The Protective Effect Of Gualou Guizhi Decoction On Neuron Injury By Promoting Microglial Cell Activation Mediated By IRF5/IRF4 Regulatory Axis

Objective:Based on the critical IRF5/IRF4 regulatory axis of microglia Gualou Guizhi Deco ction,the specific molecular mechanism of regulating the bidirectional balance of neur oinflammation after stroke and thus alleviating neuronal damage was explored,providi ng an experimental basis for the treatment of stroke.Methods:In vivo experiments:SD rats were randomly divided into sham operation group,model group,and Gualou Guizhi Decoction group,and the middle cerebral artery occlusion/reperfusion model was established.The drug was administered for 7 days after successful modeling.After the intervention,the cerebral infarction volume of rats was detected by MRI and TTC staining.The contents of pro-inflammatory factors(IL12 、 IL23)and anti-inflammatory factors(IL10、IL13)in peripheral blood of rats were detected by multiple ELISA.HE staining was used to observe the infiltration of inflammatory cells and the damage of neurons.The expression of IRF5/IRF4 and microglia-polarization-related genes CD16,MHCII,Ym-1 and Arg-1 were detected by RT-PCR.The expressions of IRF5/IRF4 and polarization-related proteins CD16,MHCII,Ym-1 and Arg-1 in microglia were detected by Western blot.The expressions of microglia activating protein(Iba-1)and neuron specific protein(MAP-2)were observed by immunohistochemistry.In vitro experiments:In vitro culture BV2 cells,the use of LPS/IL4 stimulate BV2 inflammation model is setted up,by OGD/R(Oxygen and glucose deprivation/reoxygenation,OGD/R)induced HT22 cells preparation damage model,HT22 were also conditioned with Microglia-conditioned media(MCM)of BV2 cells.Cell viability was determined by CCK8 method.The pro-inflammatory cytokines(IL12、IL23)and anti-inflammatory factors(IL10、IL13)in the supernatant of cell culture were detected by multiple ELISA.The expressions of IRF5/IRF4 and polarization-related genes and proteins CD16,MHCII,Ym-1 and Arg-1 in microglia were detected by RT-PCR and Western blot.The expression of specific marker(Neu N)of nerve cells was observed by immunofluorescence.The expression of synaptic marker protein(Tuj-1)was observed by laser confocal microscopy.Cells transfected with IRF5 si RNA knockdown IRF5 expression to verify whether GLGZD regulates bidirectional balance of post-stroke neuroinflammation and thus alleviates neuronal injury through the critical IRF5/IRF4 regulatory axis of microglia cells.Results:In vivo experiments:After intervention with Gualou Guizhi Decoction,the infarct volume of the affected side of the cerebral tissue of MCAO/R rats decreased significantly(P<0.01).Gualou Guizhi Decoction significantly decreased the contents of pro-inflammatory factors IL12 and IL23 in peripheral blood of MCAO/R rats(P<0.01),up-regulated the contents of neurotrophic factors IL10α and IL13(P<0.01 or P<0.05).The inflammatory cell infiltration and neuronal injury of cerebral cortex in infarct area of MCAO/R rats were significantly improved.GLGZD significantly down-regulated the m RNA and protein expressions of IRF5,CD16 and MHCII,and up-regulated the m RNA and protein expressions of IRF4,Ym-1 and Arg-1 in cerebral cortex tissues of infarct area(P<0.01 or P<0.05).GLGZD significantly down-regulated the positive expression of Iba-1 protein and up-regulated the positive expression of MAP-2 protein in brain tissue of rats in MCAO/R group(P<0.01).In vitro experiments:LPS(100ng/m L),IL4(20ng/m L)and GLGZD(50,100,200μg/m L)had no effect on the viability of BV2 cells.GLGZD decreased the expressio ns of inflammatory cytokines IL12 and IL23 in LPS-induced BV2 cell proinflammator y model(P<0.01 or P<0.05)and upregulated the expressions of anti-inflammatory facto rs IL10 and IL13 in IL4-induced BV2 cell proinflammatory model(P<0.01 or P<0.05).GLGZD significantly down-regulated the expressions of IRF5,CD16,MHCII m RNA a nd protein in proinflammatory cell models(P<0.01 or P<0.05)and up-regulated the exp ressions of IRF4,Ym-1,Arg-1 m RNA and protein in anti-inflammatory cell models(P<0.01 or P<0.05).GLGZD could express Neu N and Tuj-1 proteins in HT22 cells after LPS+MCM intervention(P<0.01)and Neu N and Tuj-1 proteins in HT22 cells after I L4+MCM intervention(P<0.01、P<0.05).In vitro target validation experiments:After IRF5 gene knockout,compared with LPS group,GLGZD is unable to downregulate NO expression.After IRF5 gene knockout,compared with Con group,the expression of IRF5 and CD16 protein decreased significantly(P<0.01、P<0.05).Compared with NC group,the protein expressions of IRF5 and CD16 in IRF5 si RNA group were down-regulated(P<0.01、P<0.05)while the protein expressions of IRF4 and Arg-1 were up-regulated(P<0.01、P<0.05).Compared with LPS group,IRF5 and CD16 protein expressions of GLGZD with 100 μg/ml showed no difference compared with LPS group(P>0.05),while IRF4 and Arg-1 protein expressions were up-regulated,with statistically significant differences compared with LPS group(P<0.05).Compared with LPS group IRF5 si RNA transfected with GLGZD can’t regulate the expression of Neu N protein in HT22 cells induced by microglial cell conditioned medium(P>0.05).Conclusions:GLGZD plays a protective role in nerve cell damage after stroke through IRF5/IRF4 regulating axis-mediated microglial polarization.