虫草素体外抗弓形虫的效果及其作用机制的探究

Study on the anti Toxoplasma gondii effect and mechanism of cordycepin

为探究虫草素抗弓形虫的效果及作用机制,本研究将不同浓度的虫草素与弓形虫RH株共同作用48 h后,采用CCK8法筛选对细胞安全的虫草素浓度。结果显示,以1μg/mL~200μg/mL(以对细胞的抑制率均为0判定)的虫草素为细胞的安全浓度。将巨噬细胞接种弓形虫RH-GFP株,同时分别加入1μg/mL~100μg/mL虫草素,根据弓形虫的荧光强度,计算虫草素对弓形虫的半数抑制浓度(IC50)。结果显示,虫草素对弓形虫的IC50为31.24μg/mL,且其对弓形虫的抑制作用与虫草素的浓度呈正相关。将巨噬细胞接种弓形虫RH株,同时加入100μg/mL虫草素,2 h后采用间接免疫荧光试验(IFA)检测并统计黏附与侵入细胞的弓形虫数量,分析虫草素对弓形虫黏附与侵入细胞的影响;24 h后采用吉姆萨染色后统计细胞感染率;收集上述各组细胞样品,一部分细胞采用qPCR检测细胞中弓形虫B1基因的转录水平,分析虫草素对弓形虫增殖的影响;一部分细胞采用IFA检测并统计每孔细胞100个空泡(PV)中的弓形虫速殖子的数量,分析虫草素对弓形虫复制水平的影响。结果显示,与感染RH株的对照细胞相比,虫草素处理组黏附细胞的弓形虫数量极显著上升(P<0.01),侵入细胞的弓形虫数量极显著下降(P<0.01);弓形虫对细胞的感染率及细胞中弓形虫B1基因的转录水平均极显著下降(P<0.01);虫草素处理组细胞中弓形虫速殖子数量为1或2的PV数量极显著升高,数量为4和8及以上的PV数量极显著减少(P<0.01)。上述结果表明,虫草素在体外能够显著抑制弓形虫对细胞侵袭力、细胞感染率、弓形虫的增殖及复制水平。将小鼠腹腔巨噬细胞分为3组:空白对照组(Blank control)、感染RH株的对照组(T.gondii)和虫草素与RH株共培养组(T.gondii+Cor),各组细胞培养24 h后采用ELISA试剂盒检测各组细胞上清液中各细胞因子的分泌水平;采用生化试剂盒测定各组细胞的各生化指标;采用western blot检测各组细胞中TLR4/NF-κB炎症反应信号通路相关蛋白TLR4、p-NF-κB p65和Nrf2/HO-1氧化应激信号通路相关蛋白Nrf2、HO-1的表达量。ELISA结果显示,与空白对照组相比,T.gondii组中TNF-α、IL-6和IL-1β的分泌水平均极显著升高(P<0.01);与T.gondii组相比,T.gondii+Cor组中TNF-α、IL-6和IL-1β的分泌水平均极显著降低(P<0.01)。生化指标测定结果显示,与空白对照组相比,T.gondii组中丙二醛(MDA)的含量极显著升高(P<0.01),谷胱甘肽(GSH)和超氧化物歧化酶(SOD)的含量极显著下降(P<0.01);与T.gondii组相比,T.gondii+Cor组中MDA的含量极显著降低(P<0.01),GSH和SOD的含量极显著升高(P<0.01)。Western blot结果显示,与空白对照组相比,T.gondii组中TLR4、p-NF-κB p65、Nrf2和HO-1的表达量均极显著升高(P<0.01);与T.gondii组相比,T.gondii+Cor组中TLR4和p-NF-κB p65蛋白的表达水平均极显著降低(P<0.01),Nrf2和HO-1蛋白的表达水平均极显著升高(P<0.01)。以上研究结果首次证实,虫草素在体外可能通过调控TLR4/NF-κB和Nrf2/HO-1信号通路发挥抗炎和抗氧化活性以抑制弓形虫在小鼠腹腔巨噬细胞内的增殖和复制。本研究对虫草素抗弓形虫作用及机制的初步研究及其在兽医临床中的应用提供参考依据。

