臺灣博碩士論文加值系統

在慢性發炎疼痛的過程中,免疫細胞與神經膠細胞會分泌白介素-6(Interleukin-6)與單核球化學引誘蛋白-1(chemokine monocyte chemoattractant protein-1)。在慢性發炎疼痛中，白介素-6、單核球化學引誘蛋白-1與它們的受器在背根神經節中的表現增加，進而可能加強背根神經節中的疼痛傳遞而誘導痛覺過敏(hyperalgesia)與觸覺疼(allodynia)。雖然白介素-6與單核球化學引誘蛋白-1在慢性疼痛中被認為會誘導痛覺過敏，但必須更進一步的研究白介素-6與單核球化學引誘蛋白-1在背跟神經節中促進痛覺傳遞的分子機轉。瞬時受體電位香草酸第一亞型通道(TRPV1)與1.8亞型抗河豚毒電壓依賴性鈉通道(TTX-resistant Nav1.8)選擇性地表現於背根神經節中小直徑的傳達痛覺的背根神經節神經元中，而這兩種通道被認為在背根神經節的痛覺傳遞與發炎形成或神經損傷所誘導的痛覺過敏中扮演不可或缺的角色。在本實驗中，我們假設在背根神經節中，白介素-6與單核球化學引誘蛋白-1會透過增強TRPV1與Nav1.8的功能而造成發炎性痛覺過敏。
給予白介素-6不會影響小背根神經節感覺神經元的靜止膜電位與細胞膜電容。給予小直徑背根神經節神經元白介素-6後，由TRPV1興奮劑辣椒素所引起的正電荷電流平均值明顯增加。白介素-6增加辣椒素電流的最大值但不影響TRPV1通道的開關時間過程(time-course)與半數效應濃度值(EC50 )。給予白介素-6後，TRPV1訊息核醣核酸(mRNA)在背根神經節神經元有顯著性地增加。在背根神經節神經元中，給予專一性磷脂酰肌醇-3激酶(PI-3K)抑制劑LY294002或蛋白激酶B/Akt抑制劑IV後，白介素-6無法增加辣椒素所引起正電荷電流值與TRPV1訊息核醣核酸表現。我們的結果指出白介素-6是藉由活化磷脂酰肌醇-3激酶/蛋白激酶B/Akt訊息傳遞路徑增加TRPV1的表現與增加背根神經節神經元痛覺傳遞。
給予單核球化學引誘蛋白-1不會影響小背根神經節感覺神經元的靜止膜電位與細胞膜電容。在小直徑的背根神經節神經元中，給予單核球化學引誘蛋白-1後，顯著地增加由辣椒素所引起正電荷電流平均值。單核球化學引誘蛋白-1增加辣椒素電流的最大值但對於TRPV1通道的開關時間過程與半數效應濃度值無顯著影響。給予單核球化學引誘蛋白-1後，在背根神經節神經元中的TRPV1 mRNA有明顯地增加。在背根神經節神經元中，給予專一性磷脂酰肌醇-3激酶(PI-3K)抑制劑LY294002與蛋白激酶B/Akt抑制劑IV後，單核球化學引誘蛋白-1不會增加辣椒素所引起正電荷電流值與增加TRPV1 mRNA的表現。上述結果顯示單核球化學引誘蛋白-1會透過活化磷脂酰肌醇-3激酶/蛋白激酶B訊息傳遞路徑增加TRPV1通道的活化而促進背根神經節神經元痛覺傳遞。
在小背根神經節痛覺神經元中，Nav1.8是主要的抗河豚毒電壓依賴性鈉通道。給予白介素-6或單核球化學引誘蛋白-1會顯著地增加抗河豚毒電壓依賴性鈉通道電流值。在背根神經節神經元中，給予白介素-6或單核球化學引誘蛋白-1後，Nav1.8 mRNA會顯著地增加。我們的結果顯示在背根神經節中，白介素-6或單核球化學引誘蛋白-1可能是藉由增加Nav1.8 mRNA表現引發痛覺過敏。
總結，我們的結果顯示白介素-6和單核球化學引誘蛋白-1在背根神經節神經元中，可能透過增加TRPV1與Nav1.8的功能而引起發炎性痛覺過敏。本實驗的研究結果將有助於了解發炎性慢性疼痛的病理機轉與發展慢性疼痛的治療策略。

Interleukin-6 (IL-6) and chemokine monocyte chemoattractant protein-1 (MCP-1/CCL2) are released from immune and glial cells during the chronic inflammatory pain. Expression of IL-6, CCL2 and their receptors in dorsal root ganglion (DRG) are upregulated in chronic inflammatory pain, which could induce hyperalgesia and allodynia by enhancing nociceptive transmission in DRG. Although IL-6 and CCL2 are believed to induce hyperalgesia observed in chronic pain, further study is required to investigate molecular mechanisms by which IL-6 and CCL2 facilitate nociceptive transmission in the DRG. TRPV1 and TTX-resistant Nav1.8, which are selectively expressed in small-diameter nociceptive DRG neurons, play an essential role in the nociceptive transmission in DRG and development of inflammation or nerve injury-induced hyperalgesia. In the present study, we tested the hypothesis that IL-6 and CCL2 cause inflammatory hyperalgesia by upregulating the function of TRPV1 and TTX-R Nav1.8 sodium currents in DRG nociceptive neurons.
