益气养阴活血通络方对糖尿病肾病大鼠足细胞的影响
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摘要
目的：糖尿病肾病(diabetic nephropathy, DN)是糖尿病(diabetes mellitus,DM)最常见的慢性微血管并发症之一，也是导致终末期肾病最主要的原因之一，临床上以蛋白尿为主要表现。1型和2型DM均可发生DN，严重影响DM患者的生存质量和预后，因此，探讨DN的发病机制，寻求防治DN的有效方法具有重要意义。DN的发生发展是多种因素综合作用的结果，具有复杂的病理机制，尽管国内外学者不断探索DN的防治方法，但是到目前为止，仍然缺乏防治DN的特效方法。而中医药以其独特的辨证论治体系，多靶点的整体调节功能，以及三因制宜的个性化治疗方案，在临床防治DN中显示出巨大的潜力和广阔的应用前景。课题组将中医学的病因病机理论，尤其是络病理论，与多年的临床实践经验相结合，总结DN的基本病机为气阴两虚、血瘀阻络，选用黄芪、地黄、黄精、丹参、川芎、水蛭、地龙、全蝎等配伍组成益气养阴活血通络方，临床和早期实验表明该方治疗DN效果确切。本研究以高脂饲料饲养联合小剂量链脲佐菌素(STZ)腹腔注射复制DN大鼠模型，以益气养阴活血通络方对其干预，试图以足细胞为切入点，进一步验证该方的疗效，探讨该方对DN的作用机制，并比较该方与益气养阴活血方和益气养阴通络方的作用差别，探讨该方的组方配伍机制，以效测证，探讨DN的中医证候，为临床应用提供依据。
方法：
1益气养阴活血通络方对DN大鼠的肾保护作用
健康雄性4～5周龄体重120g～150g SD大鼠55只，适应性饲养1周后随机选取10只作为正常对照组(normal control group, NG)，其余大鼠给予高脂饲料饲养。12周后高脂饲料饲养大鼠按30mg/kg腹腔注射STZ，NG注射相应体积枸橼酸缓冲液。72h后空腹血糖(fasting blood glucose,FBG)≥16.7mmol/L为DM造模成功。成模大鼠按体重随机分为模型组(model group, MG)、益气养阴活血通络方治疗组(yiqi-yangyin-huoxue-tong-luo recipe ttreated group, YHTG)、益气养阴活血方治疗组(yiqi-yangyin-huo-xue recipe treated group, YHG)、益气养阴通络方治疗组(yiqi-yangyin-tong-luo recipe treated group, YTG)各10只，YHTG给予益气养阴活血通络方由(黄芪、地黄、丹参、川芎各2袋，黄精、地龙、水蛭、全蝎各1袋中药配方颗粒组成)1.00g/kg(相当于生药5.51g/kg)、YHG给予益气养阴活血方(由黄芪、地黄、丹参、川芎各2袋，黄精1袋中药配方颗粒组成)0.92g/kg(相当于生药4.65g/kg)、YTG给予益气养阴通络方(由黄芪、地黄各2袋，黄精、地龙、水蛭、全蝎各1袋中药配方颗粒组成)0.79g/kg(相当于生药3.87g/kg)，给药量按大鼠与人体型系数折算。NG及MG给予相应的凉开水，日1次灌胃，共32周。于给药第2周末(2W)、4W、8W、16W、32W代谢笼收集24小时尿液，测量大鼠的饮水量、饲料消耗量、尿量、体重，记录24h尿量，以3500转/分离心10分钟，取上清，-20℃保存。32W同时留取随机尿标本。ELISA法检测尿白蛋白浓度，尾静脉取血测FBG和糖化血红蛋白(HbA1c)后，水合氯醛麻醉大鼠，股动脉取血，分离血清，检测血肌酐(Scr)、尿肌酐、血脂三项(总胆固醇TC、甘油三酯TG、高密度脂蛋白胆固醇HDL)，称量肾脏重量。计算内生肌酐清除率(Ccr)、尿白蛋白排泄率(ACR)、肾脏指数(KI)、低密度脂蛋白胆固醇(LDL)。留取肾皮质标本，行光镜和透射电镜检查，依据光镜切片测得的肾小球面积计算肾小球体积，从透射电镜图片上测量肾小球基底膜厚度。
2益气养阴活血通络方对DN大鼠nephrin的影响
ELISA法检测2W、8W、16W、32W24h尿白蛋白(U-alb)、24h尿nephrin(U-nephrin)和32W肾组织匀浆nephrin水平(K-nephrin)，分析U-alb和U-nephrin的相关性，以及32W U-alb、U-nephrin和K-nephrin的相关性，反转录酶多聚酶链反应(RT-PCR)检测肾皮质nephrin mRNA的表达。
3益气养阴活血通络方对DN大鼠α-辅肌动蛋白-4(α-actinin-4)和足细胞密度的影响
Western-blot和免疫组织化学法检测肾皮质α-actinin-4蛋白的表达，免疫组织化学法采用WT1C-末端标记足细胞核，计数单个肾小球内足细胞核数目，测量相应肾小球面积，计算足细胞密度，分析24h尿蛋白(U-pro)、α-actinin-4表达、足细胞密度之间的相关性。
4统计学方法
所有资料以±s表示，重复测量指标采用重复测量设计资料的单变量方差分析进行比较，各观察点进行组间两两比较；两组间比较采用t检验；多组间比较采用One-WayANOVA进行分析，两两组间比较采用LSD进行分析；变量间的相关性采用线性回归分析。所有数据分析应用SPSS16.0进行，以P<0.05为差异有显著性。
结果：
1益气养阴活血通络方对DN大鼠的肾保护作用
1.1各组大鼠不同观察点FBG的比较
与NG比较，MG和各治疗组FBG显著升高(P<0.05)；与MG比较，YHTG16W开始FBG升高显著改善(P<0.05)，YHG32W时FBG升高显著改善(P<0.05)，YTG FBG升高改善不明显(P>0.05)；治疗组间比较，各观察点FBG无显著性差异(P>0.05)。
1.2各组大鼠不同观察点饮水量的比较
与NG比较，MG和各治疗组饮水量显著增加(P<0.05)；与MG比较，YHTG2W开始饮水量增加显著改善(P<0.05)，YHG除4W、8W外其余观察点饮水量增加显著改善(P<0.05)，YTG从16W开始，饮水量增加显著改善(P<0.05)；治疗组间比较，各观察点饮水量无显著性差异(P>0.05)。
1.3各组大鼠不同观察点饲料消耗量的比较
与NG比较，MG和各治疗组饲料消耗量显著增加(P<0.05)；与MG比较，YHTG16W始饲料消耗量增加显著改善，YHG和YTG32W时饲料消耗量增加显著改善(P<0.05)；治疗组间比较，各观察点饲料消耗量无显著性差异(P>0.05)。
1.4各组大鼠不同观察点尿量的比较
与NG比较，MG和各治疗组尿量显著增加(P<0.05)；与MG比较，各治疗组尿量增加2W开始显著改善(P<0.05)；治疗组间比较，从8W开始，YHTG尿量显著少于YHG和YTG(P<0.05)。
1.5各组大鼠不同观察点体重的比较
造模前，高脂饲料组大鼠体重显著高于NG(P<0.05)；造模后，NG体重呈逐渐增加趋势，MG和各治疗组体重则逐渐下降；YHTG8W始，YHG和YTG32W时体重显著高于MG(P<0.05)；治疗组间比较，各观察点大鼠体重无显著性差异(P>0.05)。
1.632W各组大鼠HbA1c、KI、Ccr、Scr、ACR的比较
与NG相比，MG HbA1c、KI、Ccr、Scr、ACR显著升高(P<0.05)，各治疗组除Scr、YHG HbA1c、YTG KI外，其余指标也显著升高(P<0.05)；与MG相比，除YTG HbA1c下降不显著(P>0.05)外，各治疗组HbA1c、Ccr、Scr、ACR显著下降(P<0.05)，KI下降不明显(P>0.05)；治疗组间比较，YHTG对Ccr和ACR的影响大于YHG和YTG(P<0.05)，对HbA1c、KI和Scr的影响，治疗组间比较，差异不显著(P>0.05)。
1.7各组大鼠血脂四项(TC, TG, HDL, LDL)的比较
与NG比较，MG血脂四项显著升高(P<0.05)，各治疗组除TG升高不明显(P>0.05)外，TC、 HDL、LDL也显著升高(P<0.05)；与MG比较，各治疗组血脂四项升高显著改善(P<0.05)；治疗组间比较，血脂四项无显著差异(P>0.05)。
1.8各组大鼠肾小球体积的比较
与NG比较，MG和各治疗组肾小球体积显著增大(P<0.05)；与MG比较，各治疗组肾小球体积增大显著减轻(P<0.05)；治疗组间比较，肾小球体积无显著差异(P>0.05)。
1.9各组大鼠肾小球基底膜厚度的比较
与NG比较，MG和各治疗组肾小球基底膜厚度显著增厚(P<0.05)；与MG比较，各治疗组肾小球基底膜厚度增厚显著减轻(P<0.05)；治疗组间比较，肾小球基底膜厚度无显著差异(P>0.05)。
2益气养阴活血通络方对糖尿病肾病大鼠nephrin的影响
2.1各组大鼠不同观察点U-nephrin的比较
与NG比较， MG各观察点、各治疗组2W、8W U-nephrin显著增加(P<0.05)；与MG比较，各观察点各治疗组U-nephrin增加显著改善(P<0.05)；治疗组间比较，各观察点U-nephrin无显著性差异(P>0.05)。
