| 【Objective】Cl~-movement and Cl~--sensitive signal pathways play important roles in microglial survival and its phenotype conversion,and are able to participate in regulating the inflammatory brain injury following ischemic stroke,but the mechanism is still unclear.In this study,we explored the effects of Cl~-transmembrane transporter Swell1 channel activation on the survival and phenotypic transformation of microglia and its role and mechanism in cerebral ischemia-reperfusion injury.【Methods】In this study,oxygen-glucose deprivation(oxygen-glucose deprivation,OGD)and transient middle cerebral artery occlusion(t MCAO)were established to mimic in vitro and in vivo ischemic stroke,respectively.Microglial cell lines(BV2 cells)were transfected with Swell1 overexpressing adenovirus(Ad-Swell1)or Swell1 small molecule interfering RNA(Swell1 SI),or inhibited with drug DCPIB,and microglia electrophysiological testing was used to observe the changes in hypotonic conditioned Cl~-currents during microglia inhibition or overexpression of Swell1.Then we used immunofluorescence staining,real-time quantitative polymerase chain reaction(q PCR)and a multiplex cytokine/chemokine assay to assess the expression and distribution of Swell in microglia,the survival of microglia,and the level of inflammatory factors in ischemic brain tissue and serum.The level of cell viability of each group was measured by CCK-8 assay.The effect of conditioned media of Ad-Swell1 microglia on OGD-induced survival and apoptosis of neurons(HT22 cells)was assessed by flow cytometry,and 4D label-free proteomic and immunoblot assays were used to screen and verify differential protein expression following Swell1 activation under hypotonic conditions.Cre-Swell1 transgenic mice in which Swell1 was specifically overexpressed in microglia,was constructed to evaluate the effect of Swell1 on neuroprotection following 1-h t MCAO with 24-h reperfusion.TTC staining was used to assess infarction volume,neurological function scoring was used to evaluate neurological deficits,immunofluorescence staining was used to visualize the survival and distrubition of microglia and neurons,and,enzyme-linked immunosorbent assay was applied to assess the expression of cytokines and chemokines in mouse brain tissue and serum.【Results】The results of our experiments show that the expression of Swell1 was significantly increased after OGD treatment,knockdown or overexpression of Swell1 was able to inhibit or increase the volume-regulated Cl~-current in BV2 cells after hypotonic treatment,respectively.And these changes could be completely blocked by the VRAC-specific inhibitor DCPIB,suggesting the role of hypotonic activation of Cl~-currents in microglia.The vivo results showed that the microglia-specific overexpression of Swell1 was able to significantly reduce the infarct size.The overexpression of Swell1 overexpression of microglia resulted in a significant reduction in ischemic injury-induced TUNEL positive cells,which significantly reduced the loss of neurons in the ischemic brain region and significantly attenuated the neurological deficits induced by t MCAO.Alternatively,overexpression of Swell1 was able to maintain microglia survival by inhibiting ischemia-induced microglial death and promoting cell proliferation,and overexpression of Swell1 was able to increase microglial M2 type polarization,thereby alleviating neuroinflammation.The molecular mechanism is related to the opening of Swell1 leading to the FOXO3a/CREB pathway mediated by SGK1(a Cl~--sensitive kinase)and WNK1-mediated SPAK/OSR1(another Cl~--sensitive kinase).【Conclusion】Our results suggest that Swell1 is an important component of microglial VRAC,and the Swell1 channels activation exerts a neuroprotective effect by reducing ischemia-induced neuroinflammation.This study revealed that Swell1 has an important role in promoting microglial survival and phenotypic transformation,thus providing new ideas for the treatment of neuroinflammation-related ischemic brain injury. |
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