CN117257827A - 石斛多糖治疗年龄相关性黄斑病变的应用及药物 - Google Patents
石斛多糖治疗年龄相关性黄斑病变的应用及药物 Download PDFInfo
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Abstract
本发明提供一种石斛多糖治疗年龄相关性黄斑病变的应用及药物,该药物包括石斛多糖,所述石斛多糖包括氨基葡萄糖、鼠李糖、氨基半乳糖,所述石斛多糖的浓度为10‑25mg/ml,属于药物应用领域。本发明的有益效果为:为治疗年龄相关性黄斑病变提供新的治疗方案。
Description
技术领域
本发明涉及一种治疗年龄相关性黄斑病变的药物,具体涉及一种石斛多糖治疗年龄相关性黄斑病变的应用及药物。
背景技术
年龄相关性黄斑病变,又叫老年性黄斑变性AMD,为黄斑区结构的衰老性改变,AMD是一种使中心视力进行性、不可逆性丧失的疾病,严重威胁着老年人的视力,多发生于年龄>50岁的人群,并且随着年龄增大,发病率逐渐增高。随着我国经济、医疗卫生水平的提高及人均寿命的增加,AMD患病率也随之上升,其主要发病原因为RPE细胞(视网膜色素上皮细胞)的氧化损伤。因此,寻找一种天然无毒副作用,且能够修复损伤的RPE细胞的药物则对治疗年龄相关性黄斑病变至关重要。
发明内容
为解决现有技术中的问题,本发明提供一种石斛多糖治疗年龄相关性黄斑病变的应用,还提供一种治疗年龄相关性黄斑病变的药物。
本发明提供一种石斛多糖治疗年龄相关性黄斑病变的应用。
本发明作进一步改进,所述石斛多糖由氨基葡萄糖、鼠李糖、氨基半乳糖组成。
本发明作进一步改进,所述石斛多糖为铁皮石斛提取物。
本发明还提供一种治疗年龄相关性黄斑病变的药物,所述药物包括石斛多糖,所述石斛多糖包括氨基葡萄糖、鼠李糖、氨基半乳糖,所述石斛多糖的浓度为10-25mg/ml。
本发明作进一步改进,所述石斛多糖的浓度为15mg/ml。
本发明作进一步改进,所述药物的剂型为中药汤剂、熏蒸剂、眼膏剂或丸剂。
与现有技术相比,本发明的有益效果是:拓展石斛多糖治疗年龄相关性黄斑病变新应用,为治疗年龄相关性黄斑病变提供新的治疗方案,该原料能够从铁皮石斛提取,原料获取方便,对损伤的RPE细胞修复作用明显,有效的抑制了年龄相关性黄斑病变。
附图说明
为了更清楚地说明本申请或现有技术中的方案,下面将对实施例或现有技术描述中所需要使用的附图作一个简单介绍,显而易见地,下面描述中的附图是本申请的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为通过不同浓度碘酸钠处理HRPE细胞24h对细胞活性的影响示意图;
图2为根据细胞氧化损伤情况选择最适年龄相关性黄斑病变模型实验柱状图;
图3-图5为最适年龄相关性黄斑病变模型下,三次测量不同时间段加入本发明药物24H后对细胞活性的影响示意图;
图6-图8为不同浓度药物三次测量治疗效果示意图;
图9-图11为CCK8实验验证药物有效性实验效果示意图;
图12为ROS实验验证药物有效性实验效果示意图;
图13为ORAC实验验证药物有效性实验效果示意图;
图14为GSH含量测量结果示意图;
图15为溶酶体脂褐素的测量结果示意图;
图16为基因转录组学分析结果示意图;
图17和图18为SHH与CNTF基因的关系示意图;
图19和图20为CNTF基因通过JAK激酶/STAT3蛋白通路调节RPE细胞活性实验结果示意图图;
图21-23为SHH作用基因通路实验验证结果示意图;
