Effect of a natural plant ingredient — Pulsatilla saponin B4 on clinical mastitis and serum inammatory indices in dairy cows

Background: Bovine mastitis is an inammatory reaction of the breast caused by pathogenic bacteria infection or breast injury, which affect milk yield and quality and poses severe threat to the health of dairy cows. Pulsatilla chinensis has antibacterial, anti-inammatory, and immune enhancing effects, etc. However, there was no research about the therapeutic effect of it towards the treatment of cow mastitis. In the study, twelve healthy cows were selected as the control group (group A), and 36 cows with mastitis were equally and randomly divided into group B, C and D. Group A didn’t accept treatment, while group B, C and D were treated with 15, 30 and 60 mL Pulsatilla saponin B4 (a puried extract form Pulsatilla chinensis) injection by brachiocephalicus intramuscular injection once a day for 4-6 day and the optimal dose was selected from them. Then, we investigated the effect of this extract on the clearance rates of pathogenic bacteria, the regulation of somatic cell count (SCC) and inammatory factors the in appropriate dose group. Results: Pulsatilla saponin B4 was effective in all test groups and showed a dose-effect relationship. The extract had inhibitory effects on Streptococcus agalactiae, Streptococcus dysenteriae, Klebsiella species, Mycoplasma bovis, Escherichia coli and Staphylococcus aureus. Group C cured more cows than group B and D during the 6 days of treatment and was determined as the optimal dose group. The milk SCC, serum haptoglobin (HP), prostaglandin E2 (PGE2), interleukin-1 alpha (IL-1α), interleukin-1 beta (IL-1β), interleukin-2 (IL-2), interleukin-8 (IL-8), interleukin-10 (IL-10) and tumour necrosis factor alpha (TNF-α), interleukin-4 (IL-4) and leukotriene B4 (LTB4) in Group C were signicantly higher than those of healthy cows (P 0.05) on the rst day no signicant difference with group A after treatment. Conclusion: 30 mL Pulsatilla saponin B4 applied through intramuscular injection once a day for 4-6 days leaded to the signicant amelioration of bacterial infection, SCC and levels of serum inammatory factors in cows with clinical mastitis. serum SAA, HP, PGE2, IL-1α, IL-2, IL-6, IL-8, IL-10, and TNF-α than healthy cows (P < 0.01) and signicantly higher serum LTB4, CRP, IL-1β, and IL-4 than those of healthy cows (P < 0.05). This showed a declining trend within the advancement of treatment except for serum PGE2, which was still signicantly higher than healthy cows (P < 0.05), and the other indices showed no signicant difference with the healthy cows (P > 0.05). Serum HP, IL-2, and IL-10 concentrations in cows with mastitis on d 3 was signicantly lower than d 1 (P < 0.05). Serum CRP, SAA, HP, PGE2, IL-1α, IL-2, and TNF-α concentrations in cows with mastitis on d 5 were signicantly lower than d 1 (P < 0.05). Moreover, on d 7, serum IL-6, IL-4 concentrations in cows with mastitis also became signicantly lower than d 1 (P < 0.05). serum CRP, SAA, HP, IL-1α, IL-1β, IL-2, IL-6, IL-8, TNF-α of cows with clinical mastitis.


