Distribution of Cold Receptor Transient Receptor Potential Melastatin 8-Immunopositive Axons in the Mouse Dental Pulp and Periodontal Tissue

김태헌,이재식,김윤숙,배용철 대한구강생물학회 2017 International Journal of Oral Biology Vol.42 No.4

Transient receptor potential melastatin 8 (TRPM8) plays a crucial role in innocuous cool sensation, acute cold pain and cold-induced hyperalgesia during pathologic conditions. To help understand TRPM8-mediated cold perception in the dental pulp and periodontal tissues, we examined the distribution of TRPM8-immunopositive (+) axons in molar and incisor pulp and periodontal tissues using transgenic mice expressing a genetically encoded axonal tracer in TRPM8+ neurons. In the radicular pulp of the molar teeth, a small number of TRPM8+ axons were observed. TRPM8+ axons branched frequently and extensively in the core of coronal pulp, forming a network in the peripheral pulp. Some TRPM8+ axons ascended between odontoblasts and were observed in the dentinal tubule. TRPM8+ axons were linear-shaped in the radicular pulp, whereas many TRPM8+ axons showed portions shaped like beads connected with thin axonal stands at the peripheral pulp. TRPM8 was densely expressed in the bead portions. In the incisor pulp, TRPM8+ axons were occasionally observed in the core of the coronal pulp and rarely observed at the peripheral pulp. TRPM8+ axons were occasionally observed and showed a linear shape rather than a bead-like appearance in the periodontal ligament and lamina propria of the gingival tissue. These findings, showing differential distribution of TRPM8+ axons between radicular and coronal portions of the molar pulp, between incisor and molar pulp, and between dental pulp and periodontal tissues, may reflect differential cold sensitivity in these regions.

영지버섯추출물 및 에르고스테롤의 냉감 수용체 TRPM8 (Transient Receptor Potential Cation Channel Melastatin Subtype 8) 발현 유도 효과

유화선 ( Hwa Sun Ryu ),정지연 ( Jiyeon Jeong ),전원옥 ( Weon-ok Jeon ),이춘몽 ( Chun Mong Lee ),이정노 ( Jung-no Lee ),박성민 ( Sung-min Park ) 대한화장품학회 2018 대한화장품학회지 Vol.44 No.1

UV 뿐 아니라 가시광선, 적외선에 의해 발생된 열로 인해 피부 온도 상승, MMP-1의 증가에 따른 피부 노화가 진행된다. 따라서 열에 의한 노화에서 피부 온도 조절은 노화 억제에 중요한 핵심 요소이다. 일시적인 수용체 전위 통로인 TRPM8은 멘솔 수용체(CMR1)로써 25 ℃ 이하의 온도에서 활성화되고 시원한 감각을 발생시키는 냉 수용체로 보고되어 있다. TRPM8 조절을 통해 시원한 감각과 피부 온도를 조절하는 연구가 활발히 진행되고 있다. 본 연구에서는 천연물인 영지버섯을 이용하여 냉 수용체인 TRPM8 발현에 어떠한 영향을 주는지 확인하였다. 영지버섯추출물 및 용매 분획물의 TRPM8 발현에 대한 영향을 측정한 결과, 영지버섯추출물, n-hexane 분획물 및 water 분획물에서 농도의존적으로 TRPM8 발현이 증가함을 확인하였다. Hex 분획물에서 유효성분을 찾고자 크로마토그래피를 실시하여 1개의 화합물을 분리하였으며 ¹H 및 ¹³C NMR spectrum 분석을 통하여 화학구조를 동정하였다. 분리된 화합물은 에르고스테롤로 TRPM8 발현 증가에 효과가 있음을 확인하였다. 결과를 토대로, 영지버섯추출물 및 에르고스테롤은 화장품 분야에서 새로운 쿨링 소재로서 개발가능성이 있다고 사료된다. Skin-aging is accelerated by the increased expression of MMP-1 caused by the increased skin temperature induced by IR/visible light as well as UV. Thus, the control of skin temperature is important to inhibit heat-induced aging. Many studies have been conducted to lower the skin temperature through the controlling transient receptor potential melastatin 8 channel (TRPM8), which is known as the cold and menthol receptor 1 (CMR1) and is activated at temperature below 25 ℃. In this study, we first investigated the effect of Ganoderma lucidum extract (GLE) on the TRPM8 expression. Results showed that GLE, hexane (Hex) fractions and water fractions increased the TRPM8 expression in a dose dependent manner. Active compound in Hex fractions were separated by chromatography and analyzed by ¹H and ¹³C NMR spectroscopy. The isolated compounds were identified as ergosterol and it also significantly increased the TRPM8 expression. Taken together, these results strongly suggest that G. lucidum extract and ergosterol have the potential as a new cooling ingredient in the cosmetics.

