黄芪总苷对脑缺血/再灌注损伤的保护作用及其作用机制研究
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摘要
缺血性脑血管病(Ischemic Cerebrovascular Disease,ICVD)是由脑部血液循环障碍,导致以局部神经功能损伤、缺失为特征的一组疾病。ICVD具有发病率高、致残率高、死亡率高、复发率高和并发症多等特点,是引起人类致残、死亡的最主要疾病之一,给人类的身体健康造成了极大的伤害。因此,寻找与开发防治缺血性脑血管病的理想药物己日益受到国内外的广泛重视,成为当前医药学界工作者特别关注的研究课题。
黄芪是我国传统中药,为豆科植物蒙古黄芪(Astraglus membranaceus Bge. var monghaalicus Hsiao)或膜荚黄芪(A. membranaceus Bge)的干燥根,其中含有皂苷类、黄酮类及苷类、糖类、多种氨基酸及微量元素。黄芪总苷(Astragalosides,AST)作为其主要成分具有抗氧化、免疫调节、促智等作用,临床常用于防治心脑血管疾病、促智、延缓衰老、免疫功能紊乱性疾病和抗肿瘤等多种疾病。本课题采用大鼠线栓法制作局灶性脑缺血再灌注模型和海马神经元缺氧/复氧模型,从以下几个方面对黄芪总苷进行研究,以探讨其对脑缺血再灌注损伤后神经功能和学习记忆功能恢复的保护作用及其机制。
第一部分黄芪总苷对脑缺血再灌注损伤大鼠学习记忆功能的影响及其作用机制研究
在MCAO大鼠模型上,研究了AST对脑缺血再灌注后7d大鼠学习记忆功能的影响及其机制。采用Morris水迷宫,观察AST对逃避潜伏期和游泳距离的影响;采用免疫组化的方法检测NGF和BDNF蛋白的表达;采用Western blot检测p-ERK1/2、p-JNK和p-Akt活性。实验结果显示:与模型组比较,AST(80mg/kg)可以明显缩短5d、6d和7d逃避潜伏期和游泳距离,AST(40mg/kg)可以缩短6d和7d的潜伏期和5d、6d和7d的游泳距离,AST(20mg/kg)可以缩短7d的潜伏期和6d和7d的游泳距离。AST(20,40、80mg/kg)可以增加NGF和BDNF蛋白的表达;AST(40、80mg/kg)可以上调p-ERK和p-Akt的表达、下调p-JNK的表达,可能是其发挥脑保护作用的机制之一。
第二部分黄芪总苷对脑缺血再灌注大鼠的保护作用及其作用机制研究
在MCAO大鼠模型上,研究了AST对脑缺血再灌注后1d、3d、7d和14d大鼠的保护作用及其机制。观察了AST对模型大鼠体重、神经功能评分、脑指数、脑含水量和脑梗死体积的影响;检测脑组织中超氧化物歧化酶(SOD)、乳酸脱氢酶(LDH)和一氧化氮合酶(NOS)活性,丙二醛(MDA)、乳酸(LD)和一氧化氮(NO)含量;采用苏木素-伊红(HE)染色观察脑组织病理改变;采用RT-PCR检测iNOSmRNA、NGFmRNA、BDNFmRNA和p75NTRmRNA的表达;采用real-time PCR检测TrkAmRNA和TrkBmRNA的表达。
实验结果如下:
1脑缺血再灌注后1d、3d、7d和14d模型组大鼠体重均有明显降低,AST(40mg/kg,ig)3d、7d和14d可以明显升高降低的模型大鼠体重。
2模型大鼠在缺血再灌注1d时,神经功能评分最高,然后逐渐降低,14d时基本恢复正常;AST(40mg/kg,ig)3d可以明显降低模型大鼠升高的神经功能评分。
3脑缺血再灌注后1d、3d和7d模型组大鼠脑指数均有明显增高,14d模型组大鼠脑指数恢复至正常;AST(40mg/kg,ig)3d和7d可以明显降低脑指数的增加。
4脑缺血再灌注后1d模型组大鼠脑含水量已经明显增加,随着再灌注的进行,含水量逐步增加,在再灌注3d达到高峰,然后开始下降,7d仍有增加,至14d已经恢复正常。AST(40mg/kg,ig)3d和7d可以明显减轻大鼠缺血再灌注后脑组织含水量的增加。
5脑缺血再灌注后出现明显的梗死灶,缺血再灌注3d时脑梗死体积最大。AST (40mg/kg,ig)3d、7d和14d可以明显减轻大鼠缺血再灌注后脑梗死体积。
6脑缺血再灌注后1d、3d和7d模型组大鼠脑组织SOD活力明显降低和MDA含量明显升高;AST(40mg/kg,ig)3d、7d可以明显升高SOD活性、降低脑组织中MDA含量。
7脑缺血再灌注后1d、3d和7d模型组大鼠脑组织LDH活力明显下降、LD含量明显升高,至14d基本恢复到正常水平;AST(40mg/kg,ig)3d、7d可以明显升高脑组织中LDH活性、降低脑组织中LD含量。
8脑缺血再灌注后1d、3d和7d模型组大鼠脑组织NO含量、NOS活力明显升高,至14d基本恢复到正常水平;AST(40mg/kg,ig)3d、7d可以明显降低脑组织中NO含量和NOS活性。
9脑缺血再灌注后1d、3d和7d模型组大鼠脑组织iNOSmRNA表达明显升高,至14d基本恢复到正常水平;AST(40mg/kg,ig)3d、7d可以明显降低脑组织中iNOSmRNA表达。
10 AST(40mg/kg,ig)3d、7d和14d可以不同程度的减轻脑缺血再灌注大鼠脑组织的病理损害。
