藏红花酸对TGF-β1刺激的人肝星状细胞LX-2信号转导通路的影响
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摘要
目的:研究藏红花酸(crocetin)对转化生长因子-β1(TGF-β1)刺激的人肝星状细胞LX-2信号转导通路的影响。方法:体外培养LX-2细胞,随机设立空白对照组,模型组,藏红花酸低剂量组,藏红花酸中剂量组,藏红花酸高剂量组,用含10%血清的1640培养液培养48 h,MTT法测各组细胞增殖,蛋白免疫印迹测定各组细胞α-SMA蛋白的表达,实时荧光定量PCR法测各组细胞Smad2、Smad3、Smad7mRNA的表达。结果:与对照组比较,TGF-β1刺激后人肝星状细胞LX-2增殖作用明显,且上调α-SMA蛋白、Smad2mRNA、Smad3mRNA表达,下调Smad7mRNA表达(P<0.01)与模型组比较,藏红花酸组均能抑制LX-2细胞增殖,并呈剂量依赖性,高、中剂量组作用明显,能够明显下调α-SMA蛋白、Smad2mRNA、Smad3mRNA表达,上调Smad7mRNA表达,差异具有统计学意义(P<0.05)。结论:藏红花酸对TGF-β1刺激的LX-2细胞中Smad7mRNA具有上调作用,对Smad2、Smad3mRNA具有下调作用,其抗肝纤维化作用可能与抑制TGF-β1/Smads信号转导通路有关。
Objective: To study the effects of crocetin on TGF-β1 stimulated signal transduction pathway in human hepatic stellate cells(HSC-LX2). Methods: HSC-LX2 cells were cultured in vitro and randomly divided into 5 groups: blank group, model group,low-dose crocetin group, medium-dose group, high-dose crocetin group. Then cells proliferation were measured by MTT, the expression of α-SMA was measured by Western blot, the gene expression of Smad2/Smad3/Smad7 m RNA were measured by Real-time PCR. Results:Compared with control group, the model group promotes cells proliferation. It can upregulate the expression of α-SMA protein,Smad2 mRNA and Smad3 mRNA and downregulate the expression of Smad7 mRNA(P<0.01). Compared with model group, crocetin group can inhibit the effect of TGF-β1 in a dose-dependent manner(P<0.05). The high-dose and medium-dose crocetin groups can downregulate the expression of α-SMA protein, Smad2 mRNA and Smad3 mRNA and upregulate the expression of Smad7 mRNA(P<0.05).Conclusions: Crocetin can upregulate the expression of Smad7 mRNA and downregulate the expression of Smad2 mRNA and Smad3 mRNA in LX-2 cells induced by TGF-β1. Thus the anti-hepatic fibrosis mechanism of crocetin may be related to the inhibition of TGF-β1/Smads signal transduction pathway.
Objective: To study the effects of crocetin on TGF-β1 stimulated signal transduction pathway in human hepatic stellate cells(HSC-LX2). Methods: HSC-LX2 cells were cultured in vitro and randomly divided into 5 groups: blank group, model group,low-dose crocetin group, medium-dose group, high-dose crocetin group. Then cells proliferation were measured by MTT, the expression of α-SMA was measured by Western blot, the gene expression of Smad2/Smad3/Smad7 m RNA were measured by Real-time PCR. Results:Compared with control group, the model group promotes cells proliferation. It can upregulate the expression of α-SMA protein,Smad2 mRNA and Smad3 mRNA and downregulate the expression of Smad7 mRNA(P<0.01). Compared with model group, crocetin group can inhibit the effect of TGF-β1 in a dose-dependent manner(P<0.05). The high-dose and medium-dose crocetin groups can downregulate the expression of α-SMA protein, Smad2 mRNA and Smad3 mRNA and upregulate the expression of Smad7 mRNA(P<0.05).Conclusions: Crocetin can upregulate the expression of Smad7 mRNA and downregulate the expression of Smad2 mRNA and Smad3 mRNA in LX-2 cells induced by TGF-β1. Thus the anti-hepatic fibrosis mechanism of crocetin may be related to the inhibition of TGF-β1/Smads signal transduction pathway.
