摘要
目的探讨熟地黄多糖(RGP)调控长链非编码RNA(lncRNA)母系表达基因3(MEG3)对碘乙酸钠(MIA)诱导的骨关节炎(OA)软骨细胞凋亡的影响。方法培养大鼠软骨细胞,不同浓度MIA(0、1、2、4、8μmol/L)诱导建立软骨细胞损伤模型,并给予不同浓度RGP(50、100、200、400、800 mg/mL)处理筛选合适的实验浓度。实验分为正常组、MIA组、RGP组、RGP+si-NC组、RGP+si-MEG3组,Cell Counting Kit-8(CCK-8)检测细胞活力;Hoechst 33258染色及流式细胞术检测细胞凋亡情况;实时荧光定量PCR(RT-qPCR)检测细胞中MEG3、金属蛋白酶13(MMP-13)、Ⅱ型胶原α1(COL2A1)和蛋白聚糖(ACAN)mRNA水平;蛋白免疫印迹(Western blot)检测细胞中磷脂酰肌醇3激酶(PI3K)、p-PI3K、丝/苏氨酸激酶(AKT)、p-AKT、BAX、BCL-2、caspase3蛋白表达水平。结果MIA以剂量依赖性方式降低软骨细胞活力,诱导细胞凋亡,并降低MEG3和COL2A1 mRNA、ACAN mRNA水平,升高MMP-13 mRNA水平(P<0.05)。100、200、400、800 mg/mL RGP可升高软骨细胞活力及MEG3水平(P<0.05)。与正常组相比,MIA组细胞活力、MEG3、COL2A1 mRNA、ACAN mRNA和p-PI3K/PI3K、p-AKT/AKT、BCL-2蛋白水平降低,细胞凋亡率、MMP-13 mRNA和BAX、caspase3蛋白水平升高(P<0.05);与MIA组相比,RGP组细胞活力、MEG3、COL2A1 mRNA、ACAN mRNA和p-PI3K/PI3K、p-AKT/AKT、BCL-2蛋白水平升高,细胞凋亡率、MMP-13 mRNA和BAX、caspase3蛋白水平降低(P<0.05);敲低MEG3可减弱RGP对MIA诱导的软骨细胞损伤的保护作用。结论RGP可促进软骨细胞ECM合成,并抑制细胞凋亡,减轻MIA诱导的软骨细胞损伤,其作用机制可能与上调MEG3表达,诱导PI3K/AKT通路活化有关。
Objective To explore the effect of Rehmannia glutinosa polysaccharide(RGP)on apoptosis of osteoarthritis(OA)chondrocytes induced by sodium iodoacetate(MIA)via the regulation of long-chain non-coding RNA(lncRNA)maternal expression gene 3(MEG3).Methods Rat chondrocytes were cultured with MIA 0,1,2,4,and 8μmol/L to induce chondrocyte injury,and were then treated with RGP 50,100,200,400,and 800 mg/mL,respectively,to detect the appropriate experimental concentration.Rats were divided into a normal group,MIA group,RGP group,RGP+control(NC)small interfering RNA(siRNA)group,and RGP+si-MEG3 group.Cell viability was detected by Cell Counting Kit-8(CCK-8)assay and apoptosis was detected by Hoechst 33258 staining and flow cytometry.mRNA levels of MEG3,metalloproteinase 13(MMP-13),typeⅡcollagen-α1(COL2A1),and proteoglycan(ACAN)were detected by real-time quantitative polymerase chain reaction.Protein levels of PI3K,phospho(p)PI3K,serine/threonine kinase(AKT),p-AKT,BAX,BCL-2,and caspase3 were detected by Western blot.Results MIA decreased chondrocyte viability and induced apoptosis in a dose-dependent manner,decreased MEG3,COL2A1,and ACAN mRNA levels,and increased MMP-13 mRNA levels(P<0.05).RGP 100,200,400,and 800 mg/mL increased chondrocyte viability and MEG3 levels(P<0.05).Cell viability,MEG3,COL2A1,and ACAN mRNA,and p-PI3K/PI3K,p-AKT/AKT,and BCL-2 protein levels were decreased in the MIA group compared with the control group,while the apoptosis rate,MMP-13 mRNA,and BAX and caspase3 protein levels were increased(P<0.05).Cell viability,MEG3,COL2A1,and ACAN mRNA,and p-PI3K/PI3K,P-AKT/AKT,and BCL-2 protein levels were increased in the RGP group compared with the MIA group,while the apoptosis rate,MMP-13 mRNA,and BAX and caspase3 protein levels were decreased(P<0.05).Knockdown of MEG3 weakened the protective effect of RGP on MIA-induced chondrocyte injury.Conclusions RGP can promote the synthesis of chondrocyte extracellular matrix and inhibit cell apoptosis and MIA-induced chondrocyte damage,possibly acting via a mechanism related to the up-regulation of MEG3 expression and induction of PI3K/AKT pathway activation.
作者
杜晨飞
韩磊
范围
李红旗
郑福增
DU Chenfei;HAN Lei;FAN Wei;LI Hongqi;ZHENG Fuzeng(Department of Arthritis,Henan Provincial Hospital of Traditional Chinese Medicine,Zhengzhou 450053,China.;College of Orthopedics,Henan University of Traditional Chinese Medicine,Zhengzhou 450046.;Department of Rheumatology,Henan Provincial Hospital of Traditional Chinese Medicine,Zhengzhou 450053.;Department of Trauma Surgery,Henan Provincial Hospital of Traditional Chinese Medicine,Zhengzhou 450053)
出处
《中国比较医学杂志》
CAS
北大核心
2023年第7期92-99,122,共9页
Chinese Journal of Comparative Medicine
基金
河南中医药大学科研苗圃工程项目(MP2020-13)。
关键词
熟地黄多糖
长链非编码RNA
母系表达基因3
骨关节炎
软骨细胞
凋亡
Rehmannia glutinosa polysaccharide
long non-coding RNA
maternally expressed gene 3
osteoarthritis
chondrocytes
apoptosis