11脑缺血再灌注后1d、3d、7d和14d模型组大鼠脑组织NGFmRNA表达明显升高,7d达到高峰;AST(40mg/kg,ig)3d、7d和14d可以明显升高脑组织中NGFmRNA。
12脑缺血再灌注后1d、3d、7d和14d模型组大鼠脑组织BDNFmRNA表达明显升高,3d达到高峰;AST(40mg/kg,ig)3d、7d和14d可以明显升高脑组织中BDNFmRNA。
13脑缺血再灌注后1d、3d和7d模型组大鼠脑组织p75NTRmRNA表达明显升高,3d达到高峰,14d基本恢复正常;AST(40mg/kg,ig)3d和7d可以明显降低脑组织中p75NTRmRNA。
14缺血再灌注后1d、3d和7d模型组大鼠脑组织TrkAmRNA表达明显升高,7d达到高峰,14d基本恢复正常;AST(40mg/kg,ig)7d和14d可以明显升高脑组织中TrkAmRNA。
15缺血再灌注后1d、3d和7d模型组大鼠脑组织TrkBmRNA表达明显升高,7d达到高峰,14d基本恢复正常;AST(40mg/kg,ig)3d和7d可以明显升高脑组织中TrkBmRNA。
第三部分黄芪总苷对海马神经元缺氧复氧损伤的保护作用及其作用机制研究
在原代培养的海马神经元缺氧/复氧(A/R)模型上,研究了AST对A/R诱导的海马神经元损伤的保护作用及其作用机制。采用MTT和LDH释放率法,检测海马神经元的活力;检测细胞上清液中SOD活性、MDA和NO含量;采用荧光素染料Hoechst33258染色法,测定海马神经元凋亡;采用Annexin-V/PI双染法,在流式细胞仪上检测细胞凋亡;采用Fluo-3/AM染色,测定海马神经元内[Ca2+]i的变化。结果显示,缺氧复氧组海马神经元活力明显降低;缺氧前加入AST(10、20、40μg/ml)可以明显提高海马神经元的活力,缺氧后复氧前加入AST(40μg/ml)可以提高海马神经元的活力。A/R组海马神经元LDH的释放率明显升高;AST(10、20、40μg/ml)可以明显降低A/R后海马神经元的LDH释放率。A/R后海马神经元培养上清液中SOD活性明显降低,MDA、NO含量明显升高; AST(10、20、40μg/ml)可以明显升高上清液中SOD活性、降低MDA和NO含量。A/R组表现出明显的细胞凋亡;与A/R组比较,AST(10、20、40μg/ml)可以明显减轻缺氧复氧后海马神经元的凋亡率,显著降低胞浆内钙离子的平均荧光强度。
结论:本文研究了黄芪总苷对脑缺血再灌注损伤后的神经保护作用和对学习记忆功能的影响,并对其作用机制进行了探索。研究发现黄芪总苷对脑缺血再灌注损伤后的学习记忆功能有明显的保护作用,其作用机制涉及抗氧化、促进NGF、BDNF及其高亲和力受体TrkA、TrkB的表达、抑制低亲和力受体p75NTR的表达、抑制细胞凋亡、上调p-ERK和p-Akt的表达、下调p-JNK的表达等有关。
Astragalus is a traditional Chinese medicine, as legumes Mongolia astragalus or membranaceus (Fisch) Bge’s dry pods root, which contains saponins, flavonoid glycosides category, sugars, amino acids and trace elements. Astragalosides(AST) is the main component of astragalus with the function of antioxidant, immune regulation, promotes intelligence, which was commonly used in the prevention and treatment of cardiovascular and cerebrovascular diseases, aging, immune function disorders and other diseases. In this paper, the focal cerebral ischemia reperfusion (I/R) model and hippocampal neurons anoxia-reoxygenation model were established to explore the protective effects of AST on the nerve function and learning and memory function after cerebral ischemia-reperfusion injury and its mechanism.
PartⅠEffect of astragalosides on the learning and memory after brain ischemia/ reperfusion injury and its mechanism
