The Effect Of Macrophage-stimulating Protein And Receptor Tyrosine Kinase RON On Airway Inflammation Of Rats With COPD

Objective: To observe the levels of Macrophage-stimulating protein(MSP) inbronchoalveolar lavage fluid(BALF) and serum of smoking- -fumigating rats,andthe expression changes of receptor tyrosine kinase RON on the bronchpulmonarytissues and alveolar macrophages (AM) of rats at different phases of tobacco smokeexposure in the pathogenesis of COPD. To investigate the influence of MSP on theproduction of inflammatory factors and oxygen free radicals by AMs. In oder toexplore the role of MSP and receptor tyrosine kinase RON in the airwayinflammation of the chronic obstructive pulmonary disease(COPD) rats to cigarettesmoke exposure and investigate its possible mechanism.Methods: Wistar rats were randomly divided into one Normal control grouprat and 3 passive smoking groups (1 month,2 months,3months group).COPD modelwas established by exposing the rats to cigarette smoke daily. Rat alveolarmacrophages of all groups were isolated in vivo and cultured, and then challengedwith different concentrations of MSP for 24 hours. Observed the pathologicalchanges of the lung tissue of all the groups rats under light microscope, and thenanalyzed the three emphysema indexes of MLI,PAA and MAN using the imageanalysis system. The concentrations of MSP in broncho-alveolar lavage fluid(BALF)and serum, and the levels of IL-1β,TNF-α,IL-8,IL-10 in the supernatants weremeasured by ELISA. The activity of superoxide dismutase(SOD) andmalondialdehyde(MDA) content in the culture solution were measured withchromatometry method. The expression of RON mRNA in lung tissue of rats was assessed by reverse transcription-polymerase chain reaction(RT-PCR).The levels ofRON protein in the lung tissue of rats and AMs cultured in vitro were observed byimmunohistochemistry.Results: (1) the three indices MLI, MAN and PAA to reflect the pulmonaryemphysema of 1 month smoking group had no significant differences comparedto Normal control group. The three indices of 2 months smoking group started toshow differences,and the differences of the 3 months smoking group becomedmore obvious and the lung tissue pathological changes of the 3 monthssmoking group accord with the pathological diagnostic criteria of COPD. (2)The concentrations of MSP in the serum and BALF of all smoking rats wereboth higher than those in the control group with different degree,the moreexposed to cigrarette smoke,the more significant increase of the concentration.The rats of the 3months group were the highest in passive smoking rats,and alsothe highest in all groups. (3) Compared to the control group, the levels ofRON mRNA in lung tissure and the expression of RON protein in bronchialepithelium of the three smoking groups rats were all up-regulated significantlywith different degree; the levels of RON protein in the AMs cultured in vitro ofall the three smoking-fumigating groups rats are significantly higher than thoseof the contol groups; and between the Normal contol group and 1monthgroup,the expression of RON protein in AMs were in the cell membrane,wherasthe AM’s RON protein of the 2 month group and the 3months group was foundboth in membrane and in cytoplasm. The more passive smoking time, the moreexpression of RON mRNA in the lung tissue, and the more expression of RONprotein in lung tissue and AMs. The expression of RON mRNA and protein inlung tissue as well as the expression of RON protein in AMs of 3months groupwere significantly higher than those of the other groups. (4) MSP evoked the AMs isolated from the rats to generate the more content of MDA and caused areduction in activity of SOD in a dose-dependent manner,and also had acorrelation with the passive smoking time.With the prolonging of exposure tocigarette smoke, the increase of the content of MDA and the decrease of theactivity of SOD of the AM culture supernatant were all in the time-dependentmanner.The more dose of the MSP and the smoking time,the more content ofMDA and the lower activity of SOD. (5) In addition, MSP could still stimulatecytokine release from AM of all groups rats, such as TNF-α, IL-8, IL-1βandIL-10, where the effect of MSP is in a dose-dependent manner too. In cellsupernatant, the amounts of TNF-α, IL-8 and IL-1βreleased from the AM of allthe smoking groups rats without MSP stimulation or in the same dose of MSPwere higher than the control group rats,but the IL-10 production was lower thanthe control group. And with the prolonging of exposure to cigarette smoke, theincrease of the amounts of TNF-α, IL-8 and IL-1βand the decrease of IL-10 inAM supernatant were all in the time-dependent manner.Conclusion: (1) Rat COPD model can be established successfully byexposing the rats to cigarette smoke daily.(2) smoking-fumigating increasessynthesis and release of MSP ,also up-regulates RON gene transcription and thenenhances its synthesis and expression,which had a correlation with the smokingtime.(3) MSP, in a concentration-dependent manner, induces significan oxygenfree radical and cytokine release in AM from rats, futhermore which effect canbe amplified by stimulation of cigarette smoke.(4) MSP and receptor tyrosinekinase RON plays an important role in regulating airway inflammation of ratswith COPD exposed to cigarette smoke. One of the mechanism might be thatMSP promote the macrophage release more inflammatory factors and induce theincrease of the production of oxygen free radicals.