CN118178437A - 人参皂苷Rk1在制备治疗溃疡性结肠炎药物中的应用 - Google Patents
人参皂苷Rk1在制备治疗溃疡性结肠炎药物中的应用 Download PDFInfo
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Abstract
本发明公开了一种人参皂苷Rk1在制备治疗溃疡性结肠炎药物中的应用,药物包含人参皂苷Rk1在药学上能够接受的溶剂化合物中的一种或数种,以包含人参皂苷Rk1化合物为活性成份组成的药物组合物用于治疗溃疡性结肠炎。药物为片剂、丸剂、滴丸、胶囊剂、颗粒剂、散剂、粉剂、注射液、口服液或乳剂。有益效果:人参皂苷Rk1对DSS诱导的溃疡性结肠炎小鼠具有保护作用且依赖于巨噬细胞,SIN能够通过调控巨噬细胞极化而发挥体内抗炎作用。人参皂苷Rk1能够通过调控PI3K/AKT通路调节巨噬细胞极化,从而缓解炎症。为靶向EGFR磷酸化调控巨噬细胞极化治疗UC提供了理论基础,补充了人参皂苷Rk1在UC治疗方面的药用价值及潜在机制,为临床应用人参皂苷Rk1治疗UC提供理论依据。
Description
技术领域
本发明涉及一种人参皂苷Rk1在制备药物中的应用,特别涉及一种人参皂苷Rk1在制备治疗溃疡性结肠炎药物中的应用。
背景技术
目前,溃疡性结肠炎(UC)属慢性非特异性炎性肠病。UC病情反复发作、迁延不愈,具有癌变倾向并伴随多种肠外症状已经严重威胁人类健康。因其作用机制不清楚,目前尚无理想的治疗药物及治疗方法,WHO将其确定为现代难治病之一。巨噬细胞是先天免疫***的重要组成部分,巨噬细胞的极化状态是治疗UC的重中之重。从巨噬细胞极化角度研究肠道炎症的发病机理是当今研究的前沿领域。表皮生长因子受体(EGFR)在巨噬细胞广泛表达并通过其下游调控信号通路参与刺激巨噬细胞极化。研究表明EGFR磷酸化能够激活巨噬细胞M2极化表型改善肠道炎症损伤。
人参是传统名贵的补益类中药之一,性甘、味苦,入脾、肺二经。能够推动机体的气血运行,调节免疫***功能,发挥“大补元气,补脾益肺”之功效。人参皂苷Rk1是一种四环三萜类的稀有人参皂苷,具有抗炎、抗氧化、抗肿瘤及保护神经等药理活性。其生物机制与调节PI3K/AKT信号通路具有相关性。但是人参皂苷Rk1对溃疡性结肠炎的治疗作用及其机制研究未能深入,特别是与EGFR磷酸化后激活巨噬细胞M2极化,并调控下游PI3K/AKT信号通路改善溃疡性结肠炎的的相关机制尚属空白。
发明内容
本发明的目的是通过体内和体外模型确认人参皂苷Rk1治疗溃疡性结肠炎的活性,而提供的人参皂苷Rk1在制备治疗溃疡性结肠炎药物中的应用。
人参皂苷Rk1能够在制备治疗溃疡性结肠炎药物中进行应用。
药物包含人参皂苷Rk1在药学上能够接受的溶剂化合物中的一种或数种,以包含人参皂苷Rk1化合物为活性成份组成的药物组合物用于治疗溃疡性结肠炎。
药物为片剂、丸剂、滴丸、胶囊剂、颗粒剂、散剂、粉剂、注射液、口服液或乳剂。
具体证明过程如下:
动物模型及细胞模型能够详实地证明人参皂苷Rk1抗UC活性及与EGFR磷酸化和巨噬细胞极化相关机制。
利用EGFR基因缺失动物、巨噬细胞清除动物模型和基于CRISPR/Cas9基因编辑技术和siRNA干扰技术的细胞模型,在分子和基因水平上证明参皂苷Rk1对“EGFR-巨噬细胞极化”轴的作用机制。
