Objective Arsenic trioxide(ATO)is a clinically effective anticancer agent used in the treatment of leukemia.However,it exerts adverse effects on non-tumor cells,including bone marrow mesenchymal stem cells(BMSCs).This...Objective Arsenic trioxide(ATO)is a clinically effective anticancer agent used in the treatment of leukemia.However,it exerts adverse effects on non-tumor cells,including bone marrow mesenchymal stem cells(BMSCs).This study aims to investigate the protective role and molecular mechanism of traditional Chinese medicine Astragalus polysaccharides(APS)in mitigating ATO-induced apoptosis in BMSCs.Methods BMSCs exposed to ATO(0.5μmol/L)were treated with APS(20,40,100,and 200μg/mL).Cell viability,proliferation,and migration were assessed by using MTT,EdU staining,Transwell,and scratch wound healing assays.Apoptosis was evaluated via TUNEL assay,Hoechst 33258 staining,and flow cytometry.Intracellular reactive oxygen species(ROS)and mitochondrial membrane potential were measured by using DCFH-DA and JC-1 staining.Apoptotic protein expression was analyzed by Western blotting.Results ATO exposure significantly inhibited the proliferation and migration of BMSCs and induced apoptosis,while APS markedly attenuated the apoptosis of BMSCs induced by ATO,and significantly improved cell proliferation and migration(P<0.01).Mechanistically,APS effectively reduced ATO-induced ROS(P<0.01),while the protein expression of Bcl-2-associated X protein(Bax)and cleaved Caspase-3 was significantly decreased(P<0.05),and the protein expression of Bcl-2 was significantly increased(P<0.01).In addition,APS markedly decreased the protein expression of c-Jun N-terminal kinase(Jnk)and p38 in ATO-activated BMSCs(P<0.05),and significantly decreased the protein expression of p16 and p53(P<0.01),and increased the protein expression of phosphorylated protein kinase B(p-Akt)and phosphorylated extracellular signal-regulated kinase(p-Erk)(P<0.01,0.05).Conclusion Our study reveals that APS exert significant protective effects against ATO-induced apoptosis in BMSCs.The mechanisms involve suppressing ROS generation,maintaining mitochondrial membrane stability,enhancing cell viability,migration,and proliferation,as well as inhibiting Jnk and p38 mitogen-activated protein kinase(p38 MAPK)signaling pathways.The findings highlight potential molecular targets and novel strategies for the clinical prevention and treatment of ATO-related toxicity.展开更多
目的探究补肾健脾法对大鼠骨髓间充质干细胞(BMSCs)成骨分化过程中ULK1/FUNDC1介导的线粒体自噬的影响。方法将72只SD大鼠分为六组:正常组、诱导组、补肾组、健脾组、补肾健脾组和补肾健脾+3-MA组。正常组和诱导组给予超纯水,其他组给...目的探究补肾健脾法对大鼠骨髓间充质干细胞(BMSCs)成骨分化过程中ULK1/FUNDC1介导的线粒体自噬的影响。方法将72只SD大鼠分为六组:正常组、诱导组、补肾组、健脾组、补肾健脾组和补肾健脾+3-MA组。正常组和诱导组给予超纯水,其他组给予特定药物,补肾健脾组和补肾健脾+3-MA组给予相同药物。7 d后收集血液制备含药血清。BMSCs复苏后,转移到相应的培养瓶中,按照大鼠的分组进行培养,并使用含药血清的培养基培养15 d。通过CCK8实验检测细胞在24、48、72 h的增殖情况;使用ELISA法测定碱性磷酸酶(ALP)及活性氧(ROS)的表达;通过茜素红染色观察成骨细胞(OB)的矿化结节;免疫荧光染色检测线粒体远红外荧光探针(Mito-Tracker Deep Red FM)与线粒体自噬相关蛋白LC3的共定位情况,并通过Image J软件分析细胞的平均荧光强度;采用蛋白免疫印迹法(Western blot)检测ULK1、p-ULK1(Ser555)、FUNDC1、p-FUNDC1(Ser17)和LC3-Ⅱ/LC3-Ⅰ蛋白的表达。结果①在24、48、72 h,细胞增殖呈上升趋势,补肾健脾组的促进效果最为显著;②ALP表达在不同药物干预下增加、ROS降低,补肾健脾组的效果最为明显;③茜素红染色显示,各组细胞形成不同程度的矿化结节,补肾健脾组的结节面积最大,成骨效果最佳;④线粒体荧光共定位结果表明,药物干预可提高细胞线粒体自噬水平,补肾健脾组的LC3荧光强度最高;⑤线粒体自噬蛋白检测结果显示,与诱导组相比,各治疗组均能提升ULK1、p-ULK1(Ser555)、FUNDC1、p-FUNDC1(Ser17)蛋白的表达,而LC3-Ⅱ/LC3-Ⅰ的比值表明补肾健脾组的线粒体自噬水平最高。结论补肾健脾治法可能通过调节ULK1/FUNDC1介导的线粒体自噬途径,促进BMSCs的成骨分化。展开更多
