摘要
目的探究元胡改善对乙酰氨基酚(acetaminophen,APAP)诱导的急性肝损伤(acute liver injury,ALI)的作用及机制。方法SPF雄性KM小鼠60只,随机分为6组:空白对照组,APAP模型组,元胡低剂量组(2.5 g·kg^(-1)),元胡中剂量组(5 g·kg^(-1)),元胡高剂量组(10 g·kg^(-1))及阳性对照组,每组10只小鼠。空白对照组和APAP模型组小鼠灌胃等体积的生理盐水,阳性对照组小鼠灌胃N-乙酰半胱氨酸200 mg·kg^(-1),每天给药1次,连续给药7 d。于第7 d诱导损伤,除空白对照组以外,其余组小鼠禁食16 h后按照300 mg·kg^(-1)腹腔注射APAP诱导ALI模型(第7 d 17∶00~次日9∶00),给药体积20μL。造模前称量并记录小鼠体重,造模6 h后采用戊巴比妥钠麻醉后,采用眶动静脉取血法收集血液,室温离心取血清进行后续实验。在戊巴比妥腹腔注射麻醉处死小鼠后完整取出肝脏组织称量并记录肝重量,分别采用4%多聚甲醛固定和-80℃冷冻处理。通过检测小鼠肝指数观察元胡各剂量组对小鼠急性肝损伤的保护程度;测定血清学指标以观察元胡对各组小鼠血清中ALT、AST以及TNF-α、IL-1β和IL-6的影响;组织病理学观察小鼠肝脏外观形态以及HE染色观察肝脏进一步病理变化;免疫组化及Western blot探究元胡对ALI的保护作用并阐述其分子机制。结果与空白对照组(4.35±0.18)%比较,APAP模型组(6.60±0.11)%小鼠的肝指数升高明显,而经元胡干预给药的三组小鼠与APAP模型组相比,均有不同程度的降低,其中以元胡中剂量组(4.73±0.09)%更为接近阳性对照组。相比APAP模型组[ALT:(5508.42±267.85)U·L^(-1),AST:(564.26±27.04)U·L^(-1)],元胡治疗组和阳性对照组HE染色显示小鼠肝脏坏死面积明显减少,血清中ALT和AST水平下降,元胡治疗组中以元胡中剂量组较为明显[ALT:(342.95±35.29)U·L^(-1),AST:(102.68±6.96)U·L^(-1)]。相比APAP模型组[TNF-α:(856.24±21.83)pg·mL^(-1)、IL-1β:(683.37±14.93)pg·mL^(-1)、IL-6:(1856.92±58.86)pg·mL^(-1)],元胡治疗组和阳性对照组中促炎症因子(TNF-α、IL-1β和IL-6)显著下调,其元胡治疗组中以中剂量组较为明显[TNF-α:(141.70±13.92)pg·mL^(-1)、IL-1β:(310.10±8.13)pg·mL^(-1)、IL-6:(823.05±49.63)pg·mL^(-1)]。免疫组化检测小鼠肝脏巨噬细胞标记蛋白CD68和乳酸脱氢酶-A(lactate dehydrogenase-A,LDH-A)含量,与APAP模型组相比,元胡各干预组和阳性对照组CD68和LDH-A阳性染色面积均明显下降,元胡治疗组中LDH-A表达以元胡低剂量组较为明显,CD68表达以元胡中剂量组较为显著。Western blot检测结果显示小鼠肝脏元胡各治疗组核转录因子κB p65(NF-κB p65)含量明显下调,其中元胡干预组以元胡中剂量组更为接近阳性对照组,而元胡各干预组的沉默信息调节因子6(Sirt-6)蛋白含量均有所提高,其中以元胡低剂量组更为接近阳性对照组。结论元胡对急性肝损伤具有保护作用;元胡对急性肝损伤的保护作用可能是通过抑制NF-κB p65蛋白,促进Sirt-6蛋白的表达,降低LDH-A和CD68的表达起作用。
Objective To explore the effect and mechanism of Yuanhu in improving acute liver injury(ALI)induced by acetaminophen(APAP).Methods Sixty SPF male KM mice were randomly divided into six groups:blank control group,APAP model group,low-dose Yuanhu group(2.5 g·kg^(-1)),medium dose Yuanhu group(5 g·kg^(-1)),high-dose Yuanhu group(10 g·kg^(-1)),and positive control group,with 10 mice in each group.The blank control group and APAP model group mice were given an equal volume of physiological saline by gavage,while the positive control group mice were given N-acetylcysteine(NAC)200 mg·kg^(-1) by gavage once a day for 7 consecutive days.On the 7th day of injury induction,except for the blank control group,all other groups of mice were fasted for 16 hours and then intraperitoneally injected with 300 mg·kg^(-1) APAP to induce ALI model(from 17∶00 on the 7th day to 9∶00 the next day),with a dosage volume of 20μL.Weigh and record the weight of the mice before modeling.After 6 hours of modeling,anesthetize them with pentobarbital sodium and collect blood using the orbital arteriovenous sampling method.Centrifuge the serum at room temperature for subsequent experiments.After intraperitoneal injection of pentobarbital anesthesia to euthanize mice,the liver tissue was completely removed and weighed,and the liver weight was recorded.The mice were fixed with 4%paraformaldehyde and frozen at-80℃.Observing the protective effect of different doses of Yuanhu on acute liver injury in mice by detecting their liver index;Measure serological indicators to observe the effects of Yuanhu on ALT,AST,TNF-α,IL-1β,and IL-6 in the serum of mice in each group;Histopathological observation of the appearance and morphology of mouse liver,as well as HE