黄芩汤对结肠炎相关性结肠癌肠道炎症和增殖的干预机制

Intervention Mechanism of Huangqintang on Intestinal Inflammation and Proliferation in Colitis-associated Colon Cancer

目的:研究黄芩汤对结肠炎相关性结肠癌(CAC)小鼠模型的药效,并探究黄芩汤在CAC中调节免疫功能和炎症反应,抑制细胞异常增殖,延缓或抑制CAC形成的机制。方法:C57BL/6J小鼠按体质量随机分为正常组、模型组、美沙拉嗪组、黄芩汤高、低剂量组,每组12只,除正常组外其余小鼠均给予2次腹腔注射10 mg·kg^(-1)氧化偶氮甲烷(AOM),1.5%葡聚糖硫酸钠(DSS)自由饮用7 d,正常饮水2周,“DSS-饮水”重复2个循环。在给予DSS同时,正常组和模型组小鼠给予纯水等剂量灌胃;美沙拉嗪组小鼠给予150 mg·kg^(-1)·d(-1)美沙拉嗪混悬液灌胃;黄芩汤高、低剂量组小鼠分别给予黄芩汤18、9 g·kg^(-1)·d(-1)灌胃;各组均每日给药1次,直至3个循环结束。干预结束后,测量各组小鼠体质量、结肠长度、记录小鼠结肠肿瘤数目,并进行疾病活动指数(DAI)评分。酶联免疫吸附测定法(ELISA)检测血清中白细胞介素(IL)-6、肿瘤坏死因子-α(TNF-α)、IL-1β、IL-4、IL-10及胃肠道肿瘤标志物糖类抗原-199(CA199)含量;苏木素-伊红(HE)染色观察结肠病变;免疫组化法观察结肠增殖细胞相关抗原(Ki67)表达变化;流式法检测小鼠血浆中T淋巴细胞亚群(CD3^(+)、CD4^(+)、CD8^(+)、CD49b^(+))表达;荧光标记法检测小鼠血清中异硫氰酸荧光素-D(FITC-D)含量;蛋白免疫印迹法(Western blot)检测结肠组织细胞周期相关蛋白细胞周期蛋白D1(Cyclin D1)、细胞周期蛋白依赖性激酶-2(CDK2)、细胞周期蛋白依赖性激酶4(CDK4)、紧密连接相关蛋白咬合蛋白(Occludin)、闭合蛋白-1(Claudin-1)蛋白表达。结果:与正常组比较,模型组小鼠体质量下降,DAI评分明显升高,结肠长度变短,IL-6、TNF-α、IL-1β等促炎因子均升高,其中IL-6、TNF-α升高明显(P<0.05)。抑炎因子IL-4明显降低(P<0.05),IL-10降低;肿瘤标志物CA199显著升高(P<0.01);HE染色结果显示模型组小鼠结肠出现瘤性病变,肠黏膜上皮缺损伴大量炎性浸润,隐窝破坏严重,腺体排列紊乱;Ki67阳性颗粒在结肠组织大面积表达;模型组小鼠血清CD4^(+)、CD4^(+)/CD8^(+)均明显降低(P<0.05),CD8^(+)明显升高(P<0.05);模型组小鼠血浆中FITC-D含量明显增加(P<0.05);结肠组织Cyclin D1、CDK2、CDK4蛋白表达明显升高(P<0.05,P<0.01),Occludin、Claudin-1蛋白表达均显著降低(P<0.01)。与模型组比较,美沙拉嗪组和黄芩汤高、低剂量组小鼠体质量增加,DAI评分下降,结肠变长;IL-6、TNF-α、IL-1β表达下降(P<0.05,P<0.01),IL-4、IL-10差异无统计学意义;CA199含量明显降低(P<0.05);美沙拉嗪组和黄芩汤组小鼠结肠瘤性病变和炎性浸润减轻,隐窝结构破坏较轻;Ki67阳性表达降低;CD4^(+)、CD4^(+)/CD8^(+)、CD49b^(+)均有所增加,差异无统计学意义,FITC-D含量减少(P<0.05),黄芩汤高剂量组Cyclin D1、CDK2、CDK4蛋白表达下降(P<0.05,P<0.01),Claudin-1、Occludin蛋白表达增多(P<0.05)。结论:黄芩汤对AOM/DSS诱发的炎癌转化有一定延缓和抑制作用,其作用机制可能与调节免疫功能和炎症反应,抑制促炎因子释放,修复受损的肠道屏障,抑制结肠细胞异常增殖,进而干预CAC结肠肿瘤的形成和发展有关。