IL-6 pretreatment did not affect the resting membrane potential and membrane capacitance of small DRG sensory neurons. Following IL-6 pretreatment, the mean amplitude of TRPV1 agonist capsaicin-evoked cationic current was greatly increased in small-diameter DRG neurons. IL-6 pretreatment increased the maximal magnitude of capsaicin currents without affecting the time-course and EC50 value.TRPV1 mRNA level was significantly increased after pretreating DRG neurons with IL-6. In the presence of specific PI-3K inhibitor LY294002 and Akt inhibitor IV, pretreating DRG neurons with IL-6 failed to increase the magnitude of capsaicin-evoked cationic current and upregulate TRPV1 mRNA expression in DRG neurons. Our results indicated that IL-6 upregulates TRPV1 expression and enhances nociceptive transmission of DRG neurons by activating PI-3K /Akt signaling pathway.
CCL2 pretreatment failed to affect the resting membrane potential and membrane capacitance of small DRG sensory neurons. Pretreating DRG neurons with CCL2 significantly increased the mean magnitude of capsaicin-evoked cationic current in small-diameter DRG neurons. CCL2 pretreatment increased the maximal amplitude of capsaicin-evoked cationic currents without significantly affecting the time-course and EC50 value. TRPV1 mRNA level was greatly increased in DRG neurons pretreated with CCL2. In the presence of specific PI-3K inhibitor LY294002 and Akt inhibitor IV, CCL2 pretreatment did not increase the amplitude of capsaicin-evoked cationic currents and upregulate TRPV1 mRNA expression in DRG neurons. These findings suggest that CCL2 enhances TRPV1 activation and facilitates nociceptive transmission of DRG neurons by activating PI-3K /Akt pathway.
Nav1.8 is the major subtype of TTX-resistant sodium channels in small DRG nociceptive neurons. Compared to TTX-insensitive sodium currents in control small-diameter DRG neurons, pretreatment of IL-6 or CCL2 significantly increased the amplitude of TTX-resistant sodium currents. Nav1.8 mRNA level was significantly upregulated in DRG neurons pretreated with IL-6 or CCL2. These results suggest that IL-6 and CCL2 cause pain hypersensitivity in the DRG by upregulating mRNA expression of TTX-insensitive Nav1.8 sodium channels.
In summary, the present study provides evidence that both IL-6 and CCL2 cause inflammatory hyperalgesia by upregulating the function of TRPV1 and TTX-R Nav1.8 sodium currents in DRG nociceptive neurons. Our results should lead to a better understanding of pathogenic mechanism of inflammatory chronic pain and the development of therapeutic strategies for chronic pain.

