CN113274477A - 黄连膏二氧化碳超临界提取物在制备治疗皮肤病的药物中的应用 - Google Patents
黄连膏二氧化碳超临界提取物在制备治疗皮肤病的药物中的应用 Download PDFInfo
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Abstract
本发明涉及一种黄连膏二氧化碳超临界提取物在制备治疗皮肤病的药物中的应用,属于中药及产品开发应用领域。以经典名方黄连膏为基础配方,采用二氧化碳超临界萃取的方法制备其提取物,从体内及体外研究系统中多层次、全方面证实了黄连膏二氧化碳超临界提取物在制备治疗皮肤病的药物中的应用,对尘螨诱导的豚鼠湿疹皮肤模型的保护作用,对人类永生化角质形成细胞湿疹模型的作用机制,符合丝裂原活化蛋白激酶‑核转录激活蛋白氨基末端激酶信号通路,从而发挥黄连膏二氧化碳超临界提取物对皮肤屏障的修护作用。
Description
技术领域
本发明属于中药及产品开发应用领域,尤其涉及黄连膏二氧化碳超临界提取物在制备治疗皮肤病的药物中的应用,包含利用其为原料制备的具有改善和、或治疗湿疹类皮肤病的药品及健康产品。
背景技术
黄连膏最早见于外科专著《刘涓子鬼遗方》,清朝吴谦在《医宗金鉴》中形成了用于治疗皮肤疾病的中医外科方剂,其配方组成:黄连三钱,当归尾五钱,生地一两,黄柏三钱,姜黄三钱(按照现代的剂量关系换算后的配方组成是:黄连11.175g,当归尾18.625g,生地黄37.250g,黄柏11.175g,姜黄11.175g)。古书记载主要用于鼻部不适,如“此证生于鼻窍内,初觉干燥疼痛,状如粟粒,甚则鼻外色红微肿,痛似火炙。由肺经壅热,上攻鼻窍,聚而不散,致成此疮”。黄连膏近年来在临床上拓展了新用途,被广泛用于皮肤科、肛肠科、妇科、儿科、耳鼻喉科、烧伤科、肿瘤科、风湿科、普外科等,可以治疗湿热所致的烂皮、红肿、诸疥干痒,各种疮疡疔肿、烧伤、烫伤、湿疹及外伤,而且与其它药物的联合应用也较为广泛,如痔疮术后创面愈合,急性肛裂,皮疹、急性过敏性皮炎、毛囊炎、带状疱疹,湿疹糖尿病皮肤感染等,堪称中医外科之“圣药”。关于黄连膏临床研究的文献记载,同名配方不同的现象比较多,配方中经常出现的与《医宗金鉴》记载不符的药物,如黄芩、冰片等,且还有仅以黄连一味药为处方者。目前对黄连膏的研究与应用主要集中在临床方面,对机制的研究文献报道较少,如抗炎,烧伤愈合。
湿疹是一种常见的过敏性皮肤疾病,是由多种内外因素引起的表皮及真皮浅层的炎症性皮肤病。目前临床上湿疹的治疗方法包括口服或者外用激素药、免疫抑制剂和抗生素类药物等,偶见联合应用外敷中药制剂。目前研究发现,激素药被认为是治疗特应性皮炎最有效的疗法。但是激素的长期应用产生严重的副作用,导致其使用受到限制。
关于黄连膏治疗湿疹的临床研究报道很多,儿科方面如小儿湿疹,皮肤科如干性湿疹等,但是对其功效疗效作用机制的研究几近空白,仅见类似的关于加味黄连膏的报道,此报道是基于加味黄连膏香油提取物对二硝基氯苯小鼠皮肤湿疹模型的研究。Biostir-AD特应性皮炎诱导剂是一种与人类皮炎、湿疹发病相关的含有粉尘螨虫体成分的软膏,由于其所造皮炎模型与人类湿疹相似的临床特征,因此使用Biostir-AD软膏诱导的小鼠皮肤模型被广泛应用于湿疹发病机制的研究和评估治疗药物的疗效实验中。
微丝蛋白(FLG)是表皮屏障的重要组成部分,有助于维持表皮的结构、功能和水化程度。FLG中的等位基因功能丧失突变会引发特应性湿疹和继发性疾病,如哮喘、过敏性鼻炎等。在北欧,多达50%的中重度湿疹患者患病原因可归因于FLG突变。FLG是多态的,常见的等位基因变体编码由10、11或12个FLG单体Profilaggrin分子重复组成,每增加一次FLG重复就会相应降低患湿疹的风险,FLG表达量的微量增加对湿疹治疗效果也将有明细的改善。
截至目前,国内现有文献未见对黄连膏二氧化碳超临界提取物在治疗湿疹类皮肤病中的应用的报道,黄连膏及其二氧化碳超临界提取物对Biostir-AD诱导的豚鼠湿疹治疗方面的作用机制、物质基础等研究几近空白。
发明内容
本发明提供一种黄连膏二氧化碳超临界提取物在制备治疗皮肤病的药物中的应用。本发明以经典名方黄连膏为基础配方,采用二氧化碳超临界萃取的方法制备其提取物,并经气质联用色谱仪进行成分分析,首次发现黄连膏二氧化碳超临界提取物对尘螨诱导的豚鼠湿疹皮肤模型具有保护作用,对Hacat细胞湿疹模型的作用机制符合MAPK-JNK信号通路,确定了HLG对皮肤屏障的修护作用。包含利用HLG为原料制备的具有改善和/或治疗湿疹类皮肤病的药品及健康产品,特别但不限于应用于改善和/或治疗哺乳动物表皮的外用产品。
本发明采取的技术方案是:
一种黄连膏二氧化碳超临界提取物在制备治疗皮肤病的药物中的应用
