[1]王晓萍,周明旺,魏长浩,等.基于竞争性内源性RNA调控轴探讨通痹生骨方对骨髓间充质干细胞成脂分化的影响及其作用机制[J].中医正骨,2026,38(07):1-10,33.
　WANG Xiaoping,ZHOU Mingwang,WEI Changhao,et al.Investigating the effects and mechanisms of Tongbi Shenggu Fang(通痹生骨方)on the adipogenic differentiation of bone marrow mesenchymal stem cells based on the competing endogenous RNA regulatory axis[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2026,38(07):1-10,33.
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基于竞争性内源性RNA调控轴探讨通痹生骨方对骨髓间充质干细胞成脂分化的影响及其作用机制()

《中医正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第38卷
期数:
2026年07期
页码:
1-10,33
栏目:
基础研究
出版日期:
2026-07-20

文章信息/Info

Title:
Investigating the effects and mechanisms of Tongbi Shenggu Fang(通痹生骨方)on the adipogenic differentiation of bone marrow mesenchymal stem cells based on the competing endogenous RNA regulatory axis
作者:
王晓萍1; 周明旺1; 魏长浩1; 李鑫浩2; 张安乐1
1.甘肃省中医院,甘肃 兰州 730050; 2.甘肃中医药大学,甘肃 兰州 730000
Author(s):
WANG Xiaoping1; ZHOU Mingwang1; WEI Changhao1; LI Xinhao2; ZHANG Anle1
1.Gansu Provincial Hospital of TCM,Lanzhou 730050,Gansu,China; 2.Gansu University of Chinese Medicine,Lanzhou 730000,Gansu,China
关键词:
RNA; 竞争性内源性;  骨髓;  间质干细胞;  脂肪形成;  通痹生骨方;  股骨头坏死;  糖皮质激素
Keywords:
RNA; competitive endogenous;  bone marrow;  mesenchymal stem cells;  adipogenesis;  Tongbi Shenggu Fang;  femur head necrosis;  glucocorticoids
摘要:
目的:基于竞争性内源性RNA(competing endogenous RNA,ceRNA)调控轴探讨通痹生骨方对骨髓间充质干细胞(bone marrow mesenchymal stem cell,BMMSC)成脂分化的影响及其作用机制。方法:①通痹生骨方最佳干预浓度筛选。分别以含有0、50、150、300、500 μg·mL-1通痹生骨方冻干粉的Opti-MEM培养基培养空白组及50、150、300、500 μg·mL-1组人骨髓间充质干细胞(human bone marrow mesenchymal stem cell,hBMMSC),采用CCK-8法检测细胞活力,确定最佳干预浓度。②hBMMSC成脂分化及ceRNA调控轴分子表达检测。选用第3代hBMMSC,分为6组。空白对照组以hBMMSC专用培养基培养; 成脂诱导组以hBMMSC成脂诱导分化培养基培养; 成脂诱导联合通痹生骨方组采用含通痹生骨方冻干粉的hBMMSC成脂诱导分化培养基培养; hsa_circ_0028442敲低组转染hsa_circ_0028442干扰小RNA后48 h,换用hBMMSC专用培养基培养; hsa_circ_0028442过表达组转染hsa_circ_0028442过表达质粒后48 h,换用hBMMSC专用培养基培养; hsa_circ_0028442过表达联合通痹生骨方组转染hsa_circ_0028442过表达质粒后48 h,换用含通痹生骨方冻干粉的hBMMSC专用培养基培养。干预14 d后,通过油红O染色观察hBMMSC成脂分化情况; 采用实时定量PCR和Western Blot技术检测成脂分化相关基因固醇调节元件结合蛋白1(sterol regulatory element-binding protein 1,SREBP1)、脂肪酸结合蛋白4(fatty acid-binding protein 4,FABP4)、过氧化物酶体增殖物激活受体γ(peroxisome proliferator-activated receptor γ,PPARγ)的mRNA和蛋白表达水平,以及ceRNA调控轴分子hsa_circ_0028442、hsa-miR-942-5p和上皮间质相互作用1(epithelial stem interaction 1,EPSTI1)mRNA的表达水平。③hsa_circ_0028442成环特性验证。提取上述空白对照组的总RNA,等分为2份。一份加入核糖核酸酶R及其反应缓冲液(核糖核酸酶R组),另一份加入等量核糖核酸酶R反应缓冲液(模拟处理组)。采用实时定量PCR技术检测hsa_circ_0028442及EPSTI1 mRNA的表达水平。④hsa-miR-942-5p与hsa_circ_0028442、EPSTI1预测靶位点结合验证。采用双萤光素酶实验验证hsa-miR-942-5p与hsa_circ_0028442、EPSTI1之间的靶向结合关系。hsa-miR-942-5p模拟物组的野生型亚组和突变型亚组分别以hsa_circ_0028442、EPSTI1的野生型和突变型双萤光素酶报告基因质粒及hsa-miR-942-5p模拟物共转染hBMMSC; 阴性对照组的野生型亚组和突变型亚组分别以hsa_circ_0028442、EPSTI1的野生型和突变型双萤光素酶报告基因质粒及hsa-miR-942-5p模拟物阴性对照物共转染hBMMSC。结果:①通痹生骨方最佳干预浓度筛选结果。空白组及150、300 μg·mL-1组细胞活力组间两两比较,差异均无统计学意义。50 μg·mL-1组的细胞活力高于空白组(P=0.008),与150、300 μg·mL-1组细胞活力的组间差异均无统计学意义。500 μg·mL-1组细胞活力低于空白组及50、150、300 μg·mL-1组(P=0.019,P=0.000,P=0.001,P=0.006)。最终选取对细胞活力无明显影响的较小浓度150 μg·mL-1作为后期实验干预浓度。②hBMMSC成脂分化观察结果。空白对照组、hsa_circ_0028442敲低组油红O染色均呈阴性,成脂诱导组、成脂诱导联合通痹生骨方组、hsa_circ_0028442过表达组、hsa_circ_0028442过表达联合通痹生骨方组油红O染色均呈阳性。与成脂诱导组相比,成脂诱导联合通痹生骨方组脂滴数量明显减少。与hsa_circ_0028442过表达组相比,hsa_circ_0028442过表达联合通痹生骨方组脂滴数量明显减少。③ceRNA调控轴分子的表达水平及成脂分化相关基因的mRNA表达水平检测结果。