| Objective:1.To study whether the inhibitory effect of resveratrol on AOM/DSS induced colon cancer in mice were related to cell pyroptosis..2.To investigate whether the action of resveratrol on pyroptosis were though modulating of mi R-31/SATB2 pathway.Methods:(1)DSS induced colorectal cancer(CRC)experiment in mice: 30 male C57BL/6 mice were randomly divided into normal Control group,AOM group,AOM/DSS group,AOM/DSS+Res group and Res group.The modeling cycle was 70 days in total.Mice in AOM group,AOM/DSS group and AOM/DSS+Res group,At the first day of the first week,were intraperitoneally injected AOM once,and the ordinary chaw was replaced with high iron feed,and sterile water was given.In the second week,1% DSS water was given to AOM/DSS group and AOM/DSS+Res group,and then sterile water was changed in the third week,and this cycle continued for 5 cycles.The mice in AOM/DSS+Res and Res groups were given resveratrol by oral gavage.After modeling,(1)The mice were euthanized,and the colon tissues of the mice were preserved in liquid nitrogen for protein extraction.Part of the colon tissues were fixed,embedded in paraffin,sliced,and stained by HE and detected by immunohistochemistry.(2)The expressions of NLRP3 inflammasome,Caspase-1 and,IL-1β and IL-18 in colon tissues of mice were detected by Western blot and IHC.(3)Gene expression of NLRP3,Caspase-1,IL-1β and IL-18 in mouse colon tissue by q RT-PCR.(2)In vitro experiment:(1)HCT 116 colon cancer cells were treated with 10 μg/ mL Res,and the expressions of NLRP3,caspase-1,IL-18 and protein in HCT 116 cells were detected,to evaluate the relationship of Res on colon cancer to pyroptosis.(2)HCT 116 cells were transfected with mi R-31 : HCT 116 cells were divided into Control group,mi R-31 mimic group,mi R-31 mimic + Res group,mi R-31 inhibitor group,and mi R-31 inhibitor + Res groups.mi R-31 mimic group and mi R-31 mimic + Res group were transfected with mi R-31 mimic;mi R-31 inhibitor and mi R-31 inhibitor + Res group were transfected with mi R-31 inhibitor.Cells were given Res for 24 h,After 72 h of transfection,collected the cells and extracted the protein,Western blot detected the expression of NLRP3,Caspase-1,GSDMD-N,IL-18 and IL-1β in HCT 116 cells.(3)SATB2 knock-down experiment of HCT 116 cells.HCT 116 cells were divided into Control group,Res Group,SATB2-si RNA group,SATB2-si RNA + Res group.Western blot was used to detect the expression of NLRP3,Caspase-1 and IL-18 in HCT 116 cells,and to observe whether Res could affect the scorch death of intestinal cancer cells through mi R-31 target gene SATB2.Results:1.Mice colon tissue H&E staining,according to the results of AOM mice colonic tissue microscopically showed that the mucosa layer and submucosa layer had obvious inflammatory cell infiltration,and the mucosa layer with widespread shallow and small irregular ulcer,normal glandular structure of mice colon tissue in AOM/DSS group disappeared microscopically,showed disordered arrangement of cancer cells and the tumor tissue had vacuole structure.In the AOM/DSS+Res group,the thickness of colonic wall was normal and the infiltration of inflammatory cells decreased,and no tumor tissue was found,and the colon epithelial structure was roughly intact.2.The protein expression of NLRP3,Caspase-1,IL-1β and IL-18 in mouse colon tissues with IHC: IHC results showed that NLRP3(P<0.01),Caspase-1(P<0.01)and IL-18(P<0.01)and IL-1β(P<0.01)protein expressions in AOM/DSS group were significantly increased compared with Control group.There were no significant differences in NLRP3,Caspase-1,IL-18 and IL-1β protein expression levels between AOM group and Res group.Compared with AOM/DSS group,the protein expression levels of NLRP3(P<0.01),Caspase-1(P<0.01)and IL-18(P<0.01),IL-1β(P<0.01)in AOM/DSS+Res group were significantly decreased.The results showed that resveratrol could inhibit pyroptosis by inhibiting the expression of