肉苁蓉苯乙醇总苷脂质体对rrPDGF-BB诱导的肝星状细胞增殖的影响及作用机制研究
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摘要
目的探讨肉苁蓉苯乙醇总苷(CPhGs)脂质体对重组大鼠血小板衍生生长因子-BB(rrPDGFBB)诱导的大鼠肝星状细胞(HSC)增殖作用的影响及其抗肝纤维化的相关分子机制。方法取生长状态良好的对数生长期HSC用于实验,置于CPhGs脂质体浓度为117.79、58.90、29.45、14.72、7.36、3.68、1.84、0.92和0.46mg/L的完全培养液,求出半数抑制浓度(IC_(50));rrPDGF-BB刺激后,采用四甲基偶氮唑盐(MTT)法测定29.45、14.72、7.36mg/L CPhGs脂质体对HSC增殖的影响;采用实时荧光定量聚合酶链反应(qRT-PCR)检测α-平滑肌肌动蛋白(α-SMA)、Ⅰ型胶原、ATF-2mRNA的表达水平;采用Western blot法检测CPhGs脂质体对p-p38蛋白表达的影响。结果 (1)对细胞增殖的影响:对不同时间点结果分析显示,与Normal组比较,细胞培养48h时,rrPDGF-BB组HSC细胞明显增多;CPhGs脂质体各剂量组抑制rrPDGF-BB诱导的HSC增殖作用,培养48h效率最高;对不同浓度结果分析显示,与rrPDGF-BB组比较,不同浓度CPhGs脂质体组均可抑制rrPDGF-BB诱导的HSC-T6的增殖。(2)qRT-PCR结果:CPhGs脂质体各剂量组间比较,随CPhGs脂质体药物浓度增大,α-SMA、Ⅰ型胶原和ATF-2 mRNA水平显著下降,差异有统计学意义(P<0.05),其中以CPhGs脂质体29.45mg/L组抑制效果最明显。(3)Western blot分析结果:p-p38蛋白的表达水平在CPhGs脂质体不同浓度组表达均下降,差异有统计学意义(P<0.05);CPhGs脂质体各剂量组间比较,p-P38蛋白随CPhGs脂质体干预剂量增大,表达水平显著下降(P<0.05)。结论 CPhGs脂质体能抑制rrPDGF-BB诱导的大鼠HSC增殖,从而起到抗肝纤维化的作用。
Objective To investigate the effect of cistanche phenylethanoid glycosides(CPhGs)liposomes on the proliferation of rat hepatic stellate cells(HSC)induced by platelet-derived growth factor-BB(rrPDGF-BB)and the related molecular mechanisms of its anti-hepatic fibrosis.Methods HSC in logarithmic growth phase were cultured in complete medium with CPhGs liposome concentrations of 117.79,58.90,29.45,14.72,7.36,3.68,1.84,0.92 and 0.46 mg/L.And the half maximal inhibitory concentration(IC_(50))was calculated.After rrPDGF-BB stimulation,the effects of liposome concentrations of 29.45,14.72,7.36 mg/L on HSC proliferation were detected by MTT method.Real-time fluorescence quantitative PCR(qRT-PCR)was used to detect the expression ofα-SMA,collagenⅠ and ATF-2 mRNA.Western blot was used to detect the influence of CPhGs liposome on the expression of p-p38 protein.Results(1)Effect on cell proliferation:compared with the normal group,HSC in the rrPDGF-BB group increased significantly after 48 hours of cell culture.CPhGs liposome inhibited the proliferation of HSC induced by rrPDGF-BB with the highest efficiency after 48 hours of culture.CPhGs liposome inhibited the proliferation of HSC at different concentrations compared with the rrPDGF-BB group,and CPhGs liposome at different concentrations all could inhibit the proliferation of HSC-6 induced by rrPDGF-BB.(2)The results of qRT-PCR:the content ofα-SMA,CollagenⅠ and ATF-2 mRNA decreased significantly with the increase of CPhGs liposome concentration among the different dosage groups(P<0.05),and the inhibition effect of CPhGs liposome 29.45 mg/L was most effective.(3)Western blot analysis:the expression of p-p38 protein decreased in different concentration of CPhGs liposomes with statistical significance(P<0.05).Compared among different dose of CPhGs liposomes,the expression of p-p38 protein decreased significantly with the increase of CPhGs liposomes intervention dose(P<0.05).Conclusion CPhGs liposomes can inhibit the proliferation of HSC induced by rrPDGF-BB,thereby plays the antiliver fibrosis role.
