| OBJECTIVE:To investigate the effect of spearmint oil on Emphysema-like changes induced by lipopolysaccharide or porcine pancreatic elastase and increase of matrix metalloproteinase-9.METHODS:1. SD rats were randomly divided into six groups:①control group;②model group;③Spearmint oil 10 mg·kg-1 group;④Spearmint oil 30 mg·kg-1 group;⑤Spearmint oil 100 mg·kg-1 group;⑥dexamethasone 0.3 mg·kg-1 group. Emphysematous changes model was induced by intratracheal instillation of LPS (200μg) once a week for up to 8 weeks in rats. Each group was treated with saline, dexamethasone, and spearmint oil respectively for 4 weeks. Then rats were anesthetized, instrumented for the measurement of lung resistance (RL) and dynamic compliance (Cdyn). Total and different white blood cell counts in bronchoalveolar lavage fluid(BALF) were carried out. The pathologic changes of lung tissue such as alveolar structure, airway inflammation, and goblet cell metaplasia were observed by HE and AB-PAS staining. TNF-α, IL-1β, TIMP-1 content in lung tissues were detected by ELISA and expression of MMP-9 in lung tissue was measured by immunohistochemistry. The effect of Spearmint oil on these changes were determined. Statistical software SPSS16.0 was used for all data analysis.2. C57 mice were randomly divided into six groups:①control group;②model group;③Spearmint oil 14 mg·kg-1 group;④Spearmint oil 42 mg·kg-1 group;⑤Spearmint oil 141 mg·kg-1 group;⑥dexamethasone 0.42 mg·kg-1 group. Emphysematous changes model was induced by intratracheal instillation of PPE (4.5 U) for only once. Three weeks later, each group was treated with saline, dexamethasone, and Spearmint oil respectively for 3 weeks. The pathologic changes of lung tissue such as alveolar structure, airway inflammation were observed by HE staining. The expression of MMP-9,TIMP-1, TNF-α, IL-8 mRNA in lung tissues were verified by RT-PCR. The effect of Spearmint oil on these changes were determined.Statistical software SPSS16.0 was used for all data analysis.RESULTS(1) Airway resistance(RL) in model group increased by 107.6% and dynamic compliance (Cdyn) decreased by 17.9% compared with control group. Spearmint oil 10 mg·kg-1 group significantly inhibited RL increase compared with model group.(2) The leucocyte, including neutrophile, lymphocyte and macrophage numbers in BALF were significantly higher in model group than those in control group(P<0.001), Spearmint oil 100 mg·kg-1 group and dexamethasone treatment group were obviously lower than those in model group (P<0.05,P<0.01,P<0.001).(3)In model group, we observed that alveoli vary in size, alveolar structural disorder, alveolar wall thinning, varying degrees of tearing and integration into the bulla. Compared with the control group, the lung mean linear intercept (Lm) in model group significantly increased, mean alveolar number in unit area (MAN) significantly reduced(P<0.001).Compared with model group, Spearmint oil 10,30 and 100 mg·kg-1 group and dexamethasone treatment group can significantly reduce Lm and increase MAN(P<0.01, P<0.001), and protected structure of alveolar from damaging.(4) The varying degree of bronchiolitis, pulmonary interstitial inflammation and infiltration of inflammation cells were observed in model group. Spearmint oil group and dexamethasone treatment group attenuated bronchiolitis, pulmonary interstitial inflammation and partially prevented infiltration of inflammation cells and reduced the degree of pathology inflammation in lung tissue.(5) Compared with control group, goblet cell metaplasia of bronchus, bronchioles were obviously observed in model group, and goblet cell-positive% was significantly higher(P<0.001). Spearmint oil 100 mg·kg-1 group and dexamethasone treatment group significantly reduced the goblet cell metaplasia and goblet cell-positive%(P<0.05), compared with model group.(6) ELISA analysis showed that the content of TNF-a in lung homogenate were significantly higher in model group than that in control group(P<0.05), and TNF-αin Spearmint oil 30 mg·kg-1 group and dexamethasone treatment group were lower than that in model group (P<0.05, P<0.001); as to IL-1β, the content was higher in model group than that in control group, and Spearmint oil 100 mg·kg-1 group and dexamethasone treatment group were lower than that in model group (P<0.05, P<0.001).(7) Immunohistochemical image revealed that MMP-9 weakly expressed in bronchial epithelial cell and alveolar epithelial cell, but strongly expressed in inflammation cells including neutrophile and macrophage. According to mean optical density(MOD) analyzed by Image Pro-Plus 6.0, expression of MMP-9 was higher in model group than that in control group, and Spearmint oil 10,30,100 mg·kg-1 group and dexamethasone treatment group were lower than that in model group (P<0.05, P<0.01,P<0.001).(8) In model group, we observed that alveolar ducts, alveolar saccules and alveolar ectasia, alveolar wall thinning, varying degrees of tearing and integration into the bulla, but there were few inflammation cells infiltrating into the lung tissue. Compared with control group, the lung mean linear intercept (Lm) in model group significantly increased, and mean alveolar number in unit area (MAN) significantly reduced (P<0.001). Compared with model group, Spearmint oil 42 mg·kg-1 group and dexamethasone treatment group can significantly reduce Lm and increase MAN(P<0.01, P<0.001).(9) Expression of MMP-9,TIMP-1,TNF-α,IL-8 mRNA in model group were significantly higher than those in control group. Spearmint oil 14,42 mg·kg-1 group and dexamethasone treatment group can significantly reduce expression of MMP-9 mRNA; Spearmint oil 42,141 mg·kg-1 group can significantly reduce expression of TNF-a and IL-8 mRNA.CONCLUSION:(1) LPS instilled intratracheally caused emphysematous changes with chronic inflammation in rats, showed alveolar expansion, inflammatory cells infiltration in lung a(?)sues, goblet cell metaplasia, and Spearmint oil has protective effect on alveolar damage, inflammation and lung function. The mechanism may include reducing TNF-α, IL-1βcontent and decreasing MMP-9 expression in lung tissue.(2) Spearmint oil has protective effect on emphysematous changes induced by PPE in C57 mice. The mechanism may include regulating expression of MMP-9, TIMP-1, TNF-αand IL-8 mRNA.
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