CN114209840A - 一种mif抑制剂在制备治疗骨关节炎的药物中的应用 - Google Patents
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Abstract
本发明公开了一种MIF抑制剂CPSI‑1306在制备用于治疗或者辅助治疗骨关节炎的药物中的应用。本发明确立了MIF蛋白在骨关节炎发展中的重要作用，提供的小分子化合物具有抑制MIF生物学功能的作用，可以促进关节软骨细胞的合成代谢增强并抑制其分解代谢，同时该小分子化合物可以通过抑制M1巨噬细胞极化减轻炎症。根据该小分子化合物对软骨细胞的保护作用和对炎症细胞的抑制作用，在骨关节炎治疗中具有应用价值。
Description
技术领域
本发明属于医药技术领域，具体涉及MIF抑制剂CPSI-1306在制备治疗骨性关节炎药物中应用。
背景技术
骨性关节炎(osteoarthritis，OA)是一种慢性关节退行性病变，跟许多因素相关，比如衰老、大体重、创伤、劳动损伤或先天畸形。在我国有1亿以上关节炎患者，其中主要集中于老年人，严重影响老年人的身体健康和生活质量。
骨关节炎的临床症状主要表现为关节疼痛、肿胀、变形，其发病原因可能是由于慢性炎症等诱因引起软骨组织退行性改变，引起细胞外基质合成与降解紊乱及软骨细胞变性坏死，造成关节面变薄甚至完全损耗，导致严重的OA发生。目前早期OA的治疗主要以口服非甾体消炎止痛药和改善生活习惯为主，症状无法控制的晚期OA则可以采用人工关节置换术治疗。
发明内容
本发明的第一目的是为制备治疗骨性关节炎药物，而提供一种MIF抑制剂CPSI-1306在制备治疗骨性关节炎药物中的应用。
本发明的第二目的是提供一种MIF抑制剂CPSI-1306在促进软骨形成药物中的应用。
本发明的第三目的是提供一种MIF抑制剂CPSI-1306在骨关节炎炎症抑制药物中的应用。
与现有技术相比，本发明的有益效果如下：
本发明首次公开了一种MIF抑制剂CPSI-1306在制备治疗骨关节炎的药物中的应用，本发明先在体外实验采用CPSI-1306处理软骨细胞和巨噬细胞，使用qPCR检测CPSI-1306处理后软骨细胞的合成分解代谢指标的mRNA水平，以及巨噬细胞的炎症指标的mRNA水平。进一步体内实验采用内侧半月板失稳(Destabilization of Medial Meniscus，DMM)诱导的骨关节炎模型，通过向患有OA的小鼠膝关节处注射含有CPSI-1306的注射液，观察MIF抑制剂CPSI-1306对骨关节炎的治疗作用，并评估软骨损伤程度和滑膜炎症程度。研究结果表明，CPSI-1306能够有效的缓解软骨损伤和抑制炎症。
附图说明
图1为实施例1的CPSI-1306处理软骨细胞的CCK-8增殖实验的效果图；
图2为实施例1的CPSI-1306处理软骨细胞的qPCR实验的效果图；
图3为实施例2的CPSI-1306处理巨噬细胞的CCK-8增殖实验的效果图；
图4为实施例2的CPSI-1306处理巨噬细胞的qPCR实验的效果图；
图5为实施例3的Sham组、DMM组和CPSI-1306组的小鼠关节组织番红固绿染色的效果图；
图6为实施例3的Sham组、DMM组和CPSI-1306组的小鼠滑膜组织苏木素伊红染色的效果图；
具体实施方式
具体实验方法和结果如下：
实施例1
一、实验方法：
1、提取C57/BL6小鼠的原代软骨细胞：取5天龄C57/BL6乳鼠处死，75％乙醇消毒后取关节组织，用PBS冲洗软骨3次，用刀片将软骨组织剁碎。加入10ml的0.2％二型胶原酶，37℃消化18小时后液体充分吹打混合过100um筛网，取细胞悬液离心1000r/分钟5分钟，去上清液，重悬后将上清液移至10cm的培养皿中，加入10ml添加10％血清的DMEM培养基，置于37℃、5％CO2及饱和湿度条件下培养。
