Suppression of SHH Expression in Arthritis by Butea monosperma

紫矿对关节炎中 SHH 表达的抑制

• 批准号: 8508108
• 负责人: Tariq M Haqqi
• 金额: $ 38.34万
• 依托单位: NORTHEAST OHIO MEDICAL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-30 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8508108
• 关键词: Address; Animal Model; Animals; Anti-Inflammatory Agents; Anti-inflammatory; Arthritis; Attenuated; Binding; Bioavailable; Biological Assay; Biology; Birth; Butea; Cartilage; Cells; Chondrocytes; Chronic; Clinical; Code; Collagen Type II; Color; Computer Simulation; Consumption; Degenerative Disorder; Degenerative polyarthritis; Development; Differentiation and Growth; Disease; Disease Progression; Dose; Down-Regulation; Epiphysial cartilage; Erinaceidae; Exhibits; Flowers; Functional RNA; GLI gene; GLI-1; Gelatinase A; Gene Expression; Gene Expression Profile; Genes; Glycosaminoglycans; Goals; High Prevalence; Histologic; Homeostasis; Human; IL8 gene; Immunoprecipitation; In Vitro; Incidence; India; Inflammatory; Interleukin-6; Joints; Knowledge; Ligands; Matrix Metalloproteinases; Medicinal Plants; Medicine; Messenger RNA; MicroRNAs; Modeling; Molecular Profiling; Musculoskeletal Diseases; Names; Operative Surgical Procedures; Oral; Oryctolagus cuniculus; Pathogenesis; Pattern; Plants; Plasma; Population; Post-Transcriptional Regulation; Pre-Clinical Model; Prevention; Production; Property; Protein Family; Proteins; Replacement Arthroplasty; Reporter; Rheumatoid Arthritis; Role; Serum; Severities; Signal Transduction; Skeletal Development; Societies; Sonic hedgehog protein; Staging; Synovial Fluid; System; TNF gene; Testing; Therapeutic; Toxic effect; United States National Institutes of Health; Validation; Water; activating transcription factor; aged; articular cartilage; ayurveda; cartilage cell; collagenase 3; cost effective; cytokine; effective therapy; forest; human SMO protein; in vivo; insight; liquid chromatography mass spectrometry; mRNA Expression; new technology; novel; novel strategies; pre-clinical; prevent; protective effect; protein expression; public health relevance; receptor; smoothened signaling pathway; transcriptome sequencing

DESCRIPTION (provided by applicant): Osteoarthritis (OA) is the most common musculoskeletal disorder and the only effective treatment is surgical joint replacement. MicroRNAs (miRNA) are a class of non-coding RNAs regulating gene expression. Role of specific miRNAs in OA pathogenesis is yet to be defined. Our preliminary results showed that Hsa-MIR-323B-5P (miR-323b- 5p), with no known function, was downregulated several fold in IL-1¿-stimulated chondrocytes. In silico analysis identified Sonic Hedgehog (SHH) mRNA as a target of miR-323b-5p. SHH signaling is associated with the expression of MMP-13 and cartilage degeneration and inhibition of SHH attenuates the severity of OA. Butea monosperma (Lam) is widely distributed in India and water extract of Butea monosperma flowers (BME) is used to treat arthritis. Our preliminary results show that IL-1¿ stimulates the expression of SHH in OA chondrocytes. Of note, BME modulated the SHH signaling genes expression and blocked the SHH-induced expression of MMP-13 in cartilage explants and that miR-323b-5p inhibited SHH protein expression by directly targeting coding region in the mRNA. We propose to test the cartilage protective activity of BME in vitro and in vivo using well described assays and a preclinical animal model of OA. Our basic hypothesis is that "BME suppresses the IL-1¿-induced cartilage catabolic effects in OA via post-transcriptional regulation of SHH protein expression by modulating the expression of miR-323b-5p in human chondrocytes". A corollary of this hypothesis is that "bioactive constituents of BME may exert their cartilage protective effects in OA by modulating the expression of specific miRNAs that negatively regulate the expression of SHH and other catabolic factors in vivo". Specific Aim-1: Determine (a) the effect of BME on IL-1¿- induced expression of SHH and its receptor PTCH1; and (b) the effect of BME on the SHH-induced expression of PTCH1, GLI-1, and MMP-13 in human OA chondrocytes and cartilage explants in vitro. Using microarray profiling we will also (c) identify additional miRNAs whose expression is modulated by IL-1¿ in human OA chondrocytes and bioinformatically determine if additional miRNAs target SHH mRNA; and (d) validate the interactions of newly identified additional miRNAs with SHH mRNA using reporter assays. Specific Aim-2: We will analyze the effect of IL-1¿ on the mRNA expression profile (Transcriptome) in chondrocytes with altered miR-323b-5p expression and determine whether the genes with altered expression are also targets of miR-323b-5p. Specific Aim-3: Using a rabbit model of OA we will characterize the expression profile of SHH and of the miRNAs in the joints during disease induction and progression. The articular cartilage will be evaluated macroscopically and histologically. Synovial fluid and serum will be analyzed for (a) levels of inflammatory cytokines (IL-1¿, TNF-¿, IL-6); (b) expression of secreted MMP-2, -9,-13; and (c) in animals given two different doses of BME and Isobutrin we will determine the levels of BME constituents (Butein, Butrin, Isobutrin) by LC/MS. We will also examine the effect of BME and Isobutrin consumption on the expression levels of miR-323b-5p, MMP-13 and SHH mRNA and protein in the joints and correlate with disease induction and progression.

