| Objectives: Diabetic nephropathy(DN) is also known as diabetic glomer-ulosclerosis. It is one of the most common and most serious microvascular complications of diabetes mellitus(DM). With the improvement of people’s living standards, changes in diet, the incidence of DN is rising year by year and it has become the main reason leading to end stage renal disease(ESRD). Despite home and abroad medical kidney disease experts have been constantly exploring pathogenesis and effective method to prevent and treat DN, but so far pathogenesis of DN is not completely clear and there are lack of effective method. Traditional Chinese Medicine(TCM) has showed huge capacity because of its unique characteristics, overall adjustment,multi-link and multitarget effection. Whereas, TCM has showed great potential and widely appli-cation prospect. In recent years, we have a deeper underst- anding on TCM pathogenesis of DN. Stasis-blood-blocking-collaterals is the key pathogenesis of DN and runs through all stages of DN. Clinical reports that 100% of DN patients exhibit characteristics of syndromes of blood stasis. Huayu-tongluo Chinese herbs has also become the basis for medication. Its curative effect has also been widely recognized by more and more physicians. But the mecha-nism of action of Huayu Chinese herbs and Tongluo Chinese herbs is the same or not? Are there a synergistic relationship between them. There is no report on it. According to preliminary animal experiments and clinical observations, we select Huayu-tongluo Chinese herbs(chuanxiong, salvia, earthworm, leech, scorpio)as all parties, select Huayu Chinese herbs(chuanxiong, salvia) and Tongluo Chinese herbs(earthworm, leech, scorpio) as party splits. This study adopts the model of DN rats with high-sugar high-fat diet combined with low-dose streptozotocin(STZ) by intraperitoneal injection, select the stasis- blood-blocking-collaterals and podocyte-associated protein as the main entry point. Through animal experiments and cultured podocytes, from the multi-level, multi-target, characteristics of role on Huayu-tongluo Chinese herbs are studyed and discussed in order to providing the basis for clinical optimization compatibility.Methods:1The effect of different compatibility of Huayu-tongluo Chinese herbs on biological indicators of stasis-blood-blocking-collate in DN rats65 healthy male SD rats were 4~5 weeks old. After they were fed for one week. 10 rats were randomly selected as the normal group(C group). The remaining 55 rats were injected with 1% streptozotocin(35mg/kg) after feeding high-fat and high-glucose diet for four weeks. The diabetic model rats(blood glucose≥16.7mmol/L) were randomly divided into the model group(M group), Huayu-tongluo group(Q group), Huayu group(H group), Tongluo group(T group). According to the ratio of the animal body surface area, the dosage of the rats were calculated. Q Group:Salvia 1.35g/(kg?d), Chuanxiong 1.08g/(kg?d), earthworm 0.9g/(kg?d), leech 0.54g/(kg?d), Scorpion 0.54g/(kg? d). H group: Salvia 1.35g/(kg?d), Chuanxiong 1.08g/(kg?d). T group: earth dragon 0.9g/(kg?d), leech 0.54g/(kg?d), Scorpion 0.54g/(kg?d). While the left rats were given a corresponding amount of drinking water once daily for 16 weeks. At the end of the 16 th week, 24 h urinary protein was tested. Blood samples were collected by abdominall aortic method in order to test random blood glucose, blood lipids, coagulation, plateletparameters, platelet metabolites and so on. The part of renal cortex was stored in 4% neutral formalin fixative for the immunohis-tochemistry. The part of the renal cortex was stored in-80℃ultra-low temper-ature freezer for detection of molecular biology index.2 Protective effect of Huayu-tongluo Chinese herbs on kidney in rats with diabetic nephropathy10 rats were randomly selected as the normal group(C group). The remaining 40 rats were made diabetic model. The method of modeling was same as above 36 diabetic model rats were randomly divided into the model group(M group), Huayu-tongluo medicine group(Q group), irbesartan group(I group). According to the ratio of the animal body surface