The Role Of Inflammatory Response In The Pathogenesis Of Ischemic Stroke And Its Targeted Therapy

Ischemia stroke is one of the main causes that affect the health and life of many people.It has been characterized with high incidence,high mortality,high morbidity,and high recurrence rates.There is no conventional treatment against stroke damage.To identify new therapeutic targets is becoming the most important event.Recent studies indicate that inflammation is an important factor leading to ischemic brain damage.So to intervene against the inflammatory response may become a new and effective way to treat ischemic stroke.PART ONE A study of ischemic stroke patients treated by human urinary kallidinogenaseObjective:To investigate the possible mechanism and the neuroprotective effect of human urinary kallidinogenase(HUK)in ischemic stroke patients.Materials and Methods:From 2008 to 2011,ischemic stroke patients within 72 hours of onset have been collected.The patients were divided into control group and experimental group intentionally.The same foundation treatment were received.The experiment group patients received HUK once a day,for 10 days.Before treatment we compared the general information and the risk factors between the two groups.We compared the adverse reactions to evaluate the safety during therapy.The NIHSS and Barthel index were used after 12 days and 3 months to evaluated neurological deficit.Inflammatory cytokines(IL-1?,TNF-?)in patients blood serum were detected by enzyme-linked immunosorbent assay(ELISA).Results:There were no differences of the adverse reactions between the two groups.HUK significantly improve the neurofunction.The ratio of efficacy was higher in the experimental group.After treatment the expressions of inflammatory cytokines inblood serum of experimental group patients were attenuated significantly and the HUK group difference were more obvious.Conclusion:HUK may protect against ischemic stroke injury by inhibiting inflammatory response.PART TWO Inhibition of HDAC6 protects against ischemic strokeObjective:To investigate the possible mechanism and the neuroprotective effect of inhibition of HDAC6 in ischemic stroke.Materials and Methods:1)The mouse middle cerebral artery occlusion(MCAO)model was employed.We compared the general information to evaluate the safety during MCAO.We detected the expression of HDAC6 mRNA and protein using RT-PCR and Western blot.2)Four pairs of shRNA sequences targeting HDAC6 gene were designed and synthesized.The shRNA sequences were inserted into the lentiviral vector by annealing and digesting.After amplifying operation the plasmids were extracted for DNA sequencing.The correct plasmids and lentiviral packaging plasmids were transfected into 293T cells.Then the supernatant that contained the virus particles was collected and concentrated.After virus successfully infected the mouse neurons,the shRNA target sequences that had the best interference effect was selected by using RT-PCR and Western blot methods for the follow-up test.3)We used intracerebraventricular(ICV)administration of virus 72h before MCAO in mice.The reperfusion time windows were 6h,24h and 72h.The Neurological deficits were assessed by neurological severity scores(NSS),the infarct size was measured by TTC staining and brain edema was obtained by brain water content(BWC).Inflammatory factors were measured by real-time PCR and western blot.4)Activation of MAPKs,nuclear factor-kB(NF-?B)and Nrf2 was detected by western blot.Results:1)The HDAC6 expression was higher in the MCAO group.2)The HDAC6 shRNA lentivirus(LV-HDAC6 shRNA)was successfully packaged.The shRNA sequence for best interference effect was screened.3)LV-HDAC6 shRNA significantly improved neurofunction,decreased infarct size,and suppressed edema induced by ischemia.LV-HDAC6 shRNA suppressed cerebral inflammation after MCAO in mice.4)MCAO enhanced the phosphorylation of MAPK/p38 and translocation of p65.LV-HDAC6 shRNA inhibited the phosphorylation of MAPK/p38 and translocation of p65.LV-HDAC6 shRNA activated the Nrf2 pathway in this neuroprotection.Conclusion:LV-HDAC6 shRNA suppressed inflammatory responsed,which is probably associated with the inhibition of NF-?B signaling pathway and phosphorylation of MAPK/p38 and activation of Nrf2 pathway.