葛根芩连汤对KKAy糖尿病小鼠TLR4/IKKβ/NF-κB信号通路的影响

Effect of Gegen Qinliantang on TLR4/IKKβ/NF-κB Pathway in KKAy Mice with Diabetes

目的:观察经方葛根芩连汤对KKAy小鼠部分炎性因子及脂多糖介导的炎症通路中关键靶点的影响,探讨其改善2型糖尿病的作用机制。方法:选取SPF级自发性2型糖尿病动物模型KKAy小鼠65只及对照C57BL/6J小鼠13只予屏障系统、高脂饲料喂养,随机血糖≥13.9 mmoL·L^(-1),为成模小鼠44只。随机分为5组,每组11只。正常组(生理盐水20 mL·kg^(-1))、阿卡波糖组(3.9 mg·kg^(-1))、葛根芩连汤高、低剂量组(1.82、0.45 g·kg^(-1))及模型组(生理盐水20 mL·kg^(-1)),连续灌胃给药8周,1次/d,系统评价血糖及体质量。末次给药12 h后,摘眼球取血,提取血清及肌肉、肝脏组织。采用半定量酶联免疫吸附测定法(ELISA)检测炎性因子肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)及葡萄糖转运子4(GluT4)含量,蛋白免疫印迹法(Western blot)检测肌肉组织IκB磷酸化激酶β(IKKβ)蛋白、核转录因子-κB(NF-κB)及肝组织Toll样受体4(TLR4)蛋白表达水平。结果:与正常组比较,模型组体质量及血糖显著升高(P<0.01);与模型组比较,阿卡波糖组及葛根芩连汤组能够明显降低体质量及血糖水平(P<0.05,P<0.01)。ELISA结果显示,与正常组比较,模型组炎性因子TNF-α、IL-6含量显著升高(P<0.01),GluT4含量明显下降(P<0.05);与模型组比较,各给药组炎性因子TNF-α、IL-6含量均有不同程度下降(P<0.05,P<0.01),阿卡波糖组与葛根芩连汤高剂量组GluT4含量明显升高(P<0.05,P<0.01)。Western blot分析显示,与正常组比较,模型组IKKβ、NF-κB及TLR4蛋白表达均有升高(P<0.01);与模型组比较,阿卡波糖组与葛根芩连汤组IKKβ、NF-κB及TLR4蛋白表达均明显下降(P<0.05,P<0.01)。结论:葛根芩连汤可能在调节肠道菌群的基础上,通过下调TLR4通路中关键炎性因子的水平,抑制其激活,提高GluT4的转位及活性水平,从而在一定程度上抑制炎性级联效应,改善2型糖尿病。

Objective: To observe the effect of classical prescription Gegen Qinliantang(GGQLT)on inflammatory factors and key targets in the inflammatory pathways mediated by lipopolysaccharide in KKAy mice and explore its mechanism in improving spontaneous type 2 diabetes mellitus(T2DM). Method: Sixtyfive SPF KKAy mice with spontaneous T2DM and 13 C57 BL/6 J mice(control)were selected in the barrier system and fed on a high-fat diet. The model was properly induced in 44 mice in the context of random blood glucose exceeding or equal to 13.9 mmol·L^(-1). Then the mice were assigned into a normal group(20 mL·kg^(-1) normal saline),a model group(20 mL·kg^(-1) normal saline),an acarbose group(3.9 mg·kg^(-1)),and high-and low-dose GGQLT groups(1.82 and 0.45 g·kg^(-1)),with 11 mice in each group. The mice in each group were treated correspondingly by gavage for eight weeks,once per day. Blood glucose and body weight were systematically evaluated. Twelve hours after the last administration,blood samples were collected from the eyes,and the serum and muscle and liver tissues were extracted. The levels of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),and glucose transporter type 4(GluT4)were detected by semi-quantitative enzyme-linked immunosorbent assay(ELISA). The protein expression of IκB kinase β(IKKβ)and nuclear factor-κB(NF-κB)in muscle tissues and Toll-like receptor 4(TLR4)in liver tissues was detected by Western blot. Result:Compared with the normal group,the model group showed increased body weight and blood glucose(P<0.01).Compared with the model group,the acarbose group and the GGQLT groups showed reduced body weight and blood glucose(P<0.05,P<0.01). As revealed by ELISA results,compared with the normal group,the model group showed increased levels of TNF-α and IL-6(P<0.01)and deceased GluT4 level(P<0.05). Compared with the model group,the groups with drug treatment showed reduced levels of TNF-α and IL-6(P<0.05,P<0.01),and the acarbose group and the high-dose GGQLT group showed increased GluT4 level(P<0.05,P<0.01). As displayed by Western blot results, compared with the normal group, the model group showed increased protein expression of IKKβ,NF-κB,and TLR4(P<0.01). Compared with the model group,the acarbose group and the GGQLT groups showed reduced protein expression of IKKβ,NF-κB,and TLR4(P<0.05,P<0.01). Conclusion: GGQLT can inhibit the inflammatory cascade effect and improve T2DM by downregulating the levels of key inflammatory factors in the TLR4 pathway, inhibiting their activation, and increasing the translocation and activity of GluT4 on the basis of the regulation of intestinal flora.