Effect Of Chemerin On Inflammation And Apoptosis Of Mammary Epithelial Cells In Dairy Cows

Mastitis is an inflammation caused by a combination of various physiological and biochemical factors,which caused incalculable economic losses to the dairy cow industry in China and even of the world.In the past,most of the researches on this issue at home and abroad focus on the mitigation effect of antibiotic addition,but with the deepening of research,the unique advantages of cytokines in the treatment of mastitis began to be concerned.Chemerin is a cytokine with autocrine and paracrine functions and has been proved existent in various tissues such as adipose tissue,breast and liver.As a chemokine of antigen-presenting cells,Chemerin was involved in the regulation of various inflammatory responses by binding to the G protein-coupled receptor CMKLR1 to exert both anti-inflammatory and pro-inflammatory effects,which is mainly determined by the cleavage site and mode of Chemerin.Mammary epithelial cells have been shown to be an important component of breast immune defense,and apoptosis is a self-repairing method after mammary epithelial cells were damaged.As a central regulator,NF-κB activation triggers a series of changes in physiological functions,including cell proliferation,inflammation and apoptosis.Little is known about the role of Chemerin in the inflammation of large mammals,especially the mastitis of dairy cow.Therefore,in this study,the dairy cow mammary epithelial cell(DCMECs)was used as a cell model,and the recombinant protein Chemerin was applied to the DCMECs that cultured in vitro to study the relationship between Chemerin and inflammation and apoptosis,and to explore the regulation function of NF-κB pathway,so as to improve the pathogenesis of breast diseases.1.Chemerin is highly expressed in clinical mammary gland tissues of dairy cows and induced inflammatory response in DCMECsThree normal and three mastitis mammary gland tissues of dairy cows were collected.In the RNA extract of clinical mammary gland tissues,the mRNA of Chemerin and the receptor CMKLR-1 was significantly up-regulated(P<0.01).The immunoblot results of Chemerin were consistent with the transcription results,and also significantly up-regulated(P<0.01),indicated that Chemerin/CMKLR1 axis may be involved in the development of mastitis in dairy cows.Next,the DCMECs were used as experimental materials,and the cells were treated with recombinant protein Chemerin for 12h,24h and 48h to investigate the effects of Chemerin on the inflammatory response of cells at different time points.The results showed that the mRNA and protein expression of IL-1β and IL-6 in Chemerin group were significantly up-regulated in a time-dependent manner compared with the control group(P<0.05).The secretion of ROS also increased significantly in a time-dependent manner after Chemerin treatment(P<0.01).The results indicated that Chemerin promoted the inflammatory response of DCMECs by regulating the secretion of inflammatory factors IL-1β and IL-6 and the accumulation of inflammatory medium ROS.2.Chemerin activated NF-κB signaling pathway and induced apoptosis in DCMECsThe phosphorylation level of NF-κB protein in DCMECs was detected at 12h,24h and 48h after Chemerin treatment,and the effects of Chemerin on apoptosis of DCMECs at different time point was analyzed.The results showed that the phosphorylation level of NF-κB protein in DCMECs was gradually increased after Chemerin treatment for 12h(P<0.05),24h(P<0.05)and 48h(P<0.01);the results of immunofluorescence showed that the NF-κB protein in the control group was mainly located around the cytoplasm and nucleus,while the NF-κB protein was gradually transferred into the nucleus after Chemerin treatment.Western blot results further revealed that the phosphorylation level of NF-κB protein in the nucleus significantly increased in a time-dependent manner after Chemerin treatment(P<0.05),but the result in the cytoplasm was reversed(P<0.05).These results indicated that high doses of Chemerin can activate the NF-κB pathway in DCMECs.In order to analyze how Chemerin affects the cell structure after inducing inflammation in the DCMECs,the cell state was differentiated by fluorescent dyes propidium iodide(PI)and annexin V-FITC(AV),and then evaluated the apoptosis rate by flow cytometry.The results showed that Chemerin increased the apoptotic rate of DCMECs in a time-dependent manner,but there was no difference at 12h,and reached a significant level at 24h(P<0.05)and 48h(P<0.01).At the same time,the key gene of apoptosis,Bax/Bcl-2,showed a significant increase in time gradient at both transcriptional and translational levels(P<0.05).In addition,Caspase-3 was gradually increased at translation level,reaching significant levels at 24 h(P<0.01)and 48 h(P<0.01).These data supported the conclusion that Chemerin can initiate apoptosis cascades to induce apoptosis in DCMECs.In order to further accurately quantify the effect of NF-κB pathway on inflammation and apoptosis stimulated by Chemerin of DCMECs,Chemerin was selected for 24h in subsequent experiments.3.Chemerin mediated NF-κB signaling pathway to regulate inflammation and apoptosis of DCMECsIn this experiment,cells were pretreated with NF-κB inhibitor PDTC for 3 h,followed by treatment with Chemerin for 24 h.The cells were divided into four groups:Control group,Chemerin group,PDTC group and PDTC+Chemerin group.Western blot results showed that PDTC significantly reduced Chemerin-induced phosphorylation of NF-κB protein(P<0.01),indicated that Chemerin-activated NF-κB pathway can be strongly inhibited by PDTC.Based on that,further detection revealed that IL-1β and IL-6 were significantly up-regulated at both the transcriptional and translational levels after Chemerin stimulation(P<0.05),but PDTC pretreatment reversed the effect of Chemerin on inflammatory genes(P<0.05).The data of flow cytometry showed that the apoptosis rate of DCMECs in PDTC+Chemerin group was significantly lower than that in Chemerin group(P<0.05).At the same time,the transcription and translation levels of Bax/Bcl-2 in PDTC+Chemerin group also significantly decreased(P<0.05).In addition,PDTC reversed the promotion effect of Chemerin on Csapase-3 protein in DCMECs(P<0.01).The results reflected that Chemerin can regulate the transcription of related genes by activating the NF-κB pathway to promote the inflammatory response and induce apoptosis in DCMECs.