Salvianolic Acid Attenuates Experimental Cerebral Ischemia-reperfusion Injury Through Microglial P2X7/NLRP3/GSDMD Pathway

Objective: Mounts of studies have shown that salvianolic acids could improves the prognosis of ischemic stroke and exerts neuroprotective effects through a variety of pharmacological effects and mechanisms,but its potential mechanisms are further warranted to illustrate.Microglia involve in inflammatory response after cerebral infarction.While the P2X7/NLRP3/GSDMD pathway in microglia,which composed of the NLRP3 inflammasome,the upstream receptor P2X7 and the downstream molecular GSDMD,mediates the inflammatory cascades after cerebral infarction.This study aims to investigate the neuroprotective effects and molecular mechanisms of Salvianolic Acids for Injection(SAFI,which is the injection of salvianolic acids)on cerebral ischemia/ reperfusion injury via observing the inhibitory effects of SAFI on microglial P2X7/ NLRP3/ GSDMD pathway.For this purpose,we established a middle cerebral artery occlusion/ reperfusion(MCAO/R)model in rats and an oxygen-glucose deprivation / reoxygenation(OGD/R)model in co-cultured primary neurons and primary microglia.The effects of SAFI on neurological score,infarct volume,neuronal apoptosis,expression of inflammatory factors in MCAO/R model,and neuronal viability and apoptosis in OGD/R model were observed,and the effect of SAFI on the expression of related molecules in P2X7/NLRP3/GSDMD pathway in the cortex of MCAO/R model and microglia in OGD/R model were also observed.Materials and methods:1.Neuroprotective effect of SAFI on MCAO/R model rats(1)58 rats were randomly divided into 2 parts.The rats in part 1 were randomly divided into two groups: I/R group(MCAO/reperfusion-operated rats treated with vehicle)and I/R+SAFI group(MCAO/reperfusion-operated rats treated with SAFI).These rats were used to observe neurological impairments and sacrificed on days 7 after the operation to measure infarct volumes.In part 2,the rats were randomly divided into 4 groups: Control group(sham-operated rats treated with vehicle),SAFI group(sham-operated rats administrated with SAFI),I/R group(MCAO/reperfusion-operated rats treated with vehicle),I/R + SAFI group(MCAO/reperfusion-operated rats treated with SAFI).These rats were sacrificed on day 3after the operation and used for histological observations,RT-PCR,and western blot analysis.(2)A nylon filament was inserted into the stump of external carotid artery and advanced into the internal carotid artery until it reached root of the middle cerebral artery,where the filament would block arterial blood supply to cause ischemic injury.Suture was gently removed after 120 min occlusion to restore blood flow.The neuroprotective effect of SAFI on ischemia-reperfusion injury in rats was evaluated by observing the infarct volume,neurofunctional score,and histopathological injury,expression of inflammatory factors and neuronal apoptosis,which were detected using 2,3,5-triphenyltetrazolium chloride(TTC)staining,neuro-scoring,Hematoxylin & Eosin(HE)staining,immunohistochemistry and terminal deoxynucleotidyl transferase-mediated d UTP nick-end labeling(TUNEL)assay,respectively.2.Effects of SAFI on cell viability and apoptosis of microglia and neurons treated with OGD/R(1)Primary neurons and microglia were isolated and extracted from neonatal SD mice.The expression of neuron marker MAP2 and microglia marker CD11-b were detected by immunofluorescence.MTT method was used to detect the cell activity of different concentrations of SAFI on primary neurons and microglia treated with OGD to screen the best concentration of SAFI.