基于TRAF2/AP-1信号通路探讨柴苓合剂对痰瘀互结MS-IR大鼠炎症因子影响

Exploration of the Effect of Chailing Mixture(柴苓合剂)on MS-IR Rats with Phlegm Stasis Interaction Based on TRAF2/AP-1 Signal Pathway

目的基于TRAF2/AP-1信号通路探讨柴苓合剂对痰瘀互结代谢综合征胰岛素抵抗(MS-IR)大鼠糖代谢、脂代谢、炎症因子的部分作用机制。方法48只SPF级SD雄性大鼠随机分为空白组和造模组。造模组大鼠采用高脂高糖饲料喂养,注射小剂量链脲佐菌素(STZ)(35 mg/kg),同时用物理刺激方法,建立痰瘀互结型MSIR大鼠模型。造模成功后将大鼠随机分为模型组,柴苓合剂低、中、高剂量组,西药组进行干预。给药各组分别进行柴苓合剂、吡格列酮干预治疗3周,空白组、模型组给予等体积的蒸馏水灌服。生化检测大鼠空腹血糖(FBG)、空腹胰岛素(FINS)水平,计算胰岛素抵抗指数(HOMA-IR);ELISA法检测血清肿瘤坏死因子(TNF-α)、白细胞介素-6(IL-6)水平;Western Blot检测肝组织TRAF2、JNK、AP-1蛋白表达;HE染色切片,光学显微镜观察用药前后肝细胞损伤、肝细胞坏死、脂质堆积、炎症细胞浸润等情况。结果(1)糖代谢指标水平。与空白组相比,模型组大鼠FBG、FINS、HOMA-IR水平均升高(P<0.01);与模型组相比,柴苓合剂低、中、高剂量组及西药组FBG、FINS、HOMA-IR水平均降低(P<0.01);与西药组比较,柴苓合剂高剂量组FBG、FINS、HOMA-IR水平差异无统计学意义(P>0.05);与柴苓合剂低剂量组比较,柴苓合剂高、中剂量组及西药组FBG、FINS、HOMA-IR水平均降低(P<0.01);与柴苓合剂中剂量组比较,柴苓合剂高剂量组及西药组FBG、FINS、HOMA-IR水平降低(P<0.01)。(2)脂代谢指标水平。与空白组相比,模型组大鼠甘油三酯(TG)、胆固醇(CHO)水平均升高(P<0.01);与模型组相比,柴苓合剂低、中、高剂量组及西药组TG、CHO水平均降低(P<0.01);与西药组相比,柴苓合剂中、高剂量组TG、CHO水平均降低(P<0.05,P<0.01),柴苓合剂低剂量组TG、CHO水平差异无统计学意义(P>0.05);与柴苓合剂低剂量组相比,柴苓合剂中、高剂量组TG、CHO水平均降低(P<0.01);与柴苓合剂中剂量组相比,柴苓合剂高剂量组TG、CHO水平均降低(P<0.01)。(3)TNF-α、IL-6水平。与空白组相比,模型组大鼠TNF-α、IL-6水平均升高(P<0.01);与模型组相比,柴苓合剂低、中、高剂量组及西药组TNF-α、IL-6水平均降低(P<0.01);与西药组相比,柴苓合剂低、中、高剂量组TNF-α、IL-6水平均降低(P<0.01);与柴苓合剂低剂量组相比,柴苓合剂中、高剂量组TNF-α、IL-6水平均降低(P<0.05,P<0.01);与柴苓合剂中剂量组相比,柴苓合剂高剂量组TNF-α、IL-6水平均降低(P<0.01)。(4)肝组织TRAF2/JNK/AP-1信号通路蛋白表达。与空白组相比,模型组大鼠肝组织TRAF2、JNK、AP-1蛋白表达均上调(P<0.01);与模型组比较,柴苓合剂低、中、高剂量组及西药组肝组织TRAF2、JNK、AP-1蛋白表达水平均降低(P<0.05,P<0.01);与西药组比较,柴苓合剂高剂量组肝组织TRAF2、JNK、AP-1蛋白表达水平均降低(P<0.01),柴苓合剂低、中剂量组肝组织TRAF2、JNK、AP-1蛋白表达水平差异无统计学意义(P>0.05);与柴苓合剂低剂量组比较,柴苓合剂高剂量组肝组织TRAF2、JNK、AP-1蛋白表达水平均降低(P<0.01),柴苓合剂中剂量组肝组织AP-1蛋白表达差异无统计学意义(P>0.05),TRAF2、JNK蛋白表达水平均降低(P<0.01);与柴苓合剂中剂量组比较,柴苓合剂高剂量组TRAF2、JNK、AP-1蛋白表达水平均降低(P<0.05,P<0.01)。结论柴苓合剂高剂量能显著改善痰瘀互结MS-IR大鼠糖代谢、脂代谢和炎症因子,其作用机制可能是柴苓合剂通过调控TRAF2蛋白作用于JNK蛋白,引起它的下游反应,又通过JNK的磷酸化作用于AP-1,释放炎症因子,从而TRAF2作用于JNK,然后磷酸化于AP-1使炎症因子降低有关。