To explore the effect of AST on the learning and memory function and its mechanism on MCAO rats. Morris water maze was used to observe the effect of AST on the escape latencies and swimming distance. NGF and BDNF protein expression were tested by immunohistochemistry, while the activity of p-ERK、p-JNK and p-Akt were measured by western blot. The results showed that AST(80mg/kg) could significantly shorten escape latencies and swimming distance at days5, 6 and 7 after I/R; AST(40mg/kg) could significantly shorten escape latencies at days 6 and 7, prolong swimming distance at days 5, 6 and 7 after I/R; AST (20mg/kg) could significantly shorten escape latencies of at day 7 and swimming distance at days 6 and 7. Compared with model group, AST (20,40,80mg/kg) increased the expression of NGF and BDNF; AST(40,80mg/kg) increased the expression of p-ERK, p-Akt and decreased the expression of p-JNK.
PartⅡProtective Effect of Astragalosides against Focal Cerebral Ischemia-reperfusion in rat
Male SD rats received right middle cerebral artery occlusion of 120 minutes’duration. The rats were decapitated at different reperfusion time points: days 1, 3, 7, and 14 of recirculation. The effect of AST on the weight of rats, neurological function score, the index of brain, the water content and infarction volume of brain were measured; the activity of superoxide dismutase (SOD), lactate dehydrogenase (LDH) and nitric oxide synthase (NOS), and the content of malondialdehyde (MDA), lactate (LD) and a nitric oxide (NO) of brain tissue were detected too. Using hematoxylin-eosine (HE) staining to observe brain tissue pathological changes, iNOSmRNA, NGFmRNA, BDNFmRNA and p75NTR mRNA expression were measured by RT-PCR; the expression of TrkAmRNA and TrkB mRNA were measured by real-time PCR.
The results are as follows:
1 The body weight reduced at days1, 3, 7, and 14 on MCAO rats after I/R, and AST (40mg/kg) could significantly increase body weight at days 3, 7and 14.
2 The neuroolgical function score peaked at day 1, then began to decline, and returned almost to the sham level at day 14; AST (40mg/kg) could obviously reduce the neurological function score at day 3.
