丹皮酚体外对肝星状细胞增殖和凋亡的影响

目的探讨丹皮酚对大鼠肝星状细胞(HSC)增殖和凋亡的影响。方法 HSC加入丹皮酚1.66～49.8 mg·L-1作用48 h后,MTT比色法和锥虫蓝染色法检测HSC的存活率。HSC与丹皮酚33.2 mg·L-1分别作用6,12,24,36和48 h,锥虫蓝染色法检测HSC的存活率。HSC与丹皮酚1.66～69.7 mg·L-1作用48 h用细胞乳酸脱氢酶(LDH)活性测定试剂盒检测细胞上清中LDH活性。丹皮酚1.66～49.8 mg·L-1与HSC作用24 h分别用光学显微镜、Hoechst 33258染色和流式细胞术检测HSC细胞形态、细胞凋亡和凋亡率;丹皮酚1.66～49.8 mg·L-1作用48 h用流式细胞术检测细胞周期。结果 MTT实验结果表明,丹皮酚16.6～49.8 mg·L-1对HSC增殖具有明显抑制作用,且具有浓度效应关系(r=0.955,P<0.05),作用48 h时IC50值为105.4 mg·L-1。锥虫蓝染色实验结果表明,丹皮酚对HSC存活率的抑制作用不仅具有浓度效应关系(r=-0.936,P<0.05),浓度为33.2 mg·L-1时还具有时间效应关系(r=-0.965,P<0.05)。丹皮酚16.6～69.7 mg·L-1作用48 h无明显细胞毒性。流式细胞仪检测结果显示,丹皮酚8.3～49.8 mg·L-1作用24 h能显著促进HSC细胞凋亡(P<0.05),且具有浓度效应关系(r=0.920,P<0.05)。丹皮酚33.2和49.8 mg·L-1作用48 h可调节细胞周期,G0/G1期细胞增加(P<0.01),S期细胞减少(P<0.01),具有浓度依赖性(r=0.980,P<0.05)。结论丹皮酚具有促进HSC凋亡和抑制HSC增殖的作用,可能是其抗肝纤维化作用机制之一。

Effect of paeonol on proliferation and apoptosis of hepatic stellate cells in vitro

OBJECTIVE To study the effect of paeonol on cell proliferation and apoptosis of hepatic stellate cells (HSC). METHODS HSC were treated with paeonol 1.66-49.8 mg·L^-1 for 48 h, and the viability of HSC was detected with MTT assay and trypan blue staining. The viability of HSC was also determined using trypan blue staining after treatment with paeonol 33.2 mg·L^-1 for 6, 12, 24, 36 and 48 h, respectively. Cytotoxicity of paeonol was determined with lactate dehydrogenase (LDH) release assay. Leica microscope and Hoechst 33258 dyeing were used to observe morphological changes of HSC. Flow cytometry analysis was used to detect the apoptosis rate of HSC and cell cycle. RESULTSMTT assay results showed that paeonol 16.6-49.8 mg·L^-1 had significant inhibitory effect on HSC proliferation in a concentration-dependent manner (r=0.955, P<0.05), the IC₅₀ value was 105.4 mg·L^-1 after intervention for 48 h. Trypan blue staining results showed that paeonol not only reduced the survival rate of HSC in a concentration-dependent relationship (r=0.936, P<0.05), but also in a time-dependent manner (r=0.965, P<0.05) at the concentration of 33.2 mg·L^-1. Paeonol 1.66-69.7 mg·L^-1 had no obvious cytotoxic effect on HSC. Flow cytometry assay results showed that paeonol 8.3-49.8 mg·L^-1 significantly increased HSC apoptosis (P<0.05, P<0.01) in a concentration-dependent manner (r=0.920, P<0.05). Paeonol 33.2 and 49.8 mg·L^-1 arrested HSC cell cycle at G₀/G₁ phase (P<0.01), and reduced the percentage of S phase cells (P<0.01) also in a concentration-dependent manner (r=0.980, P<0.05). CONCLUSION Paeonol can inhibit HSC proliferation and induce apoptosis, which may underlie the antifibrotic effect of paeonol.