三羟异黄酮下调Apel/Ref-l对放射性肺炎防护作用的实验研究

Study of down-regulation of Ape1/Ref-1 expression by genistein is associated with mitigating the effect of radiation

目的:研究三羟异黄酮(genistein,GEN)下调Ape1/Ref-1(apurinic/apyrimidinic endonuclease 1/redox factor-1)的表达对放射性肺炎(radiation pneumonitis,RP)的保护作用及其分子机制,为防治放射性肺炎提供理论依据。方法60只8周龄C57BL/6J雌性小鼠,随机分为对照组(A组)、单纯照射组(B组)、GEN+照射组(C组)和氨磷汀(Amifostine)+照射组(D组)。A组全肺假照射0 Gy,其余各组小鼠8MV-X线全肺照射12 Gy,其剂量率为0.5 Gy/min。C组于照射开始前24h、16h、8h皮下注射GEN 200mg/Kg,D组于照射开始前30min皮下注射氨磷汀100mg/Kg。照射后1、3、7、14、28、56天,HE、Masson染色观察小鼠肺组织病理改变和胶原纤维沉积;以流式细胞术分析三羟异黄酮联合放疗对A549细胞内ROS含量的影响;用westernblot法分析Ape1/Ref-1表达变化;以EMSA法检测三羟异黄酮对放射性肺损伤小鼠及A549细胞NF-κB表达的影响;ELISA方法测定照射2周后小鼠血清及肺泡灌洗液中的IL-1β、TNF-α、IL-6、TGFβ1含量变化。结果三羟异黄酮能显著减轻照射后小鼠肺组织炎性/纤维素样渗出,明显降低A549细胞胞内ROS的表达(P<0.05),下调放疗所致的APE1蛋白及NF-ΚB表达的升高(P<0.05),进而降低小鼠血清及肺泡灌洗液中的IL-1β、TNF-α、IL-6、TGFβ1的表达水平(P<0.05)。结论:三羟异黄酮可降低放疗诱导的Ape1/Ref-1表达的早期升高,有效地降低炎性因子的表达,减轻肺组织的炎症,对放射性肺炎具有防护作用。

Purpose To investigate lethal effect of genistein, GEN） reduced apurinic/ apyrimidinic endonuclease 1/ redox factor - 1 { Apel/ Ref - 1 } expression after irradiation to provide theoretical evidence for treating radiation pneumonitis. Methods Sixty C57BL/6J female mice were assigned randomly to control group （recevied sham- irradiation, A group） ,irradition alone group（B group）,Genistein ＋ IR group （C group）and Amifostine ＋ IR group （D group）. All rats of A group recevied sham- irradiation, All rats of B, C and D groups received a single dose of 8MV- X line radiation with 12 Gy at approximately 0.5 Gy/min by the linear accelerator to the whole lung. Genistein （200 mg/kg） was administered 24h, 16h or 8h before irradiation in C group, amifostine （ 100 mg/ kg） was administered 30min before irradiation in D group. The mice were sacrificed at 1, 3 days and 1, 2, 4, 7 weeks after the irradiation, hmg tissues and venous blood were extracted to observe the pathological changes collagen fiber deposition of lung tissue by HE and Massion staining. The reactive oxygen species （ROS） level in cells were analyzed by flow cytometry. We also used western blot analysis to analyze the APE1 expression. A EMSA assay was used to measure the level of NF - KB in vivo and in vitro. A ELISA assay was used to measure the level of TGF - betal, IL - lbeta, TNF - alpha and IL - 6 in blood serum and BALF. Results Pulmonary capillary/cellulose effusion after irradiation was damped in rats receiving genistein during the phase of pneumonitis. The inhibitory effect of genistein in combination with irradiation was more significant with a significant elevation of the intracellular ROS level on A549 ceils. Genistein treatment also decreased the expression of Apel/Ref- 1 protein levels and NF - KB protein levels and the levels of the inflatmnatory cytokines TGF - betal, IL - 1beta, TNF - alpha and IL - 6 in sennn and BALF. Conclusions Genistein may decrease the expression of radiation - induced Apel/Ref- 1, Genistein treatment can provide partial protection against the early （pneumonitis） effects of lung irradiation and reduce the extent of fibrosis.