TLR4抑制剂TAK-242对高脂饮食诱导的小鼠胰岛素抵抗的干预作用

Intervention role of TLR4 inhibitor TAK-242 on insulin resistance states in mice with a high fat diet-induced insulin resistance

目的建立高脂饮食诱导的胰岛素抵抗小鼠模型,探讨干预Toll样受体4(Toll-like receptor 4,TLR4)对高脂饮食诱导的小鼠胰岛素抵抗状态的影响。方法首先建立高脂饮食诱导的胰岛素抵抗小鼠模型。选取出生21 d的雄性C57BL/6小鼠36只,随机分为2组,正常对照组12只,以普通基础饲料喂养(low fat diet,LFD),高脂饮食实验组24只,给予高脂饲料喂养(high fat diet,HFD)。待2组小鼠体质量出现显著差异时,进行葡萄糖耐量实验(glucose tolerance test,GTT)和胰岛素耐量实验(insulin tolerance test,ITT)观察小鼠胰岛素抵抗的发生。小鼠胰岛素抵抗模型建立后,观察TLR4抑制剂TAK-242对小鼠胰岛素抵抗状态的作用。LFD组小鼠继续以基础饲料喂养(即作LFD对照组);HFD组小鼠随机分为2组,每组12只小鼠,均继续以高脂饲料喂养,其中一组HFD小鼠腹腔注射TLR4抑制剂TAK-242,以0.5 mg TAK-242/kg体质量的计量给予,每周2次(即给予TAK-242的高脂饮食实验组,HFD-T组);另一组HFD小鼠作为单纯高脂饮食的胰岛素抵抗空白对照组(HFD-C组),只给予等体积的溶媒-二甲基亚砜(DMSO)。给予小鼠TLR4抑制剂5个月,进行GTT和ITT后,处死小鼠。采用EDTA-K2抗凝管收集血液标本,立即分离外周血单核细胞(peripheral blood mononuclear cell,PBMC)和血浆。应用Western blot技术检测PBMC TLR4蛋白表达水平。采用全自动生化分析仪测定血浆葡萄糖(GLU)、甘油三酯(TG)、总胆固醇(TC)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)、丙氨酸氨基转移酶(ALT)的水平。结果在小鼠胰岛素抵抗模型建立期间,与对照组相比,高脂饮食组小鼠体质量增长较快,喂养至第20周体质量出现显著差异(P<0.05);持续高脂饮食7个月,GTT和ITT结果显示,高脂饮食组小鼠对糖的调节能力受损、对胰岛素的降糖作用减低,说明高脂饮食诱导小鼠胰岛素抵抗成功。出现胰岛素抵抗的小鼠,在给予TLR4抑制剂TAK-242 5个月后,对葡萄糖的调节能力和对胰岛素的敏感性均有所改善。血浆生化指标结果显示,与对照组比较,单纯高脂组和给予TAK-242的高脂饮食干预组小鼠血浆TG、TC、LDL-C浓度均显著升高(P<0.05),GLU、HDL-C、ALT水平均有升高趋势,但无统计学意义(P>0.05)。单纯高脂组和TAK-242干预组比较,血浆GLU、TG、TC、LDL-C、HDL-C、ALT水平在2组之间不存在显著性差异(P>0.05)。Western blot实验结果发现,正常对照组小鼠PBMC未见TLR4蛋白表达,单纯高脂饮食组小鼠PBMC有高水平的TLR4蛋白表达,给予TAK-242的高脂饮食小鼠PBMC TLR4蛋白虽有表达,但表达量明显低于单纯高脂饮食组小鼠,结果提示TAK-242部分抑制了高脂饮食喂养小鼠PBMC TLR4蛋白的表达。结论高脂饮食成功诱导了小鼠产生胰岛素抵抗,抑制TLR4蛋白的表达可以改善小鼠胰岛素抵抗状态。该研究结果丰富了胰岛素抵抗发生的机制,为进一步深入研究TLR4及其信号通路在胰岛素抵抗中的作用提供了实验依据。

The role of Toll-like receptor 4（TLR4） in obesity-related insulin resistance is not well known.This study aimed to investigate the intervention role of TLR4 inhibitor TAK-242 on insulin resistance states in mice with a high fat diet-induced insulin resistance.In order to achieve this aim,a high fat diet-induced insulin resistance model was established in mice.A total of 36 male C57BL/6 mice（born 21 days） were selected and randomly divided into two groups：the control group（12 mice） was fed with general basic diet（low fat diet,LFD）,while the experimental group（24 mice） was fed with high fat diet（high fat diet,HFD）.When the two groups demonstrated significant differences in body weight,the glucose tolerance test（GTT） and insulin tolerance test（ITT） were performed for observing the occurrence of insulin resistance in mice.After the mouse model of insulin resistance was established,the role of TLR4 inhibitor TAK-242 in insulin resistance was studied.LFD mice were still fed with general basic diet（LFD control group）;HFD mice were randomly divided into 2 groups,with 12 mice in each group,and fed with high fat diet.One group of HFD mice was injected intraperitoneally with TLR4 inhibitor TAK-242（0.5 mg TAK-242/kg body weigh）,2 times a week（HFD-T group）;another group of HFD mice was designed as control group（HFD-C group）.LFD control group and HFD-C group mice were only injected with an equal volume Dimethyl sulfoxide（DMSO）.After been giving mouse TLR4 inhibitors for GTT and ITT for 5months,the mice were sacrificed.The blood samples were collected using EDTA-K_2 anticoagulant tubes,and then peripheral blood mononuclear cells（PBMC） and plasma were isolated immediately.Western blot was applied to detect PBMC TLR4 protein levels.Automatic biochemical analyzer was used to detect plasma glucose（GLU）,triglyceride（TG）,total cholesterol（TC）,high-density lipoprotein cholesterol（HDL-C）,low-density lipoprotein cholesterol（LDL-C）,alanine aminotransferase（ALT） levels.The results showed that during the period of developing insulin resistance in mice model,the weight of high fat diet mice gained more rapidly,compared with the control group,and significant difference in body weight appeared after feeding to 20 weeks（P〈0.05）.Seven months later,GTT and ITT results showed that the ability to regulate glucose in mice fed a high fat diet was impaired,and the hypoglycemic effect of insulin was reduced,which suggested that insulin resistance appeared in high fat diet group.After the mice with insulin resistance（keeping a high fat diet） had been treated by TLR4 inhibitor TAK-242 for five months,the glucose tolerance and insulin sensitivity were improved.The plasma biochemical variables results showed that,compared with LFD control group,plasma TG,TC,LDL-C levels were significantly increased（P〈0.05）in HFD-T and HFD-C groups;GLU,HDL-C and ALT levels were also increased,but no statistical significance（P〉0.05）.There was no significant difference in GLU,TG,TC,LDL-C,HDL-C,ALT levels between HFD-T and HFD-C groups（P〉0.05）.Western blot results showed that,PBMC TLR4 protein expression was not found in LFD control group;but a high level of TLR4 protein expression was detected in HFD-C group;compared with HFD-C group,the expression level of TLR4 protein was significantly lower in the HFD-T group.The results suggested that PBMC TLR4 protein is partly inhibited by TAK-242 in mice fed a high fat die,and inhibiting the expression of TLR4 protein may improve insulin resistance states in mice.