片仔癀对大鼠大脑中动脉阻断所致局灶性脑梗死的影响研究
摘要
目的
采用MCAO(大脑中动脉梗死)造模方法,建立大鼠大脑中动脉梗死模型,观察片仔癀胶囊对MCAO大鼠术后炎性损伤相关因子IL-β、IL-6、TNF-α、血脑屏障相关因子ZO—1、MMP-9及神经营养因子BDNF表达的影响,从蛋白水平、基因水平探讨片仔癀治疗脑梗死的药效及机制,阐明片仔癀具有脑保护的作用。
方法
成年健康雄性SD大鼠,体重260-280g,随机分为手术组和非手术组(生理盐水组)。手术组大鼠采用改良Zea-Longa方法制备MCAO模型,且根据醒后2小时Longa评分将纳入标准者随机分为模型组、片仔癀大剂量组(0.0486g/100g.d)、片仔癀中剂量组(0.0162g/100g.d)、片仔癀小剂量组(0.0054g/100g.d)、尼莫地平组(0.00108g/100g.d)、脑得生组(0.0486g/100g.d)及正常组。以上各组均于造模前给药3天,第4天灌胃给药后30分钟造模,造模后给药观察至第7天取材。取材前麻醉大鼠,经腹主动脉采血,取血清以备ELISA方法检测各组IL-1β、IL-6、TNF-α的表达变化；然后断头取脑,制作石蜡切片以备免疫组织化学检测各组BDNF、IL-6表达的变化；取梗死侧大脑皮层约180-200mg左后,置于液氮罐中保存,以备Western Blot方法及RQ-PCR法检测各组ZO—1、MMP-9表达变化。
结果
1大鼠MCAO模型的制备
本实验对MCAO术后神经功能学评分进行了评价。正常组无神经功能缺损症状,评分均为0分,术后模型组评分均显著高于正常组,神经功能缺损症状严重,提示造模成功。
2免疫组织化学方法检测片仔癀对大脑中动脉阻断所致局灶性脑梗死大鼠BDNF、IL-6表达的影响
本实验通过免疫组织化学法检测片仔癀胶囊对大鼠脑梗死后不同区域(脑梗死中心区、梗死周边区、梗死对侧正常区)BDNF的变化规律,结果显示：梗死周边区：模型组较假手术组显著升高(P<0.05),各用药组BDNF表达均高于模型组,其中片仔癀大剂量组、片仔癀中剂量组、尼莫地平组较模型组差异显著(P<0.01)；片仔癀大剂量组与片仔癀小剂量组相比差异显著(P<0.05)。梗死中心区：模型组BDNF表达显著低于正常组(P<0.01),用药组均高于模型组,脑得生组、尼莫地平组及片仔癀大剂量组与模型组差异显著(P<0.01或P<0.05)。正常区：模型组BDNF表达较正常组升高,各用药组均高于模型组,各组间差异无统计学意义(P>0.05)。
本实验通过免疫组织化学法检测片仔癀胶囊对大鼠脑梗死后不同区域(脑梗死中心区、梗死周边区、梗死对侧正常区)IL-6的变化规律,结果显示：梗死周边区：模型组较假手术组显著升高(P<0.01),各用药组IL-6表达均低于模型组,其中片仔癀大剂量组、脑得生组、尼莫地平组较模型组差异显著(P<0.05)；片仔癀大剂量组与片仔癀小剂量组相比差异显著(P<0.05)。梗死中心区：模型组IL-6表达显著低于假手术组(P<0.01),用药组均高于模型组、脑得生组、尼莫地平组及片仔癀大剂量组与模型组差异显著(P<0.01)。正常区：模型组IL-6表达较正常组显著升高,各用药组均低于模型组,各组间差异无统计学意义(P>0.05)。
3ELISA方法检测片仔癀对大脑中动脉阻断所致局灶性脑梗死大鼠炎性损伤相关因子IL-1β、IL-6及TNF-α的影响
ELISA法检测MCAO大鼠术后IL-1β表达的影响,结果显示：与假手术组比较,模型组IL-1β表达显著升高(P<0.01),与模型组比较,片仔癀大剂量组、片仔癀中剂量组、片仔癀小剂量组、脑得生组及尼莫地平组IL-1β表达均显著降低(P<0.01或P<0.05)。片仔癀各组之间无统计学差异。
ELISA法检测MCAO大鼠术后IL-6表达的影响,结果显示：与假手术组比较,模型组IL-6表达显著升高(P<0.01),与模型组比较,片仔癀大剂量组、脑得生组、尼莫地平组IL-6表达显著降低(P<0.01或P<0.05)。片仔癀各组之间无统计学差异。
ELISA法检测MCAO大鼠术后TNF-α表达的影响,结果显示：与假手术组比较,模型组TNF-α表达显著升高(P<0.01),与模型组比较,片仔癀大剂量组、片仔癀中剂量组、片仔癀小剂量组、脑得生组及尼莫地平组TNF-α表达均显著降低(P<0.01或P<0.05)。4实时荧光定量PCR方法检测片仔癀保护大脑中动脉阻断所致局灶性脑梗死大鼠血脑屏障损伤相关因子ZO—1、MMP-9mRNA的影响
ZO—1结果显示：模型组较假手术组显著降低(P<0.05),各用药组ZO—1表达均高于模型组,其中片仔癀大剂量组、片仔癀中剂量组、尼莫地平组较模型组差异显著(P<0.01)；片仔癀大剂量组与片仔癀小剂量组相比差异显著(P<0.05)。
MMP-9结果显示：模型组MMP-9表达显著高于假手术组(P<0.01),用药组均低于模型组,脑得生组、尼莫地平组及片仔癀大剂量组与模型组差异显著(P<0.01)。5western blot方法检测片仔癀保护大脑中动脉阻断所致局灶性脑梗死大鼠血脑屏障相关因子Z0—1、MMP-9蛋白的影响
