【作者】 郭乐；

【导师】 蔡光先；

【作者基本信息】 湖南中医药大学 ， 中西医结合内科学， 2012， 硕士

【摘要】 目的：探讨补阳还五汤对脑缺血后炎症反应NF-κB通路及细胞增殖的影响,为补阳还五汤临床应用提供理论依据和实验依据。方法：采用线栓法,建立大鼠大脑中动脉栓塞(MCAO)的脑缺血损伤动物模型,将成年大鼠随机分为空白组,假手术组,模型组,BYHWD组,PDTC组,MC组,给予不同处理,分别于7D,14D,21D处死,对大鼠进行神经功能缺失症状评分,TTC染色测脑梗死面积比,免疫组化法观察缺血脑组织NF-κB/p65,NF-κB/p50和I-κB蛋白的表达及BrdU标记的细胞新生情况结果：1.神经功能症状评分和梗死面积比的测定：正常组,假手术组均未出现神经功能缺失及梗死灶,与正常组及假手术组相比,模型组大鼠脑缺血后表现出严重的神经功能缺失症状,TTC染色显示梗死灶非常明显(P<0.01),梗死面积比随时间减少。用药7,14,21天后,与模型组相比,用药处理组均可改善神经功能症状(P<0.05或0.01),用药7,14天后,可有效地减小缺血后梗死面积(P<0.01),用药各组间差异不具有统计学意义(P>0.05),梗死面积随用药时间的增加而减少2.NF-κB/p65和NF-κB/p50表达的免疫组化测定：正常组,假手术组可见少量的NF-κB/p65和NF-κB/p50蛋白的表达,而模型组的NF-KB/p65和NF-KB/p50阳性细胞大量表达(P<0.01),NF-κB/p65和NF-KB/p50阳性细胞数随时间点有依次减少的趋势。BYHWD, MC,PDTC组,均能显著抑制NF-KB/p65和NF-KB/p50的表达(P<0.05或0.01)。用药7天,14天后,BYHWD组与MC组,PDTC组间差异无统计意义,用药21天后,NF-KB/p50蛋白的表达,BYHWD与PDTC组比较,表现出些许优势(P<0.05)。3.I-KB的表达免疫组化测定：正常组,假手术组可见大量阳性细胞I-κB的表达,模型组的I-κB阳性细胞少量表达,BYHWD,MC, PDTC组,均能显著增加I-κB的表达,差异显著(P<0.01)。用药7天后,米诺环素组效果最好(P<0.01),用药14天后,BYHWD组与MC组均优于PDTC(P<0.01),用药21天后,BYHWD组明显优于MC,PDTC组(P<0.01),表达在时间趋势上,BYHWD组和MC组21天的阳性细胞表达较14天减少4. BrdU阳性细胞数的免疫组化测定：正常组、假手术组可见少量BrdU阳性细胞,模型组可见BrdU阳性细胞增加,有随时间增加的趋势,但是增长缓慢,BYHWD,MC,PDTC组,均能显著增加BrdU阳性细胞的数量,差异显著(P<0.05或0.01),用药7,14天后,各用药组间不具有显著性差异(P>0.05)；用药21天后,BYHWD在阳性细胞数目上与同期各组比较具有显著性差异(P<0.01),BrdU阳性细胞数随用药时间的增加而增加。结论：1. BYHWD与MC, PDTC一样可减轻局灶性脑缺血后大鼠脑组织因缺血而造成的梗死和神经功能损伤。2. BYHWD与MC, PDTC一样可通过调控缺血脑组织炎症因子NF-κBp/65,NF-κB/p50和I-κB表达,从而抑制炎症反应,提示BYHWD可能是通过调节I-KB的表达调控NF-κB炎症信号通路来达到对大鼠局灶性脑缺血损伤的保护作用。3. BYHWD与MC,PDTC一样可促进脑缺血后缺血组织的细胞增殖来达到组织修复的目的,细胞增殖的趋势与NF-κB通路因子变化趋势提示,补阳还五汤可能是通过调控NF-KB通路来影响细胞增殖。4.BYHWD在恢复期作用更显著。更多还原

