The Protective Effect Of Salvianolate On Neurons By Regulating Microglia

ObjectiveAfter the acute cerebral ischemia-reperfusion injury,it will not only cause damage to neurons in the brain,but also cause polarization of microglia to cause inflammatory response,and release inflammatory factors to promote further damage of neuronal cells.Therefore,in this subject,the neuroprotective mechanism of salvianolate was mainly explored through two aspects:in vitro Oxygen and glucose deprivation(OGD)damaged neuronal cell and OGD induced microglial polarization.And combined with middle cerebral artery occlusion(MCAO)to verify the therapeutic effect of salvianolate(salvia)in vivo.Methods1.In the OGD induced neuronal cell injury model:First,OGD was applied to neuronal cells at different times(2 h,4 h,8 h)to screen the best hypoxic time.Next,the cell counting method(CCK-8)was used to determine the optimal concentration of salvia neuroprotective.After OGD damaged neuron cells,Confocal laser scanning microscope(CLSM)was used to determine the effect of salvia on the reactive oxygen species(ROS)fluorescence expression.Finally,Hoechst 33342 staining and flow cytometry were used to determine the protective effect of salvia on neuronal cell nuclear damage and apoptosis.At the same time,Western Blotting was used to determine the apoptosis signaling pathway(Caspase-3)expression.2.In the OGD induced microglia polarization model:First,OGD was applied to microglia at different times(3 h,6 h,12 h,24 h)to screen the best time to induce phenotypic polarization.Next,the CCK-8 assay was used to detect the toxicity of salvia on microglia;OGD could induce oxidative stress in microglia;The CLSM assay was used to detect microglial ROS and phenotype polarization on the different concentrations of salvia,And the expression of superoxide dismutase(SOD),catalase(CAT),and glutathione peroxidase(GSH-px).Western Blotting was used to detect activation of microglial inflammatory signaling pathway(TLR4),and mRNA expression of inflammatory factors(TNF-α,IL-6,IL-1β,IFN-γ)by using reverse transcription polymerase chain reaction(RT-qPCR)Situation;Enzyme-linked immunosorbent assay(ELISA)was used to detect the protein expression level of inflammatory factors.Finally,Salvia inhibited OGD induced microglia activation by flow cytometry and Western Blotting methods,which affected the apoptosis of neurons by microglia.3.In the rat middle cerebral artery occlusion model(MCAO):First,the effects of salvia at different concentrations on MCAO injury in rats were measured using TTC staining.Next,hematoxylin-eosin(HE)and Nissl staining methods were used to determine the pathological changes of brain slices after salvia treatment.Laser speckle contrast imaging(LSCI)was used to determine the changes of cerebral blood flow in different experimental groups;phenotypic transformation of microglial cells in different groups by CLSM assay;TUNEL staining assay was used to detect the effects of salvia on apoptosis in rats with MCAO injury;Western Blotting assay was used to detect the expression of apoptotic pathway-related proteins in brain injured tissues.Finally,Morris water maze and Rota-Rod stick experiments were used to detect the effects of salvia on cognitive and motor improvement after MCAO injury in rats.Results1.The OGD.induced neuronal cell injury model was successfully constructed through a series of experimental methods.Salvia was used to intervene in OGD induced neuronal cell damage.The results show that salvia(5 μM)can significantly decrease neuronal cell damage and reduce the expression level of ROS,and reducing the expression of LDH and NO in injured neurons.And salvia can significantly reduce the apoptosis rate of injured neurons and inhibit the expression of proteins related to Caspase-3 apoptotic signaling pathway.2.A series of experimental methods have been successfully used to construct an OGD induced microglial polarization model.The experimental results show that OGD can induce microglia to undergo oxidative stress response,leading to a large amount of ROS expression,which makes microglia change to a pro-inflammatory phenotype,and the expression levels of inflammatory factors TNF-α and IL-6 significantly increased.There was no change in the expression of IL-1β,IFN-y.Next,salvia(4 μM,16 μM,64μM)and microglia were co-incubated under OGD conditions.With the increase of salvia concentration,OGD induced the expression of ROS in microglia gradually decreased,and the expression levels of SOD,CAT and GHS-px also gradually decreased.Then the phenotype of microglia was observed by fluorescence staining.After co-incubation of salvia and microglia,the expression of CD86,a proinflammatory phenotype antibody,gradually decreased.Salvia can reduce the activation of TLR4 signaling pathway in OGD induced microglia,and reduce the mRNA and protein expression levels of the inflammatory factors TNF-α and IL-6.Finally,the conditioned medium prepared by co-incubating salvia and microglia under OGD conditions interacted with neuronal cells.As a result,it was found that salvia conditioned medium can increase the viability of neuronal cells,reduce the rate of apoptosis,and inhibit Caspase-3 signal pathway activation.ConclusionThis project mainly explores the pharmacological activity of salvia from two aspects:On the one hand,by constructing a model of OGD induced neuronal damage,it was found that salvia can reduce the expression of ROS in neuronal cells and thus inhibit the activation of Caspas-3 signaling pathway.On the other hand,the polarization model of microglial cells was induced by OGD.It was found that salvia reduced the oxidative stress response in microglia,reduced the expression of ROS,and further inhibited the activation of TLR4 signaling pathway,thereby weakening the microglial polarities.It has also been further verified at the animal level.These results show that salvia can effectively reduce acute cerebral ischemia-reperfusion injury and has a good neuroprotective effect.