3 The brain index increased beginning at day 1, peaked at day 3, and returned almost to the sham level at day 14; AST (40mg/kg) could significantly decrease of brain index at days 3 and 7.
4 The water content increased beginning at day 1, and returned almost to the sham level at day 14; AST (40mg/kg) could significantly decrease the water content at days 3 and 7.
5 The brain infarct volume increased beginning at day 1, peaked at day 3, then began to decline; AST (40mg/kg) could significantly reduce the infarct volume at days 3, 7 and 14.
6 The activity of SOD decreased and the content of MDA increased at days 1, 3 and 7; AST (40mg/kg) could significantly increase SOD activity and decrease brain tissue MDA content at days 3 and 7.
7 The activity of LDH significantly decreased and the content of LD increased at days 1, 3 and 7; AST (40mg/kg) could significantly increase LDH activity and reduce the level of LD at days 3 and 7.
8 The activity of NOS and the content of NO increased beginning at day 1, peaked at day 3, and returned almost to the sham level at day 14; AST (40mg/kg) could significantly decrease NO content and NOS activity at days 3and 7.
9 The expression of iNOSmRNA in the rat brain tissue significantly increased at day 1, peaked at day 3, and returned almost to the sham level at day 14; AST (40mg/kg) could reduce the expression of iNOSmRNA at days 3 and 7. 10 AST (40mg/kg) could relieve the brain tissue histopathologic injury at days 3,7 and 14.
11 The expression of NGFmRNA in the rat brain significantly increased at days 1,3,7 and 14, peaked at day 7; AST (40mg/kg) could increase the expression of NGFmRNA at days 3,7 and 14.
12 The expression of BDNFmRNA in the rat brain increased at days 1,3,7 and 14, peaked at day 3; AST (40mg/kg) could significantly increase the expression of BDNFmRNA at days 3,7 and 14.
13 The expression of p75NTRmRNA in the brain tissue significantly increased at days 1,3 and 7, peaked at day 3, and returned almost to the sham level at day 14; AST (40mg/kg) could obviously decrease the expression of p75NTRmRNA at days 3 and 7.
14 The expression of TrkAmRNA in the brain tissue significantly increased at days 1,3 and 7, peaked at day 7, and returned almost to the sham level at day 14; AST (40mg/kg) could increase the expression of TrkAmRNA at days 7 and 14.
15 The expression of TrkBmRNA increased at days 1,3 and 7; AST (40mg/kg) could increase the expression of TrkBmRNA at days 3 and 7.
PartⅢProtective effect of astragalosides against injury induced by anoxia/reoxygenation on hippocampal neuron and its mechanism
To observe the protective effects and its mechanism of AST on primary cultured hippocampal neurons after anoxia and reoxygenation (A/R). An A/R model was established using cultured primary hippocampal neurons which derived from 18 day embryonic rat. MTT assay method and LDH releasing rate were used to measure the cell viability, which evaluated the cellular injury. We detected the activity of SOD and the content of MDA and NO at the same time. The apoptosis rate of hippocampal neurons after A/R was measured by hoechst 33258 staining and flow cytometry with AnnexinV-PI staining. The intracellular calcium was observed with a cofocal laser - scanning microscope and determined by mean fluorescent value with Fluo-3/AM. The results showed that the cell viability of A/R group was reduced markedly, the AST(10, 20, 40μg/ml) significantly enhanced the cell viability of hippocampal neurons before anoxia and AST(40μg/ml) also enhanced it after anoxia. The LDH releasing rate of model group increased markedly, AST(10, 20, 40μg/ml) significantly reduced the LDH releasing rate. The activity of SOD decreased and the content of MDA and NO increased significantly after A/R, but AST (10, 20, 40μg/ml) could enhance the SOD activity and reduce the MDA and NO contents markedly. Compared with the A/R group, AST(10, 20, and 40μg/ml) could reduce the A/R -induced apoptosis and decrease the fluorescence intensity value of [Ca2+]i.
