The effect of sinomenine on formalin-induced pain behavior
I investigated the analgesic effect of sinomenine using formalin model as it is a reliable model for testing the analgesic effect and mechanism of a drug39-
41. Injection of 1% formalin (i.pl.) elicited typical biphasic pain behavior (Fig.
6Aa). As compared with the vehicle group, 25 mg/kg (i.p.) sinomenine did not induce statistically significant decrease in formalin-induced pain behavior (Fig.
6Ab). However, sinomenine treated group (i.p.) had a significant analgesic effect in both the first phase and second phase, at the dose of 50 mg/kg and 75 mg/kg (Fig. 6Ab). In the first phase, formalin-induced pain behavior was reduced by 45
± 9.8% at the dose of 50 mg/kg (*p<0.05) and 81.4 ± 5.1% at the dose of 75 mg/kg, respectively (***p<0.001). In the second phase, formalin-induced pain behavior was reduced by 38.5 ± 6.8% at the dose of 50 mg/kg (**p<0.01) and 96.3 ± 2% at the dose of 75 mg/kg, respectively (***p<0.001).
The expression of c-Fos protein is a measure of acute nociception following noxious stimulation42. Thus, it is commonly used to show the analgesic property of the drug43. Injection of 1% formalin (i.pl.) caused high c-Fos expression in superficial dorsal horn innervated by L4-5 DRG neurons (Fig. 6B).
As compared with the vehicle group, sinomenine (50 mg/kg i.p.) reduced the number of c-Fos positive neurons in superficial dorsal horn by 32.1 ± 10.1%
(*p=0.0292) (Fig. 6B).
Collectively, these results showed that sinomenine produces an analgesic effect in acute inflammatory pain, suggesting that peripheral nociceptive neurons might be targeted by sinomenine.
The effect of sinomenine on APs and VGSCs in small-sized DRG neurons
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I next investigated the effects of sinomenine on the excitability of small- sized DRG neurons (<20 μm diameter). 1mM sinomenine increased the spike threshold from –31 ± 1.6 mV to –26.6 ± 2.1 mV (**p=0.0023) and the current threshold by 21.5 ± 5% (**p=0.0032) (Fig 6A), and also significantly decreased AP amplitude by 18.1 ± 3.2% (**p=0.0013) and AP overshoot level by 28.1 ± 4.9% (**p=0.0021) (Fig. 7A). Concomitantly, half- width of AP was increased by 89.3 ± 29.9% (**p=0.0057) (Fig. 7A). When I examined the effect of sinomenine on a series of APs, I found that AP firing frequency was also decreased by 3 mM sinomenine and restored partially after washout (Fig. 7B).
Compared with the control, 3 mM sinomenine reduced AP firing frequency by 39.5 ± 6% (**p=0.007).
Because the VGSCs are responsible for the generation of the APs, I tested the effect of sinomenine on INa in small-sized DRG neurons by a command pulse stepped from -90 to -10 mV. As compared with the control, 3 mM sinomenine significantly inhibited INa in small-sized DRG neurons by 63.3 ± 3.2%
(***p<0.001) (Fig. 8A). INa was decreased by sinomenine in a dose-dependent manner with 0.1 mM by 7.7 ± 1%, 0.3 mM by 13 ± 2%, 1 mM by 29.9 ± 2.3%, 3 mM by 63.3 ± 3.2% and 10 mM by 83.3 ± 3.2%. (Fig. 8B, IC50 = 2.3 ± 0.2 mM).
These results suggest peripheral action of sinomenine on VGSCs in nociceptive neurons, which might be associated with analgesic effect of sinomenine on acute inflammatory pain.
Intraplantar application of sinomenine on formalin-induced pain behavior To clarify the peripheral action of sinomenine, I then examined whether peripheral injection of sinomenine indeed produces analgesic effects. I found that
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Injection of 500 μg sinomenine (i.pl.), co-administrated with 1% formalin, only reduced the first phase of formalin-induced pain behavior by 38.2 ± 5.9%
(**p=0.0057), whereas the second phase of formalin-induced pain behavior was not affected by sinomenine (Fig. 9).
