本文關(guān)鍵詞： 糖尿病胃輕癱 胃排空率 益氣養(yǎng)陰化瘀通絡(luò)中藥 氧化應(yīng)激 HO-1 細(xì)胞凋亡 Bcl-2 Bax　出處：《河北醫(yī)科大學(xué)》2014年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：目的:糖尿病(diabetes mellitus, DM)幾乎影響全身的各個(gè)器官系統(tǒng)，疾病的持續(xù)時(shí)間以及嚴(yán)重程度可能直接影響器官受累程度，其中胃輕癱(gastroparesis)是胃腸道受累的主要表現(xiàn)之一，以胃動(dòng)力受損、排空延遲為主要特征，主要臨床表現(xiàn)為早飽、腹脹、惡心、嘔吐、不穩(wěn)定的血糖等癥狀。據(jù)最近報(bào)道約27%-65%的1型糖尿病患者及約30%的2型糖尿病患者會(huì)出現(xiàn)胃排空延遲，且多見(jiàn)于女性患者。胃輕癱通常會(huì)出現(xiàn)在10年以上病史的糖尿病患者中，這些患者通常有自主功能紊亂。 糖尿病胃輕癱(diabetic gastroparesis, DGP)發(fā)病機(jī)制尚不十分明確，目前認(rèn)為高血糖狀態(tài)與自主神經(jīng)功能紊亂是DGP發(fā)病的主要因素，同時(shí)胃腸道激素異常、腸神經(jīng)系統(tǒng)神經(jīng)元減少、Cajal間質(zhì)細(xì)胞(interstitial cells ofCajal, ICC)網(wǎng)絡(luò)失調(diào)及微循環(huán)障礙等也與DGP發(fā)病密切相關(guān)。Brownlee提出高糖誘導(dǎo)線(xiàn)粒體產(chǎn)生過(guò)多超氧化陰離子而發(fā)生氧化應(yīng)激是糖尿病各種并發(fā)癥的統(tǒng)一發(fā)生機(jī)制。近年來(lái)研究證實(shí)氧化應(yīng)激介導(dǎo)細(xì)胞凋亡在糖尿病腎病、糖尿病視網(wǎng)膜病變、糖尿病神經(jīng)病變等多種糖尿病并發(fā)癥的發(fā)生、發(fā)展中都產(chǎn)生十分重要的影響。但關(guān)于氧化應(yīng)激和細(xì)胞凋亡在DGP發(fā)病中的關(guān)系及具體信號(hào)轉(zhuǎn)導(dǎo)通路等方面的研究甚少。 臨床上我們采用益氣養(yǎng)陰化瘀通絡(luò)中藥(黃芪、熟地、鱉甲、地龍、水蛭、積雪草、丹參、大黃、茯苓、砂仁等)治療DGP取得了較好的療效。本實(shí)驗(yàn)觀(guān)察高糖誘導(dǎo)的氧化應(yīng)激狀態(tài)下DGP大鼠胃排空功能、平滑肌細(xì)胞凋亡、凋亡調(diào)控基因的表達(dá)變化；闡明益氣養(yǎng)陰化瘀通絡(luò)中藥治療DGP的可能作用機(jī)理，為中藥治療DGP提供新的理論依據(jù)。 方法：健康清潔級(jí)Sprague Dawley(SD)大鼠30只，體重201-205g，普通飼料適應(yīng)性喂養(yǎng)2周后隨機(jī)分為正常對(duì)照組(NC組)、模型組(DM組)及益氣養(yǎng)陰化瘀通絡(luò)中藥組(ZY組)，每組各10只大鼠。模型組及中藥組大鼠均按50mg/kg一次性腹腔注射鏈脲佐菌素(streptozotocin, STZ)，而正常對(duì)照組大鼠腹腔注射等體積枸櫞酸緩沖液。72小時(shí)后尾尖取血測(cè)定血糖，以血糖水平≥16.7mmol/L作為糖尿病模型成功的標(biāo)志。造模成功后，中藥組給以益氣養(yǎng)陰化瘀通絡(luò)中藥10ml/(kg·d)灌胃，正常對(duì)照組及模型組給以相同體積生理鹽水灌胃，每天一次，共6周。于第6周末，給予酚紅溶液灌胃，按2ml/kg腹腔注射8%異戊巴比妥鈉麻醉大鼠后，剖開(kāi)腹腔，結(jié)扎大鼠賁門(mén)及幽門(mén)，取出整個(gè)胃部，應(yīng)用酚紅排泄定量檢測(cè)胃排空；部分標(biāo)本置于多聚甲醛固定液中，TUNEL檢測(cè)胃平滑肌細(xì)胞凋亡情況；部分標(biāo)本保存于-80℃冰箱用于制備組織勻漿、提取RNA、蛋白標(biāo)本，檢測(cè)SOD活性，MDA含量；蛋白印跡(Western blot)檢測(cè)胃組織HO-1蛋白含量；Real-time PCR檢測(cè)胃組織HO-1、Bcl-2、Bax基因表達(dá)情況。 結(jié)果： 1各組大鼠一般狀況的比較 正常對(duì)照組大鼠皮毛色澤正常，反應(yīng)敏捷，精神狀態(tài)佳，體重較實(shí)驗(yàn)開(kāi)始時(shí)增加，且實(shí)驗(yàn)期間沒(méi)有不良反應(yīng)，無(wú)大鼠死亡記錄。模型組大鼠腹腔注射鏈脲佐菌素后漸漸出現(xiàn)多飲、多食、多尿，活動(dòng)明顯減少，體重下降，反應(yīng)遲鈍，精神狀態(tài)萎靡等，且實(shí)驗(yàn)期間模型組死亡2只大鼠。益氣養(yǎng)陰化瘀通絡(luò)中藥干預(yù)后的大鼠一般狀況要優(yōu)于模型組大鼠，反應(yīng)較靈活，精神狀態(tài)可，實(shí)驗(yàn)期間中藥組沒(méi)有大鼠死亡記錄。 2各組大鼠體質(zhì)量及血糖的比較 結(jié)果顯示：與正常對(duì)照組比較，模型組和中藥組大鼠的體質(zhì)量均明顯減輕(P0.05)；中藥組大鼠體質(zhì)量與模型組相較無(wú)明顯變化(P0.05)。與正常對(duì)照組相比，模型組與中藥組大鼠血糖水平均顯著升高(P0.05)；中藥組大鼠血糖水平較模型組明顯下降(P0.05)。 3各組大鼠胃排空率的比較 結(jié)果顯示：與正常對(duì)照組相比，模型組及中藥組大鼠胃排空率均顯著下降(P0.05)；中藥組大鼠的胃排空率較模型組顯著升高(P0.05)。 