Contributed equally
Diosgenin is a steroidal saponin extract from numerous plants, including
Neuropathic pain is a major chronic condition arising from injury or disease affecting the peripheral or central nervous system (
Accumulating evidence has demonstrated that nerve injury-induced inflammatory cytokines and reactive oxygen species (ROS) serve important roles in the progress of neuropathic pain (
Diosgenin is a steroidal saponin extract from numerous plants, including
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 (
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 (
Heat hypersensitivity was tested using a plantar test (cat. no. 7370; Ugo Basile Srl, Varese, Italy) according to a method described previously (
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 (
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 (
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 (
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
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 (
The present study demonstrated that diosgenin reversed CCI-decreased mechanical withdrawal threshold and thermal withdrawal latency. Diosgenin inhibited CCI-induced increased levels of the pro-inflammatory cytokines TNF-α, IL-1β and IL-2, and suppressed oxidative stress induced by CCI in the spinal cord. Furthermore, diosgenin significantly inhibited protein expression levels of p-p38 MAPK and NF-κB in the spinal cord induced by CCI.
The CCI model is the most commonly employed neuropathic pain model of nerve damage-induced allodynia/hyperalgesia (
Increasing evidence suggests that peripheral nerve injury contributes to neuropathic pain via upregulation of pro-inflammatory cytokines (
Previous studies have indicated that CCI produces significant oxidative damage in the sciatic nerve due to the increase in lipid peroxidation and ROS concentration (
Previous studies have demonstrated that p-p38 MAPK in spinal cord glial cells after peripheral nerve injury are involved in the development of neuropathic pain (
In conclusion, the present study demonstrated that diosgenin may be effective to reduce neuropathic pain by inhibition of activation of the p38 MAPK and NF-κB signaling pathways. These results implicate diosgenin in the treatment of neuropathic pain, which merits further clinical investigation.
Effects of diosgenin on mechanical allodynia and thermal hyperalgesia. (A) PWT and (B) PWL were measured following intraperitoneal injection of 10, 20 and 40 mg/kg diosgenin. All experiments were repeated at least three times. Data are presented as the mean ± standard deviation (n=3/group). *P<0.05 vs. sham group; #P<0.05 vs. CCI group. PWT, paw withdrawal threshold; PWL, paw withdrawal latency; CCI, chronic constriction injury.
Effect of diosgenin on pro-inflammatory cytokine expression in the spinal cord following CCI. The expression levels of (A) TNF-α, (B) IL-1β and (C) IL-2 were detected by ELISA. All experiments were repeated at least three times. Data are presented as the mean ± standard deviation (n=3/group). *P<0.05 vs. sham group; #P<0.05 vs. CCI group. TNF-α, tumor necrosis factor-α; IL, interleukin; CCI, chronic constriction injury.
Effect of diosgenin on oxidative stress in the spinal cord following CCI. The rats were sacrificed 24 h after the last administration. The levels of (A) MDA and (B) GSH-PX in the lumbar spinal cords were estimated using MDA and GSH-PX kits. All experiments were repeated at least three times. Data are presented as the mean ± standard deviation (n=3/group). *P<0.05 vs. sham group; #P<0.05 vs. CCI group. CCI, chronic constriction injury; MDA, malondialdehyde; GSH-PX, glutathione peroxidase.
Effect of diosgenin on p-p38 MAPK expression levels in the spinal cord following CCI. (A) Representative western blot images and (B) quantification of protein expression levels of p-p38 MAPK in the spinal cord. GAPDH served as a loading control. All experiments were repeated at least three times. Data are presented as the mean ± standard deviation (n=3/group). *P<0.05 vs. sham group; #P<0.05 vs. CCI group. CCI, chronic constriction injury; MAPK, mitogen activated protein kinase; p, phosphorylated.
Effect of diosgenin on NF-κB activation in the spinal cord following CCI. (A) Representative western blot images and (B) quantification of p-NF-κB p65 protein expression levels in the spinal cord. GAPDH served as a loading control. Data are presented as the mean ± standard deviation (n=3/group). *P<0.05 vs. sham group; #P<0.05 vs. CCI group. CCI, chronic constriction injury; NF-κB, nuclear factor-κB; p, phosphorylated.