CN114632080A - 尿石素b在制备防治骨质疏松的药物/保健品中的用途 - Google Patents
尿石素b在制备防治骨质疏松的药物/保健品中的用途 Download PDFInfo
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Abstract
本发明公开了尿石素B在制备防治骨质疏松的药物/保健品中的用途,尿石素B(UrolithinB,UB)是石榴、核桃和浆果中富含的鞣花单宁经肠道菌群转化生成的产物,具有抗炎抗氧化、抗癌等生物活性。在本发明研究中,我们用卵巢切除术(OVX)诱导的小鼠骨质疏松模型和体外骨髓源性巨噬细胞(BMMs)和RAW264.7细胞诱导的破骨细胞分化研究UB对骨质疏松的治疗作用,并进一步探讨了UB抑制破骨细胞分化的机制。经过本发明研究发现,尿素石B在制备防治骨质疏松的药物/保健品中具有可观的应用前景,并且其安全性较高。
Description
技术领域
本发明属于防治骨质疏松的药物和保健品领域,具体公开了尿石素B在制备防治骨质疏松的药物/保健品中的用途。
背景技术
随着人口老龄化的加重,骨质疏松症已成为严重威胁老年人群身体健康的慢性疾病之一,并带来沉重的社会医疗负担。骨质疏松症是由于各种原发或继发因素如长期应用糖皮质激素、雌激素减退、炎症的刺激导致的骨稳态异常,微观表现为破骨细胞的骨吸收活动异常活跃使骨重塑负向失衡。破骨细胞是一种来源于骨髓造血干细胞单核/巨噬细胞系的多核巨细胞,各种原发或继发因素的刺激可使其过度生成及活化。现阶段临床治疗骨质疏松的主要药物有双膦酸盐、特立帕肽、地舒单抗等,但是其在缓解骨丢失进展的同时,也会干扰骨骼的正常重建,长期用药亦会产生一系列并发症,如下颌骨坏死、非典型性骨折、增加骨肉瘤风险等。开发新的更为安全的药物成为骨质疏松症的迫切需求。
发明内容
为了解决上述问题,我们公开了尿石素B在制备防治骨质疏松的药物/保健品中的用途。尿石素B(UrolithinB,UB)是石榴、核桃和浆果中富含的鞣花单宁经肠道菌群转化生成的产物,具有抗炎抗氧化、抗癌等生物活性。在本发明研究中,我们用卵巢切除术(OVX)诱导的小鼠骨质疏松模型和体外骨髓源性巨噬细胞(BMMs)和RAW264.7细胞诱导的破骨细胞分化研究UB对骨质疏松的治疗作用,并进一步探讨了UB抑制破骨细胞分化的机制。
本发明的技术方案如下:
尿石素B在制备防治骨质疏松的药物/保健品中的用途。在一些实施例中, UB能缓解OVX小鼠骨质疏松症的进展,基于这些发现,我们认为UB是一种潜在安全有效的治疗骨质疏松症的选择。
进一步的,上述尿石素B在制备防治骨质疏松的药物/保健品中的用途,所述尿石素B抑制RANKL诱导的破骨细胞分化。在一些体外研究的实施例中,UB 可通过抑制ERK/NFκB信号通路抑制破骨细胞活化。
进一步的,上述尿石素B在制备防治骨质疏松的药物/保健品中的用途,所述尿素石B抑制破骨细胞的骨吸收功能。在一些实施例中,UB显著抑制破骨细胞介导的骨吸收坑形成,呈浓度依赖性降低,表明UB可在体外抑制破骨细胞的骨吸收功能。
进一步的,上述尿石素B在制备防治骨质疏松的药物/保健品中的用途,所述尿石素B下调破骨细胞相关基因和蛋白的表达;所述相关蛋白为MMP9或CTSK;所述相关基因为c-fos或NFATc1。在一些实施例中,破骨相关功能蛋白MMP9、 CTSK的表达经UB干预后呈浓度依赖性下调,同样,破骨细胞分化相关的转录因子c-fos、NFATc1的蛋白表达也被显著抑制。
进一步的,上述尿石素B在制备防治骨质疏松的药物/保健品中的用途,所述尿石素B通过抑制ERK/NF-κB信号通路抑制rankl诱导的破骨细胞形成。在一些实施例中,通过rt-PCR实验我们发现,相对于空白组,模型组在RANKL干预2天后相应的破骨相关基因的表达显著上升,而经UB平行干预的其他组中,破骨相关基因的表达呈浓度依赖性下降。