利用磷酸化蛋白质组学技术阐述EGFR及其下游PI3K/AKT相关通路中关键蛋白质磷酸化水平和磷酸化功能修饰位点,从宏观水平上揭示人参皂苷Rk1治疗UC过程中蛋白质磷酸化修饰及信号传导的状态。
本发明的有益效果:
本发明提供的人参皂苷Rk1在制备治疗溃疡性结肠炎药物中的应用通过采用体内和体外实验证实了人参皂苷Rk1对DSS诱导的溃疡性结肠炎小鼠具有保护作用且依赖于巨噬细胞,SIN能够通过调控巨噬细胞极化而发挥体内抗炎作用。通过LPS刺激的RAW 264.7巨噬细胞炎症模型,在体外进一步证实了人参皂苷Rk1的抗溃疡性结肠作用。同时,结果表明人参皂苷Rk1能够通过调控PI3K/AKT通路调节巨噬细胞极化,从而缓解炎症。本发明多角度探究了人参皂苷Rk1的抗溃疡性结肠炎的作用,首次提出了人参皂苷Rk1能够通过靶向EGFR磷酸化调控巨噬细胞极化相关机制。本发明为靶向EGFR磷酸化调控巨噬细胞极化治疗UC提供了理论基础,补充了人参皂苷Rk1在UC治疗方面的药用价值及潜在机制,为临床应用人参皂苷Rk1治疗UC提供理论依据。以此明确人参皂苷Rk1抗UC的活性,为研发安全、有效的抗UC药物有治疗方法奠定基础;阐明人参皂苷Rk1靶向EGFR磷酸化调节巨噬细胞极化治疗UC的作用机制和相关信号通路,为临床治疗UC提供实验依据。
附图说明
图1为本发明所述的人参皂苷Rk1对“EGFR-巨噬细胞极化”轴治疗溃疡性结肠炎作用机制示意图。
具体实施方式
请参阅图1所示:
人参皂苷Rk1能够在制备治疗溃疡性结肠炎药物中进行应用。
药物包含人参皂苷Rk1在药学上能够接受的溶剂化合物中的一种或数种,以包含人参皂苷Rk1化合物为活性成份组成的药物组合物用于治疗溃疡性结肠炎。
药物为片剂、丸剂、滴丸、胶囊剂、颗粒剂、散剂、粉剂、注射液、口服液或乳剂。
具体证明过程如下:
动物模型及细胞模型能够详实地证明人参皂苷Rk1抗UC活性及与EGFR磷酸化和巨噬细胞极化相关机制。
利用EGFR基因缺失动物、巨噬细胞清除动物模型和基于CRISPR/Cas9基因编辑技术和siRNA干扰技术的细胞模型,在分子和基因水平上证明参皂苷Rk1对“EGFR-巨噬细胞极化”轴的作用机制。
利用磷酸化蛋白质组学技术阐述EGFR及其下游PI3K/AKT相关通路中关键蛋白质磷酸化水平和磷酸化功能修饰位点,从宏观水平上揭示人参皂苷Rk1治疗UC过程中蛋白质磷酸化修饰及信号传导的状态。
具体实验过程如下:
1、体内实验:
1.1.DSS肠炎小鼠模型构建、分组及给药
雄性C57BL/6N小鼠适应性饲养7天,随机分为6组(每组10只):空白组、模型组(3%DSS)、3%DSS+Rk1低剂量组、3%DSS+Rk1中剂量组、3%DSS+Rk1高剂量组和阳性对照组(巴柳氮钠胶囊混悬液10mg/kg)。除空白组外,各组小鼠的普通饮用水更换为3%(w/v)DSS饮用水。小鼠连续饮用3%DSS饮用水(7d)以诱导发生肠炎。低剂量组、高剂量组和阳性对照组小鼠分别灌胃给予10、30mg/kg Rk1和10mg/kg巴柳氮钠胶囊混悬液14天。空白组与模型组小鼠在相同时间灌胃等量蒸馏水。实验期间,对小鼠体重、粪便硬度和直肠出血情况进行每日记录。第15天,每组5只小鼠进行安乐死,取材进行后续实验。
1.2.EGFR基因缺失小鼠UC模型的建立、分组和给药