本发明所述的黄连膏二氧化碳超临界提取物通过以下方式制备:取黄连膏配方中饮片,净制,粉碎至80~100目,置于超临界萃取斧中,萃取时间40~60min,萃取压力24~32MPa,萃取温度45℃~55℃,CO2流量30~50L/h,收集提取油,即为黄连膏二氧化碳超临界提取物,提取率1.5%~3.0%,提取物呈黑棕色,浓烈芳香气味。
优选的,黄连膏配方饮片,100目粉碎,置于超临界萃取斧中,萃取时间60min,萃取压力32MPa,萃取温度55℃,CO2流量40L/h,收集提取油,即得黄连膏二氧化碳超临界提取物。
本发明所述黄连膏二氧化碳超临界提取物在治疗皮肤病中的应用,还包括利用黄连膏二氧化碳超临界提取物为原料制备的具有改善和、或治疗湿疹类皮肤病的药品及健康产品。
本发明所述湿疹类皮肤病模型是基于尘螨诱导的豚鼠湿疹皮肤模型,是由过敏性皮炎诱发试剂Biostir AD软膏(Biostir-AD)诱导的豚鼠湿疹皮肤模型。
本发明所述黄连膏二氧化碳超临界提取物在治疗湿疹类皮肤病作用方面,是通过抑制JNK信号通路,减少炎症趋化因子分泌,提高核转录激活蛋白(C-Jun)表达量,促进细胞增殖,增加微丝蛋白(FLG)分泌量,从而达到对皮肤屏障的修护作用和对湿疹类皮肤病的治疗作用。
本发明的黄连膏二氧化碳超临界提取物可以添加常用的药用辅料制成药学上可接受外用膏剂、霜剂、气雾剂、喷剂、贴剂等,且所述膏剂、霜剂、气雾剂、喷剂、贴剂等可采用相应种类制剂的常规制备方法来制得。
对于应用本发明的黄连膏二氧化碳超临界提取物制备外用健康产品没有限制。因此,所述提取物可以添加到药品、化妆品、消毒产品、日化产品、医疗器械类产品等。
本发明的有益效果:
(1)本发明应用黄连膏二氧化碳超临界提取物,开发了一项对尘螨诱导的豚鼠湿疹皮肤模型的保护作用的新应用。
(2)本发明对所制备的黄连膏二氧化碳超临界提取物的物质基础采用气质联用色谱仪进行了半定量的成分分析,主要含有34种化学成分,其中含量较高的8种成分分别是α-姜黄烯、β-姜黄烯、β-柏木烯、α-姜黄酮、顺-藁本内酯、芳姜黄酮、curlone、α-atlantone。黄连膏二氧化碳超临界提取物纯度高,质地均匀,易于使用,提取过程中仅涉及二氧化碳气体,无外来杂质引进,对人体安全无毒副作用。
(3)本发明中黄连膏二氧化碳超临界提取物对Biostir-AD诱导的豚鼠湿疹皮肤模型的治疗效果进行了较为系统的研究,该豚鼠湿疹皮肤模型更贴近人的湿疹模型,明显区别二硝基甲苯(DNCB)试剂的造模方式。
首先体内研究建立豚鼠湿疹药理模型,观察造模后皮肤病理学改变情况,同时采用酶联免疫吸附法检测豚鼠血清中炎症因子水平变化;然后采用免疫组化法考察豚鼠皮肤中C-Jun、FLG蛋白表达量的变化。结果表明黄连膏二氧化碳超临界提取物能够改善由Biostir-AD软膏诱导的皮炎皮肤损伤、炎细胞浸润及红肿现象,修复受损的皮肤屏障,且与给药剂量呈正相关。与只涂抹Biostir-AD软膏的模型组相比,黄连膏二氧化碳超临界提取物降低了尘螨诱导的炎症因子TNF-a、IFN-r、IL-6、IgE的分泌量,抑制机体炎症反应的发展;免疫组化结果显示,黄连膏二氧化碳超临界提取物升高了C-Jun、FLG蛋白表达水平,促进皮肤细胞增殖,证明黄连膏二氧化碳超临界提取物对湿疹的治疗具有较好疗效。
(4)本发明中发明人结合体内及体外研究系统对黄连膏二氧化碳超临界提取物治疗湿疹的作用机制进行了探索。首先建立体外湿疹细胞模型,采用MTT法检测Hacat细胞的存活率,确定黄连膏二氧化碳超临界提取物的给药浓度,采用ELISA法检测造模及给药后炎症趋化因子水平变化。RT-PCR法检测JNK、ERK、ICAM-1基因表达量水平变化。Western Blot蛋白质印记法考察JNK、C-Jun、Jun-B、FLG蛋白表达量水平变化。研究结果表明黄连膏二氧化碳超临界提取物能降低Hacat细胞炎症趋化因子的分泌量,明显抑制炎症介质的释放;通过调节JNK和ERK通路,降低ICAM-1的表达并减少了表皮细胞炎症反应;促进皮肤细胞的增殖与分化,恢复C-Jun与Jun-B的平衡状态,增加FLG蛋白分泌量,从而起到对皮肤屏障的修护作用。
附图说明
图1是HLG气相色谱质谱总离子流图;
图2是豚鼠湿疹皮炎评分图;
图3是肿瘤坏死因子-a(TNF-a)分泌量图;其中与空白组比较,###P<0.001;与模型组比较,*P<0.05,**P<0.01,***表示P<0.001;
图4是干扰素-r(IFN-r)分泌量图;其中与空白组比较,###P<0.001;与模型组比较,*P<0.05,**P<0.01,***表示P<0.001;
图5是免疫球蛋白E(IgE)分泌量图;其中与空白组比较,##P<0.01;与模型组比较,*P<0.05,**P<0.01,***表示P<0.001;
图6是白介素-6(IL-6)分泌量图;其中与空白组比较,###P<0.001;与模型组比较,*P<0.05,**P<0.01,***表示P<0.001;
图7是豚鼠皮肤HE染色结果(×200,↑为表皮)对照组图;
