| Clinical mastitis severely limited the development of dairy cows breeding.Recently,mastitis was mainly prevented and treated by antibiotics,causing many problems,not only antibiotic residue in milk,treatment efficiency decline,and drug resistance,but also public health and dairy safety incidences.Pulsatilla and its extraction,Pulsatilla Saponins have much efficacy like anti-inflammation,antibacterial,anti-oxidant,and immunity promotion,which were closely suitable for mastitis treatment requirement as bacteria clearance,inflammation relief,and immunity promotion for self-healing.Thus,research in the mastitis curing effects and mechanism of Pulsatilla Saponins,provide a safe,efficient,no residue,and replaceable mastitis medicine,have an important meaning in dairy cows breeding.Test 1 dairy cows clinical mastitis treatment efficiency of Pulsatilla Saponins To investigate the treatment efficiency and optimal treatment dose of Pulsatilla Saponins in dairy cows clinical mastitis,36 weighted 600 ± 47 kg,3-5 years old,2-4 parity,only diagnosed clinical mastitis lactating Holstein cows in a Semi-closed uniform feeding large-scale dairy farm in Sichuan was selected in this test.Cows were randomly grouped into 3 groups and 12 cows per group,inject Pulsatilla Saponins 15 m L(group B),30 m L(group C)and 60 m L(group D)per day,respectively.The day of the first dosing was recorded as 1 d and continue to administrate for 4-6 d.12 healthy lactating cows were chosen as the control group(group A)and give no administration.Measuring body temperature,breath frequency,heart rate,masticate per rumination,lactation,cure time,cure rate,blood routine and biochemical indicators.Results showed that Pulsatilla Saponins have an obvious curing function of dairy cows clinical mastitis,body temperature,breathing frequency,masticate per rumination,lactation,blood routine and biochemical indicators mastitis cows were obviously recovered during administration times,while there showed no effect in heart rate,indicated that Pulsatilla Saponins have no significant clinical side effect in dairy cows.Cure rate within 12 days in each test group was reached 100% and showed a dose-effect relationship,and total cure rate was 75%,75% and 91.67% respectively,average cure time was 6.33 d,6.11 d and 6.91 d.Thus,30 m L/time/day administration could have an ideal treatment effect.Test 2 Pulsatilla Saponins effects on serum cytokines and immunoglobulin in clinical mastitis cows To investigate the effects of Pulsatilla saponins on serum cytokines and immunoglobulin in clinical mastitis cows.12 healthy and 12 clinical mastitis cows were selected randomly in the test,administration Pulsatilla saponins by 30 m L/time/day i.m.based on test 1,the day of the first dosing was recorded as 1d and continue to administrate for 4-6d.Serum factors levels of clinical mastitis cows in 1 d,3 d,5 d,7 d and healthy cows in a random day(inflammation cytokines IL-1α,IL-1β,IL-2,IL-4,IL-6,IL-8,IL-10,TNF-α,LTB4,PGE2,CRP,SAA and HP,immune factors Ig A,Ig D,Ig E,Ig G and Ig M,antioxidant factors T-AOC,SOD,MDA,CAT,GSH-Px,and NOS)were measured by ELISA method.Results showed that,serum IL-1α,IL-2,IL-6,IL-8,IL-10,THF-α,PGE-2,SAA and HP levels were increased very significantly(P<0.01),IL-1β,IL-4,LTB4 and CRP raised significantly(P<0.05),showed that inflammation reaction was severe and immune function was still not destroyed during the early period of clinical mastitis,levels of anti-inflammatory factors were raised rapidly after inflammatory factors increased to regulate inflammation reaction.Inflammation factors were decreasing with the treatment,on the 7 d,indicators of mastitis groups showed no significant difference between the control group,only PGE2 levels were significantly higher than the control group(P<0.05).Indicating Pulsatilla Saponins have significant inflammation factor clearance function.Anti-inflammation factors were decreased with the inflammation in udder was relieved;Serum Ig A,Ig D,Ig E,and Ig M levels in clinical mastitis cows were very significantly increased(P<0.01),showed that the body was in positive anti-inflammation and anti-endotoxin condition,immune factor increased rapidly and seemed a trend to decrease with treatment