| Objective: Post-infectious Irritable Bowel Syndrome(PI-IBS)is a functional gastrointestinal disorder caused by immune inflammation dysregulation,depression and agitation,brain-gut axis disorder,and intestinal flora dysbiosis.With the increasing social pressure,the incidence of this gastrointestinal-related disease is increasing,the exact cause is unknown,and the current medical treatment cannot cure this disease.Through the analysis of existing studies,polymerase I and transcript release factor(PTRF)is the main protein in the formation of cell membrane and organelle membrane,and studies have shown that PTRF has an important role in regulating colorectal carcinogenesis,but the role and relationship of PTRF in PI-IBS is not clear.relationship between PTRF and PI-IBS is unclear.The aim of this experiment was to investigate the role of PTRF in PI-IBS and discuss its potential mechanism of action.Methods: This study was specifically divided into two parts: in vivo and in vitro.In vitro experiments were performed using Gram-negative lipopolysaccharide(LPS)to induce human colonic epithelial cells(HCo Epi Cs)to establish a cellular inflammatory injury model,and the cell proliferation rate was detected by MTT method.In order to investigate the effect of LPS on HCo Epi Cs cells,the protein expression of PTRF and i NOS in 0,8,12,24,48 and 72 h cells and the expression of ERK1/2,JNK1/2 and P38 of TLR4 downstream pathway in 0,30,60 and 90 min cell lines were detected by protein blotting(Western blot,WB).To investigate the role of PTRF in HCo Epi Cs cells,a cell transfection model was established and cell transfection efficiency was detected using cellular immunofluorescence assay(Immunohistochemistry,IHC)with si RNA control and si RNA-PTRF groups,respectively.The protein expression of PTRF and inducible Nitric Oxide Synthase(i NOS)and Toll-like receptor 4(TLR4)in normal and LPS-induced cells were detected by WB method in the si RNA control and si RNA-PTRF groups,respectively.Downstream extracellular regulated protein kinases1/2(ERK1/2),c-Jun N-terminal kinase1/2(JNK1/2)and P38 were expressed.The content of nitric oxide(NO)in the supernatant of cells in the above groups was measured by the Griess reagent method(Griess).The levels of interleukin-6(IL-6)and interleukin-4(IL-4)in cell supernatants were measured by Enzyme-Linked Immunosorbent Assay(ELISA).In vivo experiments of PI-IBS rat model were performed using Doe-litter separation(DS),3-Nitrobenzenesulfonic Acid Hydrate(TNBS)induction,chronic unpredictable After stimulation with TNBS,chronic unpredictable stress(CUMS)was induced by adeno-associated virus 9(AAV9),and the adeno-associated virus 9-PTRF(AAV9-PTRF)knockdown group was constructed to investigate the effects of PTRF in PI-IBS rats.The role and potential mechanism of PTRF in PI-IBS rats were investigated by observing the fecal water content,open field test,sugar-water preference,and AWR to respond to the physical signs of PI-IBS rats.PTRF protein levels in colonic epithelial cells were detected by IHC,i NOS,ERK1/2,JNK1/2,P38 protein expression levels were detected by WB assay,colonic epithelial cells were stained using hematoxylin-eosin staining(HE)and Masson staining reaction intestinal inflammation levels,double-labeled immunofluorescence staining to detect the co-localization and expression levels of PTRF and TLR4 proteins,and Elisa assay to detect the levels of cytokines such as IL-1β,IL-6and tumor necrosis factor-α(TNF-α)in colon homogenates to investigate the development of PTRF in PI-IBS.The role of PTRF in the development of PI-IBS was investigated.Results :In 5 μg/ml LPS-induced HCo Epi Cs cells,the expression of PTRF and i NOS proteins was significantly higher than that of normal cells(P<0.05),reaching a peak at 12 h,followed by a significant decrease in the expression of PTRF and i NOS(P<0.05).NO content in cell supernatant increased significantly(P<0.01),peaked secretion after 12 h,and then gradually decreased(P<0.01).In the study of HCo Epi Cs cell inflammation