骨髓间充质干细胞移植对四氯化碳大鼠肝损伤的影响及一贯煎的干预作用
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摘要
该研究观察BMSCs早期尾静脉移植对四氯化碳(carbon tetrachloride,CCl_4)肝损伤模型大鼠的影响以及一贯煎的干预作用。雌性Wistar大鼠65只,随机分为正常组,模型组,细胞移植组,一贯煎组和一贯煎加细胞移植组。造模各组大鼠按3 mL·kg~(-1)每周2次腹腔注射50%CCl_4橄榄油溶液,共9次;细胞移植组和一贯煎加细胞移植组在首次注射CCl_4后立即经尾静脉注入第3代BMSCs 1×10~6个;用药组灌胃一贯煎水煎液。结果可见细胞移植和一贯煎均可降低首次造模48 h的血清ALT和AST。造模4周后,模型组血清ALT,AST,γ-GT活性增高,TBIL含量增高;肝组织SOD活性降低,MDA和TG含量增加;肝组织病理可见大泡型脂肪变性,油红O染色可见大量脂滴,天狼猩红染色未见明显胶原增生,血清TNF-α和IL-6含量升高(P<0.05)。BMSC细胞移植组大鼠肝功能和组织病理较模型组进一步恶化,肝组织可见少量胶原增生,肝组织SOD活性更低,MDA和TG含量进一步增高(P<0.05),血清TNF-α和IL-6含量较模型组升高(P<0.05);经一贯煎干预后,各检测指标均较模型组和细胞移植组有所改善。SRY原位杂交检测显示,BMSC移植后在心、肝、脾、肺、肾均可检测到SRY阳性表达,但以肝脏最多,经一贯煎干预后,SRY阳性细胞在肝脏表达减少。Western blot结果显示正常组高表达Wnt和β-catenin,CCl_4造模后表达明显减少,细胞移植后有进一步降低的趋势,一贯煎组干预后则表达增高。该研究表明早期尾静脉BMSCs移植可加重大鼠CCl_4肝损伤,促进肝纤维化形成,一贯煎可改善BMSCs移植加重的CCl_4肝损伤,其机制与减少BMSCs归巢肝脏,上调Wnt/β-catenin信号通路有关。
The aim of this paper was to observe the function of bone marrow mesenchymal stem cell(BMSC) transplantation in process of liver injury induced by carbon tetrachloride(CCl_4) in vivo and the intervention effect of Yiguanjian(YGJ), a compound of Chinese herbal medicine. Wistar rats were randomly divided into five groups: normal group, model group, cell transplantation(CT) group, YGJ group and cell transplantation plus Yiguanjian(CTY) group. Liver injury was induced through subcutaneous injection with CCl_4 at a dose of 3 mL·kg~(-1) body weight for 4 weeks, twice a week. They were injected for a total of 9 times. After the first injection with CCl_4, rats in the CT group and CTY group were injected with the third-generation BMSCs at dose 1×10~6(suspended in 1 mL saline solution) via tail vein. Rats in the YGJ and CTY groups were also intragastrically administered with Yiguanjian once a day. Rat serum ALT and AST activities were increased significantly on the second day after injection with CCl_4, while BMSC transplantation and Yiguanjian decreased their activities. After 4 weeks of injection with CCl_4, serum ALT, AST and γ-GT activities, and serum TNF-α and IL-6 expressions were increased, while TBIL were decreased in model rats compared with normal rats. Meanwhile, liver cells edema, plasmatic loose, and numerous lipid droplets were observed in rats of the model group. BMSC transplantation aggravated liver injury compared with model rats, which was manifested by decreasing SOD activity, increased MDA, TG, TNF-α and IL-6 levels, and aggravated necrosis level of hepatocytes, fusion of lipid droplets, and collagen deposition in liver tissue. Yiguanjian decreased liver injury induced by CCl_4 alone and CCl_4 plus BMSC transplantation. SRY gene in situ hybridization method was used to detect the positive SRY expressions in heart, liver, spleen, lung and kidney, especially in liver, while Yiguangjian decreased liver SRY expression. Wnt and β-catenin showed high expressions in rats of normal group, which were decreased significantly in rats of models group, while Yiguanjian increased their expressions. In conclusion, BMSC transplantation could exacerbate liver injury, while Yiguanjian could protect liver injury induced by CCl_4 and BMSC transplantation, which was related to decreasing the homing of BMSCs to liver and up-regulating Wnt/β-catenin signaling pathway.
