| Objective: The MCAO model was established to explore the therapeutic effects of GLGZD on rats with cerebral ischemia-reperfusion injury,and to further explore whether GLGZD exerts anti-inflammatory protective effects by regulating microglial activation and phenotypic transformation through the TWEAK / Fn14 / CCL21 / CXCR3 signaling pathway,in order to explore the mechanism of GLGZD in alleviating limb spasm after stroke.Method:(1)MCAO model establishment,rat grouping and drug administration: The thread plug was used to establish MCAO rat model,and mNSS was used to evaluate the degree of neurological damage.Rats with scores of 6 to 14 were randomly divided into MCAO model group,GLGZD low,medium and high dose group and NMDP group.in addition,the Sham group was set as the blank control.All the rats were administrated once a day for 7 day.(2)The therapeutic effects of GLGZD on MCAO rats: mNSS and Ashworth were used to score rats on day 1,3,5,and 7 of drug intervention,MRI to detect the volume of cerebral infarction,HE staining to observe the histopathological changes,Nissl staining to observe the morphology and number of nerve cells,Tunel method to detect the neuronal apoptosis rate.(3)Regulation of GLGZD on microglial activation and phenotype conversion: qRT-PCR and immunofluorescence was used to detect the mRNA and protein expression of Iba-1 in ischemic cerebral cortex of rats to assess microglial activation.Western-Blot and immunefluorescence were used to detect the protein expression of CD16 and CD206,Immunohistochemistry was used to detect the protein expression of TNF-α and Arg-1,in order to analyze microglial M1 and M2 polarization levels.(4)The effects of GLGZD on TWEAK / Fn14 / CCL21 / CXCR3 Signaling Pathway Related Factor Expression: qRT-PCR was used to detect the mRNA expression of TWEAK,Fn14,cIAP1,TRAF2,NIK,p100,CCL21,CXCR3 in ischemic cerebral cortex of rats,Western-Blot to detect the total protein expression of TWEAK,Fn14,cIAP1,TRAF2,NIK,IKKα,p-IKKα,p100,CCL21,CXCR3 in ischemic cerebral cortex of rats;Immunofluorescence was used to analyse the protein expression of Fn14,CCL21,CXCR3 quantitatively and locately;Immunohistochemistry was used to detect the nuclear protein expression of p52,Rel B.Results:(1)The therapeutic effects of GLGZD on MCAO rats: GLGZD could significantly improve neurological function(P<0.05,0.01),Significantly reduced muscle tension in the affected side of the rat(P<0.05,0.01),and improve motor function(P<0.05,0.01)and pathological changes of cerebral cortex on ischemic side in rats.It also could significantly increase Nissl positive cells in ischemic cortex of MCAO rats(P<0.01),and markedly reduce cerebral infarct volume and apoptosis of ischemic cortical neurons in MCAO rats(P<0.05,0.01).(2)Regulation of GLGZD on microglial activation and phenotype conversion: GLGZD could significantly reduce the mRNA and protein expression of Iba-1 in the ischemic cortex(P<0.05,0.01),significantly reduce the protein expression of CD16,TNF-α and the polarization rate of M1-type microglia in the ischemic cortex(P<0.01),and can significantly increase the protein expression of ischemic cortex CD206,Arg-1 and the polarization rate of M2-type microglia(P<0.05,0.01).(3)The effects of GLGZD on TWEAK / Fn14 / CCL21 / CXCR3 Signaling Pathway Related Factor Expression: GLGZD could significantly down-regulate the mRNA expression of TWEAK,Fn14,NIK,p100,CCL21,CXCR3(P<0.05,0.01)and up-regulate the mRNA expression of cIAP1 and TRAF2 in the ischemic cortex(P<0.05,0.01).And could significantly reduce the total protein expression of TWEAK,Fn14,NIK,CCL21,CXCR3(P<0.05,0.01)and increase the total protein expression of cIAP1 and TRAF2 in ischemic lateral cortex(P<0.05,0.01).It also could inhibit the total protein expression of p100 while inhibiting the processing of p100,making the p100 protein content of each dose group significantly higher than the MCAO group(P<0.05,0.01).Immunofluorescence results show,Fn14 and CCL21 are mainly expressed in neuron,and CXCR3 is mainly expressed in microglia.GLGZD could significantly reduce the nuclear protein expression of p52 and Rel B(P<0.01).Conclusion: GLGZD can promote the recovery of neurological and motor functions after cerebral ischemic injury,relieve limb spasm.Having a good protective effect on cerebral ischemia-reperfusion injury,and this effect may be related to inhibit excessive inflammation by regulating microglial activation and phenotypic transformation through inhibiting the TWEAK / Fn14 / CCL21 / CXCR3 signaling pathway. |