MCAO大鼠术后ZO-1表达的影响,结果可见：模型组ZO-1表达较假手术组显著降低(P<0.01),片仔癀大剂量组、尼莫地平组及脑得生组ZO-1表达较模型组显著升高(P<0.05或P<0.01),片仔癀大剂量组较片仔癀小剂量组差异显著(P<0.05)。
MCAO大鼠术后MMP-9表达的影响,结果可见：模型组MMP-9表达较假手术组显著升高(P<0.01),片仔癀大剂量组、尼莫地平组及脑得生组MMP-9表达较模型组显著降低(P<0.05或P<0.01)；片仔癀大剂量组较片仔癀小剂量组差异显著(P<0.05)。
结论
1片仔癀可以上调脑梗死后BDNF、下调IL-6蛋白水平表达；
2片仔癀可以下调脑梗死后血清IL-1β、IL-6及TNF-α含量；
3片仔癀可以上调脑梗死后ZO—1、下调MMP-9mRNA水平表达；
4片仔癀可以上调脑梗死后ZO—1、下调MMP-9蛋白水平表达；
5片仔癀具有脑保护的作用,能够从抑制炎性损伤、改善血脑屏障多机制促进脑缺血后缺损神经元的恢复；
Objective:
Adopting the method of MCAO(middle cerebral artery occlusion) to make model,es-tablishing the rats infarction model,to observe the effect of Pien Tze Huang on infla-mmatory injury related factors of IL-1β,IL-6,TNF-α,blood brain barrier factors of Z O-1,MMP-9and neurotrophic factor of BDNF,from the protein level and gene level to discuss Pien Tze Huang in the treatment of cerebral infarction efficacy and mechanis-ms,elucidating Pien Tze Huang having neuroprotective effect.
Methods:
Adult health male SD rats,weight260-280g.Rats were randomly divided into operati-on and non-operation group(normal group).The operation group applied improved Lon-ga method to make the focal and permarent model of MCAO,and implemented EZ-L-onga evaluation after the rats woke up for2hours.According to the evaluation the ra-ts included in the standards were randomly divided into model group,Pien Tze Huang high-dose group,Pien Tze Huang middle-dose group,Pien Tze Huang small-dose group, nimodiping group,naodesheng group and normal saline group.The above.groups were ad ministered for3days before making model,on the fourth day making the MCAO,on t he seventh day were observed.after anaesthesia in rats,through the abdominal aortabloo-d.serum for ELISA detection IL-1β,IL-6,TNF-α expression changes;and then took the brain tissue depending on the cross2mm before and after to dehydrate,make paraffin section.Quickly brung out infarction brain tissue about180-200mg in liquid nitrogen to preserve,finally the expression changes of ZO-1,MMP-9would be observed by mothed-s of western blot and RT-PCR.