2、细胞增殖实验CCK-8：
将一代的软骨细胞以2000个/孔数量接种在96孔板中，24小时后分别加入浓度为0，25/8ng/ml，25/4ng/ml，25/2ng/ml，25ng/ml，50ng/ml，100ng/ml，200ng/ml和400ng/ml的CPSI-1306溶液。培养24小时后，加入20μL CCK-8试剂盒37℃孵育3小时后用荧光酶标仪检测OD值。
3.qPCR检测
取软骨细胞以3×105/well种植于12孔板中，于37℃，5％CO2培养箱中孵育过夜；设立Control组，IL-1β处理组(10ng/ml)，IL-1β+CPSI-1306处理组(浓度梯度分别为5μΜ，10μΜ，20μΜ)；干预24小时后用Trizol法提取软骨细胞的mRNA，然后使用HiFiScript cDNA第一链合成试剂盒合成cDNA，继续按照使用SYBR Green法进行qPCR操作流程检测软骨细胞内的合成代谢指标(COL2A1和ACAN)和分解代谢指标(MMP13，MMP3，Adamts4和Adamts5)的表达。
二、实验结果：
图1所示在体外，400μm及以下浓度的CPSI-1306对软骨细胞没有毒性，进一步图2采用5/10/20μΜ浓度梯度检测CPSI-1306对IL-1β诱导的软骨细胞退变的作用。图2的结果显示IL-1β处理导致了软骨细胞合成代谢指标下调(COL2A1和ACAN)，而分解代谢指标上调(MMP13，MMP3，Adamts4和Adamts5)；而CPSI-1306的处理上调合成代谢指标，下调分解代谢指标，且与浓度相关。该结果表明在体外，CPSI-1306可以有效缓解IL-1β诱导的软骨细胞退变。
实施例2
一、实验方法：
1、提取C57/BL6小鼠的原代骨髓源巨噬细胞：取8周龄C57/BL6乳鼠处死，75％乙醇消毒后取双下肢，用PBS冲洗软骨3次，用剪刀打开骨髓腔，用1ml注射器吸取培养基反复冲洗每个骨髓腔3次，取冲洗液吹打重悬细胞，加入红细胞裂解液15分钟裂解红细胞，1000r离心5min。弃上清，1ml培养基重悬细胞，移至10cm的培养皿中，加入10ml添加有50ng/ml浓度GM-CSF的10％血清α-MEM培养基，置于37℃、5％CO2及饱和湿度条件下培养。
2、细胞增殖实验CCK-8：
将一代的巨噬细胞以2000个/孔数量接种在96孔板中，24小时后分别加入浓度为0，25/8ng/ml，25/4ng/ml，25/2ng/ml，25ng/ml，50ng/ml，100ng/ml，200ng/ml和400ng/ml的CPSI-1306溶液。培养24小时后，加入20μL CCK-8试剂盒37℃孵育2小时后用荧光酶标仪检测OD值。
3.qPCR检测
将对数生长期的巨噬细胞以3×105/well种植于12孔板中，于37℃，5％CO2培养箱中孵育过夜；设立Control组，LPS处理组(100ng/ml)，LPS+CPSI-1306处理组(浓度梯度分别为5μΜ，10μΜ，20μΜ)；干预24小时后用Trizol法提取软骨细胞的mRNA，然后使用HiFiScript cDNA第一链合成试剂盒合成cDNA，继续按照使用SYBR Green法进行qPCR操作流程检测软骨细胞内的炎症指标(iNOS，IL-1β和TNF-α)的表达。
二、实验结果：