Objective To investigate the effect of cistanche phenylethanoid glycosides(CPhGs)liposomes on the proliferation of rat hepatic stellate cells(HSC)induced by platelet-derived growth factor-BB(rrPDGF-BB)and the related molecular mechanisms of its anti-hepatic fibrosis.Methods HSC in logarithmic growth phase were cultured in complete medium with CPhGs liposome concentrations of 117.79,58.90,29.45,14.72,7.36,3.68,1.84,0.92 and 0.46 mg/L.And the half maximal inhibitory concentration(IC_(50))was calculated.After rrPDGF-BB stimulation,the effects of liposome concentrations of 29.45,14.72,7.36 mg/L on HSC proliferation were detected by MTT method.Real-time fluorescence quantitative PCR(qRT-PCR)was used to detect the expression ofα-SMA,collagenⅠ and ATF-2 mRNA.Western blot was used to detect the influence of CPhGs liposome on the expression of p-p38 protein.Results(1)Effect on cell proliferation:compared with the normal group,HSC in the rrPDGF-BB group increased significantly after 48 hours of cell culture.CPhGs liposome inhibited the proliferation of HSC induced by rrPDGF-BB with the highest efficiency after 48 hours of culture.CPhGs liposome inhibited the proliferation of HSC at different concentrations compared with the rrPDGF-BB group,and CPhGs liposome at different concentrations all could inhibit the proliferation of HSC-6 induced by rrPDGF-BB.(2)The results of qRT-PCR:the content ofα-SMA,CollagenⅠ and ATF-2 mRNA decreased significantly with the increase of CPhGs liposome concentration among the different dosage groups(P<0.05),and the inhibition effect of CPhGs liposome 29.45 mg/L was most effective.(3)Western blot analysis:the expression of p-p38 protein decreased in different concentration of CPhGs liposomes with statistical significance(P<0.05).Compared among different dose of CPhGs liposomes,the expression of p-p38 protein decreased significantly with the increase of CPhGs liposomes intervention dose(P<0.05).Conclusion CPhGs liposomes can inhibit the proliferation of HSC induced by rrPDGF-BB,thereby plays the antiliver fibrosis role.
引文
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[17]冯芹,夏文凯,王现珍,等.连翘苷元对四氯化碳大鼠急性肝损伤的保护作用[J].中国药理学通报,2015,31(3):426-429.
[18]聂钱,李香丹,许东元,等.IGF-1对糖尿病大鼠肝纤维化的作用及其机制[J].中国病理生理杂志,2015,41(10):1878-1879.
[19]詹迪迪,李飞龙,张功武,等.转化生长因子-β/CTGF在肝纤维化发生机制中的作用[J].肝胆外科杂志,2016,24(3):233-237.
[20]FANG L,ZHAN S X,HUANG C,et al.TRPM7Channel regulates PDGF-BB-induced proliferation of hepatic stellate cells via PI3Kand ERK pathways[J].Toxicol Appl Pharmacol,2013,272(3):713-725.
[21]郑娜娜,岳雅伦,郑勇,等.硫化氢通过p38MAPK信号通路对肝纤维化大鼠肝细胞增殖、凋亡的影响[J].世界华人消化杂志,2015,23(6):901-906.
[22]孔令娜,占书箱,黄成,等.栀子苷对PDGF诱导的HSC-T6增殖活化的影响[J].安徽医科大学学报,2015,61(4):481-485.
[2]周光德,赵景民.不同病因致肝纤维化/肝硬化的病理特点[J].临床肝胆病杂志,2016,32(6):1086-1091.
