摘要
目的:探讨瑞博西林(Ribociclib)在脓毒症急性肾损伤(AKI)炎症反应中的作用及可能机制。方法:①按随机数字表法将20只成年雄性C57BL/6小鼠分为假手术组(Sham组;只开腹,不结扎盲肠、穿孔,假手术前12 h灌胃乳酸钠缓冲液)、瑞博西林对照组(只灌胃瑞博西林150 mg/kg)、盲肠结扎穿孔术(CLP)致脓毒症模型组(CLP组;CLP术前12 h灌胃乳酸钠缓冲液)和瑞博西林预处理组(CLP术前12 h灌胃瑞博西林150 mg/kg),每组5只。各组于术后12 h取肾组织,采用苏木素-伊红(HE)染色后观察肾组织病理学改变;采用酶联免疫吸附试验(ELISA)检测肾组织匀浆和细胞培养基上清液中炎性因子肿瘤坏死因子-α(TNF-α)和白细胞介素-6(IL-6)水平;用蛋白质免疫印迹试验(Western Blot)检测周期相关蛋白磷酸化视网膜母细胞瘤蛋白(p-Rb)、凋亡相关蛋白Bcl-2和Bax表达。②用TCMK-1肾小管上皮细胞株进行体外验证实验,将细胞分为空白对照组、瑞博西林组(5μmol/L瑞博西林处理24 h)、脂多糖(LPS)组(200 mg/L的LPS培养基处理6 h)、瑞博西林+LPS组(用5μmol/L瑞博西林处理18 h后更换为含有5μmol/L瑞博西林和200 mg/L LPS的培养基联合处理6 h)。采用ELISA法检测上清液中炎性因子水平,用Western Blot检测细胞中p-Rb、Bcl-2、Bax、自噬相关蛋白微管关联蛋白1轻链3b(LC3bⅡ、LC3bⅠ)和p62、磷酸化蛋白激酶B(p-AKT)、磷酸化哺乳动物雷帕霉素靶蛋白(p-mTOR)表达。结果:①动物实验显示,与Sham组相比,CLP可显著损伤小鼠肾脏组织,增加肾组织TNF-α、IL-6水平,降低肾组织p-Rb表达和Bcl-2/Bax比值;而瑞博西林对照组各指标与Sham组比较差异无统计学意义。与CLP组相比,瑞博西林预处理后小鼠肾组织损伤明显改善,病理学评分明显降低(分:1.48±0.16比2.68±0.16,P<0.01),肾组织炎性因子水平显著降低〔TNF-α(ng/g):340.55±34.96比745.08±58.86,IL-6(mg/g):17.33±1.01比114.20±20.49,均P<0.01〕,p-Rb表达进一步下降(p-Rb/β-tubulin:0.14±0.01比0.73±0.06,P<0.01),Bcl-2/Bax比值增加(0.89±0.06比0.62±0.10,P<0.01)。②体外实验显示,与空白对照组相比,LPS可导致TCMK-1细胞释放TNF-α和IL-6增加,p-Rb表达降低,Bcl-2/Bax比值和LC3bⅡ/Ⅰ比值减小,p62、p-AKT和p-mTOR表达升高;瑞博西林处理TCMK-1细胞可使p-Rb表达下降。与LPS组相比,瑞博西林+LPS组TCMK-1细胞释放TNF-α和IL-6减少〔TNF-α(ng/L):2.73±0.23比4.96±0.10,IL-6(ng/L):36.05±5.83比53.78±24.08,均P<0.01〕,p-Rb表达进一步降低(p-Rb/β-tubulin:0.25±0.05比0.65±0.05,P<0.01),Bcl-2/Bax比值和LC3bⅡ/Ⅰ比值增加(Bcl-2/Bax比值:1.01±0.07比0.73±0.05,LC3bⅡ/Ⅰ比值:2.08±0.31比1.04±0.01,均P<0.05),p62、p-AKT和p-mTOR表达降低(p62/β-tubulin:0.59±0.01比1.09±0.08,p-AKT/β-tubulin:0.61±0.03比1.20±0.06,p-mTOR/β-tubulin:0.50±0.05比1.15±0.08,均P<0.01)。结论:瑞博西林预处理可减轻脓毒症致AKI,mTOR/AKT通路可能参与了瑞博西林对肾脏的保护作用。
Objective To investigate the role of Ribociclib in sepsis induced-acute kidney injury(AKI)and its possible mechanisms.Methods①Twenty adult male C57BL/6 mice were divided into sham operation group(Sham group;only open the abdomen without ligating or perforating the cecum,administered with sodium lactate buffer 12 hours before the sham operation),Ribociclib control group(administered with 150 mg/kg Ribociclib),cecal ligation and puncture(CLP)group(sepsis model induced by CLP;lactate buffer was given by intragastric administration 12 hours before CLP),and Ribociclib pretreatment group(administered with 150 mg/kg Ribociclib 12 hours before CLP)according to random number table,with 5 mice in each group.Kidneys were harvested 12 hours after the operation.Pathological changes in kidney were observed by hematoxylin-eosin(HE)staining.Tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)levels in mice kidney homogenate were measured by enzyme linked immunosorbent assay(ELISA).Western Blot was used to detect the expression of cell cycle-related protein phosphorylate retinoblastoma protein(p-Rb),apoptosis-related protein Bcl-2 and Bax.