Conclusions: It was found that AST could improve the rats learning and memory function after brain ischemia/reperfusion injury, the mechanism may related with anti-oxidation, increasing the expression of NGF, BDNF, TrkA and TrkB, decreasing the expression of p75NTR, inhibiting the hippocampal neurons apoptosis, up-regulating the expression of p-ERK and p-Akt, down-regulating the expression of p-JNK.
黄芪是我国传统中药,为豆科植物蒙古黄芪(Astraglus membranaceus Bge. var monghaalicus Hsiao)或膜荚黄芪(A. membranaceus Bge)的干燥根,其中含有皂苷类、黄酮类及苷类、糖类、多种氨基酸及微量元素。黄芪总苷(Astragalosides,AST)作为其主要成分具有抗氧化、免疫调节、促智等作用,临床常用于防治心脑血管疾病、促智、延缓衰老、免疫功能紊乱性疾病和抗肿瘤等多种疾病。本课题采用大鼠线栓法制作局灶性脑缺血再灌注模型和海马神经元缺氧/复氧模型,从以下几个方面对黄芪总苷进行研究,以探讨其对脑缺血再灌注损伤后神经功能和学习记忆功能恢复的保护作用及其机制。
第一部分黄芪总苷对脑缺血再灌注损伤大鼠学习记忆功能的影响及其作用机制研究
在MCAO大鼠模型上,研究了AST对脑缺血再灌注后7d大鼠学习记忆功能的影响及其机制。采用Morris水迷宫,观察AST对逃避潜伏期和游泳距离的影响;采用免疫组化的方法检测NGF和BDNF蛋白的表达;采用Western blot检测p-ERK1/2、p-JNK和p-Akt活性。实验结果显示:与模型组比较,AST(80mg/kg)可以明显缩短5d、6d和7d逃避潜伏期和游泳距离,AST(40mg/kg)可以缩短6d和7d的潜伏期和5d、6d和7d的游泳距离,AST(20mg/kg)可以缩短7d的潜伏期和6d和7d的游泳距离。AST(20,40、80mg/kg)可以增加NGF和BDNF蛋白的表达;AST(40、80mg/kg)可以上调p-ERK和p-Akt的表达、下调p-JNK的表达,可能是其发挥脑保护作用的机制之一。
第二部分黄芪总苷对脑缺血再灌注大鼠的保护作用及其作用机制研究
在MCAO大鼠模型上,研究了AST对脑缺血再灌注后1d、3d、7d和14d大鼠的保护作用及其机制。观察了AST对模型大鼠体重、神经功能评分、脑指数、脑含水量和脑梗死体积的影响;检测脑组织中超氧化物歧化酶(SOD)、乳酸脱氢酶(LDH)和一氧化氮合酶(NOS)活性,丙二醛(MDA)、乳酸(LD)和一氧化氮(NO)含量;采用苏木素-伊红(HE)染色观察脑组织病理改变;采用RT-PCR检测iNOSmRNA、NGFmRNA、BDNFmRNA和p75NTRmRNA的表达;采用real-time PCR检测TrkAmRNA和TrkBmRNA的表达。
实验结果如下:
1脑缺血再灌注后1d、3d、7d和14d模型组大鼠体重均有明显降低,AST(40mg/kg,ig)3d、7d和14d可以明显升高降低的模型大鼠体重。
2模型大鼠在缺血再灌注1d时,神经功能评分最高,然后逐渐降低,14d时基本恢复正常;AST(40mg/kg,ig)3d可以明显降低模型大鼠升高的神经功能评分。
3脑缺血再灌注后1d、3d和7d模型组大鼠脑指数均有明显增高,14d模型组大鼠脑指数恢复至正常;AST(40mg/kg,ig)3d和7d可以明显降低脑指数的增加。
4脑缺血再灌注后1d模型组大鼠脑含水量已经明显增加,随着再灌注的进行,含水量逐步增加,在再灌注3d达到高峰,然后开始下降,7d仍有增加,至14d已经恢复正常。AST(40mg/kg,ig)3d和7d可以明显减轻大鼠缺血再灌注后脑组织含水量的增加。
5脑缺血再灌注后出现明显的梗死灶,缺血再灌注3d时脑梗死体积最大。AST (40mg/kg,ig)3d、7d和14d可以明显减轻大鼠缺血再灌注后脑梗死体积。
6脑缺血再灌注后1d、3d和7d模型组大鼠脑组织SOD活力明显降低和MDA含量明显升高;AST(40mg/kg,ig)3d、7d可以明显升高SOD活性、降低脑组织中MDA含量。
7脑缺血再灌注后1d、3d和7d模型组大鼠脑组织LDH活力明显下降、LD含量明显升高,至14d基本恢复到正常水平;AST(40mg/kg,ig)3d、7d可以明显升高脑组织中LDH活性、降低脑组织中LD含量。
8脑缺血再灌注后1d、3d和7d模型组大鼠脑组织NO含量、NOS活力明显升高,至14d基本恢复到正常水平;AST(40mg/kg,ig)3d、7d可以明显降低脑组织中NO含量和NOS活性。
9脑缺血再灌注后1d、3d和7d模型组大鼠脑组织iNOSmRNA表达明显升高,至14d基本恢复到正常水平;AST(40mg/kg,ig)3d、7d可以明显降低脑组织中iNOSmRNA表达。
10 AST(40mg/kg,ig)3d、7d和14d可以不同程度的减轻脑缺血再灌注大鼠脑组织的病理损害。
