本研究旨在分析芦丁在BV2细胞中的体外抗弓形虫作用及其潜在机制.首先通过CCK8法、间接荧光染色法等测定芦丁的细胞毒性、弓形虫抑制率及增殖情况以探究芦丁在BV2细胞中的抗弓形虫作用;随后,将细胞分成4组,分别为:空白组(Normal)、感染对照组(T.gondii)、感染后低剂量芦丁治疗组(Rut 50)和感染后高剂量芦丁治疗组(Rut 100),进行弓形虫感染及芦丁处理试验,并用ELISA等方法检测细胞培养液上清液中抗氧化酶SOD、GSH及脂质氧化产物MDA水平,炎症因子TNF-α、IL-6及IL-1β水平,用 Western blot检测BV2细胞中PI3K/AKT/Nrf2/HO-1、TLR4/NF-κB及P2X7R/NLRP3信号通路中关键蛋白表达量.结果表明,在BV2细胞中,50和100 μg·mL-1的芦丁具有明显的抗虫效果,可显著抑制弓形虫感染率、入侵能力和增殖(P＜0.05).50和100 μg·mL-1的芦丁还可显著增多上清液中的SOD、GSH含量并降低MDA含量(P＜0.05),且降低TNF-α、IL-6及IL-1β水平(P＜0.05),并显著上调抗氧化通路PI3K/AKT/Nrf2/HO-1、下调炎症通路TLR4/NF-κB及P2X7R/NLRP3中关键蛋白表达量(P＜0.05).结果提示,芦丁在BV2细胞中具有明显的抗弓形虫作用,这可能与其可以通过激活PI3K/AKT/Nrf2/HO-1通路从而增强细胞抗氧化能力、以及抑制TLR4/NF-κB及P2X7R/NLRP3通路从而减弱炎症反应有关.研究结果可为后续探究芦丁体内抗虫效果及分析其对弓形虫性脑损伤保护机制相关研究提供一定的理论参考,并为将芦丁开发成治疗人畜弓形虫病的潜在药物提供科学依据.
Study on the Anti-Toxoplasma gondii Effect of Rutin in BV2 Cells and Its Mechanism
This experiment was conducted to study the in vitro anti-Toxoplasma gondii(T.gondii)effect of rutin and its mechanism in BV2 cells.Firstly,the anti-T.gondii effect of rutin in BV2 cells was explored by detecting rutin cytotoxicity,T.gondii inhibition rate and proliferation by CCK8 and indirect fluorescence staining,etc.Subsequently,the cells were divided into four groups:normal group(Normal),T.gondii infected control group(T.gondii),and T.gondii infected groups with low-dose rutin(Rut 50)and T.gondii infected groups with high-dose Rutin(rut 100).Then T.gondii infection and rutin treatment experiments were performed,and the levels of antioxidant enzymes SOD,GSH and lipid oxidation product MDA,the levels of inflammatory factors TNF-α,1L-6 and IL-1β in the supernatant were detected by ELISA methods.The expression of key proteins in PI3K/AKT/Nrf2/HO-1,TLR4/NF-κB and P2X7R/NLRP3 signaling pathways in BV2 cells were detected by Western blot.The results showed that 50 and 100 μg·mL-1 of rutin had a significant anti-T.gondii effect in BV2 cells,as the T.gondii infection rate,invasion ability and proliferation were significantly inhibited(P＜0.05).Rutin also significantly increased the SOD and GSH activities and decreased the MDA content(P＜0.05),and decreased TNF-α,IL-6 and IL-1β levels in the supernatant(P＜0.05).And it also significantly up-regulated the expression of key proteins in the antioxidant pathway PI3K/AKT/Nrf2/HO-1,and down-regulated the inflammatory pathway TLR4/NF-κB and P2X7R/NLRP3(P＜0.05).These results indicated that rutin has a significant anti-T.gondii effect in BV2 cells in vitro,which may be related to its ability to enhance cellular antioxidant capacity through activation of the PI3K/AKT/Nrf2/HO-1 pathway,as well as inhibit the TLR4/NF-κB and P2X7R/NLRP3 pathways to attenuate inflammatory responses.These findings may provide some theoretical references for further research on the anti-T.gondii effect of rutin in vivo,as well as its protective effect in T.gondii-induced brain damage.It may also provide some theoretical basis for the development of rutin as a potential therapeutic agent for the treatment of toxoplasmosis in humans and animals.
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