area, the dosage of the rats in each group was calculated. The dosage of rats in Q group was the same as the first part. The dosage of rats in I group was 13.5mg/(kg?d). At the same time, the rats in C, M groups were given the corresponding amount of drinking water. At the end of 0th week(before modeling), 4th, 8th, 12 th and 16 th week, the rats were placed into metabolic cages for 24 hours to collect 24 h urine. Meanwhile the rats′body weight, diet intake, water intake were measured. At the end of 16 th week, blood samples were collected by abdominal-aortic method in order to test renal function. The part of renal cortex was stored so that the pathomorphological change of renal tissues was observed with light and electron microscope.3 The effect of different compatibility of Huayu-tongluo Chinese herbs on expression of podocin,CD2 AP and ZO-1 in podocytes of rats with DNIn the first part of this experiment, the partial renal cortex was preserved in 4% neutral formalin fixed solution. Podocin and CD2 AP were detected by immunohistochemistry(IHC), the partial renal cortex were harvested, stored in the frozen stored tube, quickly put into liquid nitrogen. After a brief cooling, it was stored in-80℃ultra-low temperature freezer. The expression of podocin, CD2 AP and ZO-1 were detected by Real-Time PCR.4 The effect of different compatibility of Huayu-tongluo Chinese herbs on expression of a-actinin-4 and Synaptopodin in podocytes of rats with DNIn the first part of this experiment, the partial renal cortex was harvested, stored in the frozen stored tube, quickly put into liquid nitrogen. After a brief cooling, it was stored in-80℃ ultra-low temperature freezer. The expression of a-actinin-4 and Synaptopodin were detected by Western-blot and RealTime PCR.5 The effect of Huayu-tongluo Chinese herbs on high glucose stimulating podocytesThe conditionally immortalized mouse podocytes cultured under the growth permissive condition were divided into the normal glucose group(CG group), high glucose group(HG group) and high glucose dosing group(HG+Z group). Mouse podocytes were intervened for 24 h, 48 h and 72 hours. The expression of podocin and CD2 AP was detected by immunocytochemistry(ICC), Western-blot and Real-Time PCR.Results:1The effect of different compatibility of Huayu-tongluo Chinese herbs on biological indicators of stasis-blood-blocking-collate in DN rats1.1 General condition in each groupThe rats in C group were in good spirits, agile, athletic, normal diet, a steady increase in weight. The rats in M group appeared obvious polydipsia, polyphagia, polyuria and loss of weight. Not only so the rats in M group had dull coat, thin muscles, dull reaction. With the extension of time, all the symptoms became more serious. The rats′symptoms had improved in each treated group.1.2 Comparison of random blood glucose(RBG) in each groupAt the end of 16 th week, compared with C group, RBG significantly increased in M group and all treated groups(P<0.01). Compared with M group, there was no significant difference of RBG in all treated groups(P>0.05). There was no significant difference among all treated groups(P>0.05).1.3 Comparison of 24 h urine total protein(UTP) in each groupAt the end of 16 th week, compared with C group, 24 h UTP significantly increased in M group and treated groups(P<0.01). Compared with M group, 24 h UTP significantly reduced in all treated groups(P<0.01). There was no significant difference among all treated groups(P>0.05).1.4 Comparison of blood lipid(TC, TG, LDL-C, HDL-C) in each groupAt the end of 16 th week, compared with C group, TC, TG, LDL-C and HDL-C significantly increased in M group and all treated groups(P<0.01). Compared with M group, TC, TG, LDL-C significantly reduced in all treated groups(P<0.01), HDL-C did not change significantly(P>0.05). TG in Q group was significantly lower than T group(P<0.05). There was no difference of TG between Q group and H group. LDL-C in Q, H groups was significantly lower than T group(P<0.05). There was no difference of TG between Q group and H group. There was no difference of TC and HDL-C among treated