本研究探讨了竹节参总皂苷(TSPJ)对对乙酰氨基酚(APAP)肝损伤的作用及其机制。雄性昆明小鼠随机分为空白对照组,TSPJ组(200mg·kg~(-1),ig),模型组,APAP+TSPJ低剂量组(50mg·kg~(-1),ig),APAP+TSPJ中剂量组(100mg·kg~(-1),ig),APAP+TSPJ高剂量组(200mg·kg~(-1),ig),和APAP+N-乙酰-L-半胱氨酸组(200mg·kg~(-1),ip)。给药组连续14天每天一次通过ig或ip接受相应的药物治疗。在最后一次给药一小时后,除空白对照组和TSPJ组外,各组小鼠灌胃给予500mg·kg~(-1)APAP。24小时后,小鼠血清和肝组织收集血清丙氨酸转氨酶(ALT),天冬氨酸转氨酶(AST),活性氧(ROS),肿瘤坏死因子α(TNF-α),白细胞介素-1β(IL-1β),环氧合酶-2(COX-2),IL-6,IL-4,IL-10,以及乳酸脱氢酶(LDH),谷胱甘肽(GSH),超氧化物歧化酶(SOD),过氧化氢酶(CAT),总抗氧化能力(T-AOC),丙二醛(MDA),和髓过氧化物酶(MPO)肝组织。采用苏木精-伊红染色观察肝组织形态学变化。淋巴细胞抗原6G(Ly6G)的mRNA表达水平,半乳糖凝集素3(Mac-2),TNF-α,IL-1β,通过定量实时聚合酶链反应(PCR)测定肝组织中的COX-2,IL-6,IL-4和IL-10。Westernblot检测Ly6G蛋白表达水平,Mac-2,细胞外调节蛋白激酶(ERK),磷酸化的细胞外调节蛋白激酶(p-ERK),COX-2,核因子κB蛋白α(IκBα)抑制剂,核因子κB蛋白α(p-IκBα)的磷酸化抑制剂,胞浆和肝组织细胞核中的核因子-κB亚基p65(NF-κBp65)。结果表明,TSPJ显著降低肝脏系数,血清ALT,AST,ROS,TNF-α,IL-1β,IL-6和COX-2水平,LDH,MPO,肝组织MDA含量,和TNF-α的mRNA表达,IL-1β,和IL-6在APAP诱导的肝损伤小鼠中的作用。它显著升高血清IL-4和IL-10水平,GSH,CAT,SOD,和T-AOC内容,肝组织中IL-4和IL-10的mRNA表达,改善肝脏病理损伤的程度,抑制肝组织中性粒细胞浸润和巨噬细胞募集。此外,TSPJ降低中性粒细胞标记物Ly6G的mRNA和蛋白表达,肝组织中的巨噬细胞标记物Mac-2和COX-2,p-ERK的蛋白表达,p-IκBα,核NF-κBp65,和p-ERK/ERK和p-IκBα/p-IκBα的比值以及NF-κBp65在细胞质中的蛋白表达。这些结果表明TSPJ对APAP诱导的小鼠肝损伤具有显著的保护作用,能减轻APAP诱导的氧化损伤和炎症反应。其机制可能与抑制ERK/NF-κB/COX-2信号通路的激活有关,从而抑制炎症细胞浸润,细胞因子产生,和肝细胞损伤。
This study investigated the effects and mechanisms of total saponins of Panax japonicus(TSPJ) against liver injury induced by acetaminophen(APAP). Male Kunming mice were randomly divided into a blank control group, TSPJ group(200 mg·kg~(-1), ig), model group, APAP+ TSPJ low-dose group(50 mg·kg~(-1), ig), APAP+ TSPJ medium-dose group(100 mg·kg~(-1), ig), APAP+ TSPJ high-dose group(200 mg·kg~(-1), ig), and APAP+ N-acetyl-L-cysteine group(200 mg·kg~(-1), ip). The administration group received the corresponding medications via ig or ip once a day for 14 consecutive days. After the last administration for one hour, except for the blank control group and TSPJ group, all groups of mice were given 500 mg·kg~(-1) APAP by gavage. After 24 hours, mouse serum and liver tissue were collected for serum alanine aminotransferase(ALT), aspartate aminotransferase(AST), reactive oxygen species(ROS), tumor necrosis factor alpha(TNF-α), interleukin-1 beta(IL-1β), cyclooxygenase-2(COX-2), IL-6, IL-4, IL-10, as well as lactate dehydrogenase(LDH), glutathione(GSH), superoxide dismutase(SOD), catalase(CAT), total antioxidant capacity(T-AOC), malondialdehyde(MDA), and myeloperoxidase(MPO) liver tissue. Hematoxylin-eosin staining was used to observe the morphological changes of liver tissue. The mRNA expression levels of lymphocyte antigen 6G(Ly6G), galectin 3(Mac-2), TNF-α, IL-1β, COX-2, IL-6, IL-4, and IL-10 in liver tissue were determined by quantitative real-time polymerase chain reaction(PCR). Western blot was utilized to detect the protein expression levels of Ly6G, Mac-2, extracellular regulated protein kinases(ERK), phosphorylated extracellular regulated protein kinases(p-ERK), COX-2, inhibitor of nuclear factor κB protein α(IκBα), phosphorylated inhibitor of nuclear factor κB protein α(p-IκBα), and nuclear factor-κB subunit p65(NF-κB p65) in cytosol and nucleus in liver tissue. The results manifested that TSPJ dramatically reduced liver coefficient, serum ALT, AST, ROS, TNF-α, IL-1β, IL-6, and COX-2 levels, LDH, MPO, and MDA contents in liver tissue, and mRNA expressions of TNF-α, IL-1β, and IL-6 in APAP-induced liver injury mice. It prominently elevated serum IL-4 and IL-10 levels, GSH, CAT, SOD, and T-AOC contents, and mRNA expressions of IL-4 and IL-10 in liver tissue, improved the degree of liver pathological damage, and suppressed neutrophil infiltration and macrophage recruitment in liver tissue. In addition, TSPJ lessened the mRNA and protein expressions of neutrophil marker Ly6G, macrophage marker Mac-2, and COX-2 in liver tissue, protein expressions of p-ERK, p-IκBα, and NF-κB p65 in nuclear, and p-ERK/ERK and p-IκBα/p-IκBα ratios and hoisted protein expression of NF-κB p65 in cytosol. These results suggest that TSPJ has a significant protective effect on APAP-induced liver injury in mice, and it can alleviate APAP-induced oxidative damage and inflammatory response. Its mechanism may be related to suppressing ERK/NF-κB/COX-2 signaling pathway activation, thus inhibiting inflammatory cell infiltration, cytokine production, and liver cell damage.