丹参酚酸B盐对转化生长因子β1活化的大鼠肝星状细胞内p38丝裂原活化蛋白激酶通路及其转录因子的影响
단삼분산B염대전화생장인자β1활화적대서간성상세포내p38사렬원활화단백격매통로급기전록인자적영향
Effects of salvianolic-acid B on the p38MAPK signaling pathway and its transcriptional factor induced by transforming growth factor β1 in activated rat hepatic stellate cells
  • 万方数据
    中华肝脏病杂志 중화간장병잡지
    CHINESE JOURNAL OF HEPATOLOGY
    2012年 12期 902-907 ,共6页

    宋雅芳%吕志刚%徐列明

    송아방%려지강%서렬명

    转化生长因子β1%丝裂原活化蛋白激酶类%信号传导%丹参酚%肝星状细胞 轉化生長因子β1%絲裂原活化蛋白激酶類%信號傳導%丹參酚%肝星狀細胞
    전화생장인자β1%사렬원활화단백격매류%신호전도%단삼분%간성상세포
    Transforming growth factor beta1%Mitogen-activated protein kinases%Signal transduction%Salviol%Hepatic stellate cells
    目的 探讨丹参酚酸B盐(SA-B)对转化生长因子β1(TGFβ1)活化的大鼠肝星状细胞(HSC)内p38丝裂原活化蛋白激酶(MAPK)信号传导通路的影响. 方法 分离并培养正常大鼠HSC,将TGFβ1和SA-B直接添加于原代HSC的无血清培养液中,用p38信号通路特异性阻断剂SB203580和细胞外信号调节激酶(ERK)信号通路特异性阻断剂PD98059分别阻断HSC内p38MAPK和ERK信号通路.细胞内总的P38蛋白、MKK3/6蛋白及MEF2A、MEF2C的测定分为空白对照组、SA-B组、SA-B+ TGF β1组和TGFβ1组;磷酸化P38蛋白、MKK3/6蛋白和α-SMA蛋白的测定分为空白对照组、SA-B组、SA-B+ TGF β1组、TGFβ1组、PD98059、PD98059+ SA-B组、PD98059+ TGFβ1组和SA-B+ PD98059+ TGFβ1组；SA-B对TGFβ1刺激的HSC内MEF2和Ⅰ型胶原报道基因的影响分为突变型(mt)对照组、野生型(wt)对照组、TGFβ1组、SA-B+ TGF β1组、SA-B组、SB203580+ TGF β1组、SB203580组.Western blot法检测HSC内磷酸化和总P38蛋白、MAPK激酶3/6 (MKK3/6)蛋白、肌细胞增强因子2(MEF2)A、MEF2C、α-平滑肌肌动蛋白(α-SMA)的表达；荧光素酶报道基因测定法检测MEF2报道基因和Ⅰ型胶原启动子的活性.多组间数据的多重比较用q检验.结果 SA-B组磷酸化P38蛋白相对表达量为0.33±0.05,明显低于空白对照组(q=7.08,P＜0.01); SA-B +TGF β1组的磷酸化P38蛋白相对表达量为0.46±0.04,明显低于TGFβ1组(q=10.45,P＜0.01)；SA-B组磷酸化MKK3/6蛋白相对表达量为0.11±0.07,明显低于空白对照组(q=3.944,P＜0.05)；SA-B+ TGF β1组磷酸化MKK3/6蛋白相对表达量为0.28±0.07,明显低于TGFβ1组(q=7.91,P＜0.01)；SA-B+ TGFβ1组和SB203580+ TGF β1组MEF2报道基因的相对荧光素酶活性分别为2.93±0.09和2.50±0.05,均明显低于TGFβ1组(q值分别为35.35和37.2,P值均＜0.01)；SA-B组MEF2C及MEF2A的相对表达量分别为15.82±0.97和13.00±0.40,均明显低于空白对照组(q值分别为5.18和13.32,P值均＜0.01)；SA-B+ TGF β1组MEF2C及MEF2A的相对表达量分别为13.40±0.72和20.47±0.83,均明显低于TGF β1组(q值分别为43.93和12.52,P值均＜0.01); SA-B+ TGFβ1组α-SMA相对表达量为8.76±0.44,明显低于TGFβ1组(q=20.35,P＜0.01); SA-B+ SB203580+TGF β1组α-SMA相对表达量仅为3.57±0.49,明显低于TGFβ1组(q=39.78,P＜0.01)；SA-B+ TGF β1组和SB203580+ TGF β1组Ⅰ型胶原报道基因的相对荧光素酶活性分别为1.61±0.05和1.42±0.07,较TGFβ1组明显降低(q值分别为26.4和27.62,P值均＜0.01).结论 SA-B可能通过抑制原代HSC内TGFβ 1的p38MAPK信号传导通路,抑制HSC的活化.
