雷帕霉素介导P53选择性自噬对急性肝损伤和肝纤维化大鼠的干预作用

Intervention of rapamycin in acute liver injury and fibrosis rats by mediating P53 selective autophagy

目的观察四氯化碳(carbon tetrachloride,CCl_(4))诱导的急性肝损伤和肝纤维化大鼠应用雷帕霉素处理后肝脏炎症、纤维化程度及原代肝细胞P53和自噬相关蛋白P62、LC3B表达变化,探讨雷帕霉素对急性肝损伤和肝纤维化的干预作用及可能机制。方法36只SD雄性大鼠随机分为雷帕霉素组、模型组和对照组各12只。模型组、雷帕霉素组大鼠腹腔注射含体积分数40%CCl_(4)的橄榄油溶液2mL/kg,2次/周,连续4周,制备急性肝损伤、肝纤维化模型;对照组腹腔注射等量橄榄油。雷帕霉素组自造模第1天起腹腔注射雷帕霉素1.5mg/(kg·d),连续4周;对照组和模型组腹腔注射等量生理盐水。注射CCl_(4)第6、28天,3组随机各取6只大鼠,腹腔注射质量分数3%戊巴比妥钠1mL/kg麻醉后经下腔静脉取血,检测血清谷丙转氨酶(glutamic-pyruvic transaminase,GPT)、谷草转氨酶(glutamic-oxaloacetic transaminase,GOT)水平;行肝脏原位灌注,剪取肝左叶组织行HE染色,采用Ishak评分评估肝组织炎症坏死和纤维化程度,剩余肝组织提取原代肝细胞。取3组大鼠原代肝细胞,采用Western blot法检测P53、P62、LC3B蛋白相对表达量,采用免疫荧光染色法检测P53和LC3B共表达情况。结果(1)对照组注射CCl_(4)第6、28天肝细胞结构清晰、排列规则;模型组注射CCl_(4)第6天肝细胞水肿、气球样变性,汇管区炎症明显,注射CCl_(4)第28天肝纤维化形成;雷帕霉素组注射CCl_(4)第6天肝细胞炎症坏死程度较模型组明显减轻,注射CCl4第28天肝细胞炎症坏死和纤维化程度较模型组明显减轻。(2)注射CCl_(4)第6、28天Ishak炎症坏死评分及第28天Ishak纤维化评分在模型组[(12.17±1.94)、(8.33±3.27)、(3.17±0.75)分]、雷帕霉素组[(5.17±1.47)、(5.00±1.41)、(1.50±0.83)分]均高于对照组(0、0、0)(P<0.05),在雷帕霉素组均低于模型组(P<0.05)。(3)注射CCl_(4)第6、28天血清GPT、GOT水平在模型组[GPT(445.18±157.17)、(215.85±74.79)u/L,GOT(243.86±74.37)、(140.85±39.31)u/L]、雷帕霉素组[GPT(120.54±37.70)、(40.78±17.80)u/L,GOT(95.54±31.54)、(27.45±9.48)u/L]均高于对照组[GPT(8.34±3.90)、(9.87±5.15)u/L,GOT(4.84±3.14)、(5.04±3.49)u/L](P<0.05),在雷帕霉素组均低于模型组(P<0.05)。(4)注射CCl_(4)第6、28天,模型组原代肝细胞P62(0.973±0.422、1.213±0.390)、P53(1.790±0.473、2.529±2.215)蛋白相对表达量均高于雷帕霉素组[P62(0.324±0.155、0.496±0.191),P53(0.789±0.270、0.716±0.179)]、对照组[P62(0.166±0.050、0.492±0.193),P53(0.912±0.047、0.067±0.032)](P<0.05),LC3B(0.085±0.031、0.059±0.025)均低于雷帕霉素组(0.740±0.362、0.964±0.387)、对照组(0.319±0.077、0.310±0.141)(P<0.05)。(5)与雷帕霉素组和对照组比较,模型组注射CCl_(4)第6、28天P53表达升高,LC3B表达降低,且P53与LC3B共定位增加。结论CCl_(4)诱导的急性肝损伤、肝纤维化大鼠肝细胞P53自噬水平下降;雷帕霉素通过激活肝细胞P53选择性自噬减轻急性肝损伤、肝纤维化程度。

