Therapeutic Effect And Mechanisms Of Ginsenoside Rg1 On Goat Mastitis Induced By Lipopolysaccharides

Escherichia coli(E.coli)is one of the main causes of clinical mastitis in lactating animals.It often causes problems such as reduced milk production,decreased milk quality,and increased treatment management costs.In severe cases,animals can be eliminated or even killed after Escherichia coli infection,bringing huge economic losses to dairy production.At present,dairy farms rely on antibiotics to treat and prevent cases of lactation or non-lactating mastitis which results in the emergence of drug-resistant strains.Therefore,more and more researches are focused on non-antibiotic therapy in mastitis.Ginsenoside Rgl is a protopanaxatriol saponin that binds to Toll-like receptor 4(TLR4)on mouse RAW264.7 and inhibits the recognition of E.coli lipopolysaccharides(LPS)by macrophages to down-regulate the inflammatory response.This paper focuses on the therapeutic effect of ginsenoside Rgl on caprine E.coli endotoxin-induced mastitis,and provides a new idea for the treatment of clinical E.coli mastitis.1.Rgl can block down the signaling pathway induced by LPS in goat peripheral blood monocytesObjective:To investigate the blocking effects of ginsenoside Rgl on LPS-induced inflammatory response and oxidative stress in peripheral blood monocytes of goats.Method:1)The distribution of Rgl in peripheral blood monocytes from goats was detected by immunofluorescence assay.Monocytes were incubated with Rgl,and the cells were performed after that.2)The interference effect of Rgl on LPS bingding was detected in peripheral blood monocytes from goats.Monocytes were co-incubated with Rgl and Alexa fluor 488-LPS,and the cells were performed with the immunofluorescence assay.3)The mRNA expression of tumor necrosis factor(TNF)-a,interleukin(IL)-1?,IL-6,IL-8,TLR4 and nuclear transcription factor-?B(NF-?B)in monocytes induced by co-stimulation of Rgl and LPS was detected.4)Peripheral blood monocytes were cultured with Rgl and LPS.The supernatants were collected for prostaglandin E2(PGE2)determination.5)The inhibition of Rgl on oxidative stress damage caused by LPS was detected by cultured monocytes with Rgl and LPS.The cell lysates were prepared for superoxide dismutase(SOD)and malondialdehyde(MDA)detection.Results:1)The results of immunofluorescence assay showed that ginsenoside Rgl could be observed in monocytes with red fluorescence,indicating that Rg1 can distribute in the monocytes of goats.2)The dose dependent effects of Rg1 on interference of the LPS binding in monocytes was observed in this study with decreased green fluorescence.3)Rg1 treatment could significantly inhibit the effect of LPS on the increased mRNA expression levels of IL-1?,IL-6,IL-8,TNF-?,TLR4 and NF-?B,and reduced the up-regulation of LPS-induced PGE2 in monocytes.4)With Rg1,the SOD in cells was up-regulated and the MDA was decreased.Thus,Rg1 can enter into the monocytes,inhibit the LPS-induced inflammatory meditors,reduce the oxidative stress and significantly enhance antioxidant levels in monocytes.2.Therapeutic effect of ginsenoside Rg1 on mastitis experimentally induced by lipopolysaccharide in lactating goatsObjective:To investigate the therapeutic effect of ginsenoside Rg1 on mastitis experimentally induced by LPS in goats.Method:9 lactating goats were randomly assigned into three groups.Group 1,were intramammary infused with LPS,following with saline intravenous injection,as a goat mastitis model;Group 2 were intramammary infused with LPS and intravenous injected with Rg1,as the treatment group;Group 3 were intramammary administrated and intravenous injected with saline,as a control group.Blood and milk samples were collected 3,6,9,12,15,18,21,24,48 and 72 h post LPS challenge for the measurements of milk yield,blood and milk components.White blood cells(WBC),neutrophils(NEU),lymphocytes(LYM),and serum total protein(TP),albumin(ALB),globulin(GLOB)tests,and milk somastic cell counts(SCC),lactose,N-Acetyl-Beta-D-Glucosaminidase(NAGase)activity tests were performed using blood automatic testers and milk component testers,respectively.Clinical indicators,including udder gland girth,rectal temperature and udder skim temperature,were monitored 1 to 10,12,15,18,21,24,48 and 72 h after LPS challenge.Results:1)In group 1,goats were depressed after LPS infusion.Rectal temperature test showed that the body temperature of the goat increased significantly and restored after 10 h after infection.Udder glands were swelling and painful.Milk yield decreased,and recovered after 96 h after challenge.The lactose in milk was significantly down-regulated after endotoxin infusion,and SCC and NAGase activity was significantly up-regulated.WBC,NEU,LYM and serum TP,ALB,and GLOB levels were reduced in mastitis goats.The results indicate that the damage of blood-milk barrier during LPS-induced mastitis which causes the increased flow of blood components such as WBC,TP and ALB into the interstitium or milk,and the enhanced external swelling of the gland.2)In group 2,intravenous injection of Rgl could reduce the pain in udder glands,lower the temperature of rectal and udder surface.Rgl can inhibit the reduce of milk yield caused by LPS,and