and reached healthy levels on 7 d(P>0.05).Showed that,Pulsatilla Saponins may have the ability to clear invading bacteria and endotoxin while enhancing the resistance of udder mucosa to pathogens,effectively reduce immune stress level of clinical mastitis dairy cows;Serum T-AOC,GSH-PX and NOS levels increased very significantly(P<0.01),CAT concentrations raised significantly(P<0.05),SOD and MDA levels were higher than control group but not significant,showed that compensatory stress occurred after udder tissue invaded by pathogenic bacteria and stimulate udder tissue by free radicals,inducing anti-oxidation function raise and saw a trend to decline with treatment,showed Pulsatilla Saponin could mitigate the oxidation damage in udder tissue and relief the anti-oxidation requirement.Test 3 Effect of Pulsatilla Saponins on plasma metabolites in clinical mastitis cows To discover the effect of Pulsatilla Saponins on plasma metabolites in clinical mastitis cows,this test based on the dose optimized by test(30 m L/time/day),the day of first dosing was recorded as 1 d and continue administrate for 4-6 d.12 have only clinical mastitis cows with similar lactation age,milk production,mastitis severity and treatment day(group T)and 12 healthy cows(control group,group C)were selected in this study.Collect tail venous blood of T group on the mastitis discovered day(before Pulsatilla Saponins administration),4 d,7 d and C groups in a random day,record as T1(T1-1~T1-12),T2(T2-1~ T2-12),T3(T3-1~T3-12),and C(C1~12),anticoagulated by heparin sodium,centrifuged 10 min at 3000 rpm after rest 30 min.Collect serum and full spectrum analysis each serum sample by HILIC UHPLC-Q-TOF MS technology combined with data-dependent acquisition,acquire primary and secondary mass spectrometry data,then using XCMS to extraction peak and identification metabolites form database,detect and select plasma differential metabolites for bioinformatics analysis and identification,the result showed that 6166 positive ion peaks and 3823 negative ion peaks were identified by XCMS software.69 and 57 differential metabolites were identified in positive and negative ion mode respectively,and those differential metabolites were mainly involved in amino acid and lipid metabolism.In dairy cows clinical mastitis,essential amino acid Leucine,histidine,phenylalanine,threonine and proline were significantly down-regulated,tyrosine was sawed a trend to down-regulate but have no significant difference,and phenol is significantly down-regulated in tyrosine metabolism;non-essential amino acid,citrulline and tryptophan are significantly down-regulated;Metabolites such as myristic acid,hexadecenoic acid,octadecanoic acid,and phosphatidylcholine(PC)are significantly up-regulated.These differential metabolites are widely involved in fatty acid biosynthesis,glycerophospholipid,Arachidal,linoleic acid,and alpha-linolenic acid metabolism pathways.SOPC of each comparison group were showed significant difference with treatment time,SOPC was significantly increased in the comparison if group T1 and C(FC= 1.603),and SOPC between T2-T1 and T3-T2 were significantly down-regulated(FC= 0.358 and 0.693),although group T3 was still higher than group C,but fold change was lower than group T1 and C,speculating SOPC have a more sensitive change in the cure of mastitis and Pulsatilla Saponin treatment,could be a potential dairy cows mastitis biology indicator.Taurine deoxycholate,taurochenodeoxycholate,taurodeoxycholate and taurocholic acid levels raised significantly and taucuronate have an upward trend in the taurine metabolic pathway.Suspected that Pulsatilla Saponins may improve nature immune function.,relief the inflammation caused by infection by enhancing this pathway.Test 4 Effect of Pulsatilla Saponins on plasma protein regulation in dairy cows with clinical mastitis To discover the effect of Pulsatilla saponins on plasma protein regulation in dairy cows with clinical mastitis,this test based on the dose optimized by test(30 m L/time/day),the day of first dosing was recorded as 1 d and continue to administrate for 4-6 d.12 have only clinical mastitis cows with similar lactation age,milk production,mastitis severity and treatment day(group