Objective:To investigate the efficacy of Huangqintang on mouse models of colitisassociated colon cancer(CAC)and explore the mechanism of Huangqintang in regulating immune function and inflammatory response,inhibiting abnormal cell proliferation,and delaying or inhibiting CAC formation in CAC.Method:C57BL/6J mice were randomly divided into a normal group,model group,mesalazine group,and high-and low-dose Huangqintang groups according to body weight,with 12 mice in each group.Except for the normal group,the rest of the mice were given two intraperitoneal injections of 10 mg·kg^(-1) azomethane(AOM)and allowed to drink 1.5%dextran sodium sulfate(DSS)freely for seven days and water normally for two weeks.Then,two cycles of"DSS-drinking water"were repeated.During the administration of DSS,mice in the normal group and model group were given gavage in equal doses of pure water.Mice in the mesalazine group were given 150 mg·kg^(-1)·d^(-1) mesalamine suspension for gavage,and mice in the high-and low-dose Huangqintang groups were given 18 and 9 g·kg^(-1)·d^(-1) Huangqintang for gavage,respectively.Each group was given one dose daily until the end of three cycles.After the intervention,the body weight,colon length,and number of colon tumors in each group were measured,and disease activity index(DAI)scores were performed.The serum contents of interleukin-6(IL-6),tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),interleukin-4(IL-4),interleukin-10(IL-10),and gastrointestinal tumor marker carbohydrate antigen-199(CA199)were detected by enzyme linked immunosorbent assay(ELISA).The colonic lesions were observed by hematoxylin-eosin(HE)staining.The expression of proliferative cell-associated antigen(Ki67)was observed by immunohistochemistry.The expression of T lymphocyte subsets(CD3^(+),CD4^(+),CD8^(+),and CD49b^(+))in mouse plasma was detected by flow cytometry.Fluorescein isothiocyanate-D(FITC-D)content in mouse serum was detected by fluorescent labeling method.The Western blot method was used to detect the expression of Cyclin D1,cyclin-dependent kinase 2(CDK2),cyclin-dependent kinase 4(CDK4),and tightly junction-related Occludin and Claudin-1.Result:Compared with the normal group,the body weight of mice in the model group decreased.DAI score increased significantly,and the colon became shorter.Pro-inflammatory factors such as IL-6,TNF-α,and IL-1βincreased,and IL-6 and TNF-αwere significantly increased(P<0.05).The inflammatory factor IL-4(P<0.05)and IL-10 were significantly reduced,and the tumor marker CA199 was significantly increased(P<0.01).HE staining showed that colon lesions,intestinal mucosal epithelial defects with a large number of inflammatory infiltrates,serious crypt structure damage,and glandular arrangement disorder were observed in the model group.Ki67 positive granules were expressed in large areas of colonic tissue.The serum CD4^(+)and CD4^(+)/CD8^(+)of mice in the model group decreased significantly(P<0.05),and CD8^(+)increased significantly(P<0.05).The plasma content of FITC-D in the model group was significantly increased(P<0.05),and the expression of Cyclin D1,CDK2,and CDK4 proteins in colon tissue was significantly increased(P<0.05,P<0.01).In addition,the expression of Occludin and Claudin-1 was significantly decreased.Compared with the model group,the body weight of mice in the mesalazine group and the high-and low-dose Huangqintang groups increased.DAI score decreased,and the colon became longer.IL-6,TNF-α,and IL-1βexpression decreased(P<0.05,P<0.01),but there was no significant change in IL-4 and IL-10.The content of CA199 was significantly reduced(P<0.05),and the colomatoid lesions and inflammatory infiltrates were reduced in the mesalazine group and the Huangqintang group.The crypt structure damage was lighter,and the positive expression of Ki67 was reduced.CD4^(+),CD4^(+)/CD8^(+),and CD49b^(+)increased,and the difference was not statistically significant.FITC-D content decreased(P<0.05).The expression of Cyclin D1,CDK2,and CDK4 decreased(P<0.05,P<0.01),and Claudin-1 and Occludin protein expression increased in the high-dose Huangqintang group(P<0.05).Conclusion:Huangqintang has a certain delay and inhibitory effect on AOM/DSS-induced inflammatory cancer transformation,and its mechanism of action may be related to regulating immune function and inflammatory response,inhibiting the release of pro-inflammatory factors,repairing damaged intestinal barriers,inhibiting abnormal proliferation of colon cells,and intervening in the formation and development of CAC colon tumors.