groups(P>0.05).1.5 Comparison of blood coagulation(PT, INR, APTT, TT, FIB) in each groupAt the end of 16 th week, compared with C group, PT significantlyreduced in M group and all treated groups(P<0.01). Compared with M group,PT significantly improved in all treated groups(P<0.05, P<0.01). PT in Qgroup was significantly extended than T group(P<0.05). Compared with Cgroup, INR significantly reduced in M group and all treated groups(P<0.01).Compared with M group, INR in Q group significantly improved(P<0.05).There was no significant difference of INR among all treated groups(P>0.05).Compared with C group, APTT significantly reduced in M group and alltreated groups(P<0.01). Compared with M group, APTT in Q, H groupssignificantly improved(P<0.01). Compared with T group, APTT in Q groupwas significantly improved(P<0.05). Compared with C group,FIB significan-tly increased in M group and all treated groups(P<0.05, P<0.01). Comparedwith M group, FIB in all treated groups significantly reduced(P<0.05). Therewas no significant difference of FIB among all treated groups(P>0.05).1.6 Comparison of platelet parameters(PLT, MPV, PCT%, PDW) in each groupAt the end of 16 th week, compared with C group, PLT and PDW significantly changed in M group and all treated groups(P<0.05, P<0.01). Compared with M group, PLT, MPV and PCT% significantly improved in all treated groups(P<0.05, P<0.01). Compared with M group, PDW in Q group significantly reduced(P<0.05). There was no significant difference of PLT, MPV, PCT% and PDW among all treated groups(P>0.05).1.7 Comparison of TXB2, 6-keto-PGF1 a and TXB2/6-keto-PGF1 a in each groupAt the end of 16 th week, compared with C group, TXB2, TXB2/6-ketoPGF1 a significantly increased in M group and all treated groups(P<0.05, P <0.01). Compared with M group, TXB2 and TXB2/6-keto-PGF1 a significantly reduced, 6-keto-PGF1 a significantly increased(P<0.05, P<0.01). TXB2 and TXB2/6-keto-PGF1 a in Q group was significantly lower than T group(P<0.05, P<0.01).2 Protective effect of Huayu-tongluo Chinese herbs on kidney in rats with diabetic nephropathy2.1 Comparison of body weight in each groupBody weight of rats in C group gradually increased. Compared with C group, body weight of rats in M, Q and I group significantly reduced(P<0.01) at the end of 4th, 8th, 12 th and 16 th week. There was no significant difference among M, Q, I groups(P>0.05).2.2 Comparison of diet-intake in each groupBefore modeling,there was no significant difference among all the groups(P>0.05). Compared with C group, diet-intake of rats in M, Q and I groups significantly increased(P<0.01)at the end of 4th, 8th, 12 th and 16 th week. There was no significant difference among M, Q, I groups(P>0.05).2.3 Comparison of water-intake in each groupBefore modeling, there was no significant difference among all the groups(P>0.05). Compared with C group, water-intake of rats in M, Q and I group significantly increased(P<0.05)at the end of 4th, 8th, 12 th and 16 th week. There was no significant difference among M, Q, I groups(P>0.05)at each different time point(P>0.05).2.4 Comparison of urine-output in each groupBefore modeling, there was no significant difference among all the groups(P>0.05). Compared with C group, urine-output of rats in M, Q and I groups significantly increased(P<0.05) at the end of 4th, 8th, 12 th and 16 th week. There was no significant difference among M, Q, I groups(P>0.05)at each different time point(P>0.05).2.5 Comparison of 24 h UTP in each groupBefore modeling, there was no significant difference among all the groups(P>0.05). Compared with C group, 24 h UTP of rats in M, Q and I groups signi- fycantly increased(P<0.01)at the end of 4th, 8th, 12 th and 16 th week. Compared with M group, 24 h UTP of rats in Q, I groups significantly reduced(P<0.05, P<0.01)at the end of 4th, 8th, 12 th and 16 th week. Compared with I group, 24 h UTP of rats in Q group significantly reduced(P<0.05)at the end of 16 th week.2.6 Comparison of KI, Cys-c, Scr, BUN, UA in each groupAt the end of 16 th week, compared with C group, KI, Cys-c, Scr, BUN, UA significantly increased in M, Q, Igroups(P<0.05, P<0.01). Compared with M group, Cys-c, BUN significantly reduced in Q, Igroups(P<0.05, P<0.01), Scr, KI, UA did not change significantly(P>0.05). There was no significant difference of KI, Cys-c, Scr, BUN, UA between Q group and I group(P>0.05). 