1,25(OH)_2D_3通过调节miR-146a水平抑制大鼠肝纤维化的体内和体外观察
|
摘要
目的体内体外观察1,25(OH)_2D_3通过调节mi R-146a水平抑制大鼠肝纤维化的作用机制。方法建立CCl_4诱导的大鼠肝纤维化模型,体外转染肝星状细胞(HSCs)mi R-146a模拟剂/抑制剂,观察1,25(OH)_2D_3处理对动物肝组织变化和HSC增殖和凋亡的影响。采用qPCR法检测肝组织mi R-146a水平,采用CCK8法检测细胞增殖,使用流式细胞术检测HSC凋亡。结果在干预8 w末,1,25(OH)_2D_3干预组大鼠肝纤维化程度明显减轻;1,25(OH)_2D_3干预组大鼠肝组织mi R-146a水平为(0.70±0.03),显著高于橄榄油组【(0.33±0.17,P<0.05)】;1,25(OH)_2D_3组细胞增殖率为58.8%,较DMSO组下降了15.9%,转染mi R-146a模拟剂组大鼠HSC增殖率为46.5%,较对照组下降了53.3%,转染mi R-146a抑制剂组HSC增殖率为132.8%,较对照物组升高了32.8%(P<0.05),1,25(OH)_2D_3干预组细胞凋亡率为12.6%,较DMSO组增加了5.2%,转染mi R-146a模拟剂组细胞凋亡率为16.8%,较对照组细胞凋亡率增加了8.2%,转染mi R-146a抑制剂组细胞凋亡率为6.3%,较对照组细胞凋亡率减少了2.2%(P<0.05),提示1,25(OH)_2D_3具有抑制HSC增殖、促进凋亡作用。结论 1,25(OH)_2D_3可能通过调节mi R-146a水平抑制HSC活化和抑制大鼠肝纤维化。
Objective To observe the inhibitory effect of 1,25(OH)_2 D_3 on liver fibrosis in hepatic stellate cells(HSCs) and in rat model. Method The CCl_4-induced liver fibrosis was made in rats,and the activation of HSCs were induced by 10 pmmol/L TGF-β1 incubation. The miR-146 a mimic/inhibitor were transfected in HSCs and 1,25(OH)_2 D_3 intervention were paralleled. The miR-146 a levels in liver tissues were detected by qPCR,the proliferation of cells was measured by CCK8 and the apoptosis was detected by flow cytometry. Results At the end of 8 weeks,the hepatic fibrosis in 1,25(OH)_2 D_3 intervention group was significantly milder than that in the control group;the miR-146 a level in 1,25(OH)_2 D_3 intervention group was significantly higher than in oil control group [(0.70±0.03) vs.(0.33±0.17),P<0.05];the proliferation rate in1,25(OH)_2 D_3 group was58.8%,15.91% of lower than in DMSO group,in miR-146 a mimic transfected group was 46.5%,53.3% of lower than in control,in miR-146 a inhibitor group was132.8%,32.8% of higher than in control(P<0.05);the apoptosis rate of HSCs in1,25(OH)_2 D_3 group was 12.6%,5.2% of higher than in DMSO,in miR-146 a mimic transfected group was16.8%,8.2% of higher than in control,and in miR-146 a inhibitor transfected group was 6.3%,2.2% of lower than in control(P<0.05),indicating the inhibition of 1,25(OH)_2 D_3 on proliferation and promotion on apoptosis of HSCs. Conclusion 1,25(OH)_2 D_3 might modulate miR-146 a levels to inhibit liver fibrosis.