根据前部分构建的肠炎小鼠模型,再采用腹腔注射氯膦酸二盐脂质体法清除小鼠体内巨噬细胞,以PBS脂质体(PBS-lipo)作为对照。C57BL/6N小鼠(雌雄各半)首先喂养7天,以适应环境,然后进行随机分为五组(每组10只):PBS-lipo+DSS+PBS组、PBS-lipo+DSS+Rk1组、Clod-lipo+DSS+PBS组和Clod-lipo+DSS+Rk1组。各组小鼠在第0天至第7天均给予普通饮用水,给予普通饮用水7天后,将各组小鼠的饮用水替换为3%(w/v)DSS饮用水。PBS-lipo+DSS+Rk1组与Clod-lipo+DSS+Rk1组小鼠于第0天至第13天Rk1(高剂量10mg/kg)灌胃给药。PBS-lipo+DSS+PBS组与Clod-lipo+DSS+PBS组小鼠在相同时间灌胃等量蒸馏水。在第6天、第8天和第10天,对PBS-lipo+DSS+PBS组及PBS-lipo+DSS+PLD组小鼠腹腔注射PBS-lipo,每只小鼠0.2ml;对Clod-lipo+DSS+PBS组及Clod-lipo+DSS+PLD组小鼠腹腔注射Clod-lipo,每只小鼠0.2ml。实验期期间,将小鼠体重、粪便硬度和直肠出血情况进行每日记录。第14天,所有小鼠均被安乐死,取材进行后续实验。
1.3.EGFR基因缺失UC小鼠模型的建立、分组和给药:
30只正常雄性C57BL/6N小鼠及30只C57BL/6N-EGFRem1Cya基因敲除雄性小鼠随机区组设计分为4组,每组10只。分为EGFR野生组(EGFRWT);EGFR野生模型组(EGFRWT+DSS)和EGFR野生模型给药组(EGFRWT+DSS+Rk1)。基因敲除空白组(EGFR-);基因敲除模型组(EGFR-+DSS);基因敲除给药组(EGFR-+DSS+Rk1);具体造模型方法和Rk1给药剂量同1.1。实验结束后取材用于后续实验。
1.4.人参皂苷Rk1对DSS诱导UC小鼠溃疡活动程度的影响:
每天记录小鼠体质量、精神状态、毛发色泽、饮水量、采食量,并收集新鲜粪便,加入粪便DNA保护剂于-80℃冰箱保存。按表1标准进行疾病活动指数(DAI)评分。
表1疾病活动指数(DAI)评分标准
1.5.人参皂苷Rk1对DSS诱导UC小鼠中血清及结肠组织炎症因子水平的影响:
采用ELISA法:取小鼠血清及部分结肠组织(将其剪碎后置于EP管内,取每1g组织加入10mL RIPA蛋白裂解液,冰上孵育1.5-2h后匀浆机匀浆。将组织匀浆置于冷冻离心机中,离心,提取上清液)。严格按照ELISA试剂盒说明书步骤进行操作,测定结肠组织中TNF-α、IL-6、IL-10和IL-1β的水平。
1.6.人参皂苷Rk1对DSS诱导UC小鼠结肠组织的病理学影响:
HE染色法:取4%多聚甲醛固定后的小鼠结肠组织,梯度酒精浸泡脱水、二甲苯透明、石蜡包埋、切片厚度4μm、脱蜡、苏木素染色、PBS洗涤、1%盐酸乙醇分化、伊红溶液染色30s,梯度酒精脱水,透明,中性胶密封,在光学显微镜下观察结肠组织的病理变化。
1.7.人参皂苷Rk1对DSS诱导UC小鼠结肠组织密连接蛋白表达的影响:
采用免疫荧光染色法:将新鲜的小鼠结肠组织以石蜡包埋,8μm厚度连续冰冻组织切片、脱水、固定、透膜、封闭后,室温放置1h,加入小鼠抗紧密连接蛋白Claudin-1及ZO-1多克隆抗体(用PBS替代一抗作阴性对照),4℃孵育过夜;以PBS漂洗,滴加标记红色荧光的抗兔DyLight 649,室温避光孵育1h;PBS漂洗后,滴加DAPI染核,在荧光显微镜下观察各细胞间紧密连接的分布并拍照,显示红色荧光为阳性表达。
1.8.人参皂苷Rk1对DSS诱导UC小鼠巨噬细胞极化蛋白表达的影响:
