解毒通络调肝方对脂肪细胞内质网应激和炎症的影响

Effects of Jiedu Tongluo Tiaogan formula on the endoplasmic reticulum stress and inflammation of adipocytes

目的通过观察解毒通络调肝方对内质网应激(ERS)的3T3-L1脂肪细胞肌醇依赖酶(IRE1)、c-Jun氨基末端激酶(JNK)、激活核因子B(NF-κB)蛋白表达以及白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)、C反应蛋白(CRP)和脂联素(ADPN)分泌的影响,探讨解毒通络调肝方对3T3-L1脂肪细胞内质网应激的炎症调节作用。方法将3T3-L1前脂肪细胞诱导为成熟的脂肪细胞,随机分为空白对照组、模型组、解毒通络调肝方低、中、高浓度组和二甲双胍组,除空白对照组外,各组均通过2 mg/L衣霉素诱导为ERS脂肪细胞,分别给予对应药物进行24 h干预后,采用酶联免疫吸附实验(ELISA)检测脂肪细胞上清中IL-6、TNF-α、CRP和ADPN的分泌情况,通过蛋白质印迹法(Western-blot)检测脂肪细胞内IRE1、JNK、NF-κB蛋白表达。结果 Western-blot检测显示解毒通络调肝方低、中、高浓度组的IRE1、JNK和NF-κB蛋白表达明显低于模型组,特别是中、高浓度组效果显著(P <0. 01),其差异有统计学意义。ELISA检测显示ADPN表达在解毒通络调肝方低、中、高浓度组中均有上升(P <0. 01),解毒通络调肝方中、高浓度组均能降低IL-6、TNF-α、CRP表达,其中解毒通络调肝方高浓度组效果显著(P <0. 01),对JNK和IL-6表达的下调较二甲双胍组更优(P <0. 01)。结论解毒通络调肝方可能通过抑制炎症相关因子、促进脂联素的分泌,减轻脂肪细胞炎症反应,降低UPR通路中关键蛋白IRE1的表达,以多靶点方式缓解ERS。

Objective To observe the effects of Jiedu Tongluo Tiaogan formula( Detoxifying Collateraldredging Liver-regulating Formula,JTT) on the expression of IRE1, JNK and NF-κB in 3T3-L1 adipocytes and the secretion of IL-6,TNF-α,CRP and ADPN,to investigate the inflammatory regulation of JTT formula on endoplasmic reticulum stress( ERS) of 3T3-L1 adipocytes. Methods 3T3-L1 preadipocytes were induced into mature adipocytes which were randomly divided into blank control group,model group,low,medium and high dose of JTT formula groups and metformin group. Except for the blank control group,the other groups were induced to ERS adipocytes by using 2 mg/L tunicamycin.After 24 h intervention,the secretion of IL-6,TNF-α,CRP and ADPN in the supernatant of adipocytes was detected by using enzyme-linked immunosorbent assay( ELISA). Protein expression of IRE1,JNK and NF-κB in adipocytes was measured with Western blot assay. Results Western blot assay showed that the expressions of IRE1,JNK and NF-κB in the low,medium and high dose JTT prescription groups were significantly lower than those in the model group,especially in the mid-dose and high-dose JTT groups( P < 0. 01); the difference was statistically significant. ELISA showed that the expression of ADPN increased in all doses of JTT groups( P < 0. 01). The expressions of IL-6,TNF-α and CRP were all reduced in the mid-dose and high-dose JTT groups with significant changes in the high-dose JTT group( P < 0. 01). The expressions of JNK and IL-6 were lower in the high-dose JTT group than that in the metformin group( P < 0. 01). Conclusion JTT formula may inhibit the secretion of adiponectin by inhibiting inflammation-related factors,thereby reduce the inflammatory response of fat cells and reduce the expression of the key protein IRE1 in the UPR pathway. ERS of 3T3-L1 adipocytes could thus be alleviated in a multi-target manner.