4各組大鼠胃組織中SOD活性及MDA含量的比較 結(jié)果顯示：與正常對(duì)照組相比，模型組及中藥組大鼠胃組織SOD活性均明顯降低(P0.05)；中藥組SOD活性較模型組明顯提高(P0.05)。 與正常對(duì)照組相比，模型組及中藥組大鼠胃組織MDA含量明顯升高(P0.05)；中藥組MDA含量較模型組明顯下降(P0.05)。 5各組大鼠胃組織HO-1蛋白表達(dá)的比較 結(jié)果顯示：與正常對(duì)照組相比，，模型組及中藥組大鼠胃組織HO-1蛋白含量均有升高(P0.05)與模型組相比，中藥組HO-1蛋白含量顯著升高(P0.05)。 6各組大鼠胃組織HO-1mRNA表達(dá)的比較 結(jié)果顯示：與正常對(duì)照組相比，模型組及中藥組大鼠胃組織HO-1基因表達(dá)水平升高(P0.05)；其中中藥組大鼠胃組織HO-1基因表達(dá)水平顯著高于模型組大鼠(P0.05)，這與HO-1蛋白表達(dá)趨勢(shì)一致。 7各組大鼠胃平滑肌細(xì)胞凋亡的比較 結(jié)果顯示：正常對(duì)照組大鼠胃平滑肌層甚少見(jiàn)凋亡細(xì)胞；與正常對(duì)照組相比，模型組及中藥組大鼠胃平滑肌凋亡細(xì)胞數(shù)量明顯增多(P0.05)；與模型組相比，中藥組大鼠胃平滑肌凋亡細(xì)胞數(shù)量明顯減少(P0.05)。 8各組大鼠胃平滑肌細(xì)胞凋亡相關(guān)基因Bcl-2和Bax mRNA表達(dá)的比較 結(jié)果顯示：與正常對(duì)照組相比，模型組及中藥組Bcl-2mRNA表達(dá)水平均明顯下降(P0.05)；與模型組相比，中藥組大鼠Bcl-2mRNA表達(dá)水平增高(P0.05)。 與正常對(duì)照組大鼠比較，模型組大鼠Bax mRNA表達(dá)水平明顯增高(P0.05)，中藥組大鼠Bax mRNA表達(dá)水平?jīng)]有明顯變化(P0.05)；中藥組大鼠Bax mRNA表達(dá)水平較模型組明顯下降(P0.05)。 結(jié)論： 1糖尿病胃輕癱大鼠存在明顯的胃動(dòng)力障礙；益氣養(yǎng)陰化瘀通絡(luò)中藥可明顯降低糖尿病胃輕癱大鼠血糖，促進(jìn)胃動(dòng)力，對(duì)糖尿病胃輕癱具有治療作用。 2糖尿病胃輕癱大鼠胃組織氧化應(yīng)激水平升高，氧化應(yīng)激誘導(dǎo)胃平滑肌細(xì)胞凋亡增加可能是糖尿病胃輕癱發(fā)生、發(fā)展的中心環(huán)節(jié)。 3益氣養(yǎng)陰化瘀通絡(luò)中藥可調(diào)節(jié)機(jī)體氧化應(yīng)激水平，抑制胃平滑肌細(xì)胞凋亡，此可能為其治療糖尿病胃輕癱的作用機(jī)理之一。
[Abstract]:Objective: diabetes mellitus (diabetes, mellitus, DM) affects almost every organ system of the body, the duration and severity of the disease may directly affect the organ involvement, including gastroparesis (gastroparesis) is one of the main manifestations of gastrointestinal tract involvement, with impaired gastric motility and emptying delay as the main feature, the main clinical manifestations of early satiety abdominal distension, nausea, vomiting, and other symptoms, blood glucose instability. According to recent reports about 27%-65% of patients with type 1 diabetes and 30% patients with type 2 diabetes will appear delayed gastric emptying, and more common in female patients. Diabetes Gastroparesis usually occurs in patients with a history of over 10 years, these patients usually have autonomic dysfunction.
Diabetic gastroparesis (diabetic gastroparesis, DGP) pathogenesis is not very clear, the hyperglycemia and autonomic dysfunction is a major factor in the pathogenesis of DGP, and gastrointestinal hormone abnormalities, reduce enteric nervous system neurons, interstitial cells of Cajal (interstitial cells ofCajal, ICC) network disorder and microcirculation etc. closely associated with the onset of DGP.Brownlee is proposed to produce superoxide anion and excessive oxidative stress is a unified mechanism of various complications of diabetes mitochondria