| Infection of mammary gland tissue by pathogenic microorganisms is one of the main causes of mastitis.Mastitis leads to a decrease in milk production,a decrease in the quality of dairy products,and an increase in the culling rate of dairy cows,which brings huge economic losses to the dairy industry.Staphylococcus aureus(S.aureus)is one of the most common pathogenic bacteria causing mastitis.Pattern recognition receptors(PRR)on the surface of mammary epithelial cells recognize the virulence factors of S.aureus.Macrophages,as immune cells,play a role in innate and acquired immunity against S.aureus.Luteolin is a natural flavonoid that exists in a variety of plants.It has antioxidant and anti-inflammatory effects,but its anti-inflammatory mechanism is still unclear,and its anti-mastitis efficacy has not been reported.The present study aims to enrich the research on dairy cow mastitis by exploring the anti-inflammatory mechanism of luteolin on dairy cow mastitis and provide data support and a theoretical basis for the application and promotion of Chinese herbal medicine in the treatment of mastitis.In dairy cow mastitis,the destruction of the mammary gland tissue structure,the recruitment of immune cells and the secretion of cytokines in the tissue increases.In this experiment,healthy and inflamed mammary gland tissues were collected from cattle slaughterhouses and observed by HE and immunofluorescence.Fluorescence quantitative and other molecular biological experiments revealed the changes of genes and proteins.To establish a mammary gland cell model and immune cell model of S.aureus infection,explore the mechanism of luteolin on S.aureus mastitis in animals;and establish a mouse mammary gland model to verify the research results.The main research contents and experimental results are as follows:In order to study the tissue changes in cow mastitis,we selected healthy and inflamed mammary gland tissues for histological analysis.HE section observation showed that mammary gland tissue structure was destroyed,acinar irregularity,and mammary epithelial cells shedding during inflammation.The immunofluorescence test presented that the number of macrophages in the tissues increased,and there were a large number of infiltered cells in the acini,including macrophages.The RT-q PCR and ELISA exhibited that the expression levels of cytokines IL-1β,IL-6 and TNF-α in inflammatory mammary tissue were significantly increased.Western Blot(WB)detected inflammation-related proteins in NF-κB and MAPK pathways involved in mastitis.The results displayed that the phosphorylation levels of IκB,p65 and JNK,ERK and p38 proteins in the pathway were increased.The findings of WB exhibited that the expression level of macrophage marker antigen CD68 increased,indicating that macrophage recruitment increased in inflammatory tissues.These results demonstrated that the expression of inflammatory cytokines in the inflammatory tissue of dairy cow mammary glands increased,the protein phosphorylation levels of NF-κB and MAPK pathways increased,and the number of macrophages increased.In order to explore the anti-inflammatory effect of luteolin in mammary epithelial cells and its possible regulatory pathways,here,different bacterial cell infection ratios(MOI=1:1,10:1,100:1)of S.aureus were used to treat dairy cow mammary epithelial cells.The cow mammary epithelial cells and mouse mammary epithelial cells were stimulated for 3h,6h,12 h,and 24 h.CCK-8 detected the effect of S.aureus on cell viability,and the secretion of cytokines was detected by RT-q PCR.The results showed that a good cellular inflammation model could be established by stimulating with MOI=10:1 for 12 h.Since the toxicity of luteolin was not determined,the concentration of luteolin was screened by CCK8 in this study.The results showed that 2.5,5 and 7.5μg/m L luteolin could establish a suitable cell therapeutic model.The cow mammary epithelial cells and the mouse mammary epithelial cells were stimulated with S.aureus,respectively,and then treated with luteolin.RT q PCR and ELISA exposed that S.aureus significantly increased the level of IL-1β,IL-6 and TNF-α.However,luteolin significantly inhibited the expression of its cytokines,and the higher the concentration of luteolin,the more pronounced the inhibitory effect.The target proteins of luteolin were predicted through HERB and TCMSP websites,and GO and KEGG enrichment analysis was performed on these target proteins.It was found that luteolin has multiple target proteins,of which p65 is enriched in NF-κB,and some proteins,such as ERK,are enriched in the MAPK signaling pathway.TLR2 is the PRR of S.aureus,while the NF-κB and MAPK pathways are downstream of TLR2.The mammary epithelial cells stimulated by S.aureus to verify luteolin’s effect on these pathways.In the current study,WB was used to detect the protein expression of these pathways.The results illuminated that when stimulated by S.aureus,the expression level of TLR2 protein increased,and the phosphorylation levels of IκBα,p65 in the NF-κB pathway as well as JNK,ERK,and p38 proteins in the MAPK pathway were increased,which was consistent with the expression in cow mastitis.After luteolin administration,the expression of TLR2 and phosphorylation levels of proteins of downstream pathways were decreased,and the decreased level was dose-related with luteolin concentration.Immunofluorescence revealed that the quantity of phosphorylated p65 in the nucleus decreased with the increase of luteolin concentration.The above results indicated that luteolin could inhibit S.aureus-stimulated cytokine secretion and protein phosphorylation levels in TLR2 and its downstream NF-κB and MAPK signaling pathways in a concentration-dependent manner in mammary epithelial cells.The above three experimental results prove that luteolin exerts an anti-inflammatory effect by inhibiting the protein phosphorylation level of TLR2 and its downstream NF-κB and MAPK signaling pathways.Macrophages,as immune cells,participate in the elimination of S.aureus.In this study,luteolin inhibits the migration of inflammatory macrophages and affects polarization typing and its regulatory pathway by regulating p65.Firstly,RAW264.7 cells were used as the macrophage model of S.aureus infection.Transwell and cell scratch tests were used to detect that the migration speed of macrophages was