2.2各组大鼠不同观察点U-alb的比较
与NG比较，各观察点MG和各治疗组U-alb显著增加(P<0.05)；与MG比较，YHTG从2W始、YHG和YTG从8W始，U-alb增加显著改善(P<0.05)；治疗组间比较，2W、8W时YHTG U-alb显著低于YHG和YTG(P<0.05)。
2.3MG不同观察点U-alb和U-nephrin相关性分析
在各观察点，U-alb和U-nephrin呈正相关。
2.4各组大鼠K-nephrin的比较
与NG比较，MG和YHG K-nephrin显著下降(P<0.05)，YHTG和YTGK-nephrin下降不明显(P>0.05)；与MG比较，各治疗组K-nephrin下降明显改善(P<0.05)；治疗组间比较，K-nephrin无显著差异(P>0.05)。
2.5MG大鼠32W末U-alb与U-nephrin、K-nephrin的相关性分析
U-alb和U-nephrin呈正相关(r=0.828,P=0.006)，U-alb和K-nephrin呈负相关(r=-0.845,P=0.004)，U-nephrin和K-nephrin呈负相关(r=-0.991,P=0.000)。
2.6RT-PCR检测各组大鼠肾皮质nephrin mRNA表达
与NG比较，MG和各治疗组nephrin mRNA表达显著下降(P<0.05)；与MG比较，各治疗组nephrin mRNA表达下降显著改善(P<0.05)；治疗组间比较，nephrin mRNA表达无显著差异(P>0.05)。
3益气养阴活血通络方对糖尿病肾病大鼠α-actinin-4蛋白表达及足细胞密度的影响
3.1Western-blot检测大鼠肾皮质α-actinin-4的表达
与NG比较，MG、YHG和YTG肾皮质α-actinin-4的表达显著下降(P<0.05)，YHTG肾皮质α-actinin-4的表达下降不明显(P>0.05)；与MG比较，各治疗组肾皮质α-actinin-4的表达减弱显著改善(P<0.05)；治疗组间比较，YHTG大鼠肾皮质α-actinin-4蛋白的表达明显高于YHG和YTG(P<0.05)。
3.2免疫组织化学法检测大鼠肾皮质α-actinin-4的表达
与NG比较，MG、YHG和YTG肾皮质α-actinin-4的表达显著下降(P<0.05)，YHTG肾皮质α-actinin-4的表达下降不明显(P>0.05)；与MG比较，各治疗组肾皮质α-actinin-4的表达减弱显著改善(P<0.05)；治疗组间比较，YHTG大鼠肾皮质α-actinin-4蛋白的表达明显高于YHG和YTG(P<0.05)。
3.3各组大鼠足细胞密度的比较
与NG比较，MG和各治疗组足细胞密度显著减小(P<0.05)；与MG比较，各治疗组足细胞密度减小显著改善(P<0.05)；治疗组间比较，足细胞密度无显著差异(P>0.05)。
3.4各组大鼠U-pro的比较
与NG比较，MG和各治疗组U-pro显著增加(P<0.05)；与MG比较，各治疗组U-pro增加显著改善(P<0.05)；治疗组间比较，YHTG U-pro显著低于YHG和YTG(P<0.05)。
3.5MG大鼠U-pro、肾皮质α-actinin-4/β-actin、足细胞密度的相关性分析
U-pro和α-actinin-4/β-actin呈负相关(r=-0.846,P=0.004)；U-pro和足细胞密度呈负相关(r=-0.844,P=0.002)；足细胞密度和α-actinin-4/β-actin呈正相关(r=0.815,P=0.007)。
结论：
1益气养阴活血通络方、益气养阴活血方和益气养阴通络方能显著减轻DN大鼠“三多一少”表现，改善DN大鼠脂代谢紊乱，降低DN大鼠ACR、U-pro和肾小球高滤过，减轻肾小球肥大和肾小球基底膜增厚，具有肾保护作用；
2益气养阴活血通络方、益气养阴活血方可显著改善DN大鼠糖代谢紊乱，益气养阴通络方对糖代谢紊乱改善不显著；益气养阴活血通络方、益气养阴活血方、益气养阴通络方可减少DN大鼠尿nephrin的排出、抑制肾皮质nephrin蛋白和基因表达的下调、改善α-actinin-4蛋白的下调、减轻足细胞密度的下降，对DN足细胞有保护作用；
3益气养阴活血通络方在降低ACR、减少U-pro、减少尿量、改善肾小球高滤过等方面明显优于益气养阴活血方和益气养阴通络方，可能与其改善α-actinin-4蛋白的下调优于益气养阴活血方和益气养阴通络方有关。
Objective: Diabetic nephropathy(DN) is one of popuar chronicmicrovascular complications of diabetes mellitus(DM), and also one of maincauses leading to end stage renal diaease. Proteinuria is main clinicalcharacteristic. Both type1DM and type2DM may develop into DN. DNeffects diabetic patients’ quality of live and prognosis severely. So, itsimportant to probe the pathogenesis of DN and seek effective method toprevent or treat DN. The onset and development of DN is the result of manyfactors, with complicated pathological machanism. Despite home and abroadmedical specialists expored pathogenesis and effective method to prevent ortreat DN ceaselessly, there are lack of effective method up to now. Whereas,Traditional Chinese Medicine(TCM) has showed huge capacity and widelyapplied foreground, because of its special syndrome differentiation andtreatment theoretical system, multi-targets’ entire regulation, andpersonalized treatment protocol. We combined TCM’s theory of pathogen andpathogenesis especially collateral disease theory with long-term clinicalpractise, concluded deficiency of qi and yin, stasis obstructing collaterals asbasic pathogenesis of DN, and chose Huangqi, Dihuang, huangjing, Danshen,Chuanxiong, Dilong, Shuizhi, Quanxie to organize yiqi-yangyin-huoxue-tong-luo recipe. Clinical and former studies showed the recipe had affirmativeeffects on DN. In the study, Diabetic model was reduced by high fat dietfeeding and low dose STZ ip. The model was intervened withyiqi-yangyin-huoxue-tongluo recipe. We aimed to probe the recipe’s actionmechanism further and verificate the therapeutic effects, by view of podocyte,and to compare the actional difference with yiqi-yangyin-huoxue recipe andyiqi-yangyin-tongluo recipe, as well as to explore organizational mechanism ofthe recipe, to probe the TCM essence of DN according to treated results, and to provide basis for clinical practice.
