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

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算每组雏鸡的抗球虫指数(ACI);分别于感染后4 d、6 d和8 d分别剖杀10只鸡,取盲肠组织制备病理切片,观察各组鸡盲肠组织的病变。结果显示,CA组鸡粪便卵囊排出率为22.00%,极显著低于PC组(P<0.0001),极显著高于WM组(P<0.0001);ACI为173.13,极显著高于PC组和WM组(P<0.0001),达到中效抗球虫水平;剖检病变观察可见PC组雏鸡的盲肠肠壁变薄,长度变短,高度肿胀,而CA组鸡盲肠肠壁逐渐恢复正常形态,与WM组雏鸡盲肠改善结果接近;组织病变可见,与PC组和WM组相比,CA组盲肠无炎性细胞浸润且肠绒毛肠壁形态完整,未观察到E.tenella裂殖子。以上结果首次证实柴胡皂苷在体内外均可有效抑制E.tenella对细胞的感染,并且能够改善与修复E.tenella对雏鸡盲肠的损伤,本研究为天然中药成分用于治疗E.tenella的感染提供参考依据。

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),but at 48 and 72 hours,the IL-10 transcription level gradually increased and was significantly higher than that in the other 3 groups(P<0.0001).The 13-day-old broiler chicks were divided into the above 4 groups.Except for the NC group,each group of chicks was orally infected with 2×10^(4)E.tenella sporulated oocysts.At 2 days post-infection,the chicks in CA group were orally administered with 0.0645g/L saikosaponin(1 mL/chick),and the chicks in WM group were orally administered with 0.5g/L sodium sulfadiazine(1mL/chick)for 7 days.From 4 to 8 days post-infection,the fecal oocyst excretion rate of each group was calculated using a hemocytometer.At 8 days post-infection,the chicks were weighed and slaughtered,and the cecum was collected to observe the lesions.The relative weight gain rate,survival rate,oocyst index,and cecal lesion score of the chicks were used for calculating the anti-coccidial index(ACI)of each group.Ten chicks were dissected at 4,6 and 8 days post-infection,respectively,and the cecum were taken to prepare pathological sections to observe the pathological changes of cecal tissues in each group.The results showed that the fecal oocyst excretion rate of CA group was 22.00%,which was extremely significantly lower than that of PC group(P<0.0001)and extremely significantly higher than that of WM group.The ACI of CA group was 173.13,which was extremely significantly higher than that of PC and WM groups(P<0.0001),reaching a moderate anti-coccidial level.Necropsy observations showed that the cecal wall of the chicks in PC group became thinner,shorter and severely swollen,while the cecal wall of the chicks in CA group gradually recovered to a normal morphology,similar to the improvement of the cecum of chicks in WM group.Histopathological observations showed that compared with the PC and WM groups,there was no inflammatory cell infiltration in the cecum of chicks in CA group,the intestinal villi and wall morphology were intact,and no E.tenella schizonts were observed.These results provided the first evidence that saikosaponin can effectively inhibit the infection of E.tenella in vitro and in vivo and can improve and repair its damage to the cecum of chicks,providing a reference for the use of natural Chinese herbal ingredients in the treatment of E.tenella infection.