背景:尽管刺激Wnt/β-catenin信号传导是治疗缺血性卒中的重要策略,其信号通路尚未完全阐明。最近,RSPO3(R-spondin3)/LGR4(富含亮氨酸的重复序列的G蛋白偶联受体4)信号已经解决了TLR4(toll样受体4)诱导的肺损伤炎症;然而,该信号在缺血性脑中是否重要仍未知。因此,我们研究了RSPO3/LGR4信号在缺血性脑中的作用。
方法:BALB/c小鼠暴露于永久性远端大脑中动脉和颈总动脉闭塞。检查了时间RSPO3和LGR4的表达,并将小鼠随机分配接受载体或重组RSPO3。使用小胶质细胞系和原代混合胶质-内皮-神经元和原代神经元培养物研究了潜在的机制。
结果:在缺血性大脑中,RSPO3和LGR4在内皮细胞和小胶质细胞/巨噬细胞和神经元中表达,分别。重组RSPO3刺激RSPO3/LGR4信号可以恢复神经功能缺损,第3天Il1β和iNOSmRNA减少,第9天Gap43增加。在培养的细胞中,LGR4在神经元和小胶质细胞中表达,而RSPO3促进β-catenin的核易位。在脂多糖刺激的神经胶质-内皮-神经元培养物中观察到炎性细胞因子表达降低的神经保护作用,但在谷氨酸-CoCl2-,H2O2-,或氧葡萄糖剥夺刺激的神经元培养物,表明RSPO3/LGR4可以通过调节炎性细胞因子来保护神经元。LGR4-Fc嵌合体,用于阻断内源性RSPO3/LGR4信号传导,增加LPS诱导的炎症细胞因子的产生,表明内源性RSPO3抑制炎症。RSPO3通过降低TLR4表达来降低TLR4相关的炎性细胞因子表达,而不影响M1/M2表型。RSPO3还抑制TLR2和TLR9诱导的炎症,但不抑制TLR7诱导的炎症,促进了神经突生长。
结论:RSPO3/LGR4信号在调节TLR诱导的炎症和缺血性脑中的神经突生长中起关键作用。增强该信号将是治疗缺血性中风的有希望的方法。
Although stimulation of Wnt/β-catenin signaling is an important strategy to treat ischemic stroke, its signaling pathway has not been fully clarified yet. Recently, RSPO3 (R-spondin 3)/LGR4 (leucine-rich repeat-containing G protein-coupled receptor 4) signaling has resolved TLR4 (toll-like receptor 4)-induced inflammation in lung injury; however, whether this signal is critical in the ischemic brain remains unknown. Therefore, we investigated the role of RSPO3/LGR4 signaling in the ischemic brain.
BALB/c mice were exposed to permanent distal middle cerebral artery and common carotid artery occlusion. Temporal RSPO3 and LGR4 expressions were examined, and the mice were randomly assigned to receive vehicle or recombinant RSPO3. The underlying mechanisms were investigated using microglial cell lines and primary mixed glia-endothelia-neuron and primary neuronal cultures.
In the ischemic brain, RSPO3 and LGR4 were expressed in endothelial cells and microglia/macrophages and neurons, respectively. Stimulation of RSPO3/LGR4 signaling by recombinant RSPO3 recovered neurological deficits with decreased Il1β and iNOS mRNA on day 3 and increased Gap43 on day 9. In cultured cells, LGR4 was expressed in neuron and microglia, whereas RSPO3 promoted nuclear translocation of β-catenin. Neuroprotective effects with reduced expression of inflammatory cytokines were observed in lipopolysaccharide-stimulated glia-endothelium-neuron cultures but not in glutamate-, CoCl2-, H2O2-, or oxygen glucose deprivation-stimulated neuronal cultures, indicating that RSPO3/LGR4 can protect neurons by regulating inflammatory cytokines. LGR4-Fc chimera, which was used to block endogenous RSPO3/LGR4 signaling, increased LPS-induced production of inflammatory cytokines, suggesting that endogenous RSPO3 suppresses inflammation. RSPO3 decreased TLR4-related inflammatory cytokine expression by decreasing TLR4 expression without affecting the M1/M2 phenotype. RSPO3 also inhibited TLR2- and TLR9-induced inflammation but not TLR7-induced inflammation, and promoted neurite outgrowth.
RSPO3/LGR4 signaling plays a critical role in regulating TLR-induced inflammation and neurite outgrowth in the ischemic brain. Enhancing this signal will be a promising approach for treating ischemic stroke.