Methods:
1Renal protective effects of yiqi-yangyin-huoxue-tongluo recipe on DN rats
Healthy male4～5weeks old55SD rats with body weight120g～150gafter adaptive feeding for1week were grouped randomly. Ten rats werenormal control group (NG). Other45rats were fed with high fat dietthroughout the study. After12weeks, STZ30mg/kg was adminstrated to highfat diet fed rats intraperitoneal injection, and rats in NG were ipvehic.Seventy-two hours later, the rats whose fasting blood glucose(FBG)≥16.7mmol/L were indentified as diabetic model. The diabetic rats wererandomly grouped, i.e., model group(MG), yiqi-yangyin-huoxue-tongluorecipe treated group(YHTG), yiqi-yangyin-huoxue recipe treated group(YHG),yiqi-yangyin-tongluo recipe treated group (YTG),10rats respectively. YHTGaccepted yiqi-yangyin-huoxue-tongluo recipe(consisted each two packages ofdecoction-free herbal granules of Huang Qi, Di Huang, Chuan Xiong, DanShen, as well as each one package of Huang Jing, Di Long, Shui Zhi, andQuan Xie) at dose of1.00g/kg(eaqual to crude herbs5.51g/kg), and YHGaccepted yiqi-yangyin-huoxue recipe(consisted each two packages ofdecoction-free herbal granules of Huang Qi, Di Huang, Chuan Xiong, DanShen, as well as one package of Huang Jing) at dose of0.92g/kg(eaqual tocrude herbs4.65g/kg), while YTG accepted yiqi-yangyin-tongluo recipe(consisted each two packages of decoction-free herbal granules of Huang Qi,Di Huang, as well as each one package of Huang Jing, Di Long, Shui Zhi, andQuan Xie)at dose of0.79g/kg(eaqual to crude herbs3.87g/kg). The dose wasconverted according to body type coefficient of rat to human. NG and MGwere given diluent. All rats were intragastric administrated once daily for32weeks consecutively. At the end of2weeks(2W),4W,8W,16W, and32W, ratswere placed into metabolic cages for24hours to collected24h urine. Rats′water intake, diet intake, urine output, and body weight were measured. At theend of the study, point urine samples were collected too. Urine samples werecentrifuged at speed of3500rpm for10minutes, and supernate of samples were stored at-20℃. Urinary albumin concentration was measured by ELISAmethod. FBG and HbA1c were measured by sampling from tail vein, then ratswere anesthesiaed with chloraldurate. Blood sample was collected fromfemoral artery, and serum was separated. Serum creatinine, urinary creatinine,serum lipid (including total choleaterol TC, triglyceride TG, high density lipidcholeaterol HDL) were measured by full-automatic biochemistry checker.Renal weight was weighed. Endogenous creatinine clearance rate (Ccr),urinealbumin to creatinine ratio(ACR), kidney index(KI), and low density lipidcholeaterol (LDL) were calculated. Renal cortex samples were stored for rulemicroscopy or transmission electron microscopy. Glomerular volume wascalculated by glomerular area on base of microscopes, and glomerularbasement menbrane thickness was measured by transmission electronmicroscopes.
2Effects of yiqi-yangyin-huoxue-tongluo recipe on nephrin in DN rats
Urine albumin of24h(U-alb) and urinary nephrin of24h(U-nephrin) for2W,8W,16W, and32W, as well as nephrin of kidney cortex homogenate(K-nephrin) by the end of study were measured by ELISA method. Correlationbetween U-alb and U-nephrin, as well as correlation among U-alb, U-nephrin,and K-nephrin were analysed. Expression of renal cortex nephrin mRNA wasinvestigated by reverse transcriptase polymerse chain reaction(RT-PCR).
3Effects of yiqi-yangyin-huoxue-tongluo recipe on α-actinin-4and podocytedensity in DN rats
Expression of renal cortex α-actinin-4protein was investigated byWestern-blot and immunohistochemical method. Podocytic nuclei werestained by C-terminal antibody of WT1using immunohistochemical method,and glomerular area was measured,then podocyte density was calculated.Correlation among24h urinary protein(U-pro), α-actinin-4/β-actin, andpodocyte density were analysed.
4Statistical analysis
All results were expressed as±s. Repeated measures and multivariateanalysis of variance (ANOVA) process of the general linear model was used to identify comparison among different groups and different measure timepairwise; t-test was used to compare mean values between two groups;One-Way ANOVA was used to compare mean values among groups, and LSDto compare mean values between each two group; Univariate linear regressionanalysis was used to evaluate the relationships among the variables. Analysiswas carried out using SPSS16.0. P-value of <0.05was considered to bestatistically significant.
Results:
1Renal protective effects of yiqi-yangyin-huoxue-tongluo recipe on DN rats
1.1Comparison of FBG in each group at diffent checktime
Compared with NG, FBG in MG and all treated groups increasednotably(P<0.05) at diffent checktime. Compared with MG, high FBG inYHTG improved notably from16W(P<0.05), while high FBG in YHGimproved notably at the end of32W(P<0.05), but high FBG in YTG didn′timprove notably(P>0.05). Compared among all treated groups, there was nosignificant difference of FBG in all treated groups at diffentchecktime(P>0.05).
1.2Comparison of water intake in each group at diffent checktime
Compared with NG, water intake in MG and all treated groups increasednotably (P<0.05) at diffent checktime. Compared with MG, polydipsia inYHTG improved notably from2W(P<0.05), and polydipsia in YHG improvednotably(P<0.05) except for4W and8W, while polydipsia in YTG improvednotably from16W(P<0.05). Compared among all treated groups, there was nosignificant difference of water intake in all treated groups at diffentchecktime(P>0.05).
1.3Comparison of diet intake in each group at diffent checktime
Compared with NG, diet intake in MG and all treated groups increasednotably(P<0.05) at diffent checktime. Compared with MG, polyphagia inYHTG improved notably from16W(P<0.05), while polyphagia in YHG andYTG improved notably at the end of32W(P<0.05). Compared among alltreated groups, there was no significant difference of diet intake in all treated groups at diffent checktime(P>0.05).
