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Abstract：
BACKGROUND: Mesenchymal stem cells (MSCs) have been widely studied to alleviate acute lung injury (ALI) due to their paracrine function. However, the microenvironment of inflammatory outbreaks significantly restricted the factors secreted from MSCs like keratinocyte growth factor (KGF). KGF is a growth factor with tissue-repaired ability. Is there a better therapeutic prospect for MSCs in combination with compounds that promote their paracrine function? Through compound screening, we screened out isoxazole-9 (ISX-9) to promote MSCs derived KGF secretion and investigated the underlying mechanisms of action.
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.
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.
摘要：
背景：间充质干细胞（MSCs）由于其旁分泌功能而减轻急性肺损伤（ALI）已被广泛研究。然而,炎症爆发的微环境显著限制了骨髓间充质干细胞分泌的因子，如角质形成细胞生长因子（KGF）。KGF是一种具有组织修复能力的生长因子。间充质干细胞与促进其旁分泌功能的化合物联合治疗是否有更好的治疗前景？我们筛选了异恶唑-9(ISX-9)以促进MSCs来源的KGF分泌,并研究了其潜在的作用机制。
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。
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。
