硒代蛋氨酸对猪 δ冠状病毒感染小鼠肠道损伤的保护作用及机制

Protective effect and mechanism of selenomethionine on intestinal injury in mice infected with porcine deltacoronavirus

摘要:

目的 探讨硒代蛋氨酸(Se-Met)对猪δ冠状病毒(porcine deltacoronavirus, PDCoV)感染小鼠肠道的氧化应激、肠道屏障损伤的保护作用及其潜在调节机制。 方法 40只雌性C57小鼠随机分为对照组、Se-Met组(0.3 mg/kg Se)、PDCoV组和Se-Met+PDCoV组(0.3 mg/kg Se)。饲养23 d后，PDCoV组和Se-Met+PDCoV组小鼠灌胃PDCoV HNZK-02-P5株300 μL (1×10 ⁶ TCID ₅₀)，其余2组小鼠灌胃等量的Dulbecco’s改良细胞培养基。观察所有小鼠的临床症状、食物摄取量和体重，直至28 d。接种病毒后5 d时，收集肠道组织，测定PDCoV滴度。采用苏木精-伊红染色监测肠道病理变化。检测氧化应激相关指标：丙二醛(malondialdehyde, MDA)、超氧化物歧化酶(superoxide dismutase, SOD)和谷胱甘肽过氧化物酶(glutathione peroxidase, GSH-PX)。采用2′,7′-双乙酸二氯荧光素(2′,7′-dichlorofluorescein diacetate, DCFH-DA)探针测定空肠组织中活性氧(reactive oxygen species, ROS)水平。免疫荧光法分析小肠紧密连接蛋白(ZO-1和Occludin)的变化。采用RT-qPCR法分析炎症因子(TNF-α、IL-1β、IL-6和IL-10)、肠道紧密连接蛋白(ZO-1和Occludin)及Nrf2信号通路(Nrf2、HO-1和NQO1)相关基因的表达。Western blotting分析Nrf2信号通路相关基因的蛋白表达。 结果 体重、摄食量、病理检查及不同肠道组织病毒RNA滴度的结果证实Se-Met可以增加PDCoV感染小鼠的体重，降低肠道组织病毒滴度，并减轻PDCoV诱导的肠绒毛结构损伤。Se-Met通过降低IL-1β、IL-6和TNF-α的基因表达来减轻PDCoV诱导的肠道炎症。Se-Met通过增加ZO-1和Occludin的基因表达抑制PDCoV诱导的肠黏膜屏障损伤。Se-Met通过降低ROS和MDA水平，增加GSH-PX和SOD水平，改善了PDCoV诱导的肠道氧化应激。Se-Met通过激活Nrf2信号通路抑制PDCoV诱导的氧化应激。 结论 Se-Met可能通过激活Nrf2信号通路减轻PDCoV感染小鼠肠道损伤，为预防和治疗PDCoV感染提供了理论依据。

Abstract:

Objective To explore the protective effect of selenomethionine (Se-Met) on oxidative stress and intestinal barrier damage in mice infected with porcine deltacoronavirus (PDCoV) and the potential regulatory mechanism. Methods Forty female C57 mice were randomly grouped as follows: control, Se-Met (0.3 mg/kg Se), PDCoV, and Se-Met+PDCoV (0.3 mg/kg Se). After being fed with or without Se-Met for 23 days, the mice in the PDCoV group and the Se-Met+PDCoV group were administrated with 300 μL suspension of PDCoV HNZK-02-P5 strain (1×10 ⁶ TCID ₅₀) by gavage, while those in the other two groups were administered with the same volume of Dulbecco’s Modified Eagle Medium (DMEM). All the mice were observed daily for clinical signs, food intake, and body weight changes until day 28. At five days post-inoculation (dpi), intestinal tissues were collected and PDCoV titers were determined. Hematoxylin staining and eosin staining were used to monitor pathological changes in intestinal tissues. Oxidative stress-related indicators such as malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-PX) were investigated. The level of ROS in the jejunum tissue was measured via a 2′,7′-dichlorofluorescein diacetate (DCFH-DA) probe. Immunofluorescence was used to analyze the changes of small intestinal tight junction proteins (ZO-1 and Occludin). The mRNA levels of inflammatory cytokines (TNF-α, IL-1β, IL-6, and IL-10), intestinal tight junction proteins (ZO-1 and Occludin), and the Nrf2 signaling pathway-associated factors (Nrf2, HO-1, and NQO1) were determined by RT-qPCR. Western blotting was employed to assess the protein levels of factors related to the Nrf2 signaling pathway. Results The results of body weight, food intake, pathological examination, and viral RNA titers in different intestinal tissues revealed that Se-Met might increase the body weight, decrease viral titers in intestinal tissues, and attenuate PDCoV-induced structural damage of intestinal villi in PDCoV-infected mice. Se-Met attenuated PDCoV-induced inflammation by lowering the mRNA levels of major inflammatory cytokines, such as IL-1β, IL-6, and TNFα in the jejunum. Se-Met ameliorated PDCoV-induced intestinal mucosal barrier damage by up-regulating the mRNA levels of ZO-1 and Occludin in the jejunum. Se-Met ameliorated PDCoV-induced oxidative stress by decreasing the levels of ROS and MDA and increasing the levels of GSH-PX and SOD in the jejunum. Se-Met inhibited PDCoV-induced oxidative stress by activating the Nrf2 signaling pathway. Conclusion Se-Met may attenuate the intestinal injury in mice infected with PDCoV by activating the Nrf2 signaling pathway, which provides a theoretical basis for the prevention and treatment of PDCoV infection.