Study On The Anti-inflammatory Mechanism Of Qingkailing On Cerebral Ischemia Rats Based On The Phenotypic Changes Of Microglia

Ischemic stroke is a kind of middle cerebral artery occlusion caused by embolism or thrombosis.Inflammatory reaction after ischemic stroke can aggravate secondary neuron damage,which is harmful for the recovery of brain function.Microglia are the permanent immune cells in the brain.They continuously monitor the microenvironment of the brain under physiological conditions.Once ischemia occurs,microglia are activated rapidly.Activated microglia are defined as activated microglia(M1)and selectively activated microglia(M2)according to different secretory factors and surface markers.M1 microglia mainly play a neurotoxic(anti-inflammatory)role while M2 microglia mainly play a neuroprotective role.The potential mechanism of anti-inflammatory effect of Qingkailing injection after ischemic stroke has not been fully elucidated.Based on the phenotypic changes of microglia,this study explored the anti-inflammatory mechanism of Qingkailing on cerebral ischemia rats.To further elucidate the anti-inflammatory mechanism of Qingkailing after ischemic stroke,and provide experimental basis for the study of traditional Chinese medicine.Objective(1)To prove the protective and anti-inflammatory effects of Qingkailing injection on middle cerebralartery occlusion(MCAO)rats.(2)To explore the anti-inflammatory mechanism of Qingkailing on MCAO rats based on the phenotypic changes of microglia.Methods(1)Healthy male Sprague-Dawley rats weighing 210-230 g were randomly divided intothree groups: sham-operated group,model group(0.9ml/100g)and Qingkailing group(0.9ml/100g).The model group and Qingkailing group were made MCAO model.Qingkailing group was injected intraperitoneally with 0.9 mL/100 g diluted Qingkailing injection(diluted by 2 times normal saline)at 0h and 4h after ischemia.From the next day,the drug was administered twice a day,with an interval of 12 hours.The sham-operated group and the model group were intraperitoneally injected with saline of equal volume.24 hours after MCAO,the neurological deficits were assessed by Bederson neurological function score,the volume of cerebral infarction was measured by TTC staining,and the histopathological changes around cerebral ischemic focus were observed by HE staining.The changes of IL-1?,IL-6 and TNF-a were detected by Elisa.The expression of p65/p-p65 and p-IkBa was detected by Western blot.The effect of Qingkailing injection on the signal pathway of NF-kappa B and inflammatory factors were observed after ischemic stroke.(2)Healthy male Sprague-Dawley rats weighing 210-230 g were randomly divided into three groups: sham-operated group,model group(0.9ml/100g)and Qingkailing group(0.9ml/100g).Qingkailing group was injected intraperitoneally with 0.9 mL/100 g diluted Qingkailing injection(diluted by 2 times normal saline)at 0h and 4h after MCAO.From the next day,Qingkailing injection was injected twice a day with an interval of 12 hours.The sham-operated group and the model group were intraperitoneally injected with saline of equal volume.MCAO models were prepared in model group and Qingkailing group.The expression of M1 microglia marker CD16 and M2 microglia marker CD206 was observed by double immunofluorescence staining.The expression of M1 microglia-specific protein CD16,CD32,iNOS and M2 microglia-specific protein CD206,TGF-beta were further detected by Q-PCR.The mechanism of Qingkailing's anti-inflammatory effect after ischemic stroke was revealed by regulating microglia phenotype.Results(1)Neurological Function: There was no neurological impairment in the sham-operated group.Most of the rats in the MCAO group and QKL group had left hemiplegia and body turned to the left side.Left forelimb could not be fully straightened when tail was suspended,Furthermore,left forelimb was adduction and close to the chest wall.Compared with the sham-operated group,the neurological function score of the MCAO group increased significantly(P < 0.01);after the treatment of QKL,the neurological function improved and the neurological function score decreased significantly(P < 0.01).(2)TTC staining: In the sham-operated group,the brain tissue of rats showed a uniform rose-red color.Compared with the sham-operated group,the MCAO group showed obvious ischemia area(P < 0.01);Compared with the MCAO group,the brain infarct volume of the QKL-treated group was significantly reduced(P < 0.01).(3)HE staining: In sham-operated group,the structure of brain tissue was intact,the neurons cells were arranged orderly,the cytoplasm was rich and stained lightly.The nucleus was large,the nucleolus was clear,and the structure of glial cells was intact and arranged orderly.In the MCAO group,large infarction area were observed in the brain tissue,and the number of nerve cells disappeared or decreased with disordered arrangement.Glial cells around the infarction increased significantly,the necrotic margin was narrow,and neutrophils infiltrated in large numbers.In QKL-treated group,the area of cerebral infarction area decreased significantly,the number of neurons decreased slightly,the proliferation of glial cells was not obvious,and the marginal area was more broader.(4)Influences on NF-kappa B pathway and inflammatory factors: after middle cerebral artery occlusion,TNF-a(P < 0.01),IL-1?(P < 0.01)and IL-6(P < 0.01)of MCAO group were significantly higher than those in sham-operated group.Compared with the MCAO group,QKL could inhibit the expression of TNF-a(P < 0.01),IL-1?(P < 0.05),IL-6(P < 0.05),the ratio of p-p65 to p65(P < 0.05),and the expression of p-IkBa(P < 0.05).It has been proved that QKL can inhibit the activation of NF-kappa B pathway,inhibit the secretion of inflammatory factors,and thus resist post-ischemic inflammation.(5)Immunofluorescence staining and Q-PCR: Double immunofluorescence results showed that after MCAO,QKL inhibited the expression of CD16,a marker of M1 microglia,and increased the expression of CD206,a marker of M2 microglia.Q-PCR results showed that QKL could decrease the expression of M1 microglia protein markers CD16,CD32 and iNOS,increase the expression of M2 microglia protein markers CD206 and TGF-?.Conclusions(1)QKL injection can reduce the volume of cerebral infarction in MCAO rats,reduce the neurological function score and ameliorate the pathological damage.Qingkailing injection can inhibit the activation of NF-kappa B pathway,reduce the secretion of inflammatory factors,inhibit the inflammatory response of ischemia-reperfusion injury rats,and play a protective role in brain.(2)QKL injection can inhibit the expression of CD16,a neurotoxic(pro-inflammatory)microglia marker of M1,and promote the expression of CD206,a neuroprotective(anti-inflammatory)microglia marker of M2.It can decrease the expression of CD16,CD32 and iNOS,and up-regulate the expression of CD206 and TGF-?,a microglia-specific protein of M2.It promotes the transformation of microglia into beneficial phenotype.QKL's promotion of M2 may be related to its inhibition of the activation of NF-kappa B pathway and reduction of pro-inflammatory factors.QKL can inhibit the inflammatory response and exert protective effect on brain of cerebral ischemia rats by regulating the phenotype of microglia.