Comprehensive Genome-Wide Transcriptomic Analysis of Immature Articular Cartilage following Ischemic Osteonecrosis of the Femoral Head in Piglets

ObjectiveIschemic osteonecrosis of the femoral head (ONFH) in piglets results in an ischemic injury to the immature articular cartilage. The molecular changes in the articular cartilage in response to ONFH have not been investigated using a transcriptomic approach. The purpose of this study was to perform a genome-wide transcriptomic analysis to identify genes that are upregulated in the immature articular cartilage following ONFH.MethodsONFH was induced in the right femoral head of 6-week old piglets. The unoperated femoral head was used as the normal control. At 24 hours (acute ischemic-hypoxic injury), 2 weeks (avascular necrosis in the femoral head) and 4 weeks (early repair) after surgery (n = 4 piglets/time point), RNA was isolated from the articular cartilage of the femoral head. A microarray analysis was performed using Affymetrix Porcine GeneChip Array. An enrichment analysis and functional clustering of the genes upregulated due to ONFH were performed using DAVID and STRING software, respectively. The increased expression of selected genes was confirmed by a real-time qRTPCR analysis.ResultsInduction of ONFH resulted in the upregulation of 383 genes at 24 hours, 122 genes at 2 weeks and 124 genes at 4 weeks compared to the normal controls. At 24 hours, the genes involved in oxidoreductive, cell-survival, and angiogenic responses were significantly enriched among the upregulated genes. These genes were involved in HIF-1, PI3K-Akt, and MAPK signaling pathways. At 2 weeks, secretory and signaling proteins involved in angiogenic and inflammatory responses, PI3K-Akt and matrix-remodeling pathways were significantly enriched. At 4 weeks, genes that represent inflammatory cytokines and chemokine signaling pathways were significantly enriched. Several index genes (genes that are upregulated at more than one time point following ONFH and are known to be important in various biological processes) including HIF-1A, VEGFA, IL-6, IL6R, IL-8, CCL2, FGF2, TGFB2, MMP1, MMP3, ITGA5, FN and Col6A1 were upregulated in the immature articular cartilage following ONFH. A qRTPCR analysis of selected genes confirmed the upregulated expression observed in the microarray analysis.ConclusionImmature articular cartilage responds to ONFH by the upregulation of genes involved in hypoxic stress response, angiogenesis, matrix remodeling and inflammation. This study provides novel insights into the multi-faceted role of immature articular cartilage, with inflammation as a key component, following ONFH in piglets.

Objective：仔猪股骨头缺血性坏死（ischemic osteonecrosis of the femoral head, ONFH）可导致未成熟关节软骨发生缺血性损伤。目前尚未有研究通过转录组学方法探究ONFH发生后关节软骨的分子变化。本研究旨在开展全基因组转录组分析，以鉴定ONFH诱导后未成熟关节软骨中上调表达的基因。

Methods：对6周龄仔猪的右侧股骨头构建ONFH模型，以未行手术的股骨头作为正常对照。分别于术后24小时（急性缺血缺氧损伤阶段）、2周（股骨头缺血坏死阶段）及4周（早期修复阶段）采集样本（每个时间点n=4头仔猪），从股骨头关节软骨中提取总RNA。采用Affymetrix猪基因芯片阵列（Affymetrix Porcine GeneChip Array）进行基因芯片分析。分别使用DAVID和STRING软件对ONFH诱导上调的基因进行富集分析与功能聚类分析。通过实时定量逆转录聚合酶链反应（real-time qRTPCR）验证部分候选基因的表达上调情况。

Results：与正常对照组相比，ONFH诱导后在24小时、2周和4周分别有383个、122个和124个基因出现上调表达。术后24小时，上调基因显著富集于氧化还原反应、细胞存活及血管生成相关生物学过程，且涉及HIF-1、PI3K-Akt和MAPK信号通路。术后2周，参与血管生成、炎症反应、PI3K-Akt及基质重塑通路的分泌蛋白与信号蛋白显著富集。术后4周，代表炎症细胞因子和趋化因子信号通路的基因显著富集。多个核心基因（即在ONFH后多个时间点均上调且在多种生物学过程中发挥关键作用的基因）包括HIF-1A、VEGFA、IL-6、IL6R、IL-8、CCL2、FGF2、TGFB2、MMP1、MMP3、ITGA5、FN及Col6A1，在ONFH后的未成熟关节软骨中均呈上调表达。实时qRTPCR分析结果验证了基因芯片分析中观察到的基因上调趋势。

Conclusion：未成熟关节软骨可通过上调参与缺氧应激反应、血管生成、基质重塑及炎症反应的基因来应对ONFH损伤。本研究为仔猪ONFH发生后未成熟关节软骨的多维度调控作用提供了新的见解，其中炎症反应是核心组成部分。