Objective Based on the TRAF2/AP-1 signaling pathway,to explore the partial mechanism of Chailing Mixture(柴苓合剂)on glucose metabolism,lipid metabolism and inflammatory factors in MS-IR rats with phlegm and blood stasis.Methods 48 SPF male SD rats were randomly divided into blank group and model group.Rats in the model group were fed with high-fat and high-sugar feed,injected with a small dose of STZ(35 mg/kg)and physically stimulated to establish a MS-IR rat model with phlegm and blood stasis.After the successful modeling,the rats were randomly divided into the model group,the low-dose,medium-dose and high-dose groups of Chailing Mixture,and the Western medicine group for intervention.Each drug group received Chailing Mixture and pioglitazone intervention for 3 weeks,and the blank group and model group were given equal volumes of distilled water.Biochemical detection of FBG and FINS in rats,and calculation of HOMA-IR;detection of serum tumor necrosis factor(TNF-α)levels and interleukin 6(IL-6)by ELISA;detection of TRAF2,JNK,AP-1 protein expression in liver tissue by Western Blot;HE-stained sections were used to observe liver cell injury,liver cell necrosis,lipid accumulation,and inflammatory cell infiltration before and after treatment with an optical microscope.Results(1)The level of glucose metabolism indicators.Compared with the blank group,the levels of FBG,FINS,and HOMA-IR in the model group rats increased(P<0.01);Compared with the model group,the FBG,FINS,and HOMA-IR levels in the low,medium,and high dose groups of Chailing Mixture and the Western medicine group were all reduced(P<0.01);Compared with the Western medicine group,there was no statistically significant difference in FBG,FINS,and HOMA-IR levels in the high-dose group of Chailing Mixture(P>0.05);Compared with the low-dose group of Chailing Mixture,the FBG,FINS,and HOMA-IR levels in the high and medium dose groups of Chailing Mixture,as well as the Western medicine group,were all reduced(P<0.01);Compared with the medium-dose group of Chailing Mixture,the high-dose group of Chailing Mixture and the Western medicine group showed decrease in FBG,FINS,and HOMA-IR levels(P<0.01).(2)Level of lipid metabolism indicators.Compared with the blank group,the levels of triglycerides(TG)and cholesterol(CHO)in the model group rats increased(P<0.01);Compared with the model group,the levels of TG and CHO in the low,medium,and high dose groups of Chailing Mixture and the Western medicine group were all reduced(P<0.01);Compared with the Western medicine group,the levels of TG and CHO in the medium and high dose groups of Chailing Mixture decreased(P<0.05,P<0.01),while there was no statistically significant difference in TG and CHO levels in the low-dose group of Chailing Mixture(P>0.05);Compared with the low-dose group of Chailing Mixture,the levels of TG and CHO in both the medium and high dose groups of Chailing Mixture decreased(P<0.01);Compared with the mediumdose group of Chailing Mixture,the high-dose group of Chailing Mixture showed decrease in TG and CHO levels(P<0.01).(3)TNF-α and IL-6 levels.Compared with the blank group,the model group rats showed average increase in TNF-α and IL-6 levels(P<0.01);Compared with the model group,the low,medium,and high dose groups of Chailing Mixture,as well as the Western medicine group,showed average decrease in TNF-α and IL-6 levels(P<0.01);Compared with the Western medicine group,the low,medium,and high dose groups of Chailing Mixture showed average decrease in TNF-α and IL-6 levels(P<0.01);Compared with the low-dose group of Chailing Mixture,the middle and high dose groups of Chailing Mixture showed average decrease in TNF-α and IL-6 levels(P<0.05,P<0.01);Compared with the medium-dose group of Chailing Mixture,the high dose group of Chailing Mixture showed average decrease in TNF-α and IL-6 levels(P<0.01).(4)Expression of TRAF2/JNK/AP-1 signaling pathway proteins in liver tissue.Compared with the blank group,the expression of TRAF2,JNK,and AP-1 proteins in the liver tissue of the model group rats was upregulated(P<0.01);Compared with the model group,the expression levels of TRAF2,JNK,and AP-1 proteins in liver tissue of the low,medium,and high dose groups of Chailing Mixture and the Western medicine group were all reduced(P<0.05,P<0.01);Compared with the Western medicine group,the expression levels of TRAF2,JNK,and AP-1 proteins in the liver tissue of the high-dose group of Chailing Mixture decreased(P<0.01),while there was no statistically significant difference in the expression levels of TRAF2,JNK,and AP-1 proteins in the liver tissue of the low and medium dose groups of Chailing Mixture(P>0.05);Compared with the low-dose group of Chailing Mixture,the high-dose group of Chailing Mixture showed decrease in the expression levels of TRAF2,JNK,and AP-1 proteins in liver tissue(P<0.01),while the mediumdose group of Chailing Mixture showed no statistically significant difference in AP-1 protein expression in liver tissue(P>0.05).The expression levels of TRAF2 and JNK proteins decreased(P<0.01);Compared with the medium dose-group of Chailing Mixture,the high dose-group of Chailing Mixture showed decrease in the expression levels of TRAF2,JNK,and AP-1 proteins(P<0.05,P<0.01).Conclusion High dose of Cailing Mixture can significantly improve glucose metabolism,lipid metabolism and inflammatory factors in MS-IR rats with phlegm and blood stasis.In response,the phosphorylation of JNK acts on AP-1 to release inflammatory factors,so TRAF2 acts on JNK,and then phosphorylates on AP-1 to reduce inflammatory factors.