| Mastitis of dairy cows is a common and frequent dairy mammary disease in China,and its causes are complex,diverse and widely spread.It not only affects the milk production of cows,but also reduces milk quality and even poses risks to human health,which greatly limits the development of the dairy industry.Mastitis causes a violent inflammatory response in the mammary tissue,resulting in pathological damage to the mammary tissue.Currently,clinical treatment of mastitis in dairy cows is based on antibiotics,but antibiotics are mainly bactericidal and cannot relieve the inflammatory response.It was found that the combination of antibiotics with anti-inflammatory drugs for mastitis was effective in shortening the treatment cycle and reducing the use of antibiotics,thus reducing production costs and increasing economic benefits.However,there are few anti-inflammatory drugs available for clinical treatment,so it is important to find green and safe natural products with anti-inflammatory efficacy for the treatment of mastitis of dairy cows.Menthol is a cyclic monoterpene compound extracted from the stems and leaves of peppermint,which is widely used in the medical industry and daily life.Menthol is one of the main active components of peppermint and has a variety of biological effects such as anti-inflammatory and antioxidant.However,it is not known whether menthol has an inhibitory effect on the inflammatory response of mammary tissue.Therefore,this experiment firstly screened the infection of mastitis in cattle farms in some areas of Jilin Province by epidemiological survey to clarify the main bacteria of mastitis infection in Jilin Province.Then,the anti-inflammatory effect of menthol and its mechanism were investigated in LPS-treated mammary epithelial cells（b MECs）of dairy cows.Finally,menthol cream was applied as an adjunct to the clinical treatment of bovine mastitis to clarify its effect on mastitis.First,in this study,138 milk samples of dairy cows with clinical mastitis were collected aseptically from 12 large-scale dairy farms in Changchun,Jilin and Baicheng cities of Jilin Province.In this study,Escherichia coli（E.coil）,Streptococcus agalactiae and Staphylococcus aureus infections were detected in milk samples by PCR.The results showed that the detection rate of E.coli was 57.9%,Streptococcus agalactiae was 47.8% and Staphylococcus aureus was 35.5%.The above results indicate that mastitis caused by infestation of E.coli,Streptococcus agalactiae and Staphylococcus aureus is more common in Jilin Province,with the highest rate of E.coli infection.Second,to investigate the anti-inflammatory effects of menthol,primary b MECs were isolated and purified and cultured in this study.The b MECs were treated with different concentrations of menthol（10,50,100,200 m M）for 1 h and then treated with LPS（5 mg/m L）for 12 h.The result assayed by q RT-PCR and Western blot.The results showed that LPS significantly upregulated the protein levels of cyclooxygenase-2（COX-2）and inducible nitric oxide synthase（INOS）as well as the m RNA levels of tumor necrosis factor-α（TNF-α）,interleukin-6（IL-6）and interleukin-1β（IL-1β）,while menthol was able to inhibit this effect of LPS in a dose-dependent manner.In addition,menthol was found to alleviate the reduction in milk fat and milk protein synthesis caused by LPS stimulation,as measured by milk fat staining and Western blot for milk lipid and milk protein expression.The above experiments showed that menthol was able to inhibit the expression of pro-inflammatory mediators in b MECs.In order to investigate the mechanism of action,b MECs were treated with menthol（200 m M）,and the results showed that autophagy marker protein LC3 II was significantly upregulated,p62 was significantly downregulated,m RNA levels of some autophagy-related genes were significantly upregulated,and autophagic flux was significantly enhanced.The inhibition of pro-inflammatory mediators in b MECs by menthol was significantly reduced when autophagy was blocked with the autophagy inhibitor 3-methyladenine（3-MA,5mg/m L）.This suggests that the anti-inflammatory effect of menthol may be exerted through the activation of the autophagic pathway.To clarify the mechanism by which menthol activates autophagy,this study used menthol（200 m M）to treat b MECs and found that menthol promoted phosphorylation of AMP-activated protein kinase（AMPK）and unc-5-like kinase 1（ULK1）and increased the entry of nuclear factor-E2-related factor 2（Nrf-2）into the nucleus.Menthol-induced autophagy was significantly inhibited when the AMPK pathway was blocked using AMPK inhibitor compound C（CC,10 mg/m L）.The menthol-activated autophagic pathway was also significantly inhibited by blocking Nrf-2 with the Nrf-2 inhibitor ML385（5mg/m L）.The above results suggest that menthol can promote autophagy and reduce the secretion of pro-inflammatory mediators in b MECs by activating the AMPK/ULK1/Nrf-2 signaling pathway.To further validate the therapeutic effect of menthol in clinical dairy cows.This study screened cows with mastitis caused by E.coli infection,and the therapeutic effect of menthol on clinical mastitis was analyzed by using a strategy of menthol in combination with antibiotic treatment for 7 consecutive days,and peripheral blood and milk were collected to detect pro-inflammatory mediators in serum and whey as well as somatic cell counts in milk.The results revealed that the expression levels of IL-6,IL-1β and TNF-α in whey and serum as well as the number of somatic cells in milk were significantly lower in the menthol-treated group.And the result in the menthol-treated group was lower than in the antibiotic-treated group alone.These data suggest that menthol has an adjuvant effect on the treatment of bovine mastitis.The above results indicate that mastitis caused by E.coli,Streptococcus agalactiae and staphylococcus aureus infestation is more common in Jilin Province,with mastitis caused by E.coli being the most common causative agent of mastitis found in the region in this study.At the same time,menthol can activate autophagy through AMPK and its downstream related pathways,and thus exert anti-inflammatory effects.And the results of the clinical trial showed that the antibiotic combined with menthol regimen can effectively alleviate the inflammatory response in the mammary gland and reduce the somatic cell count,providing a reference for the clinical treatment of mastitis. |
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