CN117599043A - 二氢异香豆素及其衍生物在制备用于治疗骨科疾病的药物中的应用 - Google Patents
二氢异香豆素及其衍生物在制备用于治疗骨科疾病的药物中的应用 Download PDFInfo
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- CN117599043A CN117599043A CN202311567867.0A CN202311567867A CN117599043A CN 117599043 A CN117599043 A CN 117599043A CN 202311567867 A CN202311567867 A CN 202311567867A CN 117599043 A CN117599043 A CN 117599043A
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Abstract
本发明属于医药领域，公开了二氢异香豆素及其衍生物在制备用于治疗骨科疾病的药物中的应用，二氢异香豆素及其衍生物是Compoud1‑5中的任意一种；其中，Compoud1是二氢异香豆素，Compoud2是7‑羟基香豆素，Compoud3是7‑甲氧基香豆素，Compoud4是4‑羟基香豆素，Compound 5是4,7‑二甲氧基香豆素。骨科疾病包括骨质疏松症、骨坏死、佩吉特病。Compound4的作用靶点为GZMA和ANGPTL7，Compound4下调GZMA表达，上调ANGPTL7表达。本发明Compound1‑5对骨质疏松症、骨坏死、佩吉特病等骨科疾病具有潜在的治疗作用。
Description
技术领域
本发明属于医药领域，特别涉及二氢异香豆素及其衍生物在制备用于治疗骨科疾病的药物中的应用。
背景技术
美洲大蠊是中国古代医书《神农本草》中记载的一种药用昆虫，在中国被用于治疗瘀血、溃疡、骨质疏松和烧伤等。研究表明美洲大蠊提取物中包含多巴胺、香豆素、二肽、维生素、有机酸、氨基酸及其代谢物等有机化合物，美洲大蠊提取物(PAE)作为中药用于治疗发热、疼痛、炎症、消化道疾病、慢性心力衰竭、皮肤病变等疾病已有数百年的历史。
其中，康复新液是美洲大蠊提取的单药，具有促进愈合与对皮肤细胞的增殖、对成纤维细胞的趋化和迁移、促血管生成和刺激生长因子分泌等作用，有研究表明康复新液对创面愈合、溃疡有治疗作用，并对人牙髓干细胞有促进作用，但其作用机制尚不明确。
发明内容
本发明的目的在于提供二氢异香豆素及其衍生物在制备用于治疗骨科疾病的药物中的应用。本发明从康复新液中提取出二氢异香豆素及其衍生物(Compound1-5)，通过对小鼠骨髓间质干细胞进行破骨诱导，在诱导过程中加入康复新液提取物，CCK-8法评价(Compound1-5)对破骨细胞的作用，首次发现这些化合物有抑制破骨细胞的增殖，同时我们通过转录组测序挖掘出Compound4的潜在作用靶点。骨的平衡是由成骨细胞介导的骨形成与破骨细胞介导的骨吸收的连续循环的过程，若这个平衡被打破，就会形成一系列骨科疾病，如骨质疏松症、骨坏死、佩吉特病等骨骼疾病[9]。本发明发现康复新液提取物Compound1-5对骨质疏松、骨坏死等骨科疾病的治疗具有重要意义。
为达到上述目的，本发明采用以下技术方案：
本发明提供了二氢异香豆素及其衍生物在制备用于治疗骨科疾病的药物中的应用，所述二氢异香豆素及其衍生物是Compoud1-5中的任意一种；其中，Compoud1是二氢异香豆素，Compoud2是7-羟基香豆素，Compoud3是7-甲氧基香豆素，Compoud4是4-羟基香豆素，Compound 5是4,7-二甲氧基香豆素。
在以上技术方案中，所述骨科疾病包括骨质疏松症、骨坏死、佩吉特病。
在以上技术方案中，所述Compound1-5对破骨细胞的活性产生抑制效应，对破骨细胞的分化和增殖有抑制作用。
作为其中一种优选实施例，所述二氢异香豆素及其衍生物为Compoud4，即4-羟基香豆素。