进一步的,上述尿石素B在制备防治骨质疏松的药物/保健品中的用途,所述尿石素B可缓解去势小鼠的骨丢失。在一些实施例中,UB处理过的OVX组小鼠的破骨细胞数量/骨表面(N.Oc/BS)和破骨细胞表面/骨表面(Oc.S/BS)显著降低,呈浓度依赖性。
进一步的,上述尿石素B在制备防治骨质疏松的药物/保健品中的用途,所述尿素石B的剂量为10mg/kg/2d-50mg/kg/2d。
进一步的,一种用于防治骨质疏松的药物/保健品,含有尿素石B。
进一步的,上述一种用于防治骨质疏松的药物/保健品,所述尿素石B为注射剂或口服制剂。
进一步的,上述一种用于防治骨质疏松的药物/保健品,所述尿石素是浆果中富含的鞣花单宁经肠道菌群转化生成的产物或者是化学合成的产物。
本发明具有如下有益效果:本发明首次公开了尿石素B在制备防治骨质疏松的药物/保健品中的用途,我们用卵巢切除术(OVX)诱导的小鼠骨质疏松模型和体外骨髓源性巨噬细胞(BMMs)和RAW264.7细胞诱导的破骨细胞分化研究UB 对骨质疏松的治疗作用,并进一步明确了UB抑制破骨细胞分化的机制;上述研究数据可以帮助研制包含尿石素B的用于治疗或者预防骨质疏松的药物以及保健品,用以生产新的更为有效和安全的治疗骨质疏松症的药物以及保健品,同时为进一步的研究新的具有协同作用的药物组合物打下了基础。
附图说明
附图1CCK8试验;
附图2BMMs细胞的TRAcP染色;
附图3MMP9免疫荧光染色;
附图4骨板吸收试验;
附图5Western blotting检测不同浓度UB(0、1、5、25μM)和RANKL处理后, mmp9、ctsk、c-fos、nfact1的表达;
附图6rt-PCR检测MMP9、CTSK、TRAP、ATPase、c-fos、NFATc1的表达;
附图7Western blotting检测RANKL不加/加UB刺激raw264.7细胞12h、1d、3d的mmp9、ctsk、c-fos、nfact1的表达;
附图8Western blotting检测pikbα、ikbα、pp65和p65的表达;
附图9Western blotting检测pjnk,jnk,pp38,p38,perk和erk的表达;
附图10药物干预30min时,pp65、p65、perk和erk的表达;
附图11OVX诱导的骨松模型小鼠股骨micro-CT;
附图12小鼠股骨H&E染色;
附图13小鼠股骨骨组织切片TRAcP染色;
附图14小鼠股骨MMP9和NFATc1的免疫荧光染色;
附图15ELISA法检测小鼠血清CTX-1;
附图16小鼠肝肾H&E染色。
具体实施方式
下面对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其它实施例,都属于本发明保护的范围。
本发明实施例中使用的试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规试剂产品。
材料和方法:
细胞培养
所有细胞均在37℃、5%CO2的湿润环境中培养,RAW264.7细胞(FuHeng BioLogy,ShangHai,China)在种板及药物干预前用含10%FBS (Gibco,Rockville,USA)和100u/mL青霉素-链霉素-两性霉素B(NCM Biotech, SuZhou,China)的DMEM(GE Healthcare,Pittsburgh,USA)培养基孵育传代,培养基每隔一天换一次。BMMs细胞取自6周龄的C57/BL6小鼠新鲜股骨,将麻醉处死的小鼠置于75%乙醇中消毒5min,剔净双下肢软组织并将股骨胫骨分离,用DMEM培养基漂洗两遍,在超净台内用手术剪剪开骨端,DMEM冲洗骨髓腔,冲洗液经70μm的细胞筛滤过后加入红细胞裂解液,1000r/min离心 5min,去上清并重悬,培养于含10%FBS、 50ng/mLM-CSF(R&DSystems,Minneapolis,MN,USA)和100u/mL青霉素-链霉素-两性霉素B的DMEM培养基内。
细胞活力测定