Objective To observe the inhibitory effect of 1,25(OH)_2 D_3 on liver fibrosis in hepatic stellate cells(HSCs) and in rat model. Method The CCl_4-induced liver fibrosis was made in rats,and the activation of HSCs were induced by 10 pmmol/L TGF-β1 incubation. The miR-146 a mimic/inhibitor were transfected in HSCs and 1,25(OH)_2 D_3 intervention were paralleled. The miR-146 a levels in liver tissues were detected by qPCR,the proliferation of cells was measured by CCK8 and the apoptosis was detected by flow cytometry. Results At the end of 8 weeks,the hepatic fibrosis in 1,25(OH)_2 D_3 intervention group was significantly milder than that in the control group;the miR-146 a level in 1,25(OH)_2 D_3 intervention group was significantly higher than in oil control group [(0.70±0.03) vs.(0.33±0.17),P<0.05];the proliferation rate in1,25(OH)_2 D_3 group was58.8%,15.91% of lower than in DMSO group,in miR-146 a mimic transfected group was 46.5%,53.3% of lower than in control,in miR-146 a inhibitor group was132.8%,32.8% of higher than in control(P<0.05);the apoptosis rate of HSCs in1,25(OH)_2 D_3 group was 12.6%,5.2% of higher than in DMSO,in miR-146 a mimic transfected group was16.8%,8.2% of higher than in control,and in miR-146 a inhibitor transfected group was 6.3%,2.2% of lower than in control(P<0.05),indicating the inhibition of 1,25(OH)_2 D_3 on proliferation and promotion on apoptosis of HSCs. Conclusion 1,25(OH)_2 D_3 might modulate miR-146 a levels to inhibit liver fibrosis.
引文
[1]Friedman SL.Liver fibrosis-from bench to bedside.J Hepatol,2003,38(Suppl 1):S38-S53.
[2]Albanis E,Friedman SL.Antifibrotic agents for liver disease.Am J Transplant,2006,6(1):12-19.
[3]Moreira PK.Hepatic stellate cells and liver fibrosis.Arch Pathol Lab Med,2007,131(11):1728-1734.
[4]Friedman SL.Evolving challenges in hepatic fibrosis.Nat Rev Gastroenterol Hepatol,2010,7(8),425-436.
[5]Bartel DP.MicroRNAs:genomics,biogenesis,mechanism,and function.Cell,2004,116(2),281-297.
[6]王蕾,李校天,张利,等.活性维生素D3联合LY294002体外抑制大鼠肝星状细胞增殖作用研究.实用肝脏病杂志,2016,19(2):147-150.
[7]尹燕,李校天,郭永泽,等.1,25-二羟维生素D3对肝纤维化大鼠组织HIF-1α和TREM-1表达的影响.实用肝脏病杂志,2017,20(2):148-152.
[8]Roy S,Benz F,Vargas D,et al.miR-30c and miR-193 are a part of the TGF-β-dependent regulatory controlling extracellular matrix genes in liver fibrosis.J Dig Dis,2015,16(9):513-524.
[9]Taganov KD,Boldin MP,Chang KJ,et al.NF-kappaB-dependent induction of microRNA miR-146a,an inhibitor targeted to signaling proteins of innate immune responses.Proc Natl A-cad Sci USA,2006,103(33):12481-12486.
[10]Li JF,Dai XP,Zhang W,et al.Upregulation of microRNA-146a by hepatitis B virus X protein contributes to hepatitis development by downregulating complement factor H.MBIO,2015,6(2):e0259-314.
[11]Hou ZH,Han QJ,Zhang C,et al.miR-146a impairs the IFN-induced anti-HBV immune response by down regulating STAT1in hepatocytes.Liver Int,2014,34(1):58-68.
[12]Zhang Z,Zhang Y,Sun XX,et al.microRNA-146a inhibits cancer metastasis by downregulating VEGF through dual pathways in hepatocellular carcinoma.Mol Cancer,2015,21:14:5.
[13]Sun X,Zhang J,Hou Z,et al.miR-146a is directly regulated by STAT3 in human hepatocellular carcinoma cells and involved in anti-tumor immune suppression.Cell Cycle,2015,14(2):243-252.
[14]Maubach G,Lim MC,Chen J,et al.miRNA studies in vitro and in vivo activated hepatic stellate cells.World J Gastroenterol,2001,17(22):2748-2773.