成脂诱导组、hsa_circ_0028442过表达组的hsa_circ_0028442、EPSTI1 mRNA、SREBP1 mRNA、FABP4 mRNA、PPARγ mRNA的表达水平均高于空白对照组(P=0.000,P=0.000; P=0.000,P=0.000; P=0.000,P=0.002; P=0.000,P=0.000; P=0.000,P=0.000),hsa-miR-942-5p表达水平均低于空白对照组(P=0.000,P=0.000); hsa_circ_0028442敲低组的hsa_circ_0028442、EPSTI1 mRNA、SREBP1 mRNA、FABP4 mRNA、PPARγ mRNA的表达水平均低于空白对照组(P=0.000,P=0.001,P=0.000,P=0.001,P=0.000),hsa-miR-942-5p表达水平高于空白对照组(P=0.000)。成脂诱导联合通痹生骨方组的hsa_circ_0028442、EPSTI1 mRNA、SREBP1 mRNA、FABP4 mRNA、PPARγ mRNA的表达水平均低于成脂诱导组(P=0.000,P=0.000,P=0.000,P=0.000,P=0.000),hsa-miR-942-5p表达水平高于成脂诱导组(P=0.000)。hsa_circ_0028442过表达联合通痹生骨方组的hsa_circ_0028442、EPSTI1 mRNA、SREBP1 mRNA、FABP4 mRNA、PPARγ mRNA的表达水平均低于hsa_circ_0028442过表达组(P=0.000,P=0.000,P=0.000,P=0.000,P=0.000),hsa-miR-942-5p表达水平高于hsa_circ_0028442过表达组(P=0.000)。④成脂分化相关基因的蛋白表达水平检测结果。成脂诱导组、hsa_circ_0028442过表达组的SREBP1、FABP4、PPARγ蛋白表达水平均高于空白对照组(P=0.000,P=0.000; P=0.004,P=0.003; P=0.004,P=0.004); hsa_circ_0028442敲低组的SREBP1、FABP4、PPARγ蛋白表达水平均低于空白对照组(P=0.000,P=0.034,P=0.002)。成脂诱导联合通痹生骨方组的SREBP1、FABP4、PPARγ蛋白表达水平均低于成脂诱导组(P=0.000,P=0.004,P=0.001)。hsa_circ_0028442过表达联合通痹生骨方组的SREBP1、FABP4、PPARγ蛋白表达水平均低于hsa_circ_0028442过表达组(P=0.000,P=0.004,P=0.000)。⑤hsa_circ_0028442成环特性验证结果。2组hsa_circ_0028442表达水平的差异无统计学意义。核糖核酸酶R组EPSTI1 mRNA的表达水平低于模拟处理组(P=0.000)。⑥hsa-miR-942-5p与hsa_circ_0028442、EPSTI1预测靶位点结合验证结果。hsa-miR-942-5p模拟物组的野生型hsa_circ_0028442亚组及野生型EPSTI1亚组的相对萤光素酶活性均低于阴性对照组(P=0.005,P=0.005); hsa-miR-942-5p模拟物组的突变型hsa_circ_0028442亚组及突变型EPSTI1亚组的相对萤光素酶活性与阴性对照组相比,组间差异无统计学意义。结论:通痹生骨方可抑制BMMSC成脂分化及成脂分化相关基因表达,其作用机制可能涉及一种ceRNA调控轴,具体机制可能为下调hsa_circ_0028442表达,减弱其对hsa-miR-942-5p的竞争性吸附,进而增强hsa-miR-942-5p对靶基因EPSTI1的抑制作用,实现对成脂分化的负向调控。
Abstract:
Objective:To investigate the effects of Tongbi Shenggu Fang(通痹生骨方,TSF)on the adipogenic differentiation of bone marrow mesenchymal stem cell(BMMSC),and to elucidate its underlying molecular mechanism based on the competing endogenous RNA(ceRNA)regulatory axis.Methods:①Screening for the optimal working concentration of TSF.The passage 3(P3)human bone marrow mesenchymal stem cells(hBMMSCs)were selected and divided into the blank group and the 50,150,300,and 500 μg/mL groups.The hBMMSCs in the blank group were cultured in Opti-MEM medium,while the ones in the 50,150,300,and 500 μg/mL groups were cultured in Opti-MEM medium containing 50,150,300,and 500 μg/mL TSF lyophilized powder,respectively for 24 hours.After the end of the culture,the hBMMSCs viability was assessed using the CCK-8 assay to identify the optimal working concentration.②Detecting the adipogenic differentiation of hBMMSCs and the expression of molecules in the ceRNA regulatory axis.The P3 hBMMSCs were selected and divided into blank control group,adipogenic induction group,adipogenic induction combined with TSF group,hsa_circ_0028442 knockdown group,hsa_circ_0028442 overexpression group,and hsa_circ_0028442 overexpression combined with TSF group.The P3 hBMMSCs in the blank control group was cultured with hBMMSC-specific medium; the ones in adipogenic induction group with hBMMSC adipogenic induction differentiation medium; the ones in adipogenic induction combined with TSF group with hBMMSC adipogenic induction differentiation medium con-taining TSF lyophilized powder; while the ones in the hsa_circ_0028442 knockdown group was transfected with small interfering RNA(siRNA)against hsa_circ_0028442 for 48 hours,then switched to hBMMSC-specific medium; the ones in hsa_circ_0028442 overexpression group was transfected with hsa_circ_0028442 overexpression plasmid