糖痹康干预糖尿病周围神经病变临床及作用机制研究

【作者】 吕翠岩；

【导师】 刘铜华；

【作者基本信息】 北京中医药大学 ， 中医内科学， 2014， 博士

【摘要】 [研究背景]糖尿病周围神经病变(Diabetic peripheral neuropathy,DPN)是糖尿病(Diabetes mellitus, DM)常见并发症之一,临床上常常累及DPN患者的感觉神经及植物神经,使之产生刺痛、麻木甚至导致运动及神经功能障碍,发病率较高,严重危害患者的身体健康,是糖尿病病人致残的首要因素。DPN发病机制十分复杂,非单一因素所致,尚未完全阐明,造成西医对其治疗存在一定的局限性和有效性；中医药对DPN具有多手段、多途径、多靶点整体治疗优势和明显疗效。导师刘铜华教授经过多年的临床研究,认为本病主要病机为“气阴不足,毒瘀神络”,并在国内外首次提出“益气养阴、解毒化瘀通络”的新治疗原则。本课题在前期研究基础上,通过实验及临床研究,进一步探讨中药复方糖痹康对DPN神经保护机制及作用靶点,为临床推广应用提供理论依据。[研究目的]临床研究采用自身前后对照方法,客观评价临床有效复方中药糖痹康治疗糖尿病周围神经病变的临床有效性及安全性；实验研究通过动物及细胞模型,从分子水平探讨糖痹康作用机制。[研究方法]临床研究采用自身前后对照,观察中药复方糖痹康对35例DPN患者的临床疗效。在基础治疗(如血糖、血压、血脂等)的基础上,给予35例DPN病人口服糖痹康配方颗粒,治疗8周后,对病人的各项指标(如：中医证候、实验室检查及肌电图)进行客观评价。实验研究1整体实验采用高脂饲料和小剂量链脲佐菌素诱发2型糖尿病大鼠动物模型,不同剂量的糖痹康灌胃,并与弥可保对照,每4周检测体重、空腹血糖和坐骨神经传导速度；16周后,光镜观察各组大鼠坐骨神经病理变化、电镜观察坐骨神经超微结构；采用酶法测定TC、 TG;采用免疫比浊法测定HDL-C、LDL-C;放射免疫法检测大鼠血浆β-EP、ET、FINS水平；铜试纸显色法检测大鼠血清NEFA、NO水平；免疫组织化学法和western blot检测坐骨神经NGF、BDNF、NT-3、VEGF蛋白表达；实时荧光定量RT-PCR检测NGF、BDNF. NT-3、VEGF mRNA表达。2细胞实验通过雪旺细胞株,建立高糖环境下大鼠雪旺细胞的细胞模型；用不同剂量的含药血清和正常血清培养基刺激24h、48h、72h和96h后,MTT法检测高糖环境下不同剂量的糖痹康含药血清对大鼠细胞增殖的影响；并用不同剂量的含药血清和正常血清培养基刺激48h后,采用放免法检测细胞上清中TNF-α的含量；ELISA法检测细胞上清中sVCAM-1的含量,Western blot法检测大鼠雪旺细胞P38和P-P38的表达。[研究结果]临床研究糖痹康治疗前后受试对象的双侧胫运动神经(MCv)、感觉神经(SCv)传导速度明显改善(P<0.05),且治疗前后安全性指标(血糖、糖化血红蛋白、血脂及血压)均在正常范围内,无显著性差异(P>0.05)同时未出现不良反应；总有效率达到91.14%。实验研究：1整体实验与正常组比较,模型组体重均显著降低,血糖均显著升高,坐骨神经传导速率均显著降低,NO、β-EP、FINS、HDL-C、NGF的蛋白及mRNA、BDNF的蛋白及mRNA、NT-3的蛋白及mRNA的含量均显著降低,ET、TG、TC、LDL-C、NEFA、VEGF的蛋白及mRNA的含量均显著升高。与模型组比较,糖痹康及弥可保组体重均显著升高,血糖均显著降低,坐骨神经传导速率均显著升高,NO、β-EP、FINS、HDL-C、NGF的蛋白及mRNA、BDNF的蛋白及nRNA、NT-3的蛋白及mRNA的含量均显著升高,ET、TG、 TC、LDL-C、NEFA、VEGF的蛋白及nRNA的含量均显著降低。病理检测显示,正常组大鼠坐骨神经纤维结构紧密、分布均匀、排列整齐；模型组大鼠坐骨神经纤维排列疏松、间隙变大、多处发生变性及断裂；各给药组大鼠坐骨神经纤维的变性与断裂显著减少,纤维结构紧密、排列整齐,未见变性与断裂的发生,髓鞘变厚且着色均匀。电镜结果显示,正常组大鼠坐骨神经横切面髓鞘结构完整、致密、均匀；模型组大鼠坐骨神经横切面髓鞘的板层结构发生严重分离,各层间排列紊乱,出现大量空泡及裂隙；各给药组坐骨神经中空泡及裂隙均显著降低,但髓鞘仍未恢复板层状结构,线粒体及粗面内质网结构正常,出现结构完整的雪旺细胞。2细胞实验与空白组比较,24、48、72、96小时后,50MGLU与75MGLU组细胞增殖程度均显著降低,但50M GLU与75M GLU组间并无差异；与50mM高糖对照组比较,24、48小时后,弥可保及糖痹康1:1、1：2、1：8组均可显著增高细胞增殖能力。与空白对照组比较,50mM高糖对照组的sVCAM-1、TNF-α、P38丝裂素活化蛋白激酶及磷酸化P38丝裂素活化蛋白激酶含量显著升高；与50mM高糖对照组比较,弥可保与糖痹康各剂量组上清中sVCAM-1、TNF-α、P38丝裂素活化蛋白激酶及磷酸化P38丝裂素活化蛋白激酶的含量显著降低。[结论]临床观察中药复方糖痹康可显著改善DPN患者的临床症状、提高神经传导速度、显著降低国际公认糖尿病周围神经病变积分并且总有效率达到91.14%。实验研究1整体实验：(1)中药复方糖痹康能改善STZ-DM大鼠血糖、体重及血脂；降低DPN大鼠血清游离脂肪酸水平；改善糖尿病周围神经病变大鼠坐骨神经传导速度且存在剂量与用药时间依赖关系；(2)中药复方糖痹康对DPN大鼠坐骨神经的形态具有保护作用以及对其坐骨神经髓鞘、轴索等具有修复作用；(3)中药复方糖痹康能上调DPN大鼠FINS的表达、血清中NO的表达及下调血浆中ET的表达,这些可能是其防治DPN的机制之一；(4)中药复方糖痹康能显著上调DPN大鼠血浆中p-EP的表达,这可能是其对糖尿病周围神经病变的神经保护及镇痛作用的机制之一；(5)中药复方糖痹康能显著上调DPN大鼠坐骨神经中NGF、BDNF、NT-3蛋白及基因表达,下调DPN大鼠坐骨神经中VEGF蛋白及基因表达,这些可能是其防治DPN的有效作用靶点之一。2细胞实验：(1)糖痹康组可明显改善高糖培养环境下,雪旺细胞增殖活性,糖痹康低剂量组优于西药(弥可保)组,其在高糖培养的环境下,中医复方糖痹康可促进大鼠坐骨神经雪旺细胞的增殖,可能是其防治DPN的机制之一；(2)糖痹康含药血清可下调在高糖环境下大鼠坐骨神经雪旺细胞p38MAPK及其激活形式p-p38MAPK蛋白表达,及下调大鼠坐骨神经雪旺细胞上清中TNF-α的含量,可能是其DPN神经保护及镇痛作用靶点之一；(3)糖痹康含药血清可下调大鼠坐骨神经雪旺细胞上清中sVCAM-1的含量,可能是其DPN神经保护作用靶点之一。更多还原