Abstract
Background: Bovine mastitis is an in ammatory reaction of the breast caused by pathogenic bacteria infection or breast injury, which affect milk yield and quality and poses severe threat to the health of dairy cows. Pulsatilla chinensis has antibacterial, anti-in ammatory, and immune enhancing effects, etc. However, there was no research about the therapeutic effect of it towards the treatment of cow mastitis. In the study, twelve healthy cows were selected as the control group (group A), and 36 cows with mastitis were equally and randomly divided into group B, C and D. Group A didn't accept treatment, while group B, C and D were treated with 15, 30 and 60 mL Pulsatilla saponin B4 (a puri ed extract form Pulsatilla chinensis) injection by brachiocephalicus intramuscular injection once a day for 4-6 day and the optimal dose was selected from them. Then, we investigated the effect of this extract on the clearance rates of pathogenic bacteria, the regulation of somatic cell count (SCC) and in ammatory factors the in appropriate dose group.
Results: Pulsatilla saponin B4 was effective in all test groups and showed a dose-effect relationship. The extract had inhibitory effects on Streptococcus agalactiae, Streptococcus dysenteriae, Klebsiella species, Mycoplasma bovis, Escherichia coli and Staphylococcus aureus. Group C cured more cows than group B and D during the 6 days of treatment and was determined as the optimal dose group. The milk SCC, serum haptoglobin (HP), prostaglandin E2 (PGE2), interleukin-1 alpha (IL-1α), interleukin-1 beta (IL-1β), interleukin-2 (IL-2), interleukin-8 (IL-8), interleukin-10 (IL-10) and tumour necrosis factor alpha (TNF-α), interleukin-4 (IL-4) and leukotriene B4 (LTB4) in Group C were signi cantly higher than those of healthy cows (P < 0.05) on the rst day but had no signi cant difference with group A after treatment.
Conclusion: 30 mL Pulsatilla saponin B4 applied through intramuscular injection once a day for 4-6 days leaded to the signi cant amelioration of bacterial infection, SCC and levels of serum in ammatory factors in cows with clinical mastitis.
Pulsatilla saponin B4 is the main active ingredient of Pulsatilla chinensis (Bunge) Rege and plays a therapeutic role in the case of mastitis as an anti-in ammatory, anti-bacterial, and immune regulating agent [7,8]. Recent studies showed that Pulsatilla chinensis has inhibitory effects on Staphylococcus aureus, Streptococcus dysenteriae and Salmonella typhi [9]. It inhibits the expressions of pro-in ammatory cytokines through NF-κB signaling pathway, which exhibited obvious inhibitory effect on LPS induced in ammation [10], and showd relatively high immunoregulatory activity and low cytotoxicity [11]. These pharmacological effects of Pulsatilla saponins closely coincided with the therapeutic needs of mastitis cows in clearing bacteria and alleviating in ammation. On the other hand, few attempts have been made to treat mastitis with Pulsatilla saponins.
Considered that Pulsatilla Saponin B4 has anti-bacterial and anti-in ammatory effects, it is hypothesized that this extract might be able to treat dairy cows with clinical mastitis. Accordingly, in this research, Pulsatilla saponin B4 were expected to potentially reduce infected bacteria and ameliorate the levels of in ammatory factors in cows so as to prevent and treat clinical mastitis. However, there was no vivo study of it towards treating the clinical mastitis cows. Thus, the objective of the present study was to investigate the effect of Pulsatilla saponin B4 on the clearance rates of bacterial infection, the regulation of somatic cell count and in ammatory factors in dairy cows with clinical mastitis.