【Abstract】 Objective:To explore The effects of BuyangHuanwu Decoction on the signal transduction of NF-κB factorn and cell proliferation on rats following focal cerebral ischemia to provide proof for clinic to facilitate work.Methods:Rat model of cerebral ischemia injury was created by the middle cerebral artery occlusion (MCAO) with suture method, healthy adult SD rats were randomly divided into the normal group,Sham-operated group,ischemia group and BYHWD treatment group,MC treatment group,PDTC treatment group, Animals were receiveid different processing and killed at7,14,21Day point,after operation.The nervous deficit symptoms were observed and the infarct size ratio was measured by triphenyltetrazolium chloride (TTC) staining after the cerebral ischemia. In addition, The expression of NF-KB/p65, NF-κB/p50, I-κB and BrdU were detected by immunohistochemistry technology. Result:1.Neurological function deficit symptom scores and infarct size ratio: compared with the normal group and the sham group,The model group had seriously neurological deficit and increase the cerebral infarct size ratio(P<0.01),the cerebral infarct size ratio and,neurological function deficit symptom scores decreased along the survival time increasing at7,14day point..after7,14,21days’treatment, the treatment groups all could significantly improve the grade of nerve function in comparison with the model group(P<0.05or0.01), and significantly decrease the cerebral infarct size ratio(P<0.01). the difference Between the treatment groups is not significant (P>0.05). the cerebral infarct size ratio decreased along the survival time increasing.2.The result of the expression of NF-KB/p65, NF-κB/p50by immunohistochemical stainingshow that:the normal and Sham group have a few positive NF-κB/p65, NF-κB/p50cells;In model group, the expression of NF-κB/p65, NF-κB/p50seriously increased(P<0.01), The expression of NF-κB/p65, NF-κB/p50decreased along the survival time increasing,BYHWD, MC, PDTC,treatment group, all could significantly inhibit the expression of NF-κB/p65, NF-KB/p50(P<0.05or0.01). after7,14days’treatment, the difference Between the treatment group is not significant.after21days’treatment BYHWD displays the trifle superiority in comparison with PDTC(P<0.05). 3.The result of the expression of I-κB by immunohistochemical staining show that the normal and Sham group have an massive positive I-κB; In model group, the expression of I-κB seriously decreased BYHWD, MC, PDTC, treatment group, all could significantly increase the expression of I-κB(P<0.01), after7days’treatment,MC treatment result is better than the other treatments result, after14days’treatment(P<0.01), BYHWD,MC treatment result is both better than PDTC treatment(P<0.01), after21days’treatment, BYHWD treatment result is better than MC and PDTC treatment(P<0.01), at14,21days’point the expression of I-κB at BYHWD,MC treatment group decreased along the survival time increasing.4.The result of the expression of BrdU positive cellby immunohistochemical staining show that the normal and Sham group have a few BrdU positive cell, In model group, the expression of BrdU positive cell increase,but not much, The expression increased along the survival time increasing, BYHWD, MC, PDTC, treatment group, all could significantly increase the expression of BrdU positive cell(P<0.05or0.01), after7,14days’treatment, the difference Between the treatment group is not significant (P>0.05), after21days’treatment BYHWD displays superiority in comparison with the other the treatment groups (P<0.01). Conclusion:1. BYHWD has the same efficacy as MC,PDTC on deereasing infarct size ratio and neurological function deficit symptom.2. BYHWD has significant protection on cerebral ischemia injury in rats, BYHWD has the same efficacy as MC,PDTC on control inflammatory factors expression in Protein,which may be related to the inhibition of inflammatory cytokine secretion and expression.and inducing the inflammation in brain tissue.The result maybe show that BYHWD protect brain by regulate the the expression of I-κB to control signal transduction of NF-κB factor,revealed the mechanism of inflammatory action in the view of signal transduction of NF-κB factor.3. BYHWD has the same efficacy as MC, PDTC on tissue self-repair by promote cell proliferation after focal cerebral ischemia, cell proliferation’s tendency and factor of signal transduction of NF-κB change tendency prompt that BYHWD possibly affects the cell proliferation through regulating signal transduction of NF-κB.4. the function of BYHWD to be more remarkable at the recovery period.更多还原