小鼠腹部皮肤进行酒精消毒处理,剪开腹部,暴露腹膜,使用无菌注射器吸取DMEM完全培养基,小心刺入腹膜,缓慢将培养基打入。轻轻按摩小鼠腹部缓慢吸出灌洗液,收集腹腔灌洗液。之后剪开腹膜,吸出腹腔内剩余的液体。合并收集液,离心,弃上清,用PBS缓冲液重悬细胞沉淀,接种于6孔板中培养,37℃,5%CO2中孵育3小时,使各组小鼠腹腔巨噬细胞贴壁,收集各组小鼠腹腔巨噬细胞待后续实验。采用RT-PCR法:根据All-in-one 1stStrand cDNA Synthesis SuperMix kit的操作手册,进行RNA逆转录实验。检测M1型巨噬细胞极化标志物iNOS、CD86和M2型巨噬细胞极化标志物Arg1、CD206的mRNA水平。
1.9.人参皂苷Rk1对DSS诱导UC小鼠巨噬细胞极化比例的影响:
采用流式细胞术检测巨噬细胞比例:对小鼠体结肠和腹腔灌洗液中巨噬细胞进F4/80和CD11b特异性标记。将获得的单细胞悬液进行计数,按照Anti-F4/80PE,Anti-CD11bPERCP-Cy5.5的产品说明书加入抗体,孵育、洗涤、过滤、上机分析并进行分析及结果可视化处理。
2、体外实验:
2.1.LPS诱导的RAW264.7细胞细胞模型的建立:
将RAW 264.7细胞以每孔5×105个接种到6孔板中,次日加入RK1(5μM)处理24h,然后加入LPS(1μg/ml)进行12h刺激。
2.2.细胞模型的建立分组及给药:
利用正常细胞株和EGFR基因过表达、敲低和敲除的四种细胞株。每株细胞均设置空白组、模型组和Rk1(5μM)给药组,分别进行如下试验。给药组均采用Rk1(高剂量)预处理细胞24h后,以1μg/ml LPS刺激6h。模型组均采用先等量PBS处理24h后,用LPS刺激6h后取细胞培养上清,收集细胞,以待后续实验备用。
2.3.人参皂苷Rk1对细胞活力的影响:
采用CCK8法确认人参皂苷给药的最佳浓度再按上述分组进行细胞活力检测检测:每孔加入CCK8试剂,于37℃下,孵育,用酶标仪测定450nm处测吸光度。细胞存活率=[(As-Ab)/(Ac-Ab)]×100%;抑制率=[(Ac-As)/(Ac-Ab)]×100% As为实验孔吸光度,Ac为对照孔吸光度,Ab为空白孔吸光度。
2.4.人参皂苷Rk1对细胞模型中巨噬细胞极化比例影响:
将iNOS和CD206分别作为RAW264.7细胞系M1型和M2型巨噬细胞的标志物。将获得的各组单细胞悬液进行计数,分组,固定,洗涤,标记胞内抗体按照Anti-iNOS Alexa Fluro488和Anti-CD206 APC的产品说明书,加入胞内抗体,孵育,洗涤,过滤后上机分析。
2.5.人参皂苷Rk1对细胞模型中巨噬细胞极化相关通路蛋白表达和磷酸化水平的影响:
采用Western blotting法检测蛋白的表达:提取细胞总蛋白并测定浓度,取等量蛋白样品,使用聚丙烯酰胺凝胶电泳分离,切胶,采用湿转法将蛋白条带转移至PVDF膜上,5%脱脂牛奶封闭后,分别加入一抗体孵育过夜,加入各自对应的二抗孵育后,化学发光法显色。检测的抗体有p-EGFR、EGFR、p-PI3K、PI3K、p-Akt、Akt、p-p65以及p65(信号通路相关蛋白)等。
3、实验结果:
