CN115531396A - 胆酸类物质在制备抑制破骨细胞分化的药物中的应用 - Google Patents
胆酸类物质在制备抑制破骨细胞分化的药物中的应用 Download PDFInfo
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Abstract
本发明公开了胆酸类物质在制备抑制破骨细胞分化的药物中的应用,属于医药领域。本发明证明了脱氢胆酸等胆酸类物质能有效抑制破骨细胞的生成与分化及其作用机制。本发明能够为骨质疏松患者寻求安全有效的治疗药物提供重要依据,且有重大的临床意义和广阔的市场前景。
Description
技术领域
本发明涉及胆酸类物质在制备抑制破骨细胞分化的药物中的应用,属于医药领域。
背景技术
骨质疏松是一种以单位体积内骨量减少为特点的代谢性骨病变,是由于成骨细胞介导的骨形成与破骨细胞介导的骨吸收失平衡所致。骨质疏松多发于中老年人,绝经后的女性发病率高。虽然死亡率不高,但致残率极高,严重影响患者的生活质量和身心健康。目前,骨质疏松症临床治疗多采用双膦酸盐、降钙素、选择性雌激素受体调节剂和denosumab,然而,大多数药物都有严重的副作用或不适合长期使用。因此,迫切需要研发一种安全、有效的能通过抑制破骨细胞的分化和功能来治疗骨质疏松的药物。
脱氢胆酸是胆酸合成衍生物,作为一种利胆药,多用于治疗胆囊及胆道功能失调和胆结石等疾病。迄今,脱氢胆酸在骨质疏松症中的作用尚不清楚。
发明内容
为解决现有技术存在的问题,本发明提供了胆酸类物质在制备抑制破骨细胞分化的药物中的应用。本发明首先采用抗酒石酸酸性磷酸酶染色对破骨细胞进行了鉴定,采用CCK8法检测脱氢胆酸对破骨细胞的毒性,并通过抗酒石酸酸性磷酸酶染色筛选出能有效抑制破骨细胞生成及分化的有效浓度,再通过qRT-PCR验证脱氢胆酸抑制破骨细胞分化的有效性,最后通过Western Blotting探究脱氢胆酸抑制破骨细胞分化的作用机制。
本发明提供了胆酸类物质在制备抑制破骨细胞分化的药物中的应用。
本发明还提供了胆酸类物质在制备预防或治疗骨质疏松的药物中的应用。
进一步地,上述技术方案中,将胆酸类物质制备成单一化学成分的药物制剂,或与其他药物合用制备成复方药物制剂。
进一步地,上述技术方案中,所述胆酸类物质包括脱氢胆酸(CAS号:81-23-2)。
进一步地,上述技术方案中,所述药物的剂型包括片剂、丸剂、散剂、胶囊。
进一步地,上述技术方案中,所述药物的服用剂量为:每日用量10-20mg/kg。
进一步地,上述技术方案中,在制备用于抑制与破骨细胞分化成熟相关的ACP5基因表达的药物中的应用;在制备用于抑制与骨吸收功能相关的CTSK及MMP9基因表达的药物中的应用。
进一步地,上述技术方案中,在制备用于抑制TGF-β蛋白表达药物中的应用;在制备用于提升TRAF3蛋白表达药物中的应用。
进一步地,上述技术方案中,在制备用于提升OPG蛋白表达药物中的应用;在制备用于抑制RANK蛋白表达药物中的应用。
具体方法和结果如下:
细胞毒性实验:将不同浓度的脱氢胆酸(10、50、100、200、500、1000μM)作用于破骨细胞细胞。CCK8法检测脱氢胆酸作用于破骨细胞4天的细胞毒性,筛选出最大的无毒剂量进行后续的药效研究。
药效实验:首先采用10、50、100、150、200μM浓度的脱氢胆酸作用于破骨细胞4天,采用抗酒石酸酸性磷酸酶染色筛选脱氢胆酸抑制破骨细胞生成最佳浓度。采用最大的无毒剂量作用于破骨细胞4天后提取破骨细胞RNA进行qRT-PCR检测ACP5、CTSK和MMP9 mRNA表达
探究作用机制实验:采用最大的无毒剂量作用于破骨细胞4天后提取破骨细胞蛋白,检测破骨细胞中RANK/OPG及TGF-β/TRAF3蛋白表达量,探究脱氢胆酸抑制破骨细胞分化的作用机制。
发明有益效果
本发明采用脱氢胆酸作用于提取培养的破骨细胞,证明脱氢胆酸能有效抑制破骨细胞的分化及其作用机制。本发明能够为骨质疏松患者寻求安全有效的治疗药物提供重要依据,且有重大的临床意义和广阔的市场前景。
附图说明
图1为脱氢胆酸结构图。
图2A为脱氢胆酸毒性实验,与未加脱氢胆酸组相比,**P<0.01;图2B为抗酒石酸酸性磷酸酶染色,脱氢胆酸对破骨细胞形成的抑制作用,在200μM浓度的抑制作用最强。
图3为破骨细胞中ACP5、CTSK和MMP9 mRNA水平,与未加脱氢胆酸组相比,**P<0.01。