for 48 hours,then cultured with hBMMSC-specific medium; the ones in hsa_circ_0028442 overexpression combined with TSF group was transfected with hsa_circ_0028442 overexpression plasmid for 48 hours,then switched to hBMMSC-specific medium containing TSF lyophilized powder.Following 14-day intervention,the adipogenic differentiation of hBMMSCs was evaluated by Oil Red O staining,and the mRNA and protein expression levels of adipogenic differentiation-related genes,including sterol regulatory element-binding protein 1(SREBP1),fatty acid-binding protein 4(FABP4),and peroxisome proliferator-activated receptor γ(PPARγ),as well as the expression levels of molecules in the ceRNA regulatory axis,including hsa_circ_0028442,hsa-miR-942-5p,and epithelial-stromal interaction 1(EPSTI1)mRNA,were detected by real-time quantitative PCR(RT-qPCR)and Western Blot,respectively.③Verification of the circular characteristics of hsa_circ_0028442.The total RNA was extracted from the hBMMSCs in the blank control group and divided into two aliquots.One was treated with ribonuclease(RNase)R and its reaction buffer(RNase R group),and the other with an equal volume of RNase R reaction buffer only(mock treatment group).The expression levels of hsa_circ_0028442 and EPSTI1 mRNA were detected by RT-qPCR.④Verification of the binding of hsa-miR-942-5p to the predicted target sites of hsa_circ_0028442 and EPSTI1.The targeted binding relationships between hsa-miR-942-5p and hsa_circ_0028442 or EPSTI1 were verified by a dual-luciferase reporter assay.In the hsa-miR-942-5p mimic group,the wild-type(WT)and mutant(MUT)subgroups were co-transfected with the WT and MUT dual-luciferase reporter gene plasmids of hsa_circ_0028442 and EPSTI1,respectively,together with the hsa-miR-942-5p mimics,into hBMMSCs.Similarly,in the negative control(NC)group,the WT and MUT subgroups were co-transfected with the corresponding WT and MUT dual-luciferase reporter gene plasmids and the hsa-miR-942-5p NC mimics.Results:①The optimal working concentration of TSF.Pairwise comparisons of cell viability among the blank group and the 150 and 300 μg/mL groups showed no statistically significant differences.The 50 μg/mL group exhibited higher viability than the blank group(P=0.008),but no significant differences compared to the 150 and 300 μg/mL groups.Moreover,the 500 μg/mL group showed lower viability than the blank group and the 50,150,300 μg/mL groups(P=0.019,P=0.000,P=0.001,P=0.006).Ultimately,150 μg/mL,a concentration without notable cytotoxicity,was selected as the working concentration for subsequent experiments.②The adipogenic differentiation of hBMMSCs.Oil Red O staining was negative in the blank control group and hsa_circ_0028442 knockdown group,but positive in the adipogenic induction group,adipogenic induction combined with TSF group,hsa_circ_0028442 overexpression group,and hsa_circ_0028442 overexpression combined with TSF group.Notably,the lipid droplets were markedly reduced in the adipogenic induction combined with TSF group compared to the adipogenic induction group,as well as in the hsa_circ_0028442 overexpression combined with TSF group compared to the hsa_circ_0028442 overexpression group.