咖啡因对早产鼠高氧肺损伤的影响及其与p38丝裂原活化蛋白激酶信号途径关系的研究

Effect of caffeine on hyperoxic lung injury of premature rats and its relationship with p38 motigen-activated protein kinase signal pathway

目的探讨咖啡因干预对早产鼠高氧肺损伤的影响及其与p38丝裂原活化蛋白激酶(motigen-activated protein kinase,MAPK)信号途径的关系。方法将60只Wistar早产鼠按随机数字表法分为4组(n=15):空气+生理盐水组(A+N组)、空气+咖啡因组(A+C组)、高氧+生理盐水组(H+N组)、高氧+咖啡因组(H+C组),其中H+N组和H+C组持续暴露于高氧中(氧浓度60%~70%),A+C组和H+C组于早产鼠出生后每日腹腔注射咖啡因29 mg/(kg·d),A+N组和H+N组腹腔注射同等体积的生理盐水。各组分别于第3、7、14天时随机对每组5只早产鼠肺组织取材,光镜下观察肺组织病理改变、辐射状肺泡计数(radiated alveolar count,RAC)、肺组织胶原含量;检测肺湿/干重(wet/drg ratio,W/D)比值;二步法免疫组织化学检测p38MAPK在肺组织中的分布;Western blot检测磷酸化p38MAPK(p-p38MAPK)蛋白含量变化。结果与A+N组、A+C组相比,H+N组和H+C组早产鼠在第3、7、14天肺组织均可见不同程度炎症改变,高氧暴露时间越长,改变越明显,在第14天时可见肺纤维化;咖啡因干预后有一定改善。H+N组和H+C组早产鼠RAC值较A+N组、A+C组显著降低(P<0.05),W/D比值及肺组织胶原含量显著升高(P<0.05),咖啡因干预后有明显改善(P<0.05)。二步免疫组织化学法结果显示H+N组和H+C组早产鼠肺组织中p-p38MAPK阳性细胞数表达增多,分布广泛;咖啡因干预后p-p38MAPK阳性细胞数减少;Western blot结果显示,H+N组和H+C组早产鼠肺组织中p-p38MAPK蛋白含量明显高于A+N组和A+C组(P<0.05),咖啡因干预后p-p38MAPK蛋白含量降低(P<0.05)。结论高氧可通过激活p38MAPK信号途径促进肺纤维化形成;咖啡因通过阻断p38MAPK的表达而减轻高氧暴露下肺组织纤维化,进而起保护作用。

Objective To study the effect of caffeine on hyperoxic lung injury of premature rats and its relationship with p38 motigen-activated protein kinase(MAPK) signal pathway. Methods Sixty Wistar premature rats were divided into 4 groups(n=15) according to the random number table: air + normal saline group(A+ N group), air + caffeine group(A+ C group), hyperoxia + normal saline group(H+ N group), and hyperoxia + caffeine group(H+ C group). Among them, H+ N group and H+ C group were continually exposed to hyperoxia(oxygen concentration was 60%~70%). For A+ C group and H+ C group, the premature rats were injected with caffeine of 29 mg/(kg·d) into their peritoneal cavities every day after birth.For A+ N group and H+ N group, the premature rats were injected with normal saline of the same volume into their peritoneal cavities.In each group, the lung tissues of 5 premature rats were randomly selected on the third, seventh and fourteenth day respectively.The pathological changes of lung tissue, radiated alveolar count(RAC) and collagen content in lung tissue were observed under a light microscope.The wet/dry ratio(W/D) was measured.Two-step immunohistochemistry was used to detect the distribution of p38MAPK in lung tissue.The content of phosphorylated p38MAPK(p-p38MAPK) protein was detected by western blot. Results Compared with the air groups, the lung tissues of premature rats in high oxygen exposure groups showed different degree of inflammatory changes on the third, seventh, and fourteenth day.The changes were more obvious with the prolonged exposure to hyperoxia.Pulmonary fibrosis was visible on the fourteenth day, which was improved after caffeine intervention.The RAC value of premature rats in hyperoxia exposure groups was significantly lower than that in air-exposure groups(P<0.05), and the W/D ratio and collagen content in lung tissue increased significantly(P<0.05), which were improved after caffeine intervention(P<0.05). The results of two-step immunohistochemistry showed that the number of p-p38MAPK positive cells in the lung tissue of premature rats in hyperoxia exposure groups increased and widely distributed, but decreased after caffeine intervention.The results of western blot showed that the content of p-p38MAPK protein in lung tissue of premature rats in hyperoxia exposure groups was significantly higher than that of air groups(P<0.05), but it decreased after caffeine intervention(P<0.05). Conclusion Hyperoxia can promote the formation of pulmonary fibrosis by activating p38MAPK signal pathway.Caffeine can interdict the expression of p38MAPK to alleviate the fibrosis of lung tissue exposed to hyperoxia and thus protects the lung tissue.