Abstract (Chinese) v
Abstract (English) viii
Abbreviations xi
Contents xii
Ⅰ. Introduction 1
Ⅱ. Specific aims 6
Ⅲ. Methods and Materials 7
3.1 Primary culture of dorsal root ganglion (DRG)7
3.2 Whole-cell patch-clamp recordings 7
3.3 Real-time quantitative RT-PCR assay 8
3.4 Statistics 9
Ⅳ. Results 10
4.1 Proinflammatory cytokine IL-6 enhances capsaicin
activation of TRPV1 and upregulates TRPV1 mRNA
expression 10
4.2 Chemokine CCL2 augments capsaicin activation of
TRPV1by increasing TRPV1 mRNA level 11
4.3 IL-6 upregulates TRPV1 expression and enhances
capsaicin activation of TRPV1 by activating
phosphatidylinositol 3-kinase (PI-3K) /Akt (protein
kinase B) pathway 13
4.4 CCL2 increases TRPV1 mRNA level and augments
capsaicin activation of TRPV1 through activating PI- 3K/Akt pathway 14
4.5 IL-6 and CCL2 increase the amplitude of TTX-
resistant sodium currents and upregulate Nav1.8
mRNA expression in DRG nociceptive neurons 15
Ⅴ. Discussion 17
Ⅵ. References 22
Ⅶ. Figures 28

Abbadie C. (2005) Chemokines, chemokine receptors and pain.
Trends in Immunol., 26, 529-534.
Akopian AN, Souslova V, England S, Okuse K, Ogata N, Ure J,
Smith A, Kerr BJ, McMahon SB, Boyce S, Hill R, Stanfa LC,
Dickenson AH, Wood JN.(1999) The tetrodotoxin-resistant
sodium channel SNS has a specialized function in pain
pathways. Nat Neurosci., 2, 541–548.
Amaya F, Decosterd I, Samad TA, Plumpton C, Tate S, Mannion
RJ, Costigan M, Woolf CJ.( 2000) Diversity of expression
of the sensory neuron-specific TTX-resistant voltage-
gated sodium ion channels SNS and SNS2. Mol Cell
Neurosci., 15, 331–342.
Black J.A., Liu S., Tanaka M., Cummins T.R., Waxman S.G.
(2004) Changes in the expression of tetrodotoxin-
sensitive sodium channels within dorsal root ganglia
neurons in inflammatory pain. Pain, 108, 237–247
Chuang H.H., Prescott E.D., Kong H., Shields S., Jordt
S.E., Basbaum A.I., Chao M.V. and Julius D. (2001)
Bradykinin and nerve growth factor release the capsaicin
receptor from PtdIns(4,5) P2-mediated inhibition. Nature,
411, 957–962
Cummins T.R., Sheets P.L., Waxman S. G. (2007) The roles of
sodium channels in nociception: implication for
mechanisms of pain. Pain, 131, 243-257.
Davis C.N., Chen S., Boehme S.A., Bacon K.B. and Harrison
J.K. (2003) Chemokine receptor binding and signal
transduction in native cells of the central nervous
system. Methods, 29, 4326-334.
DeLeo J.A., Colburn R.W., Nichols M. and Malhotra A. (1996)
Interleukin-6-mediated hyperalgesia/allodynia and
increased spinal IL-6 expression in a rat mononeuropathy
model. J. Interferon Cytokine Res., 16, 695-700.
Dib-Hajj S, Black JA, Felts P, Waxman SG.(1996) Down-
regulation of transcripts for Na channel alpha-SNS in
spinal sensory neurons following axotomy. Proc Natl Acad
Sci U S A. 93, 14950–14954.
Hu H.J., Bhave G. and Gereau R.W. (2002) Prostaglandin and
protein kinase A-dependent modulation of vanilloid
receptor function by metabotropic glutamate receptor 5:
potential mechanism for thermal hyperalgesia. J.
Neurosci., 22, 7444–7452.
