IL-10Regulate The Expression Of ICAM-1from Lung Fibroblast Via P38MAPK Signaling Pathway

Objective: Taking the embryo lung fibroblasts as the research object,thisstudy investigated the effect of IL-10on the expression level of ICAM-1，which was upregulated by IL-1β via P38MAPK signaling pathway，based onthe observation of both the expression level of ICAM-1and thephosphorylation level of P38MAPK.In this study， we explored thepathogenesis of lung fibrosis further in order to provide some basis for thenew method of clinical treatment.Methods: Cultivation of embryo lung fibroblasts by tissue culturetechnology:after anaesthetize the wistar parturient rats by ether, we removethe fetal rat immediately and then remove its lung tissue. Cut it into1mm3sizesmall, and then put it into DMEM medium containing20%fetal bovineserum（condition of cultivation：constant temperature37℃，95%air and5%CO2gas mixture，saturated humidity），cultule4to6hours. The passagebegan about a week later.After5or6passages，the lung fibroblast grewadherently to the wall with consistent in morphology and structure. Then usethe fibroblast in fifth or sixth generations for the next step.First，the cultured lung fibroblasts （1×105/ml）were inoculated into a6orifice with a coverglass（6mm×22mm，disinfected）;then we place it intoa cell incubater for the cellular fusion（about80%-100%）.Abandon the oldnutrient solution，then inject the new nutrient solution（free from fetal bovineserum）.About12hours later，the G0cells synchronize generally. Divide thecultured fibroblasts into3groups:(1) Control group: merely culture theembryo lung fibroblasts in DMEM nutrient solution for6hours;(2)Intervention group with IL-1β: use the nutrient solution which contains IL-1β（15ng/ml） to culture the embryo lung fibroblasts for6hours;(3) Theintervention group with IL-10and IL-1β: Intervention group with IL-1β:use the nutrient solution which contains IL-10l0ng/ml and IL-1β（15ng/ml） toculture the embryo lung fibroblasts for6hours；each group repeat for3times.RT-PCR was applied to detect the expression level of ICAM-1mRNA.Western Blot was applied to detect the expression level ofphosphor-p38MAPK.Results:1Successful primary Culture of Wister fetal rat lung fibroblastsThis experiment adopts the tissue culture technology to culture the Wisterfetal rat lung fibroblasts. Finally we obtained an ideal lung fibroblastexperimental model with satisfactory growth conditions.These cells possessevenly cytoplasm，plump soma，apparent nucleolus，distinct mitosis and acoincident cellular morphology.2Expression level of ICAM-1mRNA in lung fibroblast cellThe optical density scanning of ICAM-1mRNA electrophoretic bandshows：in control group，there is a small amount of basal expression ofICAM-1mRNA in the lung fibroblasts（0.361±0.045）; the expression quantityof the intervention group with IL-1β(0.848±0.039) is significantly higher thanthe control group（P<0.01); the expression quantity of the intervention groupwith IL-10and IL-1β(0.740±0.026) is significantly lower than theintervention group with IL-1β（P<0.01), but higher than the expressionquantity of control group (P <0.01).3Expression level of P-P38protein in lung fibroblast cellThere is a basal expression of P-P38in the lung fibroblasts in controlgroup（0.581±0.034）;the expression quantity of the intervention group withIL-1β(1.196±0.038) is significantly higher than that the control group（P<0.01); the expression quantity of the intervention group with IL-10andIL-1β(0.780±0.028) is significantly lower than the intervention group withIL-1β（P<0.01), but higher than the expression quantity of control group，showed statistically significancy（P <0.01）.Conclusion:1There is a minor amount of expression of ICAM-1mRNA in normal lung fibroblasts.2IL-1β can significantly up-regulate the expression of ICAM-1in lungfibroblasts； this up-regulation may mediated by P38MAPK signalingpathway.3IL-10may restrain the expression of ICAM-1in lung fibroblasts byinhibiting the up-regulation of ICAM-1by IL-1β via P38MAPK signalingpathway.