在以上技术方案中，Compound4的作用靶点为GZMA和ANGPTL7，Compound4下调GZMA表达，上调ANGPTL7表达。
本发明还提供了一种治疗骨科疾病的药物，所述药物中含有一种或多种二氢异香豆素及其衍生物，所述二氢异香豆素及其衍生物包括二氢异香豆素、7-羟基香豆素、7-甲氧基香豆素、4-羟基香豆素以及4,7-二甲氧基香豆素。
本发明的有益效果在于：本发明采用高效液相色谱法从康复新液中提取香豆素及其衍生物Compound1-5(二氢异香豆素、7-羟基香豆素、7-甲氧基香豆素、4-羟基香豆素、4,7-二甲氧基香豆素)。采用CCK8(cell Counting Kit-8)法和LDA实验评估细胞增殖，采用实时定量聚合酶链反应(RT-qPCR)和western blot检测破骨细胞标志物的表达水平。通过RNA测序结合在线数据库筛选出Compound4的作用靶点，并验证了其对关键基因的作用。Compound1-5对破骨细胞的增殖有抑制作用，其中Compound4(4-羟基香豆素)作用最强。筛选出Compound4(4-羟基香豆素)的潜在靶点GZMA(Granzyme A)和ANGPTL7(Angiopoietin-like 7)。Compound4下调GZMA表达，上调ANGPTL7表达。Compound1-5对骨质疏松症、骨坏死、佩吉特病等骨科疾病具有潜在的治疗作用。
附图说明
图1是Compound 1-5的化学结构式；
图2是Compound 1-5对破骨细胞的影响实验，
其中，(A)CCK-8实验检测compound 1-5对破骨细胞增殖水平的影响。(B)LDH实验检测compound 4对破骨细胞的杀伤作用。(C-E)qPCR检测compound4对破骨细胞标记物的影响。破骨细胞标志物CTSK、ACP5、TRAP相关基因mRNA表达，注:***,p<0.01；
图3是筛选Compound 4作用于破骨细胞的靶点实验，
其中，破骨细胞与compound4进行培养进行转录组测序，(A-B)差异表达基因总共有856个，其中包括496个上调基因和360个下调基因，(C)化合物compound4作用靶点与856个差异表达基因进行VENN图绘制得到73个共同基因，即为Compound 4作用于破骨细胞的靶点，(D)73个核心基因的PPI网络；
图4是Compound 4作用于破骨细胞的靶点的功能富集及验证实验，其中，(A)73个核心基因的KEGG富集分析，(B)73个核心基因的GO富集分析，(C)qPCR检测GZMA mRNA表达水平，(D)WB检测GZMA蛋白表达水平，(E)qPCR检测ANGPTL7 mRNA表达水平，(F)WB检测ANGPTL7蛋白表达，注:***,p<0.01。
具体实施方式
为了更好地说明本发明的目的、技术方案和优点，下面将结合具体实施例对本发明做进一步描述。本发明可以以许多不同的形式实施，而不应该被理解为限于在此阐述的实施例。相反，提供这些实施例，使得本公开将是彻底和完整的，并且将把本发明的构思充分传达给本领域技术人员，本发明将仅由权利要求来限定。
一实验方法
1、分离提纯的仪器以及方法
超高压液相(UltiMate 3000，THERMO)；高分辨质谱(X500R QTOF，AB SCIEX)；色谱柱(ACQUITY UPLC HSS T3 1.8μm 2.1×100mm，Waters)；乙腈(Acetonitrile，Merck，Darmstadt,Germany)；甲醇(Methanol，Merck，Darmstadt,Germany)；甲酸(Formic acid，CNW，Shanghai,China)；超纯水(Ultrapure water，Merck，Darmstadt,Germany)；乙酸铵(ammonium acetate，CNW，Shanghai,China)
2、分离提纯试剂、步骤