②Mouse renal tubular epithelial(TCMK-1)cell line was used for in vitro experiment.The cells were divided into control group,Ribociclib group(treated with 5μmol/L Ribociclib for 24 hours),lipopolysaccharide(LPS)group(treated with 200 mg/L LPS for 6 hours),Ribociclib+LPS group(replaced with the medium containing 5μmol/L Ribociclib and 200 mg/L LPS for 6 hours after exposing with 5μmol/L Ribociclib for 18 hours).Inflammatory cytokines in cell culture medium were detected by ELISA.The expression of p-Rb,Bcl-2 and Bax,autophagy-related proteins microtubule associated protein 1 light chain LC3b(LC3bⅡ,LC3bⅠ)and p62,phosphate protein kinase B(p-AKT),phosphorylated mammalian target of rapamycin(p-mTOR)were measured by Western Blot.Results①Animal experiments showed that,compared with the Sham group,the kidney tissue of mice were significantly damaged,the levels of TNF-αand IL-6 were increased,the expressions of p-Rb and Bcl-2/Bax ratio were decreased in kidney tissue in CLP group;but there was no significant difference in indexes between Ribociclib control group and Sham group.Compared with the CLP group,kidney injury in mice pretreated with Ribociclib was significantly ameliorated,the pathological score was significantly decreased(1.48±0.16 vs.2.68±0.16,P<0.01),the levels of TNF-αand IL-6 in kidney homogenate were significantly decreased[TNF-α(ng/g):340.55±34.96 vs.745.08±58.86,IL-6(mg/g):17.33±1.01 vs.114.20±20.49,both P<0.01],the expression of p-Rb was furtherly decreased(p-Rb/β-tubulin:0.14±0.01 vs.0.73±0.06,P<0.01),Bcl-2/Bax ratio was increased(0.89±0.06 vs.0.62±0.10,P<0.01).②In vitro experiments showed that,compared with the control group,the releases of TNF-αand IL-6 were increased,the expression of p-Rb was decreased,the ratios of Bcl-2/Bax and LC3bⅡ/Ⅰwere decreased,the expressions of p62,p-AKT and p-mTOR were increased in LPS group;the expression of p-Rb was decreased after Ribociclib treatment in TCMK-1 cells.Compared with the LPS group,TNF-αand IL-6 were decreased[TNF-α(ng/L):2.73±0.23 vs.4.96±0.10,IL-6(ng/L):36.05±5.83 vs.53.78±24.08,both P<0.01],the expression of p-Rb was furtherly decreased(p-Rb/β-tubulin:0.25±0.05 vs.0.65±0.05,P<0.01),the ratios of Bcl-2/Bax and LC3bⅡ/Ⅰwere increased(Bcl-2/Bax:1.01±0.07 vs.0.73±0.05,LC3bⅡ/Ⅰ:2.08±0.31 vs.1.04±0.01,both P<0.05),the expressions of p62,p-AKT and p-mTOR were decreased(p62/β-tubulin:0.59±0.01 vs.1.09±0.08,p-AKT/β-tubulin:0.61±0.03 vs.1.20±0.06,p-mTOR/β-tubulin:0.50±0.05 vs.1.15±0.08,all P<0.01)in the Ribociclib+LPS group.Conclusion Ribociclib pretreatment ameliorated sepsis-induced AKI and AKT/mTOR pathway may be involved in the protective role of Ribociclib on kidney.
作者
王倩
龚晓莹
贾佳
李国福
Wang Qian;Gong Xiaoying;Jia Jia;Li Guofu(Department of Critical Care Medicine,Shengjing Hospital of China Medical University,Shenyang 110004,Liaoning,China)
出处
《中华危重病急救医学》
CAS
CSCD
北大核心
2020年第2期204-209,共6页
Chinese Critical Care Medicine
基金
国家自然科学基金(81471847)
辽宁省自然科学基金(20180550267,2014021003)
辽宁省沈阳市科技计划项目(F14-158-9-40)。
