Resveratrol Via STAT3/BMP Pathway Regulated Astrocyte Activation To Facilitate Recovery After Spinal Cord Injury

Backgrounds and Objectives:Spinal cord injury(SCI)represents a significant health problem associated with life-long disability and a broad range of secondary complications,eventually cause patients paralysis or death.To date,there is not a accepted and the most effective treatment.A large number of data show that pharmacotherapy is now widely accepted treatment,but currently most of them have some side effect,therefore,seeking more effective drug treatment is the frontier subject of spinal cord injury research.SCI often causes incurable neurological dysfunction.After SCI,reactive astrocytes and glial scar make the injured microenvironment change to hamper the injured spinal cord recovery.Our previous study reveals that the astrocytes reactivated after SCI secrete BMP2 and inhibit the co-cultured OPCs to differentiate into OLs.It has been reported that the activation of the STAT3 is necessary to activate the astrocytes after SCI.BMP2 is the downstream target gene in the pathway of STAT3 during the astrocytogenesis.Based on this evidence,we hypothesize that STAT3 would increase the secretion of the BMP2.Therefore,the inhibition of STAT3 may provide a way to regulate the secretion of BMP2 that is produced by reactive astrocytes.Resveratrol(RES),wildely found in natural plants,has a variety of biological activity and is capable of inhibiting the activation of STAT3.This proposal is designed to use Res to treat the reactive astrocytes to regulate the activation of STAT3/BMP pathway,which may further affect the proliferation and differentiation of OPCs.Hence,this proposal attempts to establish a mechanism with key factors involved in a pathway for SCI recovery.The ultimate goal of this proposal is to provide a strategywith sound hypothesis that will be experimentally proved through molecular biology approach.Methods:We made a injury spinal cord model by spinal cord impacter;Astrocyte cultures were obtained from the SD rat of spinal cord at 7 day after injury,established an vitro model of reactive astrocytes;Primary culture SD rat astrocytes and neurons were obtained from the brain of neonatal SD rat,established SD rats neurons/astrocytes coculture.1)The NSCA and ISCA proliferative potential was determined by cell counting / Brdu Straining /Flow cytometry analysis.2)The effects of resveratrol on astrocyte proliferation were estimated by MTT and flow cytometry(FCM).3)To measured the effect of resveratrol on the expression of GFAP /STAT3/P-STAT3/BMP2 in astrocyte by Western-blot and ICC.4)The effects of resveratrol on the expression of GFAP/STAT3/BMP2 in level of transcription were detected by RT-qPCR.5)The influence of resveratrol on neurons axon length were estimated by immunofluorescence staining.6)High performance liquid chromatography(HPLC)method is applied to detect the absorption of NSC and ISC tissue,after intraperitoneal injection of resveratrol.7)Application of BBB behavioral detection observed effection of resveratrol on motor function recovery of spinal cord injury.8)The effect of resveratrol on spinal cord injury area and the expression of GFAP /STAT3/PSTAT3/BMP2 and NF-M were measured by HE staining and immunofluorescence.Results: 1)Compared to the NSCA,the proliferation ability of ISCA were enhanced,expression of GFAP and BMP2 in ISCA was stronger.2)ISCA proliferation activity is reduced,and occurred phase cycle arrest after resveratrol treatment for 48 h.3)Resveratrol downregulated the expression of GFAP/STAT3/BMP2 both in the mRNA and the protein levels.4)Resveratrol could promote neuronal neurite growth in neuron/astrocyte cocultures.5)After intraperitoneal injection of resveratrol,we can both detect the resveratrol in the the NSC and ISC.6)The BBB test scores of resveratrol treatment group was obviously higher than that of the DMSO control group;SCI-25 mm group rat BBB scores can achieve around 20 points,SCI-50 mm group rat scores only to around 11 points.7)The spinal cord injury damage area of resveratrol treatmentgroup is smaller than DMSO control group,in the damaged region,the expression of BMP2/STAT3 and P-STAT3 protein significantly lowered,but NF-M expression significantly enhanced.Conclusions: 1)Compared with SCI-25 mm group,SCI-50 mm group rat spinal cord injury was obviously,so the SCI-50 mm is highly suitable for the spinal cord injury model.2)The proliferation ability of ISCA were enhanced,the expression of GFAP and BMP2 increased,and the characteristics similar with activation of astrocytes.Activated astrocytes vitro model was successfully established.3)Resveratrol could inhibite the activated astrocytes,that mainly showed by inhibiting the proliferation of astrocytes and downregulating the protein of GFAP and BMP2.4)STAT3/BMP pathway may be the target of resveratrol regulates the activation of astrocytes,showing the effect through downregulating the protein of STAT3,P-STAT3 and BMP2.5)Resveratrol could promote neuronal neurite growth in neuron/astrocyte cocultures.It is suggested that resveratrol has potential role in promoting on regeneration and repair of nerve.6)Resveratrol can hinder the formation of glial scar and reduce the area of the spinal cord injury.7)Resveratrol promote the recovery of motor function of rats after spinal cord injury that mainly showed by BBB test.