取200ul样品，加600ul甲醇。涡旋并离心15分钟。抽干上清，加入200ul 50％甲醇水溶解。过滤样品(0.22um有机滤膜)。使用AB X500R Triple TOF质谱仪检测样品。使用IDA(Information-Dependent Acquisition)功能采集质谱数据。
3、破骨细胞诱导
将RAW 264.7细胞在含有10％胎牛血清和1％青霉素-链霉素α-MEM中培养过夜(Meilunbio,MA0411)。为了诱导破骨细胞，在培养基中加入100μg/L RANKL进行培养。
4、破骨细胞的培养以及CCK8实验
将细胞密度标准调整为3×10^4个细胞/mL，将0.1mL细胞悬液接种到标记的96孔板中。每组各设5个重复孔，在37℃、5％ CO2条件下培养。分别于24h、48h和72h后去除上清，每孔用200uL无血清培养基洗涤1次。加入10μL检测试剂(CCK-8,Cell Counting Kit 8,ab228554,Abcam,1000T)和90μL完整培养基混合物。细胞在CO2培养箱中孵育2h，在450nm处测定每孔吸光度(OD)。
5、乳酸脱氢酶(LDH)实验
为每个反应制备50μL反应混合物，向每个标准、样品孔中加入50μL反应混合物，充分混合。在37℃避光条件下，在动力学模式下测量OD，并在线性范围内选择两个时间点(T1和T2)(OD值分别为A1和A2)来计算样品的LDH活性，反应时间为60min。测试样品中LDH的活性计算如下：
LDH Activity＝(B/ΔT*xV)*D＝nmol/min/ml＝mU/ml(B＝根据标准曲线计算的样品孔中NADH的量(nmol)；ΔT＝反应时间(分钟)；V＝加入反应孔的原始样品体积(mL)；D＝样品稀释系数)
6、Western blot
利用体外培养的破骨细胞提取蛋白，用BCA试剂盒测定蛋白浓度。提取的蛋白经过SDS-PAGE、膜转移和TBST洗涤。加入抗GZMA抗体(13075-1-AP,Proteintech,100ul)、抗ANGPTL7抗体(13588-1-AP,Proteintech,100ul)，用5％脱脂牛奶4℃封闭过夜。TBST洗膜3次，加入二抗(PR30012,Proteintech,5ml)，37℃孵育2h。PVDF膜上加入ECL发光液，凝胶成像系统照相。与内参蛋白的灰度比确定目标蛋白的相对含量。
7、qPCR
从PubMed获取目标基因的mRNA序列。使用Beacon Designer设计Q-PCR引物(F和R，如表1所示)。将动物组织(25-50mg)液氮研磨成粉末，加入含有450μl Buffer Rlysis-AG的1.5ml RNase-free离心管中，并震荡混匀。在室温下放置3分钟，避免RNA降解。以12,000rpm的速度在4℃下离心3分钟，将上清转移到新的离心管中。加入无水乙醇的一半体积，混匀。使用吸附柱去除DNA，加入GT Solution和NT Solution，按照流程离心，确保去除残余乙醇。使用DEPC处理的ddH2O洗脱RNA，并将RNA保存在-70℃。使用核酸蛋白定量仪检测RNA浓度和纯度。使用琼脂糖凝胶电泳检测RNA完整性，制作凝胶，然后使用凝胶成像仪拍照。根据逆转录反应所需的总RNA量计算所需体积，确保得到2μg总RNA来合成20μl的cDNA，然后配制反应液并按流程孵育以获得cDNA。进行实时荧光定量PCR，使用设计好的引物，分析基因表达水平。
表1.qPCR引物
8、转录组组测序及生物信息学分析
下机原始数据格式为fastq，使用fastp(https://github.com/OpenGene/fastp)软件对下机原始数据进行质控，包括去除接头、重复序列和低质量序列，参数为默认参数。使用HISAT2(https://ccb.jhu.edu/software/hisat2)将测序数据比对到基因组上(Homosapiens,GRCh38)，得到文件格式为bam。使用StringTie软件(https://ccb.jhu.edu/software/hisat2)对基因或转录本进行组装并用FPKM定量(FPKM＝total_exon_fragments/mapped_reads(millions)×exon_length(kB)])，使用R包edgeR(https://bioconductor.org/packages/release/bioc/html/edgeR.html)对样本之间的差异基因进行分析，差异倍数>2倍或<0.5倍，且p value<0.05定义为。最后使用DAVID软件(https://david.ncifcrf.gov/)对基因进行GO和KEGG富集分析。
