中华医学杂志
中華醫學雜誌
중화의학잡지
National Medical Journal of China
2011年
10期
689-693
,共5页
罗琳%尤列·皮尔曼%维克多·科罗索夫%周向东
囉琳%尤列·皮爾曼%維剋多·科囉索伕%週嚮東
라림%우렬·피이만%유극다·과라색부%주향동
黏蛋白类%阿奇霉素%基质金属蛋白酶9
黏蛋白類%阿奇黴素%基質金屬蛋白酶9
점단백류%아기매소%기질금속단백매9
Mucins%Azithromycin%Matrix metalloproteinase 9
目的 探讨阿奇霉素(AZT)通过基质金属蛋白酶9(MMP9)抑制气道黏液高分泌的作用机制.方法 培养人支气管上皮细胞株HBE16细胞,以中性粒细胞弹性蛋白酶(NE)为刺激物.AZT和表皮生长因子受体(EGFR)拮抗剂BIBX1522为干扰因素,分别对NE刺激的HBE16细胞进行预处理.培养细胞分为对照组、NE组、AZT+NE组和BIBX1522+NE组.用RT-PCR检测各组细胞黏蛋白(MUC)5AC mRNA和MMP9 mRNA;ELISA检测培养上清液中MUC5AC蛋白含量;明胶酶谱法测定培养细胞内MMP9活性;Western印迹检测细胞中MMP9、前酶原MMP9(pro-MMP9)和金属蛋白酶类组织抑制剂(TIMP-1)蛋白含量及磷酸化的EGFR(p-EGFR)和磷酸化的信号末端调节激酶(p-ERK).结果 与对照组相比,NE组中MUC5AC与MMP9的基因转录(0.83±0.17,0.79±0.16)和蛋白表达水平(0.84±0.15,0.88±0.16)明显高(均P＜0.01),并且MMP9的活性指标高(392.33±18.33,P＜0.01),而pro-MMP9蛋白含量低(0.17±0.10,P＜0.01),TIMP-1含量未见明显改变,p-EGFR(0.86±0.23)和p-ERK(0.85±0.22)蛋白水平明显高(均P＜0.01);与NE组相比,AZT+NE组MUC5AC与MMP9的基因转录(0.36±0.15,0.41±0.09)和蛋白表达水平(0.30±0.08,0.37±0.14)明显低(均P＜0.01),MMP9活性也较低(295.33±14.54,P＜0.01),pro-MMP9(0.46±0.14)和TIMP-1(0.67±0.17)蛋白含量高(均P＜0.05),p-EGFR的蛋白水平未见明显改变,而p-ERK蛋白水平低(0.40±0.19,P＜0.01);与NE组相比,BIBX1522+NE组中除MUC5AC mRNA(0.37±0.14)、MMP9 mRNA(0.37±0.13)、p-EGFR(0.36±0.13)和p-ERK(0.37±0.18)明显低外(均P＜0.01),其余指标无明显改变,差异无统计学意义.结论 AZT能抑制气道上皮HBE16细胞内MMP9的活性及其产生,由此发挥抑制气道黏液高分泌的作用.
目的 探討阿奇黴素(AZT)通過基質金屬蛋白酶9(MMP9)抑製氣道黏液高分泌的作用機製.方法 培養人支氣管上皮細胞株HBE16細胞,以中性粒細胞彈性蛋白酶(NE)為刺激物.AZT和錶皮生長因子受體(EGFR)拮抗劑BIBX1522為榦擾因素,分彆對NE刺激的HBE16細胞進行預處理.培養細胞分為對照組、NE組、AZT+NE組和BIBX1522+NE組.用RT-PCR檢測各組細胞黏蛋白(MUC)5AC mRNA和MMP9 mRNA;ELISA檢測培養上清液中MUC5AC蛋白含量;明膠酶譜法測定培養細胞內MMP9活性;Western印跡檢測細胞中MMP9、前酶原MMP9(pro-MMP9)和金屬蛋白酶類組織抑製劑(TIMP-1)蛋白含量及燐痠化的EGFR(p-EGFR)和燐痠化的信號末耑調節激酶(p-ERK).結果 與對照組相比,NE組中MUC5AC與MMP9的基因轉錄(0.83±0.17,0.79±0.16)和蛋白錶達水平(0.84±0.15,0.88±0.16)明顯高(均P＜0.01),併且MMP9的活性指標高(392.33±18.33,P＜0.01),而pro-MMP9蛋白含量低(0.17±0.10,P＜0.01),TIMP-1含量未見明顯改變,p-EGFR(0.86±0.23)和p-ERK(0.85±0.22)蛋白水平明顯高(均P＜0.01);與NE組相比,AZT+NE組MUC5AC與MMP9的基因轉錄(0.36±0.15,0.41±0.09)和蛋白錶達水平(0.30±0.08,0.37±0.14)明顯低(均P＜0.01),MMP9活性也較低(295.33±14.54,P＜0.01),pro-MMP9(0.46±0.14)和TIMP-1(0.67±0.17)蛋白含量高(均P＜0.05),p-EGFR的蛋白水平未見明顯改變,而p-ERK蛋白水平低(0.40±0.19,P＜0.01);與NE組相比,BIBX1522+NE組中除MUC5AC mRNA(0.37±0.14)、MMP9 mRNA(0.37±0.13)、p-EGFR(0.36±0.13)和p-ERK(0.37±0.18)明顯低外(均P＜0.01),其餘指標無明顯改變,差異無統計學意義.結論 AZT能抑製氣道上皮HBE16細胞內MMP9的活性及其產生,由此髮揮抑製氣道黏液高分泌的作用.
