孕期及哺乳期母鼠邻苯二甲酸酯类暴露对仔鼠呼吸道过敏的影响

目的通过使孕期及哺乳期Wistar大鼠暴露于邻苯二甲酸二(2-乙基)己酯(DEHP),探讨DEHP母代暴露对子代呼吸道过敏影响。方法将36只健康2月龄雌性Wistar大鼠分成3个不同剂量组,从妊娠0天(GD0)起,各组孕鼠分别给予0、30和300 mg/(kg·d) DEHP至仔鼠出生后21天(PND21)断乳,建立母体DEHP暴露模型。仔鼠出生后,不同剂量组各笼选取1只仔鼠在PND21、PND28进行卵清白蛋白(OVA)腹腔注射致敏并在PND32、PND33和PND34连续滴鼻致敏激发,PND35与未致敏仔鼠共同取材,包括肺泡灌洗液以及肺组织。ELISA检测肺泡灌洗液中Th2型细胞因子白细胞介素(IL)-4、IL-5、IL-13的分泌情况,肺组织进行HE及PAS染色观察肺组织过敏性炎症病理变化;免疫组化检测上皮衍生因子IL-33的表达变化。结果肺泡灌洗液中,与对照组比较,DEHP0+OVA组总炎症细胞以及嗜酸性粒细胞数目上升为(131.500±25.548)×10~4、(32.000±10.079)×10~4(P<0.05);DEHP30+OVA组总炎症细胞以及嗜酸性粒细胞数目上升为(156.167±17.994)×10~4、(16.331±6.667)×10~4(P<0.05);DEHP300+OVA组总炎症细胞以及嗜酸性粒细胞数目上升为(172.167±19.994)×10~4、(55.000±17.018)×10~4(P<0.05)。加入不同剂量DEHP与OVA致敏后,与对照组比较,DEHP0+OVA组IL-4、IL-5、IL-13表达量分别增加为(38.401±6.594)pg/mL(P>0.05)、(30.026±2.756)pg/mL(P<0.05)、(13.806±4.355)pg/mL(P<0.05);DEHP30+OVA组IL-4、IL-5、IL-13表达量分别增加为(57.733±7.293)pg/mL(P<0.05)、(31.544±1.043)pg/mL(P>0.05)、(18.068±1.497)pg/mL(P<0.05);DEHP300+OVA组IL-4、IL-5、IL-13表达量分别增加为(54.943±6.049)pg/mL(P>0.05)、(32.377±3.739)pg/mL(P>0.05)、(20.168±0.939)pg/mL(P<0.05)。肺组织病理观察显示,单纯DEHP暴露组仔鼠肺组织没有明显的过敏性炎症反应,而各DEHP+OVA组仔鼠肺组织有较明显的过敏反应且随着DEHP剂量加重而加剧。免疫组化显示DEHP单纯暴露组IL-33表达无明显增加,而DEHP+OVA各组IL-33表达量增加且有一定的剂量-反应关系。结论孕期及哺乳期DEHP暴露会导致子代呼吸道过敏反应加重,其可能机制是通过增加IL-33表达量启动以Th2型细胞为主的肺部致敏反应所致。

Effects of phthalate on pulmonary allergy of offspring during intrauterin and lactating exposure

OBJECTIVE To investigate the effects of exposure to diethyl phthalate (DEHP)during pregnancy and lactation in respiratory allergy of offspring Wistar rats.METHODS To establish the maternal DEHP exposure model:36 heahhy 2-monthold female Wistar rats were divided into three different dose groups.From GD0,each group of pregnant mice were given different concentrations of DEHP (0,30,300 rag/(kg·d))until to the newborn weaning(PND21).After birth,one of offspring was selected from each cage in different dose groups.At PND21 and PND28 the offspring were sensitized by intraperitoneal injection (i.p.)OVA and continuous nasal sensitization at PND32,PND33,PND34.Bronchoalveolar lavage fluid (BALF)and lung tissue were collected at PND35 with non-sensitized groups.ELISA detected the secretion of Th2 cytokine interleukin(IL)-4,IL-5 and IL-13 in BALF,and the pathological changes of allergic inflammation in lung tissues were observed by HE and PAS staining.Expression of epithelium-derived factor IL-33 detected by immunohistochemistry.RESULTS In BALF,compared with the control group,the total number of inflammatory cells and eosinophils in the DEHP0±OVA group increased to (131.500±25.548)×10^4,(32.000± 10.079)×10^4(p<0.05);The total number of inflammatory cells and eosinophils in the DEHP30±OVA group increased to (156.167±17.994)×10^4,(16.331±6.667)×10^4 (p< 0.05);and the total number of inflammatory cells and eosinophils in the DEHP300±OVA group increased to (172.167±19.994)×10^4,(55.000±17.018)×10^4(P<0.05).After adding different doses of DEHP and OVA,compared with the control group,The expression levels of IL-4,IL-5,IL-13 in DEHP0±OVA group increased to (38.401± 6.594)pg/mL (P>0.05),(30.026±2.756)pg/mL (P<0.05),(13.806±4.355) pg/mL (P<0.05);The expression levels of IL-4,IL-5 and IL-13 in DEHP30±OVA group increased to (57.733±7.293)pg/mL (P<0.05)and (31.544±1.043)pg/mL (P>0.05),(18.068±1.497)pg/mL (P<0.05);The expression levels of IL-4,IL-5 and IL-13 in DEHP300±OVA group increased to (54.943±6.049)pg/mL (P>0.05) and (32.377±3.739)pg/mL (P>0.05) (20.168±0.939)pg/mL (P<0.05), respectively.The tissue section of all the DEHP±saline groups could be observed that no obvious allergic inflammatory reaction,meanwhile all the DEHP ±OVA groups had a relatively obvious allergic reaction and were severe with the increase of DEHP dose. Immunohistochemistry showed no significant increase in the expression of IL-33 in the DEHP±saline groups,while the expression of IL-33 in the DEHP±OVA groups increased with a certain close response.CONCLUSION Exposure to DEHP during pregnancy and lactation will aggravate the sensitization reaction of offspring,the possible mechanism that DEHP increase the Th2 type of immune response may be the overexpression of IL-33 in the epithelial cells.