11脑缺血再灌注后1d、3d、7d和14d模型组大鼠脑组织NGFmRNA表达明显升高,7d达到高峰;AST(40mg/kg,ig)3d、7d和14d可以明显升高脑组织中NGFmRNA。
12脑缺血再灌注后1d、3d、7d和14d模型组大鼠脑组织BDNFmRNA表达明显升高,3d达到高峰;AST(40mg/kg,ig)3d、7d和14d可以明显升高脑组织中BDNFmRNA。
13脑缺血再灌注后1d、3d和7d模型组大鼠脑组织p75NTRmRNA表达明显升高,3d达到高峰,14d基本恢复正常;AST(40mg/kg,ig)3d和7d可以明显降低脑组织中p75NTRmRNA。
14缺血再灌注后1d、3d和7d模型组大鼠脑组织TrkAmRNA表达明显升高,7d达到高峰,14d基本恢复正常;AST(40mg/kg,ig)7d和14d可以明显升高脑组织中TrkAmRNA。
15缺血再灌注后1d、3d和7d模型组大鼠脑组织TrkBmRNA表达明显升高,7d达到高峰,14d基本恢复正常;AST(40mg/kg,ig)3d和7d可以明显升高脑组织中TrkBmRNA。
第三部分黄芪总苷对海马神经元缺氧复氧损伤的保护作用及其作用机制研究
在原代培养的海马神经元缺氧/复氧(A/R)模型上,研究了AST对A/R诱导的海马神经元损伤的保护作用及其作用机制。采用MTT和LDH释放率法,检测海马神经元的活力;检测细胞上清液中SOD活性、MDA和NO含量;采用荧光素染料Hoechst33258染色法,测定海马神经元凋亡;采用Annexin-V/PI双染法,在流式细胞仪上检测细胞凋亡;采用Fluo-3/AM染色,测定海马神经元内[Ca2+]i的变化。结果显示,缺氧复氧组海马神经元活力明显降低;缺氧前加入AST(10、20、40μg/ml)可以明显提高海马神经元的活力,缺氧后复氧前加入AST(40μg/ml)可以提高海马神经元的活力。A/R组海马神经元LDH的释放率明显升高;AST(10、20、40μg/ml)可以明显降低A/R后海马神经元的LDH释放率。A/R后海马神经元培养上清液中SOD活性明显降低,MDA、NO含量明显升高; AST(10、20、40μg/ml)可以明显升高上清液中SOD活性、降低MDA和NO含量。A/R组表现出明显的细胞凋亡;与A/R组比较,AST(10、20、40μg/ml)可以明显减轻缺氧复氧后海马神经元的凋亡率,显著降低胞浆内钙离子的平均荧光强度。
结论:本文研究了黄芪总苷对脑缺血再灌注损伤后的神经保护作用和对学习记忆功能的影响,并对其作用机制进行了探索。研究发现黄芪总苷对脑缺血再灌注损伤后的学习记忆功能有明显的保护作用,其作用机制涉及抗氧化、促进NGF、BDNF及其高亲和力受体TrkA、TrkB的表达、抑制低亲和力受体p75NTR的表达、抑制细胞凋亡、上调p-ERK和p-Akt的表达、下调p-JNK的表达等有关。
Astragalus is a traditional Chinese medicine, as legumes Mongolia astragalus or membranaceus (Fisch) Bge’s dry pods root, which contains saponins, flavonoid glycosides category, sugars, amino acids and trace elements. Astragalosides(AST) is the main component of astragalus with the function of antioxidant, immune regulation, promotes intelligence, which was commonly used in the prevention and treatment of cardiovascular and cerebrovascular diseases, aging, immune function disorders and other diseases. In this paper, the focal cerebral ischemia reperfusion (I/R) model and hippocampal neurons anoxia-reoxygenation model were established to explore the protective effects of AST on the nerve function and learning and memory function after cerebral ischemia-reperfusion injury and its mechanism.
PartⅠEffect of astragalosides on the learning and memory after brain ischemia/ reperfusion injury and its mechanism