In order to explore the effect and mechanism of cordycepin against Toxoplasma gondii(T.gondii),this study used different concentrations of cordycepin to interact with Toxoplasma gondii RH strain for 48 hours,and then used the CCK8 method to screen the safe concentration of cordycepin on cells.The results showed that 1μg/mL-200μg/mL cordycepin was the safe concentration for cells.Macrophages were inoculated with T.gondii RH-GFP strain,and 1μg/mL-100μg/mL cordycepin was added at the same time,respectively.Based on the fluorescence intensity of T.gondii,the half inhibitory concentration(IC50)of cordycepin against T.gondii was calculated.The results showed that the IC50 of cordycepin against T.gondii was 31.24μg/mL,and the inhibitory effect of cordycepin on T.gondii was positively correlated with its concentration.Macrophages were inoculated with the RH strain of T.gondii,and 100μg/mL cordycepin was added at the same time.After 2 hours,indirect immunofluorescence assay(IFA)was used to observe and count the number of T.gondii adhering and invading cells.After 24 hours,Giemsa staining was used to count the cell infection rate.Cell samples from each group were collected,and some cells were used to detected the transcription level of T.gondii B1 gene using qPCR to analyze the effect of cordycepin on T.gondii proliferation.A portion of cells were observed under a fluorescence microscope using IFA and the number of T.gondii tachyzoites in 100 vacuoles(PV)in each well was counted to analyze the effect of cordycepin on the replication level of T.gondii.The results showed that compared with the control cells infected with RH strain,the number of T.gondii in adherent cells in the cordycepin treated group increased significantly(P<0.01),and the number of T.gondii invading cells decreased significantly(P<0.01).The infection rate of T.gondii on cells and the transcription level of Toxoplasma gondii B1 gene in cells were significantly reduced(P<0.01).The number of PV in cells with 1 and 2 tachyzoites of T.gondii in the cordycepin treated group was significantly increased,and the number of PV in cells with 4 and 8 decreased significantly(P<0.01).The above results indicate that cordycepin can significantly inhibit cell invasion by T.gondii in vitro,reduce cell infection rate,and inhibit T.gondii proliferation and replication levels.Mouse peritoneal macrophages were divide into three groups:blank control group,RH strain-infected control group(T.gondii),and cordycepin and RH strain co-culture group(T.gondii+Cor).After 24 hours,an ELISA kit was used to detect the secretion levels of various cytokines in the supernatant of each group of cells.Biochemical reagent kits were used to measure various biochemical indicators in each group of cells.Western blot was used to detect the expression levels of TLR4/NF-κB inflammatory response signaling pathway related proteins TLR4 and p-NF-κB p65 and Nrf2/HO-1 oxidative stress signaling pathway related proteins Nrf2 and HO-1 in the cells in each group.The ELISA results showed that compared to the blank control group,the secretion levels of TNF-α,IL-6 and IL-1βin the supernatant were all significantly increased in T.gondii group(P<0.01).Compared to the T.gondii group,the secretion levels of TNF-α,IL-6 and IL-1βin the supernatant were all significantly decreased(P<0.01)in the T.gondii+Cor group.The biochemical index measurement results showed that compared with the blank control group,the content of MDA in the T.gondii group was significantly increased(P<0.01),and the content of GSH and SOD was significantly decreased(P<0.01).Compared to the T.gondii group,the content of MDA in the T.gondii+Cor group was significantly reduced(P<0.01),and the content of GSH and SOD was significantly increased(P<0.01).Western blot results showed that compared to the blank control group,the expression levels of TLR4,p-NF-κB p65,Nrf2 and HO-1 were significantly increased in T.gondii group(P<0.01).Compared to the T.gondii group,the expression levels of TLR4,p-NF-κB p65,Nrf2 and HO-1 were significantly reduced(P<0.01),and the expression levels of Nrf2 and HO-1 proteins were significantly increased(P<0.01).The above research demonstrates for the first time that cordycepin may exert anti-inflammatory and antioxidant activities by regulating TLR4/NF-κB and Nrf2/HO-1 signaling pathways to inhibit the proliferation and replication of T.gondii in mouse peritoneal macrophages.This preliminary study on the anti T.gondii effect and mechanism of cordycepin provides theoretical reference for its clinical application in veterinary medicine.