熊果酸改善Ⅰ型糖尿病大鼠胰岛β细胞损伤的实验研究

Ursolic acid ameliorates pancreaticβ-cell injury in type I diabetic rats via the TLR4/NF-κB pathway and Th17/Treg cell homeostasis

目的探究熊果酸(UA)对Ⅰ型糖尿病(T1DM)大鼠TLR4/NF-κB信号通路及Th17/Treg细胞的影响。方法腹腔注射链脲佐菌素(STZ)制备T1DM大鼠模型,随机分为空白组(Control组)、模型组(Model组)、二甲双胍组(MET组)和熊果酸组(UA组)。记录大鼠体重、血糖等一般情况,灌胃6周后采集大鼠外周血、胰腺组织评估胰岛素干预情况。免疫组化观察胰腺组织病理变化;鲎试剂检测血清脂多糖(LPS)含量变化;qRT-PCR法检测胰腺TLR4、MyD88、IκBα、NF-κB p65 mRNA的表达,以及转录因子RORγt、Foxp3 mRNA的表达;Western blot法检测胰腺TLR4、MyD88、IκBα、NF-κB p65蛋白的表达,以及转录因子RORγt、Foxp3蛋白的表达;流式细胞术检测外周血Th17、Treg细胞比例变化;ELISA法检测血清TNF-α、IL-6、IL-1β含量变化。结果经STZ诱导的糖尿病大鼠经6周灌胃处理,与模型组相比,二甲双胍组大鼠和熊果酸组大鼠空腹血糖(FBG)均明显下降,体重均明显升高;胰岛β细胞炎性浸润减少;TLR4、MyD88、IκBα、NF-κB p65、RORγt mRNA和蛋白的表达明显降低;LPS含量明显下降;IκBα、Foxp3 mRNA和蛋白表达明显升高;Th17/Treg比值明显下降;TNF-α、IL-6、IL-1β含量明显下降。结论UA可通过减少LPS移位,抑制TLR4/NF-κB通路,下调RORγt并上调Foxp3的表达纠正T1DM大鼠Th17/Treg细胞比例失衡来改善大鼠症状。

Objective To investigate the effects of ursolic acid(UA)on the TLR4/NF-κB signaling pathway and Th17/Treg cells in type 1 diabetes mellitus(T1DM)rats.Methods T1DM rat models were established by intraperitoneal injection of streptozotocin(STZ)and randomly divided into blank(Control),Model,metformin(MET),and UA groups.General conditions,such as body weight and blood glucose,were recorded,and peripheral blood and pancreatic tissues were collected after 6 weeks of gavage to assess insulin treatment.Immunohistochemistry was used to observe pathological changes in pancreatic tissues.Horseshoe crab reagent was used to assess changes in serum lipopolysaccharide(LPS)content.qRT-PCR was used to measure expression of pancreatic TLR4,MyD88,IκBα,and NF-κB p65 mRNAs,and mRNA expression of transcription factors RORγt and Foxp3.Western blot was used to assess pancreatic TLR4,MyD88,IκBα,NF-κB p65,RORγt,and Foxp3.Flow cytometry was used to assess changes Th17/Treg cell ratio in peripheral blood.ELISA were used to measure serum contents of TNF-α,IL-6,and IL-1β.Results After STZ-induced diabetic rats were treated by gavage for 6 weeks,compared with the Model group,the fasting blood glucose of rats in MET and UA groups was significantly decreased and their body weights were increased.Inflammatory infiltration of pancreatic isletβ-cells was reduced.Expression of TLR4,MyD88,IκBα,NF-κB p65,and RORγt mRNAs and proteins was significantly decreased.LPS content was significantly decreased.IκBαand Foxp3 mRNA and protein expression was significantly increased.The Th17/Treg ratio was significantly decreased,and TNF-α,IL-6,and IL-1βcontents were significantly decreased.Conclusions UA improves the symptoms of rats by reducing the LPS shift,inhibiting the TLR4/NF-κB pathway,down-regulating RORγt expression,and up-regulating Foxp3 expression to correct the imbalance in the Th17/Treg cell ratio in T1DM rats.