목적 탐토아기매소(AZT)통과기질금속단백매9(MMP9)억제기도점액고분비적작용궤제.방법 배양인지기관상피세포주HBE16세포,이중성립세포탄성단백매(NE)위자격물.AZT화표피생장인자수체(EGFR)길항제BIBX1522위간우인소,분별대NE자격적HBE16세포진행예처리.배양세포분위대조조、NE조、AZT+NE조화BIBX1522+NE조.용RT-PCR검측각조세포점단백(MUC)5AC mRNA화MMP9 mRNA;ELISA검측배양상청액중MUC5AC단백함량;명효매보법측정배양세포내MMP9활성;Western인적검측세포중MMP9、전매원MMP9(pro-MMP9)화금속단백매류조직억제제(TIMP-1)단백함량급린산화적EGFR(p-EGFR)화린산화적신호말단조절격매(p-ERK).결과 여대조조상비,NE조중MUC5AC여MMP9적기인전록(0.83±0.17,0.79±0.16)화단백표체수평(0.84±0.15,0.88±0.16)명현고(균P＜0.01),병차MMP9적활성지표고(392.33±18.33,P＜0.01),이pro-MMP9단백함량저(0.17±0.10,P＜0.01),TIMP-1함량미견명현개변,p-EGFR(0.86±0.23)화p-ERK(0.85±0.22)단백수평명현고(균P＜0.01);여NE조상비,AZT+NE조MUC5AC여MMP9적기인전록(0.36±0.15,0.41±0.09)화단백표체수평(0.30±0.08,0.37±0.14)명현저(균P＜0.01),MMP9활성야교저(295.33±14.54,P＜0.01),pro-MMP9(0.46±0.14)화TIMP-1(0.67±0.17)단백함량고(균P＜0.05),p-EGFR적단백수평미견명현개변,이p-ERK단백수평저(0.40±0.19,P＜0.01);여NE조상비,BIBX1522+NE조중제MUC5AC mRNA(0.37±0.14)、MMP9 mRNA(0.37±0.13)、p-EGFR(0.36±0.13)화p-ERK(0.37±0.18)명현저외(균P＜0.01),기여지표무명현개변,차이무통계학의의.결론 AZT능억제기도상피HBE16세포내MMP9적활성급기산생,유차발휘억제기도점액고분비적작용.
Objective To investigate the mechanism of azithromycin (AZT) inhibiting the airway mucous hypersecretion through matrix metalloproteinase 9 (MMP9).Methods After culturing,the airway epithelial cells of line HBE16 were stimulated with neutrophil elastase (NE) and pretreated with AZT and epidermal growth factor receptor (EGFR) antagonist BIBX1522.Then the cells were divided into 4 groups:control group,NE-stimulated group,AZT-pretreated & NE-stimulated group and BIBX1522-pretreated & NE-stimulated group.The mucin (MUC)5AC mRNA and MMP9 mRNA levels were analyzed by RT-PCR (reverse transcription-polymerase chain reaction).And the MUC5AC protein content in supernatant was detected by ELISA (enzyme-linked immunosorbent assay).Gelatin zymogrphy was employed to assay the MMP9 activity.Western blot was used to detect the protein levels of MMP9,prozymogen MMP9 (proMMP9),tissue inhibitors of metalloproteinases 1 (TIMP-1),phosphorylated EGFR (p-EGFR) and phosphorylated external-signal regulated kinase (p-ERK).Results As compared with the control group,the levels of MUC5AC and MMP9 gene transcription (0.83 ± 0.17,0.79 ± 0.16) and protein expression (0.84 ±0.15,0.88 ±0.16) in the NE stimulated group were obviously higher than those of the control group (all P ＜ 0.01).And the activity of MMP9 increased (392.33 ± 18.33,P ＜ 0.01) while the protein level of pro-MMP9 decreased (0.17 ±0.10,P ＜0.01).But the expression of TIMP-1 showed no significant change.The protein expressions of p-EGFR and p-ERK increased (0.86 ± 0.23,0.85 ± 0.22,both P ＜0.01);as compared with the NE-stimulated group,there was a reduction of MUC5AC and MMP9 gene transcription (0.36 ± 0.15,0.41 ± 0.09,both P ＜ 0.01) and protein expression levels (0.30 ± 0.08,0.37 ±0.14,both P ＜0.01) in the AZT-pretreated and NE-stimulated group.The MMP9 activity decreased (295.33 ± 14.54,P ＜0.01),the protein levels of pro-MMP9 and TIMP-1 increased (0.46 ±0.14,0.67 ±0.17,both P ＜ 0.05),p-ERK protein level decreased (0.40 ± 0.19,P ＜ 0.01) while the expression of p-EGFR showed no significant decline.As compared with the NE-stimulated group,except for the expressions of MUC5AC mRNA (0.37 ±0.14),MMP9 mRNA (0.37 ±0.13),p-EGFR (0.36 ±0.13) and p-ERK (0.37 ±0.18) decreasing (all P ＜0.01) in BIBX1522-pretreated and NE-stimulated group,the other results had no obvious change.Conclusion AZT may suppress the activity and production of MMP9 in HBE16 cells so as to inhibit the airway mucous hypersecretion.