本实验中采用DSS诱导的结肠炎小鼠模型评价人参皂苷Rk1的抗UC活性。与空白组相比,溃疡性结肠炎小鼠试验期体重明显减轻,部分溃疡性结肠大鼠造模2天后出现严重腹泻或明显直肠出血症状。然而人参皂苷Rk1给药组出现体重减轻的趋势明显被抑制。截取近***10cm结肠段,裸眼检查显示无病变大鼠结肠(空白组)表面光滑,皱褶清晰;UC大鼠模型组结肠黏膜充血充血,黏膜上皮糜烂明显。与UC组相比,人参皂苷Rk1给药组上皮黏膜充血和结肠上皮糜烂明显减少。通过HE染色评估结肠组织的组织病理学特征:未处理DSS-UC组的组织病理学观察包括粘膜变性和坏死,隐窝炎和囊性扩张,以及与健康对照组相比,中性粒细胞和单核炎性细胞的粘膜和粘膜下浸润。而人参皂苷Rk1给药组小鼠结肠固有层均可见单核炎性细胞的浸润和聚集,上皮变性和坏死情况有所改善,小鼠结肠充血减轻,溃疡线性愈合,中性粒细胞和单核炎性细胞浸润明显减少。
人参皂苷Rk1能够改善DSS诱导UC小鼠结肠黏膜损伤,结肠组织中oclaudin、ZO-1和Tubulin-β的表达通过上调恢复肠黏膜屏障功能。Western-blot结果也显示Rk1能够上调DSS诱导UC小鼠结肠Claudin-1及JAM-1紧密连接蛋白水平。证明了Rk1提高结肠组织紧密连接蛋白表达恢复肠道屏障改善肠黏膜损伤的活性。
接下来,取样各组小鼠结肠组织并提取总蛋白,用Western Blot法检测各组细胞的总蛋白中p-EGFR、EGFR、p-PI3K、PI3K、p-Akt、Akt、p-p65以及p65的蛋白表达水平。结果表明与DSS诱导的UC模型组相比,在Rk1灌胃给药处理组中,p-EGFR/EGFR、p-PI3K/PI3K和p-AKT/AKT比例显著上调,而p-p65/p65的比例显著下降。NF-κB p65的核转位以及AKT磷酸化与巨噬细胞极化发挥抗炎功能密切相关。
为了确认人参皂苷Rk1对巨噬细胞的极化调节作用及其相关机制,采用小鼠巨噬细胞系RAW 264.7细胞并构建LPS刺激的RAW 264.7细胞炎症模型进行证明。首先,采用CCK8实验评估不同浓度Rk1对RAW264.7细胞/LPS刺激RAW264.7细胞活性的影响。2.5μM及5μM的Rk1对正常细胞活性无显著影响对模型细胞存在显著差异。因此在后续的预实验中采用了高剂量组5μM的Rk1进行后续试验。接下来,用ELISA法检测了人参皂苷Rk1对LPS刺激的RAW264.7细胞的细胞因子水平的影响。Rk1处理后均显著降低了LPS刺激的RAW 264.7细胞模型中促炎因子TNF-α和IL-6的表达,提高抗炎因子IL-10的表达。进一步,运用RT-PCR法检测了巨噬细胞极化标志物(M1型:iNOS和CD86;M2型:Arg1和CD206)的mRNA表达水平。与LPS组相比,人参皂苷Rk1显著下调iNOS和CD86的mRNA水平;显著上调了Arg1和CD206的mRNA水平。因此能够证明:Rk1在LPS刺激的RAW 264.7细胞模型中,能够抑制巨噬细胞的M1型极化,促进巨噬细胞的M2型极化。
Claims (3)
1.一种人参皂苷Rk1在制备治疗溃疡性结肠炎药物中的应用。
2.根据权利要求1所述的一种人参皂苷Rk1在制备治疗溃疡性结肠炎药物中的应用,其特征在于:所述药物包含人参皂苷Rk1在药学上能够接受的溶剂化合物中的一种或数种,以包含人参皂苷Rk1化合物为活性成份组成的药物组合物用于治疗溃疡性结肠炎。
3.根据权利要求1或2所述的一种人参皂苷Rk1在制备治疗溃疡性结肠炎药物中的应用,其特征在于:所述药物为片剂、丸剂、滴丸、胶囊剂、颗粒剂、散剂、粉剂、注射液、口服液或乳剂。
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