引文
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[19] Tang LY, Heller M, Meng Z, et al. Transforming Growth Factor-β(TGF-β)Directly Activates the JAK1-STAT3 Axis to Induce Hepatic Fibrosis in Coordination with the SMAD Pathway[J]. J Biol Chem,2017, 292(10):4302-4312
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[22] Tang Li-xia, He Rui-hua, Yang Guang, et al. Asiatic acid inhibits liver fibrosis by blocking TGF-beta/Smad signaling in vivo and in vitro[J].PLo S ONE, 2012, 7(2):e31350
[23] Schuppan D, Kim Yo. Evolving therapies for liver fibrosis[J]. J Clin Invest, 2013, 123(5):1887-1901
[24] Inagaki Y, Okazaki I. Emerging insights into Transforming growth factor beta Smad signal in hepatic fibrogenesis[J]. Gut, 2007, 56(2):284-292
[25] Xu W, Wang LW, Shi JZ, et al. Effects of RNA interference targeting transforming growth factor-beta 1 on immune hepatic fibrosis induced by Concanavalin A in mice[J]. Hepatobiliary Pancreat Dis Int, 2009, 8(3):300-308
[26] Zhang L, Liu C, Meng XM, et al. Smad2 protects against TGF-β1/Smad3-mediated collagen synthesis in human hepatic stellate cells during hepatic fibrosis[J]. Mol Cell Biochem, 2015, 400(1-2):17-28
[27] Wu Shi-Pin, Yang Zhi, Li Fu-Rong, et al. Smad7-overexpressing rat BMSCs inhibit the fibrosis of hepatic stellate cells by regulating the TGF-β1/Smad signaling pathway[J]. Exp Ther Med, 2017, 14(3):2568-2576
[28] Yang D, Li L, Qian S. Evodiamine ameliorates liver fibrosis in rats via TGF-β1/Smad signaling pathway[J]. J Nat Med, 2017
[29] Hafez MM, Hamed SS, El-Khadragy MF, et al. Effect of ginseng extract on the TGF-β1 signaling pathway in CCl4-induced liver fibrosis in rats[J]. BMC complementary and alternative medicine, 2017, 17(1):45
[2] Hansen T, Christensen E. Cirrhosis and liver fibrosis are potentially reversible[J]. Ugeskr Laeger 2015, 177(48):V06150527
[3] Tsochatzis EA, Bosch J, Burroughs AK. Liver cirrhosis[J]. Lancet,2014, 383:9930, 1749-1761
[4] Huang Y, Deng X, Liang J. Modulation of hepatic stellate cells and reversibility of hepatic fibrosis[J]. Exp Cell Res, 2017, 352(2):420-426
[5] Cui W, Jin HB, Li ZW. Mechanism of the transforming growth factor-beta induction of fibronectin expression in hepatic stem-like cells[J]. Braz J Med Biol Res, 2010, 43(1):36-42
[6] Yoshida Katsunori, Murata Miki, Yamaguchi Takashi, et al.TGF-β/Smad signaling during hepatic fibro-carcinogenesis(review)[J]. Int J Oncol, 2014, 45(4):1363-1371
[7] Dropmann A, Dediulia T, Breitkopf Heinlein K, et al.TGF-β1 and TGF-β2 abundance in liver diseases of mice and men[J]. Oncotarget,2016, 7(15):19499-19518
[8] Breitkopf K, Godoy P, Ciuclan L, et al. TGF-β/Smad Signaling in the Injured Liver[J]. Z Gastroenterol, 2006, 44(1):57-66
[9]国家重点发展的39种中药材[J].中国中医药信息杂志, 2003, 10(11):3439 species Traditional Chinese Medicine in the national major Development[J]. Chinese Journal of Information on Traditional Chinese Medicine, 2003, 10(11):34
[10] Nam KN, Park YM, Jung HJ, et al. Anti-inflammatory effects of crocin and crocetin in rat brain microglial cells[J]. European Journal of Pharmacology, 2010, 648(1-3):110-116
[11] Lee IA, Lee JH, Baek NI, et al. Antihyperlipidemic effect of crocin isolated from the fructus of Gardenia jasminoides and its metabolite Crocetin[J]. Biological&Pharmaceutical Bulletin, 2005, 28(11):2106-2110