采用CCK-8活力测定法检测UB的细胞毒性。将BMMs细胞或RAW264.7 细胞以1×104/孔的密度接种在96孔板中3-4小时诱导培养后,用不同浓度的UB (0、1、5、25、50、100、150μmol/L)干预1、2或3天,每孔加入10μL的CCK-8 缓冲液(Beyotime,Shanghai,China),在37℃恒温箱内培养1小时后,使用酶标仪 (BioTek,Vermont,USA)在450nm波长下测量吸光度。
TRAP染色
将BMMs以1×105/孔的密度接种在12孔板中,与50ng/mL的M-CSF和 50ng/mL的RANKL一起孵育,并用0、1、5、25μmUrolithinB处理5天。用PBS 洗涤两次15分钟,用多聚甲醛固定后用TRAP染色试剂盒(Sigma,Missouri,USA) 进行染色拍照。使用ImageJ软件(NIH,Bethesda,Maryland,USA)测量每孔破骨细胞(具有三个以上细胞核的细胞)的平均面积。
免疫荧光染色
将BMMs细胞以1×105/孔的密度种于12孔板并以50ng/mL RANKL、 50ng/mLM-CSF和不同浓度UB(0、1、5、25μmol/L)干预一周,分化后的破骨细胞依次用PBS洗涤3次,4%多聚甲醛固定30分钟,Triton X-100渗透10分钟。然后用抗MMP9(1:1000,ab38898,Abcam)、一抗在4℃过夜,然后用Alexa Fluor 555和Molecular Probes Alexa Fluor 488Phalloidin(1:1000,Cell Signaling Technology,Danvers,USA)在黑暗中孵育1小时。用含DAPI的封片液固定10分钟后,使用EVOS M5000细胞成像系统(Thermo Fisher Scientific,Bothell,WA, USA)成像,量化分析荧光强度和每个破骨细胞的细胞核数量。
骨吸收试验
BMMs以每孔1×105个细胞的密度植入24孔胶原涂层板中,用UrolithinB (0、1、5、25μmol/L)和50ng/mLM-CSF,50ng/mLRANKL处理细胞。5天后,用显微镜记录骨吸收区域的图像,并通过ImageJ软件进行定量分析骨吸收面积。
蛋白质印迹分析
将RAW264.7以每孔1×105个细胞的密度种入6孔板并贴壁,用含50ng/mL RANKL和不同浓度UrolithinB(0、1、5、25μmol/L)的培养基处理相应时间后,加入裂解液,提取贴壁细胞的总蛋白并测定蛋白含量。在电泳仪中分离蛋白质样品,然后转移到PVDF膜(MerckMillipore,MA,USA)上,将膜用封闭液封闭15 分钟,并在4℃下与一级抗体孵育过夜。所用抗体如下:破骨相关功能蛋白: MMP9(1:1000,ab38898,Abcam)、CTSK(1:1000,ab19027,Abcam),转录因子: c-Fos(1:1000,ab190289,Abcam)、NFATc1(1:1000,ab25916,Abcam),以及MAPK、 NFκB通路相关蛋白抗体:JNK(1:1000,ab179461,Abcam)、p-JNK(1:1000,ab32503,Abcam)、and p38(1:1000,ab170099,Abcam)、p-p38(1:1000,#4511, CST)、ERK(1:1000,#4695,CST)、p-ERK(1:1000,#4377,CST)、IκB-α(1:1000, ab32518,abcam)、p-IκBα(1:1000,#2859,CST)、P65(1:1000,ab16502,Abcam)、 p-P65(1:1000,#3031,CST)。TBST(CWBiotech,Beijing,China)清洗后,用二级抗体孵育细胞膜2小时。最后,用化学发光HRP底物(Millipore Corporation,MA, USA)测定蛋白质。
定量RT-PCR分析
