柴胡皂苷体内外抗鸡柔嫩艾美耳球虫的研究  

Effect of saikosaponin against Eimeria tenella in chickens in vitro and in vivo

作　　者：孟心茹 甘晓凤 郭丽君 张嫱 郑哲 王燕春 赵权[1,2] 蔡亚南 MENG Xin-ru;GAN Xiao-feng;GUO Li-jun;ZHANG Qiang;ZHENG Zhe;WANG Yan-chun;ZHAO Quan;CAI Ya-nan(College of Veterinary Medicine,Jilin Agricultural University,Changchun 130118,China;Key Laboratory of Animal Product Quality and Safety Ministry of Education,Changchun 130118,China)

机构地区：[1]吉林农业大学动物医学院,吉林长春130118 [2]动物生产及产品质量安全教育部重点实验室,吉林长春130118

出　　处：《中国预防兽医学报》2024年第6期621-629,共9页Chinese Journal of Preventive Veterinary Medicine

基　　金：吉林省科技发展计划项目(20220202063NC);吉林省教育厅科技研究项目(JJKH20210366KJ)。

摘　　要：为了探究柴胡皂苷对柔嫩艾美耳球虫(E.tenella)体内外感染的影响,本实验采用CCK-8法检测2倍倍比稀释(0.384 g/L~0.0645 g/L)的柴胡皂苷对MDBK细胞活力的影响,结果显示,浓度为0.0645 g/L的柴胡皂苷对MDBK细胞的活力影响最小,并且可以促进细胞的生长,因此以该浓度的柴胡皂苷进行后续试验。本研究设置阴性对照组(NC)、E.tenella感染组(PC)、磺胺嘧啶钠对照组(WM)以及柴胡皂苷治疗组(CA)。除NC组外,每组细胞加入1.6×10^(6)个E.tenella子孢子,4 h后WM组加入0.5 g/L磺胺嘧啶钠,CA组加入0.0645 g/L柴胡皂苷,继续培养至不同时间,分别提取4组细胞蛋白,通过western blot检测感染E.tenella的MDBK细胞中p65蛋白的磷酸化水平;采用荧光定量PCR(qPCR)检测4组细胞中炎症因子的转录水平。Western blot结果显示,培养至24 h时,4组细胞中p65蛋白磷酸化水平均无显著差异;感染48 h与72 h时,与WM和PC组相比,CA组细胞中p65蛋白磷酸化水平极显著降低(P<0.001),但与NC组之间无显著差异(培养48 h)和极显著高于NC组(P<0.001,培养72 h)。qPCR结果显示,培养24 h和48 h,各组细胞中各细胞因子的相对转录水平虽有不同,但在培养72 h时,与PC组和WM组相比,CA组MDBK细胞中促炎因子IL-1β、IL-8、TNF-αmRNA的转录水平极显著降低(P<0.001);24 h时,CA组细胞中抑炎因子IL-10 mRNA的转录水平极显著低于WM组(P<0.0001),但在48 h和72 h时,IL-10 mRNA的转录水平逐渐升高,并极显著高于其他3组(P<0.0001)。将13日龄白羽肉鸡设为上述4个组。除NC组外,每组雏鸡均口服感染2×10^(4)个E.tenella孢子化卵囊。感染后2 d,CA组雏鸡口服0.0645 g/L柴胡皂苷,1 mL/只,WM组雏鸡口服0.5 g/L磺胺嘧啶,1 mL/只,共服用7 d。感染后4 d~8 d,采用血球计数板计算每组鸡粪便卵囊排出率。感染后8 d将各组鸡称重后剖杀,观察盲肠的剖检病变,并通过计算各组肉鸡的相对增重率、存活率、卵囊值以及盲肠病变值计�To explore the effects of saikosaponin on the in vitro and in vivo activity of Eimeria tenella(E.tenella),the CCK-8 method was used in this study to detect the effect of 2-fold serially diluted(0.384 g/L-0.0645 g/L)saikosaponin on the viability of MDBK cells.The results revealed that the saikosaponin at a concentration of 0.0645 g/L showed the least effect on the activity of MDBK cells and could promote the cell growth.Therefore,this concentration was selected for subsequent experiments.Four groups were set up including a negative control group(NC),an E.tenella infection group(PC),a sodium sulfadiazine control group(WM),and a saikosaponin treatment group(CA).Except for the NC group,each group of cells was inoculated with 1.6×106 E.tenella oocysts.After 4 hours,the WM group was given 0.5g/L sodium sulfadiazine,and the CA group was given 0.0645g/L saikosaponin,and then cultured for different time periods.The protein levels and phosphorylation status of p65 in E.tenella-infected MDBK cells were detected by western blot.The transcription levels of inflammatory factors in the 4 groups of cells were detected by qPCR.The western blot results showed that the phosphorylation levels of p65 protein in the 4 groups were similar at 24 hours.While at 48 and 72 hours,the phosphorylation level of p65 protein in CA group was extremely significantly lower than that in WM and PC groups(P<0.001),but not significantly different from that in NC group(cultured for 48 hours)or extremely significantly higher than that in NC group(P<0.001,cultured for 72 hours).The qPCR results showed that the relative mRNA transcription levels of the tested cytokines in the four groups fluctuated at 24 and 48 hours.However,the transcription levels of the pro-inflammatory factors IL-1β,IL-8,and TNF-αin MDBK cells in CA group were extremely significantly lower than those in PC and WM groups at 72 hours(P<0.001).At 24 hours,the transcription level of the anti-inflammatory factor IL-10 in CA group was significantly lower than that in WM group(P<0.0001),bu

关 键 词：柴胡皂苷 柔嫩艾美耳球虫 p65蛋白的磷酸化 NF-ΚB信号通路 

分 类 号：S858.31[农业科学—临床兽医学]