图4为破骨细胞中RANK、OPG、TGF-β、TRAF3蛋白表达水平,与未加脱氢胆酸组相比,**P<0.01。
具体实施方式
下述非限定性实施例可以使本领域的普通技术人员更全面地理解本发明,但不以任何方式限制本发明。
实施例1
下面结合具体实施案例对本发明做进一步阐述。
1.材料
1.1实验动物
本实验中所使用的SPF级健康雄性6周龄C57BL/6小鼠(全部购自大连医科大学实验动物中心),小鼠体重20g左右。
1.2主要试剂与药品
GM-CSF购自大连美仑生物技术有限公司;RANKL购自美国MCE公司;RNA抽提试剂盒、Evo M-MLV反转录试剂盒和Green Pro Taq HS预混型qPCR试剂盒均购自湖南艾克瑞生物有限公司;TGF-β、OPG和β-actin抗体购自英国Abcam公司;RANK抗体购自中国正能生物有限公司;TRAF3抗体购自武汉三鹰生物技术有限公司;抗酒石酸酸性磷酸酶染色试剂盒购自美国GENMED公司;脱氢胆酸购自美国Panphy化学品公司。
2.方法
2.1小鼠骨髓巨噬细胞分离培养
小鼠在麻醉下颈部脱臼处死,75%酒精浸泡消毒。在无菌超净台内分离出胫骨和股骨去除胫骨和股骨上的肌肉,骨头取出来后放在PBS中。剪断骨骺端,用PBS轻轻冲洗股骨和胫骨的髓腔,直到骨头变半透明,反复吹打成细胞悬液,用100μm细胞筛网过滤至15mL离心管中,1000rpm离心5min弃液。加入5mL红细胞裂解液重悬细胞并轻轻吹打,4℃静置10min,1000rpm离心5min,弃液,加入PBS清洗2次。用DMEM培养基(10% FBS+20ng/mL GM-CSF)重悬细胞,转移到培养瓶中,37℃、5%CO2培养3天,得到骨髓巨噬细胞。
2.2细胞毒性检测
对小鼠中获取的骨髓巨噬细胞进行破骨细胞诱导分化实验。将骨髓巨噬细胞培养在DMEM(10% FBS+20ng/mL GM-CSF+50ng/mL RANKL)完全培养基中,诱导其分化为破骨细胞。实验组每孔分别加入10、50、100、200、500、1000μM的脱氢胆酸;培养4天后均加入100μLCCK-8试剂,孵育2h,应用酶标仪450nm波长下测定各孔OD值。
2.3破骨细胞的分化实验
将骨髓巨噬细胞培养在DMEM(10% FBS+20ng/mL GM-CSF+50ng/mL RANKL)完全培养基中,诱导其分化为破骨细胞。采用不同浓度的脱氢胆酸(0、10、50、100、150、200μM)分别干预细胞。在脱氢胆酸作用破骨细胞4天后通过抗酒石酸酸性磷酸酶染色试剂盒评估破骨细胞,显微镜下细胞核数量≥3的细胞则视为破骨细胞,分别统计其数量。
2.4qRT-qPCR
向破骨细胞中加入1mL TRizol裂解液使样本充分裂解,加入200μL氯仿试剂涡旋混合在手中用力震荡15s(10次),室温静置5min。12000rpm(4℃)离心15min取上层水相置于新EP管中。加500μL异丙醇,充分混匀,室温静置10min。12000rpm(4℃)离心10min。弃上清液,保留下层白色的RNA沉淀。加入1mL 75%乙醇进行洗涤,12000rpm(4℃)离心5min,弃上清。让沉淀的RNA在室温下自然干燥,待乙醇挥发后加入RNase-free水溶解沉淀,得到总RNA溶液。采用Evo M-MLV反转录试剂盒将RNA反转录为cDNA,保存于-20℃。所有引物均按照qPCR引物设计原则,由生工生物工程(上海)股份有限公司设计合成,具体引物序列如表1所示。按照Green Pro Taq HS预混型qPCR试剂盒说明书进行qPCR反应,结果采用2-△△Ct法进行分析。
表1.引物序列
2.5 Western blotting分析
采用Western blotting法检测TGF-β、TRAF3、OPG、RANK蛋白。培养至破骨细胞分化成熟,使用含有1%磷酸酶抑制剂、0.1%蛋白酶抑制剂、1% PMSF的100μLRIPA裂解液裂解细胞,提取细胞蛋白。根据凯基生物的BCA蛋白浓度测定试剂盒,测量各组蛋白的浓度,并且使用裂解液调整为统一的浓度。在蛋白样本中加入适量1×dye,加热10min使蛋白变性。配置10% SDS-PAGE分离胶,每孔加入等量总蛋白(30μg)的样品溶液,加样完成后,连接电源进行电泳,80V,30min跑浓缩胶,换120V跑分离胶,至溴酚蓝刚跑出即可终止电泳。根据目标蛋白的分子量,切胶,按照黑面-海绵-滤纸-胶-PVDF膜-滤纸-海绵-白面顺序,将转膜夹插入转膜槽,220mA在冰上转膜1h,结束取出PVDF膜。用5%脱脂奶粉封闭1h,TBST洗10min×3次,按说明书稀释一抗TGF-β、TRAF3、OPG、RANK(1:2000),内参β-actin(1∶5000),4℃孵育过夜,第二天TBST洗10min×3次后在室温下孵育二抗(1∶10000)1h,加入ECL化学发光液显影曝光。根据灰度值数据分析各组蛋白相对表达量。
