锦灯笼调控PPAR/JNK2通路防治药物性肝损伤的作用机制探讨

Exploration of the Mechanism of Physalis Calyxseuf Ructus in the Prevention and Treatment of DrugInduced Liver Injury Based on PPAR/JNK2 Pathway

目的基于PPAR/JNK2通路探讨锦灯笼防治药物性肝损伤(DILI)的作用机制。方法将50只Wistar大鼠随机分为5组:正常组、模型组、水飞蓟宾组(44.1 mg·kg^(-1))及锦灯笼高(6.67 g·kg^(-1))、低(1.67 g·kg^(-1))剂量组。采用对乙酰氨基酚(APAP,1000 mg·kg^(-1))溶液灌胃复制DILI大鼠模型,同时给予相应药物灌胃,每日1次,连续30 d。检测大鼠血清生化指标:谷丙转氨酶(ALT)、谷草转氨酶(AST)、总胆红素(TBIL)、直接胆红素(DBIL)、肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6)、γ干扰素(IFN-γ)、丙二醛(MDA)、谷胱甘肽(GSH)、谷胱甘肽过氧化物酶(GSH-PX);HE染色法观察肝组织病理变化;免疫组化法检测肝组织巨噬细胞炎性蛋白1β(MIP-1β)的表达情况;免疫荧光检测肝组织巨噬细胞炎性蛋白2(MIP-2)的表达情况;Western Blot法检测肝组织c-Jun氨基末端激酶2(JNK2)、磷酸化氨基末端激酶2(p-JNK2)蛋白表达情况;qRT-PCR法检测肝组织过氧化物酶体增殖物激活受体α(PPARα)、过氧化物酶体增殖物激活受体γ(PPARγ)和JNK2的基因表达水平。结果与正常组相比,模型组大鼠血清ALT、AST、DBIL及TBIL水平均明显升高(P<0.05,P<0.01),TNF-α、IL-6及IFN-γ水平均显著升高(P<0.01);血清MDA水平显著升高(P<0.01),GSH、GSH-PX水平显著降低(P<0.01);大鼠肝组织的肝小叶结构被严重破坏,肝细胞排列紊乱,有明显的炎性细胞浸润,并出现不同程度的坏死和变性;大鼠肝组织MIP-1β蛋白表达量显著升高(P<0.01),MIP-2蛋白阳性表达显著增强(P<0.01),p-JNK2蛋白表达显著上调(P<0.01);大鼠肝组织PPARαmRNA表达显著下调(P<0.01),PPARγ、JNK2 mRNA表达显著上调(P<0.01)。与模型组相比,水飞蓟宾组和锦灯笼高、低剂量组大鼠血清ALT、AST、DBIL、TBIL、TNF-α、IL-6及IFN-γ水平均明显降低(P<0.05,P<0.01);血清MDA水平显著降低(P<0.01),GSH、GSH-PX水平明显升高(P<0.05,P<0.01);肝损伤程度降低,肝细胞排列较为整齐,少有炎性细胞浸润,其中以锦灯笼高剂量组肝组织损伤的改善程度最为明显;大鼠肝组织MIP-1β蛋白表达量明显降低(P<0.05,P<0.01),MIP-2蛋白表达明显下调(P<0.05,P<0.01);锦灯笼高、低剂量组大鼠肝组织PPARαm RNA表达均显著上调(P<0.01),JNK2 mRNA表达显著下调(P<0.01),锦灯笼高剂量组大鼠肝组织p-JNK2蛋白表达明显下调(P<0.05),PPARγmRNA表达明显下调(P<0.05)。各组大鼠肝组织JNK2蛋白表达无明显差异(P>0.05)。结论锦灯笼可能通过调控PPAR/JNK2通路,抑制炎性因子水平,减少趋化因子产生,降低氧化应激反应,从而发挥对APAP诱导DILI大鼠的保护作用。

