转化生长因子βⅡ型受体胞外域融合蛋白的体内抗肝纤维化活性研究
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摘要
研究人血清白蛋白与转化生长因子βⅡ型受体胞外域的融合蛋白(HSA-eTGFBR2)的体内抗肝纤维化作用,以期寻找更加稳定的抗肝纤维化药物。通过CCl_4诱导构建小鼠肝纤维化模型,设正常组、模型组、阳性组、eTGFBR2治疗组、HSA-eTGFBR2治疗组和HSA组。经过苏木精-伊红染色、血清肝功能指标活性检测及Western blot等方法鉴定各组抗肝纤维化效果。结果显示:(1)CCl_4能引起肝脏结构紊乱、肝细胞坏死、胶原纤维增生,损伤肝功能,诱导纤维化;(2)HSA-eTGFBR2及其单体均能明显改善肝纤维化症状,减轻肝细胞损伤及胶原沉积,改善肝功能,降低肝脏中纤维化标志物α-SMA和COL I的表达水平,其改善肝纤维化效果与单体药物相当,而白蛋白几乎没有治疗效果;(3)与单体药物相比,HSA-eTGFBR2融合蛋白在降低给药频率的情况下能取得相当的治疗效果。综上表明HSA-eTGFBR2融合蛋白具有良好的抗肝纤维化效果,与单体药物相比,融合蛋白药物在低注射频率下也能取得较好的治疗效果,表明该融合蛋白药物半衰期更长、更稳定,具有更优的应用前景。
This study is performed to analyze the anti-liver fibrosis effect of the fusion protein of human serum albumin and extracellular domain of transforming growth factor beta type Ⅱ receptor(eTGFBR2) in vivo to looking for the more stable anti-liver fibrosis drug.The mice model of liver fibrosis was constructed by CCl_4 induction and the following groups are included in the study:the control group,CCl_4 model group,the positive control group,eTGFBR2 treatment group,HSA-eTGFBR2 treatment group,and HSA group.Hematoxylin eosin staining,serum liver function index detection,and western blot are used to identify the anti-liver fibrosis activities.The results showed that:(1) CCl_4 caused liver structure disorder,hepatocellular necrosis,collagen fibers proliferation,and induced liver fibrosis at last;(2) HSA-eTGFBR2 and its monomer drug improved the symptoms of liver fibrosis significantly,as well as reduced the damage of liver cells and collagen deposition,and recovered the liver basic structure to normal.Both of HSA-eTGFBR2 and its monomer drug improved liver function and reduced the expression level of liver fibrosis marker α-SMA and COL I.Moreover,the anti-liver fibrosis effect of the fusion protein is comparable to the monomer drug.In contrast,the albumin had no effect on therapeutic effect;(3) Reducing the injection frequency of HSA-eTGFBR2 achieved the comparable effects to the monomer drug with the normal injection frequency.In summary,the fusion protein HSA-eTGFBR2 has good anti-liver fibrosis effect.In addition,reducing the injection frequency of the fusion protein could also achieve the comparable treatment with the monomer drug,indicating that the fusion protein is stable and has longer half-lives and then a relatively positive application prospect in future.
