槲皮素预处理ALI大鼠肺组织损伤、炎症/氧化应激反应、铁死亡及Nrf2/HO-1信号通路激活情况观察

Effects of quercetin pretreatment on lung tissue damage,inflammatory/oxidative stress responses,ferroptosis,and Nrf2/HO-1 signaling pathway activation in ALI rats

目的观察槲皮素灌胃预处理的LPS诱导急性肺损伤(ALI)大鼠肺组织损伤、炎症反应、氧化应激反应、铁死亡及Nrf2/HO-1信号通路激活情况,以探讨槲皮素对ALI的预防作用及机制。方法24只SD大鼠分为槲皮素低、中、高剂量组和阳性对照组(地塞米松)、模型组、正常对照组。槲皮素低、中、高剂量组以25、50、100 mg/kg槲皮素灌胃,1次/天,连续7 d;槲皮素灌胃处理的第4天在大鼠气管内滴注5 mg/kg LPS;阳性对照组以地塞米松1.04 mg/kg灌胃,其余处理同槲皮素组;模型组以生理盐水灌胃,1次/天,连续7天,其余处理同槲皮素组;正常对照组以生理盐水连续灌胃7 d。末次灌注给药后,观察各组肺组织损伤(肺功能及肺组织病理改变、纤维组织阳性表达率、肺泡上皮细胞凋亡率)、炎症反应(肺泡灌洗液TNF-α、IL-6、IL-1β)、氧化应激反应(肺泡灌洗液SOD、GSH、MDA,肺组织ROS)、铁死亡(Fe^(2+)水平)及Nrf2/HO-1信号通路激活[肺组织核因子红细胞2相关因子2(Nfr2)、血红素加氧酶1(HO-1)、超氧化物歧化酶2(SOD2)、过氧化氢酶(CAT)蛋白]情况。结果与正常对照组比较,模型组PaCO_(2)水平升高,PaO_(2)、SaO_(2)水平降低(P均<0.01);肺组织可见肺泡上皮细胞变性坏死,纤维组织阳性表达率高;肺泡上皮细胞凋亡率高;肺泡灌洗液中TNF-α、IL-6、IL-1β水平均升高,SOD、GSH水平降低,MDA水平升高,肺组织ROS表达水平升高,肺泡灌洗液中Fe^(2+)水平升高(P均<0.05)。与模型组相比,槲皮素中、高剂量组和阳性对照组中PaCO_(2)均降低(P均<0.05),槲皮素高剂量组PaO_(2)和SaO_(2)水平升高(P均<0.05);槲皮素高剂量组与阳性对照组肺组织炎性浸润与纤维增生明显减少,肺泡恢复正常生理结构;槲皮素低、中、高剂量组和阳性对照组肺纤维组织阳性表达率均下降(P均<0.05),其中槲皮素高剂量组肺纤维组织阳性表达率最低;槲皮素低、中、高剂量组和阳性对照组肺泡上皮细胞凋亡率均降低(P均<0.01);槲皮素低、中、高剂量组和阳性对照组肺泡灌洗液中TNF-α、IL-6、IL-1β水平均降低、SOD、GSH水平升高、MDA水平降低,肺组织ROS表达水平降低(P均<0.01),肺泡灌洗液中Fe^(2+)水平降低。与正常对照组比较,模型组肺组织中CAT、HO-1、Nrf2、SOD2蛋白表达水平低(P均<0.01);与模型组比较,槲皮素中、高剂量组和阳性对照组肺组织中CAT、HO-1、Nrf2、SOD2蛋白表达水平高(P均<0.05)。结论槲皮素灌胃预处理的ALI大鼠肺组织损伤、炎症反应、氧化应激反应、铁死亡情况减轻,Nfr2/HO-1信号通路激活;槲皮素灌胃预处理可预防LPS诱导的大鼠ALI,可能通过抑制炎症反应、氧化应激反应及铁死亡而起作用;槲皮素可能通过上调Nfr2/HO-1信号通路而抑制炎症反应、氧化应激反应及铁死亡;50、100 mg/kg槲皮素均对LPS诱导的大鼠ALI起预防作用,以100 mg/kg槲皮素的作用效果更佳。

