摘要
目的评价腺苷酸活化蛋白激酶(AMPK)在小檗碱减轻肾缺血再灌注小鼠肾纤维化中的作用及其与铁死亡的关系。方法SPF级健康成年雄性C57BL/6小鼠24只,8~10周龄,体重20~25 g,采用随机数字表法分为4组(n=6):假手术组(S组)、肾缺血再灌注组(I/R组)、小檗碱组(B组)和小檗碱+AMPK抑制剂Compound C组(BC组)。采用夹闭左肾肾蒂45 min后恢复灌注的方法制备小鼠肾缺血再灌注模型。B组和BC组术前14 d连续进行小檗碱100 mg/kg灌胃,BC组术前3 d连续腹腔注射Compound C 0.2 mg/kg,S组和I/R组给予等容量生理盐水,于再灌注第14天眶静脉采集血标本,检测血清Cr和BUN浓度,随后处死小鼠取肾组织,检测Fe^(2+)、MDA含量和SOD活性,ELISA法检测IL-β和TNF-α含量,光镜下观察病理学结果,Western blot法检测p-AMPKα、长链脂酰辅酶A合成酶4(ACSL4)和谷胱甘肽过氧化物酶(GPX4)的表达。透射电镜下观察细胞线粒体超微结构。结果与S组比较,I/R组、B组和BC组血清Cr和BUN浓度、肾小管损伤评分和肾纤维化评分升高,I/R组和BC组肾组织MDA、IL-1β和TNF-α含量升高,SOD活性降低,I/R组和B组肾组织p-AMPKα和ACSL4、BC组ACSL4、B组GPX4表达上调,I/R组和BC组GPX4表达下调,I/R组和BC组Fe^(2+)含量升高(P<0.05);与I/R组比较,B组和BC组血清Cr和BUN浓度、肾小管损伤评分和肾纤维化评分降低,B组肾组织MDA、IL-1β和TNF-α含量降低,SOD活性升高,B组肾组织p-AMPKα和GPX4、BC组ACSL4表达上调,BC组肾组织p-AMPKα和GPX4、B组ACSL4表达下调(P<0.05);与B组比较,BC组血清Cr和BUN浓度、肾小管损伤评分和肾纤维化评分升高,MDA、IL-1β和TNF-α含量升高,SOD活性降低,p-AMPKα和GPX4表达下调,ACSL4表达上调,Fe^(2+)含量升高(P<0.05)。BC组肾组织病理学损伤和超微结构损伤较B组加重。结论小檗碱可明显减轻肾缺血再灌注小鼠肾纤维化,其机制与AMPK激活进而抑制铁死亡有关。
Objective To evaluate the role of Adenosine 5′-monophosphate(AMP)-activated protein kinase(AMPK)in berberine-induced reduction of renal fibrosis in a mouse model of renal ischemia-reperfusion(I/R)and the relationship with ferroptosis.Methods Twenty-four SPF healthy adult male C57BL/6 mice,aged 8-10 weeks,weighing 20-25 g,were divided into 4 groups(n=6 each)by a random number table method:sham operation group(group S),renal I/R group(I/R group),berberine group(B group),and berberine plus AMPK inhibitor Compound C group(BC group).The renal I/R model was established by clamping the renal pedicle of the left kidney for 45 min followed by reperfusion in anesthetized mice.In B and BC groups,berberine 100 mg/kg was given for continuous intragastric administration at 14 days before surgery,Compound C 0.2 mg/kg was intraperitoneally injected continuously at 3 days before surgery in group BC,and the equal volume of normal saline was given instead in S and I/R groups.Blood samples were collected from the orbital vein on the 14th day of reperfusion to measure serum Cr and BUN concentrations.Then the mice were sacrificed and kidney tissues were removed for determination of the Fe^(2+)and malondialdehyde(MDA)contents and superoxide dismutase(SOD)activity,interleukin-1beta(IL-1β)and tumor necrosis factor-alpha(TNF-α)contents(by enzyme-linked immunosorbent assay),and expression of phosphorylated AMPKα(p-AMPKα),Acyl-CoA synthetase long-chain familymember4(ACSL4)and recombinant glutathione peroxidase 4(GPX4)(by Western blot)and for examination of the pathological changes(with a light microscope)and ultrastructure of cell mitochondria(with a transmission electron microscope).The damage to the renal tubules was scored.Results Compared with group S,serum Cr and BUN concentrations,renal tubular damage score and renal fibrosis score were significantly increased in I/R,B and BC groups,the contents of MDA,IL-1βand TNF-αwere significantly increased,and SOD activity was decreased in I/R and BC groups,the expression of p-AMPKαand A
作者
孙琳琳
郝明月
盛明薇
吕景淑
翁亦齐
喻文立
杜洪印
Sun Linlin;Hao Mingyue;Sheng Mingwei;Lyu Jingshu;Weng Yiqi;Yu Wenli;Du Hongyin(School of Medical,Nankai University,Tianjin 300071,China;Tianjin First Central Hospital Clinic Institute,Tianjin Medical University,Tianjin 300192,China;Department of Anesthesiology,Tianjin First Center Hospital,School of Medical,Nankai University,Tianjin 300192,China)
出处
《中华麻醉学杂志》
CAS
CSCD
北大核心
2020年第11期1392-1396,共5页
Chinese Journal of Anesthesiology
基金
国家自然科学基金(82072219,81700659,81700569)
天津市自然科学基金(18JCYBJC27500)
天津市卫生健康委员会科技项目(ZC20177,ZC20052)。
