摘要
目的观察温胆汤对肥胖痰湿证大鼠肾周脂肪组织肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-6、IL-1β、单核细胞趋化蛋白-1(monocyte chemotactic protein 1,MCP-1)等相关炎症细胞因子,对自噬活性标志物B细胞淋巴瘤2结合蛋白-1(Beclin-1)、微管相关蛋白轻链(LC3) mRNA与蛋白水平表达,以及对脂肪细胞超微结构与自噬状态变化的影响,探讨温胆汤干预肥胖痰湿证炎症状态的作用机制。方法选用清洁级60 g左右雄性SD大鼠100只,随机分成造模组(70只)和空白组(30只),空白组采用普通饲料喂养,造模组采用高脂饲料喂养,持续喂养6周,借助文献方法制作肥胖痰湿证大鼠模型。造模成功后,按照体质量依序淘汰过轻者,共遴选出16只肥胖大鼠,随机分为模型组、温胆汤组(为本课题组前期实验已确定的最佳剂量组),每组8只;另在空白组中随机选取8只大鼠设为正常组。药物组以15 g·kg^(-1)生药量进行灌胃,模型组和正常组均予等量蒸馏水进行灌胃,1次/d,共6周。禁食不禁水12 h,麻醉后进行样本采集,检测并计算大鼠体质量、Lee’s指数和肥胖率,按试剂盒说明书采用全自动生化分析仪检测总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白(LDL-C)和高密度脂蛋白(HDL-C)水平;采用酶联免疫吸附测定(ELISA)技术检测SD大鼠肾周脂肪组织炎症细胞因子TNF-α、IL-6、IL-1β、MCP-1的表达;采用实时荧光定量聚合酶链式反应(Real-time PCR)法检测肾周脂肪组织Beclin-1、LC3 mRNA表达;采用蛋白免疫印迹法(Western Blot)检测肾周脂肪组织Beclin-1、LC3蛋白表达;采用透射电镜观测腹周脂肪组织脂肪细胞超微结构与自噬体的改变。结果高脂饲料喂养可成功复制肥胖痰湿证模型,造模组大鼠肥胖率大于20%,与空白组比较,模型组体质量、Lee’s指数呈显著升高(P<0.01,P<0.05)。与正常组比较,模型组大鼠体质量显著升高(P<0.01),血脂水平呈显著改变(P<0.01),肾周脂肪组织炎症细胞因子TNF-α、IL-6、IL-1β、MCP-1的表达水平显著升高(P<0.01),自噬基因Beclin-1、LC3 mRNA与蛋白水平显著升高(P<0.01),与模型组比较,药物组体质量、Lee’s指数显著下降(P<0.01,P<0.05),血脂水平显著变化(P<0.01),肾周脂肪组织炎症细胞因子TNF-α、IL-6、IL-1β、MCP-1表达水平显著下降(P<0.01),自噬基因Beclin-1、LC3 mRNA和蛋白表达水平显著下降(P<0.01),透射电镜观察显示,正常组的细胞结构完整,表面突起,细胞核不规则,细胞器较丰富;模型组的细胞表面突起严重,细胞核不规则,细胞器较丰富,线粒体嵴破坏显著,出现碎解和分离现象,胞浆内有空泡,自噬体较多。药物组的细胞表面稍突起,细胞核不规则,部分线粒体嵴破坏,部分胞浆内有空泡,自噬体减少。结论温胆汤可有效改善肥胖大鼠痰湿证的炎症状态,其机理可能与调节肾周脂肪细胞自噬活性有关,这为经典治痰名方温胆汤干预肥胖痰湿证提供了实验依据。
Objective To observe the effect of Wendan Decoction(温胆汤)on tumor necrosis factor-α(TNF-α),interleukin(IL)-6 and IL-1β,monocyte chemotactic protein 1(MCP-1)and other related inflammatory cytokines in perirenal adipose tissue of obese rats with phlegm-dampness syndrome,and the effect on autophagy activity markers B-cell Lymphoma 2 binding protein-1(beclin-1)and microtubule associated protein light chain(LC3).To explore the mechanism of Wendan Decoction on the inflammatory state of obesity of phlegm-dampness syndrome.Methods A total of 100 male SD rats were randomly divided into model group(70 rats)and blank group(30 rats).The blank group was fed with normal diet,and the model group was fed with high fat diet and both groups were fed continuously for 6 weeks.And then the rat model of phlegm-dampness syndrome of obesity was established by methods in the literature.After the model was established successfully,16 obese rats were selected after the underweight eliminated according to weight and were randomly divided into model group and Wendan Decoction intervention group(hereinafter referred to as"drug group",which was the best dose group determined in the previous experiment of this research group)with 8 rats in each group.Eight rats in the blank group were randomly selected as the normal group.In the drug group,15 g·kg^(-1) of crude drug was administrated to the stomach.The model group and the normal group were treated with the same amount of distilled water by gavage,once a day totally for six weeks.Three groups fasted without water for 12 h and the samples were collected after anesthesia.The rats’body weight,Lee’s index and obesity rate were measured and the levels of TC,TG,LDL-C