| [Objective]To investigate the regulation of pertussis toxin(PTx)on microglia after cerebral ischemia and its possible mechanism.[Method]divided into in vitro and in vivo experiments.In vivo:A mouse MACO stroke model was established and randomly divided into three groups of 12 rats each.Treatment group:After the establishment of a stroke model,PTx 400ng/d was given and dissolved in 1ml normal saline for intraperitoneal injection and continuous intervention for 3 days.In the control group,1ml normal saline was injected intraperitoneally once a stroke was established;Sham group:Daily Give 1 ml normal saline intraperitoneally.Behavioral scores were performed at 24,48,and 72 hours,respectively,to statistically analyze the damage and repair of neurological function in mice.Infarct size was detected by TTC staining at 72 hours.Cell apoptosis and microglia distribution and activation were detected by immunohistochemistry.At the same time,apoptosis and microglia were detected by Western blot.In vitro:Primary microglia were cultured in vitro and stimulated with lipopolysaccharide(LPS)to simulate a model of microglial injury following stroke.Randomly divided into three groups,the treatment group:PTx 50ng/ml/d intervention,continuous intervention for three days;control group and sham group daily volume of the same cell culture medium.MTT and SRB experiments were used to examine the regulation of survival and damage of microglia after stroke by PTx.ELISA was used to detect the effect of PTx on the release of cytotoxic cytokines from microglia after stroke.Immunofluorescence was used to detect the regulation of CX3CR1 expression in microglia after stroke.[Results]PTx treatment reduced the infarct size of the mice after apoplexy from 52± 12%to 34±8%(P<0.05),and could accelerate the repair of neurological impairment.At 72H,the LONGA score was determined by 3.2 points fell to 1.6 points.Immunohistochemistry showed that PTx could reduce the apoptosis of cerebral apoptotic cells and reduce the number of Caspase-3+ cells from the field of 83±18/mm2 in the control group to 52±16/mm2(P<0.05).Immunohistochemistry also showed that PTx could also reduce the aggregation and activation of microglia in the infarcted area.The number of Iba1+ cells was reduced from the 183±46/mm2 field in the control group to the field of 63±22/mm2(P<0.05).Western blot also obtained consistent results with immunohistochemistry experiments.Treatment of PTx significantly reduced the expression of caspase-3 in the affected rat brain.The band was decreased from1.2.±0.2 to 0.78±0.06(p<0.05).Treatment with PTx also reduced the aggregation of microglia in the infarct zone,and the gray scale of the band decreased from 1.1±0.16 to 0.79±0.09(p<0.05).In vitro MTT assays suggested that PTx reduced the proliferation of microglia after LPS stimulation,and the absorbance value decreased from 0.151 ±0.001 to 0.088±0.001(P<0.05).The detection of SRB also showed similar results.After PTx treatment,the absorbance value was 0.462±0.018 decreased to 0.426±0.02.ELISA results showed that PTx could also reduce the release of IL-1β and TNF-a,and the absorbance values decreased from 0.165±0.001 to 0.091 ±0.0004 and 0.75±0.003 to 0.46±0.005,respectively(P<0.05).And PTx could reduce the expression of CX3CR1 on microglia.Immunofluorescence experiments showed that the fluorescence intensity of CX3CR1 expression in microglial cells decreased after PTx treatment,from 0.043±0.003 to 0.025±0.003(p<0.05).Western blot results also suggested that the expression of CX3CR1 decreased,and the gray value decreased from 0.38 ± 0.05 to 0.29 ± 0.04(p<0.05).[Conclusion]The treatment of PTx in stroke can reduce the accumulation of microglial cells in the infarct zone by reducing the expression of chemokine CX3CR1,weakening the proliferation ability and reducing the secretion of IL-1β and TNF-α,resulting in stroke.The area of hindbrain stenosis is reduced,and the recovery of nerve function is accelerated,which protects the brain. |
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