accelerated under the effect of S.aureus.The findings of WB showed that the phosphorylation level of the p65 protein in the NF-κB signaling pathway was increased;immunofluorescence indicated that the p65 of macrophages was increased into the nucleus due to S.aureus infection.After adding luteolin,the migration rate of macrophages infected with S.aureus was significantly slower,while the phosphorylation level of p65 protein and nuclear entry was reduced.The target protein of luteolin was analyzed again through the Metascape website,and RELA(p65)was found to play a role in biological regulation as a transcription factor.To verify the relationship between luteolin and macrophage migration,this experiment first established a model S.aureus infected macrophages,and used different luteolin to intervene.Transwell and cell scratch experiments found S.aureus infection promoted cell migration.Still,cell migration was significantly inhibited by luteolin,and the migration speed was slower when the concentration of luteolin increased.In order to verify the targeting relationship between luteolin and p65 and its biological role as a transcript,this experiment constructed a p65 overexpression vector and used the p65 inhibitor BAY11-7082 to set a macrophage model of S.aureus infection;luteolin was used to reversed these effects.Transwell and cell scratch experiments found that S.aureus infection and overexpression of p65 promoted cell migration,but this migration of cells was significantly inhibited by luteolin.The Twist1 protein expression was positively correlated with macrophage migration.When macrophages migrated fast,Twist1 protein expression was high;when macrophages migrated slowly,Twist1 protein expression level decreased.The transcription factor of Twist1 was predicted on JASPAR and PROMO websites and verified by double luciferase.Double luciferase detection represented that p65 was the transcription factor of Twist1.In order to explore the effect of Twist1 on macrophage migration,we also constructed the Twist1 overexpression vector and interference vector si-Twist1.The same study as the previous method confirmations that Twist1 overexpression promotes macrophage migration,while si-Twist1 significantly inhibits this forward migration of macrophages.The above results indicate that luteolin can down-regulate the expression of Twist1 protein by regulating p65 in macrophages infected with S.aureus,thereby inhibiting macrophage migration.Finally,the same method was used to set up an experimental group to study the polarization and typing state of macrophages.The results displayed that the phosphorylation level of the p65 protein in macrophages and its nuclear translocation were significantly increased due to the infection of S.aureus.Macrophages exhibited M1-type proinflammatory polarization due to S.aureus infection and overexpression of p65.The expressions of M1-type marker proteins CD86 and i NOS were increased,and the expressions of M2 marker proteins CD206 and Arg1 were down-regulated.Increased secretion of pro-inflammatory cytokines IL-1β,IL-6 and TNF-α.After luteolin was added,the phosphorylation level of p65 protein decreased,and the nuclear entry was reduced.After luteolin or p65 inhibitor BAY11-7082,the model inhibited the expression of M1 marker CD86 and i NOS protein,decreased the secretion of proinflammatory cytokines.However,the expression of M2 markers CD206 and Arg1 increased.The results suggested that luteolin could regulate the polarization state of macrophages stimulated by S.aureus,convert them from the pro-inflammatory M1 type to the anti-inflammatory M2 type,and exert an anti-inflammatory effect.The above results indicate that luteolin plays an anti-inflammatory role by regulating p65 to inhibit macrophage migration and promote macrophage transformation from M1 to M2.In order to verify the anti-inflammatory effect of luteolin and its molecular mechanism,the mouse mammary duct was inoculated with S.aureus to establish a mouse mastitis model.The infection model was confirmed by euthanizing 24 hours after infection,and the remaining mice were injected with luteolin(25,50,75 mg/kg)every 12 hours,a total of 4 times.Mice were euthanized 12 h after the last administration,and mammary gland tissues were collected aseptically.H&E found that S.aureus caused pathological changes in mouse mammary gland tissue,similar to cow mastitis tissue results.The results showed that the mammary gland tissue structure was damaged,the acini were irregular,and the mammary epithelial cells were shed.There were many shed cells and macrophages in the acini;the pathological changes of mammary gland tissue in mice injected with luteolin were significantly alleviated and related to the concentration of luteolin.The acinar wall was relatively complete,and the acinar epithelial cells were arranged regularly and shed less.The RT-q PCR and ELISA findings revealed that the luteolin model inhibited the secretion of pro-inflammatory cytokines in mastitis.The WB displayed that the phosphorylation levels of IκBα and p65 in the NF-κB pathway and JNK,ERK and p38 in the MAPK pathway were increased in inflammatory mammary tissue,but luteolin significantly inhibited the phosphorylation levels of these proteins.Immunofluorescence and WB presented that macrophages increased in inflammatory mammary tissue,and the expression level of mouse macrophage marker F4/80 protein increased.However,with the increase of luteolin concentration,macrophages in mammary gland tissue gradually decreased,and F4/80,the marker of mouse macrophages,was significantly decreased.The above results indicate that luteolin inhibits the inflammatory response of mastitis by inhibiting the phosphorylation levels of NF-κB and MAPK pathway-related proteins,preventing the migration of macrophages to mammary tissue.Conclusion: Luteolin reduces the secretion of inflammatory factors in mammary epithelial cells and inhibits the inflammatory response of mastitis by down-regulating the phosphorylation and nuclear translocation of NF-κB and MAPK pathway-related proteins.Luteolin regulating p65 prevents macrophage migration via Twist1,reduces macrophage differentiation to M1 type,increases macrophage differentiation to M2 type,and inhibits the inflammatory response of macrophage in mammary tissue. |
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