CN116236513A - 黑沙蒿根提取物在制备抗类风湿关节炎药物中的应用 - Google Patents
黑沙蒿根提取物在制备抗类风湿关节炎药物中的应用 Download PDFInfo
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Abstract
本发明提供了一种黑沙蒿根提取物在制备抗类风湿关节炎药物中的应用,其中,所述黑沙蒿根提取物为黑沙蒿根的乙酸乙酯提取物。本发明从分子蛋白的层面阐述黑沙蒿根乙酸乙酯提取物发挥抗类风湿关节炎的作用机制以及在免疫方面产生的作用,拓宽了黑沙蒿疾病治疗的范围(其他自身免疫性疾病),促进了黑沙蒿的现代化研究。
Description
技术领域
本发明属于医药领域,涉及一种中草药的新型提取物在制备抗类风湿关节炎药物中的用途。
背景技术
类风湿关节炎(rheumatoid arthritis,RA)是一种以侵蚀性关节炎为主要临床表现的自身免疫病,其基本病理变化是关节滑膜的慢性炎症增生,形成血管翳,侵犯关节软骨、软骨下骨、韧带和肌腱等,造成关节软骨、骨和关节囊破坏,最终导致关节畸形和功能丧失,并且还可引发肺部、心血管等方面的疾病。流行病学调查显示,类风湿关节炎的全球发病率为0.5%~1%,我国发病率为0.42%,总患病人群约500万,其中男女患病比率约为1:4,且患病率呈逐年上升的趋势。
类风湿关节炎通常以隐匿的症状发作为特征,但随着时间的推移,疾病会进展并逐渐恶化,是发生在滑膜和滑液中的免疫过程。在临床发病时,早期类风湿关节炎阶段出现大量的白细胞迁移到关节处,在关节内形成的免疫复合物(ICs)可以激活补体级联,进一步激活/招募白细胞和间质细胞。其他先天免疫细胞,主要是单核细胞/巨噬细胞和自然杀伤细胞,也被招募到滑膜上到达滑膜内层。最终,长期的炎症环境导致常驻的滑膜成纤维细胞(FLSs)的生物学发生根本变化,使其具有不受控制增殖的肿瘤样特征。而这种免疫细胞的不断浸润和FLS的不断增殖,最终导致了滑膜增生。此外FLS过度增殖则会导致组织局部缺氧,产生了血管生成的强烈触发因素。与此同时,FLS、浸润性巨噬细胞局部产生促血管生成因子,会进一步促进血管翳的生成。当血管翳充分形成后,FLS等滑膜组织细胞会进一步增加促炎介质的分泌,例如细胞炎性因子、趋化因子等,进而招募更多的免疫细胞浸润,扩大炎症,产生了炎症的恶性循环。在血管翳—软骨界面处,破骨细胞被激活,滑膜组织则会分泌各种基质金属蛋白酶(MMP),溶解并入侵富含胶原蛋白的软骨,进而进入到侵蚀性类风湿关节炎阶段,产生骨破坏。总体来说,类风湿关节炎滑膜组织的病理特征为:FLS的异常增殖产生的滑膜增生、血管翳的形成、免疫细胞的浸润。
临床中已有多种药物可进行类风湿关节炎的治疗。首先是非甾体抗炎药物(如,塞莱希布、布洛芬等),通过抑制环氧合酶,减少炎症来减轻疼痛,常用于关节炎急性期反应;其次是传统合成类风湿药(包括甲氨蝶呤、来氟米特、羟氯喹等)通过抑制细胞增殖、减少自身抗体产生、减少炎症因子的释放、抑制B细胞成熟等不同途径治疗类风湿关节炎,常作为一线治疗药物。当一线治疗不耐受或无效时,则使用靶向合成类风湿药(枸橼酸托法替布等)或生物制剂类风湿药(阿达木单抗等)。而糖皮质激素(如,泼尼松、地塞米松等)可减少循环系统中单核-巨噬细胞的数量,前列腺素的合成,进而产生强有力的抗炎效果和免疫调节作用,同时还能防止炎症细胞的渗出,减少破骨细胞的生成,减少关节软骨的破坏,具有抗炎和免疫抑制作用,常用作辅助药物治疗。尽管如此,类风湿关节炎的临床治疗效果仍不理想,甚至会引发严重的不良反应,并对患者造成极大的经济负担,故而研究一种新的抗炎治疗药物手段迫在眉睫。
