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
Adiponectin receptor 1(Adipor1)deficiency has been shown to inhibit Th17 cell differentiation and reduce joint inflammation and bone erosion in antigen-induced arthritis mice.Additional emerging evidence indicates tha...Adiponectin receptor 1(Adipor1)deficiency has been shown to inhibit Th17 cell differentiation and reduce joint inflammation and bone erosion in antigen-induced arthritis mice.Additional emerging evidence indicates that Th17 cells may differentiate into pathogenic(pTh17)and non-pathogenic(npTh17)cells,with the pTh17 cells playing a crucial role in numerous autoimmune and inflammatory conditions.In the current study,we found that Adipor1 deficiency inhibited pTh17 differentiation in vitro and induced mitochondrial dysfunction in pTh17 cells.RNA sequencing demonstrated a significant increase in the expression levels of Fundc1,a gene related to mitochondrial function,in Adipor1-deficient CD4^(+)T cells.Fundc1 knockdown in Adipor1-deficient CD4^(+)T cells partially reversed the effects of Adipor1 deficiency on mitochondrial function and pTh17 differentiation.In conclusion,the current study demonstrated a novel role of Adipor1 in regulating mitochondrial function via Fundc1 to promote pTh17 cell differentiation,providing some insight into potential therapeutic targets for autoimmune and inflammatory diseases.展开更多
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 National Natural Science Foundation of China(Grant No.82071827)the Jiangsu Province Natural Science Foundation(Grant No.BK20210963).
文摘Adiponectin receptor 1(Adipor1)deficiency has been shown to inhibit Th17 cell differentiation and reduce joint inflammation and bone erosion in antigen-induced arthritis mice.Additional emerging evidence indicates that Th17 cells may differentiate into pathogenic(pTh17)and non-pathogenic(npTh17)cells,with the pTh17 cells playing a crucial role in numerous autoimmune and inflammatory conditions.In the current study,we found that Adipor1 deficiency inhibited pTh17 differentiation in vitro and induced mitochondrial dysfunction in pTh17 cells.RNA sequencing demonstrated a significant increase in the expression levels of Fundc1,a gene related to mitochondrial function,in Adipor1-deficient CD4^(+)T cells.Fundc1 knockdown in Adipor1-deficient CD4^(+)T cells partially reversed the effects of Adipor1 deficiency on mitochondrial function and pTh17 differentiation.In conclusion,the current study demonstrated a novel role of Adipor1 in regulating mitochondrial function via Fundc1 to promote pTh17 cell differentiation,providing some insight into potential therapeutic targets for autoimmune and inflammatory diseases.
