穗花杉双黄酮调控人瘢痕成纤维细胞活性及凋亡的研究

Influence of Amentoflavone on the viability and apoptosis of hypertrophic scar fibroblasts

目的探讨穗花杉双黄酮调控人增生性瘢痕成纤维细胞生长与凋亡的作用机制。方法将细胞接种于24孔板，分别加入不同浓度的穗花杉双黄酮（0、25、50、100、150、200μmol／L），48h后，酶标仪检测490波长的吸光度。选取浓度为50txmol／L和100μmol／L的穗花杉双黄酮作为后续实验的用药浓度，以0μmol／L穗花杉双黄酮为对照，加入到人增生性瘢痕成纤维细胞中作用48h，分别采用DAPI染色检测穗花杉双黄酮对人增生性瘢痕成纤维细胞凋亡的作用，Western blot检测穗花杉双黄酮对人增生眭瘢痕成纤维细胞磷酸化AKT（p-AKT）及凋亡相关蛋白，如翻译控制肿瘤蛋白、BAX、caspase3、caspase8、caspase9的影响。结果穗花杉双黄酮对人增生性瘢痕成纤维细胞的活力具有剂量依赖性抑制作用，穗花杉双黄酮浓度为50μmol／L以上与对照组相比，差异有统计学意义（P〈0．05）。DAPI染色显示，穗花杉双黄酮具有诱导人增生性瘢痕成纤维细胞凋亡的趋势，与对照组相比，加药组细胞表现为细胞核深染、核膜皱缩、有凋亡小体形成。Westernblot显示，与对照组相比，AKT磷酸化水平降低；促凋亡蛋白BAX表达升高；抗凋亡蛋白TCTP表达降低；caspase3、caspase8、caspase9表达升高。结论穗花杉双黄酮可诱导瘢痕成纤维细胞凋亡，抑制其活力，有望成为临床治疗增生胜瘢痕的一种有效药物。

Objective To investigate the effect of Amentoflavone （AF） on the viability and apoptosis of hypertrophic scar fibroblasts （HSFBs） and its underlying mechanism. Methods XTT assay was performed to assess cell viability. HSFBs were seeded at 3.0 × 104cells/well in a 24-well plate overnight, and then treated with different concentrations of AF（0 μmol/L, 25 μmol/L, 50 μmol/L, 100 μmol/L, 150 μmol/L, 200 μmol/L. After incubation for 48 h, cell absorbance was detected at 490 nm wavelength. In following experiments, 50 μmol/1 AF and 100 μmol/1 AF concentration was used. HSFBs were incubated with 50 μmol/L AF or 100 μmol/L AF or 0 μmol/L AF for 48 h. Cell apoptosis were examined by DAPI staining. Western blotting was used to analyze the expression of cell apoptosis-related proteins such as translationally controlled tumor protein （TCTP）, BAX, caspsae 3, caspase 8, caspase 9, and p-AKT in HSFB treated with AF. Results AF can inhibit the viability of HSFB dose dependently. Comparing control group （AF：0 μmol/L） with 50 μmol/L AF or higher AF groups, the difference in cell viability was significant （ P 〈 0.05 ）. DAPI staining showed AF-induced apoptosis in HSFBs. AF induces typical apoptotic features such as membrane blebbing, cell shrinkage and detachment, nuclear condensation and fragmentation. Western blot showed that the expression of AKT phosphorylation and anti-apoptotic protein TCTP were lower, while the expression of pro-apoptotic protein BAX and caspase 3, caspase 8, caspase 9 were higher compared with the control group. Conclusion Amentofla- vone could induce aptosis of scar fibroblasts so as to inhibit its activity which is expected to become effective medicine for hyperplastic scar treatment.