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（由适用提供）：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）是调节基因表达的一类非编码RNA。特定miRNA在OA发病机理中的作用尚待定义。我们的初步结果表明，没有已知功能的HSA-MIR-323B-5P（miR-323b-5p）在IL-1刺激的软骨细胞中下调了几倍。在硅分析中，Sonic刺猬（SHH）mRNA是miR-323b-5p的靶标。 SHH信号传导与MMP-13的表达和软骨变性和SHH的抑制有关，从而减轻了OA的严重程度。 Butea Monosperma（LAM）广泛分布在印度，Butea Monosperma Flowers（BME）的水提取物用于治疗关节炎。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调节了SHH信号基因的表达，并阻止了软骨外植体中SHH诱导的MMP-13表达，而miR-323b-5p通过直接靶向mRNA中的编码区来抑制SHH蛋白表达。我们建议使用精心描述的测定法和OA的临床前动物模型在体外和体内测试软骨受保护的活性。我们的基本假设是“ BME通过调节人类软骨细胞中miR-323b-5p的表达来抑制OA中IL-1诱导的软骨分解代谢作用”。该假设的必然性是“生物活性构成BME的生物活性可以通过调节特定miRNA的表达来对其在OA中受保护作用，从而负调节体内SHH和其他分解代谢因子的表达”。特定目标-1：确定（a）BME对IL-1缺的影响SHH及其受体PTCH1的表达； （b）BME对人OA软骨细胞和软骨外植体PTCH1，GLI-1和MMP-13的SHH诱导表达的影响。使用微阵列分析，我们还将（c）确定其他miRNA 其表达在人OA软骨细胞中受IL-1的调节，并且生物信息可以确定其他miRNA靶向mRNA； （d）使用记者分析验证新鉴定的其他miRNA与SHH mRNA的相互作用。具体目标-2：我们将分析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的靶标。特定的AIM-3：使用OA的兔模型，我们将在疾病诱导和进展过程中表征SHH和关节中miRNA的表达曲线。关节软骨将通过宏观和组织学评估。滑液和血清将分析（a）炎性细胞因子水平（IL-1，TNF- - ，IL-6）； （b）分泌的MMP-2，-9，-13的表达； （c）在给出两种不同剂量的BME和异丁蛋蛋白的动物中，我们将通过LC/MS确定BME构成的水平（Butein，Butrin，isobutin）。我们还将检查BME和异丁丁蛋白消耗对关节中miR-323b-5p，MMP-13和SHH mRNA和蛋白质的表达水平的影响，并与疾病诱导和进展相关。