Study On The Regulatory Effect Of Refined Qingkailing On The A1/A2 Phenotype Of OGD/R-injured Astrocytes

ObjectiveAstrocytes are the dominant non-neuronal cell group,which can show various reactive structural and functional changes in response to ischemia and hypoxia.Recent studies have divided astrocytes into different phenotypes,including A1 and A2.A1 astrocytes are mainly present in inflammatory lesions and are considered to be harmful phenotypes that cause neuronal damage,synapse loss,and impaired skipping action potential propagation.In contrast,A2 astrocytes are thought to have a stronger neuroprotective effect,which can secrete anti-inflammatory mediators,and promote neuron survival.The method of preventing astrocytes from polarizing to the A1 neurotoxic phenotype and maintaining the A2 neuroprotective phenotype has great potential to promote repair and recovery after ischemic injury.Inducible nitric oxide synthase(iNOS)and arginase 1(Argl)are regarded as markers of classical and alternately activated astrocytes,respectively.In addition,complement C3(C3)and S100 calcium binding protein A10(S100A10)are regarded as markers of A1 and A2 astrocytes,respectively.The phenotypic differentiation method of astrocytes provides a way to further study its mechanism.The results of previous studies have shown that RQKL can improve the volume of cerebral infarction in a variety of cerebral ischemia models in vivo and in vitro,improve neurological function,increase the survival of primary neurons,promote the secretion of neurotrophic factors and maintain the integrity of the BBB,and exert anti-apoptotic and oxidative effects.Various effects such as stimulation,inflammation,and anti-excitatory toxicity.Regarding astrocytes,RQKL can inhibit the excessive proliferation,surface area and volume of astrocytes in the surrounding area 7 days after MCAO model infarction.Through network pharmacology studies,it is found that RQKL can affect the response of astrocytes in IS to hypoxia,and RQKL may regulate inflammation with "IL-10,IL-6,CCL2,IL-1?" as the core The factor network exerts an anti-inflammatory effect.Therefore,we speculate whether RQKL may play a protective role in the brain by regulating the responsiveness of astrocytes to ischemia and hypoxia,or by regulating the neurotoxicity and neuroprotective phenotype of astrocytes?Therefore,this experiment intends to explore the regulatory effect of RQKL on OGD/R injury A1 neurotoxic/A2 neuroprotective astrocytes,and provide for the study of the effect and mechanism of RQKL on ischemic stroke on astrocytes.Ideas.Provide ideas for the overall study of IS damage and protection mechanisms,and for finding new targets for clinical treatment.Methods1.Firstly,isolate and culture primary astrocytes from SD rats about 3 days old in vitro,observe and record the growth status of primary and second-generation astrocytes,and use its specific marker glial fibrillary acidic protein GFAP was stained by immunofluorescence to identify the purity of the cells,laying the foundation for subsequent experiments.2.Screening the optimal protective concentration of RQKL against astrocytes:adopt the Anning Pack series for in vitro OGD/R model to simulate ischemic stroke;RQKL was diluted to 31.25?L/mL,15.63?L/mL,7.81?L according to the gradient./mL,3.91 ?L/mL,1.95?L/mL,0.98?L/mL,the experiment is divided into 9 groups,namely blank group,model group,6 groups of RQKL gradient dilution group,and edaravone positive drug group;using CCK8 method Detect the cell viability of astrocytes after injection.3.To study the regulating effect of refined Qingkailing on the A1/A2 phenotype of astrocytes after OGD/R injury:divided into blank group,OGD/R model group,and RQKL 7.81 ?L/mL group.The neurotoxic phenotype and neuroprotective phenotype of astrocytes were classified using A1/A2 markers,respectively.The following methods and indicators are used to evaluate:immunofluorescence double staining method to identify A1(C3 and iNOS labeled)and A2(Argl and S100A10 labeled)astrocyte subpopulations,Western Blot method to identify A1(C3 and iNOS labeled)and A2(Argl label),combined with enzymelinked immunosorbent assay ELISA method to detect the expression of pro-inflammatory factors TNF-?,IL-6 and anti-inflammatory cytokine IL-10.Results1.Successfully established a culture method of astrocytes in vitro,using SD rats to extract primary astrocytes from the cerebral cortex,selecting an appropriate culture concentration,and summarizing the culture and growth rules of astrocytes,Which is conducive to the consistent operation of subsequent experiments.The cells were purified by changing the medium after shaking every other day.After GFAP/DAPI immunofluorescence purity identification,the purity of astrocytes can reach more than 95%,which meets the requirements of further experiments.2.Use Anearo Pack Series to make OGD/R model of astrocyte,OGD4h/24h can achieve obvious model effect(P<0.01).RQKL in the 1.95-31.25?L/mL group can significantly improve the cell viability of astrocytes,and at the same time can improve the cell morphology after modeling.Among them,RQKL at 7.81?L/mL had the best protective effect,which was significantly different from other groups(P<0.01).Therefore,the concentration of 7.81 ?L/mLRQKL was selected for subsequent experiments.3.In Experiment 3,the use of RQKL to intervene the phenotypic changes of astrocytes A1/A2 in OGD/R injury found that RQKL can inhibit the neurotoxicity marker C3 of astrocytes after OGD/R(P<0.05)and the expression of iNOS(P<0.01),and promote the expression of neuroprotective markers Argl(P<0.01)and S100A10;at the same time,RQKL can inhibit the expression of pro-inflammatory factors TNF-? and IL-1?(P<0.01)),promote the expression of anti-inflammatory factor IL-10(P<0.01).ConclusionIt shows that RQKL can inhibit the A1/A2 phenotype of OGD/R model astrocytes.It can inhibit A1 and promote the regulation of A2.RQKL can reduce the pro-inflammatory response of astrocytes exposed to OGD/R damage and inhibit astrocytes.Astrocytes transform to the direction of A1 neurotoxicity and promote their polarization to the neuroprotective direction,thereby exerting neuroprotective effects.