近几年,随着中医药的不断发展,许多中草药材的研究也越发广泛,其中治疗类风湿关节炎的中草药物备受关注。譬如常见的雷公藤、白芍、伸筋草等均具有抗炎活性,可在一定程度上抑制白三烯、前列腺素等炎症介质的表达。黑沙蒿(Artemisia ordosicakrasch.)也称鄂尔多斯蒿,为菊科蒿属植物,味辛苦,性微温,具有杀“粘”、清热、燥黄水、愈伤、生肌、合脉止血、消肿、软便之功效。《内蒙古中草药》中记载:其根具有止血,治鼻衄、吐血、功能性子宫出血的功效。黑沙蒿全草以萜类、黄酮类、挥发油、香豆素类等化学成分为主,大多具有清热解毒、抗菌消炎、祛风除湿、提高免疫、通经活络、活血止血等功效。
发明内容
类风湿关节炎作为一种自身免疫性疾病,临床表现为体内免疫机制失衡,长期的炎症环境导致了体内相关的炎症因子呈上升趋势,为此,降低炎症因子的血清水平是治疗类风湿关节炎的一条有效通路(临床生物制剂)。此外,抑制关节部位的相关蛋白表达也可间接的控制类风湿关节炎的发生发展:例如,①STAT3是类风湿关节炎发病过程中一个关键致病因子,是JAK的下游信号蛋白,影响细胞的生存、生长和分化,且与破骨细胞形成密切相关。STAT3可抑制FLS的凋亡,促进血管的生成,其过度表达会使类风湿关节炎进一步恶化。有研究表明类风湿关节炎大鼠的滑膜组织高表达STAT3,进而促进了FLS的增殖存活。②Caspase-3在细胞凋亡中起着关键的作用。当细胞接受凋亡刺激时,Caspase-3将被活化,最终导致细胞凋亡。因此Caspase-3的表达与类风湿关节炎的发生发展密切相关。③Galectin-3可促进中性粒细胞激活、脱粒和超氧化物产生,还能促进单核细胞趋化性、巨噬细胞活化,在先天免疫反应的发展中起关键作用,是细胞黏附的调节剂。研究通过mRNA和蛋白质组学分析表明,FLS是关节内Galectin-3的重要来源,Galectin-3在类风湿关节炎和骨关节炎患者的发炎滑膜中高度表达和分泌,可刺激FLS产生IL-6、粒细胞-巨噬细胞集落刺激因子、CXCL8、MMP3等相关因子,在激活和释放炎症因子的过程中起着至关重要的作用。类风湿关节炎中FLS数量的增加也是趋化因子的丰富来源,负责关节内细胞的招募和保留,而Galectin-3有助于类风湿关节炎中FLS的激活。因此,Galectin-3过表达导致活化的FLS和滑膜巨噬细胞对凋亡不敏感。在未成熟小鼠中关节内注射Galectin-3导致肿胀和骨关节样病变,进一步强调了关节内Galectin-3的存在与滑膜炎症和软骨/骨退化相关。④S100A9蛋白主要在先天免疫细胞中表达。在炎症条件下,S100A9由中性粒细胞和单核细胞大量释放,随后诱导信号通路触发炎症反应,例如趋化性、吞噬细胞迁移和各种巨噬细胞功能的调节。类风湿关节炎病情的不断发展与关节部位炎细胞的不断浸润有密切的关系,S100A9主要由浸润在关节部位的中性粒细胞,单核细胞等白细胞释放,部分还由FLS释放产生,能增强炎症细胞的跨内皮迁移,并诱导先天免疫细胞分泌炎症和趋化因子,招募更多的炎细胞到达关节部位,产生炎症恶性循环,是关节炎起始和进展的关键参与者。研究通过直接抑制S100A9可减少小鼠LPS-CIA模型中的肿胀并保护了骨/软骨的完整性,推测抗S100A9治疗效果可能是抑制S100A9介导的促进中性粒细胞转运和单核细胞促炎细胞因子分泌,进而抑制免疫反应的放大。