图24为SHH作用通路验证实验结果示意图。
具体实施方式
除非另有定义,本文所使用的所有的技术和科学术语与属于本申请技术领域的技术人员通常理解的含义相同;本文中在申请的说明书中所使用的术语只是为了描述具体的实施例的目的,不是旨在于限制本申请;本申请的说明书和权利要求书及上述附图说明中的术语“包括”和“具有”以及它们的任何变形,意图在于覆盖不排他的包含。本申请的说明书和权利要求书或上述附图中的术语“第一”、“第二”等是用于区别不同对象,而不是用于描述特定顺序。
在本文中提及“实施例”意味着,结合实施例描述的特定特征、结构或特性可以包含在本申请的至少一个实施例中。在说明书中的各个位置出现该短语并不一定均是指相同的实施例,也不是与其它实施例互斥的独立的或备选的实施例。本领域技术人员显式地和隐式地理解的是,本文所描述的实施例可以与其它实施例相结合。
为了使本技术领域的人员更好地理解本申请方案,下面将结合附图,对本申请实施例中的技术方案进行清楚、完整地描述。
现代药理研究表明,石斛总多糖可抑制脂质过氧化、提高抗氧化能力和减少氧化产物生成纠正氧化/抗氧化失衡状态。
据此,本发明提出一种石斛多糖治疗年龄相关性黄斑病变的应用,并根据该应用提供一种包含所述石斛多糖、用于治疗年龄相关性黄斑病变的药物。然后通过实验验证本发明用于治疗年龄相关性黄斑病变的有效性。
优选的,本发明的石斛多糖选用天然的铁皮石斛提取物,铁皮石斛提取出的石斛多糖由氨基葡萄糖,鼠李糖,氨基半乳糖组成,其中,
氨基葡糖糖具有明显的抗RPE(视网膜色素上皮)细胞氧化功效;
氨基半乳糖含有作为动物细胞和组织支撑的物质糖蛋白,保护和润滑软组织;
鼠李糖具有排毒和平衡、改善肠胃的作用;
本发明通过氨基葡萄糖,鼠李糖,氨基半乳糖共同作用,鼠李糖促进肠胃吸收氨基葡萄糖与氨基半乳糖,扩大抗氧化、眼部软组织保护功能,且鼠李糖的排毒作用,有效降解氨基半乳糖对肝脏的伤害。从而三者结合,能够有效地提高RPE细胞抗氧化能力及氧化损伤修复能力,进而降低RPE细胞的氧化损伤,保证RPE细胞能够为视网膜外层组织提供营养、维持正常的新陈代谢,因为RPE细胞正常的新陈代谢,吞噬和消化光感受器外节盘膜的功能正常运行,才能够避免年龄相关性黄斑病变的形成。
本发明将通过研究不同浓度的石斛总多糖能否有效降低RPE细胞的氧化损伤,从脂质过氧化、抗氧化能力和减少氧化产物等角度研究其对AMD的治疗作用及相关机制。
本发明探索最适浓度碘酸钠伤害HRPE细胞制造AMD氧化损伤模型,然后提取铁皮石斛的石斛多糖(SHH),配成不同浓度的水剂,然后找出最适浓度的石斛总多糖(SHH)治疗AMD,本发明的具体实验方法包括如下步骤:
1、寻找最适碘酸钠伤害HRPE细胞的浓度,制造AMD模型;
2、确定HRPE细胞加入碘酸钠伤害后细胞活性稳定不变的时长;
3、探索提高HRPE细胞活性最佳浓度;
4、由步骤1探索出的最适碘酸钠损伤步骤2确定的时长,然后加入步骤3探索出的最佳活性浓度治疗,通过观察细胞活性,验证SHH治疗效果;
5、验证SHH治疗RPE细胞碘酸钠损伤模型对细胞器功能性方面产生的作用;
6、基因测序实验,验证SHH作用机理。
以下结合具体实验数据对本发明的实验步骤进行详细说明。
1、CCK8初步选定HRPE细胞制造AMD氧化损伤模型浓度范围