To explore the effect of AST on the learning and memory function and its mechanism on MCAO rats. Morris water maze was used to observe the effect of AST on the escape latencies and swimming distance. NGF and BDNF protein expression were tested by immunohistochemistry, while the activity of p-ERK、p-JNK and p-Akt were measured by western blot. The results showed that AST(80mg/kg) could significantly shorten escape latencies and swimming distance at days5, 6 and 7 after I/R; AST(40mg/kg) could significantly shorten escape latencies at days 6 and 7, prolong swimming distance at days 5, 6 and 7 after I/R; AST (20mg/kg) could significantly shorten escape latencies of at day 7 and swimming distance at days 6 and 7. Compared with model group, AST (20,40,80mg/kg) increased the expression of NGF and BDNF; AST(40,80mg/kg) increased the expression of p-ERK, p-Akt and decreased the expression of p-JNK.
PartⅡProtective Effect of Astragalosides against Focal Cerebral Ischemia-reperfusion in rat
Male SD rats received right middle cerebral artery occlusion of 120 minutes’duration. The rats were decapitated at different reperfusion time points: days 1, 3, 7, and 14 of recirculation. The effect of AST on the weight of rats, neurological function score, the index of brain, the water content and infarction volume of brain were measured; the activity of superoxide dismutase (SOD), lactate dehydrogenase (LDH) and nitric oxide synthase (NOS), and the content of malondialdehyde (MDA), lactate (LD) and a nitric oxide (NO) of brain tissue were detected too. Using hematoxylin-eosine (HE) staining to observe brain tissue pathological changes, iNOSmRNA, NGFmRNA, BDNFmRNA and p75NTR mRNA expression were measured by RT-PCR; the expression of TrkAmRNA and TrkB mRNA were measured by real-time PCR.
The results are as follows:
1 The body weight reduced at days1, 3, 7, and 14 on MCAO rats after I/R, and AST (40mg/kg) could significantly increase body weight at days 3, 7and 14.
2 The neuroolgical function score peaked at day 1, then began to decline, and returned almost to the sham level at day 14; AST (40mg/kg) could obviously reduce the neurological function score at day 3.
3 The brain index increased beginning at day 1, peaked at day 3, and returned almost to the sham level at day 14; AST (40mg/kg) could significantly decrease of brain index at days 3 and 7.
4 The water content increased beginning at day 1, and returned almost to the sham level at day 14; AST (40mg/kg) could significantly decrease the water content at days 3 and 7.
