益气养阴化瘀通络中药对糖尿病胃轻瘫大鼠SOD、MDA、HO-1及Bcl-2与Bax基因表达的影响

发布时间：2018-02-08 20:22

  本文关键词： 糖尿病胃轻瘫 胃排空率 益气养阴化瘀通络中药 氧化应激 HO-1 细胞凋亡 Bcl-2 Bax　出处：《河北医科大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的:糖尿病(diabetes mellitus, DM)几乎影响全身的各个器官系统，疾病的持续时间以及严重程度可能直接影响器官受累程度，其中胃轻瘫(gastroparesis)是胃肠道受累的主要表现之一，以胃动力受损、排空延迟为主要特征，主要临床表现为早饱、腹胀、恶心、呕吐、不稳定的血糖等症状。据最近报道约27%-65%的1型糖尿病患者及约30%的2型糖尿病患者会出现胃排空延迟，且多见于女性患者。胃轻瘫通常会出现在10年以上病史的糖尿病患者中，这些患者通常有自主功能紊乱。 糖尿病胃轻瘫(diabetic gastroparesis, DGP)发病机制尚不十分明确，目前认为高血糖状态与自主神经功能紊乱是DGP发病的主要因素，同时胃肠道激素异常、肠神经系统神经元减少、Cajal间质细胞(interstitial cells ofCajal, ICC)网络失调及微循环障碍等也与DGP发病密切相关。Brownlee提出高糖诱导线粒体产生过多超氧化阴离子而发生氧化应激是糖尿病各种并发症的统一发生机制。近年来研究证实氧化应激介导细胞凋亡在糖尿病肾病、糖尿病视网膜病变、糖尿病神经病变等多种糖尿病并发症的发生、发展中都产生十分重要的影响。但关于氧化应激和细胞凋亡在DGP发病中的关系及具体信号转导通路等方面的研究甚少。 临床上我们采用益气养阴化瘀通络中药(黄芪、熟地、鳖甲、地龙、水蛭、积雪草、丹参、大黄、茯苓、砂仁等)治疗DGP取得了较好的疗效。本实验观察高糖诱导的氧化应激状态下DGP大鼠胃排空功能、平滑肌细胞凋亡、凋亡调控基因的表达变化；阐明益气养阴化瘀通络中药治疗DGP的可能作用机理，为中药治疗DGP提供新的理论依据。 方法：健康清洁级Sprague Dawley(SD)大鼠30只，体重201-205g，普通饲料适应性喂养2周后随机分为正常对照组(NC组)、模型组(DM组)及益气养阴化瘀通络中药组(ZY组)，每组各10只大鼠。模型组及中药组大鼠均按50mg/kg一次性腹腔注射链脲佐菌素(streptozotocin, STZ)，而正常对照组大鼠腹腔注射等体积枸橼酸缓冲液。72小时后尾尖取血测定血糖，以血糖水平≥16.7mmol/L作为糖尿病模型成功的标志。造模成功后，中药组给以益气养阴化瘀通络中药10ml/(kg·d)灌胃，正常对照组及模型组给以相同体积生理盐水灌胃，每天一次，共6周。于第6周末，给予酚红溶液灌胃，按2ml/kg腹腔注射8%异戊巴比妥钠麻醉大鼠后，剖开腹腔，结扎大鼠贲门及幽门，取出整个胃部，应用酚红排泄定量检测胃排空；部分标本置于多聚甲醛固定液中，TUNEL检测胃平滑肌细胞凋亡情况；部分标本保存于-80℃冰箱用于制备组织匀浆、提取RNA、蛋白标本，检测SOD活性，MDA含量；蛋白印迹(Western blot)检测胃组织HO-1蛋白含量；Real-time PCR检测胃组织HO-1、Bcl-2、Bax基因表达情况。 结果： 1各组大鼠一般状况的比较 正常对照组大鼠皮毛色泽正常，反应敏捷，精神状态佳，体重较实验开始时增加，且实验期间没有不良反应，无大鼠死亡记录。模型组大鼠腹腔注射链脲佐菌素后渐渐出现多饮、多食、多尿，活动明显减少，体重下降，反应迟钝，精神状态萎靡等，且实验期间模型组死亡2只大鼠。益气养阴化瘀通络中药干预后的大鼠一般状况要优于模型组大鼠，反应较灵活，精神状态可，实验期间中药组没有大鼠死亡记录。 2各组大鼠体质量及血糖的比较 结果显示：与正常对照组比较，模型组和中药组大鼠的体质量均明显减轻(P0.05)；中药组大鼠体质量与模型组相较无明显变化(P0.05)。与正常对照组相比，模型组与中药组大鼠血糖水平均显著升高(P0.05)；中药组大鼠血糖水平较模型组明显下降(P0.05)。 3各组大鼠胃排空率的比较 结果显示：与正常对照组相比，模型组及中药组大鼠胃排空率均显著下降(P0.05)；中药组大鼠的胃排空率较模型组显著升高(P0.05)。 4各组大鼠胃组织中SOD活性及MDA含量的比较 结果显示：与正常对照组相比，模型组及中药组大鼠胃组织SOD活性均明显降低(P0.05)；中药组SOD活性较模型组明显提高(P0.05)。 与正常对照组相比，模型组及中药组大鼠胃组织MDA含量明显升高(P0.05)；中药组MDA含量较模型组明显下降(P0.05)。 5各组大鼠胃组织HO-1蛋白表达的比较 结果显示：与正常对照组相比，，模型组及中药组大鼠胃组织HO-1蛋白含量均有升高(P0.05)与模型组相比，中药组HO-1蛋白含量显著升高(P0.05)。 6各组大鼠胃组织HO-1mRNA表达的比较 结果显示：与正常对照组相比，模型组及中药组大鼠胃组织HO-1基因表达水平升高(P0.05)；其中中药组大鼠胃组织HO-1基因表达水平显著高于模型组大鼠(P0.05)，这与HO-1蛋白表达趋势一致。 7各组大鼠胃平滑肌细胞凋亡的比较 结果显示：正常对照组大鼠胃平滑肌层甚少见凋亡细胞；与正常对照组相比，模型组及中药组大鼠胃平滑肌凋亡细胞数量明显增多(P0.05)；与模型组相比，中药组大鼠胃平滑肌凋亡细胞数量明显减少(P0.05)。 8各组大鼠胃平滑肌细胞凋亡相关基因Bcl-2和Bax mRNA表达的比较 结果显示：与正常对照组相比，模型组及中药组Bcl-2mRNA表达水平均明显下降(P0.05)；与模型组相比，中药组大鼠Bcl-2mRNA表达水平增高(P0.05)。 与正常对照组大鼠比较，模型组大鼠Bax mRNA表达水平明显增高(P0.05)，中药组大鼠Bax mRNA表达水平没有明显变化(P0.05)；中药组大鼠Bax mRNA表达水平较模型组明显下降(P0.05)。 结论： 1糖尿病胃轻瘫大鼠存在明显的胃动力障碍；益气养阴化瘀通络中药可明显降低糖尿病胃轻瘫大鼠血糖，促进胃动力，对糖尿病胃轻瘫具有治疗作用。 2糖尿病胃轻瘫大鼠胃组织氧化应激水平升高，氧化应激诱导胃平滑肌细胞凋亡增加可能是糖尿病胃轻瘫发生、发展的中心环节。 3益气养阴化瘀通络中药可调节机体氧化应激水平，抑制胃平滑肌细胞凋亡，此可能为其治疗糖尿病胃轻瘫的作用机理之一。
[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锛

本文编号：1496190