大麻素受体2激动剂对人胚肺成纤维细胞株HLF1增殖和分化的影响及机制

Effect of cannabinoid receptor 2 agonist on proliferation and differentiation of human embryonic lung fibroblast cell line HLF1 and its mechanism

目的探讨大麻素受体2(CB2)激动剂JWH133对人胚肺成纤维细胞株HLF1增殖、分化的影响及机制。方法取对数生长期HLF1细胞株,采用细胞计数试剂-8(CCK8)法检测不同浓度(0.01、0.10、1.00、10.00及20.00μmol/L)JWH133及CB2(0.0、0.5、5.0及50.0μmol/L)拮抗剂AM630对HLF1的增殖能力影响;取对数生长期HLF1细胞分为正常(N)组、5μg/L转化生长因子β1(TGF-β1)组(即0μmol/L JWH133)、10.00μmol/L JWH133(J10)组及J10+5.0μmol/L AM630(AM630)组,培养48 h,采用Western blot和逆转录-聚合酶链反应PCR(RT-qPCR)检测各组细胞中胶原蛋白1α1(Col1α1)、α-平滑肌肌动蛋白(α-SMA)、磷酸化-AKT(p-AKT)蛋白及Col1α1、α-SMA mRNA,采用细胞免疫荧光检测各组细胞中α-SMA蛋白荧光信号及应力纤维细胞百分比。结果随着JWH133浓度的增大,HLF1细胞的吸光度(OD)值逐渐降低,0.0、0.5、5.0及50.0μmol/L AM630组HLF1存活率均在1附近(P>0.05);J10组HLF1细胞中Col1α1、α-SMA蛋白及p-AKT表达均较TGF-β1组降低(P<0.05),J10+AM630组则较J10组升高(P<0.05);J10组HLF1细胞Col1α1、α-SMA mRNA均较TGF-β1组降低(P<0.05),J10+AM630组则较J10组升高(P<0.05);J10组HLF1细胞α-SMA蛋白荧光信号及应力纤维细胞占总细胞比值均较TGF-β1组减少(P<0.05),J10+AM630组则较J10组增多(P<0.05)。结论CB2受体激动剂JWH133可抑制HLF1细胞的增殖及分化,其机制可能与下调PI3K-AKT通路有关。

Objective To investigate the effects of cannabinoid receptor 2(CB2)agonist JWH133 on proliferation and differentiation of human embryonic lung fibroblast cell line HLF1 and its effect and mechanism.Methods HLF1 Cell lines in the logarithmic growth stage were selected and treated and Cell Counting Kit-8(CCK8)was used to measure the proliferative ability of JWH133 and CB2 antagonist AM630 to HLF1 at different concentrations(0.01,0.10,1.00,10.00,and 20.00μmol/L).HLF1 Cells in the logarithmic growth stage were divided into Normal cells(N)group,5μg/L transforming growth factorβ1(TGF-β1)group(0μmol/L JWH133),10.00μmol/L JWH133(J10)group,and J10+5.0μmol/L AM630(AM630)group.All these aforementioned groups were cultured for 48 h.Western blot and reverse transcription-polymerase chain reaction(RT-qPCR)were used to detect Collagen 1 alpha 1(Col1α1),α-smooth muscle actin(α-SMA),phosphorylated-AKT(p-AKT)protein,as well as Col1α1 mRNA andα-SMA mRNA in each group of cells.Cell immunofluorescence was used to detect the fluorescence signal ofα-SMA protein and the percentage of stressed fiber cells in each group.Results With the increase of JWH133 concentration,the optical density(OD)value of HLF1 cells decreased gradually.The survival rate of HLF1 cells in 0.0,0.5,5.0,and 50.0μmol/L AM630 groups was 1(P>0.05).The expressions of Col1α1,α-SMA proteins and p-AKT in HLF1 cells in J10 group decreased when compared with TGF-β1 group(P<0.05),whereas the expressions of such markers increased in J10+AM630 group when compared with those in J10 group(P<0.05).Col1α1 mRNA andα-SMA mRNA of HLF1 in J10 group decreased when compared with those in TGF-β1 group(P<0.05),while the expressions of such markers increased in J10+AM630 group when compared with those in J10 group(P<0.05).The fluorescence signal ofα-SMA protein and the percentage of stressed fiber cells in HLF1 cells in J10 group decreased when compared with TGF-β1 group(P<0.05),while the expressions of such markers in J10+AM630 group increased when compared with J10 group(P<0.05).Conclusion CB2 receptor agonist JWH133 can inhibit the proliferation and differentiation of HLF1 cells,and its mechanism may be related to the down-regulation of PI3K-AKT pathway.