A total of 50 specific pathogen-free male Sprague Dawley rats (age, 6–8 weeks; body weight, 160–200 g) were purchased from Shanghai Sippr-BK Laboratory Animal Co., Ltd. [Shanghai, China; (certificate of approval, SCXK (Shanghai); 2008-0016]. Rats were bred in the Research Center of Zhejiang Chinese Medical University (Zhejiang, China), with the experimental license number of SYXK 2013–0184 (clean grade). Rats were maintained in the following experimental conditions: A 12-h light-dark cycle; a maintained temperature of 18–22°C with a relative humidity of 40–70%; and access to food and water ad libitum. All animal experiments were approved by Zhejiang Chinese Medical University Animal Ethics Committee (Zhejiang, China).

TS formula for one human body daily dosage is constituted of six different Chinese herbs, including Radix puerariae (15 g), astragalus (18 g), Ligustrum lucidum (15 g), Lucid ganoderma (15 g), Salvia miltiorrhiza (9 g) and Rheum rhabarbarum (6 g), which were all purchased from the Pharmacy Department of Zhejiang Provincial Hospital of Traditional Chinese Medicine (Zhejiang, China). Once herbs were combined, water (v/w) at eight times of the herbs weight was added. Subsequently, the medicinal herbs were decocted on a slow fire for 1 h and the remaining fluid was collected. The remaining pharmacological agents were continuously processed by adding water (v/w) six times for a further 1 h on the slow fire. The two remaining fluids were merged and the compounds were condensed at a final concentration of 2 g/ml and stored at 4°C. The positive agent, valsartan, was purchased from Beijing Pharma Ltd. (Beijing, China). Valsartan was dissolved in sodium carboxymethyl cellulose.

Streptozotocin (STZ) was purchased from Sigma-Aldrich (Merck Millipore, Darmstadt, Germany) and the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) iTaq™ Universal SYBR^® Green One-Step kit was purchased from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). The primary antibody of monocyte chemoattractant protein-1 (MCP-1; sc-1785; 1:800), macrophage migration inhibitory factor (MIF; sc-20,121; 1:800), nuclear factor-κB (NF-κB; sc-372; 1:500), sirtuin-1 (SIRT-1; sc-15,404; 1:300) and β-actin were all purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). The IgG-horse radish peroxidase (1:3,000; cat no. M209180) was obtained from Pierce (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Hemorheology was performed using a 7020-automatic biochemical analyzer (Hitachi, Ltd., Tokyo, Japan). Western blotting and PCR were performed using a T Personal thermocycler (Biometra GmhH, Göttingen, Germany) and Mini-PROTEAN electrophoresis system (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Prior to experiments, a total of 50 SD rats were fed for 1 week for adaptation and urine protein and blood glucose levels were detected. A total of 3 rats exhibited abnormal results and were subsequently sacrificed. The remaining 47 SD rats were intraperitoneally injected with 1% STZ at 40 mg/kg and fed high-fat glucose fodder, containing 10% egg yolk powder, 10% lard, 10% sucrose, 0.5% cholesterol and basic feed. This fodder was processed and disinfected by the Zhejiang Academy of Medical Sciences (Zhejiang, China). Another 8 rats were injected with an equal volume of vehicle (0.1 M citrate buffer; pH 4.2) to represent the normal control. A total of 72 h after the injection, tail vein blood was collected and serum glucose was detected in all rats. The diabetic rat model was successfully established with serum glucose ≥16.7 mmol/l. Diabetic rats were randomly divided into groups: Diabetic nephropathy group (DN group; n=8), valsartan group (valsartan group; n=8), and low-, medium- and high-dosage of TS formula groups (all n=8). Rats injected with vehicle were considered as the normal control group (n=8). Rats of the valsartan group were treated with valsartan solution (25 mg/kg/day) and the rats in the TS formula groups were treated with TS formula, 6.5 g/kg/day (low dose), 13 g/kg/day (medium dose) and 26 g/kg/day (high dose), respectively. The rats in these groups were observed for 12 weeks; 12 weeks following treatment, all rats were sacrificed and the renal cortex was collected for pathological observation and further investigation.

Following treatment, 24-h urine collection was performed. Under empty stomach conditions, 3% pentobarbital sodium (Sigma-Aldrich; Merck Millipore; cat no. P376; 45 mg/kg) anesthesia was administered to acquire serum, plasma and whole blood samples. Rat kidneys were weighed and either stored in 10% formalin solution for pathological detection or stored at −80°C for protein and mRNA detection.