CCK8实验意义说明:CCK-8试剂(Cell Counting Kit-8细胞计数试剂)中含有WST–8:化学名:2-(2-甲氧基-4-硝基苯基)-3-(4-硝基苯基)-5-(2,4-二磺酸苯)-2H-四唑单钠盐,它在电子载体1-甲氧基-5-甲基吩嗪硫酸二甲酯(1-Methoxy PMS)的作用下被细胞线粒体中的脱氢酶还原为具有高度水溶性的黄色甲臜产物(Formazan),生成的甲臜物的数量与活细胞的数量成正比。用酶联免疫检测仪在450nm波长处测定其光吸收值(OD),可间接反映活细胞数量,也就是OD值越高,活细胞越多。
ROS实验意义说明:ROS越高,氧化严重,说明细胞氧化损伤的越多。
ORAC实验意义说明:表示对自由基的抗氧化能力,ORAC结果越高,说明抗氧化能力越强。
本发明选择不同浓度的碘酸钠(0mM-15mM)处理HRPE细胞(10000个/孔细胞数)24h对细胞活性的影响,其中碘酸钠浓度梯度为:0mM、5mM、6mM、7mM、8mM、9mM、10mM、11mM、12mM、13mM、14M、15mM。
实验结果如图1所示,由纵坐标OD值可知,使细胞半数致死的碘酸钠浓度在0-7mM范围内,故选择该浓度范围的碘酸钠损伤模型进一步展开ROS实验。
本例选择检测碘酸钠浓度(0mM~7mM)处理HRPE细胞(10000个/孔细胞数)24h后ROS值,选定最适造模浓度。
如图2所示,本例通过ROS实验进行实验,选择最适造模浓度。纵坐标为MFI,MFI就是平均荧光强度(Mean fluorescence intensity),用于表示某个指标的流式检测最终的荧光强度,可根据MFI值的大小,表示ROS的水平。ROS越高,氧化严重,说明细胞氧化损伤的越多。图2中,浓度为5mM ROS检测出现锋值时,细胞过氧化损伤最明显。
因此,AMD模型条件为:碘酸钠浓度5mM处理HRPE细胞(10000个/孔细胞数)24h。
2、确定HRPE细胞加入碘酸钠伤害后细胞活性稳定不变的时长
本例将HRPE细胞加入NaIO35mM(0,1s,1min,5min,10min,15min,20min)不同时间段加入药,24h后对HRPE细胞(10000个/孔细胞数)活性的影响(CCK8),从而确定损伤稳定时间。
如图3-5所示,HRPE细胞(10000个/孔细胞数),加入5mM碘酸钠伤害(1min,5min,10min,15min,20min)后,于37度培养箱培养24h测CCK8,三次测量结果一致,其细胞活性稳定,故选择5mM碘酸钠伤害HRPE细胞20min后再加SHH治疗24小时。
3、探索提高HRPE细胞活性最佳浓度
本例做实验用的SHH药物用培养基直接溶解,配置成不同浓度的药物,也可以将铁皮石斛多糖标准品加水或生理盐水溶解(如果为注射或滴剂则选用生理盐水配置),分别配置成不同浓度的SHH药物,本实验分别选用0mg/ml,5mg/ml,10mg/ml,15mg/ml,20mg/ml,55mg/ml处理hRPE细胞24h对HRPE细胞(10000个/孔细胞数)活性的影响(CCK8),从而确定最适治疗浓度。
如图6-8的实验结果可知,HRPE细胞加入SHH(5mg/ml,10mg/ml,15mg/ml,25mg/ml)相比对照组(SHH浓度0mg/ml)均有所提高,且加入SHH浓度为15mg/ml时细胞活性达到最高,所以选择15mg/ml浓度的SHH作为最适治疗浓度。
4、SHH效果验证
由步骤1探索出的最适碘酸钠损伤步骤2确定的时长,然后加入步骤3探索出的最佳活性浓度治疗,通过观察细胞活性,验证SHH治疗效果,本例采用CCK8、ROS、ORAC三种实验分别进行验证。
造模:碘酸钠浓度5mM处理HRPE细胞(10000个/孔细胞数)24h;
治疗:SHH浓度为15mg/ml处理造模细胞24h;