The effect of sinomenine on motor function in rota-rod test
Because drug-induced motor function impairment may result in false positive results in nociceptive pain behavior, I carried out rota-rod test to examine the effect of sinomenine on motor function. I did not observe motor activity impairment at the dose of 25 mg/kg and 50 mg/kg, whereas there was a significant motor dysfunction at the dose of 75 mg/kg (Fig. 10). 75 mg/kg sinomenine (i.p.) significantly increased number of falls (*p=0.0267) and decreased performance time (***p=0.0002) in rota-rod test.
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Figure 6.
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Figure 6. Effects of sinomenine on formalin-induced pain
(A) Effects of sinomenine (SIN) on the time course curves of formalin-induced pain behavior (a). Formalin-induced pain behavior was divided into two phases and analyzed (b). The bar graph represented first phase (0-10 min) and second phase (10-40 min) of formalin-induced pain behavior. Intraperitoneal (i.p.) injection of sinomenine inhibited formalin-induced pain behavior in both first phase and second phase at the dose of 50 mg/kg or 75 mg/kg. *p<0.05, **p<0.01 and ***p<0.001 (one-way ANOVA followed by Tukey’s test) (B) Intraplantar injection of 1% formalin induced c-Fos expression in superficial dorsal horn of Lumbar 4-5 (L4-5) segments (a). Sinomenine (SIN, 50 mg/kg, i.p.) decreased formalin-induced c-Fos expression in superficial dorsal horn of L4-5 (b). The quantification of c-Fos expression (c). 50 mg/kg sinomenine suppressed formalin-induced c-Fos expression in superficial dorsal horn of L4-5 as compared with vehicle. *p<0.05 (unpaired t test, two-tailed), Scale bar = 200 μm, SDH; superficial dorsal horn
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Figure 7.
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Figure 7. Effects of sinomenine on action potentials in small sized dorsal root ganglion neurons
(A) The inhibitory effect of sinomenine on action potentials (APs). A representative APs trace (a). 1 mM sinomenine (SIN) increased the spike threshold (b) and the current threshold (c), and inhibited AP amplitude (d) and peak (e). After application of 1 mM sinomenine, half-width of AP was increased (f). (B) sinomenine decreased APs firing frequency. Series of APs were recorded during 500 ms depolarizing current step. 3 mM sinomenine inhibited APs firing frequency (n=4). Series of APs returned partially after washout. **p<0.01 (paired t test, two-tailed)
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Figure 8.
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Figure 8. Effects of sinomenine on voltage-gated sodium channel in small sized dorsal root ganglion neurons
(A) 3 mM sinomenine (SIN) inhibited voltage-gated sodium current (INa). Time course of effects of 3 mM sinomenine on INa (1) before, 2) during, 3) after the application of 3 mM sinomenine) (a). INa trace at the points indicated (b).
Normalized INa. As compared with control 3 mM sinomenine significantly decreased INa (n=6) (c). ***p<0.001 (repeated measures ANOVA followed by Tukey’s test) (B) INa was decreased by sinomenine in a dose dependent manner with IC50 = 2.3 ± 0.2 mM. (0.1 mM n=4, 0.3 mM n=5, 1 mM n=8, 3 mM n=6, 10 mM n=5)
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Figure 9.
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Figure 9. Intraplantar administration of sinomenine on formalin-induced pain behavior
(A) Effects of sinomenine (SIN) on the time course curves of formalin-induced pain behavior. (B) Formalin-induced pain behavior was divided into two phases and analyzed. The bar graph represented first phase (0–10 min) and second phase (10–40 min) of formalin-induced pain behavior. Intraplantar injection of sinomenine (500 μg) inhibited formalin-induced pain behavior only in the first phase. **p<0.01 (unpaired t test, two-tailed)
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Figure 10.
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Figure 10. Effects of sinomenine in rota-rod test
The performance time on rota-rod (A) and the number of falls (B) was measured 5 minutes (PRE) before and 30 minutes (POST) after intraperitoneal (i.p.) injection with vehicle or sinomenine. Sinomenine had no effect on rota-rod test at the dose of 25 mg/kg (n=6) or 50 mg/kg (n=7). 75 mg/kg sinomenine showed significant difference in rota-rod score (n=6). *p<0.05 and ***p<0.0001 (paired t test, two-tailed)
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