Ribosomal RNA(rRNA)synthesis is intricately tied to cellular growth and proliferation.Basic fibroblast growth factor(FGF2),a pivotal factor for bone marrow mesenchymal stem cells(BMSCs),can stimulates rRNA transcripti...Ribosomal RNA(rRNA)synthesis is intricately tied to cellular growth and proliferation.Basic fibroblast growth factor(FGF2),a pivotal factor for bone marrow mesenchymal stem cells(BMSCs),can stimulates rRNA transcription,though the underlying mechanism remains unknown.Here,we demonstrate that the cytoplasm-nucleus translocation of FGF2 is determined by the stable nuclear localization motif.Meanwhile,the nuclear FGF2 regulates rRNA expression and BMSCs proliferation via phase separation.Next,through FGF2 related epigenomics and 3D genomes analysis,we identified chromatin architectures during BMSCs differentiation and aging.In the process,topologically associating domains(TADs)and chromatin loops profiling revealed the attenuated genomic interaction among proximal chromosomes 13,14,15,21,and 22,where phase-separated FGF2 facilitates rDNA transcription depend on specific super-enhancers(SEs).Furthermore,we validated that FGF2 orchestrates rDNA chromatin architecture in coordination with STAT5.Together,these findings underscore the pivotal role of FGF2 in rDNA chromatin architectures,which determines BMSCs cell fate.展开更多
目的对比分析单纯股骨头髓芯减压与联合自体血清培养BMSCs移植治疗早期股骨头缺血性坏死(avascular necrosis of the femoral head,ANFH)的临床疗效。方法 2006年5月-2008年10月收治8例16髋双侧早期ANFH患者。男7例,女1例;年龄19～43岁...目的对比分析单纯股骨头髓芯减压与联合自体血清培养BMSCs移植治疗早期股骨头缺血性坏死(avascular necrosis of the femoral head,ANFH)的临床疗效。方法 2006年5月-2008年10月收治8例16髋双侧早期ANFH患者。男7例,女1例;年龄19～43岁,平均35.7岁。其中激素性2例,酒精性3例,无明显诱因3例。病程4个月～2年,平均1.1年。按左右侧随机分两组:A组采用单纯股骨头髓芯减压治疗,B组行股骨头髓芯减压结合自体血清培养的BMSCs移植治疗。按世界骨循环研究学会(ARCO)国际骨坏死分期标准:A组:Ⅱa期4髋,Ⅱb期2髋,Ⅱc期1髋,Ⅲa期1髋;B组:Ⅱa期2髋,Ⅱb期2髋,Ⅱc期3髋,Ⅲa期1髋。采用Harris评分和疼痛视觉模拟(VAS)评分对患者术前及术后进行临床随访;采用X线片及MRI进行影像学评估;检测红细胞沉降率、C反应蛋白、肝肾功、免疫球蛋白等指标,进行安全性评估。结果术后切口均Ⅰ期愈合。8例均获随访,随访时间12～42个月,平均23.5个月。术后3个月疼痛、跛行等临床症状逐渐改善。术前及术后3、6个月两组Harris评分及VAS评分比较差异均无统计学意义(P>0.05),术后12个月B组评分优于A组(P<0.05);术后各时间点组内Harris评分及VAS评分均较术前显著提高(P<0.05)。术后12个月X线片及MRI检查示,A组1例Ⅲa期患者出现股骨头塌陷;B组T1相低信号区所占股骨头体积的百分比为13.25%±2.12%,与A组(18.13%±2.59%)比较,差异有统计学意义(P<0.05);安全性评估显示所有患者术后均未出现发热、局部感染等不良反应。结论自体血清培养BMSCs移植结合髓芯减压与单纯髓芯减压治疗早期ANFH均有效,但前者缓解疼痛及延缓塌陷方面优于后者,安全可靠。展开更多
基金supported by the National Natural Science Foundation of China(No.82473925,81800784,81972117,82472477)the Young Elite Scientists Sponsorship Program by CAST(No.2019QNRC001)+8 种基金the Postdoctoral Science Foundation of China(No.2018M630370)the Key Research and Development Program of Heilongjiang Province(No.GA21C006)the Heilongjiang Postdoctoral Fund for Young Talent Program(No.LBH-TZ1020)the Natural Science Foundation of Heilongjiang Province(No.YQ2020H003)the Research Institution’s Scientific Research Expenses Project of Heilongjiang Province(No.CZKYF2022-1-C006)the Excellent Young Talents Funding(No.2019-QD-11)the CAMS Innovation Fund for Medical Sciences(No.CIFMS 2020-I2M-5-003)the Natural Science Foundation of Jiangsu Province(No.BK20210110)the Program of Science and Technology of Suzhou(No.SYG202119).
