华支睾吸虫来源的rCsHscB对小鼠炎症性结肠癌的治疗效果研究

The therapeutic effect of rCsHscB sourced from Clonorchis sinensis on colitis-related colorectal cancer in mice

目的研究华支睾吸虫来源的分子伴侣(molecular chaperones HscB,CsHscB)对氧化偶氮甲烷(azoxymethane,AOM)联合葡聚糖硫酸钠(dextran sodium sulfate,DSS)诱导的小鼠炎症性结肠癌的治疗效果及潜在的分子机制。方法C57BL/6小鼠随机分为正常对照组(NC,n=10),rCsHscB组(n=10),模型组(AOM+DSS,n=15),干预组(AOM+DSS+rCsHscB,n=15)。模型组和干预组按8 mg/kg体重的剂量经腹腔注射AOM,1次/d,连续7 d。然后给予7 d 2%DSS饮水和14 d正常饮水,共4个循环。在给予DSS饮水的第4 d、7 d,rCsHscB组和干预组小鼠按照1.25 mg/kg体重的剂量经腹腔注射rCsHscB蛋白。记录小鼠体重和生存状况,第84 d剖杀小鼠,测量结肠长度,记录结肠肿瘤个数,HE和Masson染色观察结肠病变,ELISA检测结肠组织中IL-6、MCP-1和IL-10含量,Western blot检测结肠组织中α-SMA、MMP2、Occludin表达水平和ERK1/2、JNK、P38、AKT、STAT3的磷酸化及非磷酸化水平。结果与正常对照组相比,模型组71.4%的小鼠结肠中出现肿瘤,小鼠体重和结肠长度显著降低缩短(均P<0.05),结肠组织中有大量炎症细胞浸润和纤维化形成。与模型组相比,干预组小鼠的生存率显著升高(P<0.01),体重、结肠长度和炎症纤维化均有所改善,仅有10%小鼠出现结肠肿瘤(P<0.01)。与模型组相比,干预组小鼠结肠组织中IL-6和MCP-1显著降低(均P<0.05),IL-10显著升高(P<0.0501),α-SMA和MMP2蛋白表达水平降低(均P<0.05),Occludin蛋白表达水平升高(P<0.05),ERK1/2、JNK、P38、AKT和STAT3蛋白的磷酸化和非磷酸化水平均显著降低(均P<0.05)。结论rCsHscB可能通过抑制MAPK和AKT/STAT3信号通路的活化改善AOM/DSS诱导的小鼠炎症性结肠癌。

Objective To study the effect and possible mechanism of Molecular chaperones HscB derived from Clonorchis sinensis(CsHscB)on colitis-related colorectal cancer induced by Azoxymethane(AOM)and Dextran sodium sulfate(DSS).Methods Mice with C57BL/6 background were randomly divided into normal control group(NC group,n=10),rCsHscB group(n=10),model group(AOM+DSS group,n=15),and rCsHscB intervention group(AOM+DSS+rCsHscB group,n=15).The mice in AOM+DSS group and AOM+DSS+rCsHscB group were given 8 mg/kg body weight AOM intraperitoneally once a day for 7 days.Then,mice were given drinking water with 2%DSS for 7 days,and normal drinking water for 14 weeks,which repeated four times.The mice in rCsHscB group and AOM+DSS+rCsHscB group were intraperitoneally injected with 1.25mg/kg body weight rCsHscB protein after four and seven days for 2%DSS drinking water.Body weight and the death of mice in each group were recorded.All mice were killed after 84 days for 2%DSS drinking water,and the length of colon,number of tumors were recorded.HE and Masson staining were used to observe the histopathology of colon.The levels of IL-6,MCP-1 and IL-10 in colon were detected by ELISA.Western Blot were conducted to detect the expression levels ofα-SMA,MMP2,Occludin,ERK1/2,p-ERK1/2,JNK,p-JNK,P38,p-P38,AKT,p-AKT,STAT3 and p-STAT3 in colon.Results Compared with Normal group,the occurrence of colorectal cancer was 71.4%.The body weight and the length of colon in AOM+DSS group decreased significantly(F=14.136,P0.05;F=60.875,P0.01)and inflammatory cells infiltration and fibrosis were more serious in colon.However,compared with the AOM+DSS group,the survival rate in AOM+DSS+rCsHscB group increased significantly(χ~2=6.652,P0.01);the body weight loss(F=14.136,P0.01),colon shortening(F=60.875,P0.01),inflammation and fibrosis in colon were all improved;the number of colon tumors reduced significantly(t=3.229,P0.01),only 10%of mice developed colon tumor.In addition,IL-6 and MCP-1 in colon also decreased significantly(F=35.351,P0.05;F=21.118,P0.01),while IL-10 increased in this group(F=27.351,P0.01).Western Blot analysis showed that the protein levels ofα-SMA and MMP2 decreased(F=29.361,P0.01;F=83.001,P0.01),but the Occludin increased(F=29.023,P0.05)in AOM+DSS+rCsHscB group.Moreover,the levels of p-ERK1/2/ERK1/2(F=11.565,P0.01),p-JNK/JNK(F=19.954,P0.01),p-P38/P38(F=14.725,P0.01),p-AKT/AKT(F=44.02,P0.01),p-STAT3/STAT3(F=15.411,P0.05)were significantly lower than those in the AOM+DSS group.Conclusion rCsHscB alleviates AOM/DSS-induced colitis-related colorectal cancer via inhibiting the activation of MAPK and AKT/STAT3 signaling pathways.