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 injection compared with untreated CFA mice [(F2, 76 = 12.48 and P = 0.0003), Time (F4, 76 = 9.34 and P < 0.0001), and Time × Treatment interaction (F8, 76 = 1.56 and P = 0.1525)]. This revealed that TPN alleviated CFA-induced paw edema and has a strong anti-inflammatory effect.
For thermal hyperalgesia, an analysis of behavior data after TPN treatments by two-way ANOVA revealed a significant effect of Treatment [(F2, 76 = 13.88 and P = 0.0002), Time (F4, 76 = 40.92 and P < 0.0001), and Time × Treatment interaction (F8, 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, F2, 76 = 7.94 and P < 0.0031; Time, F4, 76 = 9.60 and P < 0.0001; Interaction, F8, 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). CFA injection with vehicle treatment increased pAKT-immunofluorescence (IF) 5 days after CFA injection (P < 0.001, Fig. 2C and 2D). In TPN-treated animals, both the number of pAKT positive cells and the intensity of pAKT-IF were significantly reduced (P < 0.001, Fig. 2C and 2D), which was consistent with the Western blotting results. Double immunostaining further showed that pAKT was expressed in the DRG neurons (Fig. 2E).
TPN inhibits CFA-induced the increase of TNF-α, IL-1β, and IL-6 in the DRG
TNF-α, IL-1β and IL-6 are important proinflammatory cytokines in mediating peripheral sensitization and neuropathic pain [23–25]. To check whether the anti-nociceptive effect of TPN is associated with the downregulation of proinflammatory 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, TNF-α, 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). Both the dose of 0.5 mg/kg and 2.0 mg/kg TPN treatments significantly reduced the CFA-induced mRNA increases of TNF-α, IL-1β, and IL-6 (0.05 mg/kg: P < 0.05, P < 0.01, P < 0.05; 2.0 mg/kg: P < 0.05, P < 0.001, P < 0.05; respectively, one-way ANOVA, Fig. 3. A-C) These data indicate the inhibitory effect of TPN on CFA-induced TNF-α, IL-1β, and IL-6 expression in the DRG.
TPN attenuates LPS-induced pAKT activation, and TNF-α, IL-1β, and IL-6 expression in the ND7/23 Cells
To further confirm that TPN is able to attenuate pAKT activation in DRG neurons under inflammatory conditions, we used the classical exogenous toll-like receptor 4 ligand LPS to stimulate ND7/23 cells, a DRG neuron cell line, to mimic inflammatory conditions in vitro. We first checked pAKT expression after incubation with LPS (1 µg/mL) for 6 hours. Western blotting showed that LPS induced a rapid and dramatic increase of pAKT expression (P < 0.001, one-way ANOVA). Preincubation with TPN, 1 hour before LPS application, decreased pAKT expression by 43.2% (P < 0.05, one-way ANOVA, Fig. 4. A and B), which was consistent with the in vivo results (Fig. 2. A and B).
To examine whether TPN inhibit TNF-α, IL-1β, and IL-6 expression via pAKT, we pretreated ND7/23 Cells with AKT inhibitor, AKT inhibitor Ⅳ, 1 h before LPS application. As shown in Fig. 4C-E, the qRT-PCR results showed that AKT inhibitor Ⅳ (1 µg/mL) treatment blocked CFA-induced TNF-α, IL-1β, and IL-6 increase (P < 0.001, P < 0.01, and P < 0.001 respectively, in one-way ANOVA). In consistent with AKT inhibitor Ⅳ’s effect, pretreatment with TPN (1 µg/mL) also significantly decreased LPS-induced TNF-α, IL-1β, and IL-6 upregulation (P < 0.01, P < 0.05, and P < 0.001 respectively, one-way ANOVA). These data suggest that TPN is involved in pAKT-mediated upregulation of TNF-α, IL-1β, and IL-6 in activated ND7/23 cells by LPS.
AKT inhibitor attenuates CFA-induced pain hypersensitivity and upregulation of TNF-α, 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, F1, 33 = 5.35 and P = 0.0411; Time, F3, 33 = 4.09 and P = 0.0142; Interaction, F3, 33 = 3.26 and P = 0.0335) and thermal hyperalgesia (Treatment, F1, 33 = 5.18 and P = 0.0113; Time, F3, 33 = 4.32 and P = 0.0113; Interaction, F3, 33 = 6 and P = 0.0022) at 3 and 6 h, but not 24 h after injection (Fig. 5. A and B). As shown in Fig. 5C-5E, AKT inhibitor Ⅳ treatment for 3 h also significantly blocked the mRNA expression of TNF-α, and IL-1β (P < 0.05). Taken together, these data suggest that AKT signaling pathway is involved in the development of CFA-induced inflammatory pain, and inhibition of AKT phosphorylation reduces the upregulation of inflammatory cytokines in the DRG.