因此,本发明首先通过口服黑沙蒿根乙酸乙酯提取物进行抗类风湿关节炎的药效学研究,确定其是否具有抗类风湿关节炎的效果。随后,对关节部位STAT3、Galectin-3、S100A9、凋亡蛋白caspase-3蛋白的表达,以及血清中炎症因子TNF-α、IL-1β、IL-6、IL-17A和抑炎因子IL-10浓度进行考察,明确黑沙蒿根乙酸乙酯提取物发挥抗类风湿关节炎效果的作用机制,拓宽类风湿关节炎的药物治疗范畴(其他自身免疫疾病),促进黑沙蒿根的现代化研究。
具体的,本发明首先提供黑沙蒿根提取物在制备抗类风湿关节炎药物中的应用,其中,所述黑沙蒿根提取物为黑沙蒿根的乙酸乙酯提取物。
其中,所述黑沙蒿乙酸乙酯提取物通过以下步骤制备:取黑沙蒿根粉,并加入黑沙蒿根粉10倍重量的质量分数为95%的乙醇溶液超声提取3次,每次30 min,将获得的提取液置于60℃下浓缩干燥35min。随后将浓缩物用其10倍重量的蒸馏水溶解,依次用等体积的石油醚、乙酸乙酯和正丁醇各萃取3次,得到石油醚层萃取物、乙酸乙酯层萃取物、正丁醇层萃取物及水溶物;收集其中的乙酸乙酯层萃取物,减压浓缩(去除溶剂),98℃下干燥,即得黑沙蒿根乙酸乙酯层提取物。
优选的,所述黑沙蒿根提取物的有效施用剂量为100-800 mg/kg。更优选的,所述黑沙蒿根提取物的有效施用剂量为400 mg/kg。
进一步,所述黑沙蒿根提取物通过影响关节部位的STAT3、Caspase-3、Galectin-3和S100A9蛋白的表达,来有效抑制类风湿关节炎的病情发展。
同时,本发明还提供了一种抗类风湿关节炎的药物,其中,该药物包括上述黑沙蒿根的乙酸乙酯提取物。
再进一步的,本发明还提供黑沙蒿根提取物在制备治疗STAT3蛋白过度表达引起的疾病的药物中的应用,其中,所述黑沙蒿根提取物为上述黑沙蒿根的乙酸乙酯提取物。
再进一步的,本发明还提供黑沙蒿根提取物在制备治疗抑制Caspase-3蛋白表达的疾病的药物中的应用,其中,所述黑沙蒿根提取物为上述的黑沙蒿根的乙酸乙酯提取物。
再进一步的,本发明还提供黑沙蒿根提取物在制备治疗Galectin-3蛋白引起的疾病症状的药物中的应用,其中,所述黑沙蒿根提取物为上述的黑沙蒿根的乙酸乙酯提取物。
再进一步的,本发明还提供黑沙蒿根提取物在制备治疗S100A9蛋白引起的疾病症状的药物中的应用,其中,所述黑沙蒿根提取物为上述的黑沙蒿根的乙酸乙酯提取物。
本发明通过药效学实验证明了黑沙蒿根乙酸乙酯提取物呈剂量依赖性地发挥抗类风湿关节炎的效果,当口服剂量达到400mg/kg时,其抗类风湿关节炎效果与地塞米松(0.1 mg/kg)相接近,并由此确认了100-800mg/kg的有效剂量范围。并且,本发明还验证了口服高剂量黑沙蒿根不但对机体无损伤,还可以在一定程度上缓解类风湿关节炎造成的免疫损伤。此外,经过相关抗炎机制的研究,明确了黑沙蒿根发挥抗类风湿关节炎的作用机制。ELISA实验表明,黑沙蒿根乙酸乙酯提取物呈剂量依赖性地抑制大鼠血清中炎症因子TNF-α、IL-1β、IL-6、IL-17A的浓度,提高抗炎因子IL-10的血清高浓度,缓解了类风湿关节炎病情发展。