Male Sprague-Dawley rats (n=25) weighing 180–200 g were supplied by the Experimental Animal Centre of Zhengzhou Central Hospital Affiliated to Zhengzhou University (Zhengzhou, China). The animals were housed in a room maintained at 22±1°C with an alternating 12-h light/dark cycle, and provided food and water ad libitum. The animal experimental procedures were approved and reviewed by the Institutional Animal Care and Use Committee of Zhengzhou Central Hospital Affiliated to Zhengzhou University.

Neuropathic pain was induced in experimental animals by CCI of the sciatic nerve which was performed as previously described (15). In brief, rats were anesthetized intraperitoneally with 40 mg/kg sodium pentobarbital (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Four ligatures (silk 4–0) were tied loosely around proximal bifurcation part of the nerve with 1 mm spacing between each ligature, until a brisk twitch of the right hind limb was observed. Sham surgery was performed with the sciatic nerve exposed but not ligated in control rats (n=3 per group).

Diosgenin in doses of 10, 20 and 40 mg/kg were administered intraperitoneally to neuropathic rats once a day for two weeks, starting from the first day following the induction of neuropathic pain; the sham-operated rats received normal saline (20 µl) alone, following the same treatment procedure. The rats were sacrificed by spinal dislocation 24 h after the last administration.

Mechanical allodynia was evaluated as indicated by the paw withdrawal threshold in response to von Frey filaments using the up-down method according to previously described protocol (16). In brief, rats were placed in an inverted clear plexiglass cage (23×18×13 cm) on a 3-mm-thick glass plate, and were allowed to acclimatize for 30 min before testing. The plantar surface of each hind paw was applied with pressure from below with the electronic Von Frey filament via the mesh floor. The force applied at the time of paw withdrawal was recorded.

Heat hypersensitivity was tested using a plantar test (cat. no. 7370; Ugo Basile Srl, Varese, Italy) according to a method described previously (17). In brief, the heat source was positioned under the glass floor directly beneath the hind paw. The heat intensity was set to last for ~10 sec to produce paw withdrawal latency, and the cut-off was set at 20 sec to avoid tissue damage. Each paw was measured alternatively after >5 min.

At day 14, the rats were sacrificed by spinal dislocation. Then, the lumbar spinal cord tissues (L4/5) were rapidly removed. Proteins were extracted from the lumbar spinal cord tissues (L4/5) using RIPA Cell Lysis Buffer (Takara Biotechnology, Dalian, China). Lysates were sonicated for 5 sec on ice and centrifuged at 6,000 × g for 10 min at 4°C. Supernatants were collected and the protein concentration was quantified using a Pierce Bicinchoninic Acid Protein Assay kit (Pierce; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Equal amounts of protein (30 µg) were separated by 10% SDS-PAGE and subsequently transferred to polyvinylidene difluoride membranes. The membrane was blocked with 5% non-fat dry milk in Tris-buffered saline with 0.1% Tween-20 (TBST) for 1 h at room temperature. The membrane was then incubated with a 1:1,000 dilution of the following primary antibodies, all purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA): rabbit anti-mouse phosphorylated (p)-p38 mitogen activated protein kinase (MAPK) antibody (sc-101759; 1:3,000), rabbit anti-mouse p38 MAPK antibody (sc-535; 1:2,500), rabbit anti-mouse p-NF-κB p65 antibody (sc-33020; 1:3,000;) and rabbit anti-mouse GAPDH antibody (sc-25778; 1:2,500) overnight at 4°C. Following three washes with TBST buffer, the membrane was washed and incubated with a goat anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody (sc-2030; 1:2,500) for 1 h at 37°C. Proteins were subsequently detected by Enhanced Chemiluminescence (GE Healthcare Life Sciences, Chalfont, UK) and quantified using Gel-Pro Analyzer version 4.0 software (Media Cybernetics, Inc., Rockville, MD, USA).

The levels of TNF-α, IL-1β and IL-2 in the lumbar spinal cords were measured using commercially available rat TNF-α (RAB0479), IL-1β (RAB0277) and IL-2 (RAB0288) ELISA kits (Sigma-Aldrich; Merck KGaA) according to the manufacturer's protocol. Plates were read using an ELISA reader (Omega Bio-Tek, Inc., Norcross, GA, USA) at a wavelength of 450 nm.

The levels of malondialdehyde (MDA) and glutathione peroxidase (GSH-PX) in the lumbar spinal cords were estimated by using MDA and GSH-PX kits from the Biological Engineering Research Institute (Nanjing, China).

Analysis was performed using SPSS 16.0 software (SPSS, Inc., Chicago, IL, USA). All data are presented as the mean ± standard deviation. The data of behavioral tests were analyzed by two-way analysis of variance, while the data of cytokine assays were analyzed by one-way analysis of variance, followed by Newman-Keuls post hoc test. P<0.05 were considered to indicate a statistically significant difference.

The effects of diosgenin on mechanical allodynia and thermal hyperalgesia were examined. CCI resulted in significant development of mechanical allodynia (Fig. 1A) and thermal hyperalgesia (Fig. 1B), as compared with the sham group as assessed on day 1, 7 and 14. However, diosgenin treatment reversed CCI-induced mechanical allodynia and thermal hyperalgesia in a dose-dependent manner.

There is strong evidence that pro-inflammatory cytokines have important roles in the pathology of neuropathic pain. Thus, the present study examined the effects of diosgenin on pro-inflammatory cytokine levels in spinal cord by ELISA. The levels of TNF-α (Fig. 2A), IL-1β (Fig. 2B) and IL-2 (Fig. 2C) were significantly increased in the spinal cord of CCI rats compared with the sham group. However, diosgenin reversed CCI-increased levels of TNF-α, IL-1β and IL-2 in a dose-dependent manner.

The effects of diosgenin on oxidative stress in spinal cord were examined by ELISA. Rats in the CCI group exhibited a significant increase in the production of MDA (Fig. 3A) and decrease in the content of GSH-PX (Fig. 3B), compared with the sham group. Diosgenin treatment obviously reversed CCI-induced oxidative stress in spinal cord in a dose-dependent manner.

It has been reported that activation of p-p38 MAPK contributes to the development of inflammatory and neuropathic pain induced by nerve injury. Therefore, the effects of diosgenin on phosphorylation of p38 MAPK in spinal cord were investigated. As presented in Fig. 4, protein expression levels of p-p38 MAPK were greatly increased by CCI, compared with the sham group. However, diosgenin treatment significantly inhibited the expression level of p-p38 MAPK in the spinal cord of CCI rats.

The NF-κB signaling pathway serves a key role in regulating the expression of pro-inflammatory and pain mediators. To investigate the underlying mechanism of diosgenin in CCI-induced neuropathic pain, protein expression levels of p-NF-κB p65 in spinal cord of CCI rats were detected. Western blot analysis demonstrated that the CCI group had significantly increased levels of p-NF-κB p65, compared with the sham group. However, diosgenin markedly decreased the expression of p-NF-κB p65 in the spinal cord of CCI rats, in a dose-dependent manner (Fig. 5).