图11是豚鼠皮肤HE染色结果(×200,↑为表皮)中剂量组图;
图12是豚鼠皮肤HE染色结果(×200,↑为表皮)高剂量组图;
图13是C-Jun蛋白免疫组化结果(×100)空白组图;
图14是C-Jun蛋白免疫组化结果(×100)模型组图;
图15是C-Jun蛋白免疫组化结果(×100)阳性组图;
图16是C-Jun蛋白免疫组化结果(×100)低剂量组图;
图17是C-Jun蛋白免疫组化结果(×100)中剂量组图;
图18是C-Jun蛋白免疫组化结果(×100)高剂量组图;
图19是FLG蛋白免疫组化结果(×100)空白组图;
图20是FLG蛋白免疫组化结果(×100)模型组图;
图21是FLG蛋白免疫组化结果(×100)阳性组图;
图22是FLG蛋白免疫组化结果(×100)低剂量组图;
图23是FLG蛋白免疫组化结果(×100)中剂量组图;
图24是FLG蛋白免疫组化结果(×100)高剂量组图;
图25是TNF-α和IFN-γ不同造模浓度对Hacat细胞影响图;其中*表示P<0.05,**表示P<0.01,***表示P<0.001;
图26是HLG对Hacat细胞的细胞生存性的影响图;其中*表示P<0.05,**表示P<0.01,***表示P<0.001;
图27是HLG对体外湿疹模型的细胞存活率的影响图;其中与空白组比较,###P<0.001;与模型组比较,*P<0.05,**P<0.01,***表示P<0.001;
图28是胸腺活化调节趋化因子(TARC)分泌量图;其中*表示P<0.05,**表示P<0.01,***表示P<0.001;
图29是巨噬细胞源性趋化因子(MDC)分泌量图;其中*表示P<0.05,**表示P<0.01,***表示P<0.001;
图30是调节活化T细胞表达和分泌因子(RANTES)分泌量图;其中*表示P<0.05,**表示P<0.01,***表示P<0.001;
图31是白细胞介素-8(IL-8)分泌量图;其中*表示P<0.05,**表示P<0.01,***表示P<0.001;
图32是J NK/GAPDH mRNA基因表达水平图;图中与空白组比较,#P<0.05;与模型组比较,*P<0.05,**P<0.01,***表示P<0.001;
图33是ERK/GAPDH mRNA基因表达水平图;;图中与空白组比较,#P<0.05;与模型组比较,*P<0.05,**P<0.01,***表示P<0.001;
图34是ICAM-1/GAPDH mRNA基因表达水平图;图中与空白组比较,##P<0.01;与模型组比较,*P<0.05,**P<0.01,***表示P<0.001;
图35是P-JNK/JNK蛋白表达图;
图36是P-JNK/JNK蛋白灰度值之比分析图;图中与空白组比较,##P<0.01;与模型组比较,*P<0.05,**P<0.01,***表示P<0.001;
图37是C-Jun/C-Jun蛋白表达图;
图38是C-Jun/C-Jun蛋白灰度值之比分析图;图中与空白组比较,###P<0.001;与模型组比较,*P<0.05,**P<0.01,***表示P<0.001;
图39是P-Jun-B/Jun-B蛋白表达图;
图40是P-Jun-B/Jun-B蛋白灰度值之比分析图;图中与空白组比较,##P<0.01;与模型组比较,*P<0.05,**P<0.01,***表示P<0.001;
图41是Flg/GAPDH蛋白表达图;
图42是Flg/GAPDH蛋白灰度值之比分析图;图中与空白组比较,###P<0.001;与模型组比较,*P<0.05,**P<0.01,***表示P<0.001;
图43是豚鼠湿疹皮肤观察图。
具体实施方式
下面结合具体实施例对本发明做进一步详细的说明,但实施例并不对本发明做任何形式的限定,以下实施例中的方法、设备、材料,如果未特别说明,均为本领域常规的方法、设备和材料。
实施例1本发明一种黄连膏二氧化碳超临界提取物软膏在治疗湿疹药物中的应用。
黄连111.75g,当归尾186.25g,生地黄372.50g,黄柏111.75g,姜黄111.75g,粉碎,过100目筛,置于超临界萃取斧中,萃取时间60min,萃取压力32MPa,萃取温度55℃,CO2流量40L/时,收集提取油20.5mL,加入辅料(羊毛脂,凡士林,蜂蜡、甘油、矿油等),制成软膏剂1000g。
实施例2本发明一种黄连膏二氧化碳超临界提取物护肤乳在缓解湿疹皮肤炎症等症状中的应用。
黄连111.75g,当归尾186.25g,生地黄372.50g,黄柏111.75g,姜黄111.75g,粉碎,过80目筛,置于超临界萃取斧中,萃取时间40min,萃取压力28MPa,萃取温度50℃,CO2流量30L/时,收集提取油19.7mL,加入辅料(水、矿油、矿脂、合成蜡、甘油、聚乙烯、微晶蜡、丁二醇等),制成护肤乳20公斤。
实施例3本发明一种黄连膏二氧化碳超临界提取物在润肤皂的应用。
