三叶糖脂清DPP-4抑制成分筛选及其调控JNK/AKT信号通路改善胰岛素抵抗的机制研究

Screening of DPP-4 Inhibitors of Sanyetangzhiqing and Its Regulation of JNK/AKT Signaling Pathway Ameliorates Insulin Resistance

[目的]筛选三叶糖脂清中二肽基肽酶-4(dipeptidyl peptidase-4,DPP-4)的抑制成分,探讨三叶糖脂清及其DPP-4抑制成分对胰岛素抵抗(insulin resistant,IR)细胞的影响和作用机制。[方法]运用课题组前期建立的DPP-4抑制成分筛选方法,从三叶糖脂清中筛选DPP-4抑制成分,并对抑制率大于50%的化合物进行量效考察,通过高浓度胰岛素诱导BNL.CL2小鼠胚胎肝细胞构建IR细胞模型。通过检测葡萄糖消耗量、白细胞介素-1β(interleukin-1β,IL-1β)、白细胞介素-6(interleukin-6,IL-6)水平和锰超氧化物歧化酶(manganese superoxide dismutase,Mn-SOD)含量,考察三叶糖脂清总提取物及其DPP-4抑制成分对IR的BNL.CL2细胞的影响。以Western blot检测超氧化物歧化酶2(superoxide dismutase 2,SOD2)、磷酸化c-Jun氨基末端激酶(phospho-c-Jun N-terminal kinase,p-JNK)、c-Jun氨基末端激酶(c-Jun N-terminal kinase,JNK)、磷酸化蛋白激酶B(phospho-protein kinase B,p-AKT/PKB)、蛋白激酶B(protein kinase B,AKT/PKB)的蛋白表达。[结果]三叶糖脂清中筛选出6个化合物对DPP-4的抑制率大于50%,且在10～50μmol·L^(-1)浓度范围呈良好的量效关系。三叶糖脂清总提取物及其DPP-4抑制成分隐丹参酮能促进BNL.CL2细胞葡萄糖消耗和Mn-SOD的表达,抑制IL-1β、IL-6的分泌,降低JNK蛋白磷酸化,促进AKT蛋白磷酸化和SOD2蛋白表达。[结论]三叶糖脂清中含有DPP-4抑制作用的成分,三叶糖脂清总提取物及其成分隐丹参酮可能通过降低氧化应激和炎症反应水平来调节JNK/AKT通路,改善IR。

[Objective] To screen dipeptidyl peptidase-4(DPP-4) inhibitor from Sanyetangzhiqing(SYTZQ) formula, and investigate the effect and mechanism of SYTZQ and its DPP-4 inhibitor on insulin resistant(IR) cell. [Methods] Potential DPP-4 inhibitors were screened by using a previous fluorescent-based method. The IR BNL.CL2 cell was induced by high concentration of insulin. The glucose consumption, contents of interleukin-1 β(IL-1β), interleukin-6(IL-6) and manganese superoxide dismutase(Mn-SOD) were detected. The protein expression of superoxide dismutase 2(SOD2), phospho-c-Jun N-terminal kinase(p-JNK), c-Jun N-terminal kinase(JNK), phospho-protein kinase B(p-AKT/PKB), protein kinase B(AKT/PKB) were determined by Western blot. [Results] The inhibition rate of DPP-4 by 6 compounds in SYTZQ was more than 50%, and it showed a good dose-effect relationship at10~50μmol·L-1. SYTZQ and cryptotanshinone could increase the glucose consumption, the expression of Mn-SOD and decrease the level of IL-1 β and IL-6 in IR BNL.CL2 cells. SYTZQ and cryptotanshinone reduced JNK protein phosphorylation and promoted AKT protein phosphorylation and SOD2 protein expression.[Conclusion]Six compounds from SYTZQ showed significant inhibition against DPP-4. SYTZQ and its DPP-4 inhibitor cryptotanshinone may regulate JNK/AKT pathway to improve insulin resistance by reducing oxidative stress and inflammatory response.