    목적 탐토단삼분산B염(SA-B)대전화생장인자β1(TGFβ1)활화적대서간성상세포(HSC)내p38사렬원활화단백격매(MAPK)신호전도통로적영향. 방법 분리병배양정상대서HSC,장TGFβ1화SA-B직접첨가우원대HSC적무혈청배양액중,용p38신호통로특이성조단제SB203580화세포외신호조절격매(ERK)신호통로특이성조단제PD98059분별조단HSC내p38MAPK화ERK신호통로.세포내총적P38단백、MKK3/6단백급MEF2A、MEF2C적측정분위공백대조조、SA-B조、SA-B+ TGF β1조화TGFβ1조;린산화P38단백、MKK3/6단백화α-SMA단백적측정분위공백대조조、SA-B조、SA-B+ TGF β1조、TGFβ1조、PD98059、PD98059+ SA-B조、PD98059+ TGFβ1조화SA-B+ PD98059+ TGFβ1조；SA-B대TGFβ1자격적HSC내MEF2화Ⅰ형효원보도기인적영향분위돌변형(mt)대조조、야생형(wt)대조조、TGFβ1조、SA-B+ TGF β1조、SA-B조、SB203580+ TGF β1조、SB203580조.Western blot법검측HSC내린산화화총P38단백、MAPK격매3/6 (MKK3/6)단백、기세포증강인자2(MEF2)A、MEF2C、α-평활기기동단백(α-SMA)적표체；형광소매보도기인측정법검측MEF2보도기인화Ⅰ형효원계동자적활성.다조간수거적다중비교용q검험.결과 SA-B조린산화P38단백상대표체량위0.33±0.05,명현저우공백대조조(q=7.08,P＜0.01); SA-B +TGF β1조적린산화P38단백상대표체량위0.46±0.04,명현저우TGFβ1조(q=10.45,P＜0.01)；SA-B조린산화MKK3/6단백상대표체량위0.11±0.07,명현저우공백대조조(q=3.944,P＜0.05)；SA-B+ TGF β1조린산화MKK3/6단백상대표체량위0.28±0.07,명현저우TGFβ1조(q=7.91,P＜0.01)；SA-B+ TGFβ1조화SB203580+ TGF β1조MEF2보도기인적상대형광소매활성분별위2.93±0.09화2.50±0.05,균명현저우TGFβ1조(q치분별위35.35화37.2,P치균＜0.01)；SA-B조MEF2C급MEF2A적상대표체량분별위15.82±0.97화13.00±0.40,균명현저우공백대조조(q치분별위5.18화13.32,P치균＜0.01)；SA-B+ TGF β1조MEF2C급MEF2A적상대표체량분별위13.40±0.72화20.47±0.83,균명현저우TGF β1조(q치분별위43.93화12.52,P치균＜0.01); SA-B+ TGFβ1조α-SMA상대표체량위8.76±0.44,명현저우TGFβ1조(q=20.35,P＜0.01); SA-B+ SB203580+TGF β1조α-SMA상대표체량부위3.57±0.49,명현저우TGFβ1조(q=39.78,P＜0.01)；SA-B+ TGF β1조화SB203580+ TGF β1조Ⅰ형효원보도기인적상대형광소매활성분별위1.61±0.05화1.42±0.07,교TGFβ1조명현강저(q치분별위26.4화27.62,P치균＜0.01).결론 SA-B가능통과억제원대HSC내TGFβ 1적p38MAPK신호전도통로,억제HSC적활화.