Objective To observe the liver inflammation and fibrosis degree,and the changes of P53and autophagy related protein P62and LC3Bafter rapamycin intervention in CCl_(4)-induced acute liver injury and fibrosis rats,and to investigate the intervening role of rapamycin and potential mechanism.Methods Thirty-six male SD rats were randomly divided into rapamycin group,model group and control group,with 12rats in each group.Model group and rapamycin group were intraperitoneally injected 2mL/kg of 40%carbon tetrachloride(CCl_(4))-olive oil,twice a week,for four weeks to establish the acute liver injury and fibrosis models,while control group was intraperitoneally injected olive oil.Rapamycin group was intraperitoneally injected 1.5 mg/(kg·d)rapamycin for four weeks,while control group and model group were intraperitoneally injected the same amount of normal saline.On the 6th and 28th day of CCl_(4) injection,six rats were randomly selected from each group and were anesthetized by intraperitoneal injection of 1 mL/kg 3%pentobarbital sodium,and blood was collected via the inferior vena cava.The serum glutamic-pyruvic transaminase(GPT)and glutamic-oxaloacetic transaminase(GOT)were detected.Liver in situ perfusion was done,and the left lobe tissue was collected.Liver necroinflammation and fibrosis were assessed via Ishak score,and the remained liver tissue was extracted the primary hepatocytes.The relative expressions of P53,P62and LC3Bwere detected by Western blot,and the coexpressions of P53and LC3Bwere detected by immunofluorescent staining.Results(1)On the 6th and 28th days of CCl_(4) injection,the structure of hepatocytes in control group was clear,and arranged regularly.In model group,the hepatocytes emerged edema and ballooning degeneration,inflammation was obvious in the portal area of liver tissue on the6th day,and CCl_(4)-induced liver fibrosis occurred on the 28th day.(2)Compared with model group,the necroinflammation on the 6th day and both necroinflammation and fibrosis on the 28th day were alleviated obviously.The Ishak necroinflammation scores on the 6th and 28th days and the Ishak fibrosis scores on the 28th days were higher in model group(12.17±1.94,8.33±3.27,3.17±0.75)than those in rapamycin group(5.17±1.47,5.00±1.41,1.50±0.83)and control group(0,0,0)(P<0.05),and lower in rapamycin group than those in model group(P<0.05).(3)On the 6th and 28th days of CCl_(4) injection,the serum levels of GPT and GOT were higher in model group[GPT:(445.18±157.17),(215.85±74.79)u/L;GOT:(243.86±74.37),(140.85±39.31)u/L]and rapamycin group[GPT:(120.54±37.70),(40.78±17.80)u/L;GOT:(95.54±31.54),(27.45±9.48)u/L]than those in control group[GPT:(8.34±3.90),(9.87±5.15)u/L;GOT:(4.84±3.14),(5.04±3.49)u/L](P<0.05),and lower in rapamycin group than those in model group(P<0.05).(4)On the 6th and 28th days of CCl_(4) injection,the relative expressions of P62and P53were higher in model group(P62:0.973±0.422,1.213±0.390;P53:1.790±0.473,2.529±2.215)than those in rapamycin group(P62:0.324±0.155,0.496±0.191;P53:0.789±0.270,0.716±0.179)and control group(P62:0.166±0.050,0.492±0.193;P53:0.912±0.047,0.067±0.032)(P<0.05),and LC3Blevels were lower in model group(0.085±0.031,0.059±0.025)than those in rapamycin group(0.740±0.362,0.964±0.387)and control group(0.319±0.077,0.310±0.141)(P<0.05).(5)On the 6th and 28th days,the P53expressions increased and the LC3Bexpression decreased in model group compared with rapamycin group and control group.LC3Bco-located with P53in rapamycin group.Conclusions P53autophagy decreases in rats with CCl_(4)-induced acute liver injury and fibrosis.Rapamycin alleviates acute liver injury and fibrosis by activating P53-selective autophagy in the hepatocytes.