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

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信号通路表达有关。

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 that leptin,JAK2,and STAT3 protein expression levels were significantly upregulated in the renal fibrosis of the model group.After GDFMT intervention,the fluorescence intensity decreased,with the high-dose GDFMT group showing the lowest intensity.Real-time PCR results demonstrated that leptin,OB-R,JAK2,and STAT3 mRNA expression levels were significantly elevated in the model group compared with those in the normal group,while the high-and medium-dose GDFMT groups and the penicillamine group showed significant reductions in their expression levels(P<0.05,P<0.01).Western blot analysis revealed that TGF-β_(1) and MCP-1 expression levels were significantly increased in the model group(P<0.01),and the high-and medium-dose GDFMT groups exhibited significant reductions in their expression levels(P<0.01).Conclusion:GDFMT can alleviate renal fibrosis damage in TX mice,and its mechanism of action may be related to the regulation of leptin and the JAK/STAT signaling pathway.