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（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术。MicroRNAs （miRNA）是一类调节基因表达的非编码rna。特异性mirna在OA发病机制中的作用尚未明确。我们的初步结果显示，在IL-1刺激的软骨细胞中，没有已知功能的Hsa-MIR-323B-5P （miR-323b- 5p）下调了几倍。通过计算机分析，发现Sonic Hedgehog (SHH) mRNA是miR-323b-5p的靶标。SHH信号与MMP-13的表达和软骨退变有关，抑制SHH可减轻OA的严重程度。Butea monoosperma （Lam）广泛分布在印度，Butea monoosperma花（BME）的水提取物被用来治疗关节炎。我们的初步结果表明，IL-1¿刺激骨性关节炎软骨细胞中SHH的表达。值得注意的是，BME调节SHH信号基因的表达，阻断SHH诱导的软骨外植体中MMP-13的表达，miR-323b-5p通过直接靶向mRNA中的编码区抑制SHH蛋白的表达。我们建议在体外和体内使用描述良好的实验和OA临床前动物模型来测试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，通过转录后调控SHH蛋白表达，抑制IL-1诱导的OA软骨分解代谢作用”。这一假设的一个推论是“BME的生物活性成分可能通过调节体内负调节SHH和其他分解代谢因子表达的特异性mirna的表达，在OA中发挥软骨保护作用”。特异性Aim-1：确定(a) BME对IL-1诱导的SHH及其受体PTCH1表达的影响；(b) BME对sh诱导的体外人OA软骨细胞和软骨外植体PTCH1、gli1和MMP-13表达的影响。使用微阵列分析，我们还将(c)鉴定额外的mirna