and HDL-C were measured by using full automatic biochemical analyzer according to the instructions of the kit.The expressions of TNF-α,IL-6,IL-1βand MCP-1 in adipose tissue of SD rats were measured by enzyme-linked immunosorbent assay(ELISA)technology.The expression of beclin-1,LC3 mRNA in perirenal adipose tissue was detected by Real time PCR method.The expressions of beclin-1 and LC3 protein in perirenal adipose tissue were detected by Western blotting method.The ultrastructural changes of adipocytes and autophagy were observed by Transmission electron microscope.Result The obese model of phlegm-dampness syndrome can be successfully reproduced by high fat feed and the body mass and Lee’s index of the model group were increased significantly(P<0.01,P<0.05)compared with those of the blank group.The body weight of rats in the model group was significantly increased(P<0.01).The level of blood lipid was significantly changed(P<0.01).The expression levels of inflammatory cytokines TNF-α,IL-6,IL-1βand MCP-1 in perirenal adipose tissue were significantly increased(P<0.01).The levels of beclin-1,LC3 mRNA and protein in autophagy gene were significantly increased(P<0.01)compared with those of the normal group.The body mass and Lee’s index of the drug group were decreased significantly(P<0.01,P<0.05),and the blood lipid level changed significantly(P<0.01).The expression levels of inflammatory cytokines TNF-α,IL-6,IL-1βand MCP-1 in perirenal adipose tissue were decreased significantly(P<0.01).The expression levels of autophagy genes Beclin-1,LC3 mRNA and protein were decreased significantly(P<0.01)compared with those of the model group.It showed that the normal group had complete cell structure,protruding surface,irregular nucleus and abundant organelles by transmission electron microscope.The cell surface was severely protuberant,the nucleus was irregular,organelles were abundant,mitochondrial cristae were significantly damaged,fragmentation and separation occurred,vacuoles were found in cytoplasm,and the autophagy was more common in the model group.The cell surface was slightly protruding,the nucleus was irregular,some mitochondrial cristae were destroyed,some cytoplasm had vacuoles,and autophiles decreased in the drug group.Conclusion Wendan Decoction can effectively improve the inflammatory state of phlegm-dampness syndrome in obese rats,and the mechanism may be related to the regulation of autophagy activity of perirenal adipocytes,which provides the experimental basis for the classical phlegm-treating prescription Wendan Decoction to interfere with phlegm-dampness syndrome in obesity.
作者
喻松仁
张一文
华诗培
姚琦
王河宝
周丽
程绍民
王萍
YU Songren;ZHANG Yiwen;HUA Shipei;YAO Qi;WANG Hebao;ZHOU Li;CHENG Shaomin;WANG Ping(Jiangxi University of Traditional Chinese Medicine,Nanchang 330004,Jiangxi,China)
出处
《中华中医药学刊》
CAS
北大核心
2022年第1期14-18,I0013,共6页
Chinese Archives of Traditional Chinese Medicine
基金
国家自然科学基金(81960851)
江西省教育厅科学技术研究项目(GJJ201212)。
关键词
肥胖
痰湿证
温胆汤
炎症细胞因子
自噬
作用机制
obesity
phlegm-dampness syndrome
Wendan Decoction(温胆汤)
inflammatory cytokines
autophagy
mechanism of action