【Abstract】 [Study background]Diabetic peripheral neuropathy (Diabetic peripheral neuropathy, DPN) is a common complication of diabetes mellitus (DM). DPN often involves sensory nerve and plant nerve, causing tingling, numbness, and even lead to exercise and nerve dysfunction in clinical. With high rate of incidence, it damages to health seriously and leads primary diabetes to disability. The world health organization estimates that there will be about360million DM patients in the world by2030, most of who suffer type2diabetes mellitus DPN. The pathogenesis of DPN is very complex and have not been fully elucidated causing limitation to the western medicine for the treatment and effectiveness yet. Traditional Chinese medicine treating DPN has overall advantages and obvious curative effect for its various means and ways. In recent years, there are many useful researches about DPN disease name, pathogenesis, clinical syndrome differentiation and treatment. The holistie therapy of DPN using TCM has its advantages and obviously curative effect. Professor Tonghua Liu insist the pathogenesis of DPN were deficiency of qi yin and poison stuck the shen collateral and brings up the new therapy of nourishing yin and qi, detoxify, dispersing blood stasis and clear the collateral. This project discusses the nerve protection mechanisms and targets of Chinese herbal compound Tangbikang treating DPN on the basis of previous research, and provides clinical basis of clinical application.[Objective]To objective evaluation of clinical efficacy and safety of treatment of diabetic peripheral neuropathy by Chinese herbal compound Tangbikang, the clinical trials were adopted by self control.[Methods]Clinic trial35DPN patients conforming to the diagnostic criteria, based on the basic treatment for blood pressure, blood glucose and blood lipid, were given Chinese herbal compound Tangbikang. After curing for8weeks, we measured some indicators, like TCM syndrome, laboratory examination and electromyography.Experimental research1.vivo experimentsThe experimental diabetes mellitus rats model were induced by feeding high fat forage and injection streptozotocin. Tangbikang groups were given Tangbikang with different dose, Body weight, blood glucose and sciatic nerves conduction velocitywere detected in every4weeks. Rats unilateral sciatic nerve were cutting-up after16weeks, pathological change of rat sciatic nerve of each group was observed with the light microscope, ultrastructure of rat sciatic nerve was observed by electron microscopy; the TC and TG were determined by the enzyme analysis method, the HDL-C and LDL-Cwere determined by immunoturbidimetry method; the p-EP,ET and FINS levels of rat plasma were determined by radioimmunoassay; the NEFA and NO levels of rat serum weredetermined by the method of Cul test paper; the expressions of NGF, BDNF, NT-3and VEGFproteinof rat sciatic nerve were determined by immunohistochemical assay and western blot; the expressions of NGF, BDNF, NT-3,and VEGF mRNA were detected by real-time fluorescence quantification.2.vitro experimentschwann cell model was establish in high