间质干细胞通过 Gal-9/Tim-3 改变 Th17 细胞/Tregs 的平衡来减轻脓毒症相关性急性肾损伤。

Mesenchymal Stem Cells Attenuate Sepsis-associated Acute Kidney Injury by Changing the Balance of Th17 cells/Tregs via Gal-9/Tim-3.

机构信息

Department of Nephrology, the Affiliated Hospital of Qingdao University, Shandong 266003, China.

出版信息

Curr Stem Cell Res Ther. 2023;18(4):540-550. doi: 10.2174/1574888X17666220511151343.

DOI:10.2174/1574888X17666220511151343

PMID:35546754

Abstract

OBJECTIVE

The aim of the present study was to investigate the protective effect of MSCs on CLP-induced SA-AKI and determine the mechanisms of this effect.

METHODS

The expression of Gal-9 and Tim-3 was assayed by qPCR and western blot. IL-10, IL-17, RORγt, and FOXP3 were assayed by qPCR and TNFα, INFγ, IL-4, and IL-6 were assayed by ELISA in renal samples after CLP with or without MSCs treatment. The expression of Gal-9 in MSCs was knocked down in vivo using RNA interference, and si-Gal-9-MSCs were injected in SA-AKI mice. The effect of MSCs on the differentiation of lymphocytes into Th17 cells and Tregs was evaluated in vitro by FAC in coculture of MSCs and CD4+ T cells and after blockade of the Gal-9/Tim-3 pathway.

RESULTS

MSCs decreased serum creatinine and urea nitrogen levels and relieved tubular injury. Additionally, MSCs significantly improved the survival rate and markedly attenuated the infiltration of neutrophils and the levels of TNF-α, IFN-γ, IL-4, and IL-6 in the kidneys of septic mice (P < 0.05). Treatment with MSCs also reduced the proportion of Th17 cells and the levels of IL-17 and RORγt (P < 0.05). In contrast, MSCs increased the proportion of Tregs and the levels of IL-10 and FOXP3 related to these cells (P < 0.05). Furthermore, we determined whether Gal-9/Tim-3 and Th17 cells/Tregs are involved in the protective effects of MSCs in an SA-AKI model. The results of Western blot and real-time PCR indicated that MSCs inhibited the expression of Tim-3 and increased the expression of gal-9 (P < 0.05). Knockdown of gal-9 in MSCs using small interfering RNA blunted the therapeutic effect of MSCs, and blockade of the Gal-9/Tim-3 pathway using α-lactose or anti-Tim-3 inhibited the induction of Tregs and suppressed the inhibition of the differentiation to Th17 cells by MSCs after coculture of MSCs with CD4+ T cells (P > 0.05).

CONCLUSION

Treatment with MSCs can protect against SA-AKI. The results suggested that the relieving effect of MSCs against SA-AKI may be partially mediated by the induction of Tregs and inhibition of Th17 cells via the Gal-9/Tim-3 pathway.

摘要

目的

本研究旨在探讨间充质干细胞（MSCs）对 CLP 诱导的 SA-AKI 的保护作用，并确定其作用机制。

方法

采用 qPCR 和 Western blot 检测 Gal-9 和 Tim-3 的表达。采用 qPCR 检测肾组织中 IL-10、IL-17、RORγt 和 FOXP3 的表达，采用 ELISA 检测 TNFα、INFγ、IL-4 和 IL-6 的表达。采用 RNA 干扰体内敲低 MSCs 中的 Gal-9 表达，并在 SA-AKI 小鼠中注射 si-Gal-9-MSCs。通过 MSC 与 CD4+T 细胞共培养及阻断 Gal-9/Tim-3 通路后，评估 MSCs 对淋巴细胞向 Th17 细胞和 Treg 分化的影响。

结果

MSCs 降低了血清肌酐和尿素氮水平，并缓解了肾小管损伤。此外，MSCs 显著提高了败血症小鼠的生存率，明显减轻了中性粒细胞浸润和肾脏中 TNF-α、IFN-γ、IL-4 和 IL-6 的水平（P<0.05）。MSCs 治疗还降低了 Th17 细胞的比例和 IL-17 和 RORγt 的水平（P<0.05）。相反，MSCs 增加了 Treg 的比例和与这些细胞相关的 IL-10 和 FOXP3 的水平（P<0.05）。此外，我们确定 Gal-9/Tim-3 和 Th17 细胞/Treg 是否参与了 MSC 在 SA-AKI 模型中的保护作用。Western blot 和实时 PCR 的结果表明，MSCs 抑制了 Tim-3 的表达，增加了 Gal-9 的表达（P<0.05）。用小干扰 RNA 敲低 MSCs 中的 Gal-9 减弱了 MSCs 的治疗效果，用α-乳糖或抗 Tim-3 阻断 Gal-9/Tim-3 通路抑制了 MSC 与 CD4+T 细胞共培养后 Treg 的诱导，并抑制了 MSC 对 Th17 细胞分化的抑制作用（P>0.05）。