| Objective 1.Through clinical analysis,to explore whether ipsilateral transient ischemic attack(TIA) of anterior circulation before cerebral infraction(CI) could be work as ischemic preconditioning(IPC),to alleviate follow-up ischemic injury and analysis possible influence factors. 2.Through examine CI patients expression of peroxisome proliferator-activated receptor gamma(PPARγ), nuclear factor kappa B(NF-κB) and CD36 in peripheral blood mononuclear cells(PBMCs) at different time, to evaluate the changes of PPARγ and NF-κB in the process of IPC.Methods Collected One hundred and thirteen patients with anterior circulation CI were admitted in Neurology Department of Tianjin General Hospital from November 2014 to September 2015.113 patients were divided into cerebral infarction(CI) group(n=87) and prestroke TIA-cerebral infarction(TIA-CI) group(n=26) according to their history. 36 healthy condition matched persons without history of cerebrovascular diseases were selected as the control group. 1.Compared the differences of END, NIHSS, 3 month m RS, infarct volume, serum hs-CRP level and other risk factors between these two groups. 2.According to the lasting of TIA(<10min, 10~20min, >20min), attack times(1 times, 2 to3 times, >3 times), the first time attack before stroke(<7d, 7 ~ 14 d, >14d),the TIA-CI group divided into different subgroups. Compare END, NIHSS score, 3-month m RS score and serum hs-CRP level. 3. Collected the peripheral venous blood from three group patients at the 24 hours, 3 day, 7 day, 12 day, all blood used to extract PBMCs.(1) Blot Western method was used to detect the expression levels of NF-κB and PPARγ at different time points in PBMCs.(2) The expression levels of NF-κB and PPARγ at different time points in PBMCs were detected by immunofluorescence staining.(3) Flow cytometry was to determine the expression of CD36 at different time points.Results1、Clinical research :The incidence of END, NIHSS score, 3-month m RS score, and serum hs-CRP level were significantly lower whereas the infarct size was significantly smaller in TIA-CI group than in CI group(11.5%vs31.0%,1.85 ± 2.31 vs 3.30 ± 3.32, 0.90± 0.83 vs1.78± 1.77, 2.52±3.23mg/L vs6.21±3.23mg/L, 3.92± 8.05cm3 vs9.15± 15.07cm3,P < 0.05).TIA occurred 2~3 times on 7~14 days before ischemic stroke and lasted >10 min showed lower END, NIHSS score, 3-month m RS score, and serum hs-CRP level. Hs-CRP is positively correlated with END(r=0.311,P<0.05),NIHSS on admission(r=0.455,P<0.01), NIHSS on dismission(r=0.557, P<0.01),m RS(r=0.534, P<0.01)and infarct volume(r=0.524, P<0.01). 2、Experimental study(1) Expression and active of PPARγ 1)Detection expression of PPARγ: Western blot detection of PPARγ total protein expression.Compared with the control group, the CI group and TIA-CI group PPARγ protein expression of PBMCs lower at 24 h after infarction,but differences between the control group had no statistical significance(t = 0.830,P > 0.05; t = 1.804, P > 0.05); 3d later, two groups of PPARγ expression showed increased in a time-dependent manner and between the two groups at each time point PPARγ differentially expressed no statistical significance(t=0.871,P > 0.05; t=0.587,P > 0.05; t=0.436,P > 0.05). PPARγ expression in two groups of patients showed a downward trend after rising, and the expression between the two groups showed no significant difference. 