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研究生(外文):Chun-Ping Huang
論文名稱(外文):Investigation of Voltage-Gated Sodium Channels’ Effects by Using Low-Frequency Electro-Stimulation in Inflammatory Hyperalgesia & Syndecan-4 Promotes Epithelial Tumor Cells Spreading and PKCα Activity under Mechanical Stimulation
外文關鍵詞:Voltage-gated sodium channelsAcupunctureAnalgesiaHeparan sulfate proteoglycansMechanotransduction
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疼痛的因素主要來自於炎症、創傷、糖尿病、關節炎或腫瘤等,會造成周邊神經物性的改變,並導致神經自發性放電的增加或傳導特性的改變。電壓閘控鈉離子通道(Voltage-gated sodium channels, Navs)掌控鈉離子流入神經元並在痛覺動作電位啟動與傳導的過程中扮演一個重要角色。在所有Navs的亞型中,Nav1.7、Nav1.8與Nav1.9與痛覺傳導最具相關性,然而PKA(protein kinase A)和PKC(protein kinase C)被認為可磷酸化Nav1.8並增強其電流。針刺可以刺激神經Aδ纖維和C纖維,來調節痛覺傳導。已有研究顯示,針刺能增加體內内源性腦內啡、5-羥色胺、腺苷酸以減輕疼痛。
本實驗首先在小鼠後腳掌皮下注射鹿角菜膠(carrageenan)或佛朗氏完全佐劑(Complete Freund''s Adjuvant, CFA)建立發炎疼痛動物模式,評估電針刺緩解疼痛之可能作用機轉。發炎誘導後第4天進行行為測試,行為測試後犧牲動物取背根神經節。實驗結果顯示:低頻電針刺(2 Hz, 100 μs duration, 20 m)足三里穴位(ST36)可以減緩鹿角菜膠或CFA誘發的機械和熱痛覺敏感化(藉由von Frey、輻射熱、冷熱板實驗),而在偽穴位治療組中並沒有觀察到這種現象。發炎造成Nav1.7與Nav1.8蛋白質表現量增加,但Nav1.9蛋白質表現量並沒有顯著改變,而在偽穴位治療組中亦沒有觀察到這種現象。因此,研究結果顯示,低頻電針刺能減緩鹿角菜膠和CFA誘發的Nav1.7與Nav1.8蛋白質表現增加,而並不會改變Nav1.9蛋白質表現。進一步使用電生理技術,紀錄背根神經元中抗河豚毒素(tetrodotoxin-resistant, TTX-R)電流,結果顯示CFA組中TTX-R電流會增加,而低頻電針刺能顯著地減緩TTX-R電流增加。
為了進一步探討低頻電針刺鎮痛機制,在小鼠足三里穴位注射腺苷酸A1接受器促效劑(CPA)(劑量:0.1 mg/kg),或以腹腔注射μ接受器促效劑:內啡呔1(endomorphin 1)(劑量:10 mg/kg)。結果顯示低頻電針刺與CPA可以改善動物發炎疼痛行為,且降低COX-2蛋白表現和磷酸化蛋白激脢ε(protein kinase C epsilon, PKCε);然而,endomorphin 1僅能降低磷酸化PKCε表現。進一步使用μ接受器拮抗劑:naloxone methiodide與腺苷酸A1接受器拮抗劑(rolofylline)來確認此二者在低頻電針刺鎮痛中扮演之角色,實驗結果顯示阻斷μ接受器與腺苷酸A1接受器會造成低頻電針刺失去鎮痛作用與提高COX-2蛋白和磷酸化PKCε表現量。
在機械力活化上皮癌細胞Syndecan-4之研究中,肝素硫酸鹽蛋白聚醣(Heparan sulfate proteoglycans, HSPGs)位於細胞膜表面並且在細胞貼附、延伸、形成黏著斑與感受機械力扮演著重要的角色。Syndecans歸屬於HSPGs家族成員之一,並且高度表現於各種腫瘤細胞。其中,僅Syndecan-4 (SDC4)具有活化蛋白激脢Cα(protein kinase C alpha, PKCα)的能力,其感受機械力對於腫瘤細胞之影響尚未有深入之報導。
本實驗欲探討腫瘤細胞上SDC4承載機械力之後其訊息機轉,利用兩種上皮腫瘤細胞-人類子宮頸癌細胞(HeLa cells)與小鼠黑色素腫瘤細胞(B16-F10)-種植於表面塗覆poly-L-lysine (Pl)、 fibronectin (Fn)、抗SDC4抗體之聚二甲基矽氧烷(polydimethylsiloxane, PDMS)彈性膜上,並施予5 Ib/in2之壓力。實驗結果顯示透過PDMS膜與腫瘤細胞的SDC4結合,機械力會磷酸化focal adhesion kinase (FAK)與PKCα。進一步分析細胞軟硬度(cell contractility)的指標蛋白:第二型肌球蛋白輕鏈(myosin light chain 2, MLC2),結果顯示在接受機械力10分鐘後,MLC2的磷酸化開始增加,而在30分鐘後便降低並且隨著PKCα活性改變;然而,透過與組合蛋白鍵結結構區(integrin binding motif)之細胞則無此現象。

