Intravenous administration of triptonide attenuates CFA-induced pain hypersensitivity through inhibiting AKT signaling pathway in mice

Background: Triptonide (TPN) is a major component of Tripterygium Hook.f., and reportedly has anti-inflammatory and neuroprotective effects. Recent studies have demonstrated that the phosphatidylinositol 3-kinase (PI3K)/AKT pathway plays an important role in the pathogenesis of inflammatory pain. Here we investigated the anti-nociceptive effect of systemic treatment with TPN on mouse models of chronic inflammatory pain and explored possible mechanisms. Results: Unilateral hind paw injection of complete Freund’s adjuvant (CFA) induced paw edema and persistent pain hypersensitivity. Intravenous treatment with TPN attenuated CFA-induced paw edema, mechanical allodynia, and thermal hyperalgesia. Western blotting and immunofluorescence results showed that CFA induced AKT activation in the dorsal root ganglion (DRG) neurons, which was inhibited by TPN treatment. Furthermore, TPN treatment inhibited mRNA increase of proinflammatory cytokines [tumor necrosis factor-α (TNF-α), interleukin 1 beta (IL-1β), and Interleukin 6 (IL-6)] induced by CFA. Finally, pretreatment with AKT inhibitor, AKT inhibitor Ⅳ, attenuated CFA-induced mechanical allodynia and thermal hyperalgesia, and decreased the mRNA expression of pro-inflammatory cytokines. Conclusions: These data indicate that TPN attenuates CFA-induced pain potentially via inhibiting AKT-mediated pro-inflammatory cytokines production in DRG. TPN may be used for the treatment of chronic inflammatory pain.

Tripterygium wilfordii Hook. f. and has been used as a part of systematic medication to treat a wide variety of diseases, including cancer, lupus, rheumatoid arthritis, Alzheimer's disease, Parkinson's disease, and rheumatoid arthritis [1][2][3]. TPL can effectively relieve neuropathic pain by inhibiting the activation of microglia and astrocytes in the spinal dorsal horn [4,5]. TPL also attenuates cancer pain via suppressing the up-regulation of Chemokine (C-C motif) ligand 5 and histone deacetylases in the spinal glial cells [6,7]. In addition, TPL inhibits the activation of extracellular signal-regulated kinase (ERK) pathway and the production of inflammatory cytokines in the spinal cord dorsal horn induced by inflammation [8].
Recently, TPL was reported to have a potent anti-depressive function by its influences on hippocampal neuroinflammation in a rat model of depression comorbidity of chronic pain [2]. However, clinical reports showed that TPL exposure resulted in the injury of some organs, including liver, kidney, heart, testes, and ovary in humans [9-11]. Severe hepatotoxicity was also shown after TPL exposure in animals [9, 12, 13].
Triptonide (TPN) is another bioactive component of Tripterygium wilfordii Hook. f.. It was reported that TPN does not induce liver toxicity in animals [1,3,14]. The differences between the chemical structures of TPL and TPN are the substituent groups at C-14 position in which TPL is with C-14-hydroxyl and TPN is with C- 14-carbonyl. The metabolomics study shows that the hydroxyl group at C-14 in the molecular structure of TPL plays an important role in its hepatotoxicity [14,15]. TPN acts as a novel potent anti-cancer drugs with low toxicity [16][17][18].
Phosphoinositide 3-kinase/protein kinase B/the mammalian target of rapamycin (PI3K/AKT/mTOR) pathway could be activated in DRG neurons and spinal glial cells in different pain models [19,20]. Up-regulating spinal mTOR activity by knocking down the mTOR-negative regulator reduced morphine analgesia and produced pain 4 hypersensitivity [21]. Recent studies showed that TPN can inactivate AKT and induce caspase-dependent death in cervical cancer cells [22]. TPN has also been shown to remarkably diminish both ERK and AKT signaling pathways in lymphoma [1]. In addition, TPN can efficaciously suppress PCa growth in vitro and in vivo via inhibiting the phosphorylation of mTOR and the activities of related downstream signaling pathways [3]. Although the analgesic function of TPL has been studied, whether TPN has an analgesic effect and the underlying mechanisms on inflammatory pain remains unknown.
In this study, we investigated whether systemic treatment with TPN can attenuate nocifensive behaviors in complete Freund's adjuvant (CFA)-induced inflammatory pain model. We also explored the possible analgesic mechanisms of TPN by assaying the activation of AKT pathway and the production of inflammatory cytokines.