Jarvis M.F., Honore P., Shieh C.C., Chapman M., Joshi S.,
Zhang X.F., Kort M., Carroll C., Marron B., AtkinsonR.,
Thomas J., Liu D., Krambis M., Liu Y., McGaraughty S.,
Chu K., Roeloffs R., Zhong C., Mikusa J.P., Hernandez G.,
Gauvin D., Wade C., Zhu C., Pai M., Scanio M., Shi L.,
Drizin I., Gregg R., MatulenkoM., Hakeem A., Gross M.,
Johnson M., Marsh K., Wagoner P.K., Sullivan J.P.,
Faltynek C.R., Krafte D.S. (2007) A-803467, a potent and
selective Nav1.8 sodium channel blocker, attenuates
neuropathic and inflammatory pain in the rat. Proc. Natl.
Acad. Sci. USA., 104, 8520–8525.
Ji R.R., Kohno T., Moore K.A. and Woolf C.J. (2003) Central
sensitization and LTP: do pain and memory share similar
mechanisms? Trends in Neurosci., 26, 696-705.
Joshi SK, Mikusa JP, Hernandez G, Baker S, Shieh CC,
Neelands T, Zhang XF, Niforatos W, Kage K, Han P, Krafte
D, Faltynek C, Sullivan JP, Jarvis MF, Honore P.(2006)
Involvement of the TTX-resistant sodium channel Nav 1.8
in inflammatory and neuropathic, but not post-operative,
pain states. Pain., 123, 75–82.
Julius D. and Basbaum A.I. (2001) Molecular mechanisms of
nociception. Nature, 413, 203-210
Jung, H., Toth, P.T., White, F.A., and Miller, R.J. (2008)
Monocyte chemoattractant protein-1 functions as a
neuromodulator in dorsal root ganglia neurons. J.
Neurochem., 104, 254–263.
Kasama, K., Kawakubo, M., Suzuki, T., Nishizawa, T.,
Ishida, A., Nakayama1, J. (2007) RNA interference-
mediated knock-down of transient receptor potential
vanilloid 1 prevents forepaw inflammatory hyperalgesia in
rat. European Journal of Neuroscience, 25, 2956–2963.
Khasar SG, Gold MS, Levine JD. A tetrodotoxin-resistant
sodium current mediates inflammatory pain in the rat.
Neurosci Lett. 1998;256:17–20.
Lee J.Y., Heo J.S., Suh H.N., Lee M.Y. and Han H.J. (2007)
Interleukin-6 stimulates α-MG uptake in renal proximal
tubule cells: involvement of STAT3, PI3K/Akt, MAPKs, and
NF-κB. Am J Physiol Renal Physio., 293, F1036–F1046.
Lindia J.A., McGowan E., Jochnowitz N. and Abbadie C.
(2005) Induction of CX3CL1 expression in astrocytes and
CX3CR1 in microglia in the spinal cord of a rat model of
neuropathic pain. J. Pain, 6, 434-438.
Marchand F., Perretti M. and McMahon S.B. (2005) Role of
the immune system in chronic pain. Nature Rev. Neurosic.,
6, 521-532.
Milligan E.D., Zapata V., Chacur M., Schoeniger D.,
Biedenkapp J., O’Connor K.A., Verge G.M., Chapman G.,
Green P., Foster A.C., Naeve G.S., Maier S.F. and Watkins
L.R. (2004) Evidence that exogenous and endogenous
fractalkine can induce spinal nociceptive facilitation in
rats. Eur. J. Neurosci., 20, 2294-2302.
McMahon S.B., Cafferty W.B.J. and Marchand F. (2005) Immune
and glial cell factors as pain mediators and modulators.
Exp. Neurology, 192, 444-462.
Oh S.B., Tran P.B., Gillard S.E., Hutley R.W., Hammond D.L.
and Miller R.J. (2001) Chemokines and glycoprotein120
produce pain hypersensitivity by directly exciting
primary nociceptive neurons. J. Neurosci., 21, 5027-5035.
Raghavendra V., Tanga F.Y. and DeLeo J.A. (2004) Complete
Freund’s adjuvant-induced peripheral inflammation evokes
glial activation and proinflammatory cytokine expression
in the CNS. Eur. J. Neurosci., 20, 467-473.
Sommer C., Petrausch S., Lindenlaub T. and Toyka K.V.