Results:
1.MCAO model preparation and evaluation.
In this study,postoperative neurologic scores in MCAO were evaluated.No neurologi-cal deficit symptoms of the normal group,score O.model group scores were significant-1y higher than the normal group,severe symptoms of neurological deficit,suggesting thatt he model was successful.
2.Using immunohistochemistry to detect middle cerebral artery occlusion induced focal cerebral ischemia rats neurotrophic factor BDNF,and inflammatory injury factor IL-6expression.
In this study,using immunohistochemistry method to observe different regions(Infarcti on surrounding area, in the central area of infarction area,infarction of the contralateral normal area)of BDNF variation,the result shows:infarction of the surrounding area:them odel group compared with the sham group was significantly increased(P<0.05),treatmen t group expression of BDNF were higher than the model group,which Pien Tze Huan g high dose group,Pien Tze Huang middle dose group and nimodipine group than mo del group differences were significant(P<0.01);Pien Tze Huang high dose group and P-ien Tze Huang small dose group have a significant difference(P<0.05).Infarction of the central area:The BDNF expression of model group was significantly lower than the n-ormal group(P<0.01),the treatment group were higher than the model group,naodesheng group,nimodipine group and Pien Tze Huang high dose group have a significant diffe-rence than model group differences(P<0.01or P<0.05).Normal zone:model group,the e-xpression of BDNF increased than the normal group,the treated group were higher th-an the model group,no ignificant differences among hese groups(P>0.05).
In this study,using immunohistochemistry method to observe different regions(lnfarcti on surrounding area, in the central area of infarction area,infarction of the contral ater al normal area) of IL-6variation,the result showsrinfarction of the surrounding area:the model group compared with the sham group was significantly increased(P<0.05),treat ment group expression of IL-6were lower than the model group,which Pien Tze Hua ng high dose group,naodesheng group and nimodipine group compared with model gro up differences were significant(P<0.01);Pien Tze Huang high dose group and Pien Tze Huang small dose group have a significant difference(P<0.05).Infarction of thecentral area:The IL-6expression of model group was significantly lower than the normal grou p(P<0.01),the treatment group were higher than the model group,naode-sheng group,ni modipine group and Pien Tze Huang high dose group have a significa-nt difference th an model group differences(P<0.01or P<0.05).Normal zone:model group,the expression of IL-6increased than the normal group,the treated group werelower than the model group,no significant differences among these groups (P>0.05).
3.Using ELISA methord to detect middle cerebral artery occlusion induced focal cerebral ischemia rats inflammatory injury related factors IL-1β,IL-6and TNF-α.
Detected by ELISA methord in MCAO rats,the expression of IL-1β, the results show: the model group was significantly increased(P<0.01)compared with the sham group,Pie n Tze Huang high dose group,Pien Tze Huang middle dose group,Pien Tze Huang sm all dose group,naodesheng group and nomodiping group of IL-1β expressionwere signif icantly reduced compared with the model group(P<0.01or P<0.05).
Detected by ELISA methord in MCAO rats,the expression of IL-6,the results show:t he model group was significantly increased compared with the sham group(P<0.01),Pie n Tze Huang high dose group,naodesheng group and nomodiping group of IL-6expres sion were significantly reduced compared with the model group(P<0.01or P<0.05).
Detected by ELISA methord in MCAO rats,the expression of TNF-a,the results sho w:the model group was significantly increased(P<0.01) compared with the sham group, Pien Tze Huang high dose group,Pien Tze Huang middle dose group,Pien Tze Huang small-dose group,naodesheng group and nomodiping group of TNF-a expression were s ignificantly reduced compared with the model group(P<0.01or P<0.05).
4.Using RQ-PCR methord to detect middle cerebral artery occlusion induced focal c-erebral ischemia rats blood-brain barrier related factors ZO-1,MMP-9mRNA.
