Oxaliplatin (Oxp), a platinum-based chemotherapeutic agent with a 1,2-diaminocyclohexane-carrier ligand, is widely used as treatment for advanced or recurrent colorectal cancer. However, peripheral neuropathy is a major side effect of Oxp use. The symptoms associated with Oxp-induced neurotoxicity are characterized by two stages: acute and transient, and chronic. Cold hypersensation is an acute and transient symptom that is observed in almost all patients, whereas chronic symptoms are similar to those seen with cisplatin treatment and are the most frequent dose-limiting factor in chemotherapy 1,2. Although administration of calcium gluconate/magnesium sulfate, gabapentin, or pregabalin theoretically could be used to treat Oxp-induced peripheral neuropathy in a clinical situation, effective therapy has not yet been established. Recently, it was reported that Oxp-induced cold hypersensitivity was associated with transient receptor potential melastatin 8 (TRPM8) and transient receptor potential ankyrin 1 (TRPA1) channels, which are ion channels that are activated by cold. It has also been reported that administration of Oxp increases TRPM8 expression in the dorsal root ganglia (DRG) in vitro3 and in vivo4.
Gosha-jinki-gan (GJG) is a traditional Japanese herbal medicine containing 10 crude drugs and is used therapeutically in patients with diabetic neuropathy and neuralgia with pain and numbness. GJG was reported to be effective for Oxp-induced peripheral neuropathy in clinical 5 and animal 6 studies. A clinical trial (the GONE study) on the preventive effects of GJG was conducted in Japan 7. However, Fukazawa et al.8 reported that in patients treated with Oxp, leucovorin, and fluorouracil (modified FOLFOX6 therapy), prophylactic coadministration of GJG was better in preventing the Oxp-induced peripheral neuropathy than therapeutic coadministration was in treating the neuropathy once symptoms developed. This finding suggests that the preventive actions of GJG on Oxp-induced peripheral neuropathy occur in the acute stage of peripheral neuropathy, which is associated with hypersensitivity to cold sensation. We hypothesized that the preventive effect of GJG on cold hypersensitivity induced by Oxp may be correlated with TRPM8 and TRPA1 mRNA expression. To assess this hypothesis, we first determined whether the withdrawal response to cold stimulation in rats was altered by coadministration of GJG and Oxp, and then examined the relationship between any alteration observed in the withdrawal response and the expression of TRPM8 and TRPA1 mRNA in the DRG.
Materials and methods
Oxp was purchased from Wako Pure Chemical Industries Ltd (Osaka, Japan). It was dissolved in a 5% glucose solution at a concentration of 4 mg/ml for intraperitoneal injections. GJG was purchased from Tsumura & Co. (Tokyo, Japan) and dissolved in water at a concentration of 200 mg/ml for oral administration.
Male Wistar-ST rats (Kumagai-Shigeyasu Co. Ltd, Miyagi, Japan; body weight, 110–140 g) were housed three or four per cage with free access to food and water. The lights were on from 07:00 to 19:00 h. All animals were habituated for 1 week before the experiments. All experiments were approved by the Experimental Animal Care and Use Committee of the International University of Health and Welfare.
Thirty-four rats were divided into four groups: A (n=10), B (n=10), C (n=6), and vehicle (n=8). Group A was administered Oxp at a dose of 4 mg/kg, intraperitoneal, twice a week (days 1, 2, 8, and 9, for a total cumulative dose of 16 mg/kg) and orally administered water (5 ml/kg) repeatedly for 12 days. Administration of Oxp in group B was similar to that in group A, but the rats in group B also received an oral dose of GJG (1 g/kg/day) for 12 days. Group C was administered a 5% glucose solution, intraperitoneally, twice a week and GJG was administered at the same oral dose as group B for 12 days. Rats in the vehicle group were administered a 5% glucose solution, intraperitoneally, twice a week and orally dosed with water (5 ml/kg) for 12 days. GJG was administered between 09:00 and 11:00 h, whereas Oxp was administered between 13:00 and 15:00 h.
Assessment of cold sensation
Cold sensation was assessed at 10°C (cold allodynia) and 4°C (cold hyperalgesia) using a cold plate (MK-350HC; Muromachi Kikai Co. Ltd, Tokyo, Japan). Assessment of cold allodynia was performed on the 3rd and 10th days after starting drug administration, and cold hyperalgesia on the 5th and 12th days. Behavioral tests were performed between 13:00 and 16:00 h. Baseline measurements were performed on two separate days during the habituation period. The rats were brought into the testing room for 30 min for habituation and placed individually onto the cold plate for 6 min. The number of times either hind paw was lifted in response to the cold stimulation was counted. Counts were assessed using videotape from each cold-plate test session replayed in slow motion to ensure accuracy.
