肝豆扶木汤通过JAK/STAT信号通路对TX小鼠肾纤维化的干预作用  

Intervention Effect of Gandou Fumu Decoction on Renal Fibrosis in TX Mice Through JAK/STAT Signaling Pathway

作　　者：李祥[1,2,3] 杨文明 杨悦[2,3] 郝文杰 李佩佩 胡建鹏[2,3] LI Xiang;YANG Wenming;YANG Yue;HAO Wenjie;LI Peipei;HU Jianpeng(The First Affiliated Hospital of Anhui University of Chinese Medicine,Hefei 230031,China;Anhui University of Chinese Medicine,Hefei 230038,China;Key Laboratory of Xin'an Medicine,Ministry of Education,Anhui University of Chinese Medicine,Hefei 230038,China)

机构地区：[1]安徽中医药大学第一附属医院,合肥230031 [2]安徽中医药大学,合肥230038 [3]安徽中医药大学新安医学教育部重点实验室,合肥230038

出　　处：《中国实验方剂学杂志》2023年第18期26-35,共10页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金面上项目(U22A20366,81973825);安徽高等教育自然科学基金重点项目(KJ2020A0435);安徽省中医药大学第一附属医院优秀人才培养基金项目(校发〔2019〕118号)。

摘　　要：目的:研究肝豆扶木汤(GDFMT)对Wilson病肾纤维化小鼠的保护作用及其可能作用机制。方法:将60只成年雄性毒乳鼠(TX)随机分成模型组、GDFMT高、中、低剂量组、青霉胺组,另12只作为正常组的野生小鼠共6组。GDFMT高、中、低剂量组分别予以13.92、6.96、3.48 g·kg^(-1),青霉胺组0.1 g·kg^(-1),模型组及正常组使用等量0.9%氯化钠溶液灌胃,每天1次,连续给药4周。酶联免疫吸附测定法(ELISA)检测血清中血尿素氮(BUN),肌酐(CRE),Ⅲ型前胶原(PC-Ⅲ),Ⅳ型胶原(C-Ⅳ)的含量。苏木素-伊红(HE)和马松(Masson)染色的小鼠肾的病理形态学改变。免疫荧光法测定肾细胞内瘦素(leptin)、Janus激酶2(JAK2)及信号传导及转录激活蛋白(STAT)蛋白表达情况;实时荧光定量聚合酶链式反应(Real-time PCR)测定leptin、瘦素受体(OB-R)、JAK2及STAT mRNA的情况。蛋白免疫印迹法(Western blot)检测转化生长因子-β(1 TGF-β_(1))、单核细胞趋化蛋白-1(MCP-1)表达情况。结果:与正常组比较,模型小鼠BUN、CRE、PC-Ⅲ、C-Ⅳ水平显著增加(P<0.01),与模型组比较,GDFMT高、中剂量组和青霉胺组含量明显降低(P<0.05,P<0.01),GDFMT高剂量组显著减少(P<0.01)。肾组织病理形态结果表明,模型组肾纤维组织增生,用药干预后肾组织病理损害不同程度减轻,纤维化改善。免疫荧光结果显示,模型组leptin、JAK2及STAT3蛋白肾纤维化高表达,GDFMT干预后,荧光强度降低,GDFMT高剂量组荧光强度最低。Real-time PCR结果显示,与正常组比较,模型leptin、OB-R、JAK2、STAT3 mRNA表达水平明显升高,与模型组比较,GDFMT中、高剂量组及青霉胺组表达水平明显降低(P<0.05,P<0.01)。Western blot结果显示,与正常组比较,模型组TGF-β_(1)、MCP-1表达量显著上升(P<0.01),与模型组比较,GDFMT中、高剂量组表达水平显著降低(P<0.01)。结论:GDFMT可以减轻TX小鼠肾纤维化损伤,其机制可能是通过调控leptin及JAK/STAT信号�Objective:To investigate the protective effect and underlying mechanism of Gandou Fumu decoction(GDFMT)on renal fibrosis in a mouse model of Wilson's disease.Method:Sixty adult male toxic milk(TX)mice were randomly divided into a model group,high-,medium-,and low-dose GDFMT groups,and a positive control(penicillamine)group,and another 12 wild-type mice were assigned to the normal group.The high-,medium-,and low-dose GDFMT groups were administered GDFMT at 13.92,6.96,3.48 g·kg^(-1),respectively,and the positive control group received penicillamine at 0.1 g·kg^(-1),while the model and normal groups were given an equal volume of 0.9%saline solution by gavage once a day for 4 consecutive weeks.Enzyme-linked immunosorbent assay(ELISA)was used to measure the levels of blood urea nitrogen(BUN),creatinine(CRE),typeⅢprocollagen(PC-Ⅲ),and typeⅣcollagen(C-Ⅳ)in the serum.Histological changes in the mouse kidneys were examined by hematoxylin-eosin(HE)and Masson's trichrome staining.Immunofluorescence was used to assess the protein expression of leptin,Janus kinase 2(JAK2),and signal transducer and activator of transcription(STAT)in renal cells.Real-time polymerase chain reaction(Real-time PCR)was performed to analyze the mRNA expression levels of leptin,leptin receptor(OB-R),JAK2,and STAT.Western blot was used to detect the expression of transforming growth factor-β_(1)(TGF-β_(1))and monocyte chemoattractant protein-1(MCP-1).Result:Compared with the normal group,the model mice exhibited a significant increase in BUN,CRE,PC-Ⅲ,and C-Ⅳlevels(P<0.01).Compared with the model group,the high-and medium-dose GDFMT groups and the penicillamine groups showed significant decreases in these parameters(P<0.05,P<0.01),with the high-dose GDFMT group demonstrating the most significant reduction(P<0.01).The histological examination of renal tissue revealed fibrosis in the model group,while the fibrotic damage was mitigated to varying degrees after drug intervention,with improvement in fibrosis.Immunofluorescence results showed

关 键 词：肝豆扶木汤 肝豆状核变性 毒乳鼠(TX) 肾纤维化 Janus激酶(JAK)/信号传导及转录激活蛋白(STAT) 

分 类 号：R2-0[医药卫生—中医学] R33R692R289