Pain is associated with conditions such as inflammation, trauma, diabetes, arthritis or tumor, which can result from altered properties of peripheral nerves. As a result, this leads to increase spontaneous firing or alterations in their conduction properties. Voltage-gated sodium channels (Navs) control the influx of Na+ ions into the neurons and play an essential role in the initiation and propagation of nociception action potentials in dorsal root ganglion (DRG) neurons. Acupuncture is known to stimulate the Aδ-fibers and modulate pain sensation by activating C-fibers through the meridian. Several studies have suggested that acupuncture increases the release of endogenous opioids, serotonin, and adenosine to reduce pain.
We first established inflammatory pain animal model by injection of carrageenan or Complete Freund''s Adjuvant (CFA) in the mouse plantar surface of the hind paw. Low-frequency electroacupuncture (LFEA) (2 Hz, 100 μs duration, 20 m) at Zusanli (ST36) acupoint reliably attenuated carrageenan or CFA-induced mechanical and thermal hyperalgesia (by von Frey, radial heat, hot/cold plate test). The phenomenon was not observed in sham group. Inflammation increased the expression of Nav 1.7 and Nav1.8, but not Nav1.9. LFEA-elicited down-regulation of Nav1.7 and Nav1.8 was not observed in sham group. Accordingly, our results suggest that LFEA has the ability to ameliorate carrageenan and CFA-induced overexpression of Nav1.7 and Nav1.8, rather than Nav1.9 sodium channels. We used whole-cell recording to compare the tetrodotoxin-resistant (TTX-R) sodium currents in DRG neurons. Inflammation induced by CFA amplified the TTX-R currents and decreased in LFEA-treated group. To investigate analgesia mechanisms by LFEA, C57BL/6 male mice were further injected with CPA, adenosine A1 receptor (A1AR) agonist (0.1 mg/kg) at ST36 acupoint or endomorphin 1, μ-receptor (MOR) agonist (10 mg/kg) intraperitoneally. The results showed LFEA and CPA administration could improve animal pain behaviors and down-regulate the expression of COX-2 and phosphorylated PKCε, but endomorphin 1 administration solely reduced the phosphorylation of PKCε. Furthermore, we applied naloxone methiodide (MOR antagonist) (10 mg/kg), rolofylline (A1AR antagonist) (3 mg/kg) to confirm the analgesia and anti-inflammation effects by LFEA. The data suggested blocking of MOR and A1AR reversed the analgesia effects of LFEA.
