| Objective: Ischemia with high mortality and serious disability is the most common type of cerebral vascular disease. The pathological mechanism for ischemic injury was a perplexing cascade reaction, including inflammation, oxidative stress and perturbation of calcium homeostasis. It is generally believed that excessive immunological reaction of inflammation reside in necrosis and ischemic region, then result in inflammatory injury. Therefore, it is a main measure to treat acute cerebral infarction by alleviating inflammatory injury, however, anti-inflammation therapies have not reach the expected therapeutic effect. Looking for new inhibitors of the initiation of inflammation may bring progress in treatment of cerebral ischemia. HMGB1(high mobility group box 1)induced NF-κB (nuclear factor kappa B) activation pathway plays an important role in the death of nerve cells. The medicine which can depress the activation of HMGB1 signaling pathway are important therapeutic agents for stroke. Abundant of pharmacology and clinical research find that TanshinoneⅡA(TanⅡA) has anti-inflammatory, anti-apoptosis effects, though the mechanism underlying is poorly understood. Our experiment observed the expressions of HMGB1, RAGE (receptor for advanced glycation end products),TLR4 (toll like receptor 4) and NF-κB in rat model of focal cerebral ischemia; investigated the neuroprotective effects of TanshinoneⅡA and the relationship between its neuroprotection and inhibition of HMGB1induced NF-κB activation signaling pathwayMethods: Male, healthy Sprague-Dawley rats were used and randomly assigned to five groups: Sham (Sham operated), MCAO (Vehicle), TanⅡA high dose (MCAO+ TanⅡA 20 mg/kg), TanⅡA low dose (MCAO+OMT 10 mg/kg) and Normal (Normal control) group. MCAO model was induced by using intraluminal filament technique in rats. TanⅡA (10, 20 mg/kg) was administrated by intraperitoneal injection after cerebral ischemia. For MCAO, Sham and Normal group, equal volume saline was administered in the same manner. Neurological behavior was evaluated at 3 h, 6 h, 12 h, 24 h, 48 h, 72 h,7d and 14d after operation then rats were sacrificed. Brain water content was measured by wet-dry method; Infarct volume was analyzed with 2, 3, 5- triphenyltetrazolium chloride (TTC) staining at 24 h; HMGB1, RAGE, TLR4 and NF-κB expressions were measured by immunohistochemistry, RT-PCR, Western Blot. The expression of claudin-5 and evasion of IgG were measured using confocal technology.Results:1 Rats in MCAO,TanⅡA high dose and TanⅡA low dose group performed a left palsy. Neurological deficit score in TanⅡA high dose group was decreased compared with MCAO group (P<0.05). There was no significant difference in the neurological deficit score between MCAO group and TanⅡA low dose group.2 No infarction was observed in Sham and Normal group. Compared with MCAO group, the infarct volume was significantly reduced in TanⅡA high dose group (P<0.01) but not TanⅡA low dose group.3 Compared with MCAO group, the water content was significantly reduced in TanⅡA high dose group. There was no significant difference between MCAO group and TanⅡA low dose group.4 Immunohistochemistry, RT-PCR and Western blot were used to detect the expression of HMGB1, NF-κB, RAGE and TLR4 in brain tissue after pMCAO. We found that HMGB1, NF-κB, and TLR4 were upregulated compared with Sham group (P < 0.05), beginning at 3 h and peaking at 24 h, but RAGE was upregulated beginning at 12 h after pMCAO. The result of immunohistochemistry was coincident with RT-PCR and Western blot about RAGE, NF-κB, and TLR4; the expression of HMGB1 in immunohistochemistry was coincident with Western blot, but not affected at the mRNA level. 5 HMGB1, NF-κB, RAGE and TLR4 were examined with immunohistochemistry, Western blot and RT-PCR. Few cells were stained with HMGB1, NF-κB p65, RAGE and TLR4 in the cortex in Sham group by immunohistochemistry . In MCAO group, the number of positive cells of HMGB1, NF-κB p65, RAGE and TLR4 significantly increased in the ischemic cortex. In TanⅡA high dose group, the number of positive cells of HMGB1, NF-κB p65, RAGE and TLR4 was significantly decreased compared with MCAO group (P < 0.05). However, there were no significantly differences about the positive cells between MCAO group and TanⅡA low dose group. Then we further analyzed the protein level of cytosolic HMGB1, nuclear NF-κB p65, total RAGE and TLR4 with Western blot. We found that the expressions of cytosolic HMGB1, nuclear NF-κB p65, total RAGE and TLR4 at protein level were upregulated after pMCAO. TanⅡA high dose group significantly decreased the expressions of cytosolic HMGB1, nuclear NF-κB p65, total RAGE and TLR4 at protein level. In agreement with the results of immunohistochemistry and Western blot, the mRNA expression of NF-κB, RAGE and TLR4 were upregulated in MCAO group and significantly inhibited in TanⅡA high dose group. TanⅡA low dose group did not display this effect. HMGB1 expression was not affected by cerebral ischemia at the mRNA level.6 TanⅡA ameliorated BBB permeability. RT-PCR and Western Blot showed that TanⅡA upregulated the expression of claudin-5 in the cerebral ischemia. Immunofluorescent intensity showed that IgG evasion of brain tissues was significantly decreased in Tan II A high dose group. But no statistical significance was observed in Tan II A low dose group.Conclusions: Our study showed that the expressions of HMGB1, TLR4, RAGE, and NF-κB were up-regulated after cerebral ischemia. Systemic administration of Tan II A is effective which can decrease neurologic impairment and tissue injury under cerebral ischemic conditions and this effect may be through down-regulation ofHMGB1/HMGB1 receptors (TLR4 and RAGE)/NF-κB activation pathway, up-regulation of claudin-5 expression and reduction of extravascular IgG.
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