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
目的:探讨慢性间歇低氧(chronic intermittent hypoxia,CIH)对H9C2大鼠心肌细胞的影响,以及大麻素1型受体(cannabinoid type 1 receptor,CB1R)/AMP活化蛋白激酶(AMP-activated protein kinase,AMPK)/过氧化物酶体增殖物激活受体γ辅激...目的:探讨慢性间歇低氧(chronic intermittent hypoxia,CIH)对H9C2大鼠心肌细胞的影响,以及大麻素1型受体(cannabinoid type 1 receptor,CB1R)/AMP活化蛋白激酶(AMP-activated protein kinase,AMPK)/过氧化物酶体增殖物激活受体γ辅激活因子1α(peroxisome proliferator-activated receptor-γcoactivator-1α,PGC-1α)信号通路在其中发挥的作用。方法:将H9C2大鼠心肌细胞分为正常对照(normal control,NC)组、6 h CIH组、18 h CIH组和18 h CIH+AM251(CB1R拮抗剂)组,间歇低氧仓中制备CIH细胞模型。流式细胞术检测细胞凋亡及活性氧水平,JC-1检测线粒体膜电位,MitoTracker染色后激光共聚焦显微镜观察线粒体的形态。Western blot和细胞免疫荧光分析CB1R、AMPK、磷酸化AMPK(phosphorylated AMPK,p-AMPK)和PGC-1α的表达情况。结果:与NC组相比,6 h CIH组活性氧轻度增高,线粒体膜电位显著升高,线粒体明显增多,CB1R、AMPK、p-AMPK和PGC-1α蛋白水平显著升高(P<0.05或P<0.01);18h CIH组细胞凋亡增加,活性氧显著增高,线粒体膜电位显著降低,线粒体碎片化显著增多,CB1R表达显著增多,AMPK、p-AMPK和PGC-1α蛋白水平显著降低(P<0.05或P<0.01);与18 h CIH组相比,18 h CIH+AM251组细胞凋亡和活性氧减少,线粒体膜电位显著升高,线粒体碎片化显著减少,CB1R表达显著降低,AMPK、p-AMPK和PGC-1α蛋白水平显著升高(P<0.05或P<0.01)。结论:AMPK/PGC-1α信号通路参与了CIH对H9C2大鼠心肌细胞的影响,CB1R可作为CIH介导的H9C2大鼠心肌细胞损伤的治疗靶点。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.
文摘目的:探讨慢性间歇低氧(chronic intermittent hypoxia,CIH)对H9C2大鼠心肌细胞的影响,以及大麻素1型受体(cannabinoid type 1 receptor,CB1R)/AMP活化蛋白激酶(AMP-activated protein kinase,AMPK)/过氧化物酶体增殖物激活受体γ辅激活因子1α(peroxisome proliferator-activated receptor-γcoactivator-1α,PGC-1α)信号通路在其中发挥的作用。方法:将H9C2大鼠心肌细胞分为正常对照(normal control,NC)组、6 h CIH组、18 h CIH组和18 h CIH+AM251(CB1R拮抗剂)组,间歇低氧仓中制备CIH细胞模型。流式细胞术检测细胞凋亡及活性氧水平,JC-1检测线粒体膜电位,MitoTracker染色后激光共聚焦显微镜观察线粒体的形态。Western blot和细胞免疫荧光分析CB1R、AMPK、磷酸化AMPK(phosphorylated AMPK,p-AMPK)和PGC-1α的表达情况。结果:与NC组相比,6 h CIH组活性氧轻度增高,线粒体膜电位显著升高,线粒体明显增多,CB1R、AMPK、p-AMPK和PGC-1α蛋白水平显著升高(P<0.05或P<0.01);18h CIH组细胞凋亡增加,活性氧显著增高,线粒体膜电位显著降低,线粒体碎片化显著增多,CB1R表达显著增多,AMPK、p-AMPK和PGC-1α蛋白水平显著降低(P<0.05或P<0.01);与18 h CIH组相比,18 h CIH+AM251组细胞凋亡和活性氧减少,线粒体膜电位显著升高,线粒体碎片化显著减少,CB1R表达显著降低,AMPK、p-AMPK和PGC-1α蛋白水平显著升高(P<0.05或P<0.01)。结论:AMPK/PGC-1α信号通路参与了CIH对H9C2大鼠心肌细胞的影响,CB1R可作为CIH介导的H9C2大鼠心肌细胞损伤的治疗靶点。