文摘Objective Arsenic trioxide(ATO)is a clinically effective anticancer agent used in the treatment of leukemia.However,it exerts adverse effects on non-tumor cells,including bone marrow mesenchymal stem cells(BMSCs).This study aims to investigate the protective role and molecular mechanism of traditional Chinese medicine Astragalus polysaccharides(APS)in mitigating ATO-induced apoptosis in BMSCs.Methods BMSCs exposed to ATO(0.5μmol/L)were treated with APS(20,40,100,and 200μg/mL).Cell viability,proliferation,and migration were assessed by using MTT,EdU staining,Transwell,and scratch wound healing assays.Apoptosis was evaluated via TUNEL assay,Hoechst 33258 staining,and flow cytometry.Intracellular reactive oxygen species(ROS)and mitochondrial membrane potential were measured by using DCFH-DA and JC-1 staining.Apoptotic protein expression was analyzed by Western blotting.Results ATO exposure significantly inhibited the proliferation and migration of BMSCs and induced apoptosis,while APS markedly attenuated the apoptosis of BMSCs induced by ATO,and significantly improved cell proliferation and migration(P<0.01).Mechanistically,APS effectively reduced ATO-induced ROS(P<0.01),while the protein expression of Bcl-2-associated X protein(Bax)and cleaved Caspase-3 was significantly decreased(P<0.05),and the protein expression of Bcl-2 was significantly increased(P<0.01).In addition,APS markedly decreased the protein expression of c-Jun N-terminal kinase(Jnk)and p38 in ATO-activated BMSCs(P<0.05),and significantly decreased the protein expression of p16 and p53(P<0.01),and increased the protein expression of phosphorylated protein kinase B(p-Akt)and phosphorylated extracellular signal-regulated kinase(p-Erk)(P<0.01,0.05).Conclusion Our study reveals that APS exert significant protective effects against ATO-induced apoptosis in BMSCs.The mechanisms involve suppressing ROS generation,maintaining mitochondrial membrane stability,enhancing cell viability,migration,and proliferation,as well as inhibiting Jnk and p38 mitogen-activated protein kinase(p38 MAPK)signaling pathways.The findings highlight potential molecular targets and novel strategies for the clinical prevention and treatment of ATO-related toxicity.
文摘目的探究补肾健脾法对大鼠骨髓间充质干细胞(BMSCs)成骨分化过程中ULK1/FUNDC1介导的线粒体自噬的影响。方法将72只SD大鼠分为六组:正常组、诱导组、补肾组、健脾组、补肾健脾组和补肾健脾+3-MA组。正常组和诱导组给予超纯水,其他组给予特定药物,补肾健脾组和补肾健脾+3-MA组给予相同药物。7 d后收集血液制备含药血清。BMSCs复苏后,转移到相应的培养瓶中,按照大鼠的分组进行培养,并使用含药血清的培养基培养15 d。通过CCK8实验检测细胞在24、48、72 h的增殖情况;使用ELISA法测定碱性磷酸酶(ALP)及活性氧(ROS)的表达;通过茜素红染色观察成骨细胞(OB)的矿化结节;免疫荧光染色检测线粒体远红外荧光探针(Mito-Tracker Deep Red FM)与线粒体自噬相关蛋白LC3的共定位情况,并通过Image J软件分析细胞的平均荧光强度;采用蛋白免疫印迹法(Western blot)检测ULK1、p-ULK1(Ser555)、FUNDC1、p-FUNDC1(Ser17)和LC3-Ⅱ/LC3-Ⅰ蛋白的表达。结果①在24、48、72 h,细胞增殖呈上升趋势,补肾健脾组的促进效果最为显著;②ALP表达在不同药物干预下增加、ROS降低,补肾健脾组的效果最为明显;③茜素红染色显示,各组细胞形成不同程度的矿化结节,补肾健脾组的结节面积最大,成骨效果最佳;④线粒体荧光共定位结果表明,药物干预可提高细胞线粒体自噬水平,补肾健脾组的LC3荧光强度最高;⑤线粒体自噬蛋白检测结果显示,与诱导组相比,各治疗组均能提升ULK1、p-ULK1(Ser555)、FUNDC1、p-FUNDC1(Ser17)蛋白的表达,而LC3-Ⅱ/LC3-Ⅰ的比值表明补肾健脾组的线粒体自噬水平最高。结论补肾健脾治法可能通过调节ULK1/FUNDC1介导的线粒体自噬途径,促进BMSCs的成骨分化。
基金the open research project of State Key Laboratory of Oral Diseases(no.SKLOD2024OF03)the Key Project of Natural Science Research in Anhui Provincial Universities(no.2024AH050683)+1 种基金Anhui Province Outstanding Young Teachers Development Program(no.YQYB2024013)the National Natural Science Foundation of China(no.82201026,82125006).
