Suppression of SHH Expression in Arthritis by Butea monosperma

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 发病机制中的作用尚未明确。我们的初步结果表明，功能未知的 Hsa-MIR-323B-5P (miR-323b-5p) 在 IL-1¿ 刺激的软骨细胞中下调了数倍。计算机分析确定 Sonic Hedgehog (SHH) mRNA 为 miR-323b-5p 的靶标。 SHH 信号传导与 MMP-13 的表达和软骨变性相关，抑制 SHH 可以减轻 OA 的严重程度。紫矿（Lam）广泛分布于印度，紫矿花的水提取物（BME）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 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 mRNA 中的编码区抑制 SHH 蛋白表达。我们建议使用详细描述的测定法和 OA 临床前动物模型在体外和体内测试 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控，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 负向调节体内 SHH 和其他分解代谢因子的表达”。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； (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。使用微阵列分析，我们还将 (c) 识别其他 miRNA 其表达受人 OA 软骨细胞中 IL-1¿ 的调节，并通过生物信息学确定是否有其他 miRNA 靶向 SHH mRNA； (d) 使用报告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。具体目标 3：使用 OA 兔模型，我们将表征疾病诱导和进展期间关节中 SHH 和 miRNA 的表达谱。将对关节软骨进行宏观和组织学评估。将分析滑液和血清的 (a) 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； (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（布特因、布特林、异布特林）的水平。我们还将检查 BME 和异布精消耗对关节中 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。

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.