PAI-1引起2型糖尿病合并NAFLD小鼠肝脏炎性反应和纤维化的机制研究

目的探讨纤溶酶原激活物抑制剂-1(PAI-1)对2型糖尿病(T2DM)合并非酒精性脂肪性肝病(NAFLD)小鼠肝脏脂质沉积,炎性反应,纤维化等的影响。方法以C57BL/KSJ-Lepdb雄性小鼠作为T2DM合并NAFLD组(db/db组),同周龄C57BKS db/m雄性小鼠作为对照组(db/m组)。采用苏木素-伊红染色,油红О染色等方法检测小鼠肝组织切片脂质沉积;采用ELISA检测肝组织甘油三酯含量。以小鼠正常肝细胞NCTC1469作为研究对象,分为5组:对照组给予25 mmol/L葡萄糖(NG组)高渗对照组给予25 mmol/L葡萄糖+25 mmol/L甘露醇(HMA组)、高糖高脂组给予50mmol/L葡萄糖+0.75 mmol/L棕榈酸(HG+PA组)、高糖高脂+RNA干扰对照组(HG+PA+Si-NC组)用NC siRNA瞬时转染细胞并给予高糖高脂干预,高糖高脂+PAI-1沉默组(HG+PA+Si-PAI-1组)用PAI-1的siRNA瞬时转染细胞并给予高糖高脂干预。采用实时定量PCR和Western印迹方法检测炎性因子单核细胞趋化蛋白-1(MCP-1)、白细胞介素-1β(IL-1β),肝纤维化相关指标转化生长因子-B(TGF-3),α-平滑肌肌动蛋白(α-SMA)、Ⅰ型胶原蛋白(Col I)以及PAI-1的表达情况。结果与db/m组相比,db/db组小鼠肝组织中出现明显脂质聚集和纤维组织增生,甘油三酯含量增加,且MCP-1,IL-1β、TGF-β,a-SMA,ColⅠ和PAI-1的mRNA和蛋白水平均明显上调(均P<0.05)。与NC组细胞相比,HG+PA组MCP-1、IL-1β、TGF-β、α-SMA、Col Ⅰ、PAI-1表达量均明显上调(均P<0.05);与HG+PA组相比,HG+PA+si-PAI-1组PAI-1、MCP-1、IL-β、TGF-β、α-SMA和ColⅠ等表达量均明显下调(均P<0.05)。结论T2DM合并NAFLD小鼠模型肝组织中PAl-1表达明显增加,抑制PAI-1表达可明显改善肝组织炎性反应和纤维化水平。

Research of the effects of PAI-1 on inflammation and fibrosis in type 2 diabetic mice with NAFLD

Objective To investigate the effects of plasminogen activator inhibitor 1(PAI-1)on thehepatic lipid deposition,inflammatory reaction and fibrosis of type 2 diabetic mice with nonalcoholic fatty liv-er disease(NAFLD).Methods C57BL/KSJ lepdb mice were used as the model of T2DM combined withNAFL.D(db/db group)and c57BKS db/m mice of the same age as the control group(db/m group).Hema-toxylin eosin staining and oil red 0 staining were used to detect lipid droplets of the hepatic tissue sections ofmice.NCTC1469 normal liver cells were divided into five groups:the control group(NG)treated with 25mmol/L,glucose in the medium,the HMA group treated with 25 mmol/L glucose and 25 mmol/L mannitol,the HG+PA group treated with 50 mmol L glucose and 0.75 mmoL/L PA,HG+PA+Si-NC group treatedwith 50 mmol L glucose and 0.75 mmol/L PA after the cells transiently transfected with NC siRNA,HG+PA+Si-PAI-1 group treated with 50 mmol L.glucose and 0.75 mmol/L PA after the cells transiently trans-fected with siRNA targeting PAI-1.The expression levels of MCP-1,IL-1β,TGF-β,α-SMA,Col l andPAl-1 were detected by real-time quantitative PCR and Western blotting.Results Compared to the db/mmice,there were more accumulated lipid and fibroplasia in the hepatic tissues of db/db mice,and the mR-NA and protein levels of MCP-1,IL-1β,TGF-β,α-SMA,ColⅠand PAI-1 were increased significantly(P<0.05).Compared to the control group,the expression levels of MCP-1,IL-1β,TGF-β,α-SMA,Coll and PAI-1 were also upregulated in HG+PA group(P<0.05).Compared to HG+PA group,the ex-pression levels of PAI-1,MCP-1,IL-1β,TGF-β,α-SMA and Col l were down-regulated in HG+PA+si-PAI-1 group(P<0.05).Conclusion The expression of PAI-1 was increased in T2DM mice combinedwith NAFLD.Inhibition of PAI-1 can significantly improve the inflammation and fibrosis.