9、统计分析
利用IBM公司的SPSS21.0软件对实验数据进行分析。实验重复三次，计算平均值和标准差。两组比较采用t检验；多组采用单因素方差分析(ANOVA)。p<0.05为差异有统计学意义，p<0.01为显著差异。
二实验结果
1、Compound1-5的1H和13C NMR数据及对破骨细胞活性的影响
从结果来看，美洲大蠊体内富含小分子类物质，这与虫药本身的成分特点一致，值得注意的是，我们的实验分离得到的二氢异香豆素类化合物，首次证实这类化合物对骨吸收的过程的影响，即抑制破骨细胞的分化。
香豆素是一种在美洲大蠊中已被发现的关键化合物，也是康复新液中已被认定的关键成分，起到关键作用。本发明通过高通量质谱分析方法分离出5种香豆素衍生物Compound1-5。
Compoud1(图1A)是一种浅黄色粘稠液体，其分子式为C9H8O2，我们分析正电高分辨质谱(HRESIMS)在m/z 332.1130[M+H]+(C9H8O2,012.1011),13C的核磁共振(NMR，NuclearMagnetic Resonance)光谱学和DEPT(Distortionless Enhancement by PolarizationTransfer)技术推断出该分子式。1H的核磁共光谱学的ABX自旋模式显示[δH 6.62(s),H-5；5.01(d,J＝7.4)H-1′]，值得注意的是Compoud1的13C NMR和DEPT光谱(表2)包含一个苯环和一个吡喃环，其中吡喃环上有一个酮基，显而易见，该结构与二氢异香豆素的结构一致。通过以上检测及推断确定Compoud1的结构，即Compoud1为二氢异香豆素。另外，在实验过程中发现，在骨吸收过程中，加入Compoud1对破骨细胞的形成及分化影响较小，但其衍生物对骨吸收过程具有一定的抑制作用(图2A)。
表2.Compound1-5的1H和13C NMR谱数据(1H使用600MHz，13C使用150MHz，溶剂为甲醇-d4，化学位移以ppm表示，耦合常数以Hz表示)。
表2续表
Compoud2(图1B)其分子式为C9H6O3，是一种与二氢异香豆素具有相似的结构。二者不同之处为(表2)，分子式中的氧原子数不同：Compoud2含有3个氧原子，而二氢异香豆素只含有2个氧原子。这一结论得到了δH 4.60–4.75(m)，H-3；3.10(dd,J＝15.7Hz,2.8Hz),H-4a；2.85(dd,J＝17.0Hz,11.8Hz),H-4b；6.58(s),H-5；2.05(s),H-11a；1.55(d,J＝5.9Hz),H-12a；5.15(d,J＝7.2Hz),H-1’。功能基团不同：Compoud2含有一个羟基(-OH)取代基，位于吡喃环上的位置7。而二氢异香豆素在吡喃环上没有羟基，它的基本结构是一个酮基(-C＝O)。由此确定Compoud2的结构，即7-羟基香豆素。这是一种在自然界中常见的二氢异香豆素衍生物，但在美洲大蠊中却是首次提取，且我们的实验发现其对破骨细胞具有抑制作用(图2A)。
