超微补阳还五汤对MCAo模型大鼠神经功能缺失及Nogo-A蛋白的影响
摘要
目的:观察超微补阳还五汤对大脑中动脉阻塞(MCAo)模型大鼠神经功能缺失的改善作用,并从对Nogo-A蛋白及其受体NgR表达的影响探讨其作用机制。
方法:采用线拴法复制MCAo模型,动物存活2周后纳入本实验。120只存活动物被随机分到模型组、传统补阳还五汤临床等剂量组(以下简称传统组)和超微补阳还五汤临床1/3剂量组(以下简称超微组),另取40只同批未手术动物作为空白组。各组给药前(手术后2周),给药后2周、4周、6周四个时间点随机抽取10只动物进行实验。
1.每组动物分别在给药前,给药后2周、4周、6周进行行为学评分。共计有细横木平衡实验、前肢偏用实验、食团摄取实验、粘着去除实验、前肢放置实验、横木穿越实验等6项评分。
2.在上述各时点神经行为学评分后,动物被处死,检验Nogo-A蛋白及其受体NgR的表达。采用免疫组织化学和RT-PCR两种方法。
结果:1.模型大鼠手术后出现神经功能缺失,给药前各行为学测试结果,模型组、传统组、超微组与空白组比P均＜0.01。神经缺失表现在给药后6周仍有不同程度遗留,模型组、传统组、超微组与空白组比P均＜0.01。模型组给药后6周与给药前比P＜0.01,提示神经缺失可出现一定程度的自发性恢复。给药后6周,传统组、超微组比模型组,P＜0.01或0.01＜P＜0.05,说明补阳还五汤可以促进神经缺失的恢复。而超微组与传统组给药2周、4周、6周比较,差异无统计学意义(P＞0.05),则说明超微1/3剂与传统临床等剂量作用等效。
2.动物手术后2周时可观察到Nogo-A的mRNA与蛋白高度表达,免疫组化积分光密度结果,给药前(8.57±0.79)与空白组(0.61±0.12)比P＜0.01,RT-PCR结果,给药前(0.52±0.13)与空白组(0.10±0.02)比P＜0.01。随着生存时间的增加,Nogo-A的mRNA与蛋白表达下降,模型组6周免疫组化(5.23±1.22)、RT-PCR(0.29±0.08)与术后2周免疫组化(8.57±0.79)、RT-PCR(0.52±0.13)比,P均＜0.01。两种剂型中药能明显降低Nogo-A蛋白及mRNA转录强度,给药后6周积分光密度传统组(1.84±0.38)、超微组(1.88±0.20)较模型组(5.23±1.22)比,P＜0.01,mRNA表达传统组(0.14±0.02)、超微组(0.15±0.03)较模型组(0.29±0.08),P＜0.01。Nogo-A的受体NgR的表达及药物对其表达的影响基本与Nogo-A相同。
结论:1.MCAo模型大鼠出现不同程度的神经缺失表现。通过补阳还五汤治疗,能够减轻神经缺失症状,使神经行为学评分得到改善。超微剂型相当于传统剂型1/3量时即与传统剂型之间作用效果无差异。
2.Nogo-A及其受体NgR蛋白、mRNA的表达在手术后升高,补阳还五汤能降低其表达水平,超微剂型相当于传统剂型1/3量时即与传统剂型之间作用效果无差异。
Objectives:To observe the effect of Micropowder Buyang huanwu Decoction on neurologic deficits,Nogo-A and Nogo-A recepter NgR of MCAo model rat. Discovery the possible mechanism of the improving on neurologic deficits and the characteristic in useing.
Methods:MCAo mice models were reproduced by Intraluminal Suture.One hundred and twenty rats survival were classified into three groups after 2 weeks: model group,traditional decoction group and Micropowder-form group(the dosage is 1/3 of traditional decoction group).Rats in each group were classified into four time points:before treatment,2 weeks after treatment,4 weeks after treatment,and 6 weeks after treatment respectively(10 rats for each time point).Forty another normal rats were taken as control group.
1.Each rat was scored by neuroethology before treatment,2 weeks after treatment,4 weeks after treatment,and 6 weeks after treatment respectively.Six items were evaluated:Placing test,Beam Balance test,Adhesive Removal test,Beam-traversing test,Limb-use asymmetry test,and Single-pellet reaching test.
2.Rats were killed after neuroethology score,and were analyzed the expression of Nogo-A protein and the receptor NgR protein using the Immunochistochemistry method and RT-PC method.
