| Perioperative cerebral ischemia with high mortality rates(26%-60%)and high disability rates is paid more attention by people. Although several measures being taken, the therapeutic protective effect of cerebral ischemia (CI) was still less, mainly due to the pathogenesis of CI remains unclear. However, it was found that, after CI, the brain damage is deteriorated gradually and the local in-flammatory response is more obvious simultaneously, which playing a main important role in cerebral injury may be more worse than by cerebral ischemia its-self. How to inhibiting inflammatory response after CI has become a new target for study to protect ischemic cerebral injury.Recently, help T 17 cell (Th17) and regulatory T cell (Treg) were found that they play important roles in process of several inflammatory diseases. Th17 cells expressing retinoic acid-related orphan receptorγt (RORγt) take a critical effect in the development of autoimmunity and allergic reactions by producing IL-17,that up-regulates rate of releasing inflammatory cytokines to injury cere- bral tissue.Meanwhile, Treg cells expressing the forkhead/winged helix transcription factor (Foxp3) have anti-inflammatory properties and maintain tolerance against self components by contact-dependent suppression or releasing an-ti-inflammatory cytokines [such as interleukin (IL)-10 and transforming growth factor (TGF)-β1]. Therefore, the balance between Th17 and Treg cells play an important role in the treatment and prevention of inflammatory diseases (au-toimmune diseases, CNS's inflammatory diseases, transplant organ rejection).It was recently reported that Th17 cell get a great role in the formation and development of inflammatory disease in central nervous system (CNS), such as Multiple Sclerosis (MS). Otherwise, Th17 & IL-17 were demonstrated that there are co- relationships with cerebral ischemia. The transient or permanent cerebral ischemia, the corn of fact, would be"the injury of cerebral ischemia is led by inflammatory response after CI".Depending upon above fact ,We firstly present our hypothesized in the world that:(1) There may be an imbalance of Treg/Th17 cells existing after CI, which appears and change with ischemic cerebral injury simultaneously, and this un-control of immunological regulation by imbalance of Treg/Th17 cells would amplify the degree of inflammatory response and delay its time course, which aggravate ischemic cerebral injury.(2) Recovering the balance of Th17/Treg cells by immune interference at different levels for Th17 or IL-17 or Treg may inhibit inflammatory response after CI and reduce the extent of ischemic cerebral injury. The goal of our study would be approval a new immune interference thought to prevent ischemic ce-rebral injury. Our research project was paid attention and supported by National Natural Science Foundation of China (NSFC, No.30872445). This report is our study for first part of the project.Objective:On the model of transient middle cerebral artery occlusion (tMCAO) in mice, this study was determined to confirm an imbalance of Th17/Treg existing after transient cerebral ischemia in mice, and there is a rela-tionship between Th17/Treg imbalance and ischemic brain injury, which explore the inflammatory regulation mechanism following cerebral ischemia. That would present a new therapy method for stroke patient.Methods:70 mice were randomly divided into experimental (n=60) and sham (n=10) groups. And former were divided into six subgroups by assessing time points after surgical manipulation: They were 6 h (n=10), 12 h (n=10), 24 h (n=10), 48h (n=10), 72h (n=10) and 5 days (n=10). The model of tMCAO was established by modified monofilament method;Neurologic deficit score (NDS) was performed at every time points after tMCAO, after then sacrificed mice and measured the volume of cerebral infarction by tetrazolium chloride staining (TTC);the infiltration of Th17 cells in brain was observed by immunofluores-cence;Th17/Treg functions including cell frequencies, related cytokine secretion and key transcription factors (RORγt or Foxp3) at different levels were inves-tigated by flow cytometric (FCM) analysis, enzyme-linked immunosorbent assay (ELISA) and Western blot.Results:After cerebral ischemia, brain edema became worse and brain infarcted area enlarged gradually, and the peaks were at 48h[(44.4±3.2)%], and then the brain infarcted area slightly reduced; Neurological function improved gradually when being compared 5d (2.2±0.45, P<0.05) with 6h after CI, (4.6±0.55); FCM analysis indicated the tendency that the frequencies of Th17 cells began to increase gradually following cerebral ischemia, and appeared the maximum at 24h [(0.70±0.10) %, p<0.05, vs sham and 5d], and then it was re-turned to normal level. This tendency was consistent with that of cerebral ische-mia extent. The frequencies of Treg cells, in contrast, firstly began to reduce slowly, and arrived at the minimum level at 24h [(0.9±0.29) %], almost disap-pear, After 24h, the frequencies of Treg cells recovered step by step, At 5d ,it was near normal level or even exceeded in certain mice[(3.2±0.49) %,p<0.05]; Meanwhile, most cytokines related with Th17 and Treg cells were showed to the similar change tendency with frequencies of Th17 or Treg. But, surprisingly, IL-17A increased gradually from 6h to 5d in brain or blood serum, and arrived at the maximum at 5d (brain, 77.9±5.11pg/ml; blood serum, 29.44±3.06 pg/ml); The change tendency of Foxp3 that Treg special transcription factor was in ac-cordence with that of Treg cells frequencies, while there was no obvious change for Th17 special transcription factor RORγt; In addition, the infiltration of Th17 cells in ischemic brain hemisphere was observed by immunofluorescence, but not in contralateral hemisphere.Conclusion:It is sure that Th17/Treg functional imbalance exists in mice after transient cerebral ischemia. The change tendency of associated cytokines is consistent with that of Th17 and Treg cells. Those change tendencies are closely associated with extent of cerebral injury. So we thought that Th17/Treg cells imbalance may play an important role in the progress of ischemic cerebral injury. Correcting and maintaining Th17/Treg function balance will become a new the-rapeutic target for ischemic cerebral injury. |
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