교감신경 의존적 및 비의존적 신경병증 통증 쥐 모델 후근신경절에서 Ca++ Channel α2δ Subunit와 TRPM8 발현

한동우,권태동,이윤우,김연아,최종범 대한통증학회 2008 The Korean Journal of Pain Vol.21 No.1

Background: Peripheral nerve injury induces up-regulation of the calcium channel alpha2delta (α2δ) subunit and TRPM8 in the dorsal root ganglion (DRG) which might contribute to allodynia development. We investigated the expression of the α2δ subunit and TRPM8 in the DRG of sympathetically maintained pain (SMP) and sympathetic independent pain (SIP) rat model. Methods: For the SMP model, the L5 and L6 spinal nerves were ligated tightly distal to the DRG. For the SIP model, the tibial and sural nerves were transected, while the common peroneal nerve was spared. After a 7 day postoperative period, tactile and cold allodynia were assessed using von Frey filaments and acetone drops, respectively. Expression of the α2δ subunit and TRPM8 in the L5 and L6 DRG were subsequently examined by a Western blot. Results: There were no significant differences between the two models for the thresholds of tactile and cold allodynia. Expression of the α2δ subunit in the ipsilateral DRG to the injury was increased as determined on a Western blot as compared to that in the contralateral or sham-operated DRG of the SMP model, but there was no difference in expression seen with the use of the SIP model. There was no difference in the expression of TRPM8 in the ipsilateral DRG to the injury and the contralateral or sham-operated DRG of either model. Conclusions: Up-regulation of the α2δ subunit in injured DRG may play a role that contributes to tactile allodynia development in SMP, but not TRPM8 to cold allodynia after peripheral nerve injury. (Korean J Pain 2008; 21: 11−17) Background: Peripheral nerve injury induces up-regulation of the calcium channel alpha2delta (α2δ) subunit and TRPM8 in the dorsal root ganglion (DRG) which might contribute to allodynia development. We investigated the expression of the α2δ subunit and TRPM8 in the DRG of sympathetically maintained pain (SMP) and sympathetic independent pain (SIP) rat model. Methods: For the SMP model, the L5 and L6 spinal nerves were ligated tightly distal to the DRG. For the SIP model, the tibial and sural nerves were transected, while the common peroneal nerve was spared. After a 7 day postoperative period, tactile and cold allodynia were assessed using von Frey filaments and acetone drops, respectively. Expression of the α2δ subunit and TRPM8 in the L5 and L6 DRG were subsequently examined by a Western blot. Results: There were no significant differences between the two models for the thresholds of tactile and cold allodynia. Expression of the α2δ subunit in the ipsilateral DRG to the injury was increased as determined on a Western blot as compared to that in the contralateral or sham-operated DRG of the SMP model, but there was no difference in expression seen with the use of the SIP model. There was no difference in the expression of TRPM8 in the ipsilateral DRG to the injury and the contralateral or sham-operated DRG of either model. Conclusions: Up-regulation of the α2δ subunit in injured DRG may play a role that contributes to tactile allodynia development in SMP, but not TRPM8 to cold allodynia after peripheral nerve injury. (Korean J Pain 2008; 21: 11−17)

Dose-Dependent Cytotoxic Effects of Menthol on Human Malignant Melanoma A-375 Cells: Correlation with TRPM8 Transcript Expression

Kijpornyongpan, Teeratas,Sereemaspun, Amornpun,Chanchao, Chanpen Asian Pacific Journal of Cancer Prevention 2014 Asian Pacific journal of cancer prevention Vol.15 No.4