黄连111.75g,当归尾186.25g,生地黄372.50g,黄柏111.75g,姜黄111.75g,粉碎,过100目筛,置于超临界萃取斧中,萃取时间50min,萃取压力24MPa,萃取温度45℃,CO2流量50L/时,收集提取油22.4mL,加入辅料(棕榈油酸钠、棕榈仁油酸钠、甘油、EDTA-2Na、二氧化钛等),制成润肤皂20公斤。
本发明中的黄连膏二氧化碳超临界提取物(以下简称HLG)在治疗皮肤病中的应用,具体药理实验和作用机制研究方法如下:
1.实验方法:
1.1实验细胞与动物
Hacat细胞:购自武汉普诺赛生命科技有限公司,培养条件为MEM+15%胎牛血清+1%双抗,37℃、5%CO2培养箱。
豚鼠:购自长春市亿斯实验动物技术有限责任公司,普通级,180-200g。(合格证编号:202000033830)
1.2黄连膏二氧化碳超临界提取物(HLG)成分分析
采用气相色谱-质谱法(GC/MS)进行分析。
色谱条件:色谱柱Thermo SCIENTIFIC TG-1MS(30m×0.32mm×0.25μm);载气是99.999%高纯氦气,流速1mL/min;升温程序:初始柱温40℃,以15℃/min升至120℃,保持2min,然后以5℃/min升至250℃,再以10℃/min升至280℃,保持5min;进样方式及进样量:气相液体进样,进样量1μL。
质谱条件:EI离子源;MS传输管温度280℃;离子源温度280℃。从5min起检测,采用全扫描(Full Scan)模式鉴定衍生物结构,质量扫描范围m/z:30-500,定量分析时采用单反应离子检测(selective reaction monitoring mode,SRM)模式。
1.3豚鼠湿疹皮肤模型的建立
60只8周龄的雄性豚鼠随机分为6组,每组十只。实验前24h所有豚鼠背部脱毛并涂抹4%十二烷基硫酸钠破坏屏障。实验第1-14天为造模期,模型组、阳性药组及三个给药组每隔3天在豚鼠背部脱毛的区域涂抹Biostir-AD软膏诱发湿疹样皮损,空白对照组正常饲养并涂抹赋形剂(医用羊毛脂:医用凡士林=1:1);实验第15-28天为给药期,空白对照组(赋形剂:1次/3天:1次/天)、模型组(Biostir-AD软膏50mg/只/次:1次/3天+赋形剂:1次/天)、阳性药组(Biostir-AD软膏50mg/只/次:1次/3天+氢化可的松乳膏50mg/只/次:1次/天)、低剂量组(Biostir-AD软膏50mg/只/次:1次/3天+含生药量0.09g/g自制HLG软膏:1次/天)、中剂量组(Biostir-AD软膏50mg/只/次:1次/3天+含生药量0.18g/g自制HLG软膏:1次/天)、高剂量组(Biostir-AD软膏50mg/只/次:1次/3天+含生药量0.37g/g自制HLG软膏:1次/天),每7天进行一次拍照,观察湿疹皮损严重程度。自制HLG软膏是HLG按照超临界提取率加入与生药量相当的超临界提取油,与赋形剂(医用羊毛脂:医用凡士林=1:1)制成的软膏剂。
1.4血清采集及检测
实验第28天豚鼠麻醉,心脏取血,4℃条件离心15min,转速3500rpm/min,取血清。采用ELISA法测定血清中TNF-α,IFN-r、IgE、IL-6水平,具体步骤参考试剂盒说明书。
1.5实验皮肤采集
实验第28天豚鼠麻醉,剪取豚鼠背部湿疹皮肤1*1cm2置于盛满4%多聚甲醛固定液的表面皿中24h,再将初步固定的皮肤组织,转移到装满4%多聚甲醛的50mL离心管中继续固定。
1.6造模皮肤损伤情况观察及评分
根据湿疹面积和严重程度指数(EASI)评分系统,对湿疹的相对严重程度进行宏观评估:0-无症状;1-轻度症状;2-中度症状;3-严重症状。皮炎评分:红斑/出血、水肿、切除/糜烂和结垢/干燥评分的总和。
1.7造模皮肤组织病理学检查
固定好的皮肤组织经脱水,包埋,切片后,按照以下步骤操作:利用苏木素-伊红染色法(简称HE)常规病理染色,切片于二甲苯Ⅰ和Ⅱ内浸泡应各达15~20分钟脱腊;切片经苏木素染色后,在染伊红前,经1%酸性酒精处理2~3秒,流水冲洗至少20~30分钟。盖玻片下封固切片,95%乙醇1min×2→100%乙醇1min×2→1:1乙醇二甲苯15min→二甲苯5min×2→中性树胶封片,室温干燥保存。采用数码显微摄像系统对切片进行图像采集,每张切片先于40倍下观察全部组织,观察大体病变,选择要观察的区域采集100倍和200倍图片,观察具体病变。
1.8免疫组化
将石蜡切片在80℃下烘烤3.小时,经二甲苯脱蜡,梯度乙醇水化,蒸馏水清洗后;滴加3%.甲醇H2O2,室温处理10min,PBS洗5min(3次);将切片浸入0.01M柠檬酸盐缓冲液(PH 6.0),加热至沸腾,间隔5min后,反复1次,冷却后,PBS洗5min(2次);山羊免疫血清室温封闭20min,加入按1:300稀释的一抗,4℃过夜,回收一抗,加入二抗,37℃,孵育30min,PBS洗5min(3次);室温条件DAB显色,显微镜下监测显色作用1-2min,蒸馏水终止显色反应。苏木素复染,盐酸酒精分化,流水冲洗返蓝1min,梯度酒精脱水,二甲苯透明,中性树胶封片。