1.4Comparison of urine output in each group at diffent checktime
Compared with NG, urine output in MG and all treated groups increasednotably(P<0.05) at diffent checktime. Compared with MG, polyuria in alltreated groups improevd notably from2W(P<0.05). Compared among alltreated groups, urine output in YHTG was less than YHG and YTG from8W(P<0.05).
1.5Comparison of body weight in each group at diffent checktime
Pre-modeling, body weight in high fat diet feeding rats was higher thanthat of NG(P<0.05). Post-modeling, body weight in NG increased gradually,but body weight in MG and all treated groups decreased gradually. Bodyweight in YHTG from8W, YHG and YTG at the end of32W was higher thanthat in MG(P<0.05). Compared among all treated groups, there was nosignificant difference of body weight in all treated groups at diffentchecktime(P>0.05).
1.6Comparison of HbA1c, KI, Ccr, Scr, and ACR in each group at32W
Compared with NG, HbA1c, KI, Ccr, Scr, and ACR in MG increasednotably (P<0.05), except for Scr, HbA1c in YHG, KI in YTG, remain indicesin all treated groups also increased notably(P<0.05). Compared with MG,except for HbA1c in YTG, HbA1c, Ccr, Scr, and ACR in all treated groupsdecreased notably(P<0.05), KI didn′t decrease unnotably (P>0.05). Comparedamong all treated groups, Ccr and ACR in YHTG were lower than those inYHG and YTG(P<0.05), but, there was no significant difference of HbA1c, KI,and Scr in all treated groups (P>0.05).
1.7Comparison of serum lipid (including TC, TG, HDL, and LDL) in eachgroup
Compared with NG, serum lipid in MG increased notably(P<0.05), andTC, HDL, and LDL in all treated groups also increased notably (P<0.05), but,TG didn′t increased notably(P>0.05). Compared with MG, serum lipid in alltreated groups decreased notably (P<0.05). Compared among all treatedgroups, there was no significant difference of serum lipid in all treated groups(P>0.05).
1.8Comparison of glomerular volume in each group
Compared with NG, glomerular volume in MG and all treated groupsenlarged notably(P<0.05). Compared with MG, glomerular volume in alltreated groups didn′t enlarge notably (P>0.05). Compared among all treatedgroups, there was no significant difference of glomerular volume in all treatedgroups(P>0.05).
1.9Comparison of GBM thickness in each group
Compared with NG, GBM in MG and all treated groups thickenednotably(P<0.05). Compared with MG, GBM in all treated groups didn′tthicken notably (P>0.05). Compared among all treated groups, there was nosignificant difference of GBM thickness in all treated groups(P>0.05).
2Effects of yiqi-yangyin-huoxue-tongluo recipe on nephrin in DN rats
2.1Comparison of U-nephrin in each group at diffent checktime
Compared with NG, U-nephrin in MG at diffent checktime, and alltreated groups at end of2W, as well as8W increased notably(P<0.05).Compared with MG, increased U-nephrin in all treated groups improvednotably(P<0.05) at diffent checktime. Compared among all treated groups,there was no significant difference of U-nephrin in all treated groups at diffentchecktime(P>0.05).
2.2Comparison of U-alb in each group at diffent checktime
Compared with NG, U-alb in MG and all treated groups increasednotably(P<0.05) at diffent checktime. Compared with MG, increased U-alb inYHTG from2W, YHG and YTG from8W improved notably(P<0.05).Compared among all treated groups, U-alb in YHTG was lower than that inYHG and YTG at end of2W and8W(P<0.05).
2.3Correlation between U-alb and U-nephrin in MG at diffent checktime
There was a significant positive correlation between U-alb and U-nephrinat diffent checktime.
2.4Comparison of K-nephrin in each group
Compared with NG, K-nephrin in MG and YHG decreased notably (P<0.05), but K-nephrin in YHTG and YTG didn′t decrease notably (P>0.05).Compared with MG, decreased K-nephrin in all treated groups improvednotably(P<0.05). Compared among all treated groups, there was no significantdifference of K-nephrin in all treated groups(P>0.05).
2.5Correlation among U-alb, U-nephrin, and K-nephrin in MG at the end of32W
There was a significant positive correlation between U-alb andU-nephrin(r=0.828, P=0.006), but a negative correlation between U-alb andK-nephrin (r=-0.845, P=0.004), as well as K-nephrin and U-nephrin(r=-0.991,P=0.000).
2.6Expression of nephrin mRNA in renal cortex investigated by RT-PCR
Compared with NG, the expression of nephrin mRNA in MG and alltreated groups downregulated notably (P<0.05). Compared with MG,downregulated expression of nephrin mRNA in all treated groups improvednotably(P<0.05). Compared among all treated groups, there was no significantdifference of the expression of nephrin mRNA in all treated groups(P>0.05).
3Effects of yiqi-yangyin-huoxue-tongluo recipe on the expression ofα-actinin-4and podocyte density in DN rats
3.1The expression of α-actinin-4in renal cortex investigated by Western-blot
Compared with NG, the expression of α-actinin-4in MG, YHG, and YTGdownregulated notably (P<0.05), but in YHTG didn′t downregulate notably(P>0.05). Compared with MG, downregulated expression of α-actinin-4in alltreated groups improved notably(P<0.05). Compared among all treated groups,the expression of α-actinin-4in YHTG was higher than that in YHG andYTG(P<0.05).
3.2The expression of α-actinin-4in renal cortex investigated by immunohisto-chemistry
Compared with NG, the expression of α-actinin-4in MG, YHG, and YTGdownregulated notably (P<0.05), but in YHTG didn′t downregulate notably(P>0.05). Compared with MG, downregulated expression of α-actinin-4in alltreated groups improved notably(P<0.05). Compared among all treated groups, the expression of α-actinin-4in YHTG was higher than that in YHG andYTG(P<0.05).
3.3Comparison of podocyte density in each group
Compared with NG, podocyte density in MG and all treated groupsdecreased notably(P<0.05). Compared with MG, decreased podocyte densityin all treated groups improved notably(P<0.05). Compared among all treatedgroups, there was no significant difference of podocyte density in all treatedgroups(P>0.05).
3.4Comparison of U-pro in each group
Compared with NG,U-pro in MG and all treated groups increased notably(P<0.05). Compared with MG, increased U-pro in all treated groups improvednotably(P<0.05). Compared among all treated groups, U-pro in YHTG waslower than that in YHG and YTG(P<0.05).
3.5Correlation among U-pro, α-actinin-4/β-actin, and podocyte density inMG
There was a significant negative correlation between U-pro andα-actinin-4/β-actin (r=-0.846,P=0.004), as well as U-pro and podocytedensity(r=-0.844,P=0.002), but a positive correlation between podocytedensity and α-actinin-4/β-actin (r=0.815,P=0.007).