2.7 Comparison of changes of pathomorphology observations in renal tissue in each groupBy light microscope, in C group, kidney structure was clear, there were no obvious thickening of GBM and proliferation of mesangial cell. In M group, there were obvious hypertrophy of renal glomerulus, obvious thickening of basement membrane, severe hyperplasia of mesangial. Compared with M group, both of the treated groups were significantly improved on pathological changes.By transmission electron microscopy, in C group, the structure of glomerular basement membrane was clear. The arrangement of foot process was more orderly. In M group, the basement membrane structure was not clear,there were extensive fusion of foot process. Compared with M group, both of the treated groups were significantly improved on pathological changes.3The effect of different compatibility of Huayu-tongluo Chinese herbs on expression of podocin, CD2 AP and ZO-1 in podocytes of rats with DN3.1 The expression of podocin and CD2 AP in renal cortex by immunohistoche-mistryIn C group, the expression of podocin showed brownish yellow and localized in the glomerular. Podocin was mainly distributed on the basement membrane. There was no obvious expression in renal tubular and interstitial. In M group, the distribution of podocin in the glomerular was not uniform and the expression was significantly decreased. Compared with M group, the expression of podocin was significantly enhanced and the linear distribution along the basal membrane was recovered in all treated groups.In C group, the expression of CD2 AP showed brownish yellow and localized in the glomerular and renal tubular. In M group, the expression of CD2 AP was significantly decreased. Compared with M group, the expression of CD2 AP was significantly enhanced in all treated groups. 3.2 The expression of podocin m RNA in renal cortex by Real-Time PCR.Compared with C group, the expression of podocin m RNA was signifycantly decreased in the other groups(P<0.01). Compared with M group, the expression of podocin m RNA in all treated groups was significantly increased(P<0.01). The expression of podocin m RNA in Q group was higher than T, H groups(P<0.05). There was no difference between T group and H group(P> 0.05).3.3 The expression of CD2 AP m RNA in renal cortex by Real-Time PCR.Compared with C group, the expression of CD2 AP m RNA was significantly decreased in the other groups(P<0.01). Compared with M group, the expression of CD2 AP m RNA in all treated groups was significantly increased(P<0.01). The expression of CD2 AP m RNA in Q, T groups was higher than H group(P<0.01). There was no difference between Q group and T group(P>0.05).3.4 The expression of ZO-1 m RNA in renal cortex by Real-Time PCR.Compared with C group, the expression of ZO-1 m RNA was significantly decreased in the other groups(P<0.01). Compared with M group,the expression of ZO-1 m RNA in all treated groups was significantly increased(P<0.01). The expression of ZO-1 m RNA in Q group was higher than T, H groups(P<0.05, P<0.01). The expression of ZO-1 m RNA in T group was higher than H groups(P<0.05).4 The effect of different compatibility of Huayu-tongluo Chinese herbs on expression of a-actinin-4 and Synaptopodin in podocytes of rats with DN4.1 The expression of a-actinin-4 and Synaptopodin protein in renal cortex by Western-blotCompared with C group, the expression of a-actinin-4 protein was signifycantly decreased in the other groups(P<0.05, P<0.01). Compared with the M group, the expression of a-actinin-4 protein in all treated groups was signifycantly increased(P<0.05, P<0.01). The expression of a-actinin-4 protein in Q, T groups was higher than H group(P<0.01). There was no difference between Q group and T group(P>0.05).Compared with C group, the expression of Synaptopodin protein was significantly decreased in the other groups(P<0.05, P<0.01). Compared