Heparan sulfate proteoglycans (HSPGs) at the cell surface play an important role in cell adhesion, spreading, formation of focal adhesion complexes (FACs), and sensing mechanical stress. Syndecans are members of the HSPGs family and are highly expressed in various tumor cells. Syndecan-4 (SDC4) is a unique member of syndecans that activates protein kinase C alpha (PKCα). However, syndecan-4 in tumor cells development is not clear when receiving mechanical stress. Here we investigate the role of syndecan-4 in tumor cells spreading and its downstream kinases under mechanical stimulation. Epithelial tumor cells were seeded onto elastomeric polydimethylsiloxane (PDMS) membranes coated with poly-L-lysine (Pl), fibronectin (Fn), or anti-SDC4 antibody and stretched with a modified pressure-driven cell-stretching (PreCS) device. When cells received mechanical stimulation, engagement of syndecan-4 promoted the phosphorylation of focal adhesion kinase (FAK) at tyrosine 397 and PKCα at serine 657. Furthermore, we analyzed the cell contractility marker—myosin light chain 2 (MLC2) in 30 min time courses. The levels of phosphorylated MLC2 at serine 19 were augmented through ligations of syndecan-4 but not integrin binding motif (RGD) at 10 min mechanical stimulation and were suppressed at 30 min and this phenomenon was associated with the activity of PKCα. Our data demonstrate that syndecan-4 is essential for transmitting the mechanotransduction signals via activation of PKCα and is important for tumor cells spreading, assembly of actin cytoskeleton and cell contractility.

中文摘要 i
1.1.1. Pain 1
1.1.2. Inflammation microenvironment: Inflammatory soup 1
1.1.3. Inflammatory mediators cause hyperalgesia 1
1.1.4. Peripheral sensitization increases by inflammatory mediators 1
1.1.5. Voltage-gated sodium channels serve as nociceptive signal transducer 2
1.1.6. Protein Kinase C (PKC) activity is necessary for long-term hyperalgesia 4
1.1.7. Acupuncture relieves pain by releasing endogenous opioids, serotonin and adenosine 5
1.1.8. Analgesia mechanism of MOR and A1AR 5
1.1.9. MOR modulates immune system and inflammatory reactions 6
1.1.10. LFEA down-regulate Navs expression and currents in the inflammatory pain models 6
1.2. Syndecan-4 promotes epithelial tumor cells spreading and regulates PKCα activity under mechanical stimulation 7
1.2.1 Extracellular matrix microenvironment in tumor cells progression 7
1.2.2 Mechanotransducers deliver mechanical stimuli into biochemical information 7
1.2.3. Syndecan-4 activates PKCα when FACs developing 8
1.2.4. Rho activity maintains the contractility of tumor cells 8
2.1. Animals and LFEA treatment 10
2.2. Inflammatory pain models 10
2.3. MOR and A1AR agonist administration 11
2.4. MOR and A1AR antagonist administration 11
2.5. Animal behavior of mechanical and thermal hyperalgesia 11
2.6. Immunofluorescence staining 12
2.7. Immunoblotting assay 12
2.8. Electrophysiology 13
2.9. PDMS membrane and silanization 13
2.10. Immobilized ECM molecules and antibody coating 13
2.11. Cell culture 14
2.12. Mechanical stretch experiment 14
2.13. Tumor cells immunofluorescent staining and spreading area analysis 15
2.14. Tumor cells lysates immunoblotting 15
2.15. Sandwich enzyme-linked immunosorbent assay (ELISA) 17
2.16. Statistical analysis 17
3.1.1. Inflammatory pain models and behavior 18
3.1.2. Thermal hyperalgesia on the hot and cold plate 18
3.1.3. Immunohistochemistry expression of Navs in DRG neurons 20
3.1.4. Immunoblotting quality of Navs in DRG neurons 21
3.1.5. Functional analysis of TTX-R currents using whole-cell recording 22
3.1.6. MOR and A1AR agonist administration relieved mechanical and thermal pain 22
3.1.7. Nav1.8, COX-2 and phosphorylation of PKCε were attenuated by LFEA or CPA, but endomorphin 1 administration solely reduced the phosphorylation of PKCε 22
3.1.8. The proportion of the Nav1.8 and phosphospecific PKCε double-stained cells was reduced by LFEA, endomorphin 1 or CPA administration …………………… 22
