通塞脉方取舍怀牛膝对缺血性脑损伤模型的影响

发布时间：2018-07-04 17:07

本文选题：通塞脉 + 牛膝　；参考：《南京中医药大学》2013年硕士论文

【摘要】：通塞脉片是以《验方新编》中的四妙勇安汤为基础研制而成的治疗血栓闭塞性脉管炎的有效验方。处方由黄芪、党参、石斛、玄参、金银花、怀牛膝、当归、甘草八味中药组成。方中牛膝有引药下行的功效,临床常用来治疗下肢疾病。其用于血栓闭塞性脉管炎,可引导诸药下行,加强通塞脉活血通络、益气养阴的功效。拆方研究认为牛膝在本方中作为引经药,在治疗心脑血管疾病时,可以去除。为证实这一观点,我们采用大脑中动脉栓塞法(middle cerebral artery occlusion,MCAO)建立大鼠缺血性脑卒中模型,观察通塞脉方及去牛膝方对缺血性脑卒中的脑梗塞率、神经细胞凋亡、相关炎症因子及其信号转导的影响,探讨牛膝在方中的作用。方法及结果：1.大鼠大脑中动脉缺血再灌注模型的复制取雄性SD大鼠随机分为5组,即假手术组(NS)、模型组(NS)、尼莫地平组(0.0324g/kg)、通塞脉方组(31.74g生药/kg)、去牛膝通塞脉方组(27.77g生药/kg)。大鼠分别于术前三天灌胃给相应药物,每天1次,连续3d。第四天麻醉,分离颈总动脉、颈外动脉、颈内动脉,从颈外动脉插线进行大脑中动脉阻断,造成大鼠MCAO模型,并于2h后再灌注。2.通塞脉及去牛膝方对缺血性脑损伤模型大鼠行为学评分、梗塞率、脑含水量的影响动物于造模后4小时及24小时,用Bederson's评分法进行神经行为学观察。结果表明,脑缺血后4小时,除尼莫地平组各给药组行为学评分均有降低。24小时,尼莫地平组大鼠行为学评分下降明显,其余各给药组分值也有下降,但与模型组相比差异未见显著性。 MCAO后24h取脑,沿冠状切成5片,进行TTC染色并拍照。对照片梗塞区面积及梗塞区和正常区总面积进行计算,求梗塞率。结果表明,各给药组大鼠的脑梗塞率均明显低于模型组,通塞脉去牛膝组与全方组相比,梗塞率降低更明显。动物于造模后24小时处死取脑,入烘箱烘干求其脑含水率,并计算脑指数。结果发现,各给药组均能明显降低大鼠的脑含水率及脑指数。3.通塞脉及去牛膝方对缺血性脑损伤模型大鼠脑组织形态学的影响苏木素-伊红(hematoxylin eosin, HE)染色观察各组大鼠缺血区脑组织切片,发现给药组对大鼠大脑皮层和海马内神经元坏死、神经元数目减少、细胞核固缩、胞体缩小变形、间质水肿、血管周间隙增宽均有一定的抑制作用。去牛膝方组作用与通塞脉相当。 4.通塞脉方及去牛膝方对缺血性脑损伤模型大鼠凋亡相关指标的影响大鼠于造模后24h取脑,10%甲醛溶液固定,原位末端标记法(TUNEL)测定通塞脉方及去牛膝方对大鼠脑组织海马区细胞凋亡的影响。实验结果表明,MCAO后,大鼠脑组织海马区中凋亡细胞数明显增加,而给予通塞脉方和去牛膝方后可以显著减少凋亡细胞数。免疫组化法测定通塞脉方及去牛膝方对模型大鼠脑组织海马区Bcl-2相关X蛋白(Bcl-2associated X protein, Bax)以及抑制凋亡蛋白B细胞淋巴瘤／白血病-2(Lymphoma/leukemia-2gene B, Bcl-2)表达的影响,结果表明：通塞脉方及去牛膝方均能够增加Bcl-2的表达,抑制Bax的表达。提示通塞脉方及去牛膝方均能通过抑制相关促凋亡蛋白的表达,增加抗凋亡蛋白的表达,来减少脑细胞的凋亡,抑制脑缺血对脑组织的损伤。 5.通塞脉方及去牛膝方对缺血性脑损伤模型大鼠HMGB1相关炎症信号通路的影响放免法测定MCAO大鼠血清高迁移率组蛋白B1(HMGB1)、肿瘤坏死因子(Tumor necrosis factor-α, TNF-α)和白细胞介素-1β (Interleukin-1β, IL-1β)。实验结果发现,造模后大鼠血清HMGB1、TNF-α、IL-1β的含量较假手术组明显升高,给予通塞脉方及去牛膝方后均能明显降低大鼠血清HMGB1、TNF-α、IL-1β含量,去牛膝组对IL-1p的降低效果优于全方。免疫组化法测定通塞脉方及去牛膝方对核转录因子κB (Nuclear factor-κB, NF-κB)的影响,结果发现通塞脉方及去牛膝方均可减少NF-κB的表达。然后运用免疫印迹法(Western blot, WB)检测NF-κB的表达,也证实了这一结果。免疫印迹法检测大鼠缺血区脑组织Toll样受体4(TLR4)蛋白表达,发现通塞脉方及去牛膝方均可通过减少TLR4的表达,降低MCAO引起的炎症反应。 6.通塞脉方及去牛膝方对缺血性脑损伤模型大鼠血浆血管紧张素Ⅱ含量的影响放射免疫法检测MCAO大鼠血浆血管紧张素Ⅱ (angiotensinⅡ, AngⅡ)的含量。结果表明,造模后,大鼠血浆中AngⅡ明显升高,而给予通塞脉方和去牛膝方后可以显著降低AngⅡ水平,其中去牛膝方对AngⅡ抑制作用更为明显。结论：综上所述,在脑缺血的治疗上通塞脉方的疗效与去牛膝方并无明显差异,在降低血清IL-1β、血浆AngⅡ含量方面去牛膝方作用更强。因此牛膝作为引药下行的引经药,在脑缺血的治疗上并不能发挥作用,进一步证实了去除牛膝是可行的。通塞脉方及去牛膝方均可通过抗神经细胞凋亡,抑制与HMGB1相关的NF-κB信号通路的激活,而达到减轻MCAO引起的大鼠脑组织损伤,保护神经细胞的作用。