文摘Ribosomal RNA(rRNA)synthesis is intricately tied to cellular growth and proliferation.Basic fibroblast growth factor(FGF2),a pivotal factor for bone marrow mesenchymal stem cells(BMSCs),can stimulates rRNA transcription,though the underlying mechanism remains unknown.Here,we demonstrate that the cytoplasm-nucleus translocation of FGF2 is determined by the stable nuclear localization motif.Meanwhile,the nuclear FGF2 regulates rRNA expression and BMSCs proliferation via phase separation.Next,through FGF2 related epigenomics and 3D genomes analysis,we identified chromatin architectures during BMSCs differentiation and aging.In the process,topologically associating domains(TADs)and chromatin loops profiling revealed the attenuated genomic interaction among proximal chromosomes 13,14,15,21,and 22,where phase-separated FGF2 facilitates rDNA transcription depend on specific super-enhancers(SEs).Furthermore,we validated that FGF2 orchestrates rDNA chromatin architecture in coordination with STAT5.Together,these findings underscore the pivotal role of FGF2 in rDNA chromatin architectures,which determines BMSCs cell fate.
文摘目的对比分析单纯股骨头髓芯减压与联合自体血清培养BMSCs移植治疗早期股骨头缺血性坏死(avascular necrosis of the femoral head,ANFH)的临床疗效。方法 2006年5月-2008年10月收治8例16髋双侧早期ANFH患者。男7例,女1例;年龄19～43岁,平均35.7岁。其中激素性2例,酒精性3例,无明显诱因3例。病程4个月～2年,平均1.1年。按左右侧随机分两组:A组采用单纯股骨头髓芯减压治疗,B组行股骨头髓芯减压结合自体血清培养的BMSCs移植治疗。按世界骨循环研究学会(ARCO)国际骨坏死分期标准:A组:Ⅱa期4髋,Ⅱb期2髋,Ⅱc期1髋,Ⅲa期1髋;B组:Ⅱa期2髋,Ⅱb期2髋,Ⅱc期3髋,Ⅲa期1髋。采用Harris评分和疼痛视觉模拟(VAS)评分对患者术前及术后进行临床随访;采用X线片及MRI进行影像学评估;检测红细胞沉降率、C反应蛋白、肝肾功、免疫球蛋白等指标,进行安全性评估。结果术后切口均Ⅰ期愈合。8例均获随访,随访时间12～42个月,平均23.5个月。术后3个月疼痛、跛行等临床症状逐渐改善。术前及术后3、6个月两组Harris评分及VAS评分比较差异均无统计学意义(P>0.05),术后12个月B组评分优于A组(P<0.05);术后各时间点组内Harris评分及VAS评分均较术前显著提高(P<0.05)。术后12个月X线片及MRI检查示,A组1例Ⅲa期患者出现股骨头塌陷;B组T1相低信号区所占股骨头体积的百分比为13.25%±2.12%,与A组(18.13%±2.59%)比较,差异有统计学意义(P<0.05);安全性评估显示所有患者术后均未出现发热、局部感染等不良反应。结论自体血清培养BMSCs移植结合髓芯减压与单纯髓芯减压治疗早期ANFH均有效,但前者缓解疼痛及延缓塌陷方面优于后者,安全可靠。
