Identifying the Skeletal Benefits of SFX-01

我们的项目完全符合BBSRC的“生物科学促进健康”的优先事项。它的重点是生物科学，它将确定“健康寿命”的新扩展，以减轻医疗干预要求和世界范围的骨关节炎(OA)医疗负担。它融合了新发现的RVC在健康关节随年龄自然恶化期间的信号传递方面的见解，以及Evgen Pharma针对这一途径的化合物。我们的项目显然有强烈的动机利用生物科学来支持新的有效治疗方法，以实现互惠互利。萝卜硫素(硫磺，不稳定)能有效地诱导Nrf2(核红系相关因子2)上调保护酶并抑制促炎信号。因此，我们检查了SFX-01(稳定的Sulf)是否调节了STR/Ort小鼠体内自然形成的OA，发现它确实严重限制了症状(习惯性跛行未能形成)和骨量增加。我们的假设是：SFX-01通过NRF2介导的NF-kB阻断促进了成骨，限制了骨吸收，并限制了软骨内软骨向骨的转化(EO)，从而减缓了OA的发生。我们将致力于(每年)SFX-01靶向STR/Ort和CBA(对照小鼠)破骨细胞、成骨细胞和软骨细胞的三个独立目标，以确定对Nrf2介导的NF-kB抑制的任何依赖，并在SFX-01处理的STR/Ort和CBA(正在进行的)中寻求证实。Aim 1(Yr1)：SFX-01是否调控破骨细胞(OC)的形成/吸收？将8wk STR/ORT(OA发病前)和CBA对照组(非易感)的OC形成的骨髓单个核细胞培养并将非贴壁细胞悬浮在牙本质盘上(在培养液中24小时+M-CSF/RANKL)；附着型OC前体在加/不加Sulf或SFX-01(0-1 mM；pH 7.0，最后2d激活吸收)培养8d。检测抗酒石酸酸性磷酸酶(TRAP)活性的圆盘将用于评估OC数量和吸收面积(活性/OC)。在最佳Sulf/SFX-01水平下的研究将评估在存在或不存在Nrf2选择性抑制物(胡芦巴碱)或激活剂(RA839)的情况下NF-kB的激活情况，并在使用SFX-01治疗的Str/ORT和CBA小鼠的关节中寻求确证，如有必要，在Nrf2 KO和人类OCS中进行确认。目的2(YR 2)：SFX-01是否调节成骨细胞(OB)的增殖/分化？STR/Ort和CBA的原代OB将在有或没有Sulf或SFX-01(0-1 mm)的情况下培养；结晶紫检测细胞数量/活力，增殖细胞核抗原/BrdU标记；碱性磷酸酶(每种蛋白；BCA)和组织化学检测成骨细胞分化；qPCR和免疫印迹检测成骨细胞分化不同阶段标志物的转录本/蛋白水平；茜素红测定矿化程度。如上所述，研究将使用Nrf2抑制/激活剂、Nrf2 KO Obs和SFX-01处理的STR/Ort/CBA关节和人类原代成骨细胞(正常/骨关节炎肩关节，正在进行中)来证实Nrf2介导的NF-kB作用。将STR/Ort和CBA小鼠胚胎(E15)的跖骨分别加入或不加入Sulf或SFX-01(0-1 mm)进行培养。总/矿化区长度、生长板组织(组织学)、矿物质密度和骨量/结构(MicroCT)评估10天。将测量Wnt信号标志物、增殖、细胞死亡、EO和NF-kB激活的转录/蛋白水平，以确定SFX-01的作用机制。在Nrf2 KO小鼠以及SFX-01处理的Str/ort和CBA关节中寻求证实。

Our project fully aligns to BBSRC's 'bioscience for health' priority. It focuses on bioscience that will identify new extensions to 'healthspan' to relieve medical intervention requirements and the world-wide osteoarthritis (OA) healthcare burden. It merges newly identified RVC insights in signalling during the natural deterioration of healthy joints with age with Evgen Pharma's compounds that target this pathway. Our project is clearly and acutely motivated to use bioscience to underpin new effective therapy for mutual benefit. Sulforaphane (Sulf, unstable) potently induces Nrf2 (nuclear erythroid related factor 2) to upregulate protective enzymes and inhibit proinflammatory signalling. We thus examined if SFX-01 (stable Sulf) modulates the natural OA that develops in STR/Ort mice, to find indeed that it severely limited symptoms (accustomed limp fails to develop) and increased bone mass. We hypothesise: that Nrf2-mediated blockade of NF-kB by SFX-01 promotes osteogenesis, limits bone resorption and restricts endochondral cartilage-to-bone conversion (EO) to slow OA. We will address 3 independent aims focused (annually) on SFX-01 targeting of osteoclasts, osteoblasts and chondrocytes in STR/Ort and CBA (control mice to define any reliance on Nrf2-mediated NF-kB inhibition, and seek confirmation in SFX-01-treated STR/Ort and CBA (ongoing), and Nrf2 KO mice (collaboration).Aim 1 (yr 1): Does SFX-01 regulate osteoclast (OC) formation/resorption?OC-forming marrow mononuclear cells from 8wk STR/Ort (pre-OA onset) and CBA control (non-prone) bones will be cultured and non-adherent cells suspended on dentine discs (24hr in medium +M-CSF/RANKL); attached OC precursors cultured for 8d with/without Sulf or SFX-01 (0-1mM; pH 7.0 for final 2d to activate resorption). Discs reacted for tartrate-resistant acid phosphatase (TRAP) activity will be used to asses OC number and resorbed area (activity/OC). Studies at optimal Sulf/SFX-01 levels will evaluate NF-kB activation in presence/absence of Nrf2-selective inhibitor (trigonelline) or activator (RA839) and corroboration sought in joints from Str/ort and CBA mice treated with SFX-01 and, if necessary, confirmed in Nrf2 KO and human OCs. Aim 2 (yr 2): Does SFX-01 regulate osteoblast (OB) proliferation/differentiation?Primary OBs from STR/Ort and CBA will be cultured with/without Sulf or SFX-01 (0-1mM); cell no./viability measured by crystal violet and proliferation by PCNA/BrdU labelling; OB differentiation by alkaline phosphatase (per protein; BCA) and by histochemistry; transcript/ protein levels for markers of distinct stages of OB differentiation measured by qPCR and immunoblotting; mineralisation quantified by Alizarin Red. As above, studies will corroborate Nrf2-mediated NF-kB roles using Nrf2 inhibitor/activator, Nrf2 KO OBs and SFX-01-treated STR/Ort/CBA joints and human primary osteoblasts (normal/OA shoulders, ongoing).Aim 3 (yr 3): Does SFX-01 regulate EO? Embryo (E15) metatarsals from STR/Ort and CBA mice will be cultured with/without Sulf or SFX-01 (0-1mM). Total/mineralisation zone length, growth plate organisation (histology) and mineral density and bone mass/architecture (microCT) assessed for 10d. Transcript/protein levels for Wnt signalling markers, proliferation, cell death, EO and NF-kB activation will be measured to define mechanisms of SFX-01 action. Corroboration sought in Nrf2 KO mice and in SFX-01-treated Str/ort and CBA joints.