pyroptosis related proteins in colon tissues of mice.3.The protein expressions of NLRP3,Caspase-1 and IL-18 in mouse colon tissues with Western blot: the results showed that in AOM group,the expression of IL-18 protein was increased(P<0.05),compared with the Control group.The protein expressions of NLRP3(P<0.0001),Caspase-1(P<0.001)and IL-18(P<0.01)in AOM/DSS group were significantly increased,while the protein expressions of NLRP3,Caspase-1 and IL-18 had no significantly difference between Res group and control.The protein expression levels of NLRP3(P<0.0001),Caspase-1(P<0.0001)and IL-18(P<0.0001)in AOM/DSS+Res group were lower than those in AOM/DSS group.Res down-regulated the expression of pyroptosis associated protein in mouse colon tissue.4.The gene expression of NLRP3,Caspase-1,IL-1β and IL-18 in mouse colon cancer tissues: The expression of NLRP3,Caspase-1,IL-1β and IL-18 in colon cancer tissues of AOM group were increased compared with the Control group,In AOM/DSS group,the expression of NLRP3,IL-1β,Caspsae-1and IL-18 in colon tissues of mice was significantly increased(P<0.01).In the expression of NLRP3 and Caspase-1 between Res group and control no significantly changed.The expression level of NLRP3,IL-1β,Caspsae-1 and IL-18 in AOM/DSS + Res group was significantly lower than that in AOM/DSS group(P<0.05).These results suggest that Res can inhibit pyroptosis through down-regulate the expression of key genes of pyroptosis pathway.5.Western blot detected the expressions of NLRP3,GSDMD-N and Caspase-1 related proteins in cell pyroptosis pathway: The experimental results showed that the protein expression levels of NLRP3,Caspase-1 and GSDMD-N in the 12 h and 24 h groups showed a downward trend,but there was no statistical difference,Compared with the control(0h)group.The protein expression levels of NLRP3(P<0.01),Caspase-1(P<0.0001)and GSDMD-N(P<0.05)in 48 h group were significantly decreased.Resveratrol inhibited pyroptosis in HCT 116 cells by inhibiting the expression of pyroptotic pathway proteins.6.Resveratrol inhibited pyroptosis via down-regulating mi R-31: Western Blot results showed that the protein expression of NLRP3(P<0.01),Caspase-1(P<0.05),GSDMD-N(P<0.05),IL-18(P<0.01)and IL-1β(P<0.01)in mi R-31 mimic group was increased compared with the Control group.The protein expressions of NLRP3(P<0.05),Caspase-1(P<0.01),GSDMD-N(P<0.05),IL-18(P<0.05)and IL-1β(P<0.05)in mi R-31 inhibitor group were decreased;Compared with mi R-31 mimic+Res group,the protein expressions of NLRP3(P<0.05),Caspase-1(P<0.01),GSDMD-N(P<0.01),IL-1β(P<0.05)and IL-18(P<0.05)were decreased.Compared with mi R-31 inhibitor group,NLRP3(P<0.05),Caspase-1(P<0.01),GSDMD-N(P<0.05),IL-1β(P<0.05)and IL-18(P<0.05)were also significantly decreased in mi R-31 inhibitor+Res group.These results suggest that resveratrol can inhibit the expression of key proteins in cell pyroptosis pathway by down-regulating mi R-31 expression.7.Effects of SATB2 knockdown on the expression of NLRP3、Caspase-1 and IL-18 in HCT116 cells: Western blot results showed that compared with Control group,the expression of NLRP3,Caspase-1 and IL-18 were significantly decreased after Res treatment alone(P<0.05).The protein expressions of NLRP3,Caspase-1 and IL-18 were significantly increased after SATB2 knockdown(P<0.05).The protein expression of NLRP3,Caspase-1 and IL-18 in res-treated group after SATB2 knockdown did not change significantly.The results showed that the knockdown of SATB2 was consistent with the overexpression of mi R-31,suggesting that resveratrol inhibited pyroptosis of colorectal cancer cells by regulating taget gene SATB2.Conclusion:1.Res inhibited AOM/DSS induced colon cancer in mice by down-regulating pyroptosis pathway of colorectal cancer cells.2.The anti-pyroptosis effect of resveratrol is related to the regulation of mi R-31/SATB2 pathway. |
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