| BackgroundIntracerebral hemorrhage(ICH)is a common acute cerebrovascular disease,accounting for 20%of all strokes.Effective medical treatments for this condition are currently lacking.The disease is characterized by rapid onset and progression,high mortality,and disability,which adversely impact the lives of patients.The main causes of death and disability are a series of pathological changes caused by hematoma,such as the mass effect of hematoma and the toxic substances deriving from hematoma dissolution.Early clearance of hematoma can not only prevent further increases in the volume of the hematoma to protect the brain tissue,but also prevent the secondary cerebral damage resulting from various toxic substances dissolved from the hematoma.Therefore,effective clearance of hematoma after ICH is essential to alleviate the damage to the cerebrum and improve neurological recovery.The central nervous system(CNS)is principally composed of two types of cells:neurons and glial cells.The latter are further divided into microglia,astrocyte,and oligodendrocyte.Microglia are intrinsic macrophages of the CNS,which have the function of clearing damaged and apoptotic neurons and pruning synapses.Studies have demonstrated that microglia cleared red blood cells after ICH.In a murine model of ICH,drugs that increase the phagocytic function of microglia significantly reduce the volume of hematoma.Peroxisome proliferator-activated receptor-γ(PPAR-γ)is one kind of nuclear hormone receptors.Studies have reported that PPAR-γlimited inflammation by restraining the expression of pro-inflammatory genes and increasing the expression of anti-inflammatory and anti-oxidative genes.In addition,previous researches have demonstrated that PPAR-γcould protect neurons from damage by suppressing inflammatory responses and oxidative stress.Axl and Mer TK receptor tyrosine kinases are members of the TAM(Tyro3,Axl and Mer TK)family.They are necessary for the phagocytosis and the regulation of the innate immune response.Axl and Mer TK are generally expressed in the brain.Studies have indicated that microglia require TAM family receptors to regulate phagocytosis in the process of neurogenesis or repairing damaged brain.CD36,a kind of scavenger receptor,is involved in a variety of biological functions,such as chemotaxis of immunocyte and phagocytosis of phagocyte.In models of ICH,activated PPAR-γcan augment the expression of CD36 in microglia and boost the phagocytosis of microglia,thus accelerating the clearance of hematoma.Lysosomal associated membrane protein 2(LAMP2),a lysosomal glycoprotein,is abundant in cells.As a receptor for proteins,it can be transported directly into lysosomes,and is also a mediator for phagosome-lysosome fusion.Wogonin is a natural bioflavonoid extract of the traditional Chinese medicine of Huang‐Qin.An increasing number of researches have demonstrated that wogonin exerts anti-inflammatory,anti-oxidative,anti-tumor,and neuroprotective effects.For example,wogonin decreases the expression of IL-1βand TNF-αby inhibiting the nuclear factor NF-κB signaling pathway in alcoholic liver disease,thus alleviating inflammatory responses.Research on osteoarthritis has demonstrated that wogonin protects articular cartilage by inhibiting the production of reactive oxygen species(ROS)to restrain oxidative stress.Wogonin is often used in anti-tumor therapy to induce apoptosis and to ameliorate mitochondrial dysfunction as well as endoplasmic reticulum stress.In addition,in mouse models of traumatic brain injury,wogonin treatment ameliorated brain edema and inflammation via the toll-like receptor 4(TLR4)/NF-κB pathway and ultimately improved long-term neurological function.The present study was aimed to investigate whether wogonin could enhance the phagocytosis of microglia after ICH to accelerate hematoma clearance and improve the neuroprotective,and to explore the molecular mechanism of Wogonin in the neuroprotective effect after ICH,and ultimately to provide a new target and a new method for the treatment of ICH.MethodsPart 1Autologous blood from the left femoral artery was collected by catheter to establish the model of cerebral hemorrhage.Adult healthy male C57BL/6 mice were randomly divided into Sham group,ICH+Vehicle group,and ICH+Wogonin group.The volume of hematoma at 1,3,7,14 and 28 days after intracerebral hemorrhage was examined by magnetic resonance.The expression levels of phagocytic related proteins Axl and MERTK were detected by western blot at 1,3,5,7,14 and 28 days after intracerebral hemorrhage.The study time points were determined after intracerebral hemorrhage in mice.The distribution of Axl and Mer TK was detected by immunofluorescence,and the expression levels of phagocytic related proteins(Axl,Mer TK,CD36,LAMP2)in brain tissues of mice after intracerebral hemorrhage were detected by western