Effects of a selective inhibitor of JAK2/STAT3 signaling pathway, AG490, on the biological behavior of human keloid-derived fibroblasts and their possible mechanisms / 中华皮肤科杂志

ABSTRACT

Objective To estimate effects of AG490, a selective inhibitor of JAK2/STAT3 signaling pathway, on the biological behavior of human keloid?derived fibroblasts (HKFs), and to explore their possible mechanisms. Methods In vitro cultured human skin fibroblasts(HSFs)and HKFs were both divided into several groups to be treated with AG490 at different concentrations (12.5, 25, 50, 75, 100 μmol/L), with those receiving no treatment serving as the control group. Then, cell counting kit?8(CCK?8)assay was performed to evaluate cellular proliferative activity of HSFs and HKFs after 24?, 48?and 72?hour treatment, flow cytometry to estimate cell cycle distribution and apoptosis rate in HKFs after 24?hour treatment, reverse transcription(RT)?PCR to measure STAT3 and cyclin D1 mRNA expressions in treated HKFs as well as STAT3 mRNA expression in untreated HSFs and HKFs after 24?hour culture, and Western blot analysis to measure the protein expressions of STAT3 and p?STAT3 in HSFs and HKFs after 24?hour treatment. Results CCK?8 assay showed that the proliferation inhibition rates of both HSFs and HKFs gradually increased along with the increase in AG490 concentrations and treatment duration, and the inhibitory effects increased in both dose?and time?dependent manners(all P<0.05). Besides, when cells were treated with the same concentrations of AG490 for same durations, the proliferation of HKFs were inhibited to a greater extent than that of HSFs(all P<0.05). As flow cytometry revealed, along with the increase of AG490 concentrations, the proportion of HKFs in G1 phase and the apoptosis rate in HKFs both increased gradually(all P < 0.01), while the proportion of HKFs in G2 phase gradually decreased(all P < 0.01), and the proportion of HKFs in S phase remained insignificantly changed. RT?PCR showed that the mRNA expression of STAT3 was significantly higher in untreated HKFs than in untreated HSFs after 24?hour normal culture(P < 0.05). After 24?hour treatment with AG490, the mRNA expressions of STAT3 and cyclin D1 in HKFs gradually decreased with the increase of AG490 concentrations. Correlation analysis revealed that the mRNA expression of cyclin D1 was positively correlated with that of STAT3 in AG490?treated HKFs (r = 0.855, P < 0.01). Western blot analysis showed that the protein expressions of both STAT3 and p ? STAT3 gradually decreased in HKFs and HSFs along with the increase of AG490 concentrations(all P < 0.05), and were significantly lower in HKFs than in HSFs (both P < 0.05). Conclusion AG490 can effectively inhibit HKF proliferation by selectively blocking the JAK2/STAT3 signaling pathway.