glucose environment from schwann cells lineage.The effect of different dose Tangbikangon the proliferation of rat epidermal cells was detect by MTT method after stimulate by drug serum of different dose and serum at24h,48h,72h and96h. The content of TNF-a in supernatants were determined by radioimmunoassay after stimulate by drug serum of different dose and serum at48h; the content of sVCAM-1in supernatants were determined byELISA method; the expressions of schwann cell P38and P-P38were detected by Western blot method.[Results]Clinical trialAfter the treatment byTangbikang, the conduction velocitiesof MCv and SCvwas better than before, the difference was significant (P<0.05).Security Indexincludingblood glucose, glycosylated hemoglobin, blood lipid and blood pressurewith normal limits, the difference was insignificant (P>0.05), and there were no obvious side effects has been found during the treatment; the total effective rate of the Tangbikang was91.14%.Experimental research1.vivo experimentsCompared with normal group, model group weight was significantly reduced, the blood glucose was significantlyrisen, sciatic nerves conduction velocity were significantly reduced, the content of NO,β-EP, FINS,HDL-C, protein and mRNA of NGF, protein and mRNA of BDNF and protein and mRNA of NT-3were significantly risen; the content of ET, TG, TC、LDL-C、NEFA、protein and mRNA of VEGF were significantly reduced. Compared with model group, the rats body weight with different doses of Tangbikang were significantly increased; the content of NO,β-EP HDL-C,FINS, protein and mRNA of NGF, protein and mRNA of BDNF and protein and mRNA of NT-3were significantly reduced; the content of ET、TG、TC、LDL-C、NEFA、protein and mRNA of VEGF were significantly risen. The pathological results showed that sciatic nervefiber of normal group was hard-packed, homogeneous distribution and in alignment; and looselyarranged, larger gap and degeneration and fracture in multiple places of model group; degeneration and fracture of sciatic nerve was significantly reduced in Tangbikang group, it was hard-packed, homogeneous distribution and in alignment, but has degeneration and fracture insome places, the myelin sheath was thickened and even colouring. Electron microscopy (sem) results showed that the sciatic nervemyelin sheath was structural integrity, densification and well-distributed in normal group, the sciatic nervemyelin sheath was serious separation of lamellar structure, a disordered arrangement between each layer and a large number of vacuoles and gaps were in control group, the vacuoles and gaps of sciatic nerve were significantly reduced in Tangbikang group, but the lamellar structure was also abnormal, the structures of mitochondria and rough endoplasmic reticulum were in normality, structural integrityschwann cell was observed.2.vitro experimentCompared with blank group,50M GLU and75M GLU groups, the degree of cell proliferation was significantly reduced after24h,48h,72h