Triptonide attenuates CFA-induced inflammatory pain
To test the anti-nociceptive effect of TPN in CFA-induced inflammatory pain, different doses of TPN or vehicle were intravenously injected daily for 5 consecutive days, with the first injection of TPN given at 1 h before CFA (Fig. 1A). On Day 1, the left hind paw volume of the CFA group was increased by 2 times compared to the baseline (Fig. 1B). Figure 1B showed that TPN at 0.5 mg/kg or 2.0 mg/kg resulted in a statistically significant decrease of paw swelling from 3 days to 5 days after CFA For thermal hyperalgesia, an analysis of behavior data after TPN treatments by twoway ANOVA revealed a significant effect of Treatment [(F 2, 76 = 13.88 and P = 0.0002), Time (F 4, 76 = 40.92 and P < 0.0001), and Time × Treatment interaction (F 8, 76 = 2.12 and P = 0.0442)]. The Bonferroni post hoc tests showed that TPN at 2 mg/kg had no effect on CFA-induced thermal hyperalgesia in the first 2 days after CFA injection, but started to show a reversal effect at 3 days and maintained till 5 days (Fig. 1C). TPN at 0.5 mg/kg also significantly attenuated CFA-induced thermal hyperalgesia from 3 days to 4 days after CFA injection (Fig. 1C). Meanwhile, TPN attenuated CFA-induced mechanical allodynia (Treatment, F 2, 76 = 7.94 and P < 0.0031; Time, F 4, 76 = 9.60 and P < 0.0001; Interaction, F 8, 76 = 2.92 and P = 0.0067). TPN at 2 mg/kg or at 0.5 mg/kg attenuated mechanical allodynia at 4 days and 5 days after CFA injection (Fig. 1D). These data suggest that repeated TPN administration attenuates CFA-induced pain hypersensitivity, especially thermal hyperalgesia TPN inhibits CFA-induced AKT activation in DRG To determine whether the analgesic effects of TPN were associated with inhibition of the AKT signaling pathways, we evaluated the expression level of phosphorylation of AKT (pAKT) in the DRG. Western blotting showed that pAKT was significantly increased at 5 days after CFA injection (P < 0.01). Pretreatment with TPN (2 mg/kg) significantly decreased CFA-induced pAKT upregulation (P < 0.01, Fig. 2A and B). cytokines, we checked TNF-α, IL-1β, and IL-6 expression in the DRG after repeated TPN administration. qPCR results showed that, compared with control animals, TNFα, IL-1β, and IL-6 mRNAs were significantly increased in animals after 5 days of CFA injection (P < 0.05, P < 0.01, P < 0.05, respectively, one-way ANOVA, Fig. 3. A-C).
AKT inhibitor attenuates CFA-induced pain hypersensitivity and upregulation of TNFα, IL-1β, and IL-6 in DRG To define whether AKT is associated with CFA-induced pain hypersensitivity and the upregulation of inflammatory cytokines, we intrathecally injected the AKT inhibitor (1 µg/10 µl) 3 days after CFA injection. The behavioral results showed that the i.t. administration of AKT inhibitor Ⅳ attenuated both mechanical allodynia (Treatment, Although the pAKT pathway typically regulates cell growth and survival, increasing evidence indicates the involvement of this pathway in the development and maintenance of chronic pain [27,28]. Our data further showed that pAKT was predominantly expressed in DRG neurons after CFA injection. In agreement with our results, pAKT expression in neurons was found in the DRG following paclitaxel treatment [29]. Here, intraplantar injection of CFA induced pain hypersensitivity, and increased pAKT expression in the DRG, which was inhibited by TPN administration, suggesting that the anti-nociceptive effect of TPN may be mediated TNF-α, IL-1β and IL-6 are well-known pro-inflammatory cytokines that have been implicated in inflammatory pain [30]. Our data showed that the expression of TNF-α, IL-1β, and IL-6 was increased in DRG neurons after CFA injection, and was inhibited by TPN treatment. TNF-α was expressed in the majority of voltage-gated sodium channel (Nav) 1.3-positive or Nav1.8-positive neurons and up-regulated the expression of Nav1.3 and Nav1.8 in DRG neurons following peripheral nerve injury [23]. Cleaved IL-1β expression was significantly increased in small-sized DRG neurons after CFA injection into the hind paw [31]. IL-6 was up-regulated in the ipsilateral L4 and L5 DRG neurons and in the bilateral lumbar spinal cord following L5-ventral root transaction and contributed to the development of neuropathic pain [25]. Therefore, TPN may attenuate pain hypersensitivity via the inhibition of TNF-α, IL-1β, and IL-6-mediated neuroinflammation.
Previous studies have demonstrated that inhibition of pAKT pathway prevented the LPS-induced expression of TNF-α in human bronchial epithelial cells [32]. Diesel exhaust particles exposure can activate the AKT signaling pathway, and further upregulate IL-1β protein expression in primary human bronchial epithelial cells [32].
Studies from Caco-2 cells and fibroblast-like synoviocytes showed that IL-17mediated induction of IL-6 was transduced via activation of AKT and NF-κB [33], while mitogen-activated protein kinase (MAPK) are not likely to participate in the process [29]. It is highly likely that the p-AKT pathway acts as an upstream signaling pathway up-regulated the expression of cytokines TNF-α, IL-1β, and IL-6 after CFA.