protect udder glands.At the same time,Rgl can inhibit the increase of milk girth.Changes in milk were elevated,including the decrease of somatic cells and NAGase activity,and the increase of lactose after Rgl injection.The blood coponents,such as WBC,NEU,LYM and serum TP,ALB,GLOB,were recovered with Rgl treatment.The results showed that ginsenoside Rgl can reduce the systemic and local inflammatory responses in LPS-induced mastitis goats,protect udder glands,and restore the milk yield and milk components.3)In group 3,we did not observe clinical symptoms and significant changes in milk or blood component detection after placebo injection.3.Proteomics to elucidate the anti-inflammation action of ginsenoside Rgl against LPS-induced mastitis in goatObjective:To investigate the effect of ginsenoside Rgl on LPS-induced caprine masititic whey proteins.Method:9 Milk samples were collected from 6 h post LPS challenge as described in section 2.All the samples were prepared for tandem mass tags(TMT)proteomic analysis after defatting and removing casein from the milk.Bioinformatics analysis of the different expressed proteins were performed based on Gene ontology(GO)function and Kyoyo encyclopedia of genes and genomes(KEGG)enrnchment.Results:1)All the samples were well prepared for TMT analysis.3838 peptides were observed,and a total of 791 proteins in whey samples were identified by searching against the "Ruminantia" database from Uniprot.Among the proteins,69.5%of them were identified by tow or more peptides.Protein abundance distribution of each sample was basically normal distribution.The principal component analysis showed that the differences in each of the three groups were small.2)Significantly changed proteins were analysed between groups.In group 1 vs 3,98 proteins between groups 1(LPS + Saline)and 3(Saline + Saline)were significantly different.Group 1 than control group had significantly more inflammatory factors such as IL-6,acute phase proteins,blood coagulation factors,complement proteins,and oxidative stress markers while these factors were reduced after Rgl treated.The dramatically increased ALB in mastitic whey indicated the damage of blood-milk barrier during LPS challenge.In group 2 vs 1,34 proteins between groups 2 and 1(LPS + Rg1)were significantly different.ALB in group 2 was decreasd.Forthermore,proteins,including eukaryotic translation initiation factor 4B(EIF4B),beta-1,4-galactosyltransferase I(B4GALT1),Trefoil factor 2(TFF2),fatty acid transporter(CD36),fatty acid binding protein 5(FABP5),TIMP metallopeptidase inhibitor 2(TIMP2),in group 2 associated with peroxisome proliferator-activated receptor ?(PPARy)and restore milk fat and production were up-regulated compared to group 1.In group 2 vs 3,53 proteins between groups 2 and 3 were significantly different with increased acute phase proteins in group 2.Still,the heatmap of differential expressed proteins indicating the therapeutic effect of Rgl in LPS-indcued mastitis.3)GO enrichment analysis showed that response to stimulus was the top GO item in cellular process,with 44 differentially expressed proteins between group 1 and 3,11 differentially expressed proteins between group 2 and 1,and 20 differentially expressed proteins between group 1 and 3.More differentially expressed proteins in whey proteome during LPS-induced mastitis were involved in response to stimulus,suggesting that the stress for goat after LPS challenge.After Rgl treatment,the proteins enriched in this GO item were decreased,indicating the weaker host immune responses to LPS in goats.4)The results of KEGG pathway enrichment analysis showed that the complement and coagulation cascades were enriched with more differentially expressed proteins,including 20 up-regulated proteins in group 1 vs 3,4 down-regulated proteins in group 2 vs I,and 10 up-regulated proteins in group 2 vs 3.KEGG result suggested that the complement system was activated during LPS-induced goat mastitis which can recruit peripheral blood cells into milk and mediate bacterial apoptosis in coliform mastitis.Rg1 treament can down-regulate the complement and coagulation cascades,and inhibit the expression of proinflammatory mediators and leukocyte recruitment after challenged with LPS in goats.In conclusion,these results indicated that,in vitro,ginsenoside Rgl could interfere with the binding of LPS to monocytes,inhibit the expression of inflammatory cytokines,down-regulate TLR4/NF-?B signaling pathway and protect cells from LPS-induced oxidative damage.In vivo,intravenous injection of Rgl could inhibit the growth of girth of udder half,reduce the rectal temperature and udder skin temperature after LPS infusion.In addition,Rgl treatment could maintain milk production and lactose content,restore white blood cells and serum total proteins in peripheral blood.Finally,TMT proteomic analysis of Rgl treatment in Escherichia coli endotoxin-induced caprine mastitic whey protein suggrested that Rgl could inhibit the expression of proteins involved in acute phase reaction,and up-regulate the proteins accociated with PPARy and goat udder gland protection.Therefore,how to use ginsenoside Rgl in the treatment of coliform mastitis in lactating animals deserves further studies.