The objective of this study was to determine the role of SLC15A4 in the muramyl dipeptide(MDP)-mediated inflammatory response of bovine rumen epithelial cells(BRECs).First,changes in the m RNA expression of proinflamm...The objective of this study was to determine the role of SLC15A4 in the muramyl dipeptide(MDP)-mediated inflammatory response of bovine rumen epithelial cells(BRECs).First,changes in the m RNA expression of proinflammatory factor genes in BRECs following 10μg m L^(–1)MDP treatments were examined.RT-q PCR results showed that the expression of pro-inflammatory factor(IL-1β,IL-6,and TNF-α)m RNAs were significantly increased under MDP stimulation(P<0.001).Moreover,SLC15A4-Knockout(SLC15A4-KO)cells were obtained through lentivirus packaging,transfection,screening,and cell monoclonal culture.In order to gain further insight into the potential function of SLC15A4,we utilized transcriptome data,which revealed a change in the genes between WT-BRECs and SLC15A4-KO.Five down-regulated pro-inflammatory genes and 13 down-regulated chemokine genes related to the inflammatory response were identified.Meanwhile,the down-regulated genes were mostly enriched in the nuclear factorκB(NF-κB)and mitogen-activated protein kinase(MAPK)signaling pathways.The results of RT-q PCR also verified these detected changes.To further determine the mechanism of how WT and SLC15A4-KO BRECs are involved in inflammatory responses,we investigated the inflammatory responses of cells exposed to MDP.WT-BRECs and SLC15A4-KO were treated with a culture medium containing 10μg m L^(–1)MDP,in comparison to a control without MDP.Our results show that SLC15A4-KO BRECs had reduced the expression of genes(IL-6,TNF-α,CXCL2,CXCL3,CXCL9,and CCL2)and proteins(p-p65 and p-p44/42)from the MDP-mediated inflammatory response compared to WT-BRECs(P<0.05).In this experiment,CRISPR-Cas9 was used to KO the di/tripeptide transporter SLC15A4,and its role was confirmed via the MDP-induced inflammatory response in BRECs.This work will provide a theoretical basis for studying the pro-inflammatory mechanism of MDP and its application in the prevention and treatment of subacute rumen acidosis in dairy cows.展开更多
This study was carried out to determine the effect of replacement of forage fiber sources from alfalfa and Leymus chinensis with nonforage fiber sources(NFFS) from dried distillers grains with solubles and corn germ m...This study was carried out to determine the effect of replacement of forage fiber sources from alfalfa and Leymus chinensis with nonforage fiber sources(NFFS) from dried distillers grains with solubles and corn germ meal on calves growth, rumen development and blood parameters. 48 female and 12 male calves((110.55±15.36) kg of body weight and 12 wk of age) were assigned randomly to four dietary treatments(15 calves/treatment) in a completely randomized design. Experimental diets were: 0% NFFS(control), 9% NFFS(group 1), 18% NFFS(group 2), 27% NFFS(group 3), and contained equivalent neutral detergent fiber and total digestible nutrients, respectively. The dry matter intake was similar among diets, averaging 3.33 kg d-1, and no differences were detected for body weight, withers height, body length and heart girth. In addition, the development of rumen, reticulum, omasum and abomasum also were similar among diets. Dry matter, crude protein, and neutral detergent fiber digestibilities increased with the increasing levels of NFFS in the experimental diets, but had no significant effect. Blood urea nitrogen, total protein and glubulin were not affected by the dietary treatment, but group 3 resulted in the highest(P<0.05) concentrations of glucose and the lowest(P<0.05) concentrations of triglycerides and albumin. In conclusion, dried distillers grains with solubles(DDGS) and corn germ meal(CGM) were available and alternative fiber sources for Holstein calf diets.展开更多
In this paper, toll-like receptor expression pattern in monocytes-derived macrophages by lipopolysaccharid (LPS) stimulation was examined. Jugular venous blood samples from 4 Japanese calves were obtained and the peri...In this paper, toll-like receptor expression pattern in monocytes-derived macrophages by lipopolysaccharid (LPS) stimulation was examined. Jugular venous blood samples from 4 Japanese calves were obtained and the peripheral blood mononuclear cells (PBMC) were isolated. The PBMC were cultured for 7 d so as to collect monocytes-derived macrophages in Repcell. The PBMC were stimulated by LPS for 24 h and the mRNA expression pattern of TLR and cytokines in monocytes-derived macrophages (Mod-Mφ) was analyzed. Results showed that LPS stimulation of Mod-Mφ could increase the mRNA levels of the genes of TNF-α, IL-6, and IL-8. In addition, the mRNA levels of the genes of TNF-α and IL-6 in the group of LPS stimulation were most significantly (P < 0.01) higher than those in control group and the mRNA levels of TLR1, 3, 5, 8, and 10 were significantly (P < 0.05) decreased after LPS stimulation. There was no difference in the mRNA expressions of TLR2, 4, 6, and 7 between the groups of the control and LPS stimulation. Besides, expression of TLR9 was not found. It suggested that monocytes-derived macrophages could respond to LPS and they might take an important role in the innate immunity. The important function of the cells might contribute to better disease treatment.展开更多