The 24-h urine collected was centrifuged at 1,200 g/min for 10 min at 4°C and the supernatant was obtained. The automatic biochemical analyzer tested the blood for glucose, blood urine creatinine and serum creatinine levels and the endogenous creatinine clearance rate was calculated. The micro albuminuria and β2-microglobulin (β2-MG) levels were detected using the Rat β2-microglobulin, BMG/β2-MG (Sangon Biotech Co., Ltd., Shanghai, China; cat no. D730498) and micro albuminuria ELISA kits (Wuhan Elabscience Biotechnology Co., Ltd., Wuhan, China; cat no. E-EL-R0025). Hemorheology indices, including blood viscosity and whole reduced blood viscosity, were determined using an automatic blood rheometer.

Kidney tissues were fixed in 10% neutral formalin at 4°C overnight for dehydration. Tissues were heated at 120°C for 10 min in an autoclave for antigen retrieval and then dehydrated in an ascending grade of ethanol, washed with xylene and embedded in paraffin. The paraffin-embedded samples were cut into sections 3 µm thick for histopathological examination. Tissue sections were de-paraffinized in xylene and stained with H&E for 20 min at room temperature. For PAS staining, paraffin slides were de-paraffinized, rehydrated and washed in water. Slides were placed in 2% Periodic Acid for 15 min and stained with Schiff's reagent for 25 min at room temperature. Counter-staining with filtered hematoxylin was then conducted. Following washing three times, slides were rapidly dipped in acid alcohol twice and slowly dipped in ammonia four times. Slides were then dehydrated and cover slipped. The glomerular, renal tubular structure and matrix hyperplasia were studied under a light microscope using H&E and PAS staining.

EnVision two-step method was used for immunohistochemical staining. Following antigen retrieval, the sections were incubated with cluster of differentiation (CD) 68 primary antibody (1:50) at 4°C overnight. Samples were washed with PBS and incubated with EnVision working solution for 30 min. DAB and hematin counterstaining were performed. Images were captured from five random fields under a light microscope (magnification, ×400).

Total RNA was extracted from rat kidney tissue using TRIzol total RNA isolation reagent (Gibco; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Total RNA was reverse transcribed using the RT-PCR kit followed by qPCR detection. Primer sequences were as follows: SIRT1 (306 bp), forward 5′-CCCATTCGGTTTCCATTGTA-3′ and reverse 5′-TCATCAGCCAACAGGAGGTT-3′; NF-κB (302 bp), forward 5′-TGCCTCCAGTGAGAAGAACA-3′ and reverse 5′-GCACCAGAAGTCCAGGGTTA-3′; MCP-1 (302 bp), forward 5′-CAGCAGGTGTCCCAAAGAAG-3′ and reverse 5′-AGGCATCACATTCCAAATCAC-3′; MIF-1 (180 bp), forward 5′-CAGAACCGCAACTACAGCAA-3′ and reverse 5′-CAGCAAGACTCGAAGAACAGC-3′; and GADPH (91 bp), forward 5′-GTGCTGAGTATGTCGTGGAG-3′ and reverse 5′-GTCTTCTGAGTGGCAGTGAT-3′. The RT-qPCR system contained: Sense primer 0.5 µl (10 µM), antisense primer 0.5 µl (10 µM), cDNA (1 µl), double distilled H₂O (8 µl) and SYBR Supermix from the iTaq™ Universal Syber green One-Step kit (10 µl). Reactions were incubated at 95°C for 2 min followed by 95°C for 10 sec, 55°C for 30 sec and 72°C for 30 sec, for 40 cycles. Expression levels of mRNA were quantified by the 2^−ΔΔCq method (8).

