糖络宁治疗糖尿病周围神经病变临床及作用机制研究

【作者】 张涛静；

【导师】 高彦彬；

【作者基本信息】 北京中医药大学 ， 中医内科， 2008， 博士

【摘要】 糖尿病发病率在逐年增高,糖尿病各种并发症的研究已成为普遍关注的课题。糖尿病周围神经病变(DPN)是糖尿病最常见的慢性并发症之一,其成人糖尿病发病率达到60%,是造成糖尿病足及截肢的主要原因之一。糖尿病人一旦发生神经病变,生活质量将受到严重损害。由于DPN的高发病率和高致残率,DPN的防治已成为糖尿病研究的重要课题。其发病机制为多因素综合作用的结果,目前研究证实主要与高血糖状态引起的代谢、循环障碍有关,同时与神经营养因子减少、自身免疫性损伤以及遗传因素等相关,但至今仍未完全阐明,目前现代医学尚无有效的治疗方法。导师高彦彬教授通过阅读古今文献并结合对大量DPN患者的临床观察,认为本病主要病机为肝肾气阴两虚,络脉痹阻,治宜滋补肝肾、益气养阴、化瘀通络;糖络宁便是在这一治则指导下组方,经过临床观察治疗糖尿病周围神经病变,取得较满意疗效。本研究主要分为临床观察和实验研究两部分。临床观察采用随机对照方法观察糖络宁治疗糖尿病周围神经病变的临床疗效;实验研究则是进一步开展动物实验,从DPN发病机理的角度探讨糖络宁作用的机制。临床观察目的:观察糖络宁治疗糖尿病周围神经病变的临床疗效。方法:将符合诊断标准的DPN病人80例,按就诊顺序随机分为治疗组(40例)和对照组(40例),在控制血糖、血压、血脂的基础上,治疗组予糖络宁治疗,对照组予口服弥可保(甲钴胺)治疗。对患者的临床症状、神经传导速度及血压、血脂、血糖等一般指标进行了观察。结果:治疗组的总有效率为92.11%,对照组的总有效率为72.22%,经Ridit检验显示治疗组总有效率优于对照组(P<0.05);糖络宁治疗组对两侧胫运动神经、感觉神经传导速度改善均优于弥可保对照组(P<0.05);治疗过程中未见明显的不良反应。结论:中药糖络宁可明显改善DPN患者的临床症状、神经传导速度,总体疗效优于目前临床上常用的西药弥可保。实验研究目的:探讨糖络宁治疗糖尿病周围神经病变的作用机制。方法:预实验:清洁级SD雄性大鼠24只,随机取10只为正常对照组,其余用链脲佐菌素腹腔注射(按60mg/kg的剂量标准)造模,十天后大鼠禁食8h断尾取血,测定血糖大于16.7mmol/L者为成模,饲养8周后,麻醉大鼠,分离大鼠双下肢肌肉组织,取出两侧坐骨神经组织,进行坐骨神经组织病理观察,糖尿病模型组大鼠存在不同程度的神经病变;进一步将清洁级SD雄性大鼠120只,随机取15只为正常对照组,其余造模,造模成功后随机分为模型组、弥可保组、糖络宁高剂量组、糖络宁中剂量组、糖络宁低剂量组给药,中药糖络宁制剂低、中、高剂量组分别按生药5、10、20g/kg?d灌胃,弥可保组按175ug/kg?d剂量标准灌胃,模型组及正常组予等量生理盐水灌胃,共给药12周,给药结束后,麻醉大鼠,取出肝组织、神经组织、血液,进行各项指标测定。结果:实验一:糖化血红蛋白比较:与正常组大鼠比较,所有糖尿病组大鼠的糖化血红蛋白均显著增高(P<0.01);与模型组比较,中、高剂量糖络宁治疗组能够降低糖尿病大鼠的糖化血红蛋白(P<0.05),而弥可保组无此差异;体重比较:各组STZ大鼠12周体重与第1周体重比较均有明显增加,P<0.01;但各组相邻两周体重比较却均无明显差别,P>0.05;组间STZ大鼠体重比较,自第1周到第12周,无论是西药组与模型组比较,还是中药组高、中、低剂量组与模型组比较,体重均无显著性差异(P>0.05);血糖比较:各组STZ大鼠组间血糖值比较,糖络宁中剂量组与高剂量组在8周、12周的血糖值与模型组有显著性差异(P<0.05),两组血糖值与西药组比较在8周和12周时也有显著性差异,余组间比较均无显著性差异;热刺激反应时间比较:与正常组对相比,模型组在造模后2周时对热刺激甩尾反应时间有明显缩短(P<0.01),但随着造模时间的延长,在4周、8周和12周时的大鼠甩尾时间又明显延长(P<0.05,P<0.01,P<0.01)。西药组与模型组比较,8周、12周时热刺激反应时间均有显著差异(P<0.01),在8周、12周时可以缩短甩尾时间;中药组与模型组比较,在2周时可以延长甩尾时间,但没有统计学意义,在8周、12周时低剂量组、中剂量组、高剂量组均可以显著性缩短甩尾时间(P<0.01),与西药组比较,中剂量组在8周时可以显著性缩短甩尾时间(P<0.05);冷刺激反应时间比较:模型组与正常组对比,在2周、4周时无明显差异,而8周、12周出现甩尾时间延长现象,且具有显著性差异(P<0.01);与模型组比较,西药组在8周时有显著性缩短甩尾时间的作用(P<0.05),而中剂量组和高剂量组在8周、12周时均有显著性缩短甩尾时间的作用(P<0.05或P<0.01),两组在12周时与西药组比较也有显著性缩短甩尾时间侧作用(P<0.05)。实验二:坐骨神经传导速度比较:组间比较,与正常组比较,模型组在2周、4周、8周、12周时的MNCV均有明显减慢(P<0.01);与模型组比较,西药组在4周、8周、12周可以明显改善神经传导速度(P<0.01或P<0.05);与模型组比较,低剂量、中剂量、高剂量糖络宁治疗组在各周均能明显改善神经传导速度的作用(P<0.01或P<0.05);与西药组比较,高剂量糖络宁治疗组在12周时有更加明显的改善效果(P<0.05),余均无明显差异。实验三:坐骨神经病理比较:光学显微镜下观察坐骨神经切片,可见正常组大鼠坐骨神经纤维分布密集均匀,排列整齐,细胞结构完整,单个纤维饱满,髓鞘薄厚均匀;而模型组糖尿病大鼠坐骨神经纤维形态不规则,结构不清楚,有髓神经纤维密度明显减少,神经纤维间隙扩大,部分髓鞘密度不均匀。与模型组比较,低剂量、中剂量、高剂量糖络宁组及西药组的神经纤维变性及断裂均明显减少,其中高剂量糖络宁组改善最为明显,神经纤维断裂及变性均很少,基本恢复至正常组水平;其次是低剂量组与中剂量组,两组仍有一些神经断裂及变性;最后是西药组,虽然与模型组比较有所改善,但仍出现不少神经断裂及变性。实验四:粘附分子CD54的比较:与正常组大鼠比较,各组糖尿病大鼠单个核细胞表面CD54的表达明显增强(P<0.01);与糖尿病模型组大鼠比较,西药组与中剂量糖络宁组大鼠CD54表达均有所下降,其中中剂量糖络宁组有显著性下降(P<0.01),西药组下降无统计学意义;中剂量糖络宁组与西药弥可保组比较也有显著性下降(P<0.05);粘附分子CD62P的比较:与正常组大鼠比较,各组糖尿病大鼠血小板表面CD62P的表达均明显增强(P<0.01);与糖尿病模型组大鼠比较,西药组与中剂量糖络宁组大鼠CD62P表达均有所下降,其中糖络宁中剂量组显著性下降(P<0.01);中剂量糖络宁组与西药弥可保组比较也有显著性下降(P<0.05)。实验五:血清IGF-Ⅰ含量比较:与正常组大鼠比较,模型组大鼠血清IGF-Ⅰ含量明显下降(P<0.01);与模型组大鼠比较,西药组与中剂量糖络宁组大鼠血清中IGF-Ⅰ含量明显增高(P<0.01或P<0.05),中剂量糖络宁组与西药弥可保组比较更显示出显著差异(P<0.01)。肝组织IGF-ⅠmRNA表达比较:与正常组大鼠比较,模型组大鼠肝组织中IGF-ⅠmRNA表达明显下降(P<0.01);与模型组大鼠比较,西药组可提高大鼠肝组织中IGF-ⅠmRNA的表达(P<0.05),中剂量糖络宁组可显著提高糖尿病大鼠肝组织中IGF-ⅠmRNA的表达(P<0.01)。结论:本研究结果显示,糖络宁可以降低STZ大鼠糖化血红蛋白,降低STZ大鼠血糖,但是对于体重没有明显影响;糖络宁可以明显改善STZ大鼠的糖尿病周围神经病变症状,在痛觉过敏而使刺激反应时间缩短时,糖络宁可以延长反应时间,在痛觉迟钝而使刺激反应时间延长时,糖络宁可以缩短反应时间;糖络宁可以改善糖尿病大鼠坐骨神经传导速度;糖络宁可以改善糖尿病大鼠坐骨神经病理改变。糖络宁可以有效降低糖尿病大鼠粘附分子CD54、CD62P的表达,而西药弥可保则无此效果;糖络宁能够上调糖尿病大鼠坐骨神经组织IGF-ⅠmRNA表达,增加糖尿病大鼠血清中IGF-Ⅰ的含量;提示糖络宁可能通过影响胰岛素样生长因子、粘附分子等血管、神经营养因子等途径发挥治疗DPN的作用。更多还原