3.1.9. MOR and A1AR antagonist administration reversed the analgesia effect of mechanical and thermal pain 23
3.1.10. MOR and A1AR antagonist administration suppressed the anti-inflammation effect by LFEA, up-regulated the express of phosphospecific PKCε and COX-2 23
3.2.1. HeLa cells expanded on the fibronectin or anti-syndecan-4 antibody-coated elastomeric PDMS membranes 24
3.2.2. Mechanical stretch induced the phosphorylation of focal adhesion kinase at tyrosine 397 through the engagement of syndecan-4 24
3.2.3. Mechanical stretch induced the phosphorylation of syndecan-4 downstream kinases: PKCα, FAK, and ERK 1/2 25
3.2.4. Mechanical stretch induced the phosphorylation of MLC 2 at serine19 through the engagement of syndecan-4 26
3.2.5. The phosphorylation of MLC2 was associated with the activity of PKCα under mechanical stimulation 26
3.2.6. Syndecan-4 delivered mechanical signaling and activated PKCα in B16-F10 mouse melanoma cells 27
3.2.7. FAK activation increased at the initial stage and PKCα was important to maintain the activity of MLC2 under mechanical stimulation 28
Figure 1 Primary inflammation-induced mechanical and thermal hyperalgesia through carrageenan and CFA injection 36
Figure 2 Nav1.7 and Nav1.8 expressions were increased in ipsilateral DRGs after intraplantar carrageenan injection and further attenuated by EA at the ST36 acupoint in mice, though Nav1.9 was not different 37
Figure 3 Nav1.7 and Nav1.8 expressions were increased in ipsilateral DRGs after intraplantar CFA injection and further attenuated by EA at the ST36 acupoint in mice, though Nav1.9 was not different 38
Figure 4 Nav1.7 and Nav1.8 protein levels were increased in lumbar DRGs in both intraplantar carrageenan- and CFA-induced inflammation and further attenuated by EA at the ST36 acupoint in mice, but Nav1.9 proteins were not altered 39
Figure 5 Protein levels of Nav1.7, Nav1.8, and Nav1.9 in the L3-L5 DRG in mice in control, Car, EA, S-Acu, S-GM, CFA, EA, S-Acu, S-GM groups 40
Figure 6 Tetrodotoxin-resistant (TTX-R) sodium currents in L3-L5 DRG neurons 41
Figure 7 MOR and A1AR agonist administration relieved mechanical and thermal pain. 42
Figure 8 Nav1.8, COX-2 and phosphorylation of PKCε were attenuated by LFEA or CPA, but endomorphin 1 administration solely reduced the phosphorylation of PKCε 43
Figure 9 The proportion of the Nav1.8 and phosphospecific PKCε double-stained cells was reduced by LFEA, endomorphin 1 or CPA administration 44
Figure 10 MOR and A1AR antagonist administration reversed the analgesia effect of mechanical and thermal pain 45
Figure 11 MOR and A1AR antagonist administration suppressed the anti-inflammation effect by LFEA, up-regulated the express of phosphospecific PKCε and COX-2 46
Figure 12 LFEA reduces inflammatory pain and change the expression of Nav1.7 and Nav1.8, rather than Nav1.9, in mice DRGs 47
Figure 13 HeLa cells expanded on fibronectin or anti-syndecan-4 antibody coated elastomeric PDMS membrane in starvation condition 48
Figure 14 Mechanical stretch induced the phosphorylation of focal adhesion kinase at tyrosine 397 on the elastomeric PDMS membranes 49
Figure 15 Mechanical stretch induced the phosphorylation of syndecan-4 downstream kinases: pPKCα, pFAK and pERK 1/2 50
Figure 16 Mechanical stretch induced the phosphorylation of myosin light chain 2 at serine19 on the Fn or anti-SDC4 antibody coated PDMS membrane 51
Figure 17 Mechanical stretch promoted the phosphorylation of myosin light chain 2 and dynamic changes were associated with the activity of PKCα 52
Figure 18 The engagement of syndecan-4 delivered mechanical signaling and activated PKCα in B16-F10 mouse melanoma cells 53
Figure 19 Mechanical stretch induced FAK activation at the initial stage and PKCα was important to maintain the phosphorylation of FAK and MLC2 54
Figure 20 Summary diagram of syndecan-4 regulates the dynamic activity of kinases through PKCα under mechanical stimuli 55

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