我们将BMMs以每孔1×105个细胞的密度接种于6孔板中,并培养于含 M-CSF(50ng/mL)、FBS(10%)、100u/mL青霉素-链霉素-两性霉素B和 RANKL(50ng/mL)的DMEM中。细胞贴壁后,分别用0、1、5和25μM的UB 处理细胞,两天后使用RNA快速提取试剂盒(ESScience,ShangHai,China)从细胞中提取总RNA,再将所提RNA用逆转录试剂盒(ABM,Vancouver,Canada) 逆转录为cDNA。然后,使用SYBR Green PCR MasterMix(AppliedBiosystems Vilnius,Lithuania)进行实时定量PCR。PCR循环条件为:94℃10min,95℃15s,60℃60s,循环40次,且所有反应均设置了4个复孔以确保数据的准确性。所用的qPCR引物为:
NFATc1:
正向5'-GACCCGGAGTTCGACTTCG-3'=SEQ ID NO:1
反向5'-TGACACTAGGGGACACATAACTG-3'=SEQ ID NO:2
CTSK:
正向5'-GAAGAAGACTCACCAGAAGCAG-3=SEQ ID NO:3
反向5'-TCCAGGTTATGGGCAGAGATT-3'=SEQ ID NO:4
c-fos:
正向5'-CGGGTTTCAACGCCGACTA-3'=SEQ ID NO:5
反向5'-TTGGCACTAGAGACGGACAGA-3'=SEQ ID NO:6
TRAP:
正向5'-CACTCCCACCCTGAGATTTGT-3'=SEQ ID NO:7
反向5'-CATCGTCTGCACGGTTCTG'=SEQ ID NO:8
液泡型ATPase(V-ATPase)d2:
正向5'-CAGAGCTGTACTTCAATGTGGAC-3'=SEQ ID NO:9
反向5'-AGGTCTCACACTGCACTAGGT-3'=SEQ ID NO:10
MMP9:
正向5'-CTGGACAGCCAGACACTAAAG-3'=SEQ ID NO:11
反向5'-CTCGCGGCAAGTCTTCAGAG-3'=SEQ ID NO:12
GAPDH:
正向5'-AGGTCGGTGTGAACGGATTTG-3'=SEQ ID NO:13
反向5'-TGTAGACCATGTAGTTGAGGTCA-3'=SEQ ID NO:14
动物造模及药物干预
本研究中的动物试验均获得张家港市中医院动物护理委员会研究所批准(批准文号:00000)。实验小鼠共40只,购自中国苏州JOINN实验室(批号: 202114550),均为六周龄,平均体重约20g。我们将小鼠随机分为4组:假手术组、OVX组、OVX+低剂量UB组、OVX+高剂量UB组。OVX组和OVX+UB 组小鼠均在麻醉下切除双侧卵巢及周围脂肪组织后缝合切口,假手术组小鼠仅将卵巢暴露并切除周围脂肪组织随后纳入腹腔缝合切口。我们的药物干预操作开始于造模术后的第三周,所用的干预药物UB购自Sigma-Aldrich。OVX+低剂量 UB组小鼠每两天腹腔注射UB10mg/kg,OVX+高剂量UB组小鼠每两天腹腔注射UB50mg/kg,假手术组和OVX组小鼠腹腔注射等量的生理盐水。我们在药物干预小鼠8周后对其实眼眶取血,离心后取上层血清行ELISA实验分析骨代谢标志物CTX-1含量,并将其股骨在4%多聚甲醛中固定48小时,并使用微计算机断层扫描(micro-CT)对其进行评估,随后脱钙行组织学和免疫荧光分析。
ELISA实验
我们用小鼠Ⅰ型胶原交联羧基端肽(CTXⅠ)酶联免疫吸附测定试剂盒(Elabscience,Wuhan,China)分析血清中骨代谢标志物CTx-1的含量,按照 ELLISA说明书于标准品孔中加入标准品100μL,待测样品孔中加入样本100μL,于37℃恒温箱孵育90分钟后弃去孔内液体,然后均加入100μL生物素化抗体工作液,37℃孵育60分钟,洗板三次后每孔加入100μL HRP酶结合物工作液,37℃孵育30分钟,弃掉板内液体,洗板5次,每孔加入90μL底物溶液,37℃孵育 15分钟左右,最后每孔加入50μL终止液。以空白孔调零,用酶标仪在450nm 波长下测定吸光度(OD值),计算样品浓度。