【Abstract】 Recent years along with the rise of incident rate of Diabetes Mellitus ,the research about complications of DM has been paid more attention. Diabetic peripheral neuropathy (DPN) is one of the common chronic complications of DM. The prevalence rate of DPN has been estimated to be 60%. DPN is the main reason resulting in diabetic foot and amputations. Once DPN happened, the life quality of diabetic patients will be damaged badly. Since the high incident rate, DPN has become the important project in diabetic research. The pathogenesis of DPN is still unknown. Presently, DPN is seen as the result of a complex interplay of several factors, which include the metabolism and circulation problems due to hyperglycemia, the reduction of neuron growth factors, immune damage and gene factors. Till now there is no effective remedy for curing DPN. Through hard work and clinical experience accumulation, according to Chinese traditional medical theory professor GaoYanbin thought that DPN was caused by the deficiency of liver, kidney, Qi and Yin, the blockage of blood in small channel. The herb formula TangLuoNing(TLN) is directed by this theory which showed better result after clinical use.This research consisted two parts: clinical trials and experimental research. We observed the clinic effect of TLN through clinical trial and discussed the effect mechanism of TLN through experimental research.Clinical trial Objective is to assess the clinic effect of TLN for treating DPN. Method: 80 DPN patients conforming to the diagnostic criteria were randomly divided into the herb group (40 patients) and control group(40 patients). Based on the basic treatment for blood pressure,blood glucose and blood lipid, the herb group was given Tangluoning(for nourishing liver and kidney, benefiting qi and nourishing yin and activating blood circulation). The control group was given the Methycobal (mecobalamin). The changes of the symptoms, nerve conductive velocity were observed in the patients. Result: the total effective rate of the TLN group was 92.11% and that of control group was 72.22%. After the Ridit test, the effect of herb group was better than the control group, the difference was significant (P<0.05).The TLN group and the control group had obviously difference in improvement of symptom and the nerve conductive velocity(P<0.05).There were no obvious side effects has been found during the treatment.Conclusion: Tangluoning had significant effect for curing DNP,total effect is better than methycobal.Experimental research Objective To observe the effect mechanism of TLN for curing DPN. Method Among 24 male SD rats,pick up 10 rats randomly as normal control group, the rest were induced by STZ celiac injection (60mg/kg). Ten days after the injection, the rats with fasting blood glucose level above 16.7mmol/L