Results:
1.Model rats showed neurologic deficits after surgery.The neurologic deficits still remained in 6 weeks after surgery.The score of neurologic deficits for model group, traditional decoction group and Micropowder group were statistically different from control group(P＜0.01) before treatment or in 6 weeks after surgery.Compare with 2 week after the surgery,The neurologic deficits showed spontaneous recovery after 6 weeks'treatment.The score of neurologic deficits in 6 weeks after treatment was statistically significant different from the score of which before teatmeant(P＜0.01). Buyang huanwu Decoction can improve the recovery of neurologic deficits.The score of neurologie deficits in traditional decoction group and Micropowder group were different from model group(P＜0.01 or 0.01＜P＜0.05) in 6 weeks after treatment. The difference in the score between Micropowder group and traditional group after 2 weeks'treatment,4 weeks'treatment and 6 weeks' treatment were not significant(P＞0.05).
2.The expression of mRNA and protein for Nogo-A could be observed in 2 weeks after surgery.The difference in IOD between the baseline group(8.57±0.79) and the control group(0.61±0.12) was significant(P＜0.01).The difference in RT-PCR result between the baseline group(0.52±0.13) and the control group(0.10±0.02) was significant(P＜0.01).The expression of mRNA and protein for Nogo-A decreased along with survival time increasing.Immunochistochemistry and RT-PCR were found significantly different(P＜0.01) in model group between the time of 2 weeks(8.57±0.79,0.52±0.13) and the time of 6 weeks(5.23±1.22,0.29±0.08). Both form of Chinese medicine can reduce the expression of mRNA and protein for Nogo-A.Compared with model group(5.23±1.22),significant differences(P＜0.01) in IOD were found for traditonal group(1.84±0.38) and Micropowder group (1.88±0.20) in 6 weeks after treatment.Compared with model group(0.29±0.08), significant differences(P＜0.01) in mRNA were found for traditional group(0.14±0.06) and Micropowder group(0.15±0.04) in 6 weeks after treatment.There was not significant differences between 1/3 dosage of Micropowder-form and traditional decoction.The expression of the receptor NgR protein of Nogo-A and the effect on NgR expression of stroke brain tissue by medicine were similar to the result of Nogo-A.
Conclusions:
1.MCAo rats presented different extent symptoms of neurologic deficits.The symptoms of neurologic deficits were relieved and the neuroethology score were improved after therapy by taking Buyang huanwu Decoction.The difference in the effect between Micropowder-form(the dosage is 1/3 of traditional decoction) and traditional decoction was not significant.
2.The expression of Nogo-A protein,the receptor NgR protein and mRNA were increased after surgery in MCAo rats.Buyanghuanwu Decoction can reduce this expression,and there was not significant difference in the effect between Micropowder -form(the dosage is 1/3 of traditional decoction) and traditional decoction.
方法:采用线拴法复制MCAo模型,动物存活2周后纳入本实验。120只存活动物被随机分到模型组、传统补阳还五汤临床等剂量组(以下简称传统组)和超微补阳还五汤临床1/3剂量组(以下简称超微组),另取40只同批未手术动物作为空白组。各组给药前(手术后2周),给药后2周、4周、6周四个时间点随机抽取10只动物进行实验。
1.每组动物分别在给药前,给药后2周、4周、6周进行行为学评分。共计有细横木平衡实验、前肢偏用实验、食团摄取实验、粘着去除实验、前肢放置实验、横木穿越实验等6项评分。
2.在上述各时点神经行为学评分后,动物被处死,检验Nogo-A蛋白及其受体NgR的表达。采用免疫组织化学和RT-PCR两种方法。
结果:1.模型大鼠手术后出现神经功能缺失,给药前各行为学测试结果,模型组、传统组、超微组与空白组比P均＜0.01。神经缺失表现在给药后6周仍有不同程度遗留,模型组、传统组、超微组与空白组比P均＜0.01。模型组给药后6周与给药前比P＜0.01,提示神经缺失可出现一定程度的自发性恢复。给药后6周,传统组、超微组比模型组,P＜0.01或0.01＜P＜0.05,说明补阳还五汤可以促进神经缺失的恢复。而超微组与传统组给药2周、4周、6周比较,差异无统计学意义(P＞0.05),则说明超微1/3剂与传统临床等剂量作用等效。
2.动物手术后2周时可观察到Nogo-A的mRNA与蛋白高度表达,免疫组化积分光密度结果,给药前(8.57±0.79)与空白组(0.61±0.12)比P＜0.01,RT-PCR结果,给药前(0.52±0.13)与空白组(0.10±0.02)比P＜0.01。随着生存时间的增加,Nogo-A的mRNA与蛋白表达下降,模型组6周免疫组化(5.23±1.22)、RT-PCR(0.29±0.08)与术后2周免疫组化(8.57±0.79)、RT-PCR(0.52±0.13)比,P均＜0.01。两种剂型中药能明显降低Nogo-A蛋白及mRNA转录强度,给药后6周积分光密度传统组(1.84±0.38)、超微组(1.88±0.20)较模型组(5.23±1.22)比,P＜0.01,mRNA表达传统组(0.14±0.02)、超微组(0.15±0.03)较模型组(0.29±0.08),P＜0.01。Nogo-A的受体NgR的表达及药物对其表达的影响基本与Nogo-A相同。
结论:1.MCAo模型大鼠出现不同程度的神经缺失表现。通过补阳还五汤治疗,能够减轻神经缺失症状,使神经行为学评分得到改善。超微剂型相当于传统剂型1/3量时即与传统剂型之间作用效果无差异。
2.Nogo-A及其受体NgR蛋白、mRNA的表达在手术后升高,补阳还五汤能降低其表达水平,超微剂型相当于传统剂型1/3量时即与传统剂型之间作用效果无差异。
Objectives:To observe the effect of Micropowder Buyang huanwu Decoction on neurologic deficits,Nogo-A and Nogo-A recepter NgR of MCAo model rat. Discovery the possible mechanism of the improving on neurologic deficits and the characteristic in useing.
