Protective Effects Of Active Polypeptide GRGDS On Cerebral Ischemia Reperfusion Injury

With the development of society,the changes of life style and the aging of the population,more and more people suffer from senile neurological diseases such as stroke and Alzheimer's disease.Stroke is the most severe disease among them.About 15 million people suffer from this disease every year.It has become the secondary leading cause of human death and the main cause of global disability.With high morbidity,high disability rate,and high mortality rate on stroke,it pose a serious burden on every patient and family.There are many causes of stroke,such as obesity,high blood pressure,smoking and alcoholism,and the patient population shows a trend of rejuvenation.At present,there are few therapeutic drugs for the treatment of stroke,most of them are thrombolytic drugs.Tissue fibrinogen is a kind of thrombolytic drug for the treatment of acute cerebral ischemia that has been approved for clinical treatment.However,since the patient must be treated within 4.5 hours after the onset of the stroke,the use rate of the thrombolytic drug becomes limited.It is vital important to develop a new type of drug without the limit of time,and has better effect to the stroke.The active polypeptide GRGDS consists of five amino acids,glycine-arginine-glycine-aspartate-serine(Gly-Arg-Gly-Asp-Ser,GRGDS),which are composed of tripeptide(Arg-Gly-Asp,RGD)and other two amino acids.RGD is known to be the smallest effective sequence in fibrinogen and has shown to enhance adhesion of endothelial cells and smooth muscle cells to improve cell growth and antithrombotic function.It has reported that the active polypeptide RGD also has the adhesion function and promotes cell proliferation.However,most of the research focus on GRGDS is to use it as a target to combine with other drugs or materials,such as applying it to tissue engineering or anti-tumor drugs.At present,no researches focused on the treatment of stroke with GRGDS.Therefore,we studied the treatment effect and mechanism of GRGDS on stroke.The research mainly includes the following aspects:Methods:(1)The cerebral ischemia-reperfusion model of SD rats was made by middle cerebral artery occlusion(MCAO)method.The active peptide GRGDS was injected into the tail vein of the rats.The active peptide GRGDS was injected into the tail vein of the rats.After the ischemia and reperfusion for 24 hours,the neurological deficit of the rats was scored.2,3,5-triphenyltetrazolium chloride(TTC)staining was used to observe the cerebral infarction areas in the brain of rats;The expression of SOD,MDA,TNF-? and IL-1? were detected by ELISA method.The changes of nerve cells in rat brain were observed by H&E,Nissl and TUNEL staining.(2)The model of oxygen deprivation of PC12 cells was established,and the effect of active peptide GRGDS on nerve cells was investigated by means of glucose deficiency and hypoxia.MTT assay and flow cytometry assay were used to detect the survival and apoptosis rate of PC12 cells after oxygen-glucose deprivation.The inflammatory kits were used to detect the inflammatory factors TNF-? and IL-1? in the supernatant of PC12 cells after oxygen glucose deprivation.The expression of p-JNK,Bax and Cleaved Caspase3 were detected by western blot.Meanwhile,the expression level of these proteins were measured with the treatment of JNK inhibitor.Results:(1)The results showed that active peptide GRGDS could effectively improve the neurological score and decrease cerebral infarction area after cerebral ischemia in rats(p<0.01).The active peptide GRGDS(3mg/kg dose group)can significantly reduce the release of MDA,TNF-? and IL-1? in rat serum and increase the content of SOD.The active peptide GRGDS can significantly reduce the damage of nerve cells in brain tissue after cerebral ischemia and increase the number of cells.(2)The results showed that the active peptide GRGDS can significantly increase the survival rate and decrease the apoptosis of PC12 cells after oxygen-glucose deprivation.The active peptide GRGDS can significantly reduce the inflammatory factors TNF-? and IL-1? in the supernatant of PC12 cells after oxygen-glucose deprivation.The active polypeptide GRGDS can significantly inhibit the expression of p-JNK,Bax,and Cleaved Caspase 3.Conclusions:(1)The active polypeptide GRGDS has a protective effect on cerebral ischemia-reperfusion injury.(2)The active polypeptide GRGDS exerts protective effects on cerebral ischemia-reperfusion injury is related with the decreasing apoptosis rate of PC12 cells,decreasing levels of inflammatory cytokines(TNF-? and IL-1?),and inhibition of the p-JNK,Bax and Cleaved Caspase 3.In summary,the active polypeptide can effectively alleviate cerebral ischemia-reperfusion injury in rats,and has a good protective effect on nerve cells.This reminder us a new direction for the active peptide GRGDS exerts as a neuroprotective drug to treat cerebral ischemia.