The Neuroprotective Effect Of Rosmarinic Acid In Ischemic Stroke Via Regulating The Polarization Of Microglia

Background Stroke is a common neurological disorder—the second leading cause of death and the third leading cause of disability in adults worldwide in recent years,and ischemic stroke(IS)is the most common type of stroke.According to the latest epidemiological findings,there are about 2.4 million people suffering from new stroke each year in China,most of whom are left with varying degrees of neurological impairment,posing a serious burden to patients,families and society.The clinical treatment for IS is currently based on intravenous thrombolysis,mechanical thrombus retrieval and supplemented with neurotrophic drugs,circulation improving drugs and antiplatelet drugs.However,most patients cannot be treated manually due to the narrow time window and severe complications.Therefore,the challenge of finding new treatment strategies for IS remains a topical clinical research project.A series of secondary cascade responses occur after the onset of IS,including excitotoxicity,oxidative stress,inflammatory response,and apoptosis.Among them,cellular inflammation and apoptosis,as the core pathological processes of neuronal cell death,may lead to further injury and permanent neurological impairment if not intervened in time.The activation and polarization status of microglia,as the intrinsic immune cells of the central nervous system(CNS),is critical for the immune microenvironment after injury and disease regression after IS.In recent years,several novel drugs have been used in preclinical studies of ischemic stroke,among which natural polyphenols have attracted a lot of attention.Rosmarinic acid(RA),a natural polyphenol drug isolated from the herb rosemary,has been reported to have good antioxidant,anti-inflammatory,anti-apoptotic and proangiogenic activities.However,the mechanism of its neuroprotection after cerebral ischemia is unclear and still needs further study.Objective:The purpose of this study is to investigate the neuroprotective effects of RA in mice after cerebral ischemic injury;subsequently,to explore the mechanisms by which RA exerts neuroprotective effects at the microglia level;furthermore,to filter and validate the effect sites of RA by proteomics analysis in regulating microglia polarization and apoptosis,and to provide further theoretical support for the translation of RA to the clinic.Methods: This study is divided into three parts as follows.Part Ⅰ: Neuroprotective effects of RA on mice with IS(1)The mouse MCAO/R model and PT model were constructed,and the experiments were divided into Sham group,Injury group,Injury+RA low-dose group,Injury+RA high-dose group and Injury+positive drug Edaravone group.(2)The neurological recovery of rats in each group was evaluated by Longa score,turning stick test,turning angle test and cylinder test.(3)The size of cerebral infarct area was observed by TTC staining;neuronal death was observed by H&E and Nissl’s staining;blood-brain barrier(BBB)integrity was evaluated by dry vs.wet weight method and Evans blue staining.(4)Neuronal apoptosis and microglia polarization were detected by immunefluorescence,Western Blot and ELISA.Part Ⅱ: Regulation of microglia polarization by RA(1)OGD/R models of primary mouse microglia and BV2 cell lines were constructed,and the experiments were divided into Control group,OGD/R group and OGD/R+RA group.(2)The cell survival rate and optimal drug concentration were determined by SRB assay;the effect of RA on microglia apoptosis was verified by TUNEL staining,flow cytometry and Western Blot;the regulatory effect of RA on microglia polarization was detected by immunofluorescence,flow cytometry and Western Blot.Part Ⅲ: Exploration of the mechanism of RA regulation of microglia polarization(1)The differential proteins between OGD/R group and OGD/R+RA group of BV2 cells were screened by TMT proteomics,analyzed by GO and KEGG enrichment using bioinformatics,and validated by Western Blot.(2)The alteration of microglia apoptosis and polarization after the target protein was inhibited was detected by Western Blot and flow cytometry.Results:(1)In vivo experiments demonstrated that RA can effectively promote neurological recovery,reduce infarct area,mitigate neuronal damage and protect the BBB in ischemic stroke mice,and inhibit the activation of microglia,reduce their conversion to M1 type and increase the number of M2 type conversion in ischemic stroke mice.(2)In vitro experiments further demonstrated the modulating effect of RA on microglia polarization.RA reduced the apoptosis of OGD/R microglia and BV2 cells,inhibited their activation,and reduced the number of M1 type conversions and increased the number of M2 type conversions.(3)TMT proteomics screened 112 differential proteins,including 46 up-regulated proteins and 66 down-regulated proteins,and analyzed and screened these differential proteins,finally identified the key protein Sirt3 and its downstream key pathway PI3K/Akt/mTOR,and verified that RA inhibits autophagy through increasing Sirt3 expression,which regulates the apoptosis and polarization of microglial cell in OGD/R.Conclusion: RA effectively protects neural tissue from ischemic injury and inhibits apoptosis and activation of microglia in IS,reducing their M1-type polarization but increasing their M2-type polarization,mainly by increasing microglia Sirt3 expression and therefore inhibiting autophagy via the PI3K/Akt/mTOR pathway.