基金the National Natural Science Foundation of China(31972589)the earmarked fund for China Agriculture Research System(CARS-36)the Postgraduate Research&Practice Innovation Program of Jiangsu Province,China(KYCX21-3283)。
文摘The objective of this study was to determine the role of SLC15A4 in the muramyl dipeptide(MDP)-mediated inflammatory response of bovine rumen epithelial cells(BRECs).First,changes in the m RNA expression of proinflammatory factor genes in BRECs following 10μg m L^(–1)MDP treatments were examined.RT-q PCR results showed that the expression of pro-inflammatory factor(IL-1β,IL-6,and TNF-α)m RNAs were significantly increased under MDP stimulation(P<0.001).Moreover,SLC15A4-Knockout(SLC15A4-KO)cells were obtained through lentivirus packaging,transfection,screening,and cell monoclonal culture.In order to gain further insight into the potential function of SLC15A4,we utilized transcriptome data,which revealed a change in the genes between WT-BRECs and SLC15A4-KO.Five down-regulated pro-inflammatory genes and 13 down-regulated chemokine genes related to the inflammatory response were identified.Meanwhile,the down-regulated genes were mostly enriched in the nuclear factorκB(NF-κB)and mitogen-activated protein kinase(MAPK)signaling pathways.The results of RT-q PCR also verified these detected changes.To further determine the mechanism of how WT and SLC15A4-KO BRECs are involved in inflammatory responses,we investigated the inflammatory responses of cells exposed to MDP.WT-BRECs and SLC15A4-KO were treated with a culture medium containing 10μg m L^(–1)MDP,in comparison to a control without MDP.Our results show that SLC15A4-KO BRECs had reduced the expression of genes(IL-6,TNF-α,CXCL2,CXCL3,CXCL9,and CCL2)and proteins(p-p65 and p-p44/42)from the MDP-mediated inflammatory response compared to WT-BRECs(P<0.05).In this experiment,CRISPR-Cas9 was used to KO the di/tripeptide transporter SLC15A4,and its role was confirmed via the MDP-induced inflammatory response in BRECs.This work will provide a theoretical basis for studying the pro-inflammatory mechanism of MDP and its application in the prevention and treatment of subacute rumen acidosis in dairy cows.
基金supported by the National Key Technologies during the 12th Five-Year Plan period (2011BAD17B03)
文摘This study was carried out to determine the effect of replacement of forage fiber sources from alfalfa and Leymus chinensis with nonforage fiber sources(NFFS) from dried distillers grains with solubles and corn germ meal on calves growth, rumen development and blood parameters. 48 female and 12 male calves((110.55±15.36) kg of body weight and 12 wk of age) were assigned randomly to four dietary treatments(15 calves/treatment) in a completely randomized design. Experimental diets were: 0% NFFS(control), 9% NFFS(group 1), 18% NFFS(group 2), 27% NFFS(group 3), and contained equivalent neutral detergent fiber and total digestible nutrients, respectively. The dry matter intake was similar among diets, averaging 3.33 kg d-1, and no differences were detected for body weight, withers height, body length and heart girth. In addition, the development of rumen, reticulum, omasum and abomasum also were similar among diets. Dry matter, crude protein, and neutral detergent fiber digestibilities increased with the increasing levels of NFFS in the experimental diets, but had no significant effect. Blood urea nitrogen, total protein and glubulin were not affected by the dietary treatment, but group 3 resulted in the highest(P<0.05) concentrations of glucose and the lowest(P<0.05) concentrations of triglycerides and albumin. In conclusion, dried distillers grains with solubles(DDGS) and corn germ meal(CGM) were available and alternative fiber sources for Holstein calf diets.
基金supported by the National Natural Science Foundation of China(30871800, 30471259)the National Key Technology R&D Program of China (2006BAD04A03-07)
文摘In this paper, toll-like receptor expression pattern in monocytes-derived macrophages by lipopolysaccharid (LPS) stimulation was examined. Jugular venous blood samples from 4 Japanese calves were obtained and the peripheral blood mononuclear cells (PBMC) were isolated. The PBMC were cultured for 7 d so as to collect monocytes-derived macrophages in Repcell. The PBMC were stimulated by LPS for 24 h and the mRNA expression pattern of TLR and cytokines in monocytes-derived macrophages (Mod-Mφ) was analyzed. Results showed that LPS stimulation of Mod-Mφ could increase the mRNA levels of the genes of TNF-α, IL-6, and IL-8. In addition, the mRNA levels of the genes of TNF-α and IL-6 in the group of LPS stimulation were most significantly (P < 0.01) higher than those in control group and the mRNA levels of TLR1, 3, 5, 8, and 10 were significantly (P < 0.05) decreased after LPS stimulation. There was no difference in the mRNA expressions of TLR2, 4, 6, and 7 between the groups of the control and LPS stimulation. Besides, expression of TLR9 was not found. It suggested that monocytes-derived macrophages could respond to LPS and they might take an important role in the innate immunity. The important function of the cells might contribute to better disease treatment.