通过关节组织免疫组化实验证明了黑沙蒿根可以影响与类风湿关节炎发生发展相关的蛋白表达,如STAT3、Caspase-3、Galectin-3、S100A9蛋白表达。其中黑沙蒿根乙酸乙酯提取物通过抑制STAT3间接地抑制滑膜成纤维细胞的增殖、血管的生成以及STAT3相关的炎症级联反应;Caspase-3的显著表达表明黑沙蒿根乙酸乙酯提取物可以促进滑膜成纤维细胞的凋亡,进而缓解滑膜增生以及发挥炎症保护作用;黑沙蒿根乙酸乙酯提取物通过抑制Galectin-3进而抑制滑膜成纤维细胞释放炎症因子和趋化因子,进而缓解炎症加剧;黑沙蒿根乙酸乙酯提取物通过抑制S100A9间接地降低关节部位对炎细胞的招募以及炎细胞向关节部位的迁移,进而减少关节部位炎细胞的浸润,降低关节部位的炎症反应。综上所述,黑沙蒿根乙酸乙酯提取物通过提高血清抗炎因子浓度的同时,还能抑制血清炎症因子浓度、滑膜成纤维细胞的增殖以及免疫细胞的浸润,促进滑膜成纤维细胞凋亡等方面,协同发挥抗类风湿关节炎的效果。本发明从分子蛋白的层面阐述黑沙蒿根乙酸乙酯提取物发挥抗类风湿关节炎的作用机制以及在免疫方面产生的作用,拓宽了黑沙蒿根疾病治疗的范围(其他自身免疫性疾病),促进了黑沙蒿的现代化研究。
附图说明
图1为各组大鼠的爪肿胀变化曲线(n=5)。
图2为各组大鼠关节炎评分曲线(n=5)。
图3为各组大鼠体重变化曲线(n=5)。
图4为各组大鼠脾指数和胸腺指数(n=5)。
图5为各组大鼠的组织病理学切片。
图6为各组大鼠的相关免疫因子血清浓度(n=5)。
图7为各组大鼠关节部位STAT3表达情况。
图8为各组大鼠关节部位Caspase-3表达情况。
图9为各组大鼠关节部位Galectin-3表达情况。
图10为各组大鼠关节部位S100A9表达情况。
实施方式
为了使本发明要解决的技术问题、技术方案及有益效果更加清楚明白,以下结合实施例,对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。
1. 黑沙蒿根提取物的制备
取6 kg黑沙蒿根粉,并加入黑沙蒿根粉10倍重量的质量分数为95%的乙醇溶液超声提取3次,每次30 min,将获得的提取液置于60℃下浓缩干燥35min。随后将浓缩物用其10倍重量的蒸馏水溶解,依次用等体积的石油醚、乙酸乙酯和正丁醇各萃取3次,得到石油醚层萃取物、乙酸乙酯层萃取物、正丁醇层萃取物及水溶物;收集乙酸乙酯层萃取物,减压浓缩去除溶剂,98℃下干燥,即得黑沙蒿根乙酸乙酯层提取物。
2.黑沙蒿根乙酸乙酯提取物抗类风湿关节炎药效学试验
通过胶原蛋白诱发关节炎,建立大鼠类风湿关节炎模型进行药效学研究。向雄性Wister大鼠尾巴根部皮下注射0.1 mL的完全弗氏佐剂乳化的牛II型胶原蛋白进行初始免疫,在初次注射7天后再注射一次乳化的不完全弗洛伊德佐剂和胶原蛋白进行免疫加强。在此期间,不止一个关节出现肿胀和红斑,表明关节炎的发作。在造模第21天,相应选取正常大鼠1组,造模成功的关节炎模型大鼠随机分为5组,共6组,每组5只。即正常组(Normal)、模型组(Model)、阳性对照组(地塞米松,DXM:0.1 mg/kg)、黑沙蒿根乙酸乙酯提取物高剂量组(AOK: 400 mg/kg)、黑沙蒿根乙酸乙酯提取物中剂量组(AOK: 200 mg/kg)、黑沙蒿根乙酸乙酯提取物低剂量组(AOK: 100 mg/kg)。从第21天到51天,每日灌胃给药,正常组和模型组给予等体积的生理盐水。在整个药效学试验期间,记录大鼠进行爪(后爪)肿胀程度测定、关节炎指数评价,体重测量,结果见图1-3。
