AIM: Early calcification of atherosclerotic plaques are colocalized with macrophage and high mobility group box 1( HMGB1),a cytokine associated with biomineralizing process under physiological and pathological conditi...AIM: Early calcification of atherosclerotic plaques are colocalized with macrophage and high mobility group box 1( HMGB1),a cytokine associated with biomineralizing process under physiological and pathological conditions. Our study aims to evaluate whether HMGB1 induces ectopic mineralization via promoting the secretion of matrix vesicles( MVs) from macrophages. METHODS: HMGB1 was added to the medium of macrophages,the secretion of MVs in the supernatant was tested by flow cytometry analysis. The mineral deposition in calcifying medium was detected by Alizarin Red staining and von Kossa staining. Transmission electron microscopy showed the formation of hydroxyapatite crystals in MVs. Then we subcutaneous injection into mice with MVs to induce regional mineralization. RESULTS: HMGB1 significantly promoted secretion of MVs from macrophages as raveled by flow cytometry analysis. TNAP activity,considered as a marker of MVs maturation,was higher in HMGB1-induced MVs compared to the control-MVs. HMGB1-MVs also led to mineral deposition in an in vitro MVs-collagen mineralization model. Subcutaneous injection into mice with MVs derived from HMGB1-treated cells showed a greater potential to initiate regional mineralization. Mechanistic experiments revealed that HMGB1 activated neutral sphingomyelinase 2( n SMase2) that involved the receptor for advanced glycation end products( RAGE) and p38MAPK( upstream of n SMase2). Inhibition of n SMase2 with GW4869 or p38 MAPK with SB-239063 prevented MVs secretion and mineral deposition. CONCLUSIONS: HMGB1 induces MVs secretion from macrophages at least in part,via the RAGE / p38 MAPK /n SMase2 signaling pathway. Our findings thus reveal a novel mechanism by which HMGB1 may participated in the early calcification of atherosclerotic plaques.展开更多
本试验旨在探讨黄芪多糖(APS)对地塞米松(DEX)诱导的猪小肠上皮细胞(IPEC-J2)凋亡的保护作用。采用不同浓度的APS(0~0.8 mg/m L)或DEX(0~0.7 mg/m L)处理IPEC-J2细胞24 h,利用细胞计数试剂(CCK-8)检测细胞活性,确定药物作用浓度。对照...本试验旨在探讨黄芪多糖(APS)对地塞米松(DEX)诱导的猪小肠上皮细胞(IPEC-J2)凋亡的保护作用。采用不同浓度的APS(0~0.8 mg/m L)或DEX(0~0.7 mg/m L)处理IPEC-J2细胞24 h,利用细胞计数试剂(CCK-8)检测细胞活性,确定药物作用浓度。对照组无添加,A组添加0.4 mg/m L APS培养,D组添加0.4 mg/m L DEX培养,A+D组添加0.4 mg/m L APS+0.4 mg/m L DEX共培养。采用Calcein/PI细胞活性与细胞毒性检测试剂盒检测细胞的存活情况,流式细胞仪检测IPEC-J2细胞的凋亡情况,透射电子显微镜观察细胞的形态,实时荧光定量PCR检测凋亡基因Caspase3、Caspase9、Bid、Bcl-2 m RNA的相对表达量,Western-blotting检测Caspase3、Caspase9、Bid、Bcl-2蛋白的表达水平。结果表明,APS可以通过调控凋亡蛋白和基因的表达来抑制DEX诱导的IPEC-J2细胞的凋亡,恢复IPEC-J2细胞的活性,起到保护猪小肠上皮细胞作用。展开更多
基金Supported by the grants from the National Natural Science Foundation of China(No.81270362No.81470561)State Project For Essential Drug Research and Development(No.2013ZX09103003-001)
文摘AIM: Early calcification of atherosclerotic plaques are colocalized with macrophage and high mobility group box 1( HMGB1),a cytokine associated with biomineralizing process under physiological and pathological conditions. Our study aims to evaluate whether HMGB1 induces ectopic mineralization via promoting the secretion of matrix vesicles( MVs) from macrophages. METHODS: HMGB1 was added to the medium of macrophages,the secretion of MVs in the supernatant was tested by flow cytometry analysis. The mineral deposition in calcifying medium was detected by Alizarin Red staining and von Kossa staining. Transmission electron microscopy showed the formation of hydroxyapatite crystals in MVs. Then we subcutaneous injection into mice with MVs to induce regional mineralization. RESULTS: HMGB1 significantly promoted secretion of MVs from macrophages as raveled by flow cytometry analysis. TNAP activity,considered as a marker of MVs maturation,was higher in HMGB1-induced MVs compared to the control-MVs. HMGB1-MVs also led to mineral deposition in an in vitro MVs-collagen mineralization model. Subcutaneous injection into mice with MVs derived from HMGB1-treated cells showed a greater potential to initiate regional mineralization. Mechanistic experiments revealed that HMGB1 activated neutral sphingomyelinase 2( n SMase2) that involved the receptor for advanced glycation end products( RAGE) and p38MAPK( upstream of n SMase2). Inhibition of n SMase2 with GW4869 or p38 MAPK with SB-239063 prevented MVs secretion and mineral deposition. CONCLUSIONS: HMGB1 induces MVs secretion from macrophages at least in part,via the RAGE / p38 MAPK /n SMase2 signaling pathway. Our findings thus reveal a novel mechanism by which HMGB1 may participated in the early calcification of atherosclerotic plaques.
文摘本试验旨在探讨黄芪多糖(APS)对地塞米松(DEX)诱导的猪小肠上皮细胞(IPEC-J2)凋亡的保护作用。采用不同浓度的APS(0~0.8 mg/m L)或DEX(0~0.7 mg/m L)处理IPEC-J2细胞24 h,利用细胞计数试剂(CCK-8)检测细胞活性,确定药物作用浓度。对照组无添加,A组添加0.4 mg/m L APS培养,D组添加0.4 mg/m L DEX培养,A+D组添加0.4 mg/m L APS+0.4 mg/m L DEX共培养。采用Calcein/PI细胞活性与细胞毒性检测试剂盒检测细胞的存活情况,流式细胞仪检测IPEC-J2细胞的凋亡情况,透射电子显微镜观察细胞的形态,实时荧光定量PCR检测凋亡基因Caspase3、Caspase9、Bid、Bcl-2 m RNA的相对表达量,Western-blotting检测Caspase3、Caspase9、Bid、Bcl-2蛋白的表达水平。结果表明,APS可以通过调控凋亡蛋白和基因的表达来抑制DEX诱导的IPEC-J2细胞的凋亡,恢复IPEC-J2细胞的活性,起到保护猪小肠上皮细胞作用。