Results
Effects of Pulsatilla saponin B4 on the cure rate and cure time of clinical mastitis in dairy cows As shown in table 3, all three doses were effective in the 12-day experimental period and 9, 9 and 11 cows with clinical mastitis were cured in group B, C and D respectively. Within 6 days of usage of Pulsatilla saponin B4, there were 6, 9 and 7 mastitis cows were cured in group B, C and D, respectively. The total cure rates were 50.00, 75.00 and 58.30% and the average cure time were 5.67, 5.44 and 5.28 d respectively. After usage of extract, there was no cured cow in group C, but 3 and 4 cows in group B and D were cured.
Effects of Pulsatilla saponin B4 on the regulation of the milk SCC in the cows with clinical mastitis As shown in table 4, milk SCC in group C was signi cantly higher than that in group A and showed a declining trend with the treatment. Milk SCC of cows with clinical mastitis dropped signi cantly from d 1 to d 5 (P < 0.05) and from d 7 to d 9 (P < 0.05) and showed no signi cant difference (P > 0.05) with group A on d 11 (P > 0.05).
Effects of Pulsatilla saponin B4 on the regulation of serum in ammatory indices in the cows with clinical mastitis As shown in table 5, cows with clinical mastitis exhibited very signi cantly higher levels of serum SAA, HP, PGE2, IL-1α, IL-2, IL-6, IL-8, IL-10, and TNF-α than healthy cows (P < 0.01) and signi cantly higher serum LTB4, CRP, IL-1β, and IL-4 than those of healthy cows (P < 0.05). This showed a declining trend within the advancement of treatment except for serum PGE2, which was still signi cantly higher than healthy cows (P < 0.05), and the other indices showed no signi cant difference with the healthy cows (P > 0.05). Serum HP, IL-2, and IL-10 concentrations in cows with mastitis on d 3 was signi cantly lower than d 1 (P < 0.05). Serum CRP, SAA, HP, PGE2, IL-1α, IL-2, and TNF-α concentrations in cows with mastitis on d 5 were signi cantly lower than d 1 (P < 0.05). Moreover, on d 7, serum IL-6, IL-4 concentrations in cows with mastitis also became signi cantly lower than d 1 (P < 0.05).