药效结果显示,与正常组大鼠相比,模型组大鼠的后爪体积显著增加(p<0.01);与模型组相比,阳性对照组的爪肿胀程度从第24天开始显著降低(p<0.01);黑沙蒿根乙酸乙酯提取物高剂量组、黑沙蒿根乙酸乙酯提取物中剂量组和黑沙蒿根乙酸乙酯提取物低剂量组均能显著降低爪肿胀程度(p<0.01),且效果呈剂量依赖性(见图1)。与此同时,从图2中可以看出,在试验结束时,与模型组相比,阳性对照组、黑沙蒿根乙酸乙酯提取物高剂量组、黑沙蒿根乙酸乙酯提取物中剂量组均能显著降低关节炎评分(p<0.01),而黑沙蒿根乙酸乙酯提取物低剂量组虽能降低在一定程度上降低关节评分,但与模型组无显著性差异。此外,各组大鼠体重变化显示(见图3),正常组大鼠体重增长速率最大;模型组大鼠体重增加缓慢,与正常组相比有显著性差异(p<0.05);而剩余的阳性对照组、黑沙蒿根乙酸乙酯提取物高剂量组、黑沙蒿根乙酸乙酯提取物中剂量和黑沙蒿根乙酸乙酯提取物低剂量组均呈现出体重不断增长的趋势,与正常组相比无显著性差异,表明大鼠生存状态良好。
通过对药效学试验结束后各组大鼠的脾指数和胸腺指数的测定,进行免疫学损伤评价。处死大鼠后,取出脾脏和胸腺进行称重,通过公式计算指数:器官指数=内脏湿重(mg)/大鼠体重(g)×100%。结果如图4所示,与正常组相比,模型组的脾指数(图4B)和胸腺指数(图4A)显著升高(P<0.05),但剩余的阳性对照组、黑沙蒿根提取物高剂量组、黑沙蒿根提取物中剂量和黑沙蒿根提取物低剂量组虽有所升高但无显著性差异,表明黑沙蒿根提取物对机体免疫无损伤,而长期类风湿关节炎疾病则会对机体免疫产生损伤;与模型组相比时,阳性对照组和黑沙蒿根提取物高剂量组的脾指数和胸腺指数显著降低(P<0.05),黑沙蒿根提取物中剂量和黑沙蒿根提取物低剂量组虽有所降低,但无显著性差异,表明地塞米松和高剂量黑沙蒿根的治疗可以在一定程度上缓解类风湿关节炎对机体产生的免疫损伤。
上述试验均通过宏观指标进行抗关节炎的评价,因此需通过病理学上的微观变化作为指标对药效试验各组之间存在的差异进行考察和验证。剥离药效学试验结束后的各组大鼠的后爪和膝关节进行病理学分析。将关节固定在10%福尔马林中,随后在5%硝酸中脱钙。脱钙后,将组织脱水处理并包埋在石蜡中,随后将石蜡切片(4μm)进行脱蜡处理,并用苏木精和伊红(H&E)染色,并在光学显微镜下进行评估。根据炎症细胞浸润、滑膜增生、关节肿胀和炎症、软骨骨质破坏等病理变化检查关节炎的严重程度。结果见图5,正常组(图5A)的大鼠关节和软骨未见病理改变;与正常组相比,模型组(图5B)可见大量的炎性细胞浸润、滑膜增生、中度软骨损伤伴腔内积液;与模型组相比,当给予地塞米松(图5C)和黑沙蒿根提取物(图5D-F)30天后,炎症细胞浸润和关节破坏以及滑膜增生呈剂量依赖性的减轻,该结果与药效学实验结果一致。