The results show that ZO-l:the model group compared with the sham group was si-gnificantly lower(P<0.05).treated groups of ZO-1expression were higher than the mode1group,which Pien Tze Huang high dose group,Pien Tze Huang middle dose group an d nimodipine group were significant(P<0.01);Pien Tze Huang high dose group compare d with Pien Tze Huang low-dose group had significant difference(P<0.05).
The results show that MMP-9:the model group compared with the sham group was significantly higher(P<0.05).treated groups of MMP-9expression were lower than the model group,which Pien Tze Huang high dose group,naodesheng group and nimodipine group were significant(P<0.01);Pien Tze Huang high-dose group compared with Pien Tze Huang low-dose group had significant difference(P<0.05).
5.Using Western blot method detect middle cerebral artery occlusion induced focal c erebral ischemia rats blood-brain barrier related factors ZO-1,MMP-9protein.
The results show that ZO-l:the model group compared with the sham group was si-gnificantly lower(P<0.05).treated groups of ZO-1expression were higher than the mod el group,which Pien Tze Huang high dose group,Pien Tze Huang middle dose group a nd nimodipine group were significant(P<0.01);Pien Tze Huang high-dose group compar ed with Pien Tze Huang low-dose group had significant difference(P<0.05).
The results show that MMP-9:the model group compared with the sham group was significantly higher(P<0.05).treated groups of MMP-9expression were lower than the model group,which Pien Tze Huang high dose group,naodesheng group and nimodi-pin e group were significant(P<0.01);Pien Tze Huang high-dose group compared with Pien Tze Huang low-dose group had significant difference(P<0.05).
Conclusion:
1Pien Tze Huang can promote BDNF,reduce IL-6protein level after cerebral ischemia;
2Pien Tze Huang can reduce cerebral infarction of serum IL-1β,IL-6and TNF-α content;
3Pien Tze Huang can promote cerebral infarction of ZO-1,reduce the expression levels of MMP-9mRNA;
4Pien Tze Huang can promote cerebral infarction of ZO-1,reduce the level of MMP-9protein expression;
5Pien Tze Huang has the function of cerebral protection,inhibiting the inflammatory injury,improving the blood-brain barrier mechanism after cerebral ischemia.
采用MCAO(大脑中动脉梗死)造模方法,建立大鼠大脑中动脉梗死模型,观察片仔癀胶囊对MCAO大鼠术后炎性损伤相关因子IL-β、IL-6、TNF-α、血脑屏障相关因子ZO—1、MMP-9及神经营养因子BDNF表达的影响,从蛋白水平、基因水平探讨片仔癀治疗脑梗死的药效及机制,阐明片仔癀具有脑保护的作用。
方法
成年健康雄性SD大鼠,体重260-280g,随机分为手术组和非手术组(生理盐水组)。手术组大鼠采用改良Zea-Longa方法制备MCAO模型,且根据醒后2小时Longa评分将纳入标准者随机分为模型组、片仔癀大剂量组(0.0486g/100g.d)、片仔癀中剂量组(0.0162g/100g.d)、片仔癀小剂量组(0.0054g/100g.d)、尼莫地平组(0.00108g/100g.d)、脑得生组(0.0486g/100g.d)及正常组。以上各组均于造模前给药3天,第4天灌胃给药后30分钟造模,造模后给药观察至第7天取材。取材前麻醉大鼠,经腹主动脉采血,取血清以备ELISA方法检测各组IL-1β、IL-6、TNF-α的表达变化；然后断头取脑,制作石蜡切片以备免疫组织化学检测各组BDNF、IL-6表达的变化；取梗死侧大脑皮层约180-200mg左后,置于液氮罐中保存,以备Western Blot方法及RQ-PCR法检测各组ZO—1、MMP-9表达变化。