Discussion
Pulsatilla Decoction is famous formula prescription, and it is rich in triterpenoid saponins, such as Pulsatilla Saponin B4, Pulsatilla Saponin A3, and 23-hydroxybetulinic acid [12,13]. As the major active component of Pulsatilla chinensis, Pulsatilla saponin B4 has also received attention as a pure compound for its therapeutic potential. In our study, different doses of Pulsatilla saponin B4 were effective for treating clinical mastitis in dairy cows and showed a dose-dependent effect. No side effects were observed in the cows or injection sites after usage of the extract. Within 6 days of treatment there were 6, 9 and 7 mastitis cows cured in group B, C and D, respectively. The total cure rates were 50.0, 75.0 and 58.3% and average cure time were 5.67, 5.44 and 5.28 d, respectively. There were more cured cows in group C in a similar time and it was regarded to be the optimal dose group. Therefore, milk and blood samples from group C were selected for next detection SCC is the count of somatic cells in a millilitre of milk, and contains 75% of leukocytes and 25% of epithelial cells [14]. Mammary tissues released in ammatory factors like IL-1β, IL-6, and TNF-α when damaged or infected. This promoted the increase of vascular permeability and the detachment of epithelial cells, as well as, it stimulated the secretion of chemokines, attracting leukocytes to the infected area and causing the increase of somatic cell count in milk [15]. SCC re ectd the severity of mastitis, and was used in early diagnosis and treatment evaluation of mastitis in dairy cows [16]. Das et al. [17] found that the SCC value was signi cantly higher (P < 0.05) in clinical mastitis cows compared to subclinical mastitis and healthy cows. Our ndings are consistent with them, indicating that the immune cells were accumulated abundantly in the damaged tissues through the non-speci c and uid immune response. In our study, SCC in cows with clinical mastitis decreased during the treatment and had no signi cant differences with the healthy cows nally (P < 0.05), indicating that Pulsatilla saponin B4 may relieve the auto-immune damage by decreasing the secretion of pro-in ammatory factors.
Mastitis is an in ammatory process of the udder tissue caused mainly by Staphylococcus aureus, Escherichia coli and Streptococcus [18]. Consistent with our study, Cheng et al. [19] reported tha that Escherichia coli was the commonest organism in mastitis cases, and Streptococcus uberis continued to be a prevalent pathogen closely followed by Staphylococcus aureus, Streptococcus dysgalactiae and Streptococcus agalactiae. Pulsatilla chinensis have inhibitory effects on Staphylococcus aureus, Streptococcus dysenteriae and Salmonella typhi [9]. Shafaghat [20] reported that Pulsatilla showed moderate inhibitory activity against some certain strains in Gram-positive and Gramnegative bacteria. In agreement with the foregoing studies, we found that most of the bacteria disappeared or decreased after treatment with Pulsatilla Saponin B4, indicating that Pulsatilla chinensis is potently inhibitory against the Streptococcus agalactiae, Streptococcus dysgalactiae, Klebsiella species and Escherichia coli.
The infection-induced in ammatory response is the main cause of clinical mastitis in dairy cows, and it stimulates the cellular secretion of LTB4, PGE2, and histamine to enhance the immune response, promoting the production of in ammatory factors (interleukins, bradykinin and allergic toxins) and elevating the levels of acute-phase protein [21,22]. Our study showed that CRP, SAA and HP of dairy cows with clinical mastitis were signi cantly higher than those of healthy cows (P < 0.05), suggesting that the breasts of cows were damaged by in ammation.
After treatment, the levels of CRP, SAA and HP were signi cantly decreased (P < 0.05), indicating that the in ammatory response rapidly subsided. It was speculated that Pulsatilla saponin B4 had a highly effective anti-in ammatory effect, which regulated the in ammatory response in the early stage of mastitis and reduce its damage to breast and body tissues.
In mastitis, activated Toll-like Receptors initiated the NF-κB/MAPKs pathways, which further triggered the gene expression of TNF-α, IL-1β and IL-6 [23]. Tomala et al. [24] showed that LPS, IL-1α and TNF-α modulated PGE2 and LTB4 secreted by bovine mammary gland in both vivo and vitro studies. In agreement with the foregoing studies, we found that the serum levels of PGE2, LTB4, IL-1, IL-2, IL-6, IL-8 and TNF-α in clinical mastitis cows were signi cantly higher than healthy cows. The incidence of mastitis stimulated body cells to synthesize and release in ammatory mediators, and induce the in ammatory stress in the dairy cows [25]. After treatment, the levels of serum in ammatory factors of clinical mastitis cows showed a declining trend except PGE2, which had was silimilar to that of the healthy group on 7th day (P > 0.05). Pulsatilla saponin B4 is the main active ingredient of the Pulsatilla chinensis, which has anti-bacterial, anti-in ammatory, vasodilatory, and immunoregulatory properties, and it regulated the expression of related in ammatory cytokines via the NF-κB signalling pathway, relieving the in ammatory response with high e ciency [26,27]. Hu et al. [28] showed that Pulsatilla decoction and its active ingredients could inhibit the secretion of NO, ET-1, TNF-α, and IL-1α in LPS-induced rat intestinal microvascular endothelial cells. Pulsatilla saponin B4 signi cantly decreased the expression of NO, TNF-α, IL-1β and IL-6, improve lung tissue damage and down-regulate the protein expression of NF-κB in mice [29]. In the present research, Pulsatilla saponin B4 signi cantly reduced serum pro-in ammatory factors levels. Therefore, this extract was proposed for inhibiting the secretion of in ammatory mediators during the process of mastitis in dairy cows.
Bochniarz et al. [6] found that the levels of serum anti-in ammatory factors (IL-4 and IL-10) in cows with sub-clinical mastitis were signi cantly lower than healthy cows, but our study showed that the serum levels of IL-4 and IL-10 were signi cantly and very signi cantly higher than healthy cows respectively. The reason may be that the in ammatory symptoms are more obvious in cows with clinical mastitis, and the elevation of acute pro-in ammatory factors damaged the mammary tissues and even the entire body. As a result, the serum levels of IL-4 and IL-10 increased rapidly to antagonize the in ammation. Unlike the current researches, we found that serum levels of antiin ammatory factors sexhibited a declining trend during the treatment, it may be that the secretion of pro-in ammatory factors was more related to the cellular secretion and immune response. In the acute phase of mastitis, levels of pro-in ammatory factors elevated because of the severe in ammation, and the concentrations of anti-in ammatory factors also increased as the consequence of the body's defense mechanism. When the in ammation controlled, the pro-in ammatory factors and anti-in ammatory factors decreased sequentially. However, the reason of the changes in the levels of anti-in ammatory factors still should be distinguished. Conclusion 30 mL Pulsatilla saponin B4 injection by brachiocephalics intramuscular injection once a day for 4-6 days was the most appropriate usage for the treatment of clinical mastitis. Treatment of Pulsatilla saponin B4 can signi cantly decrease the somatic cell count, eliminate pathogenic bacteria, and down-regulate the levels of serum CRP, SAA, HP, IL-1α, IL-1β, IL-2, IL-6, IL-8, TNF-α of cows with clinical mastitis.