blot and immunohistochemistry.Part 2Adult male C57BL/6 mice were randomly divided into Sham group,ICH+Vehicle group,and ICH+Wogonin group after determining the study time point after ICH.The neurological deficit score after cerebral hemorrhage was evaluated.The protein expression levels of inflammatory cytokines(IL-1β,TNF-α)in the brain tissues of mice after cerebral hemorrhage were detected by Western Blot,and the expression levels of M1,M2 microglia in the brain tissues were detected by immunofluorescence staining,Nissl staining was used to observe the survival of neurons in basal ganglia,and transmission electron microscope was used to observe the damage of myelin sheath.Part 3In vivo experiment:Adult male C57BL/6 mice were randomly divided into Sham group,ICH+solvent(ICH+Vehicle)group,ICH+Wogonin group,ICH+PPAR-γagonist(ICH+Rosiglitazone)group and ICH+Wogonin+PPAR-γinhibitor(ICH+Wogonin+GW9662)group.In vitro experiment:BV2 cells were cultured and given100 M hemoglobin to simulate intracerebral hemorrhage in vitro.They were randomly divided into 5 groups as follows:Ctrl group,Vehicle group,Wogonin group,PPAR-γagonist and Wogonin+PPAR-γantagonist group.The transcriptional levels of phagocytosis related protein genes(Axl,Mer TK,CD36 and LAMP2)were detected by q RT-PCR,and the proportion of Axl and Mer TK positive microglia in the brain tissues of mice in each group after intracerebral hemorrhage was detected by immunofluorescence staining.PKH26 labeled red blood cells were added to different BV2 cells in a 10:1 ratio(RBC:BV2),and the expression levels of phagocytosis related proteins in BV2 cells stimulated by Oxy Hb were detected by Western blot.The phagocytosis of BV2 cells on RBC in each treatment group was analyzed by flow cytometry,and the phagocytosis of BV2 on RBC cells in each treatment group was observed by immunofluorescence.ResultsPart 1After intracerebral hemorrhage,the related protein AXL was phagocytosis in the brain tissue of mice,and Mer TK began to increase,and reached the peak on the third day.The hematoma volume in ICH+Wogonin group was significantly lower than that in ICH+Vehicle group on the 3rd day after ICH.On the 3rd day after ICH,the levels of phagocytosis related proteins AXL,Mer TK,CD36 and LAMP2 in ICH+Wogonin group were significantly higher than those in ICH+Vehicle group.In addition,Wogonin increased the proportion of AXL~+,Mer TK~+microglia.Part 2On the 3rd day after intracerebral hemorrhage,neurological function in ICH+Wogonin group was significantly improved compared with ICH+Vehicle group.The expression level of inflammatory cytokines in the ICH+Wogonin group was significantly lower than that in the ICH+Vehicle group.The number of M1-type microglia in the ICH+Wogonin group was significantly lower than that in the ICH+Vehicle group,while the number of M2-type microglia was significantly higher than that of ICH+Vehicle group.In addition,the injury degree of neurons and myelin sheath was significantly lower than that of ICH+Vehicle group.Part 3In vivo experiments,the results showed that both Wogonin and PPAR-γagonist significantly increased the proportion of Axl and Mer TK positive microglia in the brain tissues of mice after intracerebral hemorrhage,as well as increased the transcription of phagocytic genes(Axl,Mer TK,CD36 and LAMP2),compared with that in the Vehicle group.PPAR-γinhibitor GW9662 blocked the above effects of Wogonin.In vitro experiments,the results showed that the expression levels of Axl,Mer TK,CD36 and LAMP2 of BV2 cells in Oxy Hb+Wogonin group and Oxy Hb+PPAR-γagonist group were significantly higher than those in Oxy Hb+Vehicle group.The expression levels of Axl,Mer TK,CD36 and LAMP2 of BV2 cells in Oxy Hb+Wogonin+GW9662 group were significantly decreased compared with that in Oxy Hb+Wogonin group.In addition,the ability of BV2 cells to phagocyte erythrocytes in Wogonin group and PPAR-γagonist group was stronger than that in Vehicle group,while the ability of BV2cells to phagocyte erythrocytes in Wogonin+PPAR-γinhibitor GW9662 group was significantly decreased.Conclusion1.Wogonin increased the expression of phagocytosis related proteins in the microglial cells of brain tissue after intracerebral hemorrhage in mice,and accelerated hematoma clearance.2.Wogonin induced microglia cell polarization to M2 type after intracerebral hemorrhage in mice,alleviated the neuroinflammatory response and oxidative stress response after intracerebral hemorrhage in mice,and then reduced the damage degree of neurons and myelin sheath around the hematoma,and improved the neurological function of mice.3.Wogonin promotes the phagocytosis ability of microglia through PPAR-γ/CD36 signaling pathway,and promotes the removal of hematoma after intracerebral hemorrhage,thereby inhibiting the secondary brain injury. |
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