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: 9330067
• 负责人: Tariq M Haqqi
• 金额: $ 52.17万
• 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 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9330067
• 关键词: Address; Animal Model; Animals; Anti-Inflammatory Agents; Anti-inflammatory; Arthritis; Attenuated; Ayurvedic Medicine; Binding; Bioavailable; Bioinformatics; 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; GLI gene; GLI-1; Gelatinase A; Gene Expression; Genes; Glycosaminoglycans; Goals; High Prevalence; Histologic; Homeostasis; Human; IL8 gene; Immunoprecipitation; In Vitro; Incidence; India; Inflammatory; Interleukin-1; 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; Phenotype; Plants; Plasma; Population; Post-Transcriptional Regulation; Pre-Clinical Model; Prevention; Production; Property; Protein Family; Proteins; Replacement Arthroplasty; Reporter; Rheumatoid Arthritis; Role; SHH gene; Serum; Severities; Signal Transduction; Skeletal Development; Societies; Synovial Fluid; System; TNF gene; Testing; Therapeutic; Toxic effect; Treatment Efficacy; United States National Institutes of Health; Untranslated RNA; Validation; Water; activating transcription factor; aged; articular cartilage; cartilage cell; cartilage degradation; collagenase 3; cost effective; cytokine; effective therapy; forest; 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; 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。在 IL-1� 刺激的软骨细胞中，Sonic Hedgehog (SHH) mRNA 的功能被下调数倍，SHH 信号传导与 MMP-13 的表达和软骨退化相关。 SHH 的抑制可减轻 OA 的严重程度，紫矿花 (Lam) 广泛分布于印度，紫矿花 (BME) 的水提取物是我们的初步结果表明，IL-1� 刺激 OA 软骨细胞中 SHH 的表达，值得注意的是，BME 调节 SHH 信号基因的表达并阻断 SHH 诱导的软骨外植体中 MMP-13 的表达，并且 miR -323b-5p 通过直接靶向 mRNA 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体内测试 BME 的软骨保护活性。我们的基本假设是“BME 通过调节人软骨细胞中 miR-323b-5p 的表达，通过 SHH 蛋白表达的转录后调节来抑制 OA 中 IL-1� 诱导的软骨分解代谢效应”。这一假设的推论是“BME 的生物活性成分可能通过调节特定 miRNA 的表达来发挥其对 OA 的软骨保护作用，这些 miRNA 负向调节SHH 和其他体内分解代谢因子”。具体目标 1：确定 (a) BME 对 IL-1� 诱导的 SHH 及其受体 PTCH1 表达的影响；以及 (b) BME 对 SHH 诱导的表达的影响使用微阵列分析，我们还将 (c) 鉴定其他 miRNA。 其表达受人 OA 软骨细胞中的 IL-1� 调节，并通过生物信息学确定其他 miRNA 是否靶向 SHH mRNA；以及 (d) 使用报告基因特异性 Aim-2 验证新鉴定的其他 miRNA 与 SHH mRNA 的相互作用：我们将进行分析。 IL-1� 对 miR-323b-5p 表达改变的软骨细胞中 mRNA 表达谱（转录组）的影响，并确定表达改变的基因是否也是miR-323b-5p。具体目标 3：使用 OA 兔模型，我们将从宏观和组织学角度评估关节中 SHH 和 miRNA 的表达谱。并分析血清 (a) 炎症细胞因子（IL-1�、TNF-�、IL-6）的水平；(b) 分泌的 MMP-2 的表达， -9,-13；以及 (c) 在给予两种不同剂量的 BME 和异布特林的动物中，我们将通过 LC/MS 测定 BME 成分（布丁蛋白、布特林、异布特林）的水平。我们还将检查 BME 和异布特林的效果。消耗对关节中 miR-323b-5p、MMP-13 和 SHH mRNA 和蛋白质表达水平的影响，并与疾病诱导和进展相关。