图3所示在体外，400μm及以下浓度的CPSI-1306对BMMs细胞没有毒性，进一步图4采用5/10/20μΜ浓度梯度检测CPSI-1306对LPS诱导的M1型巨噬细胞的作用。图4的结果显示LPS处理导致了巨噬细胞炎症指标上调(iNOS，IL-1β和TNF-α)；而CPSI-1306的处理下调炎症指标，且与浓度相关。该结果表明在体外，CPSI-1306可以有效抑制LPS诱导的M1型巨噬细胞介导的炎症。
实施例3
1、OA动物模型的建立
将8周的C57/BL6小鼠按照5只每笼饲养于SPF级别动物房中，控制合适的温度、湿度和光照周期，给予充足的鼠粮和水。小鼠在手术前一周进行环境适应。用2％的戊巴比妥纳，剂量45mg/kg体重腹腔注射，右膝剃毛后分别用75％乙醇和碘伏消毒伤口，为手术做准备。
手术组：(1)膝髌韧带与内侧副韧带之间切开结缔组织，暴露关节囊。(2)切开关节囊，暴露关节腔，切断内侧半月板平台韧带，后切除内侧半月板。(3)逐层缝合，消毒。
假手术Sham组：只打开皮肤和关节腔，生理盐水冲洗切口后缝合。
2、注射液的制备
依序添加每种溶剂制备溶剂并作为Vehicle注射液：10％DMSO、40％PEG300、5％Tween-80、45％saline。
将CPSI-1306按照1mg/ml的浓度溶于上述溶剂中，得到CPSI-1306注射液。
3、OA动物模型的治疗实验组：使用微量注射器将CPSI-1306注射液10ul注射到手术组的C57/BL6小鼠的膝关节上，每三天注射一次，连续注射两个月；每只小鼠的单个关节腔注射的CPSI-1306注射液中的CPSI-1306质量为10μg。
对照组：使用微量注射器将Vehicle注射液10ul注射到手术组的C57/BL6小鼠的膝关节上，每三天注射一次，连续注射两个月。
3、结果分析
两个月后，将实验组、对照组和假手术组的小鼠处死，取出膝关节进行番红固绿染色和苏木素伊红染色，并在显微镜下观察软骨和滑膜组织学特征。
如图5为实验组、对照组和假手术组的小鼠膝关节番红固绿染色，如图可见对照组中软骨的番红着色较少，软骨基质流失，软骨表面破裂，实验组和假手术组的软骨的番红着色较深，软骨基质保留，软骨表面完整，说明CPSI-1306能够有效的减少软骨基质流失。如图6为实验组、对照组和假手术组的小鼠滑膜苏木素伊红染色，如图可见对照组中滑膜增生并出现大量炎症细胞浸润，实验和假手术组的滑膜增生较少且炎症细胞浸润少，说明CPSI-1306能够有效的减少OA中的滑膜炎症。
以上所述，仅为本发明优选实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到的变化或替换，都应涵盖在本发明的保护范围之内。因此，本发明的保护范围应该以权利要求的保护范围为准。
Claims (4)
1.一种如下图所示的小分子化合物巨噬细胞迁移抑制因子MIF抑制剂CPSI-1306，在制备预防和治疗骨关节炎的药物中的应用。
2.一种权利要求1所述的小分子化合物CPSI-1306，在制备巨噬细胞迁移抑制因子MIF靶向药物用于预防和治疗骨关节炎中的应用。
3.一种权利要求1所述的小分子化合物CPSI-1306，在制备软骨细胞保护药物的应用。
4.一种权利要求1所述的小分子化合物CPSI-1306，在制备骨关节炎的滑膜炎症抑制剂的药物中的应用。
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| 邝立鹏: "巨噬细胞移动抑制因子与膝骨关节炎的相关性研究", 《现代诊断与治疗》, vol. 24, no. 4, pages 726 - 728 * |
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