细胞因子在类风湿关节炎的发病机制中起重要作用。因此,对药效试验各组大鼠血清中的炎症因子TNF-α、IL-1β、IL-6、IL-17A和抗炎细胞因子IL-10的含量进行测定。将药效试验结束后的大鼠用乙醚麻醉,通过心脏血液采集大鼠血样,在室温下凝固1 h,随后在4°C,3000 rpm的离心条件下,高速离心10 min后获得血清。按照ELISA试剂盒的使用说明对血清中TNF-α、IL-1β、IL-6、IL-17A和IL-10等因子浓度进行测定。结果显示(见图6),与正常组相比,模型组的炎症因子TNF-α、IL-1β、IL-6、IL-17A(图6A-D)显著升高(P<0.001),而抗炎细胞因子IL-10(图6E)显著下降(P<0.05);与模型组相比,地塞米松组的炎症因子水平显著下降(P<0.05),抗炎因子水平显著上升(P<0.05),而不同黑沙蒿根提取物剂量组呈剂量依赖性地降低炎症因子水平,同时提高了抗炎因子水平,上述结果与药效结果实验一致。结果表明黑沙蒿根提取物通过抑制炎症因子和促进抗炎因子的血清浓度,发挥抗类风湿关节炎的效果,该作用与口服剂量呈正相关,在减轻炎症中起到重要作用。
取出各组大鼠的关节组织,进行免疫组化分析,考察各组大鼠关节部位四种不同蛋白(STAT3、Caspase-3、Galectin-3、S100A9)的表达情况。将关节组织包埋在石蜡中后,将石蜡切片(4μm)进行脱蜡处理,随后进行抗原修复,用PBS清洗3次,将一抗稀释1000倍后进行孵育,4℃过夜。待孵育结束后,用PBS清洗3次切片除去多余抗体,再用二抗孵育15min后,在显微镜下进行观察。
STAT3蛋白是类风湿关节炎发病过程中一个关键性致病因子,可抑制滑膜成纤维细胞的凋亡,促进血管生成和软骨侵蚀,还是多种细胞因子的信号靶点,包括IL-γ、IL-2、IL-4、IL-6、IL-7、IL-10、IL-12和IL-15,所以抑制STAT3的表达可有效地抑制类风湿关节炎的病情发展。通过免疫组化观察各组STAT3的表达情况,结果见图7,图中STAT3表达实际显示颜色为黄色(在黑白附图中,如,图7B中,箭头所指的中部大面积深灰色部分;图7D-F中,箭头所指的边缘处深灰色部分)。与正常组(图7A)相比,模型组(图7B)大鼠的滑膜组织显著表达STAT3,说明STAT3与类风湿关节炎的发生发展密切相关;与模型组相比,地塞米松组(图7C)STAT3的表达显著下降,几乎与正常组接近,表明地塞米松的治疗效果好,滑膜增生不显著;而不同给药剂量的黑沙蒿根组(图7D-F)则呈现出不同程度的滑膜增生以及STAT3的表达,黑沙蒿根使用剂量越高,滑膜增生越轻,STAT3的表达越少。表明地塞米松和高剂量的黑沙蒿根可以通过抑制STAT3的表达,进而抑制STAT3相关的炎症级联反应,发挥抗类风湿关节炎的作用。
Caspase-3是与细胞凋亡相关的蛋白,是细胞在凋亡过程中关键的执行分子,当细胞凋亡时会大量表达该蛋白。类风湿关节炎典型病理变化是FLS增殖而引起的滑膜增生,促进细胞的凋亡会起到炎症保护的作用,因此促进FLS凋亡是治疗的类风湿关节炎的有效方法。通过免疫组化观察各组Caspase-3的表达情况,考察各组FLS的凋亡情况,结果见图8,图中Caspase-3表达实际显示颜色为黄色,转为黑白图后,显示为箭头所指的深灰色。正常组(图8A)的滑膜可见Caspase-3显著表达;与正常组相比,模型组(图8B)的滑膜增生部位未见Caspase-3明显表达;地塞米松组(图8C)滑膜可见Caspase-3显著表达,但弱于正常组;不同给药剂量的黑沙蒿根组(图8D-F)也有Caspase-3表达,表达强度呈剂量依赖性。说明地塞米松和高剂量的黑沙蒿根可以使滑膜细胞发生凋亡,进而缓解了滑膜增生以及其引发的炎症反应,有利于类风湿关节炎的治疗。