1.9Hacat细胞体外湿疹模型的建立及炎症因子浓度的确定
采用血清饥饿法建立体外湿疹炎症模型。取对数生长期的Hacat细胞悬液,接种于96孔板中(4000个/孔),置于细胞培养箱培养24h。待细胞贴壁后更换无血清空白培养基饥饿继续培养6h。更换含有不同造模浓度细胞因子的培养基处理24h后,每孔加入5mg/mL MTT溶液20μL,37℃避光孵育4h,弃去培养基,加入150μL DMSO,利用酶标仪在490nm处,检测吸光度(OD),计算细胞存活率及LD50时造模浓度。
细胞存活率(%)=OD(药物)/OD(空白)*100
实验分组如下:
空白组:MEM空白培养液培养的Hacat细胞;
造模1组:MEM空白培养液(TNF-α10ng/mL+IFN-γ10ng/mL);
造模2组:MEM空白培养液(TNF-α20ng/mL+IFN-γ20ng/mL);
造模3组:MEM空白培养液(TNF-α30ng/mL+IFN-γ30ng/mL);
造模4组:MEM空白培养液(TNF-α40ng/mL+IFN-γ40ng/mL)。
1.10Hacat细胞毒性试验及HLG给药剂量确定
胰酶消化Hacat细胞,每孔加入100μL的细胞悬液于96孔板中(4000个/孔),置培养箱中培养。待细胞贴壁后,弃去培养基,加入无血清培养基饥饿培养6h,再更换含有不同浓度HLG(0.001μL/mL、0.01μL/mL、0.1μL/mL、0.25μL/mL)的MEM无血清培养基100μL。MEM无血清培养基作为空白对照组。HLG给药24h后,每孔加入5mg/mL MTT溶液20μL,37℃避光孵育4h,弃去培养基,加入150μL DMSO,酶标仪490nm下测定吸光度值(OD)。计算细胞存活率。
取对数生长期的Hacat细胞接种于96孔板中(4000个/孔),培养24h,待细胞贴壁后,更换无血清培养基饥饿细胞6h,弃去空培养基。按照各组不同条件加药,继续培养。
A.空白组:MEM空白培养液培养的Hacat细胞;
B.模型组:MEM空白培养液(TNF-α/IFN-γ20ng/mL);
C.剂量组:MEM空白培养液(TNF-α/IFN-γ20ng/mL)+HLG(0.001μL/mL);
D.剂量组:MEM空白培养液(TNF-α/IFN-γ20ng/mL)+HLG(0.01μL/mL);
E.剂量组:MEM空白培养液(TNF-α/IFN-γ20ng/mL)+HLG(0.1μL/mL);
F.剂量组:MEM空白培养液(TNF-α/IFN-γ20ng/mL)+HLG(0.25μL/mL)
HLG作用12h后,每孔加入5mg/mL MTT溶液20μL,37℃避光孵育4h,弃去原培养液,加入150μL DMSO,酶标仪490nm波长下测定吸光度值(OD)。计算细胞存活率。
1.11ELISA法检测炎症趋化因子含量
(1)取对数生长期的Hacat细胞接种于六孔板中(7*104个/孔),培养24h,待细胞贴壁后,更换无血清培养基饥饿细胞6h,弃去空培养基。按照各组不同条件加药,继续培养24h。
(2)收集每孔上清培养基,1000rpm/min离心5min去除细胞碎片,采用Elisa酶联免疫吸附法测定上清培养基中TARC、MDC、RANTES、IL-8的含量。
1.12荧光定量PCR
各组细胞经药物处理后,使用Trizol法提取总RNA,利用琼脂糖凝胶电泳检测RNA完整性,并通过BioSpec-nano分光光度计检测RNA纯度及浓度。使用反转录试剂盒,将总RNA反转录合成cDNA。反转录条件为温度37℃,时间15min;85℃,5sec;4℃,5min。在Primer5.0软件上设计特异引物,引物序列如下表所示。RT-qPCR利用SYBR-Green荧光染料测定Ct值,热循环条件:在95℃下初始变性30秒,然后在95℃下保持5秒,54℃下保持15秒,72℃下保持30秒进行40个循环。以GAPDH作为内参,利用2-ΔΔCt计算每个基因的表达倍数变化。
1.13BCA法蛋白含量测定
根据碧云天BCA蛋白含量检测试剂盒说明书操作步骤进行检测。
1.14Western blot检测目标蛋白的表达
各组细胞经药物处理后,于RIPA缓冲液中裂解。使用BCA法进行蛋白质定量。利用10%SDS-PAGE凝胶电泳分离蛋白,采用半干转法将蛋白转移到NC膜上,5%脱脂奶粉室温封闭1小时后,PBST洗涤三次,每次5min,加入一抗(1:1000)置于摇床4℃孵育过夜。回收一抗,PBST洗涤三次,每次5min,加入二抗(1:1000),室温孵育1h。PBST洗涤三次,每次5min,利用ECL显影试剂盒于iBright凝胶成像系统拍照并分析光密度。
1.15统计学分析