描述（由申请人提供）：骨关节炎（OA）是最常见的肌肉骨骼疾病，唯一有效的治疗方法是手术关节置换。MicroRNA（miRNA）是一类调节基因表达的非编码RNA。特定miRNA在OA发病机制中的作用。我们的初步结果表明，Hsa-MIR-323B-5P (miR-323b-5p) 尚未确定。功能，在 IL-1 中下调数倍？模拟分析发现 Sonic Hedgehog (SHH) mRNA 是 miR-323b-5p 的靶标，SHH 信号传导与 MMP-13 的表达和软骨退化有关，并且 SHH 的抑制可减轻 OA 的严重程度。 （Lam）在印度广泛分布，紫矿花的水提取物（BME）用于治疗关节炎。 IL-1??刺激 OA 软骨细胞中 SHH 的表达 值得注意的是，BME 调节 SHH 信号基因表达并阻断软骨外植体中 SHH 诱导的 MMP-13 表达，并且 miR-323b-5p 通过直接靶向软骨细胞中的编码区来抑制 SHH 蛋白表达。我们建议使用详细描述的测定法和 OA 的临床前动物模型来测试 BME 的体外和体内软骨保护活性。 “BME 通过调节人软骨细胞中 miR-323b-5p 的表达，通过转录后调节 SHH 蛋白表达来抑制 OA 中 IL-1 诱导的软骨分解代谢效应”。 BME 可能通过调节特定 miRNA 的表达来发挥其对 OA 的软骨保护作用，这些 miRNA 负向调节体内 SHH 和其他分解代谢因子的表达。 Aim-1：确定 (a) BME 对 IL-1 的影响¿ -SHH 及其受体 PTCH1 的诱导表达；以及（b）BME 对人 OA 软骨细胞和软骨外植体中 SHH 诱导的 PTCH1、GLI-1 和 MMP-13 表达的影响。还 (c) 识别其他 miRNA 其表达受 IL-1 调节在人类 OA 软骨细胞中，并通过生物信息学确定其他 miRNA 是否靶向 SHH mRNA；以及 (d) 使用报告基因检测验证新鉴定的其他 miRNA 与 SHH mRNA 的相互作用：我们将分析 IL-1¿观察 miR-323b-5p 表达改变的软骨细胞中的 mRNA 表达谱（转录组），并确定表达改变的基因是否也是 miR-323b-5p 的靶标 具体目标 3：我们将使用 OA 兔模型来表征。对关节软骨在疾病诱导和进展过程中的 SHH 和 miRNA 的表达谱进行宏观和组织学评估。分析 (a) 给予两种不同剂量的动物中炎症细胞因子（IL-1¿、TNF-¿、IL-6）的水平；(b) 分泌的 MMP-2、-9、-13 的表达；我们将通过 LC/MS 测定 BME 成分（Butein、Butrin、Isobutrin）的水平。我们还将检查 BME 和异布精的消耗对 BME 表达水平的影响。关节中的 miR-323b-5p、MMP-13 和 SHH mRNA 和蛋白质与疾病诱导和进展相关。