[Abstract]:Tongsai dike is an effective prescription for the treatment of thrombovasculitis obliterans based on the four Miao Yong'an decoction. The prescription consists of Astragalus membranaceus, Codonopsis, Dendrobium, Radix sacheria, honeysuckle, Achyranthes bidentata, angelica, licorice and eight Chinese herbs. The Achyranthes bidentata is used in the treatment of lower extremity diseases. It is used for thrombus. Occluded vasculitis, which can guide the various drugs down, strengthen the effect of activating blood circulation and dredging the blood and nourishing yin. The dismantling prescription study suggests that the Achyranthes bidentata can be removed in the treatment of cardiovascular and cerebrovascular diseases. To confirm this view, we use middle cerebral artery occlusion (MCAO) to establish a rat deficiency. The model of bloody cerebral apoplexy is to observe the effect of Tongsaimai Fang and tucai prescription on cerebral infarction, apoptosis, related inflammatory factors and signal transduction in ischemic stroke, and to explore the role of the Achyranthes bidentata in the recipe. Methods and results: the replication of ischemia reperfusion model of middle cerebral artery in 1. rats
The male SD rats were randomly divided into 5 groups, namely, the sham operation group (NS), the model group (NS), the nimodipine group (0.0324g/kg), the Tongsaimai group (31.74g raw drug /kg), the Tongsaimai group (/kg) of the Achyranthes bidentata (27.77g drug /kg). The rats were intragastric to the corresponding drugs at the three day before the operation, 1 times a day and fourth days of continuous anesthesia for the separation of the common carotid artery, the external carotid artery and internal carotid artery. Pulse, blocking the middle cerebral artery from the external carotid artery, resulting in the MCAO model of rats, and reperfusion after 2h, and the effect on the behavior score, infarct rate and brain water content in the rat model of the ischemic brain injury model,.2.
The results showed that the behavior score of all the nimodipine group decreased by.24 hours after 4 hours of nimodipine group, and the behavior score of the nimodipine group decreased obviously, and the scores of the other administration groups decreased, but the difference was poor compared with the model group at 4 hours after cerebral ischemia, 4 hours and 24 hours after modeling. There is no significant difference.
After MCAO 24h, the brain was taken and 5 slices were cut along the coronal, and TTC was stained and photographed. The infarct area and the total area of the infarct area and the normal area were calculated and the infarct rate was calculated. The results showed that the infarct rate of the rats in each group was significantly lower than that of the model group.