2)Detection active of PPARγ: ` Detection of PPARγ subcellular localization:Using Immunofluorescence detection.PPARγ of control group was mostly in the cytoplasm, in the inactivated state. Compared with the control group, As lasting of infarction time(24h, 3d, 7d, 12d), the TIA-CI group PPARγ expression in the cytoplasm gradually reduced and gradually increased accumulation within the nucleus(PPARγ nuclear translocation), suggesting that PPARγ was activated; in infarction 24 h and 3d, CI group appear PPARγ nuclear translocation, and its distribution was no significant difference with the TIA-CI group. Since the beginning of the 7d of infarction, compared with the TIA-CI group, CI group PPARγ gathered in the nucleus decreased,and cytoplasmexpression increased, suggesting that PPARγ activity decreased. TIA-CI group PPARγactivity was time-dependent increase, CI group PPARγonly increase in initial activity infarction.Compared with the CI group, the TIA-CI group was activated more and the activity duration was longer than it. a Detection CD36: Flow cytometry detection of CD36.Compared to the control group, the expression of the TIA-CI group’s CD36 was infarction time dependent increased, the difference had statistical significance(t=9.198, P < 0.01; t=13.48, P < 0.01; t=17.22, P < 0.01; t=6.096, P < 0.01); CI group’s CD36 in infarction 24 h, 3d, 7d expression gradually increased, differences statistically significant(t=6.012, P < 0.01; t=9.003, P < 0.01; t=11.62, P < 0.01), 12 d expression began to decline significantly, as compared with that in the 7d had statistical significance(t=5.063, P < 0.01), but still higher than the control group(t=5.477, P < 0.01). CD36 expression in two patient groups were higher, TIA-CI group was continuing an increasing trend, CI group was reduced after increase. Between the two patient groups in 24 h, 3d, 7d had no statistically significant difference(t = 0.106, P> 0.05; t = 0.764, P> 0.05; t = 0.257, P> 0.05), but TIA-CI group each time point was higher than CI group. But in12 d TIA-CI group CD36 expression was significantly higher than CI group, the difference was statistically significant(t = 4.147, P <0.05). Compared with the CI group, the duration of TIA-CI group CD36 high-level expression was longer.(2) Expression and active of NF-κB(p65): 1) Detection expression of NF-κB: Western blot detection of NF-κB protein expression.Compared with the control group: `CI group and TIA-CI group NF-κB expression in 24 h and 3d was increased, the difference was statistically significant(CI group vs control group: t=3.959, P <0.05;t=4.838, P <0.05; TIA-CI group vs control group: t=4.039, P<0.05; t=5.003, P<0.01);TIA-CI group 7d and 12 d NF-κB expression levels were significantly lower, and lower than the level of the control group, the difference was statistically significant(t = 5.931, P <0.01; t = 4.838, P <0.01); CI group 7d began to approach the level of the control group(t = 2.771, P> 0.05), 12 d NF-κB expression was significantly lower than the control group(t = 4.933, P <0.01). a Compared two patient groups, TIA-CI infarct 24 h, 3d, 7d, 12 d NF-κB expression were lower than CI group, the difference was statistically significant(t = 1.754, P <0.05; t = 1.858, P <0.05; t = 0.609, P <0.05; t = 0.519, P <0.05). 2)Detection active of NF-κB:Using Immunofluorescence detection NF-κB subcellular localization.NF-κB in the control group was imostly in the cytoplasm, in inactivated state. Compared with the control group, TIA-CI group and CI group ’s NF-κB in 24 h, 3d occurred to nuclear translocation, suggesting NF-κB activity increased. when 7d and 12 d, compared with the CI group, TIA-CI group expression of NF-κB in the nucleus decreased, mostly in the cytoplasm, suggesting that its activity is reduced. The CI group NF-κB is still mostly located in the nucleus.Suggesting that compared with the CI group, NF-κB of TIA-CI group in a shortetr activation status.Conclusion: 1.Prestroke TIA playes a role as a kind of IPC. TIA occurred 2 ~ 3 times on 7 ~ 14 days before ischemic stroke and lasted >10 min showed more prominent; 2.Prestroke TIA by upregulating the activity of PPARγ,inhibiti NF-κB expression and accelerate its inactivation, in order to reduce the body’s inflammatory response, play a protective role in the subsequent infarction. |
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