Conclusions:
1Yiqi-yangyin-huoxue-tongluo recipe, yiqi-yangyin-huoxue recipe, andyiqi-yangyin-tongluo recipe can attenuate polydipsia, polyphagia, polyuria,and body weight reduction notably, as well as metabolic disorder of lipid, andcan decrease ACR and U-pro, attenuate glomerular hyrerfiltration, glomerularhypertrophy, thickened GBM, so,they have a renal protective role;
2Yiqi-yangyin-huoxue-tongluo recipe and yiqi-yangyin-huoxue recipe canattenuate metabolic disorder of sugar, but yiqi-yangyin-tongluo recipe can not;Yiqi-yangyin-huoxue-tongluo recipe, yiqi-yangyin-huoxue recipe, andyiqi-yangyin-tongluo recipe can decrease U-nephrin, attenuate downregulationof K-nephrin protein and mRNA, attenuate downregulation of α-actinin-4protein, lessen podocyte density reduction notably, so, they have a podocytic protective role;
3As for amelioration ACR, U-pro, polyuria, and glomerular hyperfiltration,yiqi-yangyin-huoxue-tongluo recipe is better than YHG and YTG, which maybe related to yiqi-yangyin-huoxue-tongluo recipe improved thedownregulation of α-actinin-4more notably.
方法：
1益气养阴活血通络方对DN大鼠的肾保护作用
健康雄性4～5周龄体重120g～150g SD大鼠55只，适应性饲养1周后随机选取10只作为正常对照组(normal control group, NG)，其余大鼠给予高脂饲料饲养。12周后高脂饲料饲养大鼠按30mg/kg腹腔注射STZ，NG注射相应体积枸橼酸缓冲液。72h后空腹血糖(fasting blood glucose,FBG)≥16.7mmol/L为DM造模成功。成模大鼠按体重随机分为模型组(model group, MG)、益气养阴活血通络方治疗组(yiqi-yangyin-huoxue-tong-luo recipe ttreated group, YHTG)、益气养阴活血方治疗组(yiqi-yangyin-huo-xue recipe treated group, YHG)、益气养阴通络方治疗组(yiqi-yangyin-tong-luo recipe treated group, YTG)各10只，YHTG给予益气养阴活血通络方由(黄芪、地黄、丹参、川芎各2袋，黄精、地龙、水蛭、全蝎各1袋中药配方颗粒组成)1.00g/kg(相当于生药5.51g/kg)、YHG给予益气养阴活血方(由黄芪、地黄、丹参、川芎各2袋，黄精1袋中药配方颗粒组成)0.92g/kg(相当于生药4.65g/kg)、YTG给予益气养阴通络方(由黄芪、地黄各2袋，黄精、地龙、水蛭、全蝎各1袋中药配方颗粒组成)0.79g/kg(相当于生药3.87g/kg)，给药量按大鼠与人体型系数折算。NG及MG给予相应的凉开水，日1次灌胃，共32周。于给药第2周末(2W)、4W、8W、16W、32W代谢笼收集24小时尿液，测量大鼠的饮水量、饲料消耗量、尿量、体重，记录24h尿量，以3500转/分离心10分钟，取上清，-20℃保存。32W同时留取随机尿标本。ELISA法检测尿白蛋白浓度，尾静脉取血测FBG和糖化血红蛋白(HbA1c)后，水合氯醛麻醉大鼠，股动脉取血，分离血清，检测血肌酐(Scr)、尿肌酐、血脂三项(总胆固醇TC、甘油三酯TG、高密度脂蛋白胆固醇HDL)，称量肾脏重量。计算内生肌酐清除率(Ccr)、尿白蛋白排泄率(ACR)、肾脏指数(KI)、低密度脂蛋白胆固醇(LDL)。留取肾皮质标本，行光镜和透射电镜检查，依据光镜切片测得的肾小球面积计算肾小球体积，从透射电镜图片上测量肾小球基底膜厚度。
2益气养阴活血通络方对DN大鼠nephrin的影响
ELISA法检测2W、8W、16W、32W24h尿白蛋白(U-alb)、24h尿nephrin(U-nephrin)和32W肾组织匀浆nephrin水平(K-nephrin)，分析U-alb和U-nephrin的相关性，以及32W U-alb、U-nephrin和K-nephrin的相关性，反转录酶多聚酶链反应(RT-PCR)检测肾皮质nephrin mRNA的表达。
3益气养阴活血通络方对DN大鼠α-辅肌动蛋白-4(α-actinin-4)和足细胞密度的影响
Western-blot和免疫组织化学法检测肾皮质α-actinin-4蛋白的表达，免疫组织化学法采用WT1C-末端标记足细胞核，计数单个肾小球内足细胞核数目，测量相应肾小球面积，计算足细胞密度，分析24h尿蛋白(U-pro)、α-actinin-4表达、足细胞密度之间的相关性。
4统计学方法
所有资料以±s表示，重复测量指标采用重复测量设计资料的单变量方差分析进行比较，各观察点进行组间两两比较；两组间比较采用t检验；多组间比较采用One-WayANOVA进行分析，两两组间比较采用LSD进行分析；变量间的相关性采用线性回归分析。所有数据分析应用SPSS16.0进行，以P<0.05为差异有显著性。
结果：
1益气养阴活血通络方对DN大鼠的肾保护作用
1.1各组大鼠不同观察点FBG的比较
与NG比较，MG和各治疗组FBG显著升高(P<0.05)；与MG比较，YHTG16W开始FBG升高显著改善(P<0.05)，YHG32W时FBG升高显著改善(P<0.05)，YTG FBG升高改善不明显(P>0.05)；治疗组间比较，各观察点FBG无显著性差异(P>0.05)。
1.2各组大鼠不同观察点饮水量的比较
与NG比较，MG和各治疗组饮水量显著增加(P<0.05)；与MG比较，YHTG2W开始饮水量增加显著改善(P<0.05)，YHG除4W、8W外其余观察点饮水量增加显著改善(P<0.05)，YTG从16W开始，饮水量增加显著改善(P<0.05)；治疗组间比较，各观察点饮水量无显著性差异(P>0.05)。
1.3各组大鼠不同观察点饲料消耗量的比较
与NG比较，MG和各治疗组饲料消耗量显著增加(P<0.05)；与MG比较，YHTG16W始饲料消耗量增加显著改善，YHG和YTG32W时饲料消耗量增加显著改善(P<0.05)；治疗组间比较，各观察点饲料消耗量无显著性差异(P>0.05)。
1.4各组大鼠不同观察点尿量的比较
与NG比较，MG和各治疗组尿量显著增加(P<0.05)；与MG比较，各治疗组尿量增加2W开始显著改善(P<0.05)；治疗组间比较，从8W开始，YHTG尿量显著少于YHG和YTG(P<0.05)。
1.5各组大鼠不同观察点体重的比较
造模前，高脂饲料组大鼠体重显著高于NG(P<0.05)；造模后，NG体重呈逐渐增加趋势，MG和各治疗组体重则逐渐下降；YHTG8W始，YHG和YTG32W时体重显著高于MG(P<0.05)；治疗组间比较，各观察点大鼠体重无显著性差异(P>0.05)。
1.632W各组大鼠HbA1c、KI、Ccr、Scr、ACR的比较
与NG相比，MG HbA1c、KI、Ccr、Scr、ACR显著升高(P<0.05)，各治疗组除Scr、YHG HbA1c、YTG KI外，其余指标也显著升高(P<0.05)；与MG相比，除YTG HbA1c下降不显著(P>0.05)外，各治疗组HbA1c、Ccr、Scr、ACR显著下降(P<0.05)，KI下降不明显(P>0.05)；治疗组间比较，YHTG对Ccr和ACR的影响大于YHG和YTG(P<0.05)，对HbA1c、KI和Scr的影响，治疗组间比较，差异不显著(P>0.05)。
1.7各组大鼠血脂四项(TC, TG, HDL, LDL)的比较
与NG比较，MG血脂四项显著升高(P<0.05)，各治疗组除TG升高不明显(P>0.05)外，TC、 HDL、LDL也显著升高(P<0.05)；与MG比较，各治疗组血脂四项升高显著改善(P<0.05)；治疗组间比较，血脂四项无显著差异(P>0.05)。
1.8各组大鼠肾小球体积的比较
与NG比较，MG和各治疗组肾小球体积显著增大(P<0.05)；与MG比较，各治疗组肾小球体积增大显著减轻(P<0.05)；治疗组间比较，肾小球体积无显著差异(P>0.05)。
1.9各组大鼠肾小球基底膜厚度的比较
