黄芩苷对培养人原代成纤维细胞长波紫外线光损伤端粒的影响

Effects of baicalin on ultraviolet A-induced telomere damage in cultured human primary fibroblasts

目的探讨黄芩苷对于长波紫外线（UVA）照射导致人皮肤成纤维细胞衰老的保护作用及对端粒途径的影响。方法分离培养人原代成纤维细胞，将亚融合状态的培养细胞分为空白组、黄芩苷组、UVA组和UVA＋黄芩苷组，照光组中以10JAmzUVA进行照射，药物干预组中加入50μg/ml黄芩苷干预处理。以流式细胞仪测定细胞周期变化；以端粒重复序列扩增-酶联免疫吸附法（TRAP—EHSA）检测细胞端粒酶水平；以实时定量PCR法检测细胞端粒长度及衰老相关基因p53、p16和c—mycmRNA水平；以蛋白印迹法检测p16和c—myc蛋白水平。结果UVA引起显著G1期阻滞，G1期细胞比值由正常对照组59．94％升高至81．04％，而黄芩苷处理后G。期细胞比值下降为65．55％。UVA照射后细胞端粒长度缩短为正常组的31．2％，黄芩苷干预组的端粒长度可恢复至正常细胞的63．9％。此外与正常组相比，UVA诱导p53和p16mRNA表达水平升高为2．93±0．21和2．14±0．09，而c—mycmRNA水平下降为0．53±0．03；黄芩苷干预可抑制上述改变。照射后与正常组相比，p16蛋白水平增高为5．84±0．16，c—myc蛋白表达降低为0．35±0．04；黄芩苷处理后p16蛋白表达量下降为4．09±0．13（P〈0．05）；c—myc蛋白表达水平无明显变化（P〉0．05）。正常对照和UVA组的端粒酶活性为阴性，加入黄芩苷对端粒酶活性无影响。结论黄芩苷可延缓人成纤维细胞光老化进程，其机制可能与调控p53等老化相关基因表达有关，与端粒酶活性无关。

Objective To investigate baicalin effect against ultraviolet A （UVA） induced senescence in cultured human skin fibroblasts （HSF） and influence on telomere pathway. Methods HSF were isolated from the prepuce of neonates and cultured. Subconfluent fibroblasts were classified into blank control group （without treatment）, baicalin group （treated with baicalin of 50 μg/ml）, UVA group （irradiated with UVA of 10 J/cm2） and UVA ＋ baicalin group （irradiated with UVA of 10 J/cm2 and treated with baicalin of 50 μg/ml before and after the irradiation）. After additional culture of various durations, flow cytometry was performed to detect cell cycle, telomere repeat amplification protocol-enzyme linked immunosorbent assay （TRAP-ELISA） to measure telomerase activity, real-time quantitative PCR to determine telomere length, mRNA levels of p53, p16 and c-myc, Western blot to examine the protein expressions of p16 and c-myc. Results UVA irradiation induced cell cycle arrest in G1 phase, and the percentage of HSF at G1 phase increased from 59.94% in the blank control group to 81.04% in the UVA group, but was decreased to 65.55% in the UVA ＋ baicalin group. The length of telomere in HSF in UVA group was shortened to 31.2% of that in the blank control group, but was restored to 63.9% in HSF treated with baicalin before and after the irradiation. Compared with the blank control group, the expression level of p53 and p16 mRNA was increased to 2.93 ± 0.21 and 2.14 ± 0.09, respectively, while that of c-myc mRNA decreased to 0.53 ± 0.03 in the UVA group; baicalin could inhibit these changes. Similarly, Westem blot showed that after UVA irradiation the protein expression level of p16 increased to 5.84 ±0.16, while that of c-myc decreased to 0.35 ± 0.04 in HSF compared with that in the blank control group; baicalin treatment before and after the irradiation induced no significant changes in the protein expression of c-myc, but a decline in that of p16 （4.09 ± 0.13, P 〈 0.05）. Telomerase activity was undetected in any of these groups. Conclusions Baicalin can delay the photoaging process of HSF, which may be attributed tothe regulation of expression of senescence-related genes such as p53, but not to telomerase activity.