T)and 12 healthy cows(control group,group C)were selected in this study.Collect tail venous blood of T group on the mastitis discovered day(before Pulsatilla Saponins administration),4 d,7 d and C groups in a random day.Plasma samples from 12 healthy cows and clinical mastitis cows were randomly divided into 3 groups(3 biological replicates),and 4 plasma samples were taken from each replicate for equal mixing and marked as C1,C2,C3,T1-1,T1-2,T1-3,T2-1,T2-2,T2-3,T3-1,T3-2 and T3-3,anticoagulated by heparin sodium,centrifuged 10 min at 3 000 rpm after rest 30 min.Collect serum and detect serum protein by proteomics based on Label-free technology and select differential protein for bioinformatics analysis.Result showed that 544 protein was detected;a total of 7 significant up-regulated and 3 significant down-regulated differential protein in the initial period of dairy cows clinical mastitis,and other 13 protein was only detected in this group,in which Phagein,α-S2-casein,Ras-associated C3 botulinum toxin substrate 1(Rac1)and heat shock protein β-1 can be used as biodiagnostic markers for clinical mastitis.β-defensin 12 was significantly up-regulated in the cured cows was significantly up-regulated than initial incidence period and healthy cows,indicating that Pulsatilla saponins may have β-defensin secret promotion function;comparison to healthy cows,C1 q significantly downregulated but C2 significantly increased in the Complement and coagulation cascade pathway of cured cows,suspecting that Pulsatilla Saponins could stimulate or activate C2,then activate downstream reaction and complement system.Thus,Pulsatilla Saponins can cure clinical mastitis by stimulating defensin secretion and activating the complement system to exert antibacterial,anti-inflammatory,immune-promoting and anti-inflammatory drug functions.Conclusions:(1)Pulsatilla saponin has a significant effect on clinical mastitis in dairy cows,with no obvious side effects.The ideal clinical effect can be achieved by intramuscular injection of 30 m L/time/day.(2)Clinical mastitis cow serum inflammatory factors,immunoglobulins and some antioxidant and oxidative indicators were significantly or significantly higher than healthy cows.With the treatment,the inflammatory factors,immunoglobulins and antioxidant and oxidative indexes showed a decreasing trend,indicating that Pulsatilla saponins have the effect of reducing the pro-inflammatory factors of mastitis cows,enhance the resistance of udder mucosa to pathogens,reduce oxidative damage of breast tissue,and effectively alleviate immune stress in dairy cows with clinical mastitis.(3)The experiment used XCMS software to extract 6166 metabolite ion peaks in positive ion mode and 3823 in negative ion mode.A total of 69 and 57 differential metabolites were identified in the positive and negative ion modes of each group,mainly involved in amino acid metabolism and lipid metabolism;in the early stage of clinical mastitis,the essential amino acids leucine,histidine,phenylalanine,threonine and proline,as well as the non-essential amino acids citrulline and tryptophan were significantly down-regulated,and the tyrosine was down-regulated,but the difference is not significant,but phenol is significantly down-regulated in tyrosine metabolism;Metabolites such as myristic acid,hexadecenoic acid,octadecanoic acid,and phosphatidylcholine(PC)are significantly up-regulated.These differential metabolites are widely involved in fatty acid biosynthesis,glycerophospholipid,arachidal,linoleic acid,and alpha-linolenic acid metabolism pathways,SOPC has the potential to be a biomarker for cow mastitis;Pulsatilla saponins may enhance the body’s natural immune function by up-regulating the taurine metabolic pathway,and alleviate the inflammatory response caused by infection.(4)A total of 544 proteins were identified,of which haptoglobin,α-S2-casein,Ras-related C3 botulinum toxin substrate 1(Rac1)and heat shock protein β-1 can be used as biodiagnostic markers for clinical mastitis.Pulsatilla saponins can cure clinical mastitis by stimulating defensin secretion and activating the complement system to exert antibacterial,anti-inflammatory,immune-promoting and anti-inflammatory drug functions. |