③The expression levels of molecules in the ceRNA regulatory axis and the mRNA expression levels of adipogenic differentiation-related genes.Compared with the blank control group,the adipogenic induction group and hsa_circ_0028442 overexpression group exhibited significantly upregulated expression levels of hsa_circ_0028442,EPSTI1 mRNA,SREBP1 mRNA,FABP4 mRNA,and PPARγ mRNA(P=0.000,P=0.000; P=0.000,P=0.000; P=0.000,P=0.002; P=0.000,P=0.000; P=0.000,P=0.000),but downregulated hsa-miR-942-5p level(P=0.000,P=0.000); conversely,the hsa_circ_0028442 knockdown group showed significantly downregulated expression levels of hsa_circ_0028442,EPSTI1 mRNA,SREBP1 mRNA,FABP4 mRNA,and PPARγ mRNA(P=0.000,P=0.001,P=0.000,P=0.001,P=0.000),but upregulated hsa-miR-942-5p level(P=0.000).Furthermore,compared with the adipogenic induction group,the adipogenic induction combined with TSF group exhibited significantly decreased expression levels of hsa_circ_0028442,EPSTI1 mRNA,SREBP1 mRNA,FABP4 mRNA,and PPARγ mRNA(P=0.000,P=0.000,P=0.000,P=0.000,P=0.000),but increased hsa-miR-942-5p level(P=0.000).Similarly,compared with the hsa_circ_0028442 overexpression group,the hsa_circ_0028442 overexpression combined with TSF group also showed decreased hsa_circ_0028442,EPSTI1 mRNA,SREBP1 mRNA,FABP4 mRNA,and PPARγ mRNA levels(P=0.000,P=0.000,P=0.000,P=0.000,P=0.000),but increased hsa-miR-942-5p level(P=0.000).④Protein expression level of adipogenic differentiation-related genes.Compared with the blank control group,the protein expression levels of SREBP1,FABP4,and PPARγ were higher in the adipogenic induction group and hsa_circ_0028442 overexpression group(P=0.000,P=0.000; P=0.004,P=0.003; P=0.004,P=0.004),while lower in the hsa_circ_0028442 knockdown group(P=0.000,P=0.034,P=0.002).Furthermore,the protein expression levels of all these three markers were lower in the adipogenic induction combined with TSF group compared to the adipogenic induction group(P=0.000,P=0.004,P=0.001),and also lower in the hsa_circ_0028442 overexpression combined with TSF group compared to the hsa_circ_0028442 overexpression group(P=0.000,P=0.004,P=0.000).⑤The circular characteristics of hsa_circ_0028442.No significant difference in the expression level of hsa_circ_0028442 was observed between the RNase R group and the mock treatment group,whereas the mRNA expression level of EPSTI1 was significantly lower in the RNase R group compared to the mock treatment group(P=0.000).⑥The binding of hsa-miR-942-5p to the predicted target sites of hsa_circ_0028442 and EPSTI1.In the hsa-miR-942-5p mimic group,the relative luciferase activities of both WT hsa_circ_0028442 subgroup and WT EPSTI1 subgroup were lower than those in the NC group(P=0.005,P=0.005); while,the relative luciferase activities in the MUT hsa_circ_0028442 subgroup and MUT EPSTI1 subgroup showed no statistically significant differences compared to the NC group.Conclusion:TSF can inhibit the adipogenic differentiation of BMMSCs and suppress the expression of adipogenic differentiation-related genes.It may exert the effect via a ceRNA regulatory axis.Specifically,it may downregulate the expression of hsa_circ_0028442,thereby attenuating its competitive sponging effect on hsa-miR-942-5p,and subsequently enhance the inhibitory effect of hsa-miR-942-5p on its target gene EPSTI1,thereby achieving a negative regulation on adipogenic differentiation.

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备注/Memo

备注/Memo:
基金项目:国家自然科学基金项目(82460944,82360942)
通信作者:周明旺 E-mail:zmw2026@126.com
(收稿日期:2026-04-15 本文编辑:李晓乐)
更新日期/Last Update: 2026-07-20