Akkermansia muciniphila在结肠炎相关结肠癌中的作用和机制

Role and mechanism of Akkermansia muciniphila incolon cancer associated with colitis

目的 探究Akkermansia muciniphila在结肠炎相关结肠癌中的作用以及Akkermansia muciniphila通过影响肠道黏膜屏障延缓结肠炎癌变的机制。方法 C57BL/C小鼠共30只,分为对照组、结肠炎相关结肠癌组、结肠炎相关结肠癌+AKK组,每组10只,在建模结束后采用断头法处死小鼠,HE染色检测结肠病理结构,计算成瘤率。采用ELISA法检测3组小鼠结肠组织IL-1β、IL-6、TNF-α水平,免疫荧光双染法检测3组小鼠ZO-1和E-cadherin的表达情况,免疫组化法检测3组小鼠结肠组织Occludin-1、 N-cadherin、GPCR41、GPCR43、Bcl-2、 Bax等蛋白的水平,GC-MS法检测3组小鼠短链脂肪酸的水平。结果 HE发现结肠炎相关结肠癌组小鼠的结肠组织炎症浸润,肠腺水肿,存在不同程度的异型增生,结肠炎相关结肠癌+AKK组小鼠的结肠水肿和出血减轻,肠腺结构清晰。结肠炎相关结肠癌组中小鼠IL-1β、IL-6、TNF-α水平(pg/mL)(9.12±0.36、 6.36±0.42、 45.20±3.63)较对照组(3.80±0.38、 0.06±0.89、 2.10±0.14)升高(Tamhane’s T2分别为0.001、2.549、12.831,均P<0.05),经Akkermansia muciniphila灌胃后,IL-1β、IL-6、TNF-α水平(3.92±0.19、3.54±1.28、18.20±8.47)下降(Tamhane’s T2分别为6.553、2.414、5.743,均P<0.05)。免疫荧光双染法显示ZO-1和E-cadherin主要定位在结肠柱状上皮和肠腺,结肠炎相关结肠癌组ZO-1和Ecadherin的表达(ng/mL)(0.04±0.01、0.34±0.55)比对照组(0.10±0.18、0.48±0.13)降低(Tamhane’s T2分别为0.681、4.379,均P<0.05),而经Akkermansia muciniphila处理后显著上调小鼠结肠的ZO-1和E-cadherin的表达(0.08±0.05、1.08±0.28)(Tamhane’s T2分别为7.059、5.873,均P<0.05)。免疫组化显示结肠炎相关结肠癌组Occludin-1、 GPCR41、 GPCR43、 Bcl-2表达(ng/mL)(0.36±0.06、 0.48±0.13、 0.38±0.13、 0.34±0.55)较对照组(1.08±0.08、 0.74±0.06、 0.48±0.13、 1.64±0.11)减低(LSD-t分别为2.369、 0.304、 8.119、 2.298,均P<0.05),Bax表达(ng/mL)(1.34±0.27)较对照组(0.48±0.13)增加(LSD-t为7.727,P<0.05),与结肠炎相关结肠癌组相比,结肠炎相关结肠癌+AKK组小鼠的Occludin-1、GPCR41、GPCR43、Bcl-2表达(0.84±0.06、0.60±0.19、 1.08±0.08、 1.08±0.28)上调(LSD-t分别为1.153、 4.111、 9.472、 5.873,均P<0.05), Bax表达(0.34±0.58)下调(LSD-t为8.785,P<0.05)。短链脂肪酸检测结果显示,结肠炎相关结肠癌组丙酸丙酯水平(μg/mg)(0.000 066±0.000 025)较对照组(0.000 244±0.000 035)下降(LSD-t为7.448, P<0.05),而经Akkermansia muciniphila干预后,结肠炎相关结肠癌+AKK组的丙酸丙酯水平(0.000 276±0.000 049)上升(LSD-t为8.779, P<0.05), 3组中戊酸丙酯、异己酸丙酯、己酸丙酯的水平差异均无统计学意义。结论Akkermansia muciniphila可降低结肠炎相关结肠癌小鼠IL-1β、IL-6、TNF-α水平,可能通过短链脂肪酸介导GPCR调控ZO-1、E-cadherin、Occludin-1等结肠紧密连接蛋白的表达,增强结肠的黏膜屏障,从而降低结肠炎相关癌变的发展进程。