Background: Transient receptor potential melastatin 8 (TRPM8), a principle membrane receptor involved in calcium ion influx and cell signal transduction, has been found to be up-regulated in some cancer types, including melanomas. Efficiency of menthol, an agonist of TRPM8, in killing melanoma cancer cells has been reported previously, but the mechanisms remain unclear. We here determined whether in vitro cytotoxic effects of menthol on A-375 human malignant melanoma cells might be related to TRPM8 transcript expression. Materials and Methods: The $PrestoBlue^{(R)}$ cell viability assay was used to assess the in vitro cytotoxic effect of menthol after 24h of treatment. RT-PCR was used to quantify TRPM8 transcript expression levels in normal and menthol-treated cells. Cell morphology was observed under inverted phase contrast light microscopy. Results: TRPM8 transcript expression was found at low levels in A-375 cells and down-regulated in a potentially dose-dependent manner by menthol. Menthol exerted in vitro cytotoxic effects on A-375 cells with an $IC_{50}$ value of 11.8 ${\mu}M$, which was at least as effective as 5-fluorouracil ($IC_{50}=120{\mu}M$), a commonly applied chemotherapeutic drug. Menthol showed no dose-dependent cytotoxicity on HeLa cells, a TRPM8 non-expressing cell line. Conclusions: The cytotoxic effects on A-375 cells caused by menthol might be related to reduction of the TRPM8 transcript level. This suggests that menthol might activate TRPM8 to increase cytosolic $Ca^{2+}$ levels, which leads to cytosolic $Ca^{2+}$ imbalance and triggers cell death.

Function of the Cold Receptor (TRPM8) Associated with Voiding Dysfunction in Bladder Outlet Obstruction in Rats

전지희,강효진,Mei Hua Jin,이혜영,임영재,정현진,한상원 대한배뇨장애요실금학회 2012 International Neurourology Journal Vol.16 No.2

Purpose: Bladder outlet obstruction (BOO) causes storage and voiding dysfunction in the lower urinary tract. We investigated the expression of transient receptor potential cation channel subfamily M member 8 (TRPM8) to evaluate the relationship between TRPM8 expression and overactive bladder (OAB) in a rat model of BOO. Methods: Fifty female Sprague-Dawley rats were divided into 4 groups; normal (n=10), normal-menthol (n=10), BOO (n=15), BOO-menthol (n=15). After 3 weeks, cystometry was performed by infusing physiological saline and menthol (3 mM)into the bladder at a slow infusion rate. The histological changes and expression of TRPM8 in the bladder were investigated by Masson’s trichrome staining, immunofluorescence and reverse transcription-polymerase chain reaction. Results: Cystometry showed that the intercontraction interval (ICI; 428.2±23.4 vs. 880.4±51.2, P<0.001), micturition pressure (MP; 25.7±1.01 vs. 71.80±3.01, P<0.001), and threshold pressure (2.9±0.25 vs. 9.2±1.58, P<0.01) were significantly increased in BOO rats. The bladder wall was significantly dilated compared with the control. Detrusor muscle hypertrophy and a thick mucosa layer were observed in BOO bladder. After menthol treatment, ICIs were decreased and MPs were increased in the menthol treatment groups. TRPM8-positive cells and mRNA were predominantly increased in the bladder and dorsal root ganglia of all groups compared with the normal group. Conclusions: Increased bladder wall thickness and proportion of collagen probably affect voiding dysfunction. Furthermore, an increase of TRPM8 expression in BOO may induce entry of Ca2+ from the extracellular space or stores. The increase of Ca2+ probably causes contraction of smooth muscle in BOO. However, OAB symptoms were not observed after menthol treatment although the expression of TRPM8 was abundant in the bladder epithelium after menthol treatment. Although OAB in BOO models may be caused by complex pathways, regulation of TRPM8 presents possibilities for OAB treatment.

