麦冬皂苷D对小鼠放射性肺损伤的防护作用及机制

Protective effect and mechanism of ophiopogonin D on radiation⁃induced lung injury in mice

目的探索麦冬皂苷D对小鼠放射性肺损伤的防护作用及其机制。方法60只C57BL/6雌性小鼠按随机抽样法分为4组:健康对照组、单纯照射组、照射+麦冬皂苷D组、照射+地塞米松组,每组15只。用6 MV X射线15 Gy单次照射小鼠。于照射前3 d开始,照射+麦冬皂苷D组给予10 mg/kg麦冬皂苷D溶液腹腔注射,照射+地塞米松组给予10 mg/kg地塞米松溶液腹腔注射,健康对照组和单纯照射组给予生理盐水腹腔注射,每日1次,至照后1周。于照射后3 d、1周、6周取材,苏木精-伊红(HE)染色法和Masson染色法观察肺组织病理学变化,免疫组织化学法观察8-羟基脱氧鸟苷(8-OHdG)、p53、p53上调凋亡因子(PUMA)、半胱氨酸天冬氨酸蛋白水解酶3(Caspase-3)、Ⅰ型胶原纤维(CollagenⅠ)、Ⅲ型胶原纤维(CollagenⅢ)表达,蛋白提取及免疫印迹实验(Western blot)法进一步验证凋亡相关蛋白p53、PUMA、Caspase-3的表达,酶联免疫吸附试验(ELISA)检测转化生长因子β1(TGF-β1)和白介素6(IL-6)的表达。结果照后1周,麦冬皂苷D能减轻肺组织的出血、渗出、水肿、炎症浸润,降低了照射后肺组织8-OHdG、p53、PUMA、caspase-3氧化应激及凋亡相关蛋白的表达(t=8.39、12.60、5.92、7.00,P<0.05)。在照后3 d、1周、6周,麦冬皂苷D降低了小鼠血液中TGF-β1(t=9.32、8.97、6.83,P<0.05)和IL-6(t=8.22、7.80、8.28,P<0.05)的表达。在照射后6周,麦冬皂苷D可减轻肺间质的CollagenⅠ、CollagenⅢ生成(t=6.41、7.50,P<0.05)。结论麦冬皂苷D对放射性肺损伤有防护作用,可缓解放射性肺炎和肺纤维化早期的胶原沉积,其作用机制可能是通过减轻机体氧化应激,减少炎症相关因子的表达,抑制肺组织细胞凋亡,从而抑制胶原产生。

Objective To investigate the protective effect and mechanism of ophiopogonin D on lung injury induced by radiation in mice.Methods A total of 60 female C57BL/6 mice were randomly divided into 4 groups:control group,irradiation group,irradiation+ophiopogonin D group and irradiation+dexamethasone group,with 15 mice in each group.The mice were irradiated with a single dose of 6 MV X-rays of 15 Gy.Three days before irradiation,the mice in irradiation+ophiopogonin D group were intraperitoneally injected with 10 mg/kg ophiopogonin D solution.The mice in irradiation+dexamethasone group were intraperitoneally injected with 10 mg/kg dexamethasone solution.The mice in control group and irradiation group were intraperitoneally injected with normal saline once a day until 1 week after irradiation.Tissue samples were collected at 3 d,1 week,and 6 weeks post-irradiation.Hematoxylin-eosin(HE)staining and Masson's trichrome staining were used to observe the pathological changes of lung tissue.The expressions of 8-hydroxy-deoxyguanosine(8-OHdG),p53,p53 up-regulated apoptosis factor(PUMA),cysteine aspartate proteolytic enzyme-3(caspase-3),CollagenⅠand CollagenⅢwere observed by immunohistochemistry.Western blot was used to verify the expressions of apoptosis related proteins including p53,PUMA and caspase-3.Results HE staining of lung tissue showed that ophiopogonin D could reduce hemorrhage,exudation,edema and inflammatory infiltration in lung tissue 1 week post irradiation.Moreover,ophiopogonin D reduced the expression of 8-OHdG(t=8.39,P<0.05),the oxidative stress,and the expressions of p53,PUMA,caspase-3 apoptosis-related proteins(t=12.60,5.92,7.00,P<0.05),and inhibited the apoptosis of alveolar epithelial cells and alleviated other damage in the irradiated lung tissue 1 week post-irradiation.Ophiopogonin D also reduced collagen deposition in lung tissue 6 weeks after irradiation,and reduced the expression of transforming growth factor(TGF-β1)(t=9.32,8.97,6.83,P<0.05)and interleukin-6(t=8.22,7.80,8.28,P<0.05)in the blood of mice at 3 d,1 week,and 6 weeks after irradiation.At 6 weeks after exposure,ophiopogonin D reduced the production of CollagenⅠand CollagenⅢin the lung interstitium(t=6.41,7.50,P<0.05),and alleviated the pulmonary fibrosis in the late stage of radiation.Conclusions Ophiopogonin D has protective effects on lung injury caused by radiation,including the alleviation of early radiation pneumonia and late pulmonary fibrosis,by reducing oxidative stress,the expression of inflammation-related factors,apoptosis of lung tissue,and collagen production.