induced by high glucose. Recent studies demonstrated that oxidative stress mediated apoptosis in diabetic nephropathy, diabetic retinopathy, diabetic neuropathy and other complications of diabetes, have very important influence development. But on the relationship between oxidative stress and apoptosis in the pathogenesis of DGP and the specific signal transduction pathways There is little research in the field.
Clinically, we use the traditional Chinese medicine Yiqi Yangyin Huayu Tongluo (Huang Qi, rehmannia, turtle, earthworm, leech, Centella asiatica, salvia, rhubarb, Poria, Amomum) treatment of DGP has achieved good results. The experimental observation of gastric emptying function in DGP rats under oxidative stress induced by high glucose, apoptosis of smooth muscle cells, apoptosis regulation of gene; clarify the Yiqi Yangyin Huayu Tongluo Recipe in the treatment of DGP possible mechanism, provide a new theoretical basis for Chinese medicine treatment of DGP.
Methods: healthy Sprague Dawley (SD) 30 rats, weight 201-205g, normal diet after 2 weeks of feeding were randomly divided into normal control group (NC group), model group (DM group) and Yiqi Yangyin Huayu Tongluo medicine group (ZY group), with 10 rats in each group and model group. The Chinese Medicine group rats were 50mg/kg by intraperitoneal injection of streptozotocin (streptozotocin, STZ), and the normal control group rats were injected with citrate buffer.72 hours after the tip of the tail blood glucose determination, the blood glucose level more than 16.7mmol/L as a marker of diabetic model. After the modeling, the Chinese medicine group given Yiqi Yangyin Huayu Tongluo medicine 10ml/ (kg - D) by gavage, the normal control group and model group were given the same volume of saline, once a day for 6 weeks. At the end of the sixth, given the phenol red solution by gavage, 2ml/kg by intraperitoneal injection of 8% sodium pentobarbital anesthetized rats after open Abdominal, cardiac and pyloric ligation in rats, remove the entire stomach, using phenolsulfonphthalein excretion quantitative detection of gastric emptying; some were placed in paraformaldehyde, testing the apoptosis of gastric smooth muscle cells TUNEL; some specimens are preserved in the -80 C refrigerator for the preparation of homogenate, RNA extraction, protein samples, detection of SOD activity, MDA content; Western blot (Western blot) detection of HO-1 protein in gastric tissue; HO-1 Real-time PCR Bcl-2, detection of gastric tissue, the expression of Bax gene.
Result錛

本文編號(hào)：1496190