(1999) Neutralizing antibodies to interleukin 1-receptor
reduce pain associated behavior in mice with experimental
neuropathy. Neurosic. Lett., 270, 25-28.
S. Hensellek, P. Brell, H.G. Schaible, R. Brauer and B.G.
Segond von. (2007) The cytokine TNFalpha increases the
proportion of DRG neurones expressing the TRPV1 receptor
via the TNFR1 receptor and ERK activation, Mol Cell
Neurosci., 36,381–391.
Tadano T., Namioka M., Nakagawasai O., Tan-No K.,
Matsushima K., Endo Y. and Kisara K. (1999) Induction of
nociceptive responses by intrathecal injection of
interleukin-1 in mice. Life Sci., 65, 255-261.
Tanga F.Y., Raghavendra V. and DeLeo J.A. (2004)
Quantitative real-time RT-PCR assessment of spinal
microglial and astrocytic activation markers in a rat
model of neuropathic pain. Neurochem. Int., 45, 397-407.
Tanaka T., Minami M., Nakagawa T. and Satoh M. (2004)
Enhanced production of monocyte chemoattractant protein-1
in the dorsal root ganglia in a rat model of neuropathic
pain: possible involvement in the development of
neuropathic pain. Neurosci. Res., 48, 463-469.
Tate S, Benn S, Hick C, Trezise D, John V, Mannion RJ,
Costigan M, Plumpton C, Grose D, Gladwell Z, Kendall G,
Dale K, Bountra C, Woolf CJ.(1998) Two sodium channels
contribute to the TTX-R sodium current in primary sensory
neurons. Nat Neurosci., 1, 653–655.
Thier M., Marz P., Otten U., Weis J. and Rose-John S.
(1999) Interleukin-6 and its soluble receptor support
survival of sensory neurons. J. Neurosci. Res., 55, 411-
422.
Verge G.M., Milligan E.D., Maier S.F., Watkins L.R., Naeve
G.S. and Foster A.C (2004) Fractalkine (CX3CL1) and
fractalkine receptor (CX3CR1) distribution in spinal cord
and dorsal root ganglia under basal and neuropathic pain
conditions. Eur. J. Neurosci., 20, 1150- 1160.
Von Banchet G.S., Kiehl M. and Schaible H.G. (2005) Acute
and long-term effects of IL-6 on cultured dorsal root
ganglion neurons from adult rat. J. Neurochem., 94, 238-
248.
Wang P., Meinhardt B., Andre R., Renshaw B.R., Kimber I.,
Rothwell N.J. and Pinteaux E. (2005) The interleukin-1-
related cytokine IL-1F8 is expressed in glial cells, but
fails to induce IL-1beta signalling responses. Cytokine,
29, 245-250.
Wieseler-Frank J., Maier S.F. and Watkins L.R. (2005)
Immune-to-brain communication dynamically modulates pain:
Physiological and pathological consequences. Brain
Behavior, and Immunity, 19, 104-111.
Xu X., Hao J.X., Andell-Jonsson S., Bartfai T. and
Wiesenfeld-Halin Z. (1997) Nociceptive response in
interleukin-6-deficient mice to peripheral inflammation
and peripheral nerve section. Cytokine, 9, 1028-1033.
Xu, J.T., Tu, H.Y., Xin, W.J., Liu, X.G., Zhang, G.H.,
Zhai, C.H. (2007) Activation of phosphatidylinositol 3-
kinase and protein kinase B/Akt in dorsal root ganglia
and spinal cord contributes to the neuropathic pain
induced by spinal nerve ligation in rats. Experimental
Neurology, 206, 269–279
Zhang W. and Linden D.J. (2003) The other side of the
engram: experience-driven changes in neuronal intrinsic
excitability. Nature Rev. Neurosci., 4, 885-90
Zhang N., Inan S., Cowan A., Sun R., Wang J.M., Rogers
T.J., Caterina M. and Oppenheim J.J. (2005) A
proinflammatory chemokine, CCL3, sensitizes the heat- and
capsasin-gated ion channel TRPV1. Proc. Natl. Acad. Sci.
USA, 102, 4536-4541.