Galectin-3蛋白可由FLS分泌,刺激细胞产生炎症因子和趋化因子,可招募白细胞到达炎症部位进一步加重炎症反应,在激活和释放炎症因子的过程中起着至关重要的作用,因此抑制Galectin-3可有效地抑制类风湿关节炎的炎症反应。通过免疫组化观察各组Galectin-3的表达情况,结果见图9,图中Galectin-3表达实际显示颜色为黄色,黑白图中显示为箭头所指的深灰色。模型组(图9B)可见显著的Galectin-3表达,与模型组相比,正常组(图9A)几乎不表达Galectin-3,表明Galectin-3的表达与类风湿关节炎的发生发展息息相关;地塞米松组(图9C)和高剂量黑沙蒿根组(图9D)的滑膜层Galectin-3均呈弱阳性表达,而中等剂量(图9E)和低剂量黑沙蒿根组(图9F)Galectin-3的表达逐渐显著,且伴随着较厚的滑膜增生,表明地塞米松和高剂量黑沙蒿根可通过抑制Galectin-3的表达,进而抑制FLS释放炎症因子和趋化因子,从而发挥显著的抗类风湿关节炎效果。
S100A9蛋白主要由浸润在关节部位的中性粒细胞、单核细胞等炎性细胞释放,进而招募更多的炎细胞到达关节部位,产生炎症恶性循环。类风湿关节炎病情的不断发展与关节部位炎细胞的不断浸润有密切的关系,因此抑制S100A9的表达有益于类风湿关节炎的治疗,并可通过观察组织中S100A9的表达情况,间接地反应关节部位的炎症及炎性细胞的浸润情况。通过免疫组化观察各组S100A9的表达情况,结果见图10,图中S100A9表达实际显示颜色为黄色,转为黑白图后,显示为箭头所指的中、深灰色,其中褐色的颗粒为表达S100A9的炎细胞。正常组(图10A)可见少量的炎细胞的浸润,而模型组(图10B)可见大量的黄染和炎细胞的浸润,表明模型组的炎症较为严重;与模型组相比,中等剂量(图10E)和低剂量黑沙蒿根组(图10F)的炎细胞浸润相对减少,但也可见显著的炎细胞浸润以及黄染,显著多于正常组;而地塞米松组(图10C)和高剂量黑沙蒿根组(图10D)的炎细胞浸润相比于模型组显著减少,表明炎症的发展得到了非常好的控制。说明地塞米松和黑沙蒿根可通过减少S100A9的表达,降低关节部位对炎细胞的招募以及炎细胞向关节部位的迁移,进而减少关节部位炎细胞的浸润,降低关节部位的炎症反应。
本发明首次通过药效试验证明了黑沙蒿根乙酸乙酯提取物可以呈剂量依赖性地发挥抗类风湿关节炎的效果,且通过相关研究明确了黑沙蒿根发挥抗类风湿关节炎效果时相关的作用机制。黑沙蒿根通过影响STAT3、Caspase-3、Galectin-3、S100A9蛋白表达,抑制滑膜成纤维细胞的增殖并促进其凋亡、影响血清相关免疫因子水平、影响关节部位炎症因子和趋化因子水平以及关节部位免疫细胞的浸润等方面协同发挥抗类风湿关节炎作用。由于黑沙蒿根可以抑制Galectin-3、S100A9蛋白的表达,表明黑沙蒿根在一定程度上会抑制机体免疫系统的响应,对机体的免疫系统有调节作用,对于其他自身免疫性疾病可能也有一定的治疗效果,拓宽了黑沙蒿的疾病研究范围。
上述实施例只是为了说明本发明的技术构思及特点,其目的是在于让本领域内的普通技术人员能够了解本发明的内容并据以实施,并不能以此限制本发明的保护范围。凡是根据本发明内容的实质所作出的等效的变化或修饰,都应涵盖在本发明的保护范围内。
Claims (9)
