电针激活沉默信息调节因子1干预结直肠癌炎癌转化小鼠的机制研究

Electroacupuncture postpones growth of tumors by activating SIRT1 expression to reduce inflammatory reaction and facilitate cellular autophagy in mice with transformed colorectal carcinomas

目的:观察电针对结直肠癌炎癌转化小鼠的整体干预效应,分析电针激活沉默信息调节因子1(SIRT1)后炎性反应及自噬水平的变化,探讨电针发挥控瘤效应的作用机制。方法:C57BL/6雄性小鼠随机分为正常组、模型组、电针组、针+抑组与激动剂组,每组20只。采用诱变剂氧化偶氮甲烷和致炎剂葡聚糖硫酸钠诱导结直肠癌炎癌转化小鼠模型。电针组给予双侧"足三里"及"丰隆"电针治疗,20 min/次,每周3次,连续11周。针+抑组在电针治疗的同时采用SIRT1抑制剂EX527(5 mg/kg)腹腔注射,每周3次,连续11周。激动剂组给予SIRT1的激动剂白藜芦醇(200 mg/kg)灌胃治疗,每周3次,连续11周。每周测量1次小鼠体质量;以疾病活动指数(DAI)判定小鼠疾病状态;检测结直肠的长度及瘤体数目;HE染色法观察小鼠结直肠组织形态学改变;酶联免疫吸附法检测小鼠结直肠组织中白细胞介素(IL)-6、IL-10、IL-17含量;Western blot和荧光定量PCR法检测小鼠结直肠组织中SITR1、Beclin1、P62及LC3蛋白和mRNA的表达。结果:与正常组比较,模型组小鼠体质量明显下降(P<0.001);DAI评分明显升高(P<0.0001);结直肠长度明显缩短(P<0.001),瘤体数目明显增多(P<0.0001);结直肠组织腺体结构较紊乱,细胞形态不规则,核大且深染;促炎因子IL-6、IL-17含量明显升高(P<0.001),抑炎因子IL-10含量明显降低(P<0.001);结直肠组织中P62蛋白和mRNA的表达水平明显升高(P<0.001),SIRT1、Beclin1、LC3蛋白和mRNA表达水平明显降低(P<0.001)。与模型组比较,电针组和激动剂组小鼠体质量明显增加(P<0.01,P<0.05);DAI评分明显降低(P<0.01,P<0.05,P<0.001);结直肠长度明显增加(P<0.01),瘤体数目明显减少(P<0.001);IL-6、IL-17含量明显降低(P<0.05,P<0.01,P<0.001),IL-10含量明显升高(P<0.01,);结直肠组织中P62蛋白和mRNA的表达水平明显降低(P<0.01,P<0.001),SIRT1、Beclin1、LC3蛋白和mRNA的表达水平明显升高(P<0.01,P<0.001)。针+抑组与模型组比较,上述各指标差异均无统计学意义(P>0.05)。结论:电针一定程度上可以减少结直肠癌炎癌转化小鼠模型中瘤体数目,减轻炎性反应水平,增加小鼠体质量,降低DAI评分,其机制可能与电针激活肠道组织中SIRT1的表达,进而调控自噬有关。

Objective To observe the effect of electroacupuncture(EA)on tumor number,body conditions,inflammatory factors and expression levels of silent information regulator 1(sirtuin 1,SIRT1)and autophagy-related proteins Beclin-1,P62,and LC3 in colorectal tissues in inflammatory-transformed colorectal cancer mice,so as to explore its underlying mechanisms in resisting tumor growth.Methods A total of 100 C57 BL/6 male mice were randomly divided into normal control,model,EA,EA+SIRT1 inhibitor(EA+inhibitor)and agonist resveratrol(agonist)groups,with 20 mice in each group.EA(2 Hz,1 mA)was applied to“Zusanli”(ST36)and“Fenglong”(ST40)for 20 min every time,3 times a week for 11 weeks.Mice of the EA+inhibitor group received intraperitoneal injection of SIRT1 inhibitor EX527(5 mg/kg)at the same time of EA treatment,and those of the agonist group received gavage of resveratrol(200 mg/kg,an agonist of SIRT1),3 times a week for 11 weeks.The body mass was measured weekly.The disease activity index(DAI),colorectal length and tumor number in each group were recorded.The histopathological changes of colorectal tissues were observed by H.E.staining;the contents of interleukin 6(IL-6),IL-10,IL-17,in the colorectal tissues were detected by enzyme-linked immunosorbent assay,and the expression levels of SITR1,Beclin-1,P62,and LC3 in colorectal tissues were detected by Western blot and real-time fluorescence quantitative PCR,respectively.Results Compared with the normal control group,the body weight,length of colorectum,the contents of IL-10,and the expression levels of SIRT1,Beclin-1 and LC3 mRNAs and proteins were significantly decreased(P<0.001),whereas the DAI score,the number of tumors,the contents of IL-6 and IL-17,and the expression levels of P62 mRNA and protein were significantly increased(P<0.0001,P<0.001)in the model group.In comparison with the model group,the body weight,the length of colorectum,the contents of IL-10,and the expression levels of SIRT1,Beclin-1 and LC3 mRNAs and proteins were significantly increased(P<0.01,P<0.05,P<0.001),while the DAI scores,the numbers of tumors,the contents of IL-6 and IL-17,and the expression levels of P62 mRNA and protein were obviously decreased in the EA and agonist groups(P<0.01,P<0.05,P<0.001).No significant changes were found in all the above-mentioned indexes in the EA+inhibitor group in comparison with the model group(P>0.05).Conclusion EA can reduce the number of tumors and inflammation reaction in colorectal tissue and improve the body condition in mice with colorectal cancer,which may be related to its functions in activating the expression of intestinal SIRT1,and then facilitating cellular autophagy.