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-α in 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× 104 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.
saikosaponinEimeria tenellaphosphorylation of p65 proteinNF-κB signaling pathway
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万方数据
