【作者】 唐咸玉；

【导师】 朱章志；

【作者基本信息】 广州中医药大学 ， 中医临床基础， 2007， 博士

【摘要】 一、研究目的观察温肾健脾、祛毒活血法治疗早期DN的临床疗效、对肾功能保护作用以及对炎症因子白介素-6（IL-6）、肿瘤坏死因子-α（TNF-α）的影响，并初步探讨其作用机理。二、研究内容与方法1．临床研究将符合西医早期DN诊断标准且中医辨证符合脾肾阳虚证、气虚血瘀证者58例患者分为治疗组32和对照组26例，两组均采取相同的基础治疗，包括糖尿病健康教育、糖尿病饮食、控制血压、血糖。对照组在基础治疗加服福辛普利（蒙诺）5～10mg，po，qd；治疗组在对照组的基础上加服温肾健脾、祛毒活血法中药，水煎服，每日1剂，以1个月为一个疗程。观察治疗前后早期DN及中医证候疗效，检测空腹血糖（FBG），餐后2小时血糖（P2hBG），血脂，24小时尿蛋白排泄量（UAE），测定血清IL-6、TNF-α等指标进行比较。2．实验研究将SPF级雄性SD大鼠采用一次性腹腔注射STZ（剂量为60mg／kg）建立早期DN模型。将模型成功的糖尿病大鼠随机分组与正常组共4组：正常组、模型组、西药组、中药组，中药组按生药20g／kg·d灌胃，西药组以福辛普利4mg／kg·d灌胃，正常组及模型组均用等量生理盐水灌胃，实验时间为6周。观察各组大鼠治疗后一般状态、肾重／体重比、血糖、24h尿微量白蛋白、TG、TC、SCr、BUN变化情况；采用ELISA法检测大鼠血清IL-6、TNF-α水平；光镜下观察肾组织形态变化；电镜下观察肾组织超微结构改变；采用免疫组化法观察肾脏转化生长因子（TGF-β₁）表达情况。三、研究结果1．临床研究观察到在血糖控制的基础上，治疗组总有效率为87.5％，明显优于对照组的61.5％（P＜0.05）。两组均能减轻早期DN患者症状积分、降低FBG、P₂hBG、24hUAE及改善血脂各项指标；治疗组在改善早期DN患者临床症状、降低24UAE及改善血脂各项指标等方面均明显优于对照组（P＜0.05或P＜0.01），且能降低患者血清IL-6、TNF-α水平（P＜0.05或P＜0.01），对照组炎症指标治疗前后比较差异不明显。未发现任何毒副作用。2．模型组大鼠明显消瘦，饮水较多，小便多，大便溏，精神萎靡，反映迟钝，毛竖无光泽，动作迟缓。肾组织切片，模型组可见多数肾小球体积不同程度增大，肾小球毛细血管腔扩张，可见红细胞淤积，炎症细胞浸润，肾小球系膜区扩张，基底膜不同程度增厚，未见明显的肾小球硬化。3．与正常组相比，模型大鼠体重明显减轻（P＜0.01），肾重指数、血糖、24小时尿量、UAE、TG、TC以及STZ大鼠血清炎症标志物IL-6及TNF-α含量均显著升高（P＜0.05或P＜0.01）；经治疗后中药、西药组24小时尿量、UAE均有减轻（P＜0.05或P＜0.01），中药组明显优于西药组（P＜0.05或P＜0.01）。治疗后中药组血糖、TG、TC较模型组下降明显，差异有显著性（P＜0.05或P＜0.01），西药组则无明显变化（P＞0.05），表明中药具有一定的降低血糖、调节血脂的作用。各组大鼠SCr、BUN的比较均无统计学意义。此外，中药组IL-6及TNF-α较模型组均有明显降低，差异有显著性（P＜0.05或P＜0.01），西药组IL-6及TNF-α无明显变化。4．肾脏免疫组化示模型组大鼠肾组织TGF-β₁表达明显增强，以肾小管及肾小球较为明显，西药组及中药组肾组织TGF-β₁表达与模型组相比，程度明显减轻。半定量积分示：早期DN肾组织中TGF-β₁表达与正常组比较明显增加（P＜0.01）；经治疗后中药组有明显抑制肾组织TGF-β₁的表达作用（P＜0.01），并优于西药组（P＜0.05）。四、结论1．阳气易虚，阳虚亦可致消；脾肾阳（气）虚是早期DN的发病基础，内生邪毒是早期DN的重要因素，早期DN病机为脾肾阳（气）虚，邪毒内阻，故以温肾健脾、祛毒活血为早期DN基本治法。2．温肾健脾、祛毒活血法中药复方可减轻早期DN患者临床症状，降低尿微量白蛋白，具有较好的治疗保护作用。3．温肾健脾、祛毒活血法中药复方对早期DN大鼠具有一定降低血糖、改善血脂代谢紊乱、降低尿微量白蛋白的作用；并可减轻DN大鼠肾组织病理损害伤，抑制肾脏肥大，系膜增生及肾小球基底膜增厚；明显抑制肾组织TGF-β₁的高表达，具有一定的保护肾功能的作用。4．温肾健脾、祛毒活血中药复方能显著下调血清IL-6、TNF-α水平，其治疗早期DN的作用机理可能与调节炎症因子的产生、抑制炎症反应过程有关，通过神经—内分泌—免疫调节网络对机体进行全面的调节，阻断炎症因子TNF-α、IL-6及其受体等，抑制炎症反应，从而调节多种生长因子及细胞粘附分子的表达，纠正内皮功能紊乱，并抑制肾小球系膜的增殖和细胞外基质的产生。更多还原

