棕榈酸对转化生长因子β1刺激的肝星状细胞增殖、凋亡及Caspase-12蛋白表达的影响

Effect of palmitic acid on caspase-12 and proliferation and apoptosis of hepatic stellate cells

目的研究棕榈酸对转化生长因子β_1(TGF-β_1)刺激的肝星状细胞(HSC)增殖、凋亡及半胱氨酸天冬氨酸蛋白酶12(caspase-12)蛋白表达的影响。方法将液氮保存下的肝星状细胞株,于37℃、5%CO2孵箱中进行复苏传代培养,同步化后将细胞分为5组:空白组、TGF-β_1(5 ng/ml)组、TGF-β_1+低、中、高剂量棕榈酸组,即5 ng/ml TGF-β1刺激24 h,再分别加入50、100及200μmol/L不同剂量棕榈酸作用24 h后,用MTT比色法检测细胞增殖、流式细胞仪检测细胞凋亡率、免疫细胞化学法测定Caspase-12蛋白表达。结果不同浓度棕榈酸均能抑制细胞增殖(P<0.05),且随着剂量的增加,细胞相对增殖率(RGR)降低,低、中、高剂量组分别为76.56%、60.93%和34.37%,组间比较差异有统计学意义(P<0.05)。透视电镜发现,不同剂量棕榈酸组出现细胞核变小,细胞出现大量线粒体空泡,染色质边集等典型凋亡表现。空白组、TGF-β_1组、TGF-β_1+低、中、高剂量棕榈酸组凋亡率分别为(3.32±0.29)%、(1.70±0.14)%、(7.26±0.46)%、(11.24±0.52)%及(15.55±0.31)%,组间两两比较差异有统计学意义(P<0.05);随着棕榈酸剂量的增加,细胞Caspase-12蛋白表达增加(P<0.05)。结论棕榈酸能抑制细胞增殖,增加Caspase-12蛋白表达,促进HSC凋亡。

Objective To investigate the effect of palmitic acid （PA） on caspase-12 and proliferation andapoptosis in rat hepatic stellate cells （HSCs） when stimulated by transforming growth factor-β1 （TGF-β1）.Methods HSCs were randomly divided into five groups： Sham group, TGF-β1 group, TGF-β1 ＋ low PA group,TGF-β1 ＋ middle PA group and TGF-β1 ＋ high PA group. HSCs were stimulated by TGF-β1 （5 ng/ml） for 24hours followed by co-incubation with PA under different doses （50, 100, and 200 umol/L）. No intervention wasperformed in Sham group. Cell proliferation was detected by MTT; morphological manifestations of cells wereobtained by transmission electronmicroscope; apoptosis rate was determined by flow cytometry; expression levelsof caspase-12 was measured by Immunohistochemistry. Results Cellular proliferation was significantly inhibitedwith PA treatment ina dose dependent manner（P〈 0.05）. Transmission electronmicroscope showed typicalapoptotic features such as small nuclei, large numbers of mitochondria vacuoles and obvious edge sets ofDNA. Treatment of PA dramatically increased apoptosis rate when compared with TGF-β1 group [（7.26 ± 0.46）%vs （1.70 ± 0.14） %, 〈 0.05; （11.24 ± 0.52） % vs （1.70 ± 0.14） %, 〈 0.05; （15.55 ± 0.31） % vs （1.70 ± 0.14） %,P〈 0.05]. Expression of caspase-12 protein increased significantly with PA in dose dependent manner （P〈0.05）. Conclusion PA inhibits cellular proliferation through promoting caspase-12 mediated apoptosis in HSCs.