RNA extraction, cDNA synthesis, and real-time PCR
On the 12th day after starting drug administration, rats in each group (n=4 each) were anesthetized with diethyl-ether, and L4–L6 DRG were removed for RNA extraction. Total RNA was extracted with ISOGEN (Nippon Gene Co. Ltd, Tokyo, Japan), and cDNA was synthesized with 1 μg of total RNA, Oligo-dT12–18 primer, and SuperScript II Reverse Transcriptase (Life Technologies Corp., Carlsbad, California, USA) in a 20 μl solution according to the manufacturer’s protocol.
Real-time PCR was performed with SYBR Premix DimerEraser (TaKaRa Bio Inc., Shiga, Japan). The cDNA was diluted 100-fold for PCR. The oligonucleotide primers for TRPM8 and TRPA1 channels and β-actin were based on that described by Ta et al. 3. The sequences of oligonucleotide primers for PCR were as follows: TRPM8, 5′-gcccagtgatgtggacagta-3′ (sense), 5′-ggactcatttcccgagaagg-3′ (antisense); TRPA1, 5′-atttgcggcctgagttttt-3′ (sense), 5′-tccatcattgtcctcatcca-3′ (antisense); and β-actin, 5′-cccgcgagtacaaccttct-3′ (sense), 5′-cgtcatccatggcgaact-3′ (antisense) (Life Technologies Corp.). Reactions were incubated at 95°C for 30 s and then cycled at 95°C (15 s), 55°C (30 s), and 72°C (32 s) for 45 cycles using a 7300 Real-Time PCR System (Applied Biosystems Inc., Foster City, California, USA). To observe the melting curve, a final dissociation cycle, which had been programmed in the 7300 sequence detection system software from Applied Biosystems Inc., was added (95°C for 15 s, 60°C for 1 min, and 95°C for 15 s). The relative quantification of PCR products was performed using the
method 9. The fold change in mRNA expression levels of TRPM8 and TRPA1 channels was normalized to β-actin as the reference gene, followed by normalization to vehicle group. Data are presented as fold induction of groups A, B, and C compared with the expression level in the vehicle group.
Data are presented as the means±SD. Statistical analysis was carried out by one-way analysis of variance, followed by Bonferroni’s multiple comparison test with GraphPad Prism V5.0 (GraphPad Software, San Diego, California, USA). A P value of less than 0.05 was considered as statistically significant.
The effect of GJG on Oxp-induced cold allodynia and hyperalgesia
Figure 1 shows the effect of GJG on Oxp-induced cold allodynia (assessed at 10°C). There were no significant differences in baseline values across all groups of rats. The number of withdrawal responses to cold stimulation in group A on the 3rd and 10th days of drug administration was significantly increased as compared with those in the vehicle group. In contrast, the number of withdrawal responses in group B was significantly decreased as compared with those in group A, and comparable with the number of withdrawal responses in the vehicle group. Similar results were also obtained for the effect of GJG on Oxp-induced cold hyperalgesia (assessed at 4°C). As shown in Fig. 2, the number of withdrawal responses to cold stimulation in group A on the 5th and 12th days of drug administration was increased significantly as compared with those in the vehicle group. Moreover, the number of withdrawal responses in group B was significantly decreased as compared with those in group A. These results suggest that coadministration of GJG reduced Oxp-induced cold hypersensitivity. The number of withdrawal responses in group C was comparable with those in the vehicle group under both temperature conditions.
The effect of Oxp and GJG on the expression of TRPM8 and TRPA1 mRNA
To elucidate the mechanism of the preventive effect of GJG on Oxp-induced cold hypersensitivity, we determined the expression of TRPM8 and TRPA1 mRNA in L4–L6 DRG using real-time PCR. As shown in Fig. 3, the expression of TRPM8 mRNA in group A was significantly increased as compared with the expression in the vehicle group (P<0.05). In contrast, the expression of mRNA in group B was significantly decreased as compared with group A, but comparable with expression levels observed in the vehicle group (P<0.05, compared with group A). As shown in Fig. 4, a similar pattern was observed for the expression of TRPA1 mRNA. That is, the expression of TRPA1 mRNA in group A was significantly increased as compared with the vehicle group (P<0.01), whereas the mRNA expression level of TRPA1 in group B was significantly reduced as compared with that in group A and comparable with that in the vehicle group (P<0.01, compared with group A). The mRNA expression levels of TRPA1 and TRPM8 in group C were almost identical to those in the vehicle group.