with the M group, the expression of Synaptopodin protein in all treated groups was significantly increased(P<0.05, P<0.01). The expression of Synaptopodin protein in Q group was higher than T, H groups(P<0.05). There was no difference between T group and H group(P>0.05).4.2 The expression of a-actinin-4 and Synaptopodin m RNA in renal cortex by Real-Time PCR.Compared with C group, the expression of a-actinin-4 m RNA was significantly decreased in the other groups(P<0.01). Compared with M group, the expression of a-actinin-4 m RNA in all treated groups was significantly increased(P<0.01). The expression of a-actinin-4 m RNA in Q group was higher than T, Hgroups(P<0.05, P<0.01). The expression of a-actinin-4 m RNA in T group was higher than H group(P<0.05).Compared with C group, the expression of Synaptopodin m RNA was significantly decreased in the other groups(P<0.01). Compared with M group, the expression of Synaptopodin m RNA in all treated groups was significantly increased(P<0.01). The expression of Synaptopodin m RNA in Q group was higher than T, H groups(P<0.05, P<0.01). There was no difference between T group and H group(P>0.05).5 The effect of Huayu-tongluo Chinese herbs on high glucose stimulating podocytes5.1 The expression of podocin and CD2 AP in podocytes by immunocytochemistryBy the microscope, the expression of podocin and CD2 AP in CG group showed a strong positive expression of fine particles in the cell membrane, cytoplasm and nucleus of the cell. The expression of them were significantly decreased in HG group. Compared with HG group, the expression of them were significantly enhanced in(HG+Z) group.5.2 The expression of podocin and CD2 AP protein in podocytes by Western-blotAt 48 hours, compared with CG group, the expression of podocin and CD2 AP protein in HG group was lower(P<0.05, P<0.01). Compared with HG group, the expression of podocin and CD2 AP protein in(HG+Z)group was higher(P<0.05). However, there was no difference of expressions of podocin and CD2 AP proteins among different groups at 24 and 72 hours(P>0.05). 5.3 The expression of podocin and CD2 AP m RNA in podocytes by Real-Time PCR.Compared with CG group, at same time point, the expression of podocin and CD2 AP m RNA in other groups was lower(P<0.05). Compared with HG group, only at 48 hours, the expression of podocin and CD2 AP protein in(HG+Z) group was higher(P<0.05), however, there was no difference in expre- ssions of podocin and CD2 AP proteins among different groups at 24 and 72 hours(P>0.05).Conclusions:1 It affirmed the existence of blood-stasis-blocking-collaterals syndrome of rats with DN. Huayu-tongluo Chinese herbs could improve stasisblood-blocking-collaterals biological indicators of rats with DN.The combined application of Huayu Chinese herbs and Tongluo Chinese herbs had a synergistic effect. Moreover, Huayu Chinese herbs was superior to Tongluo Chinese herbs.2 Huayu-tongluo Chinese herbs could significantly reduce the 24 h UTP of rats with DN and this action was more stable and lasting than irbesartan.3 Huayu-tongluo Chinese herbs could reduce BUN, Cys-c of rats with DN. In addition to this, it could reduce the pathological changes of renal tissue, so they had a renal protective role.4 Huayu-tongluo Chinese herbs, Huayu Chinese herbs and Tongluo Chinese herbs could up-regulate the expression of slit diaphragm protein(podocin, CD2 AP, ZO-1) and skeletal protein(α-actinin-4, Synaptopodin) in podocytes. These proteins were one of the targets of the traditional Chinese medicine to prevent and cure DN. Moreover, Tongluo Chinese herbs was superior to Huayu Chinese herbs and they had a synergistic effect.5 High glucose stimulation had the effect of down-regulating the expression of podocin and CD2 AP. Huayu-tongluo Chinese herbs had a significant antagonistic effect on the reduction of the effects of high glucose stimulating podocytes.The results of this study need to be further confirmed by increasing the sample size, avoiding the interference factors, improving the experimental conditions and so on. |
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