Micro-CT扫描
采用SkyScan1176高分辨率微计算机断层扫描仪(SkyScan,Knotich,Belgium) 获得4组小鼠(每组n=5)左股骨的μCT图像。扫描参数为每层9μm,电压80kV,电流100mA。然后将处理后的相关三维图像导入CTAn软件 (Brukermicro-CT,Kontich,Belgium),并测量分析股骨远端骨密度(BMD,g/cm3)、骨表面/骨体积(BS/BV,1/mm)、骨表面/总体积(BS/TV,1/mm)和骨小梁数目(Tb.th, mm)。
组织学和免疫组化分析
组织学和免疫组化分析在Micro-CT扫描完成后进行,将股骨用10%EDTA 脱钙21天,随后将股骨远端包埋在石蜡中,再用切片机将其切为5μm厚的切片。然后对切片进行苏木精伊红(H&E)、抗酒石酸酸性磷酸酶(TRAP)和免疫组化(IHC) 染色。H&E染色步骤:将切片脱蜡复水后,放入苏木精染色液中10min,流水冲洗至切片颜色变蓝,0.5%伊红染色液染色5min,依次经95%乙醇、无水乙醇脱水3min,二甲苯透明2次,每次5min,最后风干封片。TRAP染色步骤:将切片脱蜡复水,用TRAP染色试剂盒(Sigma,Missouri,USA)配置孵育液,注意避光和恒温37℃,切片置于TRAP孵育液内37℃孵育50min,蒸馏水漂洗,苏木素复染5min,流水冲洗10min,依次经95%乙醇、无水乙醇、二甲苯中5min脱水透明,最后风干封片。ELPS法(Enhance Labeled Polymer System)IHC染色步骤:将切片脱蜡复水,100μL过氧化氢阻断溶液孵育10min,PBS洗三次,每次5min,于100μL 5%BSA封闭液中孵育20min,加入50μL的MMP9(MA5-32705, Invitrogen)、NFATc1(A1539,ABclonal)一抗孵育12h,PBS清洗3次,每次5min,于50μL的二抗孵育30min,PBS洗三次,每次5min,加入DAB显色,流水冲洗后用苏木素复染30s,流水冲洗5min,盐酸酒精分化1s,流水冲洗反蓝10min,依次经95%乙醇、无水乙醇5min脱水,二甲苯浸泡三次,每次5min,最后封片。染色切片均使用Nikon Eclipse Ci-L光学显微镜(Nikon,Tokyo,Japan)拍照。使用全景组织细胞定量分析系统(TissueFAXS Plus,TissueGnostics GmbH,Austria) 对骨骼进行组织形态学分析。
统计学方法
本实验采用SPSS 19.0软件进行统计分析。所有实验数据均用均数±标准差 (M±SD)表示。两组间比较采用t检验,两组以上数据的比较采用单因素方差分析(ANOVA)。P<0.05表示差异具有统计学意义。
实施例1
UB抑制RANKL诱导的破骨细胞分化
我们通过CCK8试验发现,当UB浓度大于等于50μM时,细胞活力收到显著影响(图1)。接下来用UB(0,1,5和25μM)分别干预RANKL诱导的BMMs 细胞。TRAcP染色如(图2)所示,UB干预组的破骨细胞面积显著小于对照组,且呈浓度依赖性。通过免疫荧光染色我们也发现多核破骨细胞的形成受到UB干预的显著抑制(图3),RANKL组中的肌动蛋白环呈巨大的典型环状结构,而UB 高浓度组肌动蛋白环明显小且环内细胞核数量显著少于RANKL组。
实施例2
UB抑制破骨细胞的骨吸收功能
骨板吸收试验结果如图所示(图4),UB显著抑制破骨细胞介导的骨吸收坑形成,RANKL组与UB(0,1,5和25μM)干预组的骨吸收坑面积相对百分比分别为(51.25±8.07、29.44±5.39、18.83±5.52和4.84±2.70),呈浓度依赖性降低,表明UB可在体外抑制破骨细胞的骨吸收功能。
实施例3
UB下调破骨细胞相关基因和蛋白的表达
