Male six-week-old Sprague Dawley rats (n=86; body weight, 231–267 g) were provided by Vital River Laboratory Animal Technology Co., Ltd. [Beijing, China; certificate no. SCXK (Beijing) 2007-0001], and were bred in the Experimental Animal Center (specific pathogen-free level) of the Peking Union Medical College Hospital (Beijing, China). Rats were acclimated for two week before the commencement of the experiment. This study was carried out in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee of the Peking Union Medical College Hospital.

The diabetic rat model was induced as previously described (20). After a two-week adaptation period, rats were randomly divided into the normal and diabetic model (DM) groups. After a 12-h fast, a 0.45% streptozotocin (60 mg/kg) solution (Sigma, St. Louis, MO, USA) prepared in 0.1 mol/l citrate buffer (pH 4.5), was intraperitoneally injected in rats of the DM group, while rats in the normal group were injected with the same volume of 0.1 mol/l citrate buffer. Blood glucose was measured from the tail tip 72 h later using a blood glucose meter (MediSense® Optium™; Abbott Laboratories, Chicago, IL, USA). Blood glucose levels ≥16.7 mmol/l indicated model success, and the success rate was 92.1% (70/76). Consequently, 70 successfully modeled diabetic rats were randomly divided into five groups (n=14/group): DM control, JMT low-dosage (JMT-L), JMT medium-dosage (JMT-M), JMT high-dosage (JMT-H) and vitamin C (VC). The normal control group (Con) included 10 rats with blood glucose levels <7.0 mmol/l. A rat of the JMT-L group died in the initial stage of modeling. Twelve weeks after modeling, eight rats died: three in the DM group, one in each of the JMT-L, -M and -H groups and two in the VC group. Sixteen weeks after modeling, one rat in the JMT-H group died from unsuccessful gavage surgery.

JMT [composed of milkvetch root (Radix Astragali), raw rehmannia root (Radix Rehmanniae), red sage root (Rhizoma Corydalis), kudzuvine root (Herba Asari), leech (Hirudo), dodder seed (Semen Cuscutae), grossy privet fruit (Fructus Ligustri Lucidi), cassia bark tree branchlet (Ramulus Cinnamomi) and Asarum sieboldi] is a medical preparation that is used at the Peking Union Medical College Hospital of the Chinese Academy of Medical Sciences, and is manufactured by Beijing Kowloon Pharmaceutical Co., Ltd. (Beijing, China) (18). Each capsule contained 0.35 g crude drug (lot no. 061019). The rationale (18) behind the use of the various herbs in the JMT formulation according to their use in Chinese medicine is the following: Dodder seed calms yang and nourishes yin of the kidney, secures essence (retains jing or the ‘essence’ of the kidney), improves vision and alleviates diarrhea; glossy privet fruit nourishes yin of the liver and kidney and clears empty-heat (too much yang or heat pushed up from the kidney); leech and red sage root break blood (improve blood flow), expel stasis and relieve pain; milkvetch root and raw rehmannia root calm qi and nourish yin; cassia twig and kudzuvine root warm and unblock channels and vessels, and promote qi and blood circulation.

As an antioxidant, vitamin C can improve oxidative stress in diabetics (21), and it was therefore used as a positive control. Vitamin C (0.1 g/tablet) was purchased from Beijing Double-Crane Pharmaceutical Co., Ltd. (Beijing, China; lot no. 080213).

Following model success, the JMT-L group received 0.44 g/kg/day JMT, the JMT-M group received 0.88 g/kg/day and the JMT-H group received 1.75 g/kg/day. These doses were calculated based on the crude drug content of the capsules. The VC group received 0.05 g/kg/day vitamin C. These drugs were prepared in distilled water. The DM and Con groups were orally fed distilled water (10 ml/kg/day). The rats were treated for 16 weeks. Body weight and tail tip blood glucose levels were determined prior to JMT administration, and at weeks 4, 8, 12 and 16.

After 16 weeks of treatment, prior to the sacrifice of the rats, mechanical pain threshold assessment was performed using the von Frey Pain Measurement Instrument (cat. no. 2391; IITC Life Science Inc., Woodland Hills, CA, USA). The rats were placed in an elevated metal net, and covered with transparent organic glass. After a 15-min adaptation period, the middle of the hind foot of each rat was vertically stimulated with the electronic Von Frey probe, making it appear slightly S-shaped, and the paw withdrawal response was observed. A quick flinching reaction immediately subsequent to stimulation was considered to be a positive reaction, and the values (g) were recorded. A paw withdrawal reaction caused by physical activity was not reported as positive.