5 The brain infarct volume increased beginning at day 1, peaked at day 3, then began to decline; AST (40mg/kg) could significantly reduce the infarct volume at days 3, 7 and 14.
6 The activity of SOD decreased and the content of MDA increased at days 1, 3 and 7; AST (40mg/kg) could significantly increase SOD activity and decrease brain tissue MDA content at days 3 and 7.
7 The activity of LDH significantly decreased and the content of LD increased at days 1, 3 and 7; AST (40mg/kg) could significantly increase LDH activity and reduce the level of LD at days 3 and 7.
8 The activity of NOS and the content of NO increased beginning at day 1, peaked at day 3, and returned almost to the sham level at day 14; AST (40mg/kg) could significantly decrease NO content and NOS activity at days 3and 7.
9 The expression of iNOSmRNA in the rat brain tissue significantly increased at day 1, peaked at day 3, and returned almost to the sham level at day 14; AST (40mg/kg) could reduce the expression of iNOSmRNA at days 3 and 7. 10 AST (40mg/kg) could relieve the brain tissue histopathologic injury at days 3,7 and 14.
11 The expression of NGFmRNA in the rat brain significantly increased at days 1,3,7 and 14, peaked at day 7; AST (40mg/kg) could increase the expression of NGFmRNA at days 3,7 and 14.
12 The expression of BDNFmRNA in the rat brain increased at days 1,3,7 and 14, peaked at day 3; AST (40mg/kg) could significantly increase the expression of BDNFmRNA at days 3,7 and 14.
13 The expression of p75NTRmRNA in the brain tissue significantly increased at days 1,3 and 7, peaked at day 3, and returned almost to the sham level at day 14; AST (40mg/kg) could obviously decrease the expression of p75NTRmRNA at days 3 and 7.
14 The expression of TrkAmRNA in the brain tissue significantly increased at days 1,3 and 7, peaked at day 7, and returned almost to the sham level at day 14; AST (40mg/kg) could increase the expression of TrkAmRNA at days 7 and 14.
15 The expression of TrkBmRNA increased at days 1,3 and 7; AST (40mg/kg) could increase the expression of TrkBmRNA at days 3 and 7.
PartⅢProtective effect of astragalosides against injury induced by anoxia/reoxygenation on hippocampal neuron and its mechanism
To observe the protective effects and its mechanism of AST on primary cultured hippocampal neurons after anoxia and reoxygenation (A/R). An A/R model was established using cultured primary hippocampal neurons which derived from 18 day embryonic rat. MTT assay method and LDH releasing rate were used to measure the cell viability, which evaluated the cellular injury. We detected the activity of SOD and the content of MDA and NO at the same time. The apoptosis rate of hippocampal neurons after A/R was measured by hoechst 33258 staining and flow cytometry with AnnexinV-PI staining. The intracellular calcium was observed with a cofocal laser - scanning microscope and determined by mean fluorescent value with Fluo-3/AM. The results showed that the cell viability of A/R group was reduced markedly, the AST(10, 20, 40μg/ml) significantly enhanced the cell viability of hippocampal neurons before anoxia and AST(40μg/ml) also enhanced it after anoxia. The LDH releasing rate of model group increased markedly, AST(10, 20, 40μg/ml) significantly reduced the LDH releasing rate. The activity of SOD decreased and the content of MDA and NO increased significantly after A/R, but AST (10, 20, 40μg/ml) could enhance the SOD activity and reduce the MDA and NO contents markedly. Compared with the A/R group, AST(10, 20, and 40μg/ml) could reduce the A/R -induced apoptosis and decrease the fluorescence intensity value of [Ca2+]i.
Conclusions: It was found that AST could improve the rats learning and memory function after brain ischemia/reperfusion injury, the mechanism may related with anti-oxidation, increasing the expression of NGF, BDNF, TrkA and TrkB, decreasing the expression of p75NTR, inhibiting the hippocampal neurons apoptosis, up-regulating the expression of p-ERK and p-Akt, down-regulating the expression of p-JNK.
引文
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