结果
1大鼠MCAO模型的制备
本实验对MCAO术后神经功能学评分进行了评价。正常组无神经功能缺损症状,评分均为0分,术后模型组评分均显著高于正常组,神经功能缺损症状严重,提示造模成功。
2免疫组织化学方法检测片仔癀对大脑中动脉阻断所致局灶性脑梗死大鼠BDNF、IL-6表达的影响
本实验通过免疫组织化学法检测片仔癀胶囊对大鼠脑梗死后不同区域(脑梗死中心区、梗死周边区、梗死对侧正常区)BDNF的变化规律,结果显示：梗死周边区：模型组较假手术组显著升高(P<0.05),各用药组BDNF表达均高于模型组,其中片仔癀大剂量组、片仔癀中剂量组、尼莫地平组较模型组差异显著(P<0.01)；片仔癀大剂量组与片仔癀小剂量组相比差异显著(P<0.05)。梗死中心区：模型组BDNF表达显著低于正常组(P<0.01),用药组均高于模型组,脑得生组、尼莫地平组及片仔癀大剂量组与模型组差异显著(P<0.01或P<0.05)。正常区：模型组BDNF表达较正常组升高,各用药组均高于模型组,各组间差异无统计学意义(P>0.05)。
本实验通过免疫组织化学法检测片仔癀胶囊对大鼠脑梗死后不同区域(脑梗死中心区、梗死周边区、梗死对侧正常区)IL-6的变化规律,结果显示：梗死周边区：模型组较假手术组显著升高(P<0.01),各用药组IL-6表达均低于模型组,其中片仔癀大剂量组、脑得生组、尼莫地平组较模型组差异显著(P<0.05)；片仔癀大剂量组与片仔癀小剂量组相比差异显著(P<0.05)。梗死中心区：模型组IL-6表达显著低于假手术组(P<0.01),用药组均高于模型组、脑得生组、尼莫地平组及片仔癀大剂量组与模型组差异显著(P<0.01)。正常区：模型组IL-6表达较正常组显著升高,各用药组均低于模型组,各组间差异无统计学意义(P>0.05)。
3ELISA方法检测片仔癀对大脑中动脉阻断所致局灶性脑梗死大鼠炎性损伤相关因子IL-1β、IL-6及TNF-α的影响
ELISA法检测MCAO大鼠术后IL-1β表达的影响,结果显示：与假手术组比较,模型组IL-1β表达显著升高(P<0.01),与模型组比较,片仔癀大剂量组、片仔癀中剂量组、片仔癀小剂量组、脑得生组及尼莫地平组IL-1β表达均显著降低(P<0.01或P<0.05)。片仔癀各组之间无统计学差异。
ELISA法检测MCAO大鼠术后IL-6表达的影响,结果显示：与假手术组比较,模型组IL-6表达显著升高(P<0.01),与模型组比较,片仔癀大剂量组、脑得生组、尼莫地平组IL-6表达显著降低(P<0.01或P<0.05)。片仔癀各组之间无统计学差异。
ELISA法检测MCAO大鼠术后TNF-α表达的影响,结果显示：与假手术组比较,模型组TNF-α表达显著升高(P<0.01),与模型组比较,片仔癀大剂量组、片仔癀中剂量组、片仔癀小剂量组、脑得生组及尼莫地平组TNF-α表达均显著降低(P<0.01或P<0.05)。4实时荧光定量PCR方法检测片仔癀保护大脑中动脉阻断所致局灶性脑梗死大鼠血脑屏障损伤相关因子ZO—1、MMP-9mRNA的影响
ZO—1结果显示：模型组较假手术组显著降低(P<0.05),各用药组ZO—1表达均高于模型组,其中片仔癀大剂量组、片仔癀中剂量组、尼莫地平组较模型组差异显著(P<0.01)；片仔癀大剂量组与片仔癀小剂量组相比差异显著(P<0.05)。
MMP-9结果显示：模型组MMP-9表达显著高于假手术组(P<0.01),用药组均低于模型组,脑得生组、尼莫地平组及片仔癀大剂量组与模型组差异显著(P<0.01)。5western blot方法检测片仔癀保护大脑中动脉阻断所致局灶性脑梗死大鼠血脑屏障相关因子Z0—1、MMP-9蛋白的影响
MCAO大鼠术后ZO-1表达的影响,结果可见：模型组ZO-1表达较假手术组显著降低(P<0.01),片仔癀大剂量组、尼莫地平组及脑得生组ZO-1表达较模型组显著升高(P<0.05或P<0.01),片仔癀大剂量组较片仔癀小剂量组差异显著(P<0.05)。
MCAO大鼠术后MMP-9表达的影响,结果可见：模型组MMP-9表达较假手术组显著升高(P<0.01),片仔癀大剂量组、尼莫地平组及脑得生组MMP-9表达较模型组显著降低(P<0.05或P<0.01)；片仔癀大剂量组较片仔癀小剂量组差异显著(P<0.05)。
结论
1片仔癀可以上调脑梗死后BDNF、下调IL-6蛋白水平表达；
2片仔癀可以下调脑梗死后血清IL-1β、IL-6及TNF-α含量；
3片仔癀可以上调脑梗死后ZO—1、下调MMP-9mRNA水平表达；
4片仔癀可以上调脑梗死后ZO—1、下调MMP-9蛋白水平表达；
5片仔癀具有脑保护的作用,能够从抑制炎性损伤、改善血脑屏障多机制促进脑缺血后缺损神经元的恢复；
Objective:
Adopting the method of MCAO(middle cerebral artery occlusion) to make model,es-tablishing the rats infarction model,to observe the effect of Pien Tze Huang on infla-mmatory injury related factors of IL-1β,IL-6,TNF-α,blood brain barrier factors of Z O-1,MMP-9and neurotrophic factor of BDNF,from the protein level and gene level to discuss Pien Tze Huang in the treatment of cerebral infarction efficacy and mechanis-ms,elucidating Pien Tze Huang having neuroprotective effect.
Methods:
Adult health male SD rats,weight260-280g.Rats were randomly divided into operati-on and non-operation group(normal group).The operation group applied improved Lon-ga method to make the focal and permarent model of MCAO,and implemented EZ-L-onga evaluation after the rats woke up for2hours.According to the evaluation the ra-ts included in the standards were randomly divided into model group,Pien Tze Huang high-dose group,Pien Tze Huang middle-dose group,Pien Tze Huang small-dose group, nimodiping group,naodesheng group and normal saline group.The above.groups were ad ministered for3days before making model,on the fourth day making the MCAO,on t he seventh day were observed.after anaesthesia in rats,through the abdominal aortabloo-d.serum for ELISA detection IL-1β,IL-6,TNF-α expression changes;and then took the brain tissue depending on the cross2mm before and after to dehydrate,make paraffin section.Quickly brung out infarction brain tissue about180-200mg in liquid nitrogen to preserve,finally the expression changes of ZO-1,MMP-9would be observed by mothed-s of western blot and RT-PCR.
Results:
1.MCAO model preparation and evaluation.
In this study,postoperative neurologic scores in MCAO were evaluated.No neurologi-cal deficit symptoms of the normal group,score O.model group scores were significant-1y higher than the normal group,severe symptoms of neurological deficit,suggesting thatt he model was successful.
2.Using immunohistochemistry to detect middle cerebral artery occlusion induced focal cerebral ischemia rats neurotrophic factor BDNF,and inflammatory injury factor IL-6expression.
In this study,using immunohistochemistry method to observe different regions(Infarcti on surrounding area, in the central area of infarction area,infarction of the contralateral normal area)of BDNF variation,the result shows:infarction of the surrounding area:them odel group compared with the sham group was significantly increased(P<0.05),treatmen t group expression of BDNF were higher than the model group,which Pien Tze Huan g high dose group,Pien Tze Huang middle dose group and nimodipine group than mo del group differences were significant(P<0.01);Pien Tze Huang high dose group and P-ien Tze Huang small dose group have a significant difference(P<0.05).Infarction of the central area:The BDNF expression of model group was significantly lower than the n-ormal group(P<0.01),the treatment group were higher than the model group,naodesheng group,nimodipine group and Pien Tze Huang high dose group have a significant diffe-rence than model group differences(P<0.01or P<0.05).Normal zone:model group,the e-xpression of BDNF increased than the normal group,the treated group were higher th-an the model group,no ignificant differences among hese groups(P>0.05).