The aim of this paper was to observe the function of bone marrow mesenchymal stem cell(BMSC) transplantation in process of liver injury induced by carbon tetrachloride(CCl_4) in vivo and the intervention effect of Yiguanjian(YGJ), a compound of Chinese herbal medicine. Wistar rats were randomly divided into five groups: normal group, model group, cell transplantation(CT) group, YGJ group and cell transplantation plus Yiguanjian(CTY) group. Liver injury was induced through subcutaneous injection with CCl_4 at a dose of 3 mL·kg~(-1) body weight for 4 weeks, twice a week. They were injected for a total of 9 times. After the first injection with CCl_4, rats in the CT group and CTY group were injected with the third-generation BMSCs at dose 1×10~6(suspended in 1 mL saline solution) via tail vein. Rats in the YGJ and CTY groups were also intragastrically administered with Yiguanjian once a day. Rat serum ALT and AST activities were increased significantly on the second day after injection with CCl_4, while BMSC transplantation and Yiguanjian decreased their activities. After 4 weeks of injection with CCl_4, serum ALT, AST and γ-GT activities, and serum TNF-α and IL-6 expressions were increased, while TBIL were decreased in model rats compared with normal rats. Meanwhile, liver cells edema, plasmatic loose, and numerous lipid droplets were observed in rats of the model group. BMSC transplantation aggravated liver injury compared with model rats, which was manifested by decreasing SOD activity, increased MDA, TG, TNF-α and IL-6 levels, and aggravated necrosis level of hepatocytes, fusion of lipid droplets, and collagen deposition in liver tissue. Yiguanjian decreased liver injury induced by CCl_4 alone and CCl_4 plus BMSC transplantation. SRY gene in situ hybridization method was used to detect the positive SRY expressions in heart, liver, spleen, lung and kidney, especially in liver, while Yiguangjian decreased liver SRY expression. Wnt and β-catenin showed high expressions in rats of normal group, which were decreased significantly in rats of models group, while Yiguanjian increased their expressions. In conclusion, BMSC transplantation could exacerbate liver injury, while Yiguanjian could protect liver injury induced by CCl_4 and BMSC transplantation, which was related to decreasing the homing of BMSCs to liver and up-regulating Wnt/β-catenin signaling pathway.
引文
[1] Qu M, Yuan X, Liu D, et al. Bone marrow-derived mesenchymal stem cells attenuate immune-mediated liver injury and compromise virus control during acute hepatitis B virus infection in mice[J]. Stem Cells Dev, 2017, 26(11): 818.
[2] 皇甫铭一, 李黎明, 刘惠娜, 等. 复合骨髓间充质干细胞的多肽-透明质酸支架的构建与表征[J]. 中国现代应用药学, 2017, 34(12): 1647.
[3] Chang Y J, Liu J W, Lin P C, et al. Mesenchymal stem cells facilitate recovery from chemically induced liver damage and decrease liver fibrosis[J]. Life Sci, 2009, 85(13/14): 517.
[4] Dahlke M H, Popp F C, Bahlmann F H, et al. Liver regeneration in a retrorsine/CCl4-induced acute liver failure model: do bone marrow-derived cells contribute?[J]. J Hepatol, 2003, 39(3): 365.
[5] Forbes S J, Russo F P, Rey V, et al. A significant proportion of myofibroblasts are of bone marrow origin in human liver fibrosis[J]. Gastroenterology, 2004, 126(4): 955.
[6] 慕永平, 刘平, 王磊, 等. 一贯煎影响CCl4大鼠肝硬化形成期肝组织基因表达谱的效应机制研究[J]. 世界科学技术——中医药现代化, 2007, 9(3): 43.
[7] 朱英, 刘平. 一贯煎对DMN肝纤维化大鼠肝卵圆细胞增殖分化的影响[J]. 大连医科大学学报, 2011, 33(1): 11.
[8] 谢斌,谢雄,饶斌,等.一贯煎对肝癌JAK1/STAT1信号通路及其下游凋亡相关蛋白表达的影响[J].中国实验方剂学杂志,2017,23(18):100.
[9] Zhu H, Guo Z K, Jiang X X, et al. A protocol for isolation and culture of mesenchymal stem cells from mouse compact bone[J]. Nat Protoc, 2010, 5(3): 550.
[10] 熊全, 冯吉, 王军, 等. 3种不同途径移植骨髓间充质干细胞治疗肝硬化模型大鼠的效果比较[J]. 第三军医大学学报, 2011, 33(8): 804.
[11] Li J, Zhang L, Xin J, et al. Immediate intraportal transplantation of human bone marrow mesenchymal stem cells prevents death from fulminant hepatic failure in pigs[J]. Hepatology, 2012, 56(3): 1044.
[12] Sakaida I, Terai S, Yamamoto N, et al. Transplantation of bone marrow cells reduces CCl4-induced liver fibrosis in mice[J]. Hepatology, 2004, 40(6): 1304.
[13] Russo F P, Alison M R, Bigger B W, et al. The bone marrow functionally contributes to liver fibrosis[J]. Gastroenterology, 2006, 130(6): 1807.
[14] Popp F C, Slowik P, Eggenhofer E, et al. No contribution of multipotent mesenchymal stromal cells to liver regeneration in a rat model of prolonged hepatic injury[J]. Stem Cells, 2007, 25(3): 639.
