The Effect Of Xionggui Prescription And BMSCs-exosomes On The Intervention Of Post-stroke Neuroinflammation

BackgroundIschemic stroke will produce a series of inflammatory reactions,but excessive inflammatory activation aggravates the damage of neurons.At present,the treatment of neuroinflammation has not achieved satisfactory results.Bone marrow mesenchymal stem cells(BMSCs derived exosomes,BMSCs-exos)carry a variety of important information,such as proteins,and participate in cell-cell communication.Although most cell therapies can be replaced by exosomes,exosomes still have many problems,such as low targeting ability,low half-life and low concentration of functional molecules.Therefore,improving the therapeutic efficiency of exosomes is a new stage of cell therapy and regenerative medicine.Xionggui formula is composed of chuanxiong and Danggui,which is commonly used in clinical treatment of ischemic stroke.Shunaoxin dropping pill,a listed Chinese patent medicine,is also widely used in the treatment of stroke.The previous study of the team found that the intervention of shunaoxin on ischemic stroke model rats can effectively improve the recovery of neurological function,synaptic remodeling and angiogenesis after brain injury,and reduce the expression of inflammatory factors to a certain extent,suggesting that shunaoxin has a regulatory role in the inflammatory microenvironment after stroke.Therefore,to explore whether Xionggui decoction combined with BMSCs exos can improve the neuroinflammation of ischemic stroke and its mechanism is worthy of further study.ObjectiveXionggui prescription(SNX)and BMSCs-exos intervened tMCAO rats model,to evaluate its effect on inflammatory microenvironment of brain injury in stroke rats,and to explore whether it can improve neuroinflammation and its mechanism.Methods1.The middle cerebral artery occlusion(tMCAO)model of SD rats was established by thread occlusion method.After 1.5 hours of ischemia,the rats were randomly divided into four groups,model group(tMCAO),BMSCs exosomes group(tMCAO+BMSCs-exosomes),xionhgui prescription group(tMCAO+SNX)and synergistic treatment grou P(tMCAO+BMSCs-exosomes+SNX).In the model group,500 ul PBS was injected into the tail vein on 1d,3d,5d and 7d after modeling.In the Xionggui prescription group,shunaoxin dropping pills were mixed with normal saline into 4.5mg/ml solution,and each rat was given a dose of 45mg/kg/d by gavage.Exosomes group were injected with exosomes 400 ug/kg by tail vein on the 1d,3d,5d and 7d after modeling.The synergetic group was treated with the combination of SNX and exosome.Sham group(Sham)only separated the corresponding vessels after anesthesia,without ligation or embolus.2.Neurological function score and rotating rod test were performed on the 1d、3d and 7d after modeling.The neurological function score was used to evaluate the neurological deficit of rats,and the rotating rod test was used to evaluate the motor coordination ability of rats3.HE staining and Nissl staining were used to observe the edema of penumbra and neuron damage after ischemic brain injury.4.Immunofluorescence staining was used to detect the infiltration of MPO~+inflammatory cells in penumbra after ischemic brain injury.5.Immunofluorescence staining was used to detect the expression of CD86~+/TMEM119~+、CD206~+/TMEM119~+cells in penumbra after ischemic brain injury.6.Detection the m RNA expression levels of M1/m2 microglia surface marker(TNF-α、i NOS、TGF-β、Arg-1)and pro-inflammatory and anti-inflammatory cytokines(IL-1β、IL-6,IL-13)Result1.Xionggui prescription and BMSCs-exosomes can significantly improve the neurological deficit,motor function,sensory ability,reflex function,balance ability,and motor coordination ability of rats with ischemic brain injury.The results showed that the neurological function of rats began to improve from the third day,and the neurological function and motor coordination ability improved significantly on the 7d.2.HE staining and Nissl staining showed that xionggui prescription and BMSCs-exosomes could reduce brain edema and neuron damage.On the 3d and 7dafter operation,he results showed that brain edema and neuron damage increased continuously,brain tissue was seriously broken,intercellular vacuoles increased,neurons arranged disorderly and swollen;Nissl’s results showed that the boundary of the nucleus of the neurons was blurred,the nucleolus pyknosis was serious,the neurons had many vacuole like changes,and the Nissl bodies in the cytoplasm decreased significantly.Xionggui decoction combined with BMSCs exosomes can effectively improve brain edema and neuronal necrosis from the third day,and the effect is more significant on the 7d.3.Immunofluorescence results showed that Xionggui prescription combined with BMSCs exosomes could reduce the infiltration of inflammatory cells in penumbra of rats after ischemic brain injury.MPO immunofluorescence showed that inflammatory cells began to diffuse infiltration from the third day,and the damaged brain tissue began to be exposed to the inflammatory environment.This inflammatory state continued to the seventh day,and gradually aggravated.Xionggui prescription and BMSCs-exosomes can inhibit the recruitment and infiltration of inflammatory cells,and effectively improve the inflammatory response of the brain after ischemic stroke.4.Immunofluorescence results showed that Xionggui prescription and BMSCs exosomes could reduce the expression of M1 microglia marker CD86+/tmem119+cells and increase the expression of M2 microglia marker cd206+/tmem119+cells.From the 3rd day,the number of M1 microglia began to decrease and continued to the7th day,and the number of M2 microglia increased from the 3rd day to the 7th day,and increased significantly on the 7d.5.The expression level of surface marker of M1 microglia cells can be reduced and the m RNA expression level of M2 microglia surface marker can be increased by Xionggui prescription recipe in cooperation with BMSCs-exosomes.It decreased the expression of Pro-inflammatory cytokines and increased the m RNA expression of anti-inflammatory cytokines.Conclusion1.Xionggui prescription combined with BMSCs-exosomes can improve the neurological damage,promote the recovery of motor function,and improve the inflammatory microenvironment of brain tissue in rats with ischemic stroke.2.Xionggui prescription combined with BMSCs exosomes can promote the dynamic balance of M1 type microglia and M2 type microglia,and reduce the neuroinflammatory reaction in the acute stage of stroke.