清肝健脾活血方通过调控M1/M2型巨噬细胞治疗肝纤维化大鼠的作用机制分析  

Analysis of Mechanism of Qinggan Jianpi Huoxue Prescription in Treatment ofHepatic Fibrosis Rats by Regulating M1/M2 Macrophages

作　　者：潘富珍 曹洪欣[3] 张永生[1] 吴夏秋[1] 郑卫方 刘定[4] PAN Fuzhen;CAO Hongxin;ZHANG Yongsheng;WU Xiaqiu;ZHENG Weifang;LIU Ding(School of Public Health,School of Basic Medical Sciences,Zhejiang Chinese Medical University,Hangzhou 310053,China;Lanxi Hospital of Traditional Chinese Medicine,Jinhua 321100,China;China Association of Chinese Medicine,Beijing 100029,China;Heilongjiang University of Chinese Medicine,Harbin 150040,China)

机构地区：[1]浙江中医药大学公共卫生学院,基础医学院,杭州310053 [2]兰溪市中医院,浙江金华321100 [3]中华中医药学会,北京100029 [4]黑龙江中医药大学,哈尔滨150040

出　　处：《中国实验方剂学杂志》2023年第21期94-102,共9页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：黑龙江省自然科学基金项目(LH2019H109);浙江省基础公益研究计划项目(LZ22H270001)。

摘　　要：目的:基于四氯化碳(CCl_(4))大鼠肝纤维化模型,探究清肝健脾活血方对肝纤维化大鼠M1/M2型巨噬细胞极化的影响。方法:按剂量2.0 mL·kg^(-1)给大鼠腹腔注射40%CCl_(4)-橄榄油混悬液,每周2次,连续注射8周,建立大鼠肝纤维化模型,造模成功后随机分为模型组、清肝健脾活血方组(32.084 g·kg^(-1))、鳖甲煎丸组(0.9255 g·kg^(-1)),每组12只。空白组腹腔注射等量橄榄油。给药组按剂量灌胃给予相应药液,空白组和模型组给予相同剂量纯净水,1次/d,连续给药4周后取大鼠肝组织进行苏木素-伊红(HE)、马松(Masson)染色观察病理改变,并检测各组血清丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)水平,采用酶联免疫吸附测定法(ELISA)测定肝组织白细胞介素(IL)-6、IL-12、IL-10、IL-1β、转化生长因子-β_(1)(TGF-β_(1))和肿瘤坏死因子-α(TNF-α)水平,免疫组化法(IHC)检测巨噬细胞CD86、CD206蛋白表达,蛋白免疫印迹法(Western blot)、实时荧光定量聚合酶链式反应(Real-time PCR)检测肝组织诱导型一氧化氮合酶(iNOS)、精氨酸酶1(Arg-1)、磷酸化p38丝裂原活化蛋白激酶(p-p38 MAPK)、核转录因子-κB p65(NF-κB p65)蛋白及mRNA表达水平。结果:与空白组比较,模型组肝细胞板排列不规整,局部可见炎症细胞浸润、纤维增生,血清ALT、AST水平显著升高(P<0.01),肝组织炎症因子IL-1β、IL-6、IL-12、TGF-β_(1)、TNF-α水平,CD86、CD206蛋白表达水平,iNOS、NF-κB p65蛋白及mRNA,p-p38 MAPK蛋白、p38 MAPK mRNA水平均明显升高(P<0.05,P<0.01),炎症因子IL-10,Arg-1蛋白及mRNA水平明显降低(P<0.05,P<0.01);与模型组比较,清肝健脾活血方组肝细胞纤维化程度减轻,血清ALT、AST水平显著降低(P<0.01)、肝组织炎症因子IL-1β、IL-6、IL-12、TGF-β_(1)、TNF-α水平,CD86表达水平,iNOS、NF-κB p65蛋白及mRNA水平,p-p38 MAPK蛋白及p38 MAPK mRNA水平明显降低(P<0.05,P<0.01),炎症因子IL-10,CD206表达水平Objective:To observe the effect of Qinggan Jianpi Huoxue prescription(QGJPHXP)on the polarization of M1/M2 macrophages in rats with hepatic fibrosis induced by carbon tetrachloride(CCl_(4)).Method:A rat hepatic fibrosis model was established by intraperitoneal injection of 40%CCl_(4)-olive oil suspension twice a week at the dosage of 2.0 mL·kg^(-1) for 8 weeks.After the model was successfully established,these rats were randomly divided into the model group,QGJPHXP group(32.084 g·kg^(-1))and Biejiajian pills(BJJP)group(0.9255 g·kg^(-1)),with 12 rats in each group.The blank group was injected intraperitoneally with the same amount of olive oil.The rats in the administration groups were given the corresponding solution according to the dose,and the blank and model groups were given the same dose of purified water,once a day.After 4 weeks of continuous administration,the liver tissues of rats were taken and stained with hematoxylineosin(HE)and Masson to observe the pathological changes.The serums were collected to detect the alanine aminotransferase(ALT)and aspartate aminotransferase(AST)levels.Interleukin(IL)-6,IL-12,IL-10,IL-1β,transforming growth factor-β_(1)(TGF-β_(1))and tumor necrosis factor-α(TNF-α)levels in liver tissues were measured by enzyme-linked immunosorbent assay(ELISA).The expression levels of CD86 and CD206 were detected by immunohistochemistry(IHC).Western blot and real-time fluorescence quantitative polymerase chain reaction(Real-time PCR)were used to detect the protein and mRNA expression levels of inducible nitric oxide synthase(iNOS),arginase-1(Arg-1),phosphorylated p38 mitogen-activated protein kinase(p-p38 MAPK),nuclear transcription factor-κB p65(NF-κB p65)in liver tissues of rats.Result:Compared with the blank group,the hepatic cell plate was irregularly arranged,and local inflammatory cell infiltration and fibrous hyperplasia were observed,while the serum levels of ALT and AST were significantly increased in the model group(P<0.01),and IL-1β,IL-6,IL-12,TGF-β_(1),TNF-α,CD86,CD

关 键 词：清肝健脾活血方 免疫调控 巨噬细胞极化 肝纤维化 炎症因子 鳖甲煎丸 病理变化 

分 类 号：R22[医药卫生—中医基础理论] R28[医药卫生—中医学] R575R96