Methods:MCAo mice models were reproduced by Intraluminal Suture.One hundred and twenty rats survival were classified into three groups after 2 weeks: model group,traditional decoction group and Micropowder-form group(the dosage is 1/3 of traditional decoction group).Rats in each group were classified into four time points:before treatment,2 weeks after treatment,4 weeks after treatment,and 6 weeks after treatment respectively(10 rats for each time point).Forty another normal rats were taken as control group.
1.Each rat was scored by neuroethology before treatment,2 weeks after treatment,4 weeks after treatment,and 6 weeks after treatment respectively.Six items were evaluated:Placing test,Beam Balance test,Adhesive Removal test,Beam-traversing test,Limb-use asymmetry test,and Single-pellet reaching test.
2.Rats were killed after neuroethology score,and were analyzed the expression of Nogo-A protein and the receptor NgR protein using the Immunochistochemistry method and RT-PC method.
Results:
1.Model rats showed neurologic deficits after surgery.The neurologic deficits still remained in 6 weeks after surgery.The score of neurologic deficits for model group, traditional decoction group and Micropowder group were statistically different from control group(P＜0.01) before treatment or in 6 weeks after surgery.Compare with 2 week after the surgery,The neurologic deficits showed spontaneous recovery after 6 weeks'treatment.The score of neurologic deficits in 6 weeks after treatment was statistically significant different from the score of which before teatmeant(P＜0.01). Buyang huanwu Decoction can improve the recovery of neurologic deficits.The score of neurologie deficits in traditional decoction group and Micropowder group were different from model group(P＜0.01 or 0.01＜P＜0.05) in 6 weeks after treatment. The difference in the score between Micropowder group and traditional group after 2 weeks'treatment,4 weeks'treatment and 6 weeks' treatment were not significant(P＞0.05).
2.The expression of mRNA and protein for Nogo-A could be observed in 2 weeks after surgery.The difference in IOD between the baseline group(8.57±0.79) and the control group(0.61±0.12) was significant(P＜0.01).The difference in RT-PCR result between the baseline group(0.52±0.13) and the control group(0.10±0.02) was significant(P＜0.01).The expression of mRNA and protein for Nogo-A decreased along with survival time increasing.Immunochistochemistry and RT-PCR were found significantly different(P＜0.01) in model group between the time of 2 weeks(8.57±0.79,0.52±0.13) and the time of 6 weeks(5.23±1.22,0.29±0.08). Both form of Chinese medicine can reduce the expression of mRNA and protein for Nogo-A.Compared with model group(5.23±1.22),significant differences(P＜0.01) in IOD were found for traditonal group(1.84±0.38) and Micropowder group (1.88±0.20) in 6 weeks after treatment.Compared with model group(0.29±0.08), significant differences(P＜0.01) in mRNA were found for traditional group(0.14±0.06) and Micropowder group(0.15±0.04) in 6 weeks after treatment.There was not significant differences between 1/3 dosage of Micropowder-form and traditional decoction.The expression of the receptor NgR protein of Nogo-A and the effect on NgR expression of stroke brain tissue by medicine were similar to the result of Nogo-A.
Conclusions:
1.MCAo rats presented different extent symptoms of neurologic deficits.The symptoms of neurologic deficits were relieved and the neuroethology score were improved after therapy by taking Buyang huanwu Decoction.The difference in the effect between Micropowder-form(the dosage is 1/3 of traditional decoction) and traditional decoction was not significant.
2.The expression of Nogo-A protein,the receptor NgR protein and mRNA were increased after surgery in MCAo rats.Buyanghuanwu Decoction can reduce this expression,and there was not significant difference in the effect between Micropowder -form(the dosage is 1/3 of traditional decoction) and traditional decoction.
引文
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