Experiment animals and treatment
The Chinese Holstein cows were obtained from Sichuan Ninggang Animal Husbandry Co., Ltd. Twelve healthy cows and 36 cows with rstclass clinical mastitis with similar date of parturition and milk production were selected form 50 healthy and 50 cows with mastitis (with weight 612 ± 47 kg, 3-4 years of age, and 2-3 parity) in a semi-closed uni ed dairy farm. Twelve healthy cows were placed into control group (group A), the other 36 cows with clinical mastitis were divided into 3 groups (group B, C, and D) equally and randomly by simple randomizaton. In group B, C and D, the cows were applied with Pulsatilla saponin B4 Injaction through brachiocephalicus intramuscular injection with 15, 30, and 60 mL respectively, and the rst day of the experiment was recorded as day 1, then administrated the same doses continuously for 4-6 days until the recovery of cows. The control group didn't receive any administration. Our research was a eld trial. All cows were diagnosed as clinical mastitis for the rst time and didn't receive antibiotic treatment for 14 days before diagnosis. All the groups were under observation for 12 days. All experimental cows had the same feeding and management procedure without other diseases occurred and antibiotic treatment during the experimental period. We selected a small sample size because the Pulsatilla saponin B4 was evaluated for the rst time in the present study, the initial intention was to gather basic evidence regarding the usage of this drug in the further reseaches.
Sample collection 10 mL of tail venous blood of experimental cows were collected before feeding at 8 am. The sampling was done on day 1, 3, 5, and 7 in group A, B, C, and D. The blood was loaded in a centrifuge tube without anticoagulant and centrifuged with a centrifuge (Sigma, Germany) at 3 000 rpm for 10 min to separate serum after 1 hour deposition at room temperature (20-25 ℃), and the upper serum was transferred to EP tube, stored at -70 ℃ in refrigerator (Haier, China). Washed the udder by warm water, and sterilized by 75% ethanol. Discarded several streams of milk, then collected 5 mL milk and measured the SCC on day 1, 3, 5, 7, 9, and 11 in group A and group C. All sampling process were administrated before treatment.

Animals treatment after experimentation
Cows in group A and cured cows in group B, C, and D were under the normal breeding management again after the study, and the rest were treated by other medicine until cured. All cows are raised according to animal welfare principles.

SCC measurement and cure rate calculation
Detected the SCC of milk in group A and the optimal dose group by milk somatic cell detector (De Laval, Sweden), observed the clinical symptoms of the cows with mastitis every day during the experiment period, and then calculated the cure time, effective rate, and cure rate.

Criteria of PCR results
The criteria of PCR results were shown in table 2.

Data Analysis
Regarded the average of the data in group A as the control and compared it with the optimal dose group. The data was normally distributed and distinguished the differences and correlations between groups by independent sample t-test and Person relation analysis by SPSS 19.0 (IBM SPSS statistics for Windows, version 21.0). All data was recorded as`X ± SD. For each cow, three different investigators were involved as follows: a rst investigator administered the treatment and collected samples based on the randomization table. This investigator was the only person aware of the treatment group allocation. A second investigator was responsible for the measurement of somatic cell count, in ammatory factors, and qPCR and the calculation of cure rate. Finally, a third investigator (also unaware of treatment) analyzed the above results.