Objective To explore the role of Akkermansia muciniphila(A.muciniphila)in colitis-related colon cancer(CC)and the mechanism of its effect on intestinal mucosal barrier in delaying colitis cancerosis.Methods A total of 30 C57BL/C mice were divided into blank control group,colitis associated CC group and colitis associated CC+AKK group,with 10 mice in each group.After modeling,the mice were sacrificed with decapitated method.HE was used to detect the pathological structure of colon,and the tumor formation rate was calculated.The levels of IL-1β,IL-6 and TNF-αin colon tissues in the 3 groups were detected with ELISA,and the expressions of ZO-1 and E-cadherin in the 3 groups were detected with immunofluorescence double staining.The protein levels of Occludin-1,N-cadherin,GPCR41,GPCR43,Bcl-2 and Bax in the colon tissue in the 3 groups were detected using immunohistochemistry,and the levels of short-chain fatty acids in the 3 groups were detected with GC-MS.Results HE found that colitis related CC group had inflammatory infiltration of colon tissue,intestinal glandular edema,and different degrees of dysplasia.Colitis related CC+AKK group had reduced colonic edema and bleeding,and intestinal glandular structure was clear.The contents of IL-1β,IL-6 and TNF-αin colitis associated CC group(pg/mL)(9.12±0.36,6.36±0.42,45.20±3.63,respectively)were higher than those in blank control group(3.80±0.38,0.06±0.89,2.10±0.14,respectively)(Tamhane's T2 were:0.001,2.549,12.831,all P<0.05),and those of IL-1β,IL-6 and TNF-αdecreased after inoculated with A.muciniphila(respectively:3.92±0.19,3.54±1.28,18.20±8.47)(Tamhane's T2 was 6.553,2.414,5.743,all P<0.05).Immunofluorescence double staining showed that ZO-1 and E-cadherin were mainly located in colon columnar epithelium and intestinal gland.The expressions of ZO-1 and E-cadherin in colitis related CC group(ng/mL)(0.04±0.01,0.34±0.55,respectively)were lower than those in blank control group(0.10±0.18,0.48±0.13)(Tamhane's T2 was:0.681,4.379,all P<0.05),and A.muciniphila treatment significantly up-regulated the expressions of ZO-1 and E-cadherin in the colon of mice(0.08±0.05,1.08±0.28,respectively)(Tamhane's T2 were:7.059,5.873,all P<0.05).Immunohistochemistry showed that the expression of Occludin-1,GPCR41,GPCR43 and Bcl-2 in colitis related CC group(ng/mL)(0.36±0.06,0.48±0.13,0.38±0.13,0.34±0.55,respectively)was lower than those in blank control group(1.08±0.08,0.74±0.06,0.48±0.13,1.64±0.11)(LSD-t were:2.369,0.304,8.119,2.298,P<0.05),while that of Bax(1.34±0.27)increased compared with that in blank control group(0.48±0.13)(LSD-t was:7.727,P<0.05).Compared with colitis related CC group,the expressions of Occludin-1,GPCR41,GPCR43 and Bcl-2 in colitis related CC+AKK group(ng/mL)(respectively:0.84±0.06,0.60±0.19,1.08±0.08,1.08±0.28)were up-regulated(LSD-t were:1.153,4.111,9.472,5.873,all P<0.05),while Bax expression was down-regulated(0.34±0.58,LSD-t was:8.785,P<0.05).The test results of short-chain fatty acid onate showed that the level of Propylpropionate in colitis related CC group(μg/mg)(0.000066±0.000025)was lower than that in control group(0.000244±0.000035)(LSD-t was:7.448,P<0.05),and after the intervention of A.muciniphila,the level of propionic acid(0.000276±0.000049)increased in colitis related CC+AKK group(LSD-t was:8.779,P<0.05).There was no significant differences in the level of Propylvalerate,Propylisohexanoate and Propylhexanoate among the three groups.Conclusion A.muciniphila can reduce the contents of IL-1β,IL-6 and TNF-αin colitis related CC mice,and may regulate the tight junction proteins of ZO-1,E-cadherin and Occludin-1 through GPCR mediated by short chain fatty acids,thus enhancing the mucosal barrier of colon,thereby reducing the progression of colitis-related cancers.