| Objective:1. To learn the role of leukotrienes(LTs) in the pathogenesis of adenotonsillarhypertrophy(AH) in children with obstructive sleep apnea/hypopnea syndrome(OSAHS).2.Analysis the inflammatory response in children with SDB (1＜AHI≤5),expecting to provide reference basis for revision of criteria for the diagnosis ofpediatric OSAHS in China.3.Explore the mechanism of leukotriene modifier(LTM) and inhaledcorticosteroid(ICS) therapy on reducing the adenoid size and alleviating the clinicalsymptoms in children with OSAHS.4. Investigate the effects of ICS on the metabolism of LTs in children withOSAHS, expecting to provide reference for the anti-inflammatory strategies andcombination therapy in children with OSAHS.Methods：In vivo study1.Compare the levels of LTs between the children with OSAHS(including OAHI＞5group and1＜OAHI≤5group）and recurrent tonsillitis(RI): Surgically removedtonsils/adenoids, then detected the expression of leukotriene C4synthase (LTC4S)mRNA in tonsils/adenoids by fluorescent quantitative PCR (FQPCR) and theconcentrations of cysteinyl leukotrienes (CysLTs) by using commercially availableenzyme-linked immunosorbent assay (ELISA) kit.2.To learn the effects of ICS on the metabolism of LTs in children with OSAHS（OAHI＞5）:Surgically removed tonsils/adenoids, then, detected the levels of LTC4SmRNA in tonsils/adenoids by FQPCR and the concentrations of CysLTs by usingcommercially available ELISA kit.In vitro study1.The effects of ICS on the tonsillar cellular proliferation: Tonsils weresurgically removed from the children with OSAHS（OAHI＞1）.Whole cell tonsillar cultures were established in the medium and plus different concentrations ofbudesonide(BUD). Cellular proliferation were evaluated by MTS after incubating in a5%CO2incubator at37℃for48h.2.The effects of ICS on LTC4S mRNA and CysLTs: Tonsils were surgicallyremoved from the children with OSAHS（OAHI＞1）.Whole cell tonsillar cultureswere established in the medium and plus different concentrations of BUD. Cellularproliferation were evaluated by MTS after incubating in a5%CO2incubator at37℃for48h. The concentrations required to achieve25%,35%and50%reduction inproliferation were defined as low dose BUD, middle dose BUD and high dose BUD,respectively. Then, cell cultures with low, middle, high dose BUD respectively for48h, detected the expression of LTC4S mRNA in cells by FQPCR and theconcentrations of CysLTs in supernatant by using ELISA kit.3.The effects of LTM on the tonsillar cellular proliferation: Tonsils weresurgically removed from the children with OSAHS（OAHI＞1）.Whole cell tonsillarcultures were established in the medium and plus different concentrations ofmontelukast (MON). Cellular proliferation were evaluated by MTS after incubating ina5%CO2incubator at37℃for48h.Results：1.A total of33tonsills/adenoids were surgically removed from children withOSAHS or RI,7children were enrolled as RI group,9children were enrolled asOSAHS (1＜OAHI≤5) group[mean OAHI is3.19/h（1.40～5.00/h）],10childrenwere enrolled as OSAHS (OAHI＞5) group[mean OAHI is11.73/（h5.40～23.77/h）],7children were enrolled as OSAHS-ICS (OAHI＞5) group [mean OAHI is14.27/h（5.60～32.06/h）].2．The expression of LTC4S mRNA in tonsils/adenoids in OSAHS(OAHI＞5)group was higher than those in the RI group(Z=-2.150,P=0.032). There was nostatistical differences in the espression of LTC4S mRNA between OSAHS(OAHI＞5)group and (1＜OAHI≤5) group(Z=-1.103,P=0.270). The expression of LTC4S mRNAin OSAHS(1＜OAHI≤5) group was higher than those in the RIgroup(Z=-1.960,P=0.050).3. The concentrations of CysLTs in tonsils/adenoids in OSAHS(OAHI＞5) group,OSAHS(1＜OAHI≤5) group and RI group were6808.59±4297.12pg/ml，2389.96±1341.04pg/ml,1313.54±610.54pg/ml, respectively. The levels of CysLTs inOSAHS(OAHI＞5) group were higher than those in RI group(t=-3.328,P=0.005) andOSAHS(1＜OAHI≤5)(t=2.951,P=0.009). There were no