All rats were sacrificed after 16 weeks of treatment. The rats were intraperitoneally injected with 12% urethane (10 ml/kg) and were fixed on the operating table in the prone position. The skin was cut between the biceps femoris and semimembranosus, along the outside line of the femoral shaft and entering into the vastus lateralis muscle. Using glass needles, blunt dissection was performed to expose the sciatic nerve along the two shafts. A total of 2 cm nerve was fixed using 4% polyformaldehyde, and the remaining nerve tissue was frozen in liquid nitrogen for polymerase chain reaction (PCR) and western blot analyses.

Total mRNA was extracted from sciatic nerve tissue using TRIzol (Tiangen Biotech Co., Ltd., Beijing, China), according to the manufacturer's instructions. RNA purity was determined using absorbance at 260 and 280 nm (A260/280). RNA integrity was verified by electrophoresis on formaldehyde gels. Total RNA was reverse-transcribed into cDNAs using oligo (dT) 12–18 primer (Takara, Dalian, China) and Moloney Murine Leukemia Virus reverse transcriptase (Takara).

Primer Express® 5.0 software (Applied Biosystems, Foster City, CA, USA) and Oligo 6.0 biological software (Molecular Biology Insights, Inc., Colorado Springs, CO, USA) were used to design the primers shown in Table I. mRNA quantification was performed via qPCR using the BioEasy SYBR Green I Real-Time PCR kit (Bioer Technology Co., Ltd., Hangzhou, China) in a LineGene real-time PCR detection system (Bioer Technology Co., Ltd.). The qPCR reaction program was as follows: Pre-incubation at 95°C for 2 min; amplification at 95°C for 20 sec, 59°C for 25 sec and 72°C for 30 sec, for 45 cycles. The melting curve showed increments of 0.5°C/sec between 65 and 95°C, for a total of 20 sec. The threshold cycle (Ct) value was defined as the number of PCR cycles in which the fluorescence signal exceeded the detection threshold value. ΔCt was initially calculated using the equation Ct_Gene-Ct_β-actin, and then 2^−ΔCt was calculated to represent the relative mRNA expression of the target genes. β-actin was used as a reference gene.

Paraffin sections (5-µm) were made from sciatic nerves. The sections were dewaxed, washed with 0.01 mmol/l phosphate-buffered saline (PBS) for 5 min and boiled in citric acid (pH 6.0) for 2 min. Subsequent to cooling, the sections were rinsed three times for 2 min in 0.01 mmol/l PBS. Endogenous peroxidase was neutralized with 3% H₂O₂ for 30 min, and the sections were rinsed three times for 2 min in 0.01 mmol/l PBS. Bovine serum albumin (3%) was added and incubated for 20 min at room temperature. The following primary antibodies were used for incubation at 4°C, overnight: Rabbit anti-8-OHdG monoclonal antibody (1:100; Japan Institute for the Control of Aging, Nikken SEIL Co., Ltd., Shizuoka, Japan; cat.no. MOG-020P), rabbit anti-NADPH oxidase subunit p22^phox polyclonal antibody (1:400; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA; cat.no. SC20781), rabbit anti-Bcl-2 polyclonal antibody (1:200; Santa Cruz Biotechnology, Inc.; cat.no. SC783) or rabbit anti-caspase 3 (17 kDa) polyclonal antibody (1:100; Bioworld Technology Inc., Nanjing, China; cat.no. BS7004). The negative control was 0.01 mmol/l PBS. The sections were rinsed three times for 2 min in 0.01 mmol/l PBS prior to the addition of the secondary antibody (horseradish peroxidase (HRP)-conjugated goat anti-rabbit, cat.no. PV9001; Zhongshan Goldenbridge Biotechnology Co., Ltd., Beijing, China) and incubated at room temperature for 20 min. Subsequent to rinsing three times for 2 min in 0.01 mmol/l PBS, 50 µl 3,3′-diaminobenzidine solution (Zhongshan Goldenbridge Biotechnology Co., Ltd.) was added. The sections were then counterstained with hematoxylin and observed using a Leica DM3000 microscope and the Leica image acquisition system (Leica Microsystems, Wetzlar, Germany). Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA) was used for image analysis. Immunohistochemistry results were measured using the integrated optical density.

