CN115837039A - 一种防治酒精性肝损伤的兰坪被毛孢核苷提取物 - Google Patents
一种防治酒精性肝损伤的兰坪被毛孢核苷提取物 Download PDFInfo
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Abstract
本发明公开了一种兰坪被毛孢(Hirsutella lanpingensis)核苷提取物的新用途,即其在制备防治酒精性肝损伤药物中的应用,实验结果显示,兰坪被毛孢核苷提取物对酒精诱导的小鼠肝损伤具有较好的保护作用,可降低酒精性肝损伤模型小鼠的血清谷丙转氨酶(ALT)和谷草转氨酶(AST)水平;降低模型小鼠肝组织中丙二醛(MDA)含量,提高超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH)活性。此外,兰坪被毛孢核苷提取物还能减轻肝脏炎症,减轻酒精诱导的肝组织病理改变。因此,兰坪被毛孢核苷提取物可成为开发抗酒精性肝损伤的潜在候选药物。
Description
技术领域
本发明属于药学技术领域,具体涉及兰坪被毛孢(Hirsutella lanpingensis)核苷提取物在制备防治酒精性肝损伤药物中的应用。
背景技术
酒精(乙醇)是一种具有高渗透性的小分子有机物,极易被细胞吸收而诱导产生大量自由基,引起细胞氧化应激损伤。肝脏是酒精代谢的主要器官,也是酒精性损伤的主要靶器官。酒精性肝病(ALD)是长期大量饮酒所导致的,临床上根据病变组织是否伴有炎症反应和纤维化,将其分为酒精性脂肪肝、酒精性肝炎、酒精性肝纤维化和酒精性肝硬化,随着疾病的进展,部分患者会发展为肝癌。据WHO统计,全球范围内过量饮酒每年可造成330万人死亡,48%的肝硬化是由酒精导致的。虽然中国尚缺少全国性大规模的ALD流行病学调查,但地区性调查结果显示,中国饮酒人群占比及ALD患病率逐年上升,酒精已经成为我国继病毒性肝炎后导致肝损伤的第二大病因。ALD发病机制涉及乙醇代谢、氧化应激损伤、免疫损伤及肠道微生态紊乱等,由于其发病机制的复杂性,目前仍然缺乏针对性的治疗药物。因此,ALD带来的健康危害和经济负担不容小觑,新的治疗药物的开发迫在眉睫。
酒精对肝细胞损害的机制尚未完全阐明,可能涉及酒精代谢物乙醛与蛋白结合物所致肝损伤,酒精代谢导致肝小叶缺氧、肝内代谢紊乱、肝脏微循环障碍,酒精导致肠道菌群易位等多种机制。正是其机制的复杂性,使得天然药物在治疗ALD中体现出明显优势。天然产物具有多成分、多靶点、多环节综合作用的特点,可以通过多种机制保护肝脏,因此在天然产物中寻找开发抗酒精性肝损伤的活性成分具有重要意义。
兰坪被毛孢(Hirsutella lanpingensis H.Yu & Z.H.Chen sp.nov.)是线虫草科(Ophiocordyceps)线虫草属(Ophiocordyceps)物种,主要分布于喜马拉雅山脉东部的横断山区,具有药食同源性,现代药理学研究表明兰坪被毛孢具有增强免疫力、抗疲劳、抑制氧化应激等功效,并认为主要活性成分为多糖类物质。而我们的研究发现兰坪被毛孢核苷核苷提取物类成分对小鼠酒精性肝损伤具有较好的保护作用,具有开发成治疗酒精性肝损伤药物的潜力和前景。
发明内容
本发明提供了兰坪被毛孢(Hirsutella lanpingensis)核苷提取物的一种新用途,即兰坪被毛孢核苷提取物在制备防治酒精性肝损伤药物中的应用。
