Mechanism Of Scutellarin Regulating Activation Of A1 Type Astrocytes After Cerebral Ischemia

Objective(s): Ischemic stroke is a clinical syndrome caused by a series of factors that lead to cerebral circulation disorders,ischemic and hypoxic necrosis of brain tissue,and neurological dysfunction.It is the leading cause of death and disability among adults in China,with a high incidence,disability rate,mortality rate,and recurrence rate.The inflammatory response following cerebral ischemia is the main cause of neuronal death and brain damage.Astrocytes are the main inflammatory cells in the central nervous system.When ischemic stroke occurs,astrocytes are activated and participate in and mediate a series of inflammatory responses.Activated astrocytes,called reactive astrocytes,are divided into two main subtypes: the pro-inflammatory A1 phenotype and the anti-inflammatory A2 phenotype,which include cytokines and chemokines that participate in neuroinflammation and mediate pro-inflammatory or anti-inflammatory effects,playing neurotoxic or neuroprotective roles.A1 astrocytes play an important role in ischemic stroke,and regulating astrocytes towards A1 activation is a potential therapeutic target with significant value.Scutellarin(SCU)is a flavonoid compound extracted from the plant Erigeron breviscapus,which has multiple pharmacological activities,including anti-inflammatory,antioxidant,and antidiabetic properties.SCU has been widely studied in ischemic cerebrovascular diseases,involving neuronal protection,antioxidant effects,and regulation of microglial activation,but its effect on astrocyte activation has not been reported.Therefore,we will explore the effects and mechanisms of SCU on astrocyte activation after ischemia,providing new ideas for the treatment of ischemic stroke.Methods: In vivo part: Firstly,a C57BL/6 transgenic mouse model of cerebral ischemia-reperfusion(MCAO)was established.The mice were randomly divided into three groups.Treatment group: SCU100mg/kg was injected intraperitoneally 2hours before stroke model establishment,and then continued to receive SCU100mg/kg intraperitoneal injection for three consecutive days.Model group: 1 ml of saline was intraperitoneally injected 2 hours before and for three consecutive days after stroke model establishment.Sham group: 1 ml of saline was intraperitoneally injected daily.On the third day,behavioral assessments were performed to analyze the neurological function of the mice.The brain infarct volume was evaluated using TTC staining 72 hours after stroke.Immunofluorescence was used to detect the activation of A1 astrocytes,and Western blot was used to quantitatively detect the expression of A1 astrocytes.In vitro part: Primary astrocytes were cultured and subjected to OGD/R to simulate brain ischemic injury.The cells were randomly divided into three groups.Treatment group: SCU50μmmol/L,SCU100μmmol/L,and SCU200μmmol/L were used to pretreat cells for 2 hours before OGD/R,and then the cells were exposed to OGD/R for 3 hours and incubated in sugar-free medium for 24 hours in a CO2 incubator.Ischemia group: The same volume of complete medium was given before and after OGD/R.Normal control group: The same volume of complete medium was given.The astrocyte culture medium from each group was collected to stimulate PC-12 cells.CCK-8 and MTT were used to detect the toxicity of scutellarin on astrocytes and the expression of neuronal apoptosis.Q-PCR was used to detect the relative expression of C3 m RNA,a specific marker for astrocytes.Immunofluorescence and Western blot were used to quantify the expression of A1 astrocyte markers C3 and neuronal apoptosis proteins.Western blot was used to explore the effect of scutellarin on the expression of proteins related to the NF-κB signaling pathway.Results: The use of scutellarin in the treatment of cerebral apoplexy decreased the cerebral infarction size from 33.86±10.73% to 16.47±7.39%(P < 0.01),and also alleviated neurological impairment.LONGA score decreased from 2.4 to 1.5 on the third day.These results indicated that the neurological function of mice was improved after treatment with scutellarin.In vivo,scutellarin can reduce the expression of C3 protein.The results of immunofluorescence and Western blot showed that after MCAO,the expression of C3 protein in the MCAO group was increased compared with that in the Sham group,while after scutellarin treatment,the expression of C3 protein in the MCAO group was decreased compared with that in the MCAO group.This suggested that scutellarin could inhibit the activation of astrocytes into the A1 type.In vitro,Q-PCR was used to detect the expression of C3 m RNA in the A1 astrocyte marker,and the results showed that compared with the normal control group,the expression of C3 m RNA in the OGD/R group was significantly increased,indicating that astrocytes were activated to A1 type after anoxia in vitro,and after treatment with scutellarin,Compared with OGD/R group,C3 m RNA expression in the three scutellarin concentration treatment groups was decreased,indicating that the activation of astrocytes to type A1 could be reduced after scutellarin treatment,and the difference was statistically significant.The results of cell immunofluorescence and Western blot showed that the expression of OGD/R histone C3 increased compared with the normal control group,and the expression of histone C3 decreased after scutellarin treatment compared with OGD/R group,indicating that scutellarin could reduce the activation of astrocytes to type A1 after hypoxia,and the difference was statistically significant.The expression of neuronal apoptosis protein Caspase-3 in the astrocyte medium was detected by immunofluorescence.The results showed that compared with the normal group,the expression of Caspase-3-induced neuronal apoptosis protein in primary astrocyte medium after OGD/R treatment was significantly increased,while the expression of Caspase-3 in primary astrocyte medium after scutellarin treatment was significantly decreased compared with OGD/R group.These results indicated that astrocytes could reduce neuronal apoptosis after scutellarin treatment,and the difference was statistically significant.MTT was used to detect the effect of astrocyte medium on neurons.The results showed that,compared with the normal group,the absorbance OD value of the OGD/R group was significantly decreased,while the absorbance OD value of astrocyte medium after scutellarin treatment was increased compared with OGD/R group,indicating that astrocytes treated with scutellarin could reduce the death of neurons.The difference was statistically significant.The changes of key proteins of the NF-κB signaling pathway were detected by Western blot assay.The results showed that OGD/R and scutellarin had no significant effect on the changes of non-phosphorylated IκBα and P65 protein,but significantly regulated the phosphorylated IκBα and P65 protein.After OGD/R,the expression levels of P-IκBα and P-P65 protein were significantly increased compared with the normal group,and the expression of C3 protein was up-regulated.After treatment with scutellarin,the expression levels of P-IκBα and P-P65 protein decreased significantly,along with the down-regulation of C3 protein.The expression levels of P-IκBα and P-P65 were significantly inhibited by BAY 11-7082,an inhibitor of the NF-κB signaling pathway,and C3 expression was further down-regulated.These results suggest that scutellarin can directly regulate astrocyte activation by inhibiting the NF-κB pathway.Conclusion(s): Scutellarin in the treatment of stroke can reduce the transformation of astrocytes to type A1 by inhibiting the phosphorylation of P65 in the NF-κB pathway,thereby reducing the cerebral infarction volume after stroke,accelerating the recovery of nerve function,reducing neuronal apoptosis,and playing a neuroprotective role.