与NG比较，MG和各治疗组肾小球基底膜厚度显著增厚(P<0.05)；与MG比较，各治疗组肾小球基底膜厚度增厚显著减轻(P<0.05)；治疗组间比较，肾小球基底膜厚度无显著差异(P>0.05)。
2益气养阴活血通络方对糖尿病肾病大鼠nephrin的影响
2.1各组大鼠不同观察点U-nephrin的比较
与NG比较， MG各观察点、各治疗组2W、8W U-nephrin显著增加(P<0.05)；与MG比较，各观察点各治疗组U-nephrin增加显著改善(P<0.05)；治疗组间比较，各观察点U-nephrin无显著性差异(P>0.05)。
2.2各组大鼠不同观察点U-alb的比较
与NG比较，各观察点MG和各治疗组U-alb显著增加(P<0.05)；与MG比较，YHTG从2W始、YHG和YTG从8W始，U-alb增加显著改善(P<0.05)；治疗组间比较，2W、8W时YHTG U-alb显著低于YHG和YTG(P<0.05)。
2.3MG不同观察点U-alb和U-nephrin相关性分析
在各观察点，U-alb和U-nephrin呈正相关。
2.4各组大鼠K-nephrin的比较
与NG比较，MG和YHG K-nephrin显著下降(P<0.05)，YHTG和YTGK-nephrin下降不明显(P>0.05)；与MG比较，各治疗组K-nephrin下降明显改善(P<0.05)；治疗组间比较，K-nephrin无显著差异(P>0.05)。
2.5MG大鼠32W末U-alb与U-nephrin、K-nephrin的相关性分析
U-alb和U-nephrin呈正相关(r=0.828,P=0.006)，U-alb和K-nephrin呈负相关(r=-0.845,P=0.004)，U-nephrin和K-nephrin呈负相关(r=-0.991,P=0.000)。
2.6RT-PCR检测各组大鼠肾皮质nephrin mRNA表达
与NG比较，MG和各治疗组nephrin mRNA表达显著下降(P<0.05)；与MG比较，各治疗组nephrin mRNA表达下降显著改善(P<0.05)；治疗组间比较，nephrin mRNA表达无显著差异(P>0.05)。
3益气养阴活血通络方对糖尿病肾病大鼠α-actinin-4蛋白表达及足细胞密度的影响
3.1Western-blot检测大鼠肾皮质α-actinin-4的表达
与NG比较，MG、YHG和YTG肾皮质α-actinin-4的表达显著下降(P<0.05)，YHTG肾皮质α-actinin-4的表达下降不明显(P>0.05)；与MG比较，各治疗组肾皮质α-actinin-4的表达减弱显著改善(P<0.05)；治疗组间比较，YHTG大鼠肾皮质α-actinin-4蛋白的表达明显高于YHG和YTG(P<0.05)。
3.2免疫组织化学法检测大鼠肾皮质α-actinin-4的表达
与NG比较，MG、YHG和YTG肾皮质α-actinin-4的表达显著下降(P<0.05)，YHTG肾皮质α-actinin-4的表达下降不明显(P>0.05)；与MG比较，各治疗组肾皮质α-actinin-4的表达减弱显著改善(P<0.05)；治疗组间比较，YHTG大鼠肾皮质α-actinin-4蛋白的表达明显高于YHG和YTG(P<0.05)。
3.3各组大鼠足细胞密度的比较
与NG比较，MG和各治疗组足细胞密度显著减小(P<0.05)；与MG比较，各治疗组足细胞密度减小显著改善(P<0.05)；治疗组间比较，足细胞密度无显著差异(P>0.05)。
3.4各组大鼠U-pro的比较
与NG比较，MG和各治疗组U-pro显著增加(P<0.05)；与MG比较，各治疗组U-pro增加显著改善(P<0.05)；治疗组间比较，YHTG U-pro显著低于YHG和YTG(P<0.05)。
3.5MG大鼠U-pro、肾皮质α-actinin-4/β-actin、足细胞密度的相关性分析
U-pro和α-actinin-4/β-actin呈负相关(r=-0.846,P=0.004)；U-pro和足细胞密度呈负相关(r=-0.844,P=0.002)；足细胞密度和α-actinin-4/β-actin呈正相关(r=0.815,P=0.007)。
结论：
1益气养阴活血通络方、益气养阴活血方和益气养阴通络方能显著减轻DN大鼠“三多一少”表现，改善DN大鼠脂代谢紊乱，降低DN大鼠ACR、U-pro和肾小球高滤过，减轻肾小球肥大和肾小球基底膜增厚，具有肾保护作用；
2益气养阴活血通络方、益气养阴活血方可显著改善DN大鼠糖代谢紊乱，益气养阴通络方对糖代谢紊乱改善不显著；益气养阴活血通络方、益气养阴活血方、益气养阴通络方可减少DN大鼠尿nephrin的排出、抑制肾皮质nephrin蛋白和基因表达的下调、改善α-actinin-4蛋白的下调、减轻足细胞密度的下降，对DN足细胞有保护作用；
3益气养阴活血通络方在降低ACR、减少U-pro、减少尿量、改善肾小球高滤过等方面明显优于益气养阴活血方和益气养阴通络方，可能与其改善α-actinin-4蛋白的下调优于益气养阴活血方和益气养阴通络方有关。
Objective: Diabetic nephropathy(DN) is one of popuar chronicmicrovascular complications of diabetes mellitus(DM), and also one of maincauses leading to end stage renal diaease. Proteinuria is main clinicalcharacteristic. Both type1DM and type2DM may develop into DN. DNeffects diabetic patients’ quality of live and prognosis severely. So, itsimportant to probe the pathogenesis of DN and seek effective method toprevent or treat DN. The onset and development of DN is the result of manyfactors, with complicated pathological machanism. Despite home and abroadmedical specialists expored pathogenesis and effective method to prevent ortreat DN ceaselessly, there are lack of effective method up to now. Whereas,Traditional Chinese Medicine(TCM) has showed huge capacity and widelyapplied foreground, because of its special syndrome differentiation andtreatment theoretical system, multi-targets’ entire regulation, andpersonalized treatment protocol. We combined TCM’s theory of pathogen andpathogenesis especially collateral disease theory with long-term clinicalpractise, concluded deficiency of qi and yin, stasis obstructing collaterals asbasic pathogenesis of DN, and chose Huangqi, Dihuang, huangjing, Danshen,Chuanxiong, Dilong, Shuizhi, Quanxie to organize yiqi-yangyin-huoxue-tong-luo recipe. Clinical and former studies showed the recipe had affirmativeeffects on DN. In the study, Diabetic model was reduced by high fat dietfeeding and low dose STZ ip. The model was intervened withyiqi-yangyin-huoxue-tongluo recipe. We aimed to probe the recipe’s actionmechanism further and verificate the therapeutic effects, by view of podocyte,and to compare the actional difference with yiqi-yangyin-huoxue recipe andyiqi-yangyin-tongluo recipe, as well as to explore organizational mechanism ofthe recipe, to probe the TCM essence of DN according to treated results, and to provide basis for clinical practice.