统计学分析
使用GraphPad Prism 8.4.3,SPSS 24.0软件进行数据分析及作图。用t检验分析两组间的显著性差异。P<0.05为差异有统计学意义。
3结果
3.1脱氢胆酸抑制破骨细胞的生成
为了研究脱氢胆酸抑制破骨细胞生成的有效浓度,首先进行了细胞毒性检测,如图2A所示,结果显示脱氢胆酸在浓度达到500μM时,细胞活力显著降低。之后采用10、50、100、150、200μM药物浓度分别干预破骨细胞的生成与分化。抗酒石酸酸性磷酸酶染色结果如图2B所示,与未加脱氢胆酸组相比,脱氢胆酸在低浓度(10μM)已开始抑制破骨细胞的形成,在200μM浓度的抑制作用最强,抑制作用呈剂量依赖性。
3.2脱氢胆酸抑制破骨细胞分化及功能相关基因的表达
我们选取200μM脱氢胆酸干预破骨细胞分化,采用qRT-PCR技术检测破骨细胞特异性基因ACP5、CTSK和MMP9的表达水平;结果如图3所示,与未加脱氢胆酸组相比,200μM脱氢胆酸干预组有效抑制与破骨细胞分化成熟相关的ACP5和与骨吸收功能相关的CTSK及MMP9基因的表达。
3.3脱氢胆酸通过调控TGF-β/TRAF3蛋白表达,抑制破骨细胞分化
采用Western Blot检测TGF-β、TRAF3蛋白表达量,结果如图4所示,与未加脱氢胆酸组相比,200μM脱氢胆酸干预组显著抑制TGF-β蛋白表达,而TRAF3蛋白表达量显著升高。
3.4脱氢胆酸通过调控RANK/OPG信号通路,抑制破骨细胞分化
通过Western Blot检测RANK/OPG信号通路相关蛋白RANK、OPG表达情况。结果如图4所示,与未加脱氢胆酸组相比,200μM脱氢胆酸干预组OPG蛋白表达量显著升高,并且脱氢胆酸能够抑制RANK蛋白的表达。
结论
骨质疏松是老年人常见的一种代谢性骨骼疾病。随着人口老龄化趋势日益明显,骨质疏松发病率亦不断提高。骨质疏松是由于破骨细胞功能亢进,引起骨量失衡,导致骨质疏松。尽管各种相关诊疗观念和技术不断改进,现有药物仍不能达到理想的治疗效果。小鼠骨髓巨噬细胞分离培养以及破骨细胞分化实验,是目前研究抑制破骨细胞分化机制的常用体外细胞实验。应用脱氢胆酸作用于破骨细胞后,可明显抑制破骨细胞的生成及破骨细胞分化与功能相关基因的表达,能通过调控RANK/OPG及TGF-β/TRAF3信号通路,抑制破骨细胞分化,根据以上实验结果,证实脱氢胆酸具有抑制破骨细胞生成及分化的作用。
上述实施例只是用于对本发明的举例和说明,而非意在将本发明限制于所描述的实施例范围内。此外本领域技术人员可以理解的是,本发明不局限于上述实施例,根据本发明的教导还可以做出更多种的变型和修改,这些变型和修改均落在本发明所要求保护的范围内。
Claims (9)
1.胆酸类物质在制备抑制破骨细胞分化的药物中的应用。
2.胆酸类物质在制备预防或治疗骨质疏松的药物中的应用。
3.根据权利要求1或2所述的应用,其特征在于,将胆酸类物质制备成单一化学成分的药物制剂,或与其他药物合用制备成复方药物制剂。
4.根据权利要求1或2所述的应用,其特征在于,所述胆酸类物质包括脱氢胆酸。
5.根据权利要求1或2所述的应用,其特征在于,所述药物的剂型包括片剂、丸剂、散剂、胶囊。
6.根据权利要求1或2所述的应用,其特征在于,所述药物的服用剂量为:每日用量10-20mg/kg。
7.根据权利要求1或2所述的应用,其特征在于,在制备用于抑制与破骨细胞分化成熟相关的ACP5基因表达的药物中的应用;在制备用于抑制与骨吸收功能相关的CTSK及MMP9基因的表达的药物中的应用。
8.根据权利要求1或2所述的应用,其特征在于,在制备用于抑制TGF-β蛋白表达药物中的应用;在制备用于提升TRAF3蛋白表达药物中的应用。
9.根据权利要求1或2所述的应用,其特征在于,在制备用于提升OPG蛋白表达药物中的应用;在制备用于抑制RANK蛋白表达药物中的应用。
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