內生性一氧化氮藉由mitogen-activated protein kinase 途徑調控氣喘 病人先趨細胞增生能力之探討
Endogenous nitric oxide (NO) modulates the proliferation capacity via mitogen-activated protein kinase pathway in progenitor cells of asthmatics
中文摘要
我們過去的研究發現，來自氣喘患者週邊血液的CD34+先驅細胞(progenitor cells)，其誘導性一氧化氮合成酶(inducible nitric oxide synthase, iNOS)明顯上升，
給予L-NAME (NG-nitro-L-arginine methyl ester)抑制 NO (一氧化氮)合成，會促 進先驅細胞增生，株落形成大量增加。NO 也會引起細胞凋亡(apoptosis)，影響 Bcl-2 的表現。MAPK (Mitogen-activated protein kinases)是細胞膜與細胞核間訊號 傳遞的一個重要介質，與細胞的死亡和分化息息相關，而一氧化氮會調節 MAPK 的活化。本論文主要在探討 NO 影響氣喘患者週邊血液 CD34+先驅細胞
增生，是否經由MAPK 之訊號傳遞路徑，改變 Bcl-2 的表現，進一步影響先驅
細胞的增生。從26 個氣喘患者與 32 個正常人的週邊血液先驅細胞給予不同的濃
度L-NAME、SNP (sodium nitorprusside)、PD98059 (MEK 抑制劑)與 SB203580 (p38 MAPK 抑制劑)經 24 小時處理後，以流式細胞計數儀(flow cytometery)分析 Bcl-2 的蛋白質表現與 Annexin-V 的表現，分析先驅細胞細胞凋亡的變化。經 14 天細胞培養，計算細胞株落形成的數目，進一步了解各種處理對先驅細胞生長 的影響；並分析細胞內DNA 含量的變化，以探討細胞生長週期(cell cycle)的變 化。我們的結果發現，L-NAME 明顯地增加氣喘患者先驅細胞 Bcl-2 的表現，而
正常人則無影響。相反的，當以NO 提供者 SNP 處理氣喘病人及正常人先驅細
胞 24 小時後，發現其 Bcl-2 表現並沒有受到影響。以 Annexin-V 的表現分析細胞 凋亡的變化可發現，L-NAME 明顯地減少氣喘病人先驅細胞 Annexin-V 的表現，
於正常人則否。而SNP 對於氣喘病人及正常人先驅細胞之細胞凋亡完全沒有影響
為了觀察MAPK 是否會影響 Bcl-2 的表現，於氣喘病人之先驅細胞加入
SB203580 (10 μM)及 PD98059 (18 μM)培養 24 小時，發現 SB203580 及 PD98059 都會明顯的減少其Bcl-2 表現。相反地，不論 SB203580 或 PD98059 都不會影響
正常人先驅細胞Bcl-2 之表現。當以 L-NAME 處理氣喘病人先驅細胞，測量其
ERK 的磷酸化變化，發現在 2 分鐘時，L-NAME 會促進 ERK 的磷酸化表現達到 最大值，但對正常人先驅細胞則沒有影響。SNP 對氣喘病人及正常人先驅細胞 的ERK 磷酸化，則完全沒有影響。以 L-NAME (1mM)處理先驅細胞經 14 天培養 後，氣喘病人株落(colony)形成相對於對照組明顯地增加，給予 SNP 株落形成 減少，而L-NAME 及 SNP 對正常人則無影響。氣喘病人先驅細胞經培養 14 天後 發現，PD98059 明顯抑制株落形成，由對照組的 55.8 ± 4.9 個(n=12)減少至 46.9
± 4.7 個(n=12, p<0.05)，而 SB203580 則減少至 34.3 ± 4.9 個(n=12, p<0.01)，同時 加入L-NAME 與 PD98059 抑制株落形成更明顯，對正常人則無影響。然而，L-
NAME 抑制內生性 NO 並不影響先驅細胞之細胞週期，但 PD98059 則會明顯減
少S phase，而抑制細胞生長。由以上的結果發現，內生性一氧化氮會抑制氣喘
病人先驅細胞的增生，其可能是經由抑制ERK 及 p38 MAPK 的路徑，使 Bcl-2 的表現減少，因而導致先驅細胞的細胞凋亡之增加所產生。
英文摘要
Our previous report has demonstrated that progenitor cells from asthmatics possess higher expression of induced nitric oxide synthase (iNOS). Inhibition of endogenous of nitric oxide (NO) promotes the progenitor cells proliferation and increases colony formation by NG-nitro-L-arginine methyl ester (L-NAME). NO can induce cell apoptosis and affect Bcl-2 expression. Mitogen-activated protein kinases (MAPK) play an important mediator of signal transduction in regulation of cell death and proliferation. NO may modulate the activation of MAPK. It is unknown whether NO would modulate the proliferation and apoptosis of peripheral blood progenitor cells from asthmatic patient via MAPK signaling transduction pathway.
Peripheral blood progenitor cells isolated from 26 asthmatics and 32 normal subjects were treated by L-NAME or SNP (sodium nitroprusside, NO donor) or PD98059 (MEK inhibitor) or SB203580 (p38 MAPK inhibitor) for 24 hours. The expression of Bcl-2 protein and Annexin-V was analysed to measure cell apoptosis by flow
cytometry. Colonies of progenitor cells were scored to determine the capacity of cell proliferation after 14-day culture. Using flow cytometric analyses, cell cycle of progenitor cells was detected by DNA contents of propidium iodide staining. In asthmatics, inhibition of endogenous NO by L-NAME dose-dependently increased the expression of Bcl-2 protein in progenitor cells (1mM 345.2 ± 37.0, 10mM 415.2 ± 44.5 mean fluorescence intensity, MFI, respectively, n=10, p<0.01) compared to vehicle control (323.0 ± 37.3, MFI, n=10), while there was no effect in normal subjects. In contrast, SNP did not affect the Bcl-2 expression of progenitor cells either from asthmatics or normal subjects. In addition, L-NAME significantly decreased the Annexin-V expression of progenitor cells from asthmatics, but not normal subjects.
Treatment with SNP showed no difference in the Annexin-V expression of progenitor cells from asthmatics or normal subjects. To study effect of MAPK on cell apoptosis, both PD98059 (18μM) and SB203580 (10μM) significantly decreased the Bcl-2 expression (PD98059, 45.6 ± 12.1 MFI; SB203580, 50.6 ± 12.7 MFI, n=8, p<0.05, respectively) of progenitor cells from asthmatics compared to vehicle control (86.2 ± 19.3 MFI, n=8). There were no significant differences in normal subjects. In
asthmatic group, L-NAME significantly enhanced ERK phosphorylation at 2 minutes (180.9 ± 18.5 MFI, p<0.05, n=6). L-NAME potentiated and SNP inhibited the
colonies growth. In normal subjects there were no effect. Both PD98059 and
SB203580 significantly inhibited the colony formation from 55.2 ± 6.8 to 46.9 ± 6.7 (PD98059, n=12, p<0.05) and 34.3 ± 6.0 (SB203580 n=12, p<0.05). Combined L- NAME with PD98059 had a significant synergic effect on the colony formation.
There was no significant difference in normal subjects. L-NAME did not affect cell cycle of progenitor cells, while the PD98059 decreased the S phase cell cycle to inhibit cell growth.
We concluded that NO decreased the Bcl-2 expression and accelerated cell apoptosis of progenitor cells from asthmatics, which may be through signaling pathway of MAPK, in turn contributing to the inhibition of cell proliferation.
