METHODS: Compounds that promote KGF secretion were screened by a dual-luciferase reporter gene assay. The TMT isotope labeling quantitative technique was used to detect the differential proteins upon ISX-9 administrated to MSCs. The expressions of NGFR, ERK, TAU, and β-catenin were detected by Western blot. In the ALI model, we measured the inflammatory changes by HE staining, SOD content detection, RT-qPCR, immunofluorescence, etc. The influence of ISX-9 on the residence time of MSCs transplantation was explored by optical in vivo imaging.
RESULTS: We found out that ISX-9 can promote the expression of KGF in MSCs. ISX-9 acted on the membrane receptor protein NGFR, upregulated phosphorylation of downstream signaling proteins ERK and TAU, downregulated phosphorylation of β-catenin, and accelerated β-catenin into the nucleus to further increase the expression of KGF. In the ALI model, combined ISX-9 with MSCs treatments upgraded the expression of KGF in the lung, and enhanced the effect of MSCs in reducing inflammation and repairing lung damage compared with MSCs alone.
CONCLUSIONS: ISX-9 facilitated the secretion of KGF from MSCs both in vivo and in vitro. The combination of ISX-9 with MSCs enhanced the paracrine function and anti-inflammatory effect of MSCs compared with MSCs applied alone in ALI. ISX-9 played a contributive role in the transplantation of MSCs for the treatment of ALI.
方法:通过双荧光素酶报告基因测定筛选促进KGF分泌的化合物。TMT同位素标记定量技术用于检测ISX-9施用至MSC后的差异蛋白。NGFR的表达式,ERK,TAU,Westernblot检测β-catenin。在ALI模型中,我们通过HE染色测量炎症变化,SOD含量检测,RT-qPCR,免疫荧光,等。通过光学活体成像探索ISX-9对MSCs移植停留时间的影响。
结果:我们发现ISX-9可以促进MSCs中KGF的表达。ISX-9作用于膜受体蛋白NGFR,下游信号蛋白ERK和TAU的上调磷酸化,β-连环蛋白磷酸化下调,并加速β-catenin进入细胞核,进一步增加KGF的表达。在ALI模型中,ISX-9与MSCs联合治疗可提高KGF在肺中的表达,与单独使用MSCs相比,增强了MSCs减轻炎症和修复肺损伤的作用。
结论:ISX-9在体内和体外均促进MSCs分泌KGF。与单独应用于ALI的MSCs相比,ISX-9与MSCs的组合增强了MSCs的旁分泌功能和抗炎作用。ISX-9在MSCs移植治疗ALI中发挥了重要作用。