The brain was killed 24 hours after the model was made, and the brain water content was calculated in the oven and the brain index was calculated. The results showed that the water content of the brain and the brain index.3. in the rats were significantly reduced.
The hematoxylin eosin (HE) staining was used to observe the cerebral tissue sections of the ischemic regions of the rats. It was found that the group had some inhibitory effects on the necrosis of neurons in the cerebral cortex and hippocampus, the number of neurons in the hippocampus and the hippocampus, the nucleus retraction, the contraction of the cell body, the interstitial edema and the widening of the perivascular space. The plug is equal.
Effects of 4. Tongsai Mai recipe and the decoction of removing the Achyranthes bidentata on apoptosis related indexes in rats with ischemic brain injury
The rat brain, 10% Formaldehyde Solution fixation, and in situ terminal labeling (TUNEL) were used to determine the effect of Tongsai Mai Fang and yoxu Fang on the hippocampal cell apoptosis in rat brain tissue. The experimental results showed that after MCAO, the number of apoptotic cells in the hippocampus of the rat brain increased significantly after MCAO, and it could be significantly reduced after the Tongsai Fang and the Achyranthes bidentata side. Number of apoptotic cells.
The effects of Tongsai Mai Fang and Yamu Fang on the expression of Bcl-2 related X protein (Bcl-2associated X protein, Bax) and the inhibition of apoptotic protein B cell lymphoma / leukemia -2 (Lymphoma/leukemia-2gene B, Bcl-2) expression in the hippocampus of the model rats were determined by immunohistochemical method. The results showed that both Tongsai Mai Fang and Achyranthes bidentata could increase Bcl-2. The expression and inhibition of the expression of Bax suggest that both Tongsaimai recipe and Achyranthes bidentata can inhibit the expression of related apoptotic proteins and increase the expression of anti apoptotic proteins to reduce the apoptosis of brain cells and inhibit the damage of cerebral ischemia to brain tissue.
Effects of 5. Tongsai Mai Fang and "Du Gu Fang" on HMGB1 related inflammatory signaling pathway in rats with ischemic brain injury
The serum high mobility group protein B1 (HMGB1), tumor necrosis factor (Tumor necrosis factor- alpha, TNF- alpha) and interleukin -1 beta (Interleukin-1 beta, IL-1 beta) were measured by radioimmunoassay. The experimental results showed that the serum HMGB1, TNF- a, and beta content of MCAO rats were significantly higher than those of sham operation group. It can obviously reduce the content of HMGB1, TNF-, IL-1 and beta in serum of rats, and the effect of IL-1p on the Achyranthes bidentata group is better than that in the whole recipe.
The effects of Tongsai Mai Fang and Ku Ku square on nuclear factor kappa B (Nuclear factor- kappa B, NF- kappa B) were determined by immunohistochemical method. The results showed that the expression of NF- kappa B was reduced by both Tongsai Fang and Ku Ku square, and the expression of NF- kappa B was detected by Western blot (Western blot, WB), and the result was confirmed.
The expression of Toll like receptor 4 (TLR4) protein in the brain tissue of rat ischemic region was detected by Western blot. It was found that both Tongsaimai Fang and Achyranthes bidentata could reduce the expression of TLR4 and reduce the inflammatory reaction caused by MCAO.
Effects of 6. Tongsai Mai recipe and the decoction of removing the Achyranthes root on plasma angiotensin II in rats with ischemic brain injury
The content of plasma angiotensin II (angiotensin II, Ang II) in plasma of MCAO rats was detected by radioimmunoassay. The results showed that after the model, the plasma level of Ang II was significantly increased in rats, and the level of Ang II could be significantly reduced after giving the Tongsai Fang and the Achyranthes bidentata side, and the inhibition of Ang II was more obvious in the Achyranthes bidentata side.
Conclusion:
To sum up, the curative effect of Tongsaimai recipe in the treatment of cerebral ischemia is not significantly different from that of the Achyranthes bidentata side, and the effect of reducing the serum IL-1 beta and plasma Ang II content is stronger. Therefore, the medicine of the Achyranthes bidentata as the medicine under the medicine can not play a role in the treatment of cerebral ischemia, and further confirms that the removal of the Achyranthes is feasible. Both Si Mai Fang and Achyranthes bidentata can inhibit the activation of NF- kappa B signaling pathway related to HMGB1 by inhibiting the apoptosis of nerve cells, and can reduce the injury of brain tissue caused by MCAO and protect the role of neural cells.
【学位授予单位】：南京中医药大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R-332;R285.5

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