Westernblotting实验结果显示,破骨相关功能蛋白MMP9、CTSK的表达经 UB干预后呈浓度依赖性下调(图5)。同样,破骨细胞分化相关的转录因子c-fos、 NFATc1的蛋白表达也被显著抑制。通过rt-PCR实验我们发现,相对于空白组,模型组在RANKL干预2天后相应的破骨相关基因的表达显著上升,而经UB平行干预的其他组中,破骨相关基因的表达呈浓度依赖性下降(图6)。此外,我们也分别用RANKL和RANKL+UB(25μM)干预raw264.7细胞12h、1d、3d,结果显示,随着时间的延长,UB对RANKL诱导破骨相关蛋白的表达抑制愈加显著(图7)。
实施例4
UB通过抑制ERK/NF-κB信号通路抑制rankl诱导的破骨细胞形成
鉴于既往关于鞣花酸抑制破骨细胞活化的通路靶点为MAPKs和NF-κB通路 (在破骨细胞活化中的经典作用),我们也对MAPKs(包括ERK、JNK和P38)和 NF-κB通路相关蛋白的表达进行研究。定量分析表明,RANKL组加药后 phospho-P65表达水平上升并迅速达到高峰,而RANKL+UB组的phospho-P65表达水平显著低于平行时间的RANKL组且高峰期向后延迟。此外,我们也发现NFκB 的抑制蛋白IκBα的磷酸化和降解受到UB的显著抑制(图8)。接下来我们观察了MAPK通路相关蛋白的表达,在RANKL诱导的RAW264.7细胞中,phospho-ERK,phospho-JNK和phospho-p38的表达水平随干预时间的延长而升高,其中UB对 phospho-ERK的表达有显著下调的作用(图9)。此外,我们也看到在药物干预 30min时,ERK、P65和IκBα的磷酸化水平随着UB浓度的升高而降低(图10)。综上所述,UB可能通过ERK/NFκB信号通路抑制破骨细胞形成。
实施例5
UB可缓解去势小鼠的骨丢失
为了研究UB对去势小鼠的骨丢失的缓解效果,我们建立了OVX诱导的骨松模型。在造模开始及药物干预8周后,各组小鼠的体重差异均没有统计学意义,且药物干预组肝肾切片未见药物毒性所致病损,其中模型组小鼠8周后股骨远端的骨密度显著低于空白组(图11)。我们通过micro-CT对sham组、OVX组和 OVX+UB组小鼠的股骨远端结构参数进行分析。结果显示,OVX组相比于sham组小鼠股骨远端各项参数显著降低(BMD in g/cm3:1.11±0.05vs 1.55±0.05, BV/BV in%:5.55±0.85vs 16.55±1.55,Tb.th in mm:0.05±0.005vs 0.15±0.015,respectively),而UB干预组小鼠各项参数显著增加,呈浓度依赖性(图11)。H&E染色结果显示(图12),与假手术组相比,OVX组骨表面(BS) 明显减少(BS inmm2:15±0.005vs 5±0.015),而OVX+UB组骨表面(BS)呈浓度依赖性升高。骨组织切片TRAcP染色显示,OVX组较假手术组破骨细胞数量 /骨表面(N.Oc/BS)和破骨细胞表面/骨表面(Oc.S/BS)显著增加(N.Oc/BS in 1/ mm2:15.55±0.85vs 6.55±1.55;Oc.S/BS in%:5.55±0.85vs 16.55±1.55), UB处理过的OVX组小鼠的破骨细胞数量/骨表面(N.Oc/BS)和破骨细胞表面/骨表面(Oc.S/BS)显著降低,呈浓度依赖性,(图13)。破骨细胞功能蛋白MMP9和相关转录因子NFATc1的免疫荧光染色分析结果示:与假手术组相比,OVX组MMP9- 和NFATc1阳性细胞数量显著增加,而OVX+UB组与OVX组相比,MMP9-和NFATc1 阳性细胞数量显著减少(图14)。我们亦通过ELISA法检测小鼠血清骨吸收指标 CTX-1并进行组间比较,发现UB干预组血清CTX-1含量较模型组显著降低(图 15),各组间差异均有统计学意义。
总结:
综合实施例1-5可知