CCK8实验验证结果如图9-11所示,HRPE细胞加入5mM碘酸钠造模20min后加入浓度为15mM的SHH,可获得最佳治疗结果。
如图12所示,ROS实验验证结果中,纵坐标为MFI(平均荧光强度,Meanfluorescence intensity),通过MFI值的大小,可以看出,在碘酸钠溶液中,细胞氧化损伤最高,在碘酸钠加SHH溶液中,细胞氧化损伤降低,说明加入SHH后,能够提高细胞的氧化损伤修复能力。
如图13所示,ORAC实验验证结果中,SHH药物中,纵坐标ORAC值最高,说明自由基的抗氧化能力最强。
通过上述实验可知,本例SHH能够有效地提高RPE细胞抗氧化能力及氧化损伤修复能力,进而降低RPE细胞的氧化损伤,从而可以用于治疗因RPE细胞的氧化损伤造成的年龄相关性黄斑病变。本例除了加水或生理盐水直接制成水剂外,还可以制备成口服丸剂,还可以制备成熏蒸剂、滴眼剂,直接作用于人眼,上述剂型采用传统的制备方法即可,在此不再赘述。
5、验证SHH治疗RPE细胞碘酸钠损伤模型对细胞功能性分子GSH、细胞器等方面产生的作用
(1)GSH含量测量
如图14所示,当加入SSH药物后,细胞功能性分子GSH的含量由2.8提升到4.8,含量显著增加,可以说明当SHH作用氧化损伤的RPE细胞时,可增加其抗氧化能力。
(2)溶酶体脂褐素的测量
脂褐素也叫衰老素,脂褐素降低表示抗氧化能力增加。
如图15所示,通过三种柱状图对比可知,c为对照组Control的缩写,n为碘酸钠NaIO3的缩写,n+s为碘酸钠NaIO3+SSH的缩写,当加入SSH药物后,最后一个柱状的脂褐素lipo含量明显降低,说明SHH做用于氧化损伤的RPE细胞可增加其抗氧化、抗衰老能力。
6、基因测序实验,验证SHH作用机理
(1)基因测序实验
本发明通过将对照组、损伤模型组(NaIO3)、治疗组(NaIO3+SSH)进行转录测序实验,首次筛选调控RPE细胞活性的差异基因,查询结果如表1:
表1调控RPE细胞活性的差异基因列表
基因转录组学分析结果见表1和图16,CNTF基因的基因差异最大,达到两倍多,测序结果,筛选调控RPE细胞活性的CNTF基因进行QPCR(Real-time Quantitative PCRDetecting System,即实时荧光定量核酸扩增检测***)及WB实验验证。
QPCR实验结果如图17可知,纵坐标代表基因层面mRNA差异,横坐标代表样品的类别,通过验证可知,与Control相比较,NaIO3组CNTF基因水平mRNA表达量增多,NaIO3+SSH组CNTF基因水平mRNA表达量降低。
WB实验是通过聚丙烯酰胺凝胶电泳(PAGE)将混合蛋白质样品分离后,利用特殊虹吸或电场装置印迹至固相介质(例如PVDF膜)上,再以固相载体上的蛋白质或多肽作为抗原。与相对应的第一抗体特异性结合,之后再与酶或同位素标记的第二抗体相结合,最终通过底物显色或放射自显影来检测特异性目的基因表达的蛋白成分含量。如图18所示,纵坐标表示密度差异,通过验证可知,与Control相比较,NaIO3组CNTF蛋白表达量减少,NaIO3+SSH组CNTF蛋白表达量增多。
通过上述两个实验可知,SHH治疗碘酸钠损伤模型时,CNTF蛋白增加,CNTF基因mRNA降低,维持CNTF蛋白表达量在人体中稳定状态。即SHH对细胞中CNTF实施负反馈调节。
(2)CNTF基因通过JAK激酶/STAT3蛋白通路调节RPE细胞活性,并对通路进行验证
