Bacteriostatic Effect Of Tea Tree Oil And Its Antagonism On Dairy Cow Mastitis Challenged By LPS

Cow mastitis is one of the biggest challenges to dairy farming and dairy products industry,which restricts the healthy development of dairy industry.Effective prevention and treatment of cow mastitis can provide a good guarantee for the health of cows and the production of high-quality dairy products.With the problem of drug resistance becoming increasingly prominent,how to effectively prevent and treat cow mastitis and find new antibiotic substitutes have become the current research hotspot.Tea tree oil has a wide antibacterial spectrum and strong antibacterial activity,which can effectively treat the related diseases caused by fungi,bacteria or viruses.It gradually enters the field of vision of researchers and is expected to become a new generation of effective antibacterial substances after further research and development.In this study,the antibacterial effect of tea tree oil and its antagonistic effect on LPS in Mac-T were studied.In this study,the antibacterial effect of tea tree oil in vitro was tested to determine whether tea tree oil has a certain antibacterial effect on common cow mastitis pathogens.In order to simulate the mastitis of dairy cows caused by most Gram-negative bacteria infecting the mammary tissue of dairy cows,the cell model of mastitis of dairy cows induced by LPS was constructed.By adding different concentrations of tea tree oil to the constructed mastitis cell model,the antagonistic effect of tea tree oil on LPS was explored,and the appropriate concentration of tea tree oil was determined.The above experiments explored the feasibility of tea tree oil in the treatment of cow mastitis,and provided a reference for the development of tea tree oil as an effective antibacterial material to cow mastitis.The results of this study are as follows:1.Bacteriostatic effect of tea tree oil on common pathogenic bacteria of cow mastitis:Five common pathogenic bacteria of mastitis in dairy cattle,including Staphylococcus aureus,Klebsiella pneumonia,Pseudomonas aeruginosa,eschia Ferguson and Bacillus cereus,were isolated from local pastures.The strains on good condition were selected for the drug sensitivity test of tea tree oil.The results showed that the five strains were moderately sensitive to 20%concentration of tea tree oil,and the sensitivity to 10%concentration of tea tree oil was between moderate sensitivity and low sensitivity,while the sensitivity to 5%concentration of tea tree oil was only low sensitivity.The mic concentration of tea tree oil was 1.6%for Staphylococcus aureus,Klebsiella pneumonia,e.Ferguson and Bacillus cereus,and 1.8%for Pseudomonas aeruginosa.The results showed that tea tree oil had a certain bacteriostatic effect on Staphylococcus aureus,Klebsiella pneumonia,Pseudomonas aeruginosa,Escherichia Ferguson and Bacillus cereus2.The establishment of the cell model of dairy cow mastitis induced by LPS:the recovery,subculture and cryopreservation of MAC-T.LPS of 50 μg/ml,100 μg/ml,200 μg/ml,500μg/ml and 1000 μg/mL were added to the cell culture,and the related indexes were detected.Firstly,CCK-8 method was used to detect the cell proliferation activity.The results showed that the activity of the cells began to decline after 12 hours of LPS induction at 100 μg/mL Flow cytometry was used to detect apoptosis.The results showed that there was not a large number of apoptosises in 12 hours after LPS induced at 100 μg/mL.However,about 46%of the cells showed early apoptosis and late apoptosis after 12 hours of LPS treatment.The expression of inflammatory factors and apoptotic factors were detected by qPCR.The results showed that the expression of TNF-α was 16 times of that of the blank control group,the expression of IL-6 was 18 times of that of the blank control group(P<0.01),and the expression of STAT1 related to apoptosis was 6 times of that of the blank control group(P<0.01).The expression of inflammatory protein and apoptosis protein was detected by ELISA.The results showed that the expression of NF-κB,MAPK and caspase-3 were about 2.1 times,4.4 times and 7.7 times respectively(P<0.01).The above results show that:in this experiment,200 μg/mL LPS can be used as the construction condition of cow mastitis cell model for 12 hours.3.The antagonistic effect of tea tree oil on LPS in dairy cow mastitis cell model:0.0002%,0.0004%,0.0006%.0.0008%,0.001%,0.002%,0.004%,0.006%,0.008%and 0.01%tea tree oil were added to the cow mastitis cell model induced by 200μg/mL LPS for 12 hours.Flow cytometry was used to detect apoptosis.The results showed that when tea tree oil concentration was 0.0004%,0.0006%and 0.0008%,the apoptosis rate of the cells would be decreased,and the group with tea tree oil concentration of 0.0006%had the most obvious protective effect.The proportion of living cells was 71.95%,the proportion of early apoptotic cells was 22.15%,and the proportion of late apoptotic cells was 5.11%.Compared with the mastitis model group without tea tree oil,the proportion of living cells increased by about 22%.Then the expression of cytokines and apoptotic factors were detected by qPCR in three groups with protective effect.The results showed that with the increase in tea tree oil concentration,TNF-αexpression decreased more,IL-6 expression decreased less(P<0.01),STAT1 expression slightly increased when tea tree oil concentration was 0.0004%.At 0.0006%and 0.0008%concentrations,the expression of tea tree oil decreased slightly,and the expression of tea tree oil with 0.0006%concentration was the lowest(P<0.05).The results showed that the expression of NF-κB,MAPK and caspase-3 were significantly decreased in the three groups of tea tree oil added.The expression of inflammatory response protein in 0.0006%tea tree oil group was the lowest than that in the other concentration groups,about 50%of that in the blank control group(P<0.05).However,the expression of apoptosis response related proteins in the three groups was almost the same,about 55%of that in the blank control group(P<0.05).The above results showed that tea tree oil with concentration of 0.0004%-0.0008%in MAC-T had antagonistic effect on LPS,and tea tree oil with concentration of 0.0006%had the most obvious antagonistic effect on LPS.In conclusion,tea tree oil has certain bacteriostatic effect on several common pathogenic bacteria of cow mastitis.In the appropriate concentration range,it has a certain antagonistic effect on LPS,which can reduce the proportion of apoptosis,increase the survival proportion of normal cells,and down regulate the expression of cytokines and pathway proteins.