were diabetes model group for observation. After feeding for 8 weeks, the morphology of sciatic nerves was investigated by HE staining under microscope. It showed that the diabetic model rats were suffering from neuropathy. Then, pick up 15 rats randomly as normal control group among 120 male SD rats,the rest for diabetic model group. After induced by STZ, the rats which successfully became diabetic model were divide into diabetic model group, methycobal group, TLN high dose group, TLN middle dose group and TLN low dose group.Methycobal was given by 175ug/kg?d, TLN was given by 5、10、20g/kg?d herb according to low、middle、high dose. After curing for 12 weeks, we measured some items.Experiment 1: Glycosylated hemoglobin: Compared with normal rats group, glycosylated hemoglobin levels in all diabetic groups were significantly higher (P<0.01). Compared with diabetic model group, glycosylated hemoglobin levels in the middle and high dose TLN were lower (P<0.05). But Methycobal group had no this effect; The weight of STZ rat: The weight of all diabetic rats was increased significantly (P<0.01), but the weight increasing during two weeks had no such significant change. Compared with diabetic model group, the rats weight of little, middle, high dose TLN group and Methycobal group had no significant difference(P>0.05); The plasma sugar: Similar to the glycosylated hemoglobin, compared with diabetic model group, the plasma sugar levels in the middle and high dose TLN were lower (P<0.05). The Methycobal group had no significant difference(P>0.05); The thermal response latency: Compared with diabetic model group, methycobal group had significant difference in week 2, week 4, week 8, week 12.(P<0.01,or P<0.05) The middle, high dose TLN group had significant difference only in week 8, week12 , compared with diabetic model group(P<0.01,or P<0.05). But the middle dose TLN group had significant difference in week 8 compared with methycobal group (P<0.05). The cold response latency: Compared with diabetic model group, methycobal group only had significant difference in week 8.(P<0.05) The middle, high dose TLN group had significant difference in week 8, week12 , compared with diabetic model group(P<0.01,or P<0.05). Compared with methycobal group the middle, high dose TLN group also had significant difference in week 8, week12.(P<0.05)Experiment 2: Sciatic nerve conduction velocity: Compared with normal group, the MNCV of model group decreased significantly in week 2, week 4, week 8, week 12.(P<0.01). Compared with diabetic model group, methycobal group had significant increace difference in week 4, week 8, week 12(P<0.01,or P<0.05). Compared with diabetic model group, The low, middle and high dose TLN group had significant amelioration in week4, week 8, week12 (P<0.01,or P<0.05). Only the high dose TLN group had significant difference in week 12 compared with methycobal group (P<0.05).Experiment 3: There was significant injury in aciatic nerve morhpometrics of diabetic rats, which could be alleviated by TNL in different extent. The frequency of structurally abnormal fibril was significantly increased and the axon diameter of