Sanchez Christelle, Hemmer Kathrin, Krömmelbein Natascha, Seilheimer Bernd, Dubuc Jean-Emile, Antoine Christophe, Henrotin Yves
MusculoSKeletal Innovative Research Lab, University of Liège, Center for Interdisciplinary Research on Medicines, Liège, Belgium.
Heel GmbH, Baden-Baden, Germany.
Front Pharmacol. 2021 May 11;12:635034. doi: 10.3389/fphar.2021.635034. eCollection 2021.
Zeel T (Ze14) is a multicomponent medicinal product. Initial preclinical data suggested a preventive effect on cartilage degradation. Clinical observational studies demonstrated that Ze14 reduced symptoms of osteoarthritis (OA), including stiffness and pain. This study aimed to explore these effects further to better understand the mode of action of Ze14 on human OA chondrocytes . Primary chondrocytes were obtained from the knees of 19 OA patients and cultured either as monolayers or in alginate beads. The cultures were treated with 20% or 10% (v/v) Ze14 or placebo. For RNA-seq, reads were generated with Illumina NextSeq5000 sequencer and aligned to the human reference genome (UCSC hg19). Differential expression analysis between Ze14 and placebo was performed in R using the DESeq2 package. Protein quantification by ELISA was performed on selected genes from the culture medium and/or the cellular fractions of primary human OA chondrocyte cultures. In monolayer cultures, Ze14 20% (v/v) significantly modified the expression of 13 genes in OA chondrocytes by at least 10% with an adjusted -value < 0.05: EGR1, FOS, NR4A1, DUSP1, ZFP36, ZFP36L1, NFKBIZ, and CCN1 were upregulated and ATF7IP, TXNIP, DEPP1, CLEC3A, and MMP13 were downregulated after 24 h Ze14 treatment. Ze14 significantly increased (mean 2.3-fold after 24 h, = 0.0444 and 72 h, = 0.0239) the CCN1 protein production in human OA chondrocytes. After 72 h, Ze14 significantly increased type II collagen pro-peptide production by mean 27% ( = 0.0147). For both time points CCN1 production by OA chondrocytes was correlated with aggrecan (r = 0.66, = 0.0004) and type II collagen pro-peptide (r = 0.64, = 0.0008) production. In alginate beads cultures, pro-MMP-13 was decreased by Ze14 from day 7-14 (from -16 to -25%, p < 0.05) and from day 17-21 (-22%, = 0.0331) in comparison to controls. Ze14 significantly modified the expression of DUSP1, DEPP1, ZFP36/ZFP36L1, and CLEC3A, which may reduce MMP13 expression and activation. Protein analysis confirmed that Ze14 significantly reduced the production of pro-MMP-13. As MMP-13 is involved in type II collagen degradation, Ze14 may limit cartilage degradation. Ze14 also promoted extracellular matrix formation arguably through CCN1 production, a growth factor well correlated with type II collagen and aggrecan production.
Zeel T（Ze14）是一种多成分药物产品。最初的临床前数据表明其对软骨降解有预防作用。临床观察研究表明，Ze14可减轻骨关节炎（OA）的症状，包括僵硬和疼痛。本研究旨在进一步探索这些作用，以更好地了解Ze14对人OA软骨细胞的作用模式。从19例OA患者的膝关节获取原代软骨细胞，并将其培养成单层或包埋在藻酸盐珠中。培养物分别用20%或10%（v/v）的Ze14或安慰剂处理。对于RNA测序，使用Illumina NextSeq5000测序仪生成读数，并与人类参考基因组（UCSC hg19）进行比对。在R中使用DESeq2软件包对Ze14和安慰剂之间进行差异表达分析。通过ELISA对人OA软骨细胞原代培养物的培养基和/或细胞组分中选定基因进行蛋白质定量分析。在单层培养中，20%（v/v）的Ze14处理24小时后，显著改变了OA软骨细胞中13个基因的表达，至少上调或下调10%，校正P值<0.05：EGR1、FOS、NR4A1、DUSP1、ZFP36、ZFP36L1、NFKBIZ和CCN1上调，而ATF7IP、TXNIP、DEPP1、CLEC3A和MMP13下调。Ze14显著增加了人OA软骨细胞中CCN1蛋白的产生（24小时后平均增加2.3倍，P = 0.0444；72小时后，P = 0.0239）。72小时后，Ze14显著增加了II型胶原蛋白前肽的产生，平均增加27%（P = 0.0147）。在两个时间点，OA软骨细胞中CCN1的产生均与聚集蛋白聚糖（r = 0.66，P = 0.0004）和II型胶原蛋白前肽（r = 0.64，P = 0.0008）的产生相关。在藻酸盐珠培养中，与对照组相比，Ze14从第7 - 14天（从-16%至-25%，P < 0.05）和第17 - 21天（-22%，P = 0.0331）降低了前MMP - 13的水平。Ze14显著改变了DUSP1、DEPP1、ZFP36/ZFP36L1和CLEC3A的表达，这可能会降低MMP13的表达和激活。蛋白质分析证实Ze14显著降低了前MMP - 13的产生。由于MMP - 13参与II型胶原蛋白的降解，Ze14可能会限制软骨降解。Ze14还可能通过产生CCN1促进细胞外基质形成，CCN1是一种与II型胶原蛋白和聚集蛋白聚糖产生密切相关的生长因子。