(2)According to the cell types,the cells were divided into the neurons cultured group and the microglia + neurons co-cultured group.The neurons cultured group was divided into three subgroups: Neuron group(neurons were cultured under normal conditions),Neuron + OGD/R group(neurons were treated with OGD/R),and Neuron + OGD/R + SAFI group(neurons were treated with OGD/R and SAFI).Protocols: in the Neuron + OGD/R + SAFI group,the primary neurons were cultivated on the culture plate,pretreated with SAFI for 24 hours,and the cells were changed to glucose-free DMEM in 95% N2 and 5% CO2 environment(the medium contained the same concentration of SAFI)for 3 hours,then the cells were cultured under normal culture conditions for 24 hours and the cells were harvested for further assays;the Neuron + OGD/R group was cultured without SAFI,and the Neuron group was cultured without SAFI under normal conditions.The microglia + neurons co-cultured group was divided into three subgroups: Neuron + Microglia group(neurons and microglia were cocultured under normal conditions),Neuron + Microglia + OGD/R group(neurons and microglia were co-cultured and undergone OGD/R treatment),Neuron + Microglia + OGD/R+ SAFI group(neurons and microglia were co-cultured and undergone OGD/R and SAFI treatment).Protocols: in Neuron + Microglia + OGD/R + SAFI group,primary microglia and neurons were cultured in a Transwell co-cultured system.The neurons were cultured in the lower chamber and the microglia were grown in the upper chamber.Cells were pretreated with SAFI under normal culture conditions for 24 h,then cells were cultured with in glucosefree DMEM in the environment of 95% N2 + 5% CO2 at 37?.After OGD for 3 h,the cells were transferred to normal medium under normoxic conditions for 24 h.Then neurons and microglia were harvested for further experiments.Neuron + Microglia + OGD/R group was cultured without SAFI according to the protocols as before.Neuron + Microglia group was cultured without SAFI under normal culture conditions.(3)The protective effect of SAFI on OGD/R model on neurons and neurons co-cultured with microglia was observed by detecting the level of LDH in culture medium to reflect cell injury,the viability of neurons in each group was examined by CCK-8 assay,and the apoptosis of neurons was detected by flow cytometry.3.Effects of SAFI on NLRP3 inflammasome activation and pyroptosis related protein GSDMD in MCAO/R model and microglia in OGD/R model.(1)The modeling and grouping of animals are the same as the above.The co-cultured cells were divided into four groups: Control group(neurons and microglia were co-cultured under normal conditions),SAFI group(neurons and microglia co-cultured under normal conditions and added with SAFI),OGD/R group(co-cultured neurons and microglia were subjected to OGD/R),OGD/R + SAFI group(co-cultured neurons and microglia were treated with OGD/R and SAFI administration).(2)In OGD/R + SAFI group,the primary microglia and primary neurons were cultured in Transwell co-culture system.The neurons were grown in the lower chamber of 6 wells,and the microglia were inoculated in the upper chamber.The cells were pretreated with SAFI of50ug/ml concentration under normal culture conditions for 24 h,then the cells were changed to glucose-free DMEM(the medium contained the same concentration of SAFI)in 95% N2,5% CO2 environment and cultured at 37? for 3 h to establish OGD model.The neurons were collected after culturing under normal condition for 24 h.The cells in OGD/R group were cultured without SAFI as the above procedures.SAFI group was cultured with SAFI under normal cultured conditions.Control group was cultured without SAFI under normal culture conditions.(3)After the MCAO/R model and co-cultured OGD/R model were established.The expression of NLRP3 in microglia of the cortex was observed by immunofluorescence.The levels of proteins related NLRP3 inflammasome activation and pyroptosis related protein GSDMD in the brain tissue and cultured microglia were detected by western blot.The expression levels of m RNA related NLRP3 inflammasome and GSDMD in brain tissue and cultured microglia were detected using RT-PCR.(4)To investigate inhibitory effects of SAFI on activation of NLRP3 infammasome,nigericin and monosodium uric acid(MSU)were utilized as the NLRP3 inflammasome activators.The microglia were cultured at plates and primed with lipopolysaccharide(LPS).After LPS was removed by washing with phosphate-bufered saline(PBS),the cells were administrated with SAFI.After that,the microglia were cultured in a serum-free medium supplemented with nigericin or MSU to activate NLRP3 infammasome.Then,the cells were lysed and subjected to western blot assay.4.Effect of SAFI on the expression of membrane ion channel P2X7 which located in upstream NLRP3 inflammasome activation and molecular docking between P2X7 and main components.