| Objective: To investigate the effect and mechanism of the aldose reductase(AR)inhibitor Epalrestat(EPS)on ulcerative colitis(UC).Methods: Sixty SPF-grade SD rats were arbitrarily divided: control group,model group,EPS(5,10,20mg/kg)three dose groups,and mesalazine(MES)(200mg/kg)dose group,each group n=10.UC rat models were prepared by feeding 3% dextran sodium sulfate(DSS)for one week,followed by a 2% acetic acid rectal perfusion and free drinking water for one week.After the mold is completed,continuous gavage administration is given for two weeks.The disease activity index(DAI)and colon mucosal damage index(CMDI)were performed on 1 day before administration and on days 7 and 14 after administration.The ELISA method measured plasma levels of interleukin-1β(IL-1β),interleukin-6(IL-6),and NOD-like receptor thermal protein domain-associated protein 3(NLRP3).HE staining observed the degree of histopathological damage of the colon and performed tissue damage index(TDI)score.Immunohistochemistry measured colon tissue mucin 2(MUC2)protein expression levels.Culture colonic epithelial cells in vitro.In cell experiments,there were six groups: Control group,EPS(100μmol/L)alone group,lipopolysaccharide(LPS,20μg/m L)group,and EPS(1,10 and 100μmol/L),and the cells were first treated with LPS for 1h and then with EPS for 48 h.The protein expression of AR,hypoxia-inducible factor 1α(HIF-1α),Bax and Bcl-2,phosphorylation level and nuclear transfer of NF-κB p65 protein were detected by immunofluorescence.Hoechst staining and flow cytometry to observe colonic epithelial cell apoptosis.RT-q PCR detects the expression of AR,HIF-1α,IL-1β,IL-6,NLRP3,Caspase-3,Bax,Bcl-2 m RNA in colonic tissue and colonic epithelial cells.Western blots measured the expression levels of colonic tissue and colonic epithelial cells such as AR,HIF-1α,IL-1β,IL-6,NLRP3,cleaved-caspase-3,Bax,Bcl-2,p-IκBα,IκBα,p-NF-κB p65,NF-κB p65 protein and cytoplasmic NF-κB p65 protein.Results: In vivo,(1)Compared with the Control group before administration,the weight of the Model group decreased significantly(P<0.01);On the 7th day of administration,the weight of the EPS(10 and 20 mg/kg)group and the MES group increased significantly compared with the Model group(P<0.05).On day 14 of administration,rats in the EPS(5,10and 20 mg/kg)group and MES group gained further body weight compared with the Model group.(2)Compared with the Control group before administration,the DAI value of the Model group was significantly increased(P<0.01);On the 7th day of administration,the DAI value of the EPS 20mg/kg group and the MES group decreased significantly(P<0.05 or P<0.01)compared with the Model group.On day 14 of dosing,DAI values were further significantly reduced(P<0.05 or P<0.01)in the EPS(5,10 and 20 mg/kg)group and MES group compared with the Model group.(3)Compared with the Control group,the colonic mucosal injury in the Model group was serious,the CMDI score value was significantly increased(P<0.01),the colonic mucosal injury in the EPS(5,10,20mg/kg)dose group and the MES group was reduced to varying degrees,and the CMDI score value was significantly reduced(P<0.05 or P<0.01).(4)Compared with the Control group,the pathological damage was severe and the TDI was significantly increased in the Model group(P<0.01),and compared with the Model group,the pathological damage in the EPS(5,10 and 20mg/kg)group and the MES group was reduced to varying degrees,and the TDI was significantly reduced(P<0.01).(5)The immunohistochemical results showed that compared with the Control group,the positive expression of MUC2 protein in rat colon tissue epithelial cells in the Model group was significantly reduced;Compared with the Model group,the positive expression of MUC2 protein in epithelial cells of colonic tissue was increased to varying degrees in the EPS(5,10 and 20mg/kg)group and the MES group.(6)The ELISA results showed that compared with the Control group,the contents of IL-1β,IL-6 and NLRP3 in the Model group were significantly increased(P<0.01);Compared with the Model group,the contents of IL-1β,IL-6 and NLRP3 in the EPS(5,10,20mg/kg)group and MES group were significantly reduced(P<0.05 or P<0.01).