PI3K/AKt/mTOR信号通路介导黄芩苷抑制增生性瘢痕组织成纤维细胞的增殖

黄芩苷作为一种黄酮类成分可通过抑制细胞增殖、促进凋亡发挥抗肿瘤作用，但它是否对异常增生的瘢痕具有抑制增生的作用尚不清楚.本研究探讨黄芩苷抑制人增生性瘢痕组织成纤维细胞增殖的分子机制. 采用MTT比色法检测不同浓度的黄芩苷（2.24×10-2 ～ 2.24×102 mmol/L）对体外培养的增生性瘢痕组织成纤维细胞增殖的抑制作用.发现浓度为2.24×100～2.24×102 mmol/L黄芩苷处理组明显抑制增生性瘢痕组织成纤维细胞的增殖（P<0.05）.转染后的荧光素酶报告基因活性检测、RT-PCR及Western印迹分析技术检测其mRNA水平及细胞的帽状依赖翻译的表达.2.24×102 mmol/L黄芩苷处理后,黄芩苷作用组的mRNA水平并无明显差异（P>0.05）；增生性瘢痕成纤维细胞的帽状依赖结构的翻译明显被黄芩苷所抑制.采用Western印迹分析检测被黄芩苷干预的增生性瘢痕组织成纤维细胞的增殖相关的蛋白的表达；m7GTP琼脂糖珠沉淀结合蛋白4E-BP1与eIF4E的变化.发现增殖相关的蛋白mTOR、p70S6K、S6、4EBP1、eIF4E及其上游的AKT表达明显下调（P<0.05），而PTEN表达明显上调.p-AKT(Ser473)、p-mTOR(Ser2448)、p-S6(Ser235/236)、p-4EBP1(Thr37/ 46)、p-PTEN（T380/S382/383）磷酸化水平下降（P<0.05）.在黄芩苷作用下的增生性成纤维细胞中的游离的4E-BP1明显减少（P<0.05），而与eIF4E结合的4E-BP1明显增加（P<0.05）黄芩苷诱导游离的4E-BP1与eIF4E结合，从而抑制帽状依赖蛋白翻译.以上结果说明,黄芩苷可通过抑制PI3K/AKT/mTOR信号通路抑制人增生性瘢痕组织成纤维细胞的增殖.

PI3K/AKT/mTOR Signaling Mediates Baicalin-inhibited Proliferation in Hypertrophic Scar Fibroblast

Baicalin as a flavonoid derivative exerts its anti-tumor effect through inhibiting cell proliferation and inducing apoptosis, however, whether it can suppress abnormal hyperplasia scar is unknown. To determine the inhibitory effect of bacailin on the proliferation of hypertrophic scar fibroblast and its underlying mechanism, hypertrophic scar fibroblasts were primarily cultured in vitro and stimulated by baicalin at different concentrations of 2.24×10-2 to 2.24×102 mmol/L. MTT assay was performed to assess the cell proliferation. We identified that baicalin at the concentration of 2.24×102 mmol/L resulted in significantly inhibitory effect on hypertrophic scar fibroblast proliferation (P<0.05). Synthesis of hyperplasia of fibroblasts on the cap-dependent translation was significantly inhibited by baicalin (P<0.05). Cells were primarily transfected with a luciferase reporter plasmid to detect cap-dependent translation by luciferase reporter assay and RT-PCR was performed to detect the expression of mRNA. Moreover cell lysates were precipitated with m7GTP Sepharose beads followed by immunoblotting of 4E-BP1 and eIF4E. Western blotting was used to analyze the expression of cell proliferation related proteins in hypertrophic scar fibroblast treated with baicalin. We identified that the level of mRNA had no significant difference in baicalin group.The protein expression of mTOR, p70S6K, S6, 4EBP1, eIF4E and the upstream protein of AKT were significantly down-regulated by the baicalin (2.24×102 mmol/L) (P<0.05), as well as the expression of PTEN was significantly up-regulated. The phosphorylation of AKT, PTEN, mTOR, S6 and 4E-BP1 were suppressed by the baicalin (2.24×102 mmol/L) in hypertrophic scar fibroblasts (P<0.05). The free 4E-BP1 decreased significantly (P<0.5), and eIF4E combined with 4E-BP1 significantly increased (P<0.05) in hypertrophic scar fibroblasts. In addition, baicalin induced binding of 4E-BP1 to the eIF4E-mRNA complex, and inhibited cap-dependent translation. These results suggest that baicalin can inhibit the proliferation of hypertrophic scar fibroblast though PI3K/AKT/mTOR signaling pathways.