Compoud3(图1C)为黄色胶体。通过分析其正电高分辨质谱(HRESIMS)m/z 376.57[M+H]+(C10H8O3,111.0427)，推导出其分子式为C10H8O30， 1H的NMR谱表现出典型的ABX自旋模式[δH 5.02(d，J＝7.6Hz)，H-1′；8.31(dd，J＝8.589Hz)，H-6′；6.86(d，J＝8.2Hz)。13C NMR和DEPT谱显示该分子由一个苯环和一个呋喃环组成，它们通过一个羰基(C＝O)连接在一起。在呋喃环的位置7，有一个甲氧基(CH3O)取代基。在A环中的取代模式不同，Compoud3存在一个甲氧基取代基。δH 4.51–4.63(m)，H-3；3.00(dd,J＝16.6,3.4Hz)，H-4a；2.91(dd,J＝16.4,11.4Hz)，H-4b；6.68(s)，H-5；4.78(d,J＝11.5Hz)，H-11a；4.64(d,J＝11.7Hz)，H-11b；172.2(C-1)，81.5(C-2)，34.2(C-3)，107.2(C-4)。从而确定Compoud3的结构，即7-甲氧基香豆素。最早，它是在植物中发现的一种天然化合物，也是首次从美洲大蠊中提取，作为二氢异香豆素衍生物的一种，被证实具有抗肿瘤作用。本发明的实验拓展了该化合物的作用，即对骨吸收具有抑制作用(图2A)。
Compoud4(图1A)为白色针状结晶，在m/z 205.0654[M+H](C9H6O3,215.0792)的正电高分辨质谱(HRESIMS)础上具有C9H6O3的分子式。1H的NMR谱包含了ABX自旋体系[δH 3.10(dd,J＝15.9,2.8Hz，H-4a)；2.80(dd,J＝17.0,12.2Hz,H-4b)；6.65(s)H-5]的核磁共振谱。13C的NMR和DEPT谱显示了9种碳，包括3种亚甲基、2种甲基和4种非质子化碳。详细的核磁共振解释表明，4个样品的核磁共振数据与二氢异香豆素的数据相似，从而确定Compoud4的结构，即4-羟基香豆素。值得注意的是，本研究中Compoud4对骨吸收的影响是几种衍生物中最强的，可明显抑制破骨细胞形成(图2A)。同时，我们通过LDH实验发现Compoud4对破骨细胞具有明显的杀伤作用(图2B)。
Compound 5(图1E)的分子式由其正电高分辨质谱(HRESIMS)在m/z222.059[M+H]+提供，为C11H10O4。我们确定为4,7-二甲氧基香豆素。对典型ABX自旋系统的检测支持了这种改变[δH3.20(dd,J＝16.5,3.0Hz,H-4a)；2.90(dd,J＝18.5,11.8Hz,H-4b)]。且我们的实验发现其对破骨细胞具有抑制作用(图2A)。
2、Compoud4对破骨标记物的影响
破骨细胞影响骨吸收和溶解，骨微环境的动态平衡是通过破骨细胞和成骨细胞在机体整个生命过程中的合作实现的[11]。因此，我们研究Compoud4对骨吸收和骨溶解的影响。采用qPCR方法检测结果表明，与对照组相比，Compoud4处理组的破骨细胞标记物CTSK、ACP5、TRAP等相关基因的mRNA表达得到抑制(图2C-E)。因此，这些结果表明Compoud4抑制骨吸收和溶解。
3、预测Compound4抑制对破骨细胞中的关键基因的影响
我们将Compoud4与破骨细胞培养，通过转录组测序发现(图3A-B)，加入Compoud4后，破骨细胞中MMP3、LGALS2、CXCL10、CD27、CXCL6、MMP1、GZMA、CXCL9、JCHAIN和CXCL13的表达下降，提示我们这些基因可能受到Compoud4的抑制。ZBTB16、ADH1B、ADIPOQ、PLIN1、ADH1C、GPD1、APOD、FABP4、PCK1和ANGPTL7表达上调，提示我们这些基因可能受到Compoud4的激动。
为了进一步筛选Compoud4作用于破骨细胞的靶点，我们通过4个化合物靶点预测数据库(http://ctdbase.org/、http://www.swisstargetprediction.ch/、http://bindingdb.org/bind/index.jsp、http://targetnet.scbdd.com/home/index/)预测Compoud4可有79个靶点基因。将79个基因与我们转录组测序的得到的差异基因进行VENN，得到73个核心基因(图3C)，即核心集群。值得注意的是，在核心集群中，GZMA和ANGPTL7这两个基因也在我们转录组测序结果中均呈现显著差异。这提示我们，GZMA和ANGPTL7可能是Compoud4作用于破骨细胞的关键靶点。有研究发现GZMA有调控促炎因子以及细胞凋亡的作用[11,12]；ANGPTL7的缺乏会抑制造血干细胞的再生能力[13]。但GZMA和ANGPTL7缺乏在破骨细胞中的相关研究。同时，通过STRING数据库，我们预测了这73个基因的蛋白互作关系(图3D)。
