Suppression of SHH Expression in Arthritis by Butea monosperma

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。MicroRNAs(MiRNA)是一类调控基因表达的非编码RNA。特异性miRNAs在骨性关节炎发病机制中的作用尚不明确。我们的初步结果表明，在IL-1β刺激的软骨细胞中，功能未知的HSA-Mir-323b-5p(miR-323b-5p)的表达下调了数倍。在计算机分析中发现Sonic Hedgehog(SHH)mRNA是miR-323b-5p的靶标。SHH信号与基质金属蛋白酶-13的表达和软骨退变有关，抑制SHH可减轻骨性关节炎的严重程度。丁香花(LAM)广泛分布于印度，其水提物(BME)用于治疗关节炎。我们的初步结果表明，IL-1可刺激骨性关节炎软骨细胞SHH的表达。值得注意的是，BME调节了SHH信号基因的表达，并阻断了SHH诱导的基质金属蛋白酶-13的表达，miR-323b-5p通过直接靶向mRNA的编码区而抑制了SHH蛋白的表达。我们建议使用描述良好的方法和临床前的OA动物模型来测试BME在体外和体内的软骨保护活性。我们的基本假设是“BME通过调控人软骨细胞中miR-323b-5p的表达，转录后调节SHH蛋白的表达，从而抑制IL-1β诱导的骨关节炎软骨分解代谢效应”。这一假说的推论是“BME的生物活性成分可能通过调节体内特定miRNAs的表达来发挥其对软骨的保护作用，这些miRNAs负向调节SHH和其他分解代谢因子的表达”。特异性目的-1：测定(A)BME对IL-1β诱导的人骨性关节炎软骨细胞和软骨组织中SHH及其受体ptch1表达的影响；(B)BME对SHH诱导的人骨性关节炎软骨细胞和软骨组织中ptch1、GLI-1和MMP-13表达的影响。使用微阵列分析，我们还将(C)确定其他miRNAs 其在人骨关节炎软骨细胞中的表达受IL-1的调节，并通过生物信息学确定额外的miRNAs是否针对SHH mRNA；以及(D)使用报告分析验证新发现的额外miRNAs与SHH mRNA的相互作用。特异性目的-2：我们将分析IL-1β对miR-323b-5p表达改变的软骨细胞的mRNA表达谱(Transcriptome)的影响，并确定表达改变的基因是否也是miR-323b-5p的靶点。具体目的-3：使用兔OA模型，我们将描述SHH和miRNAs在疾病诱导和发展过程中关节中的表达谱。将对关节软骨进行大体和组织学评估。我们将分析滑液和血清中炎性细胞因子(IL-1β、TNF-β、IL-6)的水平；(B)分泌的MMP-2、-9、-13的表达；以及(C)在给予两种不同剂量的BME和Isobutrin的动物中，我们将用LC/MS测定BME成分(Butein、Butrin、Isobutrin)的水平。我们还将研究BME和Isobutrin的摄入对关节miR-323b-5p、MMP-13和SHH mRNA和蛋白表达的影响，并与疾病的诱导和进展相关。

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.