摘要
目的研究铁死亡是否参与大鼠骨髓间充质干细胞(BMMSCs)减轻肝硬化的过程。方法2021年6月至12月,选取10只SD大鼠(军事医学科学院动物实验中心提供)腹腔注射橄榄油12周作为对照组,另30只SD大鼠腹腔注射含40%四氯化碳(CCl4)的橄榄油混合液8周建立大鼠肝硬化模型,采用随机数表法分为模型组、BMMSCs组及小檗碱组(n=10),分别注射磷酸缓冲盐溶液(PBS)、BMMSCs及小檗碱灌胃处理4周,干预结束后检测肝脏功能,苏木精-伊红(HE)及天狼星红染色检测肝脏病理,实时定量聚合酶链反应(RT-qPCR)检测肝脏组织Ⅳ型胶原蛋白基因α1(COL4a1)、透明质酸酶1(Hyal1)mRNA,RT-qPCR、蛋白质免疫印迹法(Western blot)及免疫组织化学检测长链脂酰辅酶A合成酶4(ACSL4)、铁蛋白重链1(FTH1)、谷胱甘肽过氧化物酶4(GPX4)的mRNA及蛋白表达,检测丙二醛(MDA)、还原型谷胱甘肽(GSH)及Fe^(2+)含量变化,两组间比较采用t检验。结果采用符合标准的BMMSCs,并成功建立大鼠肝硬化模型。BMMSCs组Ishak评分及分期均低于模型组[评分:(4.00±1.00)比(11.33±2.08)分、分期:(2.33±0.58)比(5.67±0.58),t=-5.500、-7.071,P<0.05],肝功能较模型组显著改善[ALT:(44.07±0.60)U/L比(897.47±14.25)U/L、AST:(65.83±1.39)U/L比(2158.73±36.85)U/L、ALB:(39.13±1.29)g/L比(29.47±1.62)g/L,t=-103.610、-98.309、8.096,P<0.05]。铁死亡相关结果显示,BMMSCs组较模型组ACSL4、FTH1蛋白表达均升高[ACSL4:(1.17±0.14)比(0.49±0.12)、FTH1:(0.94±0.07)比(0.38±0.07),t=6.458、9.667,P<0.05],但GPX4蛋白表达升高[(0.75±0.06)比(0.41±0.09),t=5.253,P<0.05],mRNA水平与蛋白表达呈现一致;BMMSCs组较模型组MDA及Fe^(2+)的生成均增加[MDA:(0.41±0.03)μmol/mg比(0.21±0.03)μmol/mg、Fe^(2+):(5.84±0.26)nmol/mg比(2.64±0.14)nmol/mg,t=8.464、18.700,P<0.05],GSH的生成减少[(102.08±1.30)μg/ml比(220.11±1.68)μg/ml,t=-96.163,P<0.05],差异均有统计学意义,铁死亡指标表达上小檗碱组与BMMSCs组相似。结论铁死亡参与了BMMSCs改善大鼠肝硬化过程,其机制可能与BMMSCs破坏过氧化与抗过氧化平衡相关。
Objective To study whether ferroptosis is involved in the process of bone marrow mesenchymal stem cells(BMMSCs)alleviating liver cirrhosis in rats.Methods From June to December 2021,10 SD rats were selected as control group by intraperitoneal injection of olive oil for 12 weeks.Another 30 SD rats were seleted to establish liver cirrhosis models by intraperitoneal injection of olive oil containing 40%carbon tetrachloride(CCl4)for 8 weeks,and randomly divided into model group,BMMSCs group and berberine group(n=10).Phosphate buffer salt solution(PBS),BMMSCs and berberine were given by injection or gavage for 4 weeks respectively.After intervention,liver function was measured,liver pathology was detected by hematoxylin-eosin(HE)and Sirius red staining.Real-time quantitative polymerase chain reaction(RT-qPCR)was used to detect type IV collagen(COL4a1),hyaluronidase 1(Hyal1)mRNA,RT-qPCR,Western blot and immunohistochemistry to detect mRNA and protein expression of long-chain acyl-CoA synthetases 4(ACSL4),ferritin heavy chain 1(FTH1)and glutathione peroxidase 4(GPX4)in liver tissues.Malondialdehyde(MDA),reduced glutathione(GSH)and Fe^(2+)were detected.T-test was used for comparison between two groups.Results In this experiment,BMMSCs were compliant,and cirrhotic rat model was successfully established.Ishak score and staging in BMMSCs group were significantly lower than model group[score:(4.00±1.00)vs.(11.33±2.08),staging:(2.33±0.58)vs.(5.67±0.58),t=-5.500,-7.071,P<0.05],liver function in model group was significantly improved compared with model group[ALT:(44.07±0.60)U/L vs.(897.47±14.25)U/L,AST:(65.83±1.39)U/L vs.(2158.73±36.85)U/L,ALB:(39.13±1.29)g/L vs.(29.47±1.62)g/L,t=-103.610,-98.309,8.096,P<0.05].Ferroptosis index showed that expression of ACSL4 and FTH1 protein in BMMSCs group was higher than model group[ACSL4:(1.17±0.14)vs.(0.49±0.12),FTH1:(0.94±0.07)vs.(0.38±0.07),t=6.458,9.667,P<0.05],but expression of GPX4 protein increased[(0.75±0.06)vs.(0.41±0.09),t=5.253,P<0.05],mRNA level was consistent with protein.compared with model group,productions of MDA and Fe^(2+)in BMMSCs group increased[MDA:(0.41±0.03)μmol/mg vs.(0.21±0.03)μmol/mg,Fe^(2+):(5.84±0.26)nmol/mg vs.(2.64±0.14)nmol/mg,t=8.464,18.700,P<0.05],while GSH decreased[(102.08±1.30)μg/ml vs.(220.11±1.68)μg/ml,t=-96.163,P<0.05],berberine group was similar to BMMSCs group.Conclusion Ferroptosis is involved in the improvement of liver cirrhosis by BMMSCs in rats,and its mechanism may be related to BMMSCs destroying balance of peroxidation and anti-peroxidation.
作者
王玉鑫
袁梦淑
田小荣
翟浩宇
林玲
吴龙龙
左怀文
宋红丽
Wang Yuxin;Yuan Mengshu;Tian Xiaorong;Zhai Haoyu;Lin Ling;Wu Longlong;Zuo Huaiwen;Song Hongli(First Center Clinical College,Tianjin Medical University,Tianjin 300192,China;The Medical College,Nankai University,Tianjin 300074,China;Department of Organ Transplantation,Tianjin First Central Hospital,Tianjin Key Laboratory of Organ Transplantation,Tianjin Organ Transplantation Clinical Medical Research Center,Tianjin 300192,China)
出处
《中华实验外科杂志》
CAS
北大核心
2022年第10期1879-1883,共5页
Chinese Journal of Experimental Surgery
基金
国家自然科学基金(82070639、81670574)。
关键词
铁死亡
骨髓间充质干细胞
肝硬化
小檗碱
大鼠
Ferroptosis
Bone marrow mesenchymal stem cells
Liver cirrhosis
Berberine
Rat
作者简介
通信作者:宋红丽,Email:hlsong26@163.com。