(1)The modeling and grouping are the same as the above.(2)RT-PCR,western blot and immunofluorescence methods were used to observe the effect of SAFI on the expression of P2X7 in the cerebral cortex in MCAO/R model and primary microglia co-cultured with neurons in OGD/R model.(3)The three-dimensional structure file of P2X7 X-ray crystal of rats was downloaded from RCSB database.The amino acid residues in the pocket of the protein binding to ATP through hydrogen bond and ion interaction were obtained from literature reports.The ligands of protein were deleted by Discovery Studio 4.2 software,and the hydrogenation,charge calculation and conversion format were processed by Auto Dock 4.2.6 software.The molecular structures of the main components of SAFI were obtained from TCMSP database,ZINC database and Pub Chem database.The semi-flexible docking is carried out by using Auto Dock 4.2.6 software.The docking results were visually analyzed by Discovery Studio4.2 software.The binding ability to P2X7 of salvianolic acid B,salvianolic acid D,salvianolic acid Y,lithospermic acid and rosmarinci acid,the main components of SAFI,was observed by computer molecular docking method.Results:1.SAFI improved the neurological deficit and reduced the volume of cerebral infarction,and reduced the pathological injury of brain tissue in cerebral I/R model in rats.2.SAFI reduced the expression of inflammatory cytokines ICAM-1,IL-1 ?,IL-18 and TNF-?in the cerebral cortex in MCAO/R model,and reduced the neuronal apoptosis in the cerebral cortex in MCAO/R model.3.SAFI reduced the cell injury of neurons,improved cell viability and reduced the rate of apoptosis treated with OGD/R either in cultured independently or co-cultured with microglia.4.Under the condition of co-culturing and subjected to OGD/R treatment,microglia may exert cytotoxic effect on neurons,while SAFI may reduce the cytotoxic effect of microglia on neurons.5.SAFI reduced the expression of NLRP3 in microglia in the brain tissue of rats after I/R injury,and inhibited the expression of pro-inflammatory factors such as IL-1 ? and IL-18.6.SAFI inhibited the NLRP3 inflammasome activation in the brain after cerebral I/R injury and in microglia co-cultured with neurons and treated with OGD/R.7.SAFI inhibited the cleavage of pyroptosis related protein GSDMD in brain after I/R injury and microglia co-cultured with neurons and treated with OGD/R.8.SAF reduced the m RNA expression of NLRP3,ASC,caspase1 and IL-1 ? in brain tissue after cerebral I/R injury and microglia treated with OGD/R.9.SAFI reduced the number of microglia double-labeled with Iba1 and P2X7 in the cerebral cortex in MCAO/R model,and reduced the protein and m RNA expression of P2X7 in the cerebral cortex in MCAO/R model and microglia co-cultured with neurons and treated with OGD/R.10.The active components such as salvianolic acid D,salvianolic acid Y and lithospermic acid contained in SAFI may have good binding abilities with P2X7 receptor.Conclusion:1.Inhibiting inflammatory response and reducing neuronal apoptosis may be the partial pharmacological basis of neuroprotective effects of SAFI.Inhibiting inflammatory response may be one aspect to embody efficacy of promoting blood circulation and removing blood stasis of SAFI in treating cerebral ischemic stroke.2.SAFI exerts neuroprotective effects on neurons subjected to OGD/R injury through direct way and probable indirect way via antagonizing the microglial toxicity to neurons.3.The active components such as salvianolic acid D,salvianolic acid Y and lithospermic acid contained in SAFI maybe have good binding ability to P2X7 receptor,and these components might antagonize the activation of P2X7 receptor.4.SAFI alleviates cerebral ischemia/reperfusion injury in part probably via inhibiting the activation of NLRP3 inflammasome and pyroptosis in microglia.5.SAFI alleviates the expansion of downstream inflammatory cascades probably by inhibiting the activation of NLRP3 inflammasome and pyroptosis through the P2X7/NLRP3/GSDMD pathway in microglia,thereby exerting the neuroprotective effects on experimental cerebral ischemia/reperfusion injury.