Total proteins were extracted from the rat sciatic nerves using the Pierce Cell Lysis reagents (Thermo Fisher Scientific, Inc., Waltham, MA, USA) for protein extraction. The resulting solution was centrifuged at 10,000 xg and 4°C, for 10 min. The bicinchoninic acid (BCA) method (BCA Protein Assay kit; Thermo Fisher Scientific, Inc.) was used to determine the protein concentrations. The same quantity of proteins (50 µg) was separated by 6% SDS-PAGE, and transferred onto polyvinylidene fluoride membranes (GE Healthcare, Waukesha, WI, USA). Non-specific sites were blocked with 5% powdered milk diluted in Tris-buffered saline with 0.05% Tween 20 (TBST) for 30 min at room temperature. Proteins were detected overnight at 4°C, using the following primary antibodies: Anti-cleaved-PARP-1 (89 kDa) polyclonal antibody (Cell Signaling Technology, Inc., Danvers, MA, USA; cat.no. 9542s) and β-actin (Sigma, St. Louis, MO, USA; cat.no. A3853). Subsequent to being washed with TBST, the membranes were incubated with HRP-conjugated immunoglobulin G (Zhongshan Goldenbridge Biotechnology Co., Ltd.) for 1 h at room temperature. The membranes were then further washed with TBST, and the proteins were detected using an enhanced chemiluminescence reagent (Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions, using a FluorChem IS-8800 imaging system (Alpha Innotech Corp., San Leandro, CA, USA). The bands were quantified using the AlphaEaseFC™ software (Alpha Innotech Corp.).

SPSS 13.0 software (SPSS Inc., Chicago, IL, USA) was used for the statistical analysis. The one-sample Kolmogorov-Smirnov Z-test was used to determine if the variables were normally distributed. Normally-distributed data are expressed as the mean ± standard deviation. Multi-group independent samples were compared using one-way analysis of variance, with the least significant difference post hoc test. Non-normally-distributed data were analyzed with non-parametric tests. P<0.05 was considered to indicate a statistically significant difference.

Fasting blood glucose levels in all the diabetic rats were higher than those in the control group rats at all time-points (Table II, all P<0.01). No significant differences in fasting blood glucose levels were found among the DM, VC and JMT groups at any time-points (Table II, all P>0.05).

No significant differences in body weight were observed at baseline among the different groups (all P>0.05). After 4 weeks of treatment, the body weight of the diabetic rats was lower than that of the control group rats (all P<0.01). No significant differences in body weight were found among the DM, VC and JMT groups at any time-points (Table III, all P>0.05).

The mechanical pain threshold values are listed in Table IV. Compared with the normal controls, the mechanical pain threshold values were significantly lower in the DM group (P<0.01). The mechanical pain threshold values of each treatment group were higher than those of the DM group (all P<0.01). Among the JMT groups, the mechanical pain threshold was significantly higher in the JMT-M group compared with that in the JMT-L, JMT-H and VC groups (all P<0.01).

The DM group exhibited a 2.1-fold increase in 8-OHdG levels compared with the Con group (P<0.01). Compared with the DM group, 8-OHdG was decreased in each treatment group (JMT-L, −11.2%; JMT-M, −30.0%; JMT-H, −22.3% and VC, −20.2%; all P<0.01) (Fig. 1). The decrease in the JMT-M group was more notable than that in the JMT-L, JMT-H and VC groups (all P<0.01).

A marked increase in NADPH p22^phox mRNA and protein levels was observed in the DM group compared with the Con group (77.6- and 7.6-fold, respectively, both P<0.01). The increased NADPH p22^phox mRNA and protein levels in the DM group were then decreased in each treatment group. The results for the protein levels were as follows: JMT-L, −56.1%; JMT-M, −83.1%; JMT-H, −67.8% and VC, −41.4% (all P<0.05) (Fig. 2A and B).

In the DM group, a strong decrease in Bcl-2 mRNA and protein levels was induced compared with the Con group (−79.2 and −87.2%, respectively, both P<0.01). Compared with the DM group, the Bcl-2 levels were increased in each treatment group. The results for the protein levels were as follows: JMT-L, 4.0-fold; JMT-M, 4.3-fold; JMT-H, 2.5-fold and VC, 1.3-fold (all P<0.01) (Fig. 3A and B). The increase was most evident in the JMT-M group compared with the JMT-L, JMT-H and VC groups (all P<0.01).

Caspase 3 mRNA and protein levels in the DM group were markedly increased compared with those in the Con group (11.7- and 5.9-fold, respectively, both P<0.01). This increase was then decreased in each treatment group. The results for the protein levels were as follows: JMT-L, −36.6%; JMT-M, −77.6%; JMT-H, −48.3% and VC, 23.9% (all P<0.01) (Fig. 4A and B).

The DM group exhibited a significant increase in cleaved-PARP-1 levels compared with the Con group (62.6-fold, P<0.01). The cleaved-PARP-1 levels were decreased in each treatment group compared with those in the DM group (JMT-L, −25.4%; JMT-M, −62.5%; JMT-H, −39.4% and VC, 27.0%; all P<0.05) (Fig. 5). The decrease in the JMT-M group was more notable than that in the JMT-L, JMT-H and VC groups (all P<0.01).