骨稳态失衡所致的骨质疏松症已成为当下不可忽视的公共健康问题,传统治疗骨质疏松症药物作用靶点主要为破骨细胞,但此类药物的长期应用也会带来副作用开发新的更为安全的药物成为骨质疏松症的迫切需求。天然化合物如甘草酸、葛根素等由于副作用小,正逐渐成为治疗骨质疏松症的潜在疗法。同样,从肠道菌群的代谢产物中寻找潜在的药物分子近年来也逐渐成为热点,如Dylan Dodd 等发现小鼠肠道内的孢子梭菌可通过代谢芳香族氨基酸产生吲哚丙酸,调节机体的免疫系统,也有学者通过研究发现肠道菌群代谢产物N-酰基酰胺是G蛋白偶联受体的配体类似物,并可以调节糖稳态。本发明通过研究石榴、核桃等富含鞣花丹宁的水果食物经肠道菌群代谢的产物UB对破骨细胞形成及活性的影响,以求为骨质疏松症的治疗提供新的潜在方向。
破骨细胞是人体中负责骨吸收的细胞,其骨吸收活动分为骨吸附、细胞骨架重组、囊泡运输等步骤。当破骨细胞黏附到骨表面后,通过细胞骨架重组形成的肌动蛋白环与骨表面紧密连接并构成一个酸性的骨吸收微环境。接下来,破骨细胞将破骨相关功能蛋白如CTSK、MMP9等通过囊泡运输至酸性吸收微环境内,再通过囊泡将骨降解产物内化。由此可见,破骨细胞吸附于骨表面的数量、面积及破骨相关功能蛋白的分泌对骨吸收活动同等重要。而在本研究中,我们发现 UB可浓度依赖性的抑制RANKL诱导的破骨细胞的形成,通过TRAcP染色可看到破骨细胞数量和面积随着干预药物UB浓度的升高显著减少。而通过免疫荧光染色我们也可以清楚看到破骨细胞肌动蛋白环的形成被UB显著抑制,同时功能蛋白MMP9的荧光信号也因药物干预减弱。WB与PCR实验结果也与此一致,相关功能蛋白的表达呈UB浓度依赖性降低,而骨板吸收试验也更直接的表明了 UB对破骨活性的抑制。综上所述,我们的结果证实UB可在体外抑制破骨细胞的形成及活化以抑制骨吸收活动。
NFκB和MAPK通路的激活对破骨细胞的形成和活化至关重要,在本研究中,Westernblotting结果示UB可以抑制IκBα的磷酸化及降解,同时p65的磷酸化也受到抑制。IκBα是IκB家族的典型成员,具有IκB蛋白的典型特征。典型的 p65/p50异源二聚体大部分与IκBα结合并处于未激活状态,而快速信号诱导的 IκBα蛋白的磷酸化及后续的降解是NF-κBp65核定位与DNA结合所必需的。除此以外,我们也观察到UB可以浓度依赖性地抑制MAPK通路中ERK蛋白的磷酸化以及下游破骨相关转录因子NFATc1、c-fos的表达。ERK通路中的各种上游刺激因子正调控破骨细胞的分化过程,多种药物及天然化合物如双膦酸盐、金丝桃素,已被报道可通过抑制ERK信号通路抑制破骨细胞形成。在UB对MAPK 通路的观察中,ERK的磷酸化水平在RANKL干预后15min达到高峰,而在UB 的药物作用下ERK的磷酸化水平被显著下调。可见,UB通过抑制ERK/NFκB 信号通路抑制破骨细胞的形成及活化。
雌激素减退导致破骨细胞的异常活跃可导致骨的快速丢失,本研究通过卵巢切除术诱导的小鼠骨质疏松模型验证了UB在体内对骨吸收活性的抑制作用。体内实验结果显示模型组小鼠股骨远端的骨密度显著低于空白组,表明本研究中 OVX小鼠骨丢失造模的成功,而对股骨远端的形态学和组织学观察进一步证实了体内的破骨细胞的形成及活化受到UB的抑制。同样,LIN等人通过腹腔注射干预的方式验证了鞣花酸可缓解卵巢切除术诱导的小鼠骨质疏松症,然而也有学者认为鞣花酸的生物利用率较低,对其潜在全身活性的研究应集中在生物利用率更高的衍生物UB上。UB已被证实在体内有较高的利用率,并可在体内达到对骨骼肌、白细胞、心肌细胞良性干预的相应浓度,而在本研究中药物治疗组OVX 小鼠经UB治疗后骨丢失得到明显缓解,血清中骨吸收特异性指标CTx-1的含量显著降低,且肝肾切片未见药物毒性病变(图16)。可见,UB在体内对破骨吸收活动的抑制安全有效。
综上所述,我们的体外研究表明,UB可通过抑制ERK/NFκB信号通路抑制破骨细胞活化。体内实验表明,UB能缓解OVX小鼠骨质疏松症的进展。基于这些发现,我们认为UB是一种潜在安全有效的治疗骨质疏松症的选择。