and96h, but there were no significantly difference between50M GLU and75M GLU group. Compared with50mM model group, the degree of cell proliferation wassignificantly risenin Mecobalamin: Tangbikang1:1group, Mecobalamin:Tangbikang1:2group, Mecobalamin:Tangbikang1:8group.Compared with blank group,the contents of sVCAM-1, TNF-a. P38mitogen activated protein kinase and phosphorylated P38mitogen activated protein kinase were significantly risen, compared with50mM model group, the contents of sVCAM-1, TNF-a, P38mitogen activated protein kinase and phosphorylated P38mitogen activated protein kinase were significantly reduced in Mecobalamin:Tangbikanggroups.[Conclusion]Clinical trialChinese herbal compound Tangbikangcould improve of symptom, the nerve conductive velocity and the diabetic peripheral neuropathy integralsobviously, the total effective rate of the Tangbikang was91.14%.1.vivo experiments:1.1Chinese herbal compound Tangbikangcould improveblood glucose, body weight and blood lipid of STZ-DM rats; and reduce the content of non-esterified fatty acid in DPN rats serum; improvesciatic nerve conduction velocity of diabetic peripheral neuropathy rats, and it showed dose and time dependent.1.2Chinese herbal compound Tangbikang could protect the morphology ofsciatic nervein DPN rats, and repair its sciatic nerve myelin sheath and neuraxon.1.3Chinese herbal compound Tangbikang could rise the expression of FINS and NO in serum of DPN rats, reduce the expression of ET in plasma, these may be one of the mechanism of Tangbikang prevention and cure DPN.1.4Chinese herbal compound Tangbikang could rise the expression of P-EP in plasma of DPN rats, these may be one of the mechanism of Tangbikangneuroprotection and abirritation of DPN.1.5Chinese herbal compound Tangbikang could rise the expression of NGF, BDNF and NT-3protein and gene of sciatic nerve in DPN rats, reduce the expression of VEGF protein and gene of sciatic nerve in DPN rats, these may be one of the effective targets of Tangbikang prevention and cure DPN.2.vitro experiment2.1Tangbikang could proliferative activity of schwann cells in high sugar cultivation environment, Tangbikang is more effective then mecobalamin, Chinese herbal compound Tangbikangcould promoteproliferative activity of the rat sciatic nerve schwann cells,these may be one of the mechanism of Tangbikang neuroprotection and abirritation of DPN.2.2Tangbikang containing serum could reduce the expression of p38MAPK and activate the expression of p-p38MAPK protein of the rat sciatic nerve schwann cells in high sugar cultivation environment, and reduce the content of TNF-a protein in the rat sciatic nerve schwann cells supernatant, these may be one of the effective targets of Tangbikang neuroprotection and abirritation of DPN.2.3Tangbikang containing serum could reduce the content of sVCAM-1in the rat sciatic nerve schwann cells supernatant, this may be one of the effective targets of Tangbikang neuroprotection of DPN.更多还原