Samples were rinsed twice with cold PBS and then lysed on ice in radio immunoprecipitation assay lysis buffer and subsequently homogenized and centrifuged (4°C; 9,600 × g for 5 min) to collect the total protein. Protein content was determined using the BCA method. Supernatants were then transferred, mixed and boiled in sample buffer. Total protein (40 µg/lane) were separated by 12% SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The membrane was incubated in blocking buffer consisting of 5% fat-free milk dissolved in 1X Tris-buffered saline with Tween 20 (TBST; 10 mM Tris-Base, pH 7.5, 100 mM NaCl and 1% Tween 20) for 1 h at room temperature followed by incubation with monocyte chemoattractant protein-1 (MCP-1; sc-1785; 1:800), macrophage migration inhibitory factor (MIF; cat no. sc-20121; 1:800), nuclear factor-κB (NF-κB; cat no. sc-372; 1:500) and sirtuin-1 (SIRT-1; cat no. sc-15404; 1:300) primary antibodies at 4°C overnight and β-actin (cat no. sc-58673; 1:2,000) at room temperature for 1 h. The membrane was subsequently washed with TBST and incubated at 37°C with IgG-horse radish peroxidase for 1 h. The blot was visualized using an enhanced chemiluminescence reagent kit (GE Healthcare Life Sciences, Chalfont, UK) following four washes with TBS.

All data were presented as the mean ± or +standard error of the mean. Differences between multiple groups were analyzed by one-way analysis of variance followed by Duncan's multiple range test. Differences between two groups were measured by Student's t-test, using SPSS 16.0 software (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

Following STZ injection for 72 h, the condition observed in DN rats gradually declined as the rats exhibited dull fur, weight loss, were apathetic, unresponsive, had an increased appetite, thirst and frequency of urination and urine protein level, compared with the control group. Local ulceration, including foot ulcers, and deep skin ulcers were observed. Furthermore, the DN group displayed the most severe psychotic symptoms such as manic behavior. To some extent, groups with treatment exhibited less complications.

As indicated in Table I, following modeling with STZ and high-fat glucose fodder, the body weight of rats significantly decreased when compared with the normal control (P<0.05). Moreover, TS formula improved this condition in a dose-dependent manner, which was similar to the therapeutic effect exhibited by valsartan treatment (P<0.05). Conversely, kidney weight and kidney hypertrophy index were significantly increased in the DN group compared with the control; however, this was improved in a dose-dependent manner in the groups, which were treated with different doses of TS formula (P<0.05; Table I). Furthermore, these groups exhibited a similar weight and hypertrophy index as the valsartan treated group (Table I).

Fasting blood glucose (FBG), urinary albumin excretion rate (UAER), β2-MG and creatinine clearance rate (Ccr) were evaluated following TS formula or valsartan treatment. The present results indicated that a high dose of TS formula significantly decreased FBG (P<0.01), UAER (P<0.05) and β2-MG (P<0.05) levels. Furthermore, a high dose of TS formula significantly improved Ccr compared with the DN group and the rate was similar to the Ccr exhibited in the valsartan group (P<0.05; Table II). The whole blood low-, medium- and high-shear reductive viscosity and the blood viscosity were determined. The present results revealed that TS formula significantly reduced these hemorheology indices when compared with the DN group and a similar response was exhibited in the valsartan group (P<0.05; Table III).

PAS staining indicated clear glomerular and renal tubular structures in the control group, with opened glomerular capillaries. Conversely, the glomerular volume was significantly increased (P=0.025) and the basement membrane was thickened in the DN group, with mesangial cell hyperplasia, expansion and renal tubule vacuolization observed. In the TS formula and valsartan treatment groups, these pathological changes were markedly decreased (Fig. 1). In addition, immunohistochemical results from all TS treatment groups indicated that TS formula significantly ameliorated macrophage infiltration that was exhibited in the DN group and the valsartan group (TS^Low vs. DN group, P=0.006; TS^Med vs. DN group, P=0.004; TS^Hi vs. DN group, P=0.003; Fig. 2, Table IV).

RT-qPCR and western blotting results indicated that MIF and MCP-1 were significantly increased in the DN group compared with the normal control (P<0.01; Figs. 3 and 4). TS formula groups exhibited significantly decreased levels of these inflammatory factors, both at the protein level (Fig. 3) and mRNA level (Fig. 4), in a dose-dependent manner when compared with the DN group (P<0.01).

Furthermore, the SIRT1 and NF-κB expression levels following different treatments were studied. Protein (Fig. 5) and mRNA (Fig. 6) expression levels of SIRT1 were significantly decreased and NF-κB expression levels were significantly increased in the DN group when compared with the normal control (P<0.01). Conversely, treatment with either low-medium- or high-dose TS formula reversed this trend, with the high-dose TS group exhibiting similar expression levels exhibited in the valsartan group.