以上所述实施例仅表达了本发明的有限几种优选实施方式,其描述较为具体和详细,但并不能因此而理解为对本发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。
SEQUENCE LISTING
<110> 张家港市中医院
<120> 尿石素B在制备防治骨质疏松的药物/保健品中的用途
<130> 2022
<160> 14
<170> PatentIn version 3.5
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Claims (10)
1.尿石素B在制备防治骨质疏松的药物/保健品中的用途。
2.根据权利要求1所述的尿石素B在制备防治骨质疏松的药物/保健品中的用途,其特征在于,所述尿石素B抑制RANKL诱导的破骨细胞分化。
3.根据权利要求1所述的尿石素B在制备防治骨质疏松的药物/保健品中的用途,其特征在于,所述尿素石B抑制破骨细胞的骨吸收功能。
4.根据权利要求1所述的尿石素B在制备防治骨质疏松的药物/保健品中的用途,其特征在于,所述尿石素B下调破骨细胞相关基因和蛋白的表达;所述相关蛋白为MMP9或CTSK;所述相关基因为c-fos或NFATc1。
5.根据权利要求1所述的尿石素B在制备防治骨质疏松的药物/保健品中的用途,其特征在于,所述尿石素B通过抑制ERK/NF-κB信号通路抑制rankl诱导的破骨细胞形成。
6.根据权利要求1所述的尿石素B在制备防治骨质疏松的药物/保健品中的用途,其特征在于,所述尿石素B可缓解去势小鼠的骨丢失。
7.根据权利要求6所述的尿石素B在制备防治骨质疏松的药物/保健品中的用途,其特征在于,所述尿素石B的剂量为10mg/kg/2d-50mg/kg/2d。
8.一种用于防治骨质疏松的药物/保健品,其特征在于,含有尿素石B。
9.根据权利要求8所述的一种用于防治骨质疏松的药物/保健品,其特征在于,所述尿素石B为注射剂或口服制剂。
10.根据权利要求8所述的一种用于防治骨质疏松的药物/保健品,其特征在于,所述尿石素是浆果中富含的鞣花单宁经肠道菌群转化生成的产物或者是化学合成的产物。
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US20160045472A1 (en) * | 2013-01-18 | 2016-02-18 | Procell Sprl | Urolithin b for muscle growth |
US20200397747A1 (en) * | 2018-02-21 | 2020-12-24 | Daicel Corporation | Osteoclast differentiation inhibitor containing urolithin |
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US20160045472A1 (en) * | 2013-01-18 | 2016-02-18 | Procell Sprl | Urolithin b for muscle growth |
US20200397747A1 (en) * | 2018-02-21 | 2020-12-24 | Daicel Corporation | Osteoclast differentiation inhibitor containing urolithin |
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YAJUN LI等: "Urolithin B suppressed osteoclast activation and reduced bone loss of osteoporosis via inhibiting ERK/NF-κB pathway" * |
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