2,CBP and TLR4,and inhibited the expression of p53 and NF-?B.These results revealed that Shh in epithelial cells activates and regulates the expression of p53 and NF-?B signaling pathways in macrophages.PTCH in the Shh signaling pathway is positively correlated with p53,while CBP in the p53 signaling pathway is negatively correlated with NF-?B.(2)In the BCG intraperitoneal injection mouse model,there was no significant morphological change in the lungs of the mice,and inflammatory cell infiltration occurred in HE staining;the spleen became larger and congestion.The expression of p53 and NF-?B protein in mouse alveolar epithelial cells was stronger than that of the blank control group and the negative control group.Through the WB test analysis of lung tissue,we was found that Shh,p53,MDM2,CBP,TLR4,TRAF6 and NF-?B were up-regulated in the epithelial cells of mouse lung;inflammatory factors TNF-?,INF-? in serum of peripheral blood of mice IL-6 and IL-8 were significantly up-regulated.The above experimental results indicate that the three signaling pathways of Shh,p53 and NF-?B in the lungs after BCG infection are up-regulated to participate in the immune regulation of the body against BCG infection.Summary,when MTB is infected,the relevant signaling molecules of Shh,p53 and NF-?B signaling pathways are activated in cell and mouse models,and the secretion of inflammatory cytokines is regulated to counteract the immunoregulatory effect of BCG infection.For further study the immune regulation between Shh,p53 and NF-?B signaling pathways in the body of MTB infection.