4、核心集群基因的功能富集分析
KEGG富集分析显示，这73个基因富集于Nitrogen metabolism、PI3K-Aktsignaling pathway、MAPK signaling pathway等信号通路(图4A)，GO分析结果发现这73个基因与protein serine/threonine/tyrosine kinase activity、positive regulationof kinase activity和protein autophosphorylation等相关(图4B)。
5、Compoud4抑制GZMA表达并上调ANGPTL7表达
接下来，我们分析Compoud4对关键靶点GZMA和ANGPTL7的作用。我们发现破骨细胞中加入Compoud4后，GZMA的表达水平明显受到的抑制(图4C-D)，而ANGPTL7的表达水平明显上调(图4E-F)。这提示我们Compoud4作用的潜在靶点是GZMA和ANGPTL7。
三结论
康复新液提取物成分含有香豆素(Coumarins)，香豆素是一类天然产物，属于苯并吡喃酮类化合物。它们在自然界中广泛存在于植物中，具有多种生物活性和药理作用,如抗氧化、抗炎、抗菌、抗血小板凝聚等。香豆素在药学和草药学中被广泛应用。它们可能对人体健康有益，可用于调节血液循环、抑制炎症反应、促进心血管健康等。本发明通过香豆素及其衍生物对破骨细胞的作用，对骨科疾病的防治与治疗有重要意义。
本发明从康复新液中提取出香豆素及其衍生物(compound1-5)等化合物，CCK8结果表明，Compound1-5对破骨细胞的活性产生抑制效应。这可能通过抑制破骨细胞的分化和活性，减少骨重吸收过程，从而对骨质丧失和骨疾病的进展产生积极的影响。其中compound4对破骨细胞的作用最强，本发明将Compoud4与破骨细胞培养后，通过转录组测序筛选出潜在作用靶点GZMA和ANGPTL7，在抑制破骨细胞后，GZMA表达下调，ANGPTL7表达上调，从而得出GZMA与骨吸收呈正相关，而ANGPTL7与骨吸收呈负相关。
显然，上述实施例仅仅是为清楚地说明所作的举例，而并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本发明创造的保护范围之中。
Claims (6)
1.二氢异香豆素及其衍生物在制备用于治疗骨科疾病的药物中的应用，其特征在于：所述二氢异香豆素及其衍生物是Compoud1-5中的任意一种；其中，Compoud1是二氢异香豆素，Compoud2是7-羟基香豆素，Compoud3是7-甲氧基香豆素，Compoud4是4-羟基香豆素，Compound 5是4,7-二甲氧基香豆素。
2.根据权利要求1所述应用，其特征在于：所述骨科疾病包括骨质疏松症、骨坏死、佩吉特病。
3.根据权利要求1所述应用，其特征在于：所述Compound1-5对破骨细胞的活性产生抑制效应，对破骨细胞的分化和增殖有抑制作用。
4.根据权利要求1所述应用，其特征在于：所述二氢异香豆素及其衍生物为Compoud4，即4-羟基香豆素。
5.根据权利要求4所述应用，其特征在于：Compound4的作用靶点为GZMA和ANGPTL7，Compound4下调GZMA表达，上调ANGPTL7表达。
6.一种治疗骨科疾病的药物，其特征在于：所述药物中含有一种或多种二氢异香豆素及其衍生物，所述二氢异香豆素及其衍生物包括二氢异香豆素、7-羟基香豆素、7-甲氧基香豆素、4-羟基香豆素以及4,7-二甲氧基香豆素。
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