[15] 周伟, 陈鹏飞, 吴小翎, 等. 骨髓间充质干细胞对实验性肝纤维化大鼠的作用及其机制[J]. 中国生物制品学杂志, 2012, 25(2): 176.
[16] 葛建彬, 卢红建, 宋新建, 等. 枸杞多糖对小鼠脑缺血再灌注损伤的保护作用及其抑制NF-κB,TNF-α,IL-6和IL-1β表达的机制[J].中国中药杂志, 2017, 42(2): 326.
[17] 王凯, 眭丹娟, 王长锁, 等. 5种石斛对四氯化碳致小鼠急性肝损伤的保护作用[J].中国中药杂志, 2017, 42(10): 1945.
[18] Yang Y Y, Lee P C, Huang Y T, et al. Involvement of the HIF-1alpha and Wnt/beta-catenin pathways in the protective effects of losartan on fatty liver graft with ischaemia/reperfusion injury[J]. Clin Sci (Lond), 2014, 126(2): 163.
[2] 皇甫铭一, 李黎明, 刘惠娜, 等. 复合骨髓间充质干细胞的多肽-透明质酸支架的构建与表征[J]. 中国现代应用药学, 2017, 34(12): 1647.
[3] Chang Y J, Liu J W, Lin P C, et al. Mesenchymal stem cells facilitate recovery from chemically induced liver damage and decrease liver fibrosis[J]. Life Sci, 2009, 85(13/14): 517.
[4] Dahlke M H, Popp F C, Bahlmann F H, et al. Liver regeneration in a retrorsine/CCl4-induced acute liver failure model: do bone marrow-derived cells contribute?[J]. J Hepatol, 2003, 39(3): 365.
[5] Forbes S J, Russo F P, Rey V, et al. A significant proportion of myofibroblasts are of bone marrow origin in human liver fibrosis[J]. Gastroenterology, 2004, 126(4): 955.
[6] 慕永平, 刘平, 王磊, 等. 一贯煎影响CCl4大鼠肝硬化形成期肝组织基因表达谱的效应机制研究[J]. 世界科学技术——中医药现代化, 2007, 9(3): 43.
[7] 朱英, 刘平. 一贯煎对DMN肝纤维化大鼠肝卵圆细胞增殖分化的影响[J]. 大连医科大学学报, 2011, 33(1): 11.
[8] 谢斌,谢雄,饶斌,等.一贯煎对肝癌JAK1/STAT1信号通路及其下游凋亡相关蛋白表达的影响[J].中国实验方剂学杂志,2017,23(18):100.
[9] Zhu H, Guo Z K, Jiang X X, et al. A protocol for isolation and culture of mesenchymal stem cells from mouse compact bone[J]. Nat Protoc, 2010, 5(3): 550.
[10] 熊全, 冯吉, 王军, 等. 3种不同途径移植骨髓间充质干细胞治疗肝硬化模型大鼠的效果比较[J]. 第三军医大学学报, 2011, 33(8): 804.
[11] Li J, Zhang L, Xin J, et al. Immediate intraportal transplantation of human bone marrow mesenchymal stem cells prevents death from fulminant hepatic failure in pigs[J]. Hepatology, 2012, 56(3): 1044.
[12] Sakaida I, Terai S, Yamamoto N, et al. Transplantation of bone marrow cells reduces CCl4-induced liver fibrosis in mice[J]. Hepatology, 2004, 40(6): 1304.
[13] Russo F P, Alison M R, Bigger B W, et al. The bone marrow functionally contributes to liver fibrosis[J]. Gastroenterology, 2006, 130(6): 1807.
[14] Popp F C, Slowik P, Eggenhofer E, et al. No contribution of multipotent mesenchymal stromal cells to liver regeneration in a rat model of prolonged hepatic injury[J]. Stem Cells, 2007, 25(3): 639.
[15] 周伟, 陈鹏飞, 吴小翎, 等. 骨髓间充质干细胞对实验性肝纤维化大鼠的作用及其机制[J]. 中国生物制品学杂志, 2012, 25(2): 176.
[16] 葛建彬, 卢红建, 宋新建, 等. 枸杞多糖对小鼠脑缺血再灌注损伤的保护作用及其抑制NF-κB,TNF-α,IL-6和IL-1β表达的机制[J].中国中药杂志, 2017, 42(2): 326.
[17] 王凯, 眭丹娟, 王长锁, 等. 5种石斛对四氯化碳致小鼠急性肝损伤的保护作用[J].中国中药杂志, 2017, 42(10): 1945.
[18] Yang Y Y, Lee P C, Huang Y T, et al. Involvement of the HIF-1alpha and Wnt/beta-catenin pathways in the protective effects of losartan on fatty liver graft with ischaemia/reperfusion injury[J]. Clin Sci (Lond), 2014, 126(2): 163.