In this study,using immunohistochemistry method to observe different regions(lnfarcti on surrounding area, in the central area of infarction area,infarction of the contral ater al normal area) of IL-6variation,the result showsrinfarction of the surrounding area:the model group compared with the sham group was significantly increased(P<0.05),treat ment group expression of IL-6were lower than the model group,which Pien Tze Hua ng high dose group,naodesheng group and nimodipine group compared with model gro up differences were significant(P<0.01);Pien Tze Huang high dose group and Pien Tze Huang small dose group have a significant difference(P<0.05).Infarction of thecentral area:The IL-6expression of model group was significantly lower than the normal grou p(P<0.01),the treatment group were higher than the model group,naode-sheng group,ni modipine group and Pien Tze Huang high dose group have a significa-nt difference th an model group differences(P<0.01or P<0.05).Normal zone:model group,the expression of IL-6increased than the normal group,the treated group werelower than the model group,no significant differences among these groups (P>0.05).
3.Using ELISA methord to detect middle cerebral artery occlusion induced focal cerebral ischemia rats inflammatory injury related factors IL-1β,IL-6and TNF-α.
Detected by ELISA methord in MCAO rats,the expression of IL-1β, the results show: the model group was significantly increased(P<0.01)compared with the sham group,Pie n Tze Huang high dose group,Pien Tze Huang middle dose group,Pien Tze Huang sm all dose group,naodesheng group and nomodiping group of IL-1β expressionwere signif icantly reduced compared with the model group(P<0.01or P<0.05).
Detected by ELISA methord in MCAO rats,the expression of IL-6,the results show:t he model group was significantly increased compared with the sham group(P<0.01),Pie n Tze Huang high dose group,naodesheng group and nomodiping group of IL-6expres sion were significantly reduced compared with the model group(P<0.01or P<0.05).
Detected by ELISA methord in MCAO rats,the expression of TNF-a,the results sho w:the model group was significantly increased(P<0.01) compared with the sham group, Pien Tze Huang high dose group,Pien Tze Huang middle dose group,Pien Tze Huang small-dose group,naodesheng group and nomodiping group of TNF-a expression were s ignificantly reduced compared with the model group(P<0.01or P<0.05).
4.Using RQ-PCR methord to detect middle cerebral artery occlusion induced focal c-erebral ischemia rats blood-brain barrier related factors ZO-1,MMP-9mRNA.
The results show that ZO-l:the model group compared with the sham group was si-gnificantly lower(P<0.05).treated groups of ZO-1expression were higher than the mode1group,which Pien Tze Huang high dose group,Pien Tze Huang middle dose group an d nimodipine group were significant(P<0.01);Pien Tze Huang high dose group compare d with Pien Tze Huang low-dose group had significant difference(P<0.05).
The results show that MMP-9:the model group compared with the sham group was significantly higher(P<0.05).treated groups of MMP-9expression were lower than the model group,which Pien Tze Huang high dose group,naodesheng group and nimodipine group were significant(P<0.01);Pien Tze Huang high-dose group compared with Pien Tze Huang low-dose group had significant difference(P<0.05).
5.Using Western blot method detect middle cerebral artery occlusion induced focal c erebral ischemia rats blood-brain barrier related factors ZO-1,MMP-9protein.
The results show that ZO-l:the model group compared with the sham group was si-gnificantly lower(P<0.05).treated groups of ZO-1expression were higher than the mod el group,which Pien Tze Huang high dose group,Pien Tze Huang middle dose group a nd nimodipine group were significant(P<0.01);Pien Tze Huang high-dose group compar ed with Pien Tze Huang low-dose group had significant difference(P<0.05).
The results show that MMP-9:the model group compared with the sham group was significantly higher(P<0.05).treated groups of MMP-9expression were lower than the model group,which Pien Tze Huang high dose group,naodesheng group and nimodi-pin e group were significant(P<0.01);Pien Tze Huang high-dose group compared with Pien Tze Huang low-dose group had significant difference(P<0.05).
Conclusion:
1Pien Tze Huang can promote BDNF,reduce IL-6protein level after cerebral ischemia;
2Pien Tze Huang can reduce cerebral infarction of serum IL-1β,IL-6and TNF-α content;
3Pien Tze Huang can promote cerebral infarction of ZO-1,reduce the expression levels of MMP-9mRNA;
4Pien Tze Huang can promote cerebral infarction of ZO-1,reduce the level of MMP-9protein expression;
5Pien Tze Huang has the function of cerebral protection,inhibiting the inflammatory injury,improving the blood-brain barrier mechanism after cerebral ischemia.
引文
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