[3]盛婷,傅念,阳学风,等.肝纤维化发病机制中细胞和分子机制的研究进展[J].现代生物医学进展,2015,15(12):2358-2362.
[4]LI Y,SHI Y,SUN Y,et al.Restorative effects of hydroxysafflor yellow A on hepatic function in an experimental regression model of hepatic fibrosis induced by carbon tetrachloride[J].Mol Med Rep,2017,15(1):47-56.
[5]SPENGLER U.Hepatic microcirculation:a critical but neglected factor for the outcome of viral hepatitis[J].J Hepatol,2009,50(3):631-633.
[6]LIU Y W,CHIU Y T,FU S L,et al.Osthole ameliorates hepatic fibrosis and inhibits hepatic stellate cell activation[J].J Biomed Sci,2015,22(1):63.
[7]YANG F R,WEN D S,FANG B W,et al.Prevention of extract from cistanche salsa on hepatic fibrosis induced by Carbon tetrachloride in rats[J].Chinese Herbal Medicines,2013,5(3):199-204.
[8]屠鹏飞,姜勇,郭玉海,等.发展肉苁蓉生态产业推进西部荒漠地区生态文明[J].中国现代中药,2015,17(4):297-301.
[9]SERCOMBE L,VEERATI T,MOHEIMANI F,et al.Advances and challenges of liposome assisted drug delivery[J].Front Pharmacol,2015,6(3):286.
[10]LEE Y A,WALLACE M C,FRIEDMAN S L.Pathobiology of liver fibrosis:a translational success story[J].Gut,2015,64(5):830-841.
[11]WANG Y,GAO J C,ZHANG D I,et al.New insights into the antifibrotic effects of sorafenib on hepatic stellate cells and liver fibrosis[J].J Hepatol,2010,53(1):132-144.
[12]LI Y,SHI Y,SUN Y,et al.Restorative effects of hydroxysafflor yellow A on hepatic function in an experimental regression model of hepatic fibrosis induced by Carbon tetrachloride[J].Mol Med Rep,2017,15(1):47-56.
[13]孔德松,郑仕中,陆茵,等.肝内肌成纤维细胞的来源及其在肝纤维化中作用的研究[J].中国药理学通报,2011,27(3):297-300.
[14]SCHUPPAN D,KIM Y O.Evolving therapies for liver fibrosis[J].J Clin Invest,2013,123(5):1887-1901.
[15]FRIEDMAN S L.Evolving challenges in hepatic fibrosis[J].Nat Rev Gastroenterol Hepatol,2010,7(8):425-436.
[16]陈宵瑜,杨长青.肝纤维化发生机制研究新进展[J].实用肝脏病杂志,2016,19(1):121-124.
[17]冯芹,夏文凯,王现珍,等.连翘苷元对四氯化碳大鼠急性肝损伤的保护作用[J].中国药理学通报,2015,31(3):426-429.
[18]聂钱,李香丹,许东元,等.IGF-1对糖尿病大鼠肝纤维化的作用及其机制[J].中国病理生理杂志,2015,41(10):1878-1879.
[19]詹迪迪,李飞龙,张功武,等.转化生长因子-β/CTGF在肝纤维化发生机制中的作用[J].肝胆外科杂志,2016,24(3):233-237.
[20]FANG L,ZHAN S X,HUANG C,et al.TRPM7Channel regulates PDGF-BB-induced proliferation of hepatic stellate cells via PI3Kand ERK pathways[J].Toxicol Appl Pharmacol,2013,272(3):713-725.
[21]郑娜娜,岳雅伦,郑勇,等.硫化氢通过p38MAPK信号通路对肝纤维化大鼠肝细胞增殖、凋亡的影响[J].世界华人消化杂志,2015,23(6):901-906.
[22]孔令娜,占书箱,黄成,等.栀子苷对PDGF诱导的HSC-T6增殖活化的影响[J].安徽医科大学学报,2015,61(4):481-485.
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