本发明所有数据进行3次生物学重复,统计学分析应用GraphPad Prism6软件完成,定量数据以means+SD的形式展示,只比较两组间差异应用t-test检验。*P<0.05表示有显著性差异。
2实验结果
2.1HLG成分分析结果
本发明对所制备的黄连膏二氧化碳超临界提取物的物质基础采用气质联用色谱仪进行了半定量的成分分析,总离子流图见图1,主要含有34种化学成分,其中含量较高的8种成分分别是α-姜黄烯、β-姜黄烯、β-柏木烯、α-姜黄酮、顺-藁本内酯、芳姜黄酮、curlone、α-atlantone。黄连膏二氧化碳超临界提取物纯度高,质地均匀,易于使用,提取过程中仅涉及二氧化碳气体,无外来杂质引进,对人体安全无毒副作用。
2.2HLG对豚鼠湿疹皮肤形态学变化的影响及严重程度评分
每周评估一次豚鼠湿疹皮肤宏观观察如图43所示,造模2周后,豚鼠肉眼可见皮损改变具有急性湿疹皮疹的特征,表现为明显红肿、小丘疹、渗液、血痂、抓痕、轻微糜烂、部分表面粗糙干燥、上覆鳞屑。
给药2周后,与空白组相比,模型组皮损无明显恢复,且愈加严重,背部皮肤变厚,出现严重红斑,出血结痂,并伴随组织渗液(P<0.01);与模型组相比,阳性药物组的湿疹皮肤红肿基本消失,皮肤无明显增厚,鳞屑现象(P<0.001);给药组(0.09g/g;0.18g/g;0.37g/g)豚鼠湿疹皮肤血痂基本痊愈,丘疹样红疹明显缓解,没有明显的皮肤增厚和鳞屑,皮损恢复程度与HLG浓度具有剂量依赖性(P<0.001)。表1为豚鼠湿疹皮肤评分表,图2豚鼠湿疹皮炎评分图。
表1豚鼠湿疹皮肤评分表
2.3HLG对豚鼠血清炎症因子水平的影响
(1)对TNF-a水平的影响
豚鼠血清中TNF-a水平如图3所示,模型组豚鼠发生炎症反应导致TNF-a表达明显上调;与模型组相比,阳性药组(氢化可的松)显著减轻了皮肤炎症状态,且效果与高剂量组相似;给药组从数值上看均降低了TNF-a的表达量,且具有剂量依赖性。
(2)对IFN-r水平的影响
豚鼠血清中IFN-r水平如图4所示,模型组豚鼠发生炎症反应导致IFN-r表达明显上调;阳性药组(氢化可的松)与模型组相比,降低了皮肤炎症状态,但是效果没有给药组明显;给药组从数值上看均降低了IFN-r的表达量,且具有剂量依赖性。
(3)对IgE水平的影响
豚鼠血清中IgE水平如图5所示,模型组豚鼠发生炎症反应导致IgE表达明显上调;阳性药组(氢化可的松)与模型组相比,明显降低皮肤炎症状态与高剂量组效果相似;给药组从数值上看均降低了IgE的表达量,且具有剂量依赖性。
(4)对IL-6水平的影响
豚鼠血清中IL-6水平如图6所示,模型组豚鼠发生炎症反应导致IL-6表达明显上调;阳性药组(氢化可的松)与模型组相比,明显降低皮肤炎症状态与高剂量组效果相似;给药组从数值上看均降低了IL-6的表达量,且具有剂量依赖性。
2.4HLG对豚鼠湿疹皮肤恢复的组织病理学观察
各组豚鼠背部皮肤HE染色结果见图7—图12。在空白组中,皮肤的表皮层完整且薄,并且在真皮层中没有炎性细胞的浸润;模型组皮肤的表皮层明显增厚、角化层明显,且大量炎细胞浸润真皮层,表皮偶见破损;阳性药组的表皮厚度与中、高剂量组相似,但真皮炎细胞浸润明显高于中,高剂量组;低剂量组表皮略厚于阳性药组及中高剂量组,真皮层炎细胞浸润情况明显好于模型组。
2.5HLG对豚鼠湿疹皮肤中C-Jun、FLG蛋白水平的影响
根据黄连膏所含药材与湿疹共有靶点蛋白质相互作用(PPI)网络图可知,节点最多的JUN靶点在湿疹的发病与治疗中起着关键作用,结合KEGG通路富集分析结果并查阅相关文献可知,C-Jun通过MAPK-JNK信号通路参与细胞增殖,最终影响FLG的表达量。本次检测结果阴性细胞表现为蓝色,底物表现为白色,阳性细胞表现为黄色或者棕色,c-Jun和FLG阳性产物主要分布在细胞质中,细胞质阳性结果呈棕黄色。
(1)C-Jun蛋白表达分析
C-Jun蛋白免疫组化结果见图13—图18。与空白组相比,模型组皮肤细胞增殖被抑制,C-Jun蛋白表达率降低;阳性药组与模型组相比C-Jun表达量增加,但其作用效果弱于中,高剂量组,因此从蛋白表达角度来看,HLG中,高剂量组对于改善机体炎症反应,促进修复皮肤屏障方面显示了更好的活性。
(2)FLG蛋白表达分析
FLG蛋白免疫组化结果见图19-图24。与空白组相比,模型组皮肤细胞增殖被抑制,FLG蛋白表达率降低;阳性药组与模型组相比C-Jun表达量有部分增加,但其作用效果弱于给药组,因此从蛋白表达角度来看,黄连膏超临界二氧化碳提取物高剂量组对于改善机体炎症反应,促进修复皮肤屏障方面显示了更好的活性。
2.6HLG对体外湿疹模型细胞的保护作用
(1)TNF-α和IFN-γ细胞因子的最适造模浓度
采用重复测量设计的方差分析,比较不同浓度TNF-α和IFN-γ细胞因子对Hacat细胞存活率差异有统计学意义(P<0.05)。细胞存活率随TNF-α和IFN-γ细胞因子造模浓度的升高而逐渐降低,并存在显著性差异,如图25所示当空白培养基中TNF-α和IFN-γ终浓度为20ng/mL时,细胞存活率为50%,此浓度即为Hacat细胞湿疹模型的LD50。