추위에 노출된 슬관절염 모델에서 내슬안, 외슬안 직접구가 통증행동과 TRPM8 발현에 미치는 영향

지병욱 ( Byeong Uk Ji ),김이꽃 ( Yiquot Kim ),이지은 ( Ji Eun Lee ),구성태 ( Sungtae Koo ) 대한경락경혈학회 2016 Korean Journal of Acupuncture Vol.33 No.4

Objectives : The aim of the study is to investigate the effects of moxibustion on the pain behavior and expression of TRPM8 in the dorsal root ganglion(DRG) in the rat model of ambient cold(AC) exposed osteoarthritis(OA). Methods : OA was induced by the injection of 50 μl of 2% monosodium iodoacetate(MIA) into the knee joint cavity. To examine the level of pain, weight bearing forces(WBFs) of affected limb was measured. For the AC exposure, the animals were housed in 6 h/day at 4℃ for 14 days after MIA injection. Moxibustion treatment was performed at EX-LE4 and EX-LE5 with 5 cons(1, 7 or 10 mg) per day for 13 days from 5 days after MIA injection. The expressions of TRPM8 in DRG were measured by western blotting analysis. Results : The WBFs of MIA-AC group were decreased significantly compared to MIA group at 2, 3, 6, 7, 8 and 9 days after arthritis induction. After the first 6 h-AC exposure, expressions of TRPM8 in MIA-AC group were increased significantly compared to those of naive group. After moxibustion treatment, only the WBFs of 7 mg treated group were restored significantly. Moreover, the over-expressions of TRPM8 were attenuated by the moxibustion treatment in AC exposed rats. Conclusions : The data suggest that AC can increase arthritic knee pain via up-regulated TRPM8 and moxibustion treatment improve the arthritic pain via modulation of TRPM8 expression in DRG in the rat model of AC exposed MIA induced arthritis.

교감신경 의존적 및 비의존적 신경병증 통증 쥐 모델 후근신경절에서 Ca⁺⁺ Channel α2δ subunit와 TRPM8 발현

한동우,권태동,김연아,최종범,이윤우,Han, Dong Woo,Kweon, Tae Dong,Kim, Yeon A,Choi, Jong Bum,Lee, Youn Woo 대한통증학회 2008 The Korean Journal of Pain Vol.21 No.1

Background: Peripheral nerve injury induces up-regulation of the calcium channel alpha2delta (${\alpha}2{\delta}$) subunit and TRPM8 in the dorsal root ganglion (DRG) which might contribute to allodynia development. We investigated the expression of the ${\alpha}2{\delta}$ subunit and TRPM8 in the DRG of sympathetically maintained pain (SMP) and sympathetic independent pain (SIP) rat model. Methods: For the SMP model, the L5 and L6 spinal nerves were ligated tightly distal to the DRG. For the SIP model, the tibial and sural nerves were transected, while the common peroneal nerve was spared. After a 7 day postoperative period, tactile and cold allodynia were assessed using von Frey filaments and acetone drops, respectively. Expression of the ${\alpha}2{\delta}$ subunit and TRPM8 in the L5 and L6 DRG were subsequently examined by a Western blot. Results: There were no significant differences between the two models for the thresholds of tactile and cold allodynia. Expression of the ${\alpha}2{\delta}$ subunit in the ipsilateral DRG to the injury was increased as determined on a Western blot as compared to that in the contralateral or sham-operated DRG of the SMP model, but there was no difference in expression seen with the use of the SIP model. There was no difference in the expression of TRPM8 in the ipsilateral DRG to the injury and the contralateral or sham-operated DRG of either model. Conclusions: Up-regulation of the ${\alpha}2{\delta}$ subunit in injured DRG may play a role that contributes to tactile allodynia development in SMP, but not TRPM8 to cold allodynia after peripheral nerve injury.

Topical cryosim-1 (selective TRPM8 agonist) gel on itching

( Seok Young Kang ),( Min Je Jung ),( Young Won Choi ),( Bo Young Chung ),( Hye One Kim ),( Chun Wook Park ) 대한피부과학회 2019 대한피부과학회 학술발표대회집 Vol.71 No.2

