本文選題：通塞脈 + 牛膝�。� 參考：《南京中醫(yī)藥大學(xué)》2013年碩士論文

【摘要】：通塞脈片是以《驗方新編》中的四妙勇安湯為基礎(chǔ)研制而成的治療血栓閉塞性脈管炎的有效驗方。處方由黃芪、黨參、石斛、玄參、金銀花、懷牛膝、當歸、甘草八味中藥組成。方中牛膝有引藥下行的功效,臨床常用來治療下肢疾病。其用于血栓閉塞性脈管炎,可引導(dǎo)諸藥下行,加強通塞脈活血通絡(luò)、益氣養(yǎng)陰的功效。拆方研究認為牛膝在本方中作為引經(jīng)藥,在治療心腦血管疾病時,可以去除。為證實這一觀點,我們采用大腦中動脈栓塞法(middle cerebral artery occlusion,MCAO)建立大鼠缺血性腦卒中模型,觀察通塞脈方及去牛膝方對缺血性腦卒中的腦梗塞率、神經(jīng)細胞凋亡、相關(guān)炎癥因子及其信號轉(zhuǎn)導(dǎo)的影響,探討牛膝在方中的作用。方法及結(jié)果：1.大鼠大腦中動脈缺血再灌注模型的復(fù)制 取雄性SD大鼠隨機分為5組,即假手術(shù)組(NS)、模型組(NS)、尼莫地平組(0.0324g/kg)、通塞脈方組(31.74g生藥/kg)、去牛膝通塞脈方組(27.77g生藥/kg)。大鼠分別于術(shù)前三天灌胃給相應(yīng)藥物,每天1次,連續(xù)3d。第四天麻醉,分離頸總動脈、頸外動脈、頸內(nèi)動脈,從頸外動脈插線進行大腦中動脈阻斷,造成大鼠MCAO模型,并于2h后再灌注。2.通塞脈及去牛膝方對缺血性腦損傷模型大鼠行為學(xué)評分、梗塞率、腦含水量的影響 動物于造模后4小時及24小時,用Bederson's評分法進行神經(jīng)行為學(xué)觀察。結(jié)果表明,腦缺血后4小時,除尼莫地平組各給藥組行為學(xué)評分均有降低。24小時,尼莫地平組大鼠行為學(xué)評分下降明顯,其余各給藥組分值也有下降,但與模型組相比差異未見顯著性。 MCAO后24h取腦,沿冠狀切成5片,進行TTC染色并拍照。對照片梗塞區(qū)面積及梗塞區(qū)和正常區(qū)總面積進行計算,求梗塞率。結(jié)果表明,各給藥組大鼠的腦梗塞率均明顯低于模型組,通塞脈去牛膝組與全方組相比,梗塞率降低更明顯。 動物于造模后24小時處死取腦,入烘箱烘干求其腦含水率,并計算腦指數(shù)。結(jié)果發(fā)現(xiàn),各給藥組均能明顯降低大鼠的腦含水率及腦指數(shù)。3.通塞脈及去牛膝方對缺血性腦損傷模型大鼠腦組織形態(tài)學(xué)的影響 蘇木素-伊紅(hematoxylin eosin, HE)染色觀察各組大鼠缺血區(qū)腦組織切片,發(fā)現(xiàn)給藥組對大鼠大腦皮層和海馬內(nèi)神經(jīng)元壞死、神經(jīng)元數(shù)目減少、細胞核固縮、胞體縮小變形、間質(zhì)水腫、血管周間隙增寬均有一定的抑制作用。去牛膝方組作用與通塞脈相當。 4.通塞脈方及去牛膝方對缺血性腦損傷模型大鼠凋亡相關(guān)指標的影響 大鼠于造模后24h取腦,10%甲醛溶液固定,原位末端標記法(TUNEL)測定通塞脈方及去牛膝方對大鼠腦組織海馬區(qū)細胞凋亡的影響。實驗結(jié)果表明,MCAO后,大鼠腦組織海馬區(qū)中凋亡細胞數(shù)明顯增加,而給予通塞脈方和去牛膝方后可以顯著減少凋亡細胞數(shù)。 免疫組化法測定通塞脈方及去牛膝方對模型大鼠腦組織海馬區(qū)Bcl-2相關(guān)X蛋白(Bcl-2associated X protein, Bax)以及抑制凋亡蛋白B細胞淋巴瘤／白血病-2(Lymphoma/leukemia-2gene B, Bcl-2)表達的影響,結(jié)果表明：通塞脈方及去牛膝方均能夠增加Bcl-2的表達,抑制Bax的表達。提示通塞脈方及去牛膝方均能通過抑制相關(guān)促凋亡蛋白的表達,增加抗凋亡蛋白的表達,來減少腦細胞的凋亡,抑制腦缺血對腦組織的損傷。 5.通塞脈方及去牛膝方對缺血性腦損傷模型大鼠HMGB1相關(guān)炎癥信號通路的影響 放免法測定MCAO大鼠血清高遷移率組蛋白B1(HMGB1)、腫瘤壞死因子(Tumor necrosis factor-α, TNF-α)和白細胞介素-1β (Interleukin-1β, IL-1β)。實驗結(jié)果發(fā)現(xiàn),造模后大鼠血清HMGB1、TNF-α、IL-1β的含量較假手術(shù)組明顯升高,給予通塞脈方及去牛膝方后均能明顯降低大鼠血清HMGB1、TNF-α、IL-1β含量,去牛膝組對IL-1p的降低效果優(yōu)于全方。 免疫組化法測定通塞脈方及去牛膝方對核轉(zhuǎn)錄因子κB (Nuclear factor-κB, NF-κB)的影響,結(jié)果發(fā)現(xiàn)通塞脈方及去牛膝方均可減少NF-κB的表達。然后運用免疫印跡法(Western blot, WB)檢測NF-κB的表達,也證實了這一結(jié)果。 免疫印跡法檢測大鼠缺血區(qū)腦組織Toll樣受體4(TLR4)蛋白表達,發(fā)現(xiàn)通塞脈方及去牛膝方均可通過減少TLR4的表達,降低MCAO引起的炎癥反應(yīng)。 6.通塞脈方及去牛膝方對缺血性腦損傷模型大鼠血漿血管緊張素Ⅱ含量的影響 放射免疫法檢測MCAO大鼠血漿血管緊張素Ⅱ (angiotensinⅡ, AngⅡ)的含量。結(jié)果表明,造模后,大鼠血漿中AngⅡ明顯升高,而給予通塞脈方和去牛膝方后可以顯著降低AngⅡ水平,其中去牛膝方對AngⅡ抑制作用更為明顯。 結(jié)論： 綜上所述,在腦缺血的治療上通塞脈方的療效與去牛膝方并無明顯差異,在降低血清IL-1β、血漿AngⅡ含量方面去牛膝方作用更強。因此牛膝作為引藥下行的引經(jīng)藥,在腦缺血的治療上并不能發(fā)揮作用,進一步證實了去除牛膝是可行的。通塞脈方及去牛膝方均可通過抗神經(jīng)細胞凋亡,抑制與HMGB1相關(guān)的NF-κB信號通路的激活,而達到減輕MCAO引起的大鼠腦組織損傷,保護神經(jīng)細胞的作用。
[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.
【學(xué)位授予單位】：南京中醫(yī)藥大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R-332;R285.5

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