(2)HLG实验浓度筛选
如图26所示,当浓HLG度达到0.25μL/mL时,对细胞的生长出现抑制作用;HLG在0.001μL/mL至0.1μL/mL浓度范围内,对Hacat细胞无细胞毒性,经实验最终确定HLG浓度0.001μL/mL、0.01μL/mL、0.03μL/mL作为实验浓度。
(3)HLG对体外湿疹模型的有效性
如图27所示,模型组细胞存活率与空白组比较具有统计学意义(P<0.001);给药后,HLG 0.01μL/mL组和0.03μL/mL组细胞存活率分别升高至101%和105%(P<0.001),具有统计学意义;HLG 0.001μL/mL组对造模细胞也具有较弱的增殖作用,细胞的存活率为73%。证明HLG对TNF-α和IFN-γ损伤的Hacat细胞有保护作用。
2.7HLG对体外湿疹模型细胞分泌的炎症趋化因子水平的影响
(1)对TARC水平的影响
以TNF-α和IFN-γ(TI)刺激的Hacat细胞为研究对象,如图28所示,TI处理的Hacat细胞产生的TARC明显高于溶剂对照处理的细胞(P<0.05)。与此形成对照的是,HLG以剂量依赖性的方式抑制了TI刺激的TARC的产生,具有统计学意义。
(2)对MDC水平的影响
TI处理的Hacat细胞产生的MDC与溶剂对照组相比,有显著的统计学意义,如图29所示,HLG对TI诱导的MDC分泌量增加有明显的抑制作用。
(3)对RANTES水平的影响
如图30所示,TI处理细胞产生的RANTES高于溶剂对照组(P<0.1)。0.001μL/mL和0.01μL/mL浓度HLG对TI处理的Hacat细胞具有微弱抑制作用,0.03μL/mL HLG对RANTES有较强抑制作用(P<0.01)。
(4)对IL-8水平的影响
HLG以浓度依赖性的方式,显著降低白介素-8(IL-8)的水平,如图31所示,HLG对TI造成的皮肤细胞损伤有较好的恢复作用,经不同浓度HLG作用后,其炎症病变的相应指标得到了极大的改善,保护作用增强,使受损皮肤细胞恢复正常水平,其中加入0.03μL/mL提取油时IL-8分泌量最低。
2.8HLG对体外湿疹模型细胞JNK/ERK信号通路核酸表达的影响
各种免疫细胞如单核细胞,中性粒细胞和活化的T细胞从血液中渗透到皮肤是炎症性皮肤病发展的关键步骤之一。在肿瘤坏死因子(TNF-a)和干扰素(IFN-r)的促炎刺激下,角质形成细胞可通过JNK、ERK信号通路表达各种促炎细胞因子/趋化因子和粘附分子,如细胞间粘附分子-1(ICAM-1),这有助于免疫细胞进入皮肤炎症部位,加速了对湿疹皮肤的有害作用。
(1)JNK基因的mRNA表达分析
运用qRT-PCR法,以GAPDH为内参基因,对HLG作用Hacat细胞24h后JNK的mRNA表达量进行分析,基因表达水平如图32所示,模型组由于TNF-α和IFN-γ刺激导致JNK基因表达明显上调(P<0.05);与模型组相比阳性药组(氢化可的松乳膏1mg/mL)与HLG 0.001μL/mL组作用效果相似,JNK基因表达量明显下降;HLG 0.001μL/mL、0.01μL/mL和0.03μL/mL组JNK的mRNA表达量逐渐递减,呈现剂量依赖性,与模型组相比较差异均有统计学意义(P<0.05)。
(2)ERK基因的mRNA表达分析
ERK基因表达水平见图33,模型组由于TNF-α和IFN-γ刺激导致ERK基因表达明显上调(P<0.05);HLG组0.001μL/mL、0.01μL/mL和0.03μL/mL组ERK的mRNA表达量逐渐递减,呈现剂量依赖性;阳性药组(氢化可的松乳膏1mg/mL)与HLG0.001μL/mL组作用效果相似,JNK基因表达量明显下降;与模型组相比较差异均有统计学意义(P<0.05)。
(3)ICAM-1基因的mRNA表达分析
细胞间粘附分子(ICAM)-1属于粘附分子免疫球蛋白超家族,在炎症发生早期起着关键作用。在正常机体中其表达很微弱,当有炎症存在的组织中其表达明显增强。ICAM-1基因表达水平见图34,模型组由于TNF-α和IFN-γ刺激导致ICAM-1基因表达明显上调(P<0.01);HLG 0.001μL/mL、0.01μL/mL和0.03μL/mL组ICAM-1的mRNA表达量逐渐递减,呈现剂量依赖性;阳性药组(氢化可的松乳膏1mg/mL)与HLG0.001μL/mL组作用效果相似,ICAM-1基因表达量明显下降;与模型组相比较差异均有统计学意义(P<0.05)。
2.9HLG对体外湿疹模型细胞JNK、C-Jun、Jun-B、Flg蛋白的影响