[15]Li F,Zhang A,Shi Y,et al.1α,25-Dihydroxyvitamin D3 prevents the differentiation of human lung fibroblasts via microRNA-27b targeting the vitamin D receptor.Int J Mol Med,2015,36(4):967-974.
[16]Ma Y,Hu Q,Luo W,et al.1ɑ,25(OH)2D3 differentially regulates miRNA expression in human bladder cancer cells.JSteroid Biochen Mol Biol,2015,148(4):166-171.
[17]李艳芳,唐夕岚,凌文华.1,25-,二羟基维生素D3对小鼠非酒精性脂肪性肝纤维化进程的抑制作用.世界华人消化杂志,2012,20(33):3191-3198.
[2]Albanis E,Friedman SL.Antifibrotic agents for liver disease.Am J Transplant,2006,6(1):12-19.
[3]Moreira PK.Hepatic stellate cells and liver fibrosis.Arch Pathol Lab Med,2007,131(11):1728-1734.
[4]Friedman SL.Evolving challenges in hepatic fibrosis.Nat Rev Gastroenterol Hepatol,2010,7(8),425-436.
[5]Bartel DP.MicroRNAs:genomics,biogenesis,mechanism,and function.Cell,2004,116(2),281-297.
[6]王蕾,李校天,张利,等.活性维生素D3联合LY294002体外抑制大鼠肝星状细胞增殖作用研究.实用肝脏病杂志,2016,19(2):147-150.
[7]尹燕,李校天,郭永泽,等.1,25-二羟维生素D3对肝纤维化大鼠组织HIF-1α和TREM-1表达的影响.实用肝脏病杂志,2017,20(2):148-152.
[8]Roy S,Benz F,Vargas D,et al.miR-30c and miR-193 are a part of the TGF-β-dependent regulatory controlling extracellular matrix genes in liver fibrosis.J Dig Dis,2015,16(9):513-524.
[9]Taganov KD,Boldin MP,Chang KJ,et al.NF-kappaB-dependent induction of microRNA miR-146a,an inhibitor targeted to signaling proteins of innate immune responses.Proc Natl A-cad Sci USA,2006,103(33):12481-12486.
[10]Li JF,Dai XP,Zhang W,et al.Upregulation of microRNA-146a by hepatitis B virus X protein contributes to hepatitis development by downregulating complement factor H.MBIO,2015,6(2):e0259-314.
[11]Hou ZH,Han QJ,Zhang C,et al.miR-146a impairs the IFN-induced anti-HBV immune response by down regulating STAT1in hepatocytes.Liver Int,2014,34(1):58-68.
[12]Zhang Z,Zhang Y,Sun XX,et al.microRNA-146a inhibits cancer metastasis by downregulating VEGF through dual pathways in hepatocellular carcinoma.Mol Cancer,2015,21:14:5.
[13]Sun X,Zhang J,Hou Z,et al.miR-146a is directly regulated by STAT3 in human hepatocellular carcinoma cells and involved in anti-tumor immune suppression.Cell Cycle,2015,14(2):243-252.
[14]Maubach G,Lim MC,Chen J,et al.miRNA studies in vitro and in vivo activated hepatic stellate cells.World J Gastroenterol,2001,17(22):2748-2773.
[15]Li F,Zhang A,Shi Y,et al.1α,25-Dihydroxyvitamin D3 prevents the differentiation of human lung fibroblasts via microRNA-27b targeting the vitamin D receptor.Int J Mol Med,2015,36(4):967-974.
[16]Ma Y,Hu Q,Luo W,et al.1ɑ,25(OH)2D3 differentially regulates miRNA expression in human bladder cancer cells.JSteroid Biochen Mol Biol,2015,148(4):166-171.
[17]李艳芳,唐夕岚,凌文华.1,25-,二羟基维生素D3对小鼠非酒精性脂肪性肝纤维化进程的抑制作用.世界华人消化杂志,2012,20(33):3191-3198.