This study is performed to analyze the anti-liver fibrosis effect of the fusion protein of human serum albumin and extracellular domain of transforming growth factor beta type Ⅱ receptor(eTGFBR2) in vivo to looking for the more stable anti-liver fibrosis drug.The mice model of liver fibrosis was constructed by CCl_4 induction and the following groups are included in the study:the control group,CCl_4 model group,the positive control group,eTGFBR2 treatment group,HSA-eTGFBR2 treatment group,and HSA group.Hematoxylin eosin staining,serum liver function index detection,and western blot are used to identify the anti-liver fibrosis activities.The results showed that:(1) CCl_4 caused liver structure disorder,hepatocellular necrosis,collagen fibers proliferation,and induced liver fibrosis at last;(2) HSA-eTGFBR2 and its monomer drug improved the symptoms of liver fibrosis significantly,as well as reduced the damage of liver cells and collagen deposition,and recovered the liver basic structure to normal.Both of HSA-eTGFBR2 and its monomer drug improved liver function and reduced the expression level of liver fibrosis marker α-SMA and COL I.Moreover,the anti-liver fibrosis effect of the fusion protein is comparable to the monomer drug.In contrast,the albumin had no effect on therapeutic effect;(3) Reducing the injection frequency of HSA-eTGFBR2 achieved the comparable effects to the monomer drug with the normal injection frequency.In summary,the fusion protein HSA-eTGFBR2 has good anti-liver fibrosis effect.In addition,reducing the injection frequency of the fusion protein could also achieve the comparable treatment with the monomer drug,indicating that the fusion protein is stable and has longer half-lives and then a relatively positive application prospect in future.
引文
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[2] Huang YH,Shi MN,Zheng WD,et al.Therapeutic effect of interleukin-10 on CCl4-induced hepatic fibrosis in rats[J].World J Gastroenterol,2006,12(9):1386-1391.
[3] Nakamuta M,Morizono S,Tsuruta S,et al.Remote delivery and expression of soluble type II TGF-beta receptor in muscle prevents hepatic fibrosis in rats[J].Int J Mol Med,2005,16(1):59-64.
[4] Zhao LM,Wang SZ.Therapeutic applications of small molecule kinase inhibitors in liver fibrosis[J].J China Pharm Univ(中国药科大学学报),2018,49(2):147-157.
[5] Bataller R,Brenner DA.Liver fibrosis[J].J Clin Invest,2005,115(2):209-218.
[6] George J,Roulot D,Koteliansky VE,et al.In vivo inhibition of rat stellate cell activation by soluble transforming growth factor beta type II receptor:a potential new therapy for hepatic fibrosis[J].Proc Natl Acad Sci U S A,1999,96(22):12719-12724.
[7] Wan AN,Miao YN,Peng L,et al.Binding and biologic characterization of recombinant human serum albumin-eTGFBR2 fusion protein expressed in CHO cells[J].Bioengineered,2017,8(5):600-612.
[8] Kawaida K,Matsumoto K,Shimazu H,et al.Hepatocyte growth factor prevents acute renal failure of accelerates renal regeneration in mice[J].Proc Natl Acad Sci U S A,1994,91(10):4357-4361.
[9] Kobayashi K.Summary of recombinant human serum albumin development[J].Biologicals,2006,34(1):55-59.
[10] Wan AN,Xu DS,Liu KD,et al.Efficient expression of stable recombinant human insulin-like growth factor-1 fusion with human serum albumin in Chinese hamster ovary cells[J].Prep Biochem Biotechnol,2017,47(7):678-686.
[11] Wan AN,Xu DD,Cai YF,et al.Anti-liver fibrosis activities of human insulin-like growth factor-1 in vitro[J].J China Pharm Univ(中国药科大学学报),2017,48(4):476-482.
[12] Clichici S,Olteanu D,Filip A,et al.Beneficial effects of silymarin after the discontinuation of CCl4-induced liver fibrosis[J].J Med Food,2016,19(8):789-797.
[13] Cao LB,Li B,Li ZJ,et al.The effect and mechanism of silymarin on liver fibrosis in mice[J].Chin Pharmacol Bull(中国药理学通报),2009,25(6):794-796.
[14] Yata Y,Gotwals P,Koteliansky V,et al.Dose-dependent inhibition of hepatic fibrosis in mice by a TGF-β soluble receptor:implications for antifibrotic therapy[J].Hepatology,2002,35(5):1022-1030.
[15] Castilla-Cortazar I,Garcia M,Muguerza B,et al.Hepatoprotective effects of insulin-like growth factor I in rats with carbon tetrachloride-induced cirrhosis[J].Gastroenterology,1997,113(5):1682-1691.