Objective To observe the effects of quercetin pretreatment through intragastric administration on lung tissue injury,inflammatory response,oxidative stress response,ferroptosis,and the activation of the Nrf2/HO-1 signaling pathway in rats with lipopolysaccharide(LPS)-induced acute lung injury(ALI)in order to investigate quercetin's preven tive effect and mechanism on ALI.Methods Twenty-four Sprague-Dawley rats were divided into the low-dose,mediumdose,and high-dose quercetin groups,positive control group(dexamethasone),model group,and normal control group,respectively.Rats in the low-dose,medium-dose,and high-dose quercetin groups received quercetin via gavage at doses of 25,50,and 100 mg/kg,respectively,once a day for 7 consecutive days.On the fourth day of quercetin treatment,5 mg/kg LPS was instilled into the trachea of the rats.Rats in the positive control group received dexamethasone at a dose of 1.04 mg/kg via gavage,following the same treatment regimen as the quercetin groups.Rats in the model group received normal saline via gavage once a day for 7 consecutive days,with the same additional treatments as the quercetin groups.Rats in the normal control group received normal saline via gavage for 7 consecutive days.After the final administration,lung tissue injury(lung function,pathological changes,positive expression rate of fibrous tissues,and apoptosis rate of alveolar epithelial cells),inflammatory response[levels of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),and interleukin-1β(IL-1β)]in bronchoalveolar lavage fluid,oxidative stress response[levels of superoxide dismutase(SOD),glutathione(GSH),malondialdehyde(MDA)in bronchoalveolar lavage fluid,and ROS in lung tissue],ferroptosis(Fe^(2+)levels),and activation of the Nrf2/HO-1 signaling pathway[levels of nuclear factor erythroid 2-related factor 2(Nrf2),heme oxygenase-1(HO-1),superoxide dismutase 2(SOD2),and catalase(CAT)proteins in lung tissues]were observed in each group.Results Compared with the normal control group,PaCO_(2) level increased,PaO_(2) and SaO_(2) levels decreased,alveolar epithelial cells were degenerated and necrotic in lung tissues,the positive expression rate increased in fiber tissues,the apoptosis rate increased,the levels of TNF-α,IL-6,and IL-1βin alveolar lavage fluid increased,SOD and GSH levels decreased,MDA and Fe^(2+)levels increased,and ROS levels in lung tissues increased in the model group(all P<0.01).Compared with the model group,PaCO_(2) in the medium-dose and high-dose quercetin groups and positive control group decreased(all P<0.05),and PaO_(2) and SaO_(2) levels increased in the high-dose quercetin group(all P<0.05);the inflammatory infiltration and fibrous hyperplasia of lung tissue were significantly reduced in the high-dose quercetin group and positive control group,and the physiological structure of the alveolar was restored to normal;the positive fiber expression rates of the low-,medium-,and high-dose quercetin groups and the positive control group decreased(all P<0.05)with the lowest positive fiber expression rate in the high-dose quercetin group;the apoptosis rates decreased(all P<0.01),the levels of TNF-α,IL-6,and IL-1βin alveolar lavage fluid decreased,the levels of SOD and GSH increased,the levels of MDA and Fe^(2+)decreased,and the levels of ROS decreased in the low-,medium-,and high-dose quercetin groups and positive control group(all P<0.01).Compared with the normal control group,the protein expression levels of CAT,HO-1,Nrf2,and SOD2 in the lung tissues of the model group were lower(all P<0.01);compared with the model group,the protein expression levels of CAT,HO-1,Nrf2,and SOD2 in the lung tissues of the medium-dose and high-dose quercetin groups and the positive control group were higher(all P<0.05).Conclusions Quercetin pretreatment via gavage reduces lung tissue injury,inflammatory response,oxidative stress response,and ferroptosis,and activates the Nrf2/HO-1 signaling pathway in rats with LPS-induced ALI.Quercetin pretreatment can prevent LPS-induced ALI in rats,possibly by inhibiting inflammatory response,oxidative stress response,and ferroptosis,and the protective effects of quercetin may be mediated through up-regulation of the Nrf2/HO-1 signaling pathway.Both 50 mg/kg and 100 mg/kg doses of quercetin can effectively prevent LPS-induced ALI in rats,with the better effect in the 100 mg/kg dose.