statistical differences in thelevels of CysLTs between OSAHS(1＜OAHI≤5) group and RIgroup(t=-1.960,P=0.070), but the levels of CysLTs in OSAHS(1＜OAHI≤5) groupwere higher.4.The expression of LTC4S mRNA in tonsils/adenoids in OSAHS-ICS group waslower than those in OSAHS-control group(Z=-3.027,P=0.002).The concentrations of CysLTs in OSAHS-ICS group and OSAHS-control group were5184.45±2901.39pg/ml,6808.59±4297.12pg/ml, respectively. There were no statistical differences inthe levels of CysLTs(t=0.867,P=0.400), but the levels of CysLTs in OSAHS-ICSgroup were lower.5.The effects of ICS on tonsillar cellular proliferation:13tonsills weresurgically removed from children with OSAHS(OAHI＞1).Compared with thecontrol group,the absorbance value in BUD(10-4M) group, BUD(5×10-5M) group,BUD(5×10-6M) group, BUD(5×10-7M) group and BUD(5×10-8M) group weredecreased, in other words, the tonsillar cellular proliferation was inhibited(t was4.858,4.281,3.131,3.077,3.213,respectively; P was0.000,0.000,0.005,0.005,0.004,repectively).The absorbance value was negatively correlated with BUDconcentration(r=-0.319,P=0.009)，instead, BUD exhibited dose-dependent reductionsin tonsillar cellular proliferation.6. The effects of ICS on LTC4S mRNA and CysLTs:13tonsils were surgicallyremoved from children with OSAHS(OAHI＞1).The results showed that theconcentrations of BUD required to achieve25%,35%and50%reduction inproliferation were5×10-13M、5×10-10M and5×10-5M,respectively; instead, lowdose BUD, middle dose BUD and high dose BUD was5×10-13M、5×10-10M and5×10-5M,respectively. The expression of LTC4S mRNA in high dose BUD group wassignificantly higher than then control group(Z=-3.257,P=0.001).There was nostatistical differences among the middle dose group, low dose group and control group（Z was-0.795,-0.898; P was0.427,0.369,respectively）.The levels of CysLTs incontrol group, low dose group, middle dose group and high dose group were53.75±18.07pg/ml，41.03±12.58pg/ml，49.93±16.67pg/ml and58.47±19.25pg/ml,respectively. There were no statistical differences among the four groups(F=2.504,P=0.070).7.The effects of LTM on tonsillar cellular proliferation:7tonsils were surgicallyremoved from children with OSAHS(OAHI＞1).Compared with the control group,theabsorbance value in MON（10-4M）group was significantly decreased, in other words,the tonsillar cellular proliferation was inhibited(t=6.888,P=0.000).There were nostatistical differences among the MON (10-5M) group, MON (10-6M) group, MON(10-7M)group, MON (10-8M)group and control group.(t was-0.495,-0.774,-0.082,0.082；P was0.629,0.454,0.936,0.936,respectively)Conclusion：1.The expression of LTC4S mRNA and the concentrations of CysLTs in thetonsils/adenoids in children with OSAHS(OAHI＞5) who matched the diagnosticcriteria in China were significantly higher, suggesting that LTs contribute to thepathogenesis of AH in pediatric OSAHS.2．The expression of LTC4S mRNA in the tonsils/adenoids in children withOSAHS(1＜OAHI≤5) who didn’t match the diagnostic criteria in China but werediagnosed as mild OSAHS as AASM were significantly higher, and the levels of CysLTs were also higher, showing inflammatory reaction similar to OSAHS(AHI＞5).The results suggested of that our diagnostic criteria for pediatric OSAHS shouldconsider the children with1＜OAHI≤5as mild OSAHS.3．LTM and ICS could inhibit the tonsillar cellular proliferation, which maycontribute to the mechanism of reducing adenoids size and alleviating the clinicalsymptoms in children with OSAHS.4.The results in vitro revealed that BUD couldn’t inhibit the synthesis of LTs intonsillar cells. However, we found that intranasal BUD for a long time could decreasethe levels of LTs in vivo. The results emphasize the importance of longer-termspecification using ICS, and provide the basis for rational use of drugs in pediatricOSAHS. |
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