| Objective:Through observing the serum concentration of TGF-β1,IL-4and IFN-γ,and the expression of Ras,ERK in Rats asthmatic model,to invest-tigate the inhibitory mechanism of simvastatin in asthmatic mice,the objective as:(1)Simvastatin can inhibit the activation of Ras-MAPK signaling cascade.(2)Simvastatin can reduce airway inflammation through effecting on Th1/Th2balance.(3)Simvastatin can decrease serum concentration of TGF-β1and the expression of collagen deposition to reduce airway remodeling.Method:Forty male SD rats(weight range100-200g)were randomly divided into five groups:control group,asthma model group,Simvastatin20mg/kg treat group,Simvastatin40mg/kg treat group,Simvastatin60mg/kg treat group. The asthma model group and simvastatin groups were sensitized and challenged with ovalbumin. The control group rats used normal saline instead of OVA. Animals of simvastatin groups were also given intragastric administration with simvastatin (the dosage is respectively20mg/kg,40mg/kg,60mg/kg) besides be sensitized and challenged.Pulmonary function was measured in24hours when rats were anesthetized by1%pentobarbital after the last OVA challenge.Then we collected bronchoalveolar lavage fluids (BALF),the serum and lung tissue of each group rats.The level of IL-4, IFN-γ and TGF-β1in serum were assayed by ELISA.The total and differential cell counts in BALF were counted.The lung tissue sections were stained with HE and manssion staining method.We used image analysis system to measure the parameters such as thickness of airway wall and smooth muscle layer thick-ness.The expression of ras and ERK were measured by immunohistochemical method combined with microimage analysis.Results:1Result of pulmonary function:With concentration of Ach increased, the average expiratory peak was decreased remarkably in model group and simvastatin groups,but the average expiratory peak in simvastatin groups was higher than in model group(P<0.05).2The total cell and the eosinophils in BAL fluid:The total cell and the eosinophils in BAL fluid of the asthma group were significantly increased as compared with the other four groups (F=604.126P<0.01,F=133.766P<0.01). The total cell and the eosinophils in BAL fluid of the control group were lower than these of the simvastatin groups (P<0.01). Simvastatin(20mg/kg,40mg/kg and60mg/kg) substantially reduced the total cell number as compared with asthma group,which was mainly due to a obvious reduction in eosinophils in the simvastatin-treated rats in a dose-dependent manner.3Pathological findings:We can detect that inflammatory cells infiltrat-ing,the smooth muscle cell proliferating,airway wall thickness and mucus hypersecre-tion in the lung tissue of asthmatic rats.The three simvastatin groups can markedly attenuate those variations. Observing the thickness of airway wall and airway smooth muscle in these five groups showed that the control group(59.07±10.95,12.85±2.47) were the lowest among the five groups (F=34.553P<0.01, F=100.925P<0.01). Simvastatin groups [20mg/kg (80.64±14.30,31.28±2.61),40mg/kg(73.87±11.97,24.53±2.73) and60mg/kg (66.88±11.47,17.05±2.65)] significantly reduced the thickness of airway wall and airway smooth muscle as compared with countrol group(59.07±10.95,12.85±2.47). The higher the dose of simvastatin we give, the lower the thickness of airway wall and airway smooth muscle we get.4Result of collagen deposition:The expression of collagen of asthma group (0.0634±0.0071) and simvastatin groups [20mg/kg(0.0463±0.0058),40mg/kg (0.0416±0.0058),60mg/kg(0.0276±0.006)] was obviously higher than that of control group(0.0184±0.0034)(F=65.778,P<0.01).And that was lower in sim-vastatin groups than asthma group (P<0.01). The higher the dose of simvasta-tin we give,the lower the expression of collagen we get.5Result of the level of IL-4, IFN-γ and TGF-β1in serum:The level of IL-4and TGF-β1in serum of asthma group(475.71±55.56,11.86±3.17) and simvastatin groups [20mg/kg(362.91±37.59,162.03±15.51),40mg/kg (284.62±34.15,136.87±9.55) and60mg/kg (225.18±26.98,7.07±8.88)] were signi-ficantly higher than these of control group (174.34±40.64,56.83±10.62)(F=64.617P<0.01, F=124.41P<0.01).And these were lower in simvastatin groups than asthma group (P<0.01). The higher the dose of simvastatin we give,the lower the level of IL-4and TGF-β1in serum we get.The level of IFN-γ in serum of asthma group(11.86±3.17) and simvastatin groups [20mg/kg (22.11±4.82),40mg/kg (34.52±4.95) and60mg/kg(44.70±4.16)] was significantly lower than control group (51.26±8.59)(F=61.541, P<0.01). And that was higher in simvastatin groups than asthma group (P<0.05). The higher the dose of simvastatin we give,the higher the level of IFN-y in serum we get.6Result of immunohistochemistry:The average light density values of Ras protein and ERK protein in the asthma group(0.4963±0.078,0.2515±0.0417) and simvastatin groups[20mg/kg(0.3955±0.084,0.2138±0.0299),40mg/kg(0.3069±0.076,0.1727±0.031) and60mg/kg(0.2162±0.077,0.1320±0.0258)] were significantly higher than control group (0.1263±0.058,0.0926±0.016)(F=27.531P<0.01,F=31.334P<0.01).And these were lower in simvastatin groups than asthma group (P<0.05). The higher the dose of simvastatin we give,the lower the average light density values of Ras protein and ERK protein in rats airway we get.7Result of correlation analysis:the average light density values of ERK protein was positively correlated with the expression of collagen, the level of IL-4and TGF-β1in asthmatic rats.(r=0.861P<0.05,r=0.901P<0.01,r=0.822P<0.05). It was a significant negative correlation between the average light density values of ERK protein and IFN-γ(r=-0.822, P<0.05).Conclusion:1The expression of Ras protein and ERk protein in lung tissues of asthmatic model group were significantly increased as compared with the control group. It was implicated that the Ras-MAPK signaling cascade plays a pivotal role in the develepment of asthma.The expression of Ras protein and ERk protein of simvastatin groups were obviously decreased compared with the asthma group, and those were increased compared with the control group.It was implicated that simvastatin may have therapeutic potential role for the treatment of asthma through inhibition of Ras-MAPK signaling pathway.2When used simvastatin, in the asthmatic rats,the serum concentration of IL-4and TGF-β1were significantly decreased and the serum concentration of IFN-y was obviously increased.3In the asthmatic rats, the expression of collagen, the serum level of IL-4and TGF-β1were all positively correlated with the airway expression of ERK protein,but the serum level of IFN-y was negative correlated with the airway expression of ERK protein.It was indicated that simvastatin can inhibit the airway remodeling by decreasing the expression of collagen and the serum level of TGF-β1. Simvastatin can reduce airway inflammation by decreasing the serum level of IL-4and increasing the serum level of IFN-y.This effect can be due to the decrease in the airway expression of ERK protein.4The therapeutic effect of higher dose of simvastatin was better than that of lower dose.It was indicated that simvastatin may have a dose-dependent manner for the treatment of asthma. |
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