staining to observe further pathological changes in the liver;Immunohistochemistry and Western blot were used to investigate the protective effect of Yuanhu on ALI and elucidate its molecular mechanism.Results Compared with the blank control group(4.35±0.18)%,it was found that the liver index of the APAP model group(6.60±0.11)%mice increased significantly.However,the liver index of the three groups of mice treated with Yuanhu intervention decreased to varying degrees compared with the APAP model group.Among them,the results of the Yuanhu medium dose group(4.73±0.09)%were closer to the positive control group.Compared with the APAP model group[ALT:(5508.42±267.85)U·L^(-1),AST:(564.26±27.04)U·L^(-1)],HE staining of the Yuanhu treatment group and positive control group showed a significant reduction in liver necrosis area and a decrease in serum ALT and AST levels.Among the Yuanhu treatment group,the Yuanhu medium dose group was more significant[ALT:(342.95±35.29)U·L^(-1),AST:(102.68±6.96)U·L^(-1)].Compared with the APAP model group[TNF-α:(856.24±21.83)pg·mL^(-1),IL-1β:(683.37±14.93)pg·mL^(-1),IL-6:(1856.92±58.86)pg·mL^(-1)],pro-inflammatory factors(TNF-α,IL-1β,and IL-6)were significantly downregulated in the Yuanhu treatment group and positive control group.Among the Yuanhu treatment groups,the moderate dose group showed more significant downregulation[TNF-α:(141.70±13.92)pg·mL^(-1),IL-1β:(310.10±8.13)pg·mL^(-1),IL-6:(823.05±49.63)pg·mL^(-1)].Immunohistochemistry was used to detect the levels of macrophage marker protein CD68 and lactate dehydrogenase-A(LDH-A)in mouse liver.Compared with the APAP model group,the positive staining areas of CD68 and LDH-A in each intervention group and positive control group of Yuanhu were significantly reduced.In the Yuanhu treatment group,LDH-A expression was more pronounced in the low-dose Yuanhu group,and CD68 expression was more pronounced in the medium dose Yuanhu group.The Western blot analysis results showed that the nuclear transcription factor kappa B p65(NF-κB p65)content in the liver of mice treated with Yuanhu was significantly downregulated.Among them,the Yuanhu intervention group was closer to the positive control group in the Yuanhu medium dose group,while the protein content of silencing information regulatory factor 6(Sirt-6)was increased in all Yuanhu intervention groups.Among them,the Yuanhu low-dose group was closer to the positive control group.Conclusion Yuanhu has a protective effect on acute liver injury.Yuanhu s protective effect on acute liver injury may be achieved by inhibiting NF-κB p65 protein,promoting Sirt-6 protein expression,and reducing LDH-A and CD68 expression.
作者
崔陇星
王阳阳
杨石磊
黄峰
李宏
宋忠兴
CUI Longxing;WANG Yangyang;YANG Shilei;HUANG Feng;LI Hong;SONG Zhongxing(First Clinical School of Shaanxi University of Traditional Chinese Medicine,Xianyang 712000,China;Affiliated Hospital of Shaanxi University of Traditional Chinese Medicine,Xianyang 712000,China;Shaanxi University of Traditional Chinese Medicine Pharmaceutical Factory,Xianyang 712000,China;Collaborative Innovation Center for Traditional Chinese Medicine at Shaanxi University of Traditional Chinese Medicine,Xianyang 712000,China)
出处
《现代中医药》
2025年第5期143-151,共9页
Modern Chinese Medicine
基金
陕西省重点研发计划(2020ZDLSF05-09)。
