痛泻要方调控慢性应激下结肠癌NKG2DL表达促进NK细胞抗肿瘤免疫效应及机制  

Tongxie Yaofang Regulates Expression of NKG2DL to Enhance Anti-tumor Effect of NK Cells in Colon Cancer under Chronic Stress

作　　者：胡艳娥 黄渝清 杨懿 蒋义芳 付西 由凤鸣[1,2] HU Yan'e;HUANG Yuqing;YANG Yi;JIANG Yifang;FU Xi;YOU Fengming(Traditional Chinese Medicine(TCM)Regulating Metabolic Diseases Key Laboratory of Sichuan Province,Hospital of Chengdu University of TCM,Chengdu 610075,China;Cancer Institute,Chengdu University of TCM,Chengdu 610072,China)

机构地区：[1]成都中医药大学附属医院代谢性疾病中医药调控四川省重点实验室,成都610075 [2]成都中医药大学肿瘤研究所,成都610072

出　　处：《中国实验方剂学杂志》2024年第1期103-111,共9页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金面上项目(82074315)。

摘　　要：目的:探讨痛泻要方对慢性应激下肿瘤相关自然杀伤(NK)细胞功能的影响及其可能的分子机制。方法:将50只SPF级BABL/C雄性小鼠,随机分成正常组、模型组、痛泻要方低、中、高剂量组,每组10只。除空白组,其余各组进行慢性束缚应激干预7 d,进行强迫游泳及悬尾实验行为学检测来评价造模是否成功,成功后痛泻要方组分别给予痛泻要方低、中、高剂量(6.825、13.65、27.3 g·kg^(-1))灌胃,其余组予生理盐水灌胃,14 d后对每组小鼠进行结肠癌皮下瘤接种,构建慢性应激结肠癌小鼠模型。采用苏木素-伊红(HE)染色观察各组小鼠肿瘤组织病理形态变化;采用流式细胞术检测小鼠外周血及瘤体组织中CD49b阳性细胞的含量;采用酶联免疫吸附测定法(ELISA)检测小鼠外周血中NK细胞活化相关分子的含量;采用蛋白免疫印迹法(Western blot)检测小鼠瘤体组织中主要组织相容性复合物Ⅰ类多肽相关序列A和B(MICA+MICB)、UL-16结合蛋白1(ULBP1)蛋白表达。结果:与正常组比较,模型组血清中5-羟色胺(5-HT)含量降低及皮质酮(CORT)含量升高(P<0.05);与模型组比较,痛泻要方各组5-HT含量上升、CORT含量下降(P<0.05,P<0.01)。与正常组比较,模型组瘤体体积、质量增大(P<0.05);与模型组比较,痛泻要方各组瘤体体积降低,但差异无统计学意义。与正常组比较,模型组肿瘤细胞排列紧密、不规则,大小不均,异型性明显,多见核分裂,坏死面积较小,且肿瘤细胞及周围血管丰富。与模型组比较,痛泻要方各组肿瘤细胞排列稀疏,肿瘤组织内有不同程度的片状坏死区,坏死部分可见核碎裂、溶解和核碎屑。与正常组比较,模型组外周血及瘤体组织中CD49b阳性细胞数显著降低(P<0.01);与模型组比较,痛泻要方中剂量组上升(P<0.01);痛泻要方高剂量组明显上升(P<0.05,P<0.01)。与正常组比较,模型组血清中颗粒酶-B(Gzms-B)、穿孔素(PF)、γ干扰素(IFNObjective:To observe the effect of Tongxie Yaofang on the function of tumor-related natural killer(NK)cells under chronic stress and explore the possible molecular mechanism.Method:Fifty SPF-grade BABL/C male mice were randomized into normal,model,and low-,medium-,and high-dose(6.825,13.65,and 27.3 g·kg^(-1),respectively)Tongxie Yaofang groups,with 10 mice in each group.Other groups except the blank group were subjected to 7 days of chronic restraint stress,and then forced swimming and tail suspension tests were carried out to evaluate the modeling performance.After the successful modeling,rats in Tongxie Yaofang groups were administrated with low-,medium-,and high-doses of Tongxie Yaofang by gavage,while those in the other groups were administrated with normal saline by gavage.After 14 days,each group of mice was inoculated with subcutaneous colon cancer to establish the model of colon cancer under chronic stress.The pathological changes of the tumor tissue in each group of mice were observed using hematoxylin-eosin(HE)staining.The content of CD49b-positive cells in the peripheral blood and tumor tissue of mice was measured by flow cytometry.Enzyme-linked immunosorbent assay(ELISA)was employed to measure the content of molecules associated with NK cell activation in the peripheral blood.Western blot was employed to determine the protein levels of major histocompatibility complex classⅠpolypeptide-related sequences A and B(MICA+MICB)and UL-16-binding protein 1(ULBP1)in the tumor tissue.Result:Compared with the normal group,the model group showed a decrease in 5-hydroxytryptamine(5-HT)content and an increase in corticosterone(CORT)content in the serum(P<0.05).Compared with the model group,Tongxie Yaofang increased the 5-HT content and decreased the CORT content(P<0.05,P<0.01).Compared with the normal group,the modeling increased the tumor volume and weight(P<0.05),while Tongxie Yaofang inhibited such increases with no statistical significance.The tumor cells in the model group presented neat arrangement,irregul

关 键 词：痛泻要方 结肠癌 慢性应激 肿瘤相关性自然杀伤(NK)细胞 NKG2DL 

分 类 号：R2-0[医药卫生—中医学] R22R242R285.5R735.35