Conclusion
Our present study demonstrated that intravenous treatment with TPN significantly attenuated CFA-induced pain hypersensitivity, which was associated with decreased TNF-α, IL-1β, and IL-6 expression, and decreased pAKT activation in DRG neurons.
These data suggest that TPN could attenuate inflammatory pain via the inhibition of pAKT/TNF-α-IL-6-IL-1β signaling pathway axis in DRG neurons. TPN may be of therapeutic value in inflammatory pain.

Animals
Adult ICR male mice (6 to 8 weeks) were obtained from the Animal Care and Use Committee of Nantong University. Animals were kept under a 12/12 h light/dark cycle at a temperature of 23 ± 2 ℃, humidity (50%-60%) with free-feeding. All behavioral experiments were performed between 9 am and 6 pm. All in vivo studies were performed in accordance with the UK Animals Scientifc Procedures Act (1986) and were approved by the Animal Care and Use Committee of Nantong University.

CFA pain model and drug administration
The animals were randomly assigned to three groups of mice. An experimental timeline is shown in Fig. 1A

Behavioral analysis
For the von Frey test, the animals were put in boxes on an elevated metal mesh floor daily for at least 2 d before baseline testing and allowed 30 min for habituation before the examination. The plantar surface of the left hind paw was stimulated with a series of von Frey hairs with logarithmically incrementing stiffness (0.02-2.56 g, Stoelting). The 50 % paw withdrawal threshold was determined using Dixon's up-down method. For the Hargreaves test, the animals were put in a plastic box placed on a glass plate, and the plantar surface was exposed to a beam of radiant heat through a transparent glass surface (Life Science). The baseline latencies were adjusted to 10-14 s with a maximum of 20 s as a cutoff to prevent potential injury. All the behavioral experimenters were done by individuals who were blinded to the treatment of the mice.

Real-time qPCR
Total RNA was extracted from L4-6 DRG with the Trizol reagent (Invitrogen, Carlsbad, CA, USA). One microgram of total RNA was converted into cDNA using

Quantification and statistics
All data were analyzed by researchers blinded to the reagents used. All data were analyzed by GraphPad Prism (version 5.01) and presented as mean ± SEM. P < 0.05 was considered statistically significant. Behavioural data were analysed using twoway repeated measures ANOVA. Western blotting, immunofluorescence density and qPCR data were compared using one-way ANOVA. Student's t-test (2-tailed) were used to analyse qPCR data if only 2 groups were applied.

Availability of data and materials
The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request. AKT inhibitor attenuates CFA-induced pain hypersensitivity and production of TNF-α, IL-1β, an