| 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 ratsHealthy 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 ratsUrine 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 ratsExpression 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 analysisAll 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 rats1.1Comparison of FBG in each group at diffent checktimeCompared 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 checktimeCompared 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 checktimeCompared 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 checktimeCompared 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 checktimePre-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 at32WCompared 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 eachgroupCompared 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 groupCompared 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 groupCompared 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 rats2.1Comparison of U-nephrin in each group at diffent checktimeCompared 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 checktimeCompared 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 checktimeThere was a significant positive correlation between U-alb and U-nephrinat diffent checktime.2.4Comparison of K-nephrin in each groupCompared 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 of32WThere 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-PCRCompared 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 rats3.1The expression of α-actinin-4in renal cortex investigated by Western-blotCompared 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-chemistryCompared 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 groupCompared 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 groupCompared 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 inMGThere 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. |
PDF Full Text Request