induced by LPS,the phosphorylation levels of ERK1/2,JNK1/2 and P38 proteins increased significantly(P<0.05)after 30 min of induction,indicating that LPS could lead to inflammatory damage in HCo Epi Cs cells,and the damage was most serious when induced for 60 minutes.In the cell transfection model,the sh RNA-mediated PTRF knockdown rate was above 80%,and the expression of PTRF and i NOS histones in si RNA-PTRF was significantly reduced by WB analysis(P<0.05),while the expression of PTRF and i NOS proteins in the si RNA-PTRF group + LPS induction and si RNA-negative control group + LPS induction was significantly increased in PTRF and i NOS proteins(P <0.05),indicating that successful knockdown of PTRF and LPS can activate the TLR4 downstream pathways ERK1/2,JNK1/2,and P38 proteins.In vivo experiments,it was found that the rats in the model group had dull coat color,mental malaise,and poor perianal hair cleanliness compared with the normal group,and the rats had a significantly lower body weight,preference for sugar water,total distance of exercise in the central area of the open field and open field,AWR score,rat grip was significantly lower than that of the normal group(P<0.05),and the water content of feces was significantly higher than that of the normal group(P<0.05).It is worth noting that with the deepening of the modeling process of PI-IBS rats,the expression of PTRF protein increased significantly(P<0.05),and compared with the 15 th day of modeling,the expression of PTRF protein on the 50 th and 78 th days of modeling was significantly increased(P<0.05),indicating that the PTRF signaling pathway could be activated by mother-child separation in the neonatal stage,TNBS and CUMS stimulation.In the knockdown study of PTRF pathway in vivo,immunohistochemistry showed that the amount of PTRF protein in the AAV9-PTRF group was significantly reduced compared with the normal group(P<0.05)in the normal group in the normal group,indicating that the knockdown rate of PTRF in vivo was higher,and the detection of colonic epithelial cells by histoimmunodouble stain found that PTRF and TLR4 had a strong colocalization effect,and the knockdown of PTRF significantly reduced the expression of TLR4 protein(P<0.05),indicating that the protein expression of TLR4 was regulated by PTRF.Further study of the relationship between PTRF and i NOS in PI-IBS rats by WB method,it was found that the protein expression of PTRF and i NOS in the AAV9-negative control group was significantly higher than that of the normal group(P<0.05),and the expression of PTRF and i NOS protein in the AAV9-PTRF group was significantly reduced(P<0.05),indicating that the expression of i NOS in vivo is regulated by PTRF,and inflammation activates the PI-IBS rat PTRF and i NOS pathways.Through the quantitative analysis of ERK1/2,JNK1/2 and P38 proteins,the expression of the above proteins in the AAV9-PTRF group was significantly lower than that in the AAV9-negative control group(P<0.05),indicating that PTRF also has a certain regulatory effect on the TLR4 downstream pathways(including ERK1/2,JNK1/2,P38).The results of pathological HE staining and Masson staining showed that inflammatory cell infiltration,tissue edema,cell atrophy were clearly visible in the colonic mucosa of rats in the model group,and slight colonic epithelial cell damage appeared in the AAV9-PTRF group,indicating that PTRF had a promoting effect on intestinal inflammation in PI-IBS rats,and the factors such as IL-6,IL-1β,TNF-α in the serum of rats in the AAV9-PTRF group were also significantly lower than those in the AAV9-negative control group(P<0.05).Conclusion:In vitro experimental results showed that PTRF enhanced LPS-induced HCo Epi Cs cell inflammatory damage through MAPK/i NOS signaling pathway,and inhibition of PTRF could alleviate the symptoms of PI-IBS rats and inhibit the protein expression of TLR4,which provided new ideas and insights for elucidating the role of PTRF in gastrointestinal diseases and the treatment of PI-IBS. |
PDF Full Text Request