通过查阅相关文献(Vigneswara,Vasanthy et al.“Combined suppression ofCASP2and CASP6 protects retinal ganglion cells from apoptosis and promotesaxon regeneration through CNTF-mediated JAK/STAT signalling.”Brain:a journalof neurology vol.137,Pt 6(2014):1656-75.doi:10.1093/brain/awu037)可知,CNTF基因能够通过JAK/STAT3通路调节RPE细胞活性,以下对其通路进行QPCR、WB实验验证。
QPCR实验结果如图19所示,加入CNTF外源性后QPCR结果显示JAK1与JAK2均上调,且JAK1更为明显,故选定JAK1与STAT3(P)进行WB蛋白验证。WB实验结果如图20所示,加入外源性CNTF后WB结果显示JAK1与STAT3(P)均上调,通过两个实验结果验证可知,QPCR与WB结果一致,即CNTF作用通路为:CNTF-JAK1-STAT3(p)。
(3)验证SHH作用于基因,通过上述通路调节RPE细胞活性
本发明同样分别通过QPCR和WB实验进行验证。所述QPCR实验结果如图21所示,加入SHH后QPCR结果显示JAK1与JAK2均上调,且JAK1更为明显,WB实验结果如图22和图23所示,加入外源性SHH后WB结果显示JAK1、JAK2与STAT3(P)均上调,通过两个实验结果验证可知,QPCR与WB结果一致,且JAK1激酶变化更为明显,故选择
SHH-CNTF-JAK1-STAT3(P)进行WB验证,结果如图24所示,SHH治疗后,CNTF、JAK1、STAT3(P)同时增加,由此可以得出,本发明SHH作用的通路为:SHH-CNTF-JAK1-STAT3(P)。
综上所述,本发明通过实验验证SHH能够治疗损伤的RPE细胞,有效的抑制了年龄相关性黄斑病变,并通过基因实验验证了其治疗原理,从而本发明寻找到了一种天然无毒副作用的物质用于治疗年龄相关性黄斑病变新应用,效果明显,相比较西医或手术治疗,本发明安全性更高,为治疗年龄相关性黄斑病变提供新的治疗方案。
以上所述之具体实施方式为本发明的较佳实施方式,并非以此限定本发明的具体实施范围,本发明的范围包括并不限于本具体实施方式,凡依照本发明所作的等效变化均在本发明的保护范围内。
Claims (8)
1.一种石斛多糖治疗年龄相关性黄斑病变的应用。
2.根据权利要求1所述的应用,其特征在于:所述石斛多糖由氨基葡萄糖、鼠李糖、氨基半乳糖组成。
3.根据权利要求2所述的应用,其特征在于:所述石斛多糖为铁皮石斛提取物。
4.一种治疗年龄相关性黄斑病变的药物,其特征在于:所述药物包括石斛多糖,所述石斛多糖包括氨基葡萄糖、鼠李糖、氨基半乳糖,所述石斛多糖的浓度为10-25mg/ml。
5.根据权利要求4所述的药物,其特征在于:所述石斛多糖的浓度为15mg/ml。
6.根据权利要求4所述的药物,其特征在于:所述药物的剂型包括水剂、熏蒸剂、滴眼剂或丸剂。
7.根据权利要求4所述的药物,其特征在于:所述药物对CNTF基因产生负反馈调节,通过所述CNTF基因调节视网膜色素上皮细胞的细胞活性。
8.根据权利要求7所述的药物,其特征在于:所述CNTF基因通过JAK/STAT3通路调节RPE细胞活性,所述药物的作用通路为:SHH-CNTF-JAK1-STAT3。
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