【Abstract】 ObjectiveTo investigate the effects of the method of warming kidney and invigoratingspleen, removing toxicity and activating blood （WIRA） on the inflammatoryfactor serum interleukin-6 （IL-6） and tumor necrosis factor-α（TNF-α） inearly-stage diabetic nephropathy （DN）, and to explore its therapeuticmechanism.Methods1.58 cases of patients with early-stage DN with deficiency of spleen-Yangand kidney-yang, deficiency Qi and blood stasis syndrome were selected andrandomly divided into treating group（n=32） and control group（n=26）. 2 groupswere given the same basal treatment including education course for diabetes,diabetic diet, and hypoglycemic and hypotension agent in routine. Patientsin the control group attended to the fosinopril（5～10mg, per os）, once a day.And to those in the treating group, WIRA therapy was given additionally, onedose taken in twice a day. After 1 month’s treatment, the curative effectand Chinese medical syndromes were observed. Moreover, fasting bloodglucose（FBG）, post-prandial 2-hour blood glucose（P2hBG）, blood lipid, urinaryalbumin excretion rate（UAE） and serum IL-6, and TNF-αwere measured andcompared before and after treatment.2. In experimental study, SPF male Sprague Dawley （SD） rats were selected,The diabetic model rats were made by a single intraperitoneal injection of60mg/kg streptozotocin（STZ）. The rats were randomly assigned to four groups: normal group, model （early-stage DN）group, western medicine（WM） group withfosinopril(4mg·kg^-1·d^-1 by garage) and traditional Chinese medicine（TCM）group with WIRA complex prescription (20g·kg^-1·d^-1 by garage). The courseof treatment lasted 6weeks.The rats general status（psychosis, diet, haircolor, weight amplitude, etc）, renal weight/body weight ratio, blood glucose,24hUAE, triglyceride（TG）, total cholesterol（TC）, serum creatinine（SCr）,blood urea nitrogen（BUN）. The serum IL-6, and TNF-αwere measured of ratsby enzyme linked immunosorbent assay（ELISA）, and renal pathologic lesion wereobserved through light microscopy and electron microscopy. The expression oftransforming growth factor-β₁（TGF-β₁） in renal cortex of each group wereobserved by immunohistochemicl staining.Results1. Based on controlling the blood glucose availability, the total ratioof effective of TCM group was 87.5%, western medicine group is 61.5%. Comparedwith two groups, TCM group is obviously excel to western medicine group （P＜0.05）. The clinical symptom score, FBG, P₂hBG and 24hUAE lowered as well asimproved the blood lipid in both groups. In TCM group, the clinical symptomscore, 24hUAE, TC, TG, LDL-C, TNF-αand IL-6 significantly reduced, the effectwas better than that in the WM group（P＜0.05 or P＜0.01）. There are no toxicityand side-effect in tow groups.2. The model group rats’ symptoms are energy cachexia, drinking waterincreased, urinate manifold and loose stool, lowness reaction, slow action,hair stand and lackluster. Renal slices of model group rats can be seen amajority of glomerulus increasing, swelling, expanding of capillary lumen andred blood cell depositing in it. Inflammatory cell infiltrating and mesangialwidth increasing, glomerular basement membrane（GBM） incrassation. There havenot glomerular sclerosis obviously.3. Compared with normal group, the body weight of model rats was relievedobviously（P＜0.01）, Ratio of kidney weight （KW） to Body weight（BW）, bloodglucose 24Huae, TC, TG, TNF-αand IL-6 significantly increased obviously（P＜0.05 or P＜0.01）. the urinary volume and UAE in TCM group and WM group weredecreased, and there was better effective in TCM group. Compared with modelgroup, blood glucose, TC, TG, TNF-αand IL-6 were decreased significantlyin TCM group（P＜0.05 or P＜0.01）, and there was no marked change in bloodglucose, TC, TG, TNF-αand IL-6 in WM group. 4. Compared with control group, the expression of TGF-β₁ in renal cortexof model group rats were significantly increased, espcialy in the renal tubuleand glomerular epithelium, which were all significantly inhibited by WIRAcomplex prescription. The effect of suppression over-expression of TGF-β₁ ofrenal cortex in TCM group was better than that of WM group（P＜0.05）.Conclusions:1. YangQi of human body is deficient easy, and the deficiency of YangQican also incur to diabetes. Deficiency of spleen-Yang（Qi） and kidney-yang（Qi）is the onset-base of early stage DN, and internus toxicity was the importantfactor of early-stage DN. We believe that deficiency of spleen-Yang（Qi） andkidney-Yang（Qi）, internus toxicity is the main pathogenesis of early-stageDN, so warming kidney and invigorating spleen, removing toxicity andactivating blood （WIRA）is the main method in curing of early-stage DN.2. The method of WIRA has better function of therapy and protection ofrelieving clinical symptom and reducing UAE of patients with early-stage DN.3. The method of WIRA has a certain effect on reducing blood glucose,regulating serum blood lipid metabolism reducing the urinary protein excretion,ameliorating pathologic change of nephridial tissue and inhibiting renalhypertrophy, mesenterium cell proliferation and thickening of GBM inexperimental diabetic rats obviously, it can also significantly inhibitedover-expression of TGF-β₁ of renal cortex.4. The method of WIRA has certain effect of anti-inflammatory onearly-stage DN, it can reduce serum TNF-αand IL-6 significantly. Thetherapeutic mechanism for early-stage DN is not depend on the controlling bloodglucose availability, but is probably pathway of relating to regulate to theproduction of inflammatory factors and anti-inflammatory reaction. It canregulate organism all round through neuro-endocrinc-immunomodulation network,and block up inflammatory factor TNF-α、IL-6 and their receptors, accordinglyregulate the expression of growth factors and cell adhesionmolecule,andinhibit multiplication of glomerular mesangium and production of ECM.更多还原