调节皮肤细胞的增殖,分化需要大量的基因协同表达,而转录因子活化蛋白-1(AP-1)在JNK通路下游具有调控这些基因表达的作用,如外皮蛋白(involucrin)、兜甲蛋白(loricrin)、丝聚合蛋白(Flg)。Jun-B和C-Jun是AP-1的重要组成成员:C-Jun通过调节某些细胞周期调控蛋白来促进细胞增殖,而Jun-B通过激活p16INK4a抑制分子及减少周期蛋白的表达对细胞生长进行负调控,Jun-B和C-Jun的生物学作用相反且两者间的平衡决定了表皮的动态稳定。
(1)JNK蛋白表达分析
WB法检测了JNK通路相关蛋白的表达水平,JNK蛋白电泳结果如图35所示,蛋白灰度值分析结果如图36所示,与空白组相比,模型组P-JNK呈高表达。阳性药组(氢化可的松乳膏1mg/mL)可降低P-JNK与JNK比值至与空白组持平,给药后,0.001μL/mL组P-JNK与JNK蛋白灰度值之比有所降低,0.01μL/mL和0.03μL/mL组P-JNK与JNK的比值明显降低,综合分析表明HLG对TNF-α和IFN-γ刺激的Hacat细胞有保护作用。
(2)C-Jun蛋白表达分析
C-Jun蛋白电泳结果如图37所示,蛋白灰度值分析结果如图38所示,HLG能促进Hacat细胞增殖与分化,显著升高P-C-Jun/C-Jun灰度值之比,且呈剂量依赖性(P<0.001);阳性药组(氢化可的松乳膏1mg/mL)对于降低TNF-α和IFN-γ刺激产生的炎症反应和促进细胞的增殖方面显示出更好的活性(P<0.001)。
(3)Jun-B蛋白表达分析
Jun-B蛋白电泳结果如图39所示,蛋白灰度值分析结果如图40所示,模型组与空白组相比,细胞凋亡率升高,炎症损伤情况加重(P<0.01);阳性药组(氢化可的松乳膏1mg/mL)可降低P-Jun-B/Jun-B灰度值比值;HLG组能够明显降低细胞凋亡率及炎症损伤情况,0.03μL/mL组显示出更好的活性(P<0.001)。
(4)Flg蛋白表达分析
Flg蛋白电泳结果如图41所示,蛋白灰度值分析结果如图42所示,模型组与空白组相比Flg蛋白表达量明显减少,这与湿疹状态皮肤受损情况相似(P<0.001);给药后,0.001μL/mL组Flg蛋白分泌量比模型组更低,0.01μL/mL和0.03μL/mL组与模型组比较Flg蛋白分泌量有所增加,但没有阳性药组(氢化可的松乳膏1mg/mL)高,就恢复湿疹皮肤创面,升高Flg蛋白表达量方面阳性药组效果最好。
3小结
(1)黄连膏超临界二氧化碳提取物能够改善由Biostir-AD软膏诱导的皮炎皮肤损伤、炎细胞浸润及红肿现象,修复受损的皮肤屏障。
(2)黄连膏超临界二氧化碳提取物通过对炎症因子TNF-α、IFN-γ、IL-6、IgE表达量的调控抑制机体炎症反应的发生,从而起到对皮肤的保护作用。
(3)黄连膏超临界二氧化碳提取物可以降低炎症趋化因子的分泌量,且与剂量呈正相关,可明显抑制炎症介质的释放,减轻表皮细胞炎症反应,促进皮肤屏障恢复。
(4)黄连膏超临界二氧化碳提取物通过对JNK和ERK通路的调节降低ICAM-1的表达量,促进皮肤细胞的增殖与分化,恢复C-Jun与Jun-B的平衡状态,增加Flg蛋白分泌量,从而起到对皮肤屏障的保护作用。
Claims (7)
1.黄连膏二氧化碳超临界提取物在制备治疗皮肤病的药物中的应用。
2.如权利要求1所述的黄连膏二氧化碳超临界提取物在制备治疗皮肤病的药物中的应用,其特征在于:所述皮肤病为湿疹类皮肤病。
3.如权利要求1所述的黄连膏二氧化碳超临界提取物在制备治疗皮肤病的药物中的应用,其特征在于:所述的黄连膏二氧化碳超临界提取物通过以下方式制备:取黄连膏配方中饮片,净制,粉碎至80~100目,置于超临界萃取斧中,萃取时间40~60min,萃取压力24~32MPa,萃取温度45℃~55℃,CO2流量30~50L/h,收集提取油,即为黄连膏二氧化碳超临界提取物。
4.如权利要求3所述的黄连膏二氧化碳超临界提取物在制备治疗皮肤病的药物中的应用,其特征在于:取黄连膏配方饮片,100目粉碎,置于超临界萃取斧中,萃取时间60min,萃取压力32MPa,萃取温度55℃,CO2流量40L/h,收集提取油,即得黄连膏二氧化碳超临界提取物。
5.如权利要求1所述的黄连膏二氧化碳超临界提取物在制备治疗皮肤病的药物中的应用,其特征在于:包含利用其为原料制备的具有改善和、或治疗湿疹类皮肤病的药品及健康产品。
6.如权利要求2所述的黄连膏二氧化碳超临界提取物在制备治疗皮肤病的药物中的应用,其特征在于:湿疹类皮肤病模型是基于尘螨诱导的豚鼠湿疹皮肤模型,由过敏性皮炎诱发试剂Biostir-AD软膏诱导的豚鼠湿疹皮肤模型。
7.如权利要求2或6所述的黄连膏二氧化碳超临界提取物在制备治疗皮肤病的药物中的应用,其特征在于:黄连膏二氧化碳超临界提取物在治疗湿疹类皮肤病作用方面,是通过抑制JNK信号通路,减少炎症趋化因子分泌,提高核转录激活蛋白C-Jun表达量,促进细胞增殖,增加微丝蛋白FLG分泌量,从而达到对皮肤屏障的修护作用和对湿疹类皮肤病的治疗作用。
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