CysLT₂R Antagonist HAMI 3379 Ameliorates Post-Stroke Depression Through NLRP3 Inflammasome/Pyroptosis Pathway In Meriones Unguiculatus

Part Ⅰ Effect of HAMI 3379 on Poststroke Depression in Meriones UnguiculatusObjectives A transient global cerebral ischemia（tGCl）model was developed by clamping bilateral carotid arteries combined with binding stress stimulation to establish a Post-stroke depression（PSD）model.To explore the regulatory mechanism of Cysteine-leukotriene receptor 2（CysLT₂R）antagonist HAMI 3379 on nerve injury in PSD meriones unguiculatus,in order to provide a new treatment method and experimental basis for post-stroke depression.Methods 1.To establish PSD model of meriones unguiculatus:（1）A tGCI model was prepared by clamping bilateral carotid arteries,and the meriones unguiculatus were subjected to binding force stimulation for 4 h every day for 12 consecutive days starting from the second day after tGCI to establish a post-stroke depression model.（2）The changes of local cerebral blood flow during the establishment of tGCI model were monitored by laser doppler flowmeter.（3）The establishment of post-stroke depression model in meriones unguiculatus was tested by sugar water preference experiment and forced swimming experiment.2.To explore the therapeutic effect of intraperitoneal HAMI 3379（0.1 mg/kg/day）on nerve injury caused by PSD:（1）Neural impairment in meriones unguiculatus was assessed by the mNSS neurological impairment score.（2）Depression in meriones unguiculatus was observed by sugar water preference experiment and forced swim experiment.（3）Neuronal morphology and quantitative changes were observed by Nissl staining.（4）Changes in activated microglia and neuronal cells were observed by immunofluorescence histochemistry.3.Explore the mechanism of nerve injury regulated by HAMI 3379 in PSD meriones unguiculatus:（1）HAMI 3379 regulated the activation of NLRP3 inflammasome by immunefluoresce staining（2）HAMI 3379 regulated the expression of NLRP3 inflammasome related protein and GSDMD-N,the signature of pyrosis,by Western Blot analysis4.To explore the regulatory effects of NLRP3 inflammasome activation and ischemic stroke depression model after pyroptosis by combining HAMI 3379 intervention with shRNA-CysLT₂R and LV-CysLT₂R,respectively:（1）CysLT₂R expression was verified as successfully down-regulated or overexpressed,as determined by both RT-qPCR analysis and immunofluorescence histochemistry.（2）Neuronal morphology and quantitative changes were observed by Nissl staining.（3）Depression in meriones unguiculatus gerbils was observed by the sugar water preference experiment and forced swim experiment.Results 1.tGCI model combined with constraint stress stimulation was successfully used to construct PSD model of meriones unguiculatus.Compared with the sham operation group,cerebral blood flow of meriones unguiculatus in the group leader of the model decreased significantly,and neurological function was damaged.Compared with sham operation group,the model group of meriones unguiculatus showed significant depression-like behavior through sugar water preference experiment and forced swimming experiment.2.HAMI 3379 ameliorates nerve damage in PSD Mongolian gerbils by reducing neurological impairment,improving depression-like behavior,reducing neuronal loss,inhibiting microglia activation,and inhibiting cell apoptosis in the cerebral cortex.3.Obvious expression of NLRP3 inflammasome in microglia was observed in the cerebral cortex of PSD meriones unguiculatus,and significant activation of NLRP3 inflammasome and pyrosis related proteins was detected in the cerebral cortex of PSD meriones unguiculatus.Treatment with HAMI 3379 significantly reduced the activation of NLRP3 inflammasome and pyrosis.4.The mechanism of action of damage protecting PSD induced by HAMI 3379 by inhibiting the expression of the NLRP3 inflammasome and the development of pyroptosis depends on the regulation of CysLT₂R.Conclusion The effect of the CysLT₂R-selective antagonist HAMI 3379 in ameliorating neurological impairment after PSD in meriones unguiculatus was related to the inflammatory mechanisms that inhibit the NLRP3 inflammasome and pyroptosis.Part Ⅱ HAMI 3379 Improved Microglial Hyperactivation by Inhibiting NLRP3 Inflammasome Activation/PyrogenesisObjectives In vitro experiments,CysLT₂R antagonist HAMI 3379 was used to treat OGD/R-induced mouse small glioma cell line BV2 to explore the inflammatory mechanism of HAMI 3379 on OGD/R-induced BV2 cell damage.To investigate whether NLRP3 is involved in the regulation of inflammatory nerve injury,HAMI 3379 was used in combination with NLRP3 activator.Methods 1.For BV2 of mouse microglioma cells cultured in vitro,and the effect of reperfusion on BV2 cell viability for 48 h at different times of OGD（1 h,2 h and 4 h）:（1）To verify the effect of reperfusion for 48 h on BV2 cell morphology at different times of OGD.（2）The cellular activity of OGD for 48 h by CCK8 kit.（3）The amount of LDH release from reperfusion for 48 h by OGD at different times by LDH kit.2.To investigate the effect of HAMI 3379 on BV2 cell activity and LDH release.The effects of HAMI 3379 at different concentrations（0.01,0.1,1 μM）on BV2 cell activity and LDH release were tested using CCK8 and LDH kits.3.To investigate the injury mechanism of BV2 cell OGD/R after HAMI 3379 intervention:（1）The effect of HAMI 3379 on the activation of BV2 cells was observed by immunofluorescence histochemistry（2）Western Blot and immunofluorescence staining were used to detect the expression of NLRP3 inflammasome and GSDMD-N,a landmark protein of pyrosis,after the intervention of HAMI 3379 in BV2 cells4.The regulatory relationship between HAMI 3379 and NLRP3 was verified by the combined stimulation of the agonists Nigericin and Lipopolysaccharide（LPS）supplemented with NLRP3.Results 1.OGD 1 h/R48 h cell morphology was moderately changed and stable,and there was no decrease in cell activity and increased LDH release,which is more consistent with the moderate microglial activation of the overall experiment.2.The dose of HMAI 3379 had no effect on cell activity or LDH release,but it ameliorated the OGD/R-induced decreased BV2 cell activity and the increase in LDH release.3.OGD/R-induced activation and pyroptosis of the NLRP3 inflammasome in BV2 cells could be improved by HAMI 3379.4.The NLRP3 inflammasome activation and pyroptosis induced by the combination of Nigericin and LPS stimulation showed a significantly reduced expression level in response to HAMI 3379.Conclusion Amelioration of OGD/R-induced microglial damage by the CysLT₂R-selective antagonist HM3379 was closely associated with the inhibition of its NLRP3 inflammasome activation and the occurrence of pyroptosis.