(7)RT-q PCR results showed that compared with the Control group,the m RNA expression levels of AR,HIF-1α,IL-1β,IL-6,NLRP3,Caspase-3 and Bax in the Model group were significantly up-regulated in colon tissue(P<0.01),while the m RNA expression of Bcl-2 was significantly down-regulated(P<0.01);Compared with the Model group,the expression levels of AR,HIF-1α,IL-1β,IL-6,NLRP3,Caspase-3 and Bax in the EPS(5,10 and 20mg/kg)group and the MES group were down-regulated to varying degrees,while the expression levels of Bcl-2 m RNA were significantly up-regulated(P<0.05 or P<0.01).(8)Western blots results showed that compared with the Control group,the protein expression levels of AR,HIF-1α,IL-1β,IL-6,NLRP3,cleaved-caspase-3 and Bax in the Model group increased significantly,while the protein expression level of Bcl-2 proteins decreased significantly,and the phosphorylation levels of IκBα and NF-κB p65 and NF-κB p65 nuclear transfer increased significantly(P<0.01);Compared with the Model group,the protein expression levels of AR,HIF-1α,IL-1β,IL-6,NLRP3,cleaved-caspase-3 and Bax proteins in the EPS(5,10 protein 20mg/kg)group and MES group were significantly reduced,while the protein expression level of Bcl-2 was significantly increased,the phosphorylation levels of IκBα and NF-κB p65 and the degree of NF-κB p65 nuclear transfer were significantly reduced(P<0.05 or P<0.01).In vitro,(1)RT-q PCR and Western blots detected that compared with the Control group,there was no significant difference in the expression levels of IL-1β,IL-6 and NLRP3 m RNA and protein in the EPS alone dose group,while the expression levels of IL-1β,IL-6 and NLRP3 m RNA and protein in the LPS group were significantly increased.Compared with the LPS group,the expression levels of IL-1β,IL-6,NLRP3 m RNA and protein in the EPS(1,10 and 100 μmol/L)group were reduced to varying degrees(P<0.05 or P<0.01).(2)The results of Hoechst staining and flow cytometry showed that compared with the Control group,there was no significant apoptosis difference in the cells in the EPS dose group alone,and the cells in the LPS group were significantly apoptosis;Compared with the LPS group,apoptosis in the EPS(1,10,100μmol/L)dose group was reduced to varying degrees.RT-q PCR,Western blots and immunofluorescence detection showed that there was no significant difference in the expression levels of Caspase-3,cleaved-caspase-3,Bax and Bcl-2 m RNA and/or protein in the EPS alone dose group compared with the Control group,and Caspase-3,cleaved-caspase-3and Bax in the LPS group m RNA and/or protein expression levels were significantly increased,while Bcl-2 m RNA and protein expression levels were significantly decreased(P<0.01).Compared with the LPS group,Caspase-3,cleaved-caspase-3 and Bax m RNA and/or protein expression levels were significantly reduced in the EPS(1,10 and 100 μmol/L)group,while Bcl-2 m RNA and protein expression levels were significantly increased(P<0.05 or P<0.01).(3)RT-q PCR,Western blots and immunofluorescence detection showed that compared with the Control group,there were no significant changes in the expression levels of AR and HIF-1α,IκBα,NF-κB p65 phosphorylation and NF-κB p65 nuclear transfer in the EPS alone group,while the expression levels of AR,HIF-1α m RNA and protein in the LPS group were significantly increased,and the phosphorylation levels of IκBα and NF-κB p65 and NF-κB p65 nuclear transfer were significantly increased(P<0.01).Compared with the LPS group,the expression levels of AR,HIF-1α m RNA and protein in the EPS(1,10 and 100μmol/L)group were significantly reduced,and the phosphorylation levels of IκBα and NF-κB p65 and the degree of NF-κB p65 nuclear transfer were significantly reduced(P<0.05 or P<0.01).Conclusion: The AR inhibitor Epalrestat has a certain relieving effect on ulcerative colitis through anti-inflammatory and anti-apoptotic effects.The mechanism may be related to its inhibition of AR-mediated expression of HIF-1α,thereby inhibiting the expression of NF-κB-induced inflammatory factors IL-6,NLRP3 and IL-1β,upregulating Bcl-2 expression,down-regulating the expression of Bax,cleaved-caspase-3 and caspase-3. |
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