Methods:
1Renal protective effects of yiqi-yangyin-huoxue-tongluo recipe on DN rats
Healthy male4～5weeks old55SD rats with body weight120g～150gafter adaptive feeding for1week were grouped randomly. Ten rats werenormal control group (NG). Other45rats were fed with high fat dietthroughout the study. After12weeks, STZ30mg/kg was adminstrated to highfat diet fed rats intraperitoneal injection, and rats in NG were ipvehic.Seventy-two hours later, the rats whose fasting blood glucose(FBG)≥16.7mmol/L were indentified as diabetic model. The diabetic rats wererandomly grouped, i.e., model group(MG), yiqi-yangyin-huoxue-tongluorecipe treated group(YHTG), yiqi-yangyin-huoxue recipe treated group(YHG),yiqi-yangyin-tongluo recipe treated group (YTG),10rats respectively. YHTGaccepted yiqi-yangyin-huoxue-tongluo recipe(consisted each two packages ofdecoction-free herbal granules of Huang Qi, Di Huang, Chuan Xiong, DanShen, as well as each one package of Huang Jing, Di Long, Shui Zhi, andQuan Xie) at dose of1.00g/kg(eaqual to crude herbs5.51g/kg), and YHGaccepted yiqi-yangyin-huoxue recipe(consisted each two packages ofdecoction-free herbal granules of Huang Qi, Di Huang, Chuan Xiong, DanShen, as well as one package of Huang Jing) at dose of0.92g/kg(eaqual tocrude herbs4.65g/kg), while YTG accepted yiqi-yangyin-tongluo recipe(consisted each two packages of decoction-free herbal granules of Huang Qi,Di Huang, as well as each one package of Huang Jing, Di Long, Shui Zhi, andQuan Xie)at dose of0.79g/kg(eaqual to crude herbs3.87g/kg). The dose wasconverted according to body type coefficient of rat to human. NG and MGwere given diluent. All rats were intragastric administrated once daily for32weeks consecutively. At the end of2weeks(2W),4W,8W,16W, and32W, ratswere placed into metabolic cages for24hours to collected24h urine. Rats′water intake, diet intake, urine output, and body weight were measured. At theend of the study, point urine samples were collected too. Urine samples werecentrifuged at speed of3500rpm for10minutes, and supernate of samples were stored at-20℃. Urinary albumin concentration was measured by ELISAmethod. FBG and HbA1c were measured by sampling from tail vein, then ratswere anesthesiaed with chloraldurate. Blood sample was collected fromfemoral artery, and serum was separated. Serum creatinine, urinary creatinine,serum lipid (including total choleaterol TC, triglyceride TG, high density lipidcholeaterol HDL) were measured by full-automatic biochemistry checker.Renal weight was weighed. Endogenous creatinine clearance rate (Ccr),urinealbumin to creatinine ratio(ACR), kidney index(KI), and low density lipidcholeaterol (LDL) were calculated. Renal cortex samples were stored for rulemicroscopy or transmission electron microscopy. Glomerular volume wascalculated by glomerular area on base of microscopes, and glomerularbasement menbrane thickness was measured by transmission electronmicroscopes.
2Effects of yiqi-yangyin-huoxue-tongluo recipe on nephrin in DN rats
Urine albumin of24h(U-alb) and urinary nephrin of24h(U-nephrin) for2W,8W,16W, and32W, as well as nephrin of kidney cortex homogenate(K-nephrin) by the end of study were measured by ELISA method. Correlationbetween U-alb and U-nephrin, as well as correlation among U-alb, U-nephrin,and K-nephrin were analysed. Expression of renal cortex nephrin mRNA wasinvestigated by reverse transcriptase polymerse chain reaction(RT-PCR).
3Effects of yiqi-yangyin-huoxue-tongluo recipe on α-actinin-4and podocytedensity in DN rats
Expression of renal cortex α-actinin-4protein was investigated byWestern-blot and immunohistochemical method. Podocytic nuclei werestained by C-terminal antibody of WT1using immunohistochemical method,and glomerular area was measured,then podocyte density was calculated.Correlation among24h urinary protein(U-pro), α-actinin-4/β-actin, andpodocyte density were analysed.
4Statistical analysis
All results were expressed as±s. Repeated measures and multivariateanalysis of variance (ANOVA) process of the general linear model was used to identify comparison among different groups and different measure timepairwise; t-test was used to compare mean values between two groups;One-Way ANOVA was used to compare mean values among groups, and LSDto compare mean values between each two group; Univariate linear regressionanalysis was used to evaluate the relationships among the variables. Analysiswas carried out using SPSS16.0. P-value of <0.05was considered to bestatistically significant.
Results:
1Renal protective effects of yiqi-yangyin-huoxue-tongluo recipe on DN rats
1.1Comparison of FBG in each group at diffent checktime
Compared with NG, FBG in MG and all treated groups increasednotably(P<0.05) at diffent checktime. Compared with MG, high FBG inYHTG improved notably from16W(P<0.05), while high FBG in YHGimproved notably at the end of32W(P<0.05), but high FBG in YTG didn′timprove notably(P>0.05). Compared among all treated groups, there was nosignificant difference of FBG in all treated groups at diffentchecktime(P>0.05).
1.2Comparison of water intake in each group at diffent checktime
Compared with NG, water intake in MG and all treated groups increasednotably (P<0.05) at diffent checktime. Compared with MG, polydipsia inYHTG improved notably from2W(P<0.05), and polydipsia in YHG improvednotably(P<0.05) except for4W and8W, while polydipsia in YTG improvednotably from16W(P<0.05). Compared among all treated groups, there was nosignificant difference of water intake in all treated groups at diffentchecktime(P>0.05).
1.3Comparison of diet intake in each group at diffent checktime
Compared with NG, diet intake in MG and all treated groups increasednotably(P<0.05) at diffent checktime. Compared with MG, polyphagia inYHTG improved notably from16W(P<0.05), while polyphagia in YHG andYTG improved notably at the end of32W(P<0.05). Compared among alltreated groups, there was no significant difference of diet intake in all treated groups at diffent checktime(P>0.05).
1.4Comparison of urine output in each group at diffent checktime
Compared with NG, urine output in MG and all treated groups increasednotably(P<0.05) at diffent checktime. Compared with MG, polyuria in alltreated groups improevd notably from2W(P<0.05). Compared among alltreated groups, urine output in YHTG was less than YHG and YTG from8W(P<0.05).
1.5Comparison of body weight in each group at diffent checktime
Pre-modeling, body weight in high fat diet feeding rats was higher thanthat of NG(P<0.05). Post-modeling, body weight in NG increased gradually,but body weight in MG and all treated groups decreased gradually. Bodyweight in YHTG from8W, YHG and YTG at the end of32W was higher thanthat in MG(P<0.05). Compared among all treated groups, there was nosignificant difference of body weight in all treated groups at diffentchecktime(P>0.05).
