研究珠子参总皂苷(total saponins of Panax japonicus，TSPJ)对对乙酰氨基酚(acetaminophen，APAP)诱导小鼠肝损伤的影响及作用机制。将雄性昆明小鼠随机分为空白组、TSPJ组(200 mg·kg-1，ig)、模型组、APAP+TSPJ低剂量组(50 mg·kg-1，ig)、APAP+TSPJ中剂量组(100 mg·kg-1，ig)、APAP+TSPJ高剂量组(200 mg·kg-1，ig)和APAP+N-乙酰半胱氨酸组(200 mg·kg-1，ip)。给药组每天ig或ip相应的药物，每天1 次，连续14 d。末次给药1 h后，除空白组和TSPJ组外，各组小鼠均灌胃给予 500 mg·kg-1 APAP，24 h 后收集小鼠血清与肝脏组织进行血清中丙氨酸氨基转移酶(alanine aminotrans-ferase，ALT)、天冬氨酸氨基转移酶(aspartate aminotransferase，AST)、活性氧(reactive oxygen species，ROS)、肿瘤坏死因子(tumor necrosis factor，TNF)-α、白细胞介素(interleukin，IL)-1β、环氧合酶(cyclooxygenase，COX)-2、IL-6、IL-4、IL-10 及肝组织中乳酸脱氢酶(lactate dehydrogenase，LDH)、谷胱甘肽(glutathione，GSH)、超氧化物歧化酶(superoxide dismutase，SOD)、过氧化氢酶(catalase，CAT)、总抗氧化能力(total antioxidant capacity，T-AOC)、丙二醛(malondialdehyde，MDA)、髓过氧化物酶(myeloperoxidase，MPO)的水平，苏木精-伊红染色观察肝组织形态学改变;实时定量PCR法测定肝组织中淋巴细胞抗原 6G(lymphocyte antigen 6G，Ly6G)、半乳糖凝集素 3(galectin 3，Mac-2)、TNF-α、IL-1β、COX-2、IL-6、IL-4、IL-10 mRNA表达，Western blot法测定肝组织中Ly6G、Mac-2、细胞外调节蛋白激酶(extracellular regulated protein kinase，ERK)、磷酸化细胞外信号调节激酶(phosphorylated extracellular regulated protein kinase，p-ERK)、COX-2、核因子κB抑制因子α(inhibitor of nu-clear factor κB protein α，IκBα)、磷酸化核因子 κB 抑制因子 α(phosphorylated inhibitor of nuclear factor κB protein α，p-IκBα)、胞浆和胞核中核因子κB亚基p65(nuclear factor κB subunit p65，NF-κB p65)蛋白表达。结果显示，TSPJ可显著降低APAP诱导小鼠肝脏系数、血清中ALT、AST、ROS、TNF-α、IL-1β、IL-6、COX-2 水平及肝组织LDH、MPO、MDA水平和肝组织中TNF-α、IL-1β、IL-6 mRNA表达，升高血清中IL-4、IL-10 含量及肝组织中GSH、CAT、SOD、T-AOC水平和肝组织中IL-4、IL-10 mRNA表达;改善肝脏病理损伤程度，抑制肝组织中性粒细胞浸润和巨噬细胞募集;降低肝组织中中性粒细胞标记物Ly6G、巨噬细胞标记物Mac-2 和COX-2 mRNA和蛋白表达，降低p-ERK、p-IκBα、胞核NF-κB p65 蛋白表达和p-ERK/ERK、p-IκBα/IκBα比率，升高胞浆NF-κB p65 蛋白表达。以上结果表明，TSPJ对APAP诱导的小鼠肝损伤有显著保护作用、可减轻APAP引起的氧化损伤与炎症反应，其作用机制与抑制ERK/NF-κB/COX-2 信号通路激活，进而抑制炎性细胞浸润、炎症因子生成和抑制肝细胞损伤密切相关。
Mechanism of total saponins of Panax japonicus against liver injury induced by acetaminophen in mice based on ERK/NF-κB/COX-2 signaling pathway
This study investigated the effects and mechanisms of total saponins of Panax japonicus(TSPJ)against liver injury in-duced 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 re-ceived 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),phos-phorylated 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 tis-sue.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 in-jury 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 protec-tive 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 infiltra-tion,cytokine production,and liver cell damage.
total saponins of Panax japonicusacetaminophenoxidative damageinflammatory reactionextracellular regulated protein kinases/nuclear factor-kappa B/cyclooxygenase-2 signaling pathway
NETL
NSTL
万方数据