nerve fibril was reduced remarkably in 12 weeks diabetic rats. There were significant decrease in the frequency of sturctrually abnormal fibril and increase in the axon diameter of nerve fibril in TLN treated group compared with that in Methycobal group.Experiment 4: Adhesion molecule CD54: Compare with normal rats group, CD54 activities in all diabetic groups were significant higher (P<0.01). CD54 activities in middle dose TLN group and Methycobal group were lower. But only the activity of middle dose TLN group were significantly lower than the diatetic model group(P<0.01).The activity of middle dose TLN group was also significantly lower than the Methycobal group(P<0.05). Adhesion molecule CD62P: Compare with normal rats group, CD62P activities in all diabetic groups were significant higher (P<0.01). CD62P activity in middle dose TLN group and Methycobal group were lower. But only the activity of middle dose TLN group was significantly lower than the diatetic model group(P<0.01).The activity of middle dose TLN group was also significantly lower than the Methycobal group(P<0.05).Experiment 5:Insulin-like growth factor (IGF-Ⅰ) concentration in serum: Compare with normal rats group, IGF-Ⅰconcentration of serum in all diabetic groups was significant lower (P<0.01). Compare with diabetic model group, IGF-Ⅰconcentration in serum is higher in Methycobal group and TLN group (P<0.05 or P<0.01). Middle dose TLN group is significant better than Methycobal group (P<0.01). Expression of IGF-ⅠmRNA in hepatic tissue: Compare with normal rats group, expression level of IGF-ⅠmRNA in hepatic tissue in all diabetic groups was significant lower (P<0.01). Compare with diabetic model group, Methycobal group can promote the expression level of IGF-ⅠmRNA in hepatic tissue (P<0.05), middle dose TLN group can significantly promote the expression level of IGF-ⅠmRNA in hepatic tissue (P<0.01).Conclusion:Through the rats experiment, it showed that TLN can reduce the plasma sugar and Glycosylated hemoglobin. It can also ameliorate the disesthesia and hyperaesthesia symptoms of STZ DM-rats. TLN can reduce the activities of adhesion molecule CD54 and CD62P. Meanwhile, TLN can promote the IGF-Ⅰconcentration in serum and the expression level of IGF-ⅠmRNA in hepatic tissue of diabetic rats induced by STZ. It indicated that the effect mechanism of TLN for treating DPN is achieved by depressing the vessel factor and improving the nerve nurtritional fator. TLN (as a herb formula) can significantly improve symptoms and nerve conductive velocity in patients with DPN, and showed better effect than Methycobal and has no side effect. Meanwhile, through rats experiment TLN can reduce the adhesion molecule CD54 and CD62P activity in diabetic rats’sciatic serum which showed that the depression of vessel inflammation is one of main effect mechanism of TLN. TLN can promote the IGF-Ⅰconcentration in serum and the expression level of IGF-ⅠmRNA in hepatic tissue of diabetic rats induced by STZ which indicated that improvement of nerve nurtritional factor is another effective pathway of TLN for treating DPN. So, TLN is effective and safe for curing DPN.更多还原