    Objective To investigate the effects of Salvianolic-acid B on p38MAPK signaling pathway and its transcriptional factor activated by Transforming growth factor β1 in rat hepatic stellate cells.Methods Hepatic stellate cells were isolated from normal rat by in situ perfusion and Nycodenz density-gradient centrifugation method.TGFβ1 (10 ng/ml),PD98059(50 μmol/L),SB203580(10 μmol/L) and SA-B (10-6 mol/L) were directly added to the medium of the isolated HSCs.Groups:(1)The detection of total p38,MKK3/6,MEF2A and MEF2C induced by TGFβ1 in HSC:include control group,SA-B group,SA-B + TGFβ1 group and TGFβ1 group.(2)The detection of the phosphorylation of p38,MKK3/6 and α-SMA induced by TGFβ1 in HSC:include control group,SA-B group,SA-B + TGFβ1 group,TGFβ1 group,PD98059 group,PD98059 + SA-B group,PD98059 + TGFβ1 group and SA-B + PD98059 + TGFβ1 group.(3)The effects of SA-B on activity of MEF2 reporter and collagen α 1(1) reporter induced by TGFβ1 in HSC:include mt group,wt group,TGFβ1 group,SA-B + TGFβ1 group,SA-B group,SB203580 + TGFβ1 group and SB203580 group.Total and phosphorylated p38 and MKK3/6,MEF2A,MEF2C and α-SMA were assayed by Western blot.HSCs were transfected with either MEF2 or collagen α1(1) luciferase reporter gene by Lipofectamine 2000 transfection method,Cellular extracts were assayed for both MEF2 and collagen α1(Ⅰ) luciferase activities.Comparisons between groups were performed with Student-Newman-Keuls test.Results The relative expression level of the phosphorylation of p38 of SA-B group is 0.33 ± 0.05,obviously lower than control group(q =7.08,P＜ 0.01); SA-B + TGFβ1 group is 0.46 ± 0.04,obviously lower than TGF β1 group(q =10.45,P ＜ 0.01); The relative expression level of the phosphorylation of MKK3/6 of SA-B group is 0.11 ± 0.07,obviously lower than control group(q =3.944,P ＜ 0.05); SA-B + TGF β1 group is 0.28 ± 0.07,obviously lower than TGFβ1 group (q =7.91,P＜ 0.01); The relative luciferase activity of MEF2 reporter of SA-B + TGFβ1 group and SB203580 + TGF β1 group is 2.93 ± 0.09 and 2.50 ± 0.05 respectively,both obviously lower than TGFβ1 group(q =35.35 and 37.2,P ＜ 0.01); The relative expression level of MEF2C and MEF2A of SA-B group is 15.82 ± 0.97 and 13.00 ± 0.40 respectively,obviously lower than control group(q is 5.18 and 13.32,both P＜ 0.01); SA-B + TGF β1 group is 13.40 ± 0.72 and 20.47 ± 0.83 respectively,obviously lower than TGFβ1group(q is 43.93 and 12.52,both P ＜ 0.01); The relative expression level of α-SMA of SA-B + TGFβ1 group is 8.76 ± 0.44,obviously lower than TGFβ1 group(q =20.35,P ＜ 0.01); SA-B + SB203580 + TGFβ1 group is only 3.57 ± 0.49,obviously lower than TGFβ1 group(q =39.78,P＜ 0.01); The relative luciferase activity of collagen α1(Ⅰ) reporter of SA-B + TGF β1 group and SB203580 + TGFβ1 group is 1.61 ± 0.05 and 1.42 ± 0.07 respectively,obviously lower than TGFβ1 group(q =26.4 and 27.62,bothP＜ 0.01).Conclusion SA-B could inhibit activation of HSC induced by TGFβ1 through inhibiting p38MAPK signaling pathway in hepatic stellate cells.
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