Background: Itching and repetitive scraping damage the skin and lead to additional inflammation, resulting in a vicious cycle. Treatment of itching means to prevent the deterioration of the disease beyond relieving the symptoms. Objectives: TRPM8 is a major receptor for temperaturesensitive nerve fibers. Cryosim-1 acts as a selective agonist of TRPM and acts to inhibit skin sensation inconveniences by making the skin feel cold. We would like to evaluate the effect of cryosim-1 on itching relief. Methods: The study includes two study designs. Prospective, randomized, double-blinded, and placebocontrolled study was performed to compare 2 hours and 1 week after application in study A. In study B, patients were further recruited for the study ointment only, and 5-d itching scales were compared at 1-week intervals. Results: Twenty-four patients in the study group and 24 patients of control group completed the clinical trials. Study group showed significant decrease of NRS for pruritus 2 hours after application. Comparisons between baseline and week 1 showed a significant decrease in both groups. Twenty-two patients were enrolled in study B. The 5-D itching scale showed a significant decrease after one week compared to the baseline. In the subgroup there was statistically significant decrease in urticaria. Conclusion: The present study evaluated the effect of cryosim-1 on relief of itching. We confirmed that cryosim-1 could be a new alternative component to immediate and dramatic itch relievers.

Effects of Nefopam on Streptozotocin-Induced Diabetic Neuropathic Pain in Rats

Nam, Jae Sik,Cheong, Yu Seon,Karm, Myong Hwan,Ahn, Ho Soo,Sim, Ji Hoon,Kim, Jin Sun,Choi, Seong Soo,Leem, Jeong Gil The Korean Pain Society 2014 The Korean Journal of Pain Vol.27 No.4

Background: Nefopam is a centrally acting non-opioid analgesic agent. Its analgesic properties may be related to the inhibitions of monoamine reuptake and the N-methyl-D-aspartate (NMDA) receptor. The antinociceptive effect of nefopam has been shown in animal models of acute and chronic pain and in humans. However, the effect of nefopam on diabetic neuropathic pain is unclear. Therefore, we investigated the preventive effect of nefopam on diabetic neuropathic pain induced by streptozotocin (STZ) in rats. Methods: Pretreatment with nefopam (30 mg/kg) was performed intraperitoneally 30 min prior to an intraperitoneal injection of STZ (60 mg/kg). Mechanical and cold allodynia were tested before, and 1 to 4 weeks after drug administration. Thermal hyperalgesia was also investigated. In addition, the transient receptor potential ankyrin 1 (TRPA1) and TRP melastatin 8 (TRPM8) expression levels in the dorsal root ganglion (DRG) were evaluated. Results: Pretreatment with nefopam significantly inhibited STZ-induced mechanical and cold allodynia, but not thermal hyperalgesia. The STZ injection increased TRPM8, but not TRPA1, expression levels in DRG neurons. Pretreatment with nefopam decreased STZ-induced TRPM8 expression levels in the DRG. Conclusions: These results demonstrate that a nefopam pretreatment has strong antiallodynic effects on STZ-induced diabetic rats, which may be associated with TRPM8 located in the DRG.

Effects of Nefopam on Streptozotocin-Induced Diabetic Neuropathic Pain in Rats

남재식,정유선,감명환,안호수,심지훈,김진선,최성수,임정길 대한통증학회 2014 The Korean Journal of Pain Vol.27 No.4

Background: Nefopam is a centrally acting non-opioid analgesic agent. Its analgesic properties may be related to the inhibitions of monoamine reuptake and the N-methyl-D-aspartate (NMDA) receptor. The antinociceptive effect of nefopam has been shown in animal models of acute and chronic pain and in humans. However, the effect of nefopam on diabetic neuropathic pain is unclear. Therefore, we investigated the preventive effect of nefopam on diabetic neuropathic pain induced by streptozotocin (STZ) in rats. Methods: Pretreatment with nefopam (30 mg/kg) was performed intraperitoneally 30 min prior to an intraperitoneal injection of STZ (60 mg/kg). Mechanical and cold allodynia were tested before, and 1 to 4 weeks after drug administration. Thermal hyperalgesia was also investigated. In addition, the transient receptor potential ankyrin 1 (TRPA1) and TRP melastatin 8 (TRPM8) expression levels in the dorsal root ganglion (DRG) were evaluated. Results: Pretreatment with nefopam significantly inhibited STZ-induced mechanical and cold allodynia, but not thermal hyperalgesia. The STZ injection increased TRPM8, but not TRPA1, expression levels in DRG neurons. Pretreatment with nefopam decreased STZ-induced TRPM8 expression levels in the DRG. Conclusions: These results demonstrate that a nefopam pretreatment has strong antiallodynic effects on STZ-induced diabetic rats, which may be associated with TRPM8 located in the DRG.