Objective To explore the mechanism of Physalis Calyxseuf Ructus in the prevention and treatment of drug-induced liver injury(DILI) based on PPAR/JNK2 pathway.Methods Fifty Wistar rats were randomly divided into 5 groups:normal group,model group,silydianin group(44.1 mg·kg^(-1)),and Physalis Calyxseuf Ructus high-(6.67 g·kg^(-1)) and low-(1.67 g·kg^(-1)) dose groups. The DILI rat model was replicated by gavage with acetaminophen(APAP,1 000 mg·kg^(-1)) and the corresponding drugs were administered once daily for 30 consecutive days. Serum biochemical parameters were measured: alanine transaminase(ALT), aspartate aminotransferase(AST), total bilirubin(TBIL),direct bilirubin(DBIL),tumor necrosis factor α(TNF-α),interleukin 6(IL-6),interferon γ(IFN-γ),malonaldehyde(MDA),glutathione(GSH),glutathione peroxidase(GSH-PX);HE staining was used to observe the pathological changes in liver tissue;immunohistochemistry was used to detect the expression of macrophage inflammatory protein 1β(MIP-1β) in liver tissue;immunofluorescence was used to detect the expression of macrophage inflammatory protein 2(MIP-2) in liver tissue;Western Blot was used to detect the protein expressions of c-Jun amino-terminal kinase 2(JNK2),phosphorylated amino-terminal kinase 2(p-JNK2)in liver tissue;qRT-PCR was performed to detect the gene expression levels of peroxisome proliferator-activated receptor α(PPARα), peroxisome proliferator-activated receptor γ(PPARγ) and JNK2 in liver tissue.Results Compared with the normal group,the serum ALT,AST,DBIL and TBIL levels were significantly increased(P<0.05,P<0.01),TNF-α,IL-6 and IFN-γ levels were significantly increased(P<0.01);serum MDA level was significantly increased(P<0.01), GSH and GSH-PX levels were significantly decreased(P<0.01). The liver lobular structure of rat liver tissue was severely disrupted, with disorganized hepatocyte arrangement, obvious inflammatory cell infiltration and different degrees of necrosis and degeneration;the protein expression of MIP-1β in rat liver tissue was significantly increased(P<0.01),the positive expression of MIP-2 protein was significantly enhanced(P<0.01),and the protein expression of p-JNK2 was significantly up-regulated(P<0.01);the mRNA expression of PPARα was significantly down-regulated(P<0.01) and mRNA expressions of PPARγ and JNK2 were significantly up-regulated(P<0.01) in rat liver tissue. Compared with the model group,the serum ALT,AST,DBIL,TBIL,TNF-α,IL-6 and IFN-γ levels were significantly decreased in the silydianin group and the Physalis Calyxseuf Ructus high-and low-dose groups(P<0.05, P<0.01);the serum MDA levels were significantly decreased(P<0.01),and the GSH and GSH-PX levels were significantly increased(P<0.05,P<0.01);the level of liver injury was reduced,hepatocytes were more neatly arranged and less infiltrated by inflammatory cells,among which the improvement of liver tissue injury was most obvious in the high-dose group of Physalis Calyxseuf Ructus;the protein expression of MIP-1β in rat liver tissue was significantly reduced(P<0.05,P<0.01) and protein expression of MIP-2 was significantly down-regulated(P<0.05, P<0.01);the mRNA expression of PPARα was significantly up-regulated(P<0.01) and mRNA expression of JNK2 was significantly down-regulated(P<0.01) in the liver tissue of both Physalis Calyxseuf Ructus high-and low-dose groups of rats. The protein expression of p-JNK2 was significantly down-regulated(P<0.05) and mRNA expression of PPARγ was significantly down-regulated(P<0.05) in the liver tissue of the high-dose group. There was no significant difference in the protein expression of JNK2 in the liver tissue in each group of rats(P<0.05).Conclusion Physalis Calyxseuf Ructus may exert a protective effect against APAP-induced DILI rats by regulating the PPAR/JNK2 pathway,inhibiting inflammatory factor levels,reducing chemokine production and decreasing oxidative stress.