Therapeutic Effect Of Pulsatilla Saponin Injection On Bovine Subclinical Mastitis

Subclinical mastitis is a common inflammatory disease in dairy cows.In addition to treatment by down-regulating the expression of inflammatory factors and inhibiting the activity of inflammatory-related pathways,maintaining the integrity of the blood-milk barrier is also of great significance for alleviating breast inflammatory injury.Anemoside B4 is a triterpenoid saponin extracted from the roots of Pulsatilla chinensis,which has anti-inflammatory,anti-viral,anti-tumor and other activities.Many studies have shown that anemoside B4 can significantly improve the blood-brain barrier and intestinal mucosal barrier damage.Based on this,we presume that it may also have the effect of improving the blood-milk barrier.Therefore,this research explored the therapeutic effect of anemoside B4 on subclinical mastitis in dairy cows and its potential mechanism in vivo and in vitro.Experiment 1:In order to observe the therapeutic effect of Pulsatilla saponin injection on subclinical mastitis in dairy cows,103 dairy cows with subclinical mastitis were randomLy divided into Pulsatilla saponin injection high dose group 0.08 mL/(kg·d),middle dose group 0.05 mL/(kg·d),low dose group 0.01 mL/(kg·d),positive drug control group 0.10 mL/(kg·d)and positive control group 0.05 mL/(kg·d).The dosage was calcuated according to the weight of each dairy cows.And each group was administered by intramuscular injection for 5 consecutive days.HMT was used to determine the degree of subclinical mastitis in each group of cows 1 d before administration,3 d after administration and 5 d after administration.The somatic cell count and physical and physicochemical indexes of milk were measured 1 d before and 5 d after administration,and blood samples were collected for detection of blood routine,inflammatory factors and immune factors.The results showed that after treatment,the total effective rates of the high,medium and low dose groups of Pulsatilla saponin injection were 90.91%,77.42%and 77.27%,which were significantly higher than the total effective rates of the positive control group which is 33.33%(P<0.01).The total effective rates of the high and medium dose groups of Pulsatilla saponin injection were significantly higher than the total effective rates of the Shuangding injection group which is 46.67%(P<0.05).Compared with pre-treatment data and positive control group,the scores of subclinical mastitis inflammation degree and the number of milk somatic cells in high,medium and low dose groups of Pulsatilla saponin injection were significantly decreased(P<0.01)and significantly decreased(P<0.05).After treatment,there was no significant difference in the test results of milk composition in each group.The results of blood routine indexes,inflammatory factors such as IL-1β,IgG and immune indexes in serum before and after the treatment showed that the content of IL-8 in serum of dairy cows in highdose Pulsatilla saponin injection group was significantly higher than that in positive control group(P<0.01),and there was no significant change in other indexes.The results showed that Pulsatilla saponin injection had good curative effect on bovine subclinical mastitis,and was better than Shuangding injection,but had little effect on the basic physiological metabolism of cows with subclinical mastitis.Experiment 2:In this experiment,we used LPS to induce the inflammation model of bovine mammary epithelial cells and bovine aortic endothelial cells.In order to study the antiinflammatory effect and molecular mechanism of anemoside B4 in bovine mammary epithelial cells and bovine aortic endothelial cells,ELISA,RT-PCR,Western blot and immunofluorescence assays were used to detect the expression of inflammatory factors,key proteins in the TLR-4/MyD88/NF-κB signaling pathway,and tight junction proteins(ZO-1,Occludin,Claudin-1 and Claudin-3).The results showed that the viability of bovine mammary epithelial cells was not inhibited after being stimulated for 12 h by LPS at the concentration of less than 400 μg/mL and anemoside B4 at the concentration of less than 800 μg/mL.But the viability of bovine aortic endothelial cells was inhibited after being stimulated for 12 h by LPS at the concentration of 50 μg/mL,200 μg/mL and anemoside B4 at the concentration of higher than 50 μg/mL.After stimulating these two kinds of cells by LPS with concentration higher than 25 μg/mL for 12 h,the expression of tight junction protein was significantly decreased and the expression of inflammatory factors(IL-1β,IL-6 and TNF-α)was dramatically up-regulated in a dose-dependent manner.While anemoside B4 with concentration between 1.25 μg/mL and 5μg/mL can significantly decrease the expression of TLR4,MyD88,p-P65,p-IκBα and inflammatory factors,inhibit the activity of TLR-4/MyD88/NF-κB signaling pathway,alleviate the inflammatory injury in two kinds of cells induced by LPS.Anemoside B4 also significantly up-regulated the expression of tight junction proteins such as ZO-1,Occludin,Claudin-1 and Claudin-3,thus alleviating the barrier damage of bovine mammary epithelial cells and bovine aortic endothelial cells induced by LPS.In this study,we indicated that Pulsatilla saponins injection has good clinical therapeutic effect on bovine subclinical mastitis,and the mechanism is related to the anemoside B4 which can inhibit TLR-4/MyD88/NF-κB signaling pathway,decrease the expression of inflammatory factors and improve the integrity of blood-milk barrier.