| Objective: To study the effect of ephedrine on morphology,proliferation and expression of Eotaxin and IL-8in human bronchialepithelial cells (16HBE) cultured in vitro, and to explore the mechanism ofephedra in the treatment of asthma. Methods: The16HBE strain weresubcultured in cell culture flask and the fourth or fifth generation cells ingood state were choosed for the experiment:(1) The16HBE were seeded in6-well plates and96-well plates, and randomly divided into five groups,and then interfered sequentially with ephedrine at different concentration (0,150,300,600,1200μg/mL) for24,48,72hours. The morphological andgrowth status of16HBE were observed by inverted phase contrastmicroscope. The cell proliferation was detected by CCK-8assay.(2) The16HBE were inoculated in6-well plates and randomly divided into sixgroups:①Control group (group A):16HBE were incubated in completemedium for18h;②Ephedrine group (group B):16HBE were incubated incomplete medium containing300μg/mL ephedrine for18h;③TNF-αgroup (group C):16HBE were incubated in complete medium containing20ng/mL TNF-α for18h;④Ephedrine plus TNF-α group (group D): 16HBE were pre-incubated with300μg/mL ephedrine for1hour beforebeing added to100ng/mL TNF-α for18h;⑤Dexamethasone plus TNF-αgroup (group E):16HBE were pre-incubated with1.4μg/mL dexame-thasone for1h before being added to100ng/mL TNF-α for18h;⑥Ephedrine plus dexamethasone plus TNF-α group (group F):16HBE werepre-incubated with300μg/mL ephedrine and1.4μg/mL dexamethasone for1hour before being added to100ng/mL TNF-α for18h. The finalconcentration of TNF-α in group D, E, F was20ng/mL.(3) The expressionof Eotaxin and IL-8mRNA in16HBE was determined by fluorescentquantitation PCR; The expression of Eotaxin and IL-8protein was detectedby immunofluorescence; The concentration of Eotaxin and IL-8protein incell culture supernatant was quantified by ELISA. Results:(1) Aftertreatment with different ephedrine concentration (150,300,600,1200μg/mL) for24,48,72hours, the morphology and growth status were notdifferent from the control group (0μg/mL ephedrine) in16HBE.(2) TheCCK-8indicated that after treatment with different ephedrine concentration(0,150,300,600,1200μg/mL) for24hours, the OD value of16HBE was0.97±0.17,0.99±0.24,1.14±0.25,1.11±0.18,0.95±0.18, respectively.There was no statistic significantly difference among different groups(P=0.504); After treatment with different ephedrine concentration (0,150,300,600,1200μg/mL) for48hours, the OD value of16HBE was 1.09±0.43,1.16±0.99,1.16±0.87,1.18±0.56,1.11±0.30, respectively. Therewas no statistic significantly difference among different groups (P=0.282);After treatment with different ephedrine concentration (0,150,300,600,1200μg/mL) for72hours,the OD of16HBE was1.40±0.13,1.39±0.10,1.39±0.16,1.41±0.01,1.40±0.01,respectively. There was no statisticsignificantly difference among different groups (P=0.18).⑶Fluorescentquantitative PCR showed that the expression of Eotaxin mRNA(RQ) ingroup A to F was0.99±0.01,0.98±0.02,3.03±0.32,1.82±0.23,1.78±0.02,0.97±0.01,respectively. There was statistical significance among differentgroups (P<0.01). Compared with group A, the expression of EotaxinmRNA increased obviously in group C and there was statistic significancebetween them (P<0.01)．Compared with group C,the expression of EotaxinmRNA was all decreased in group D,E,F and the most significant decreasedgroup was group F. There was statistic significance between them (allP<0.01). The expression of IL-8mRNA(RQ) in16HBE in group A to Fwas0.95±0.05,0.93±0.14,7.18±0.31,4.44±0.24,4.11±0.19,2.97±0.39,respectively. There was statistical significance among different groups(P<0.01).Compared with group A, the expression of IL-8mRNA wasincreased obviously in group C and there was statistic significance betweenthem (P<0.01). Compared with group C, the expression of IL-8mRNAwas all decreased in group D, E, F and the most significant decreased group was group F. There was statistic significance between them (all P<0.01).⑷Immunofluorescence showed that the expression of Eotaxin and IL-8protein was mainly in the cytoplasm in16HBE.And the expression was thehighest in group C. Compared with group C, it was lower in group D, E, Fand the lowest in group F.⑸ELISA showed that the concentration ofEotaxin in16HBE cell culture supernatant was1.72±0.16,1.60±0.17,4.05±0.20,3.70±0.21,3.33±0.33,2.76±0.15, respectively. There wasstatistical significance among different groups (P<0.01). Compared withgroup A, the concentration of Eotaxin was increased obviously in group Cand there was statistic significance between them (P<0.01). Compared withgroup C, the concentration of Eotaxin was all decreased in group D, E, Fand the most significant decreased group was group F. There was statisticsignificance between them (all P<0.01).The concentration of IL-8in16HBE culture supernatant was17.45±2.32,18.78±1.05,27.24±1.91,23.62±3.40,19.25±2.36,14.12±3.14, respectively. There was statisticalsignificance among different groups(P<0.01). Compared with group A, theconcentration of IL-8was increased obviously in group C and there wasstatistic significance between them (P<0.01).Compared with group C, theconcentration of IL-8were all decreased in group D, E, F and the mostsignificant decreased group was group F. There was statistic significancebetween them (P<0.05, P<0.01, P<0.01). Conclusion：⑴In0-1200μg/mL concentration, ephedrine had no effect on the proliferation and morphologyof16HBE in vitro;⑵Ephedrine can inhibit the expression of Eotaxin andIL-8induced by TNF-α in16HBE in vitro, this may be one of themechanisms of ephedra which was used for the treatment of asthma;⑶There is a synergistic interaction between ephedrine and dexamethasoneon the inhibitory effect of the expression of Eotaxin and IL8in16HBE. |
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