In this study, we hypothesized that the preventive effect of GJG on cold hypersensitivity induced by Oxp may be associated with TRPM8 and TRPA1 mRNA expression. To assess this hypothesis, we first determined whether coadministration of GJG and Oxp in rats altered their withdrawal response to cold stimulation and then examined the relationship between any alteration observed in the withdrawal response and the expression of TRPM8 and TRPA1 mRNA. We found that coadministration of Oxp and GJG significantly reduced the number of withdrawal responses to cold stimulation as compared with Oxp administration alone. We also found that coadministration of Oxp and GJG significantly suppressed the overexpression of TRPM8 and TRPA1 mRNA induced by Oxp administration. There are several reports that Oxp-induced cold hypersensitivity is associated with TRPM8 and TRPA1 channels in animals. Ta et al.3 reported that Oxp increased the expression of TRPM8 mRNA in mouse trigeminal ganglion. In contrast, Zhao et al.10 reported in a behavioral study with TRPA1 null mic that acute cold hypersensitivity induced by Oxp is caused by the enhanced responsiveness of TRPA1. Moreover, Kono et al.11 reported that TRPM8 was involved in Oxp-induced acute cold hypersensitivity in vivo by evaluating the changes in the cold sensation detection threshold before and after Oxp administration. These results suggest that Oxp-induced cold hypersensitivity is involved in overexpression of TRPM8 and/or TRPA1 mRNA, and the anti-cold hypersensitive effect of GJG is involved in the expression of TRPM8 and TRPA1 mRNA increased by Oxp administration. Our results suggest that GJG improves the Oxp-induced cold hypersensitivity by suppressing the expression level of TRPM8 and TRPA1. Recently, Kawashiri et al.4 reported that L-type calcium channel blockers improved Oxp-induced cold sensitivity by suppressing the expression of TRPM8. In that report, the behavior was evaluated using the acetone test. Topical acetone-induced evaporative cooling is commonly used to assess cold allodynia 12,13. We evaluated cold allodynia and cold hypersensitivity using a cold plate. Taking all this into account, TRPA1 is likely involved in cold hyperalgesia in the acute stage. To further assess the relevance between the expression of TRPA1 mRNA and cold hyperalgesia, additional studies using TRPA1 antagonists, such as HC-030031, will be necessary 14.
GJG is a Japanese herbal medicine containing 10 crude drugs and is reported to exert antiplatelet aggregatory effects by increasing NO production 15 and to exert antinociceptive effects by stimulation of the κ-opioid receptor through dynorphin release in streptozotocin-induced diabetic mice 16. Recently, Imamura et al.17 reported that pretreatment of GJG partially suppressed the overexpression of TRPV1 and P2X3 purine receptors induced by acetic acid in rat urinary bladder. TRPV1 is a member of the same TRP superfamily as TRPM8 and TRPA1 and is activated by heat (>43°C) and by capsaicin 18. Although the suppressive effect of GJG on the expression of TRPM8 and TRPA1 mRNA is a novel finding, GJG might act to reduce the expression of sensory receptors containing channels in the TRP superfamily. Furthermore, it will be necessary to analyze the effect of GJG on the expression of other sensory receptors.
We showed that GJG suppresses Oxp-induced cold hypersensitivity by decreasing the mRNA expression levels of TRPM8 and TRPA1 channels. These findings indicate that GJG is a medicine useful for reducing the adverse effects of Oxp administration, and thus to improve the quality of life of patients treated with Oxp.
The authors thank T. Takeuchi, K. Miyagawa, M. Tsuji, and H. Takeda for technical support on the behavioral study. They also thank C. Tetsuka and T. Fujita for experimental support.
This work was supported by the Advanced Education and Research Center for Kampo Medicine, Department of Pharmaceutical Sciences, International University of Health and Welfare.
Conflicts of interest
There are no conflicts of interest.
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Keywords:© 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins
cold hypersensation; gosha-jinki-gan; oxaliplatin; peripheral neuropathy; TRPA1; TRPM8