[12] He SY, Qian ZY, Wen N, et al. Influence of Crocetin on experimental atherosclerosis in hyperlipidamic-diet quails[J]. European Journal of Pharmacology, 2007, 554(2-3):191-195
[13]石磊.西红花酸体内外抗氧化作用的研究[J].中国医药指南, 2012,10(15):118-120Shi Lei. Study on the antioxidant effect of crocetin in vitro and in vivo[J]. Guide of China Medicine, 2012, 10(15):118-120
[14] Moradzadeh M, Sadeghnia HR, Tabarraei A. Anti-tumor effects of crocetin and related molecular targets[J]. J Cell Physiol, 2017
[15]朱艳虹,陈真,钱之玉,等.西红花酸对乙醛诱导的肝星状细胞增殖和胶原合成的影响[J].中国临床药理学与治疗学, 2013, 18(08):841-847Zhu Yan-hong, Chen Zhen, Qian Zhi-yu, et al. Effects of crocetin on proliferation and collagen synthesis of hepatic stellate cells induced by acetaldehyde[J]. Chin J Clin Pharmacol Ther, 2013, 18(08):841-847
[16]胡慧,钱之玉,成文媛,等.西红花酸对心肌成纤维细胞增殖和胶原合成的影响[J].中国临床药理学与治疗学, 2012, 17(03):282-288Hu Hui, Qian Zhi-yu, Cheng Wen-yuan, et al. Effects of crocetin on proliferation and collagen synthesis of cardial fibroblast cells[J]. Chin J Clin Pharmacol Ther, 2012, 17(03):282-288
[17]周忠启.西红花酸对单侧输尿管大鼠肾组织结缔组织生长因子表达的影响[J].牡丹江医学院学报, 2008, 29(04):34-36Zhou Zhong-qi. Effects of crocetin on expression of connective tissue growth factor in UUO rats with renal tubulointerstitial fibrosis[J].Journal of Mudanjiang Medical College, 2008, 29(04):34-36
[18] Delire Bénédicte, St覿rkel Peter, Leclercq Isabelle. Animal Models for Fibrotic Liver Diseases:What We Have, What We Need, and What Is under Development[J]. J Clin Transl Hepatol, 2015, 3(1):53-66
[19] Tang LY, Heller M, Meng Z, et al. Transforming Growth Factor-β(TGF-β)Directly Activates the JAK1-STAT3 Axis to Induce Hepatic Fibrosis in Coordination with the SMAD Pathway[J]. J Biol Chem,2017, 292(10):4302-4312
[20] Wang Y, Zhang L, Lei R, et al. Effects of blocking two sites of transforming growth factor-β/Smads signaling on the formation of scar-related proteins in human skin fibroblasts[J]. Zhonghua Shao Shang Za Zhi, 2015, 31(5):372-377
[21] Fabregat I, Moreno-Càceres J, Sánchez A, et al. TGF-βsignalling and liver disease[J]. FEBS J, 2016, 283(12):2219-2232
[22] Tang Li-xia, He Rui-hua, Yang Guang, et al. Asiatic acid inhibits liver fibrosis by blocking TGF-beta/Smad signaling in vivo and in vitro[J].PLo S ONE, 2012, 7(2):e31350
[23] Schuppan D, Kim Yo. Evolving therapies for liver fibrosis[J]. J Clin Invest, 2013, 123(5):1887-1901
[24] Inagaki Y, Okazaki I. Emerging insights into Transforming growth factor beta Smad signal in hepatic fibrogenesis[J]. Gut, 2007, 56(2):284-292
[25] Xu W, Wang LW, Shi JZ, et al. Effects of RNA interference targeting transforming growth factor-beta 1 on immune hepatic fibrosis induced by Concanavalin A in mice[J]. Hepatobiliary Pancreat Dis Int, 2009, 8(3):300-308
[26] Zhang L, Liu C, Meng XM, et al. Smad2 protects against TGF-β1/Smad3-mediated collagen synthesis in human hepatic stellate cells during hepatic fibrosis[J]. Mol Cell Biochem, 2015, 400(1-2):17-28
[27] Wu Shi-Pin, Yang Zhi, Li Fu-Rong, et al. Smad7-overexpressing rat BMSCs inhibit the fibrosis of hepatic stellate cells by regulating the TGF-β1/Smad signaling pathway[J]. Exp Ther Med, 2017, 14(3):2568-2576
[28] Yang D, Li L, Qian S. Evodiamine ameliorates liver fibrosis in rats via TGF-β1/Smad signaling pathway[J]. J Nat Med, 2017
[29] Hafez MM, Hamed SS, El-Khadragy MF, et al. Effect of ginseng extract on the TGF-β1 signaling pathway in CCl4-induced liver fibrosis in rats[J]. BMC complementary and alternative medicine, 2017, 17(1):45