Anti-inflammatory Effect Of RANKL In Dairy Cow Mammary Epithelial Cells

Mastitis has a significant impact on the milk production and milk quality of dairy cows,which has been a major blow to dairy farming for a long time.Gram-positive bacteria is one of the main pathogens causing acute clinical mastitis.Lipopolysaccharide(LPS)is the outer wall of gramnegative bacteria.It is an effective inducer of inflammatory response.Receptor activator of the NF-κB ligand(RANKL)is an important member of the TNF superfamily.RANKL may have antiinflammatory properties,but the mechanism is unknown.Whether RANKL plays an antiinflammatory role in dairy cow mammary epithelial cells(DCMECs)or its mechanism is unknown.To obtain the gene expression profile of dairy cow mammary epithelial cells with RANKL overexpression,transcriptome sequencing was performed on cow mammary epithelial cells with RANKL overexpression.Compared with the control group,a total of 3725 differential genes(including 2740 up-regulated genes and 985 down-regulated genes)were found in 1213 pathways,many of which are related to inflammation.Seventeen inflammatory related differential genes were selected from DEGs and verified by qRT-PCR.The fluorescence quantitative results were consistent with the transcriptome results.To establish the inflammation model of dairy cow mammary epithelial cells,the mammary epithelial cells were treated with LPS of 0,5,10,20,50 μg/m L,respectively.The cell viability was detected by MTT assay,and the m RNA expression levels of inflammatory cytokines IL1β,IL6,iNOS and TNF-α were detected by qRT-PCR.The results showed that the cell viability was significantly down-regulated and the m RNA expressions of IL1β,IL6,iNOS and TNF-α were significantly upregulated when treated with 10 μg/m L LPS(P<0.05),indicating that the inflammation model of dairy cow mammary epithelial cells was successfully established.To explore the anti-inflammatory effect and possible mechanism of RANKL in dairy cow mammary epithelial cells,an inflammatory model of dairy cow mammary epithelial cells was used,dairy cow mammary epithelial cells were treated with LPS or transfected with RANKL overexpression vector,or treated with both(LPS+RANKL),respectively.Cells were collected after24 h.The results were as follows:(1)The expressions of IL1β,IL6,iNOS and TNF-α were detected by qRT-PCR.Compared with the control group,the expressions of IL1β,IL6,iNOS and TNF-α in LPS group were significantly up-regulated(P<0.05),but the expressions of IL1β,IL6,iNOS and TNF-α were significantly lower in LPS+RANKL group than those in LPS group(P<0.05).Therefore,RANKL could reduce the m RNA expression of inflammatory cytokines.(2)Flow cytometry was used to detect the apoptosis of dairy cow mammary epithelial cells.The results showed that there was no significant difference in RANKL overexpression group compared with the control group(P>0.05);the LPS group significantly increased the apoptosis of dairy cow mammary epithelial cells(P<0.05).Compared with the LPS group,the LPS+RANKL group significantly decreased apoptosis of dairy cow mammary epithelial cells(P<0.05).Therefore,RANKL could decrease the apoptosis induced by LPS in dairy cow mammary epithelial cells.(3)NF-κB-driven luciferase was used to detect the effect of RANKL overexpression on NF-κB phosphorylation.The results showed that the cells of the NF-κB-driven luciferase report gene showed an increase in luciferase activity under the action of LPS,but significantly decreased in the LPS+RANKL group(P<0.05).Therefore,RANKL inhibited the effect of LPS-induced NF-κB phosphorylation.(4)Western blot was used to detect inflammatory-related proteins TLR4,My D88,TRAF6,TNF-α,and P-65,p-P65,RANK and RANKL in NF-κB pathways.The results confirmed that the protein expression of LPS inflammatory-related proteins(TLR4,My D88,TRAF6,TNF-α)and pP65 in the LPS group was significantly increased than that of the control group(P<0.05).The protein expression of TLR4,My D88,TRAF6,TNF-α and p-P65 was significantly decreased than that of the LPS group(P<0.05).It was further explained that RANKL could reduce the inflammatory pathway of NF-κB which induced by LPS.(5)Co-IP was used to verify the interaction between RANK and TRAF6,and the interaction between TRAF6 and TLR4.The results showed that co-precipitation occurs between RANK and TRAF6,and co-precipitation occurs between TRAF6 and TLR4.It showed that RANKL may have an effect on the LPS/TLR4 signaling pathway,which may inhibit the LPS-TLR4 classic inflammatory pathway.In conclusion,RANKL plays an anti-inflammatory role in dairy cow mammary epithelial cells.After being activated by RANKL,RANK can reduce expression of TLR4,My D88,TNF-α,and pP65 in the NF-κB pathway.RANK may inhibit LPS/TLR4 classical inflammatory pathway by binding to TRAF6.