Effect And Mechanism Of Resveratrol On LPS Induced Mastitis In Mice

Bovine mastitis is an inflammatory disease on account of the action of various pathogenic factors invading milk cow mammary tissue.Common pathogenic factors include pathogenic microorganism infection,physical injury,chemical stimulation and improper feeding management.When dairy cows get mastitis,the milk yield and quality of dairy products will be greatly reduced,which will cause great economic losses to the dairy cattle industry.Lipopolysaccharide(LPS)is the main component of the cell wall of gram-negative bacteria and one of the most important virulence factors of this kind of pathogen.The mastitis mouse model established by LPS has the same symptoms and pathogenesis as the clinical mastitis in dairy cows,and is often used as a research tool in the research of dairy cow mastitis for the exploration of the pathogenesis of dairy cow mastitis and the screening of prevention and treatment drugs.Resveratrol is a natural structural compound of polyphenols in multifold plants.A large amount of data have confirmed that resveratrol has anti-inflammatory,antioxidant,antibacterial and immunomodulatory pharmacological activities.However,the role and mechanism of resveratrol in mouse mastitis have not been reported.In this paper,resveratrol was taken as the research object.Firstly,a mouse mastitis model was established by LPS to observe and verify the effect of resveratrol on mouse mastitis.Then the mechanism of action of resveratrol was preliminarily revealed on mouse mammary epithelial cells through in vitro experiments.The purpose of this paper is to test whether resveratrol has a protective effect on LPS-stimulated mastitis in mice through in vivo and in vitro experiments and to provide some experimental data for the screening of leading compounds with anti-inflammatory activity.First,female mice lactating for 7 days were randomly divided into 5 groups(4 mice in each group): blank control,LPS,LPS+resveratrol(10mg/kg,20mg/kg,30mg/kg).The blank control group received intraperitoneal injection of normal saline of the same volume as the resveratrol group.Mice in the LPS group were treated with LPS perfusion through milk ducts and intraperitoneal injection of normal saline of the same volume as that in the resveratrol group at 1h before that.Mice in the LPS+resveratrol group were pretreated with intraperitoneal injection of resveratrol 1h before LPS lactation catheter perfusion.Since LPS perfused for 24 h,mice was executed by cervical dislocation method,and open the mice skin along the medioventral line,macroscopic observation found that blank control group mice mammary gland was milk-white,no obvious change,LPS group of mice mammary gland swelling,there is a clear mammary gland swelling in LPS + resveratrol group,mammary gland of mice have significant reduction compared with LPS group.Mice mammary tissue samples were removed and collected,and HE staining sections showed that,compared with the blank control group,the acinar wall of the LPS group was significantly thickened and there was a large amount of inflammatory infiltration,while the inflammatory infiltration of the LPS+resveratrol group was significantly reduced and the thickness of the acinar wall was close to that of the blank group.The mRNA levels of IL-1? and TNF-? in mammary tissues were detected by qRT-PCR.The mRNA levels of the two cytokines in the LPS group were significantly higher than those in the blank control group(p<0.05),while those in the LPS+ resveratrol group were significantly lower than those in the LPS group(p<0.05)and showed a dose-dependent effect.The expression of IL-1? and TNF-? is regulated by the MAPK and NF-?B signaling pathways,whether the decrease of IL-1? and TNF-? in the mammary gland tissues of mice is related to the effect of these two signaling pathways.Therefore,the activation of the MAPK and NF-?B signaling pathways in the mammary gland tissues of mice was detected by Western blotting in this paper.In the LPS group,protein levels of p-I?B,p-p65,p-p38 and p-ERK were significantly higher than those in blank control group(p < 0.05),while in LPS+resveratrol group protein levels of p-I?B,p-p65,p-p38 and p-ERK were significantly lower than those of LPS group(p < 0.05),and showed a dose-dependent effect.These results suggest that resveratrol inhibits MAPK and NF-?B signaling pathways in mice mammary tissue and cuts the the expression level of IL-1? and TNF-?.The above results confirmed that resveratrol has anti-inflammatory effect on LPS-induced mice mastitis.In order to further reveal the mechanism of action of resveratrol,the safe dose range of resveratrol on mouse mammary epithelial cells was firstly determined by MTT,and the results showed that resveratrol had no toxic effect on mouse mammary epithelial cells in the concentration range of 12.5~50?M.The mouse mammary epithelial cells were divided into blank control,LPS,LPS+resveratrol(12.5?M,25?M,50?M)with 3 replicates in each group.Cells were culture with medium for cultivating blank group,cells of LPS group using were stimulated with 1.0?g/mL LPS for 24 h,the cells of LPS+resveratrol group were pretreated with resveratrol for1 h,then were incubated with12.5?M,25?M,50?M resveratrol and 1.0?g/mL LPS for 24 h.The mRNA expression level of IL-1? and TNF-? were detected by qRT-PCR,the result showed that resveratrol inhibited the mRNA expression level of IL-1? and TNF-? in dose-dependent.Extraction of proteins of mammary epithelial cells in mice,Western blotting test protein levels of p-I?B,p-p65,p-p38 and p-ERK,results showed that these four protein levels of LPS group were significantly higher than the blank control group(p < 0.05),while in LPS+resveratrol group these four protein levels are significantly lower than those of LPS group(p < 0.05).This result suggested resveratrol can inhibit LPS stimulation in mammary epithelial cells in mice though the inhibiting effect on activation of MAPK and NF-?B signaling pathways.These results confirmed that resveratrol inhibited activation of MAPK and NF-?B signaling pathways and the expression levels of IL-1? and TNF-?,therefore resveratrol showed the protective effects on mouse mammary epithelial cells.In this paper,in vivo and in vitro experiments confirmed that resveratrol can reduce the hyperemia of swollen mammary gland in mice and the expression levels of IL-1? and TNF-? in mammary gland and mouse mammary epithelial cells though inhibiting activation of MAPK and NF-?B signaling pathways.Therefore,resveratrol shows the protective effects on LPS-stimulated mammary gland and mammary epithelial cells in mice.This paper can provide a reference for the further research and development of resveratrol.