1.6Comparison of HbA1c, KI, Ccr, Scr, and ACR in each group at32W
Compared with NG, HbA1c, KI, Ccr, Scr, and ACR in MG increasednotably (P<0.05), except for Scr, HbA1c in YHG, KI in YTG, remain indicesin all treated groups also increased notably(P<0.05). Compared with MG,except for HbA1c in YTG, HbA1c, Ccr, Scr, and ACR in all treated groupsdecreased notably(P<0.05), KI didn′t decrease unnotably (P>0.05). Comparedamong all treated groups, Ccr and ACR in YHTG were lower than those inYHG and YTG(P<0.05), but, there was no significant difference of HbA1c, KI,and Scr in all treated groups (P>0.05).
1.7Comparison of serum lipid (including TC, TG, HDL, and LDL) in eachgroup
Compared with NG, serum lipid in MG increased notably(P<0.05), andTC, HDL, and LDL in all treated groups also increased notably (P<0.05), but,TG didn′t increased notably(P>0.05). Compared with MG, serum lipid in alltreated groups decreased notably (P<0.05). Compared among all treatedgroups, there was no significant difference of serum lipid in all treated groups(P>0.05).
1.8Comparison of glomerular volume in each group
Compared with NG, glomerular volume in MG and all treated groupsenlarged notably(P<0.05). Compared with MG, glomerular volume in alltreated groups didn′t enlarge notably (P>0.05). Compared among all treatedgroups, there was no significant difference of glomerular volume in all treatedgroups(P>0.05).
1.9Comparison of GBM thickness in each group
Compared with NG, GBM in MG and all treated groups thickenednotably(P<0.05). Compared with MG, GBM in all treated groups didn′tthicken notably (P>0.05). Compared among all treated groups, there was nosignificant difference of GBM thickness in all treated groups(P>0.05).
2Effects of yiqi-yangyin-huoxue-tongluo recipe on nephrin in DN rats
2.1Comparison of U-nephrin in each group at diffent checktime
Compared with NG, U-nephrin in MG at diffent checktime, and alltreated groups at end of2W, as well as8W increased notably(P<0.05).Compared with MG, increased U-nephrin in all treated groups improvednotably(P<0.05) at diffent checktime. Compared among all treated groups,there was no significant difference of U-nephrin in all treated groups at diffentchecktime(P>0.05).
2.2Comparison of U-alb in each group at diffent checktime
Compared with NG, U-alb in MG and all treated groups increasednotably(P<0.05) at diffent checktime. Compared with MG, increased U-alb inYHTG from2W, YHG and YTG from8W improved notably(P<0.05).Compared among all treated groups, U-alb in YHTG was lower than that inYHG and YTG at end of2W and8W(P<0.05).
2.3Correlation between U-alb and U-nephrin in MG at diffent checktime
There was a significant positive correlation between U-alb and U-nephrinat diffent checktime.
2.4Comparison of K-nephrin in each group
Compared with NG, K-nephrin in MG and YHG decreased notably (P<0.05), but K-nephrin in YHTG and YTG didn′t decrease notably (P>0.05).Compared with MG, decreased K-nephrin in all treated groups improvednotably(P<0.05). Compared among all treated groups, there was no significantdifference of K-nephrin in all treated groups(P>0.05).
2.5Correlation among U-alb, U-nephrin, and K-nephrin in MG at the end of32W
There was a significant positive correlation between U-alb andU-nephrin(r=0.828, P=0.006), but a negative correlation between U-alb andK-nephrin (r=-0.845, P=0.004), as well as K-nephrin and U-nephrin(r=-0.991,P=0.000).
2.6Expression of nephrin mRNA in renal cortex investigated by RT-PCR
Compared with NG, the expression of nephrin mRNA in MG and alltreated groups downregulated notably (P<0.05). Compared with MG,downregulated expression of nephrin mRNA in all treated groups improvednotably(P<0.05). Compared among all treated groups, there was no significantdifference of the expression of nephrin mRNA in all treated groups(P>0.05).
3Effects of yiqi-yangyin-huoxue-tongluo recipe on the expression ofα-actinin-4and podocyte density in DN rats
3.1The expression of α-actinin-4in renal cortex investigated by Western-blot
Compared with NG, the expression of α-actinin-4in MG, YHG, and YTGdownregulated notably (P<0.05), but in YHTG didn′t downregulate notably(P>0.05). Compared with MG, downregulated expression of α-actinin-4in alltreated groups improved notably(P<0.05). Compared among all treated groups,the expression of α-actinin-4in YHTG was higher than that in YHG andYTG(P<0.05).
3.2The expression of α-actinin-4in renal cortex investigated by immunohisto-chemistry
Compared with NG, the expression of α-actinin-4in MG, YHG, and YTGdownregulated notably (P<0.05), but in YHTG didn′t downregulate notably(P>0.05). Compared with MG, downregulated expression of α-actinin-4in alltreated groups improved notably(P<0.05). Compared among all treated groups, the expression of α-actinin-4in YHTG was higher than that in YHG andYTG(P<0.05).
3.3Comparison of podocyte density in each group
Compared with NG, podocyte density in MG and all treated groupsdecreased notably(P<0.05). Compared with MG, decreased podocyte densityin all treated groups improved notably(P<0.05). Compared among all treatedgroups, there was no significant difference of podocyte density in all treatedgroups(P>0.05).
3.4Comparison of U-pro in each group
Compared with NG,U-pro in MG and all treated groups increased notably(P<0.05). Compared with MG, increased U-pro in all treated groups improvednotably(P<0.05). Compared among all treated groups, U-pro in YHTG waslower than that in YHG and YTG(P<0.05).
3.5Correlation among U-pro, α-actinin-4/β-actin, and podocyte density inMG
There was a significant negative correlation between U-pro andα-actinin-4/β-actin (r=-0.846,P=0.004), as well as U-pro and podocytedensity(r=-0.844,P=0.002), but a positive correlation between podocytedensity and α-actinin-4/β-actin (r=0.815,P=0.007).
Conclusions:
1Yiqi-yangyin-huoxue-tongluo recipe, yiqi-yangyin-huoxue recipe, andyiqi-yangyin-tongluo recipe can attenuate polydipsia, polyphagia, polyuria,and body weight reduction notably, as well as metabolic disorder of lipid, andcan decrease ACR and U-pro, attenuate glomerular hyrerfiltration, glomerularhypertrophy, thickened GBM, so,they have a renal protective role;
2Yiqi-yangyin-huoxue-tongluo recipe and yiqi-yangyin-huoxue recipe canattenuate metabolic disorder of sugar, but yiqi-yangyin-tongluo recipe can not;Yiqi-yangyin-huoxue-tongluo recipe, yiqi-yangyin-huoxue recipe, andyiqi-yangyin-tongluo recipe can decrease U-nephrin, attenuate downregulationof K-nephrin protein and mRNA, attenuate downregulation of α-actinin-4protein, lessen podocyte density reduction notably, so, they have a podocytic protective role;
3As for amelioration ACR, U-pro, polyuria, and glomerular hyperfiltration,yiqi-yangyin-huoxue-tongluo recipe is better than YHG and YTG, which maybe related to yiqi-yangyin-huoxue-tongluo recipe improved thedownregulation of α-actinin-4more notably.
引文
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