胡黄连苷Ⅱ对大鼠脑缺血再灌注损伤的干预机制
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摘要
目的：研究胡黄连苷Ⅱ对大鼠脑缺血再灌注后损伤的干预机制。方法：应用线栓法建立大鼠大脑中动脉闭塞再灌注(middle cerebral artery occlusion reperfusion, MCAO/R)模型,经尾静脉注射胡黄连苷Ⅱ(10mg/kg-1)和丹参素钠(10mg/kg-1)干预治疗,Bederson评分标准评价大鼠的神经行为功能,氯化三苯基四氮唑(tetrazolium chloride, TTC)染色,观察脑梗死体积。HE染色观察神经细胞的结构。甲苯胺蓝染色观察神经元中尼氏体的形态。免疫组织化学检测神经细胞Toll样受体4(toll-like receptor 4, TLR4),核转录因子κB (nuclear factor kappaB,NFκ,B)和NFκB的抑制因子(Inhibitor kappaB, IκB)的表达。酶联免疫吸附试验(enzyme-linked immunosorbent assay, ELISA)检测脑组织匀浆中NFκB和IκB的含量,原位TUNEL染色检测神经细胞凋亡。结果：假手术组神经行为功能正常,无脑梗死。HE染色可见神经元形态完整。甲苯胺蓝可见神经元的尼氏体形态正常。假手术组免疫组化示大鼠脑组织TLR4、NFκB和IκB弱表达,TUNEL阳性细胞数量较少,散在分布。阴性对照组神经行为功能评分、脑梗死体积较假手术组有明显差异(P<0.05),HE染色可见神经元胞核固缩,甲苯胺蓝染色示神经元尼氏体崩解,免疫组化示大鼠脑组织TLR4, NFκB和IκB表达增强,脑组织匀浆NFκB和IκB增高,TUNEL阳性细胞数量增多,较假手术组有明显差异(P<0.05)。阳性对照组和胡黄连苷Ⅱ组神经行为功能、脑梗死体积低于阴性对照组(P<0.05)。阳性对照组和胡黄连苷Ⅱ组HE染色可见部分神经细胞形态完整,甲苯胺蓝染色可见部分神经细胞的尼氏体形态正常。阳性对照组和胡黄连苷Ⅱ组免疫组化示大鼠脑组织TLR4、NFκB和IκB表达及脑组织匀浆NFκB、IκB和TUNEL阳性细胞数量均低于阴性对照组(P<0.05)。阳性对照组与胡黄连苷Ⅱ组比较,各指标均无显著差异(P>0.05)。结论：胡黄连苷Ⅱ通过下调TLR4、NFκB和IκB的表达,抑制脑缺血／再灌注损伤的炎症反应,减轻脑缺血再灌注的损伤。
Objective:To study the effects of picrosideⅡon cerebral ischemic reperfusion injury in rats.
Methods:Intraluminal thread methods were applied to establish the middle cerebral artery occlusion reperfusion models in rat. PicrosideⅡ(10mg/kg-1) and Salvianic acid A sodium(10mg/kg-1) were injected from the tail vein for treatment. The nervous behavioral function was evaluated with Bederson's test. The cerebral infarction volume was observed with tetrazolium chloride(TTC) staining. The structure of cells were oberserved with Hematoxylin-Eosin. The structure of nissy body of cells were determined by toluidin blue. The expressions of (toll-like receptor4, TLR4), (nuclear kappaB, NFκB) and (inhibitor kappaB, IκB) were determined by immunohistochemical assay. The concentration of NFκB and IκB in brain tissue was determined by ELISA. TUNEL positive cells were counted by immunofluoresence assay. Results:The nevous behavioral malfunction was normal and there was no focus infarction in sham operative group. The structure of cells and the structure of the nissy body were normal. The expression of TLR4、NFκB and IκB were weakly and the apoptotic cells were scattering in brain tissue in the sham operative group. In the negative control group, the nevous behavioral malfunction and focus infarction was higher than those in sham operative group. Damaged neurons exhibit features including pyknosis, karyorhexis and shrunken cell bodies in the negative control. In the negative control group, the number of TUNEL positive cells and the expression of TLR4、NFκB and IκB increased, the concentration were significantly higher than those in the shame operative group(P<0.05). While in the positive control and picrosideⅡgroups, the nevous behavioral malfunction, the focus infarction, the expressions and concentration of TLR4、NFκB、IκB and the the number of TUNEL positive cells were significantly lower than those in the negative control group (P<0.05). There was no significant difference between the positive control group and PicrosideⅡgroup (P>0.05). Conclusion:PicrosideⅡmight downregulate the expression of TLR4、NFκB and IκB to inhibit neuronal apoptosis induced by inflammation after cerebral ischemia reperfusion injury in rats.
Objective:To study the effects of picrosideⅡon cerebral ischemic reperfusion injury in rats.
Methods:Intraluminal thread methods were applied to establish the middle cerebral artery occlusion reperfusion models in rat. PicrosideⅡ(10mg/kg-1) and Salvianic acid A sodium(10mg/kg-1) were injected from the tail vein for treatment. The nervous behavioral function was evaluated with Bederson's test. The cerebral infarction volume was observed with tetrazolium chloride(TTC) staining. The structure of cells were oberserved with Hematoxylin-Eosin. The structure of nissy body of cells were determined by toluidin blue. The expressions of (toll-like receptor4, TLR4), (nuclear kappaB, NFκB) and (inhibitor kappaB, IκB) were determined by immunohistochemical assay. The concentration of NFκB and IκB in brain tissue was determined by ELISA. TUNEL positive cells were counted by immunofluoresence assay. Results:The nevous behavioral malfunction was normal and there was no focus infarction in sham operative group. The structure of cells and the structure of the nissy body were normal. The expression of TLR4、NFκB and IκB were weakly and the apoptotic cells were scattering in brain tissue in the sham operative group. In the negative control group, the nevous behavioral malfunction and focus infarction was higher than those in sham operative group. Damaged neurons exhibit features including pyknosis, karyorhexis and shrunken cell bodies in the negative control. In the negative control group, the number of TUNEL positive cells and the expression of TLR4、NFκB and IκB increased, the concentration were significantly higher than those in the shame operative group(P<0.05). While in the positive control and picrosideⅡgroups, the nevous behavioral malfunction, the focus infarction, the expressions and concentration of TLR4、NFκB、IκB and the the number of TUNEL positive cells were significantly lower than those in the negative control group (P<0.05). There was no significant difference between the positive control group and PicrosideⅡgroup (P>0.05). Conclusion:PicrosideⅡmight downregulate the expression of TLR4、NFκB and IκB to inhibit neuronal apoptosis induced by inflammation after cerebral ischemia reperfusion injury in rats.
引文
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[2]Toshchakov v, Jones BW, Perera PY, et al. TLR, but not TLR2, mediates IFN-beta-incuced STATlalapha/beta depent gene expression in macrophages[J]. Nat Immunol,2002,3(4):392-398.
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[4]Caso J, Pradillo J, Hurtado O, Lorenzo P, Moro M, Lizasoain I. Toll-like receptor 4 is involved in brain damage and inflammation after experimental[J]. Stroke Circulation,2007, 115,(12):1599-1608.
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