1.黑沙蒿根提取物在制备抗类风湿关节炎药物中的应用,其特征在于,所述黑沙蒿根提取物为黑沙蒿根的乙酸乙酯提取物。
2.根据权利要求1所述的黑沙蒿根提取物在制备抗类风湿关节炎药物中的应用,其特征在于,所述黑沙蒿根提取物通过以下步骤制备:
称取黑沙蒿粉,并加入黑沙蒿根粉10倍重量的、质量分数为95%的乙醇溶液超声提取3次,每次30 min,将获得的提取液置于60℃下浓缩干燥35min;随后将浓缩物用其10倍重量的蒸馏水溶解,依次用等体积的石油醚、乙酸乙酯和正丁醇各萃取3次,得到石油醚层萃取物、乙酸乙酯层萃取物、正丁醇层萃取物及水溶物;收集乙酸乙酯层萃取物,减压浓缩,98℃下干燥,即得黑沙蒿根乙酸乙酯层提取物。
3.根据权利要求1或2所述的黑沙蒿根提取物在制备抗类风湿关节炎药物中的应用,其特征在于,所述黑沙蒿根提取物的有效剂量为100-800 mg/kg。
4.根据权利要求1-3任一所述的黑沙蒿根提取物在制备抗类风湿关节炎药物中的应用,其特征在于,所述黑沙蒿根提取物通过影响关节部位的STAT3、Caspase-3、Galectin-3和S100A9蛋白的表达,来有效抑制类风湿关节炎的病情发展。
5.黑沙蒿根提取物在制备治疗STAT3蛋白过度表达引起的疾病的药物中的应用,其特征在于,所述黑沙蒿根提取物为黑沙蒿根的乙酸乙酯提取物。
6.黑沙蒿根提取物在制备治疗抑制Caspase-3蛋白表达的疾病的药物中的应用,其特征在于,所述黑沙蒿根提取物为黑沙蒿根的乙酸乙酯提取物。
7.黑沙蒿根提取物在制备治疗Galectin-3蛋白引起的疾病症状的药物中的应用,其特征在于,所述黑沙蒿根提取物为黑沙蒿根的乙酸乙酯提取物。
8.黑沙蒿根提取物在制备治疗S100A9蛋白引起的疾病症状的药物中的应用,其特征在于,所述黑沙蒿根提取物为黑沙蒿根的乙酸乙酯提取物。
9.一种抗类风湿关节炎的药物,其特征在于,该药物包括根据权利要求1-4任一所述的应用中的黑沙蒿根乙酸乙酯提取物。
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CN1814064A (zh) * | 2005-11-28 | 2006-08-09 | 蓝子花 | 一种黑沙蒿活络伤湿止痛膏 |
CN110960565A (zh) * | 2018-09-28 | 2020-04-07 | 肖斌 | 一种黑沙蒿根提取物及其制备方法和应用 |
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CN1814064A (zh) * | 2005-11-28 | 2006-08-09 | 蓝子花 | 一种黑沙蒿活络伤湿止痛膏 |
CN110960565A (zh) * | 2018-09-28 | 2020-04-07 | 肖斌 | 一种黑沙蒿根提取物及其制备方法和应用 |
Non-Patent Citations (1)
Title |
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郝俊生: "基于网络药理学研究黑沙蒿活性部位及其治疗风湿性关节炎的作用机制", 中国优秀硕士学位论文全文数据库(电子期刊), pages 057 - 607 * |
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