本申请中涉及的兰坪被毛孢(Hirsutella lanpingensis)菌株在本发明申请日之前已经在非专利文献中公开过,即在“响应面分析法优化超声提取兰坪被毛孢多糖和D-甘露醇.食品工业科技,2015,(08):294-297”中公开过,申请人保证从申请日起二十年内向公众发放生物材料。
本发明目的通过以下技术方案实现:
1、活化后的兰坪被毛孢菌种采用RSP培养基培养,获得兰坪被毛孢菌丝体;
2、将收集的菌丝粉碎,过筛,85-90℃水浸提3-5次,每次30min,收集合并提取液,提取液浓缩,在浓缩液中加入无水乙醇,搅拌混匀,4℃静置过夜,过滤收集滤液,滤液中再加入无水乙醇4℃静置过夜,重复至液体中不产生沉淀,将液体旋干,蒸馏水溶解即得兰坪被毛孢核苷提取物;
3、兰坪被毛孢核苷提取物通过药理实验验证其对酒精诱导的小鼠肝损伤的保护作用,兰坪被毛孢核苷提取物的药理活性研究中,兰坪被毛孢核苷提取物对酒精诱导的小鼠肝损伤具有明显的保护作用;
在肝部形态学上,与正常小鼠相比,酒精诱导的肝损伤小鼠肝部严重肿大,组织病理学检查显示,模型组的肝细胞肿胀、杂乱、发生炎性细胞浸润,表明酒精作用后,小鼠肝脏中发生酒精性炎性病变。与酒精诱导的肝损伤小鼠相比,兰坪被毛孢核苷提取物灌胃后,可显著缓解肝脏代谢异常,维持小鼠肝细胞中较为正常的丙二醛(MDA)水平,提高谷胱甘肽(GSH)水平,降低小鼠血清中血清谷草转氨酶(AST)和谷丙转氨酶(ALT),改善酒精引起的肝脏细胞氧化应激、脂质过氧化和炎症,从而达到保肝、护肝的功效,研究表明兰坪被毛孢核苷提取物是保护和缓解酒精性肝损伤的活性物质,具有重要应用开发潜力。
附图说明
图1兰坪被毛孢核苷核苷提取物对酒精诱导的小鼠肝损伤肝指数的影响;兰坪被毛孢核苷核苷提取物低剂量组(OLPL)和高剂量组(OLPH),与空白组比较,# P<0.05,## P<0.01;与模型组比较,*P<0.05,**P<0.01;
图2兰坪被毛孢核苷核苷提取物对酒精诱导的小鼠肝损伤肝脏的组织病理学影响,H&E染色(200×);
图3兰坪被毛孢核苷核苷提取物对酒精性肝损伤小鼠的血清生物标志物AST(上图)、ALT(下图)的影响;兰坪被毛孢核苷核苷提取物低剂量组(OLPL)和高剂量组(OLPH),与空白组比较,# P<0.05,## P<0.01;与模型组比较,*P<0.05,**P<0.01;
图4兰坪被毛孢核苷核苷提取物对酒精性肝损伤小鼠肝组织氧化应激水平的影响;兰坪被毛孢核苷核苷提取物低剂量组(OLPL)和高剂量组(OLPH),与空白组比较,# P<0.05,## P<0.01;与模型组比较,*P<0.05,**P<0.01;
图5兰坪被毛孢核苷核苷提取物对酒精诱导的肝损伤小鼠炎症反应的影响;兰坪被毛孢核苷核苷提取物低剂量组(OLPL)和高剂量组(OLPH),与空白组比较,# P<0.05,## P<0.01;与模型组比较,*P<0.05,**P<0.01。
具体实施方式
以下为本发明的实施事例,所述的实施例是为了进一步描述本发明,而不是限制本发明,实施例所描述的具体实验流程及其结果仅用于说明本发明。实施例中的方法如无特殊说明均为常规方法,使用的试剂如无特殊说明均为常规市售试剂或按常规方法配置的试剂。
实施例1:兰坪被毛孢核苷提取物的制备
1、兰坪被毛孢的活化
将兰坪被毛孢菌丝体接种至种子培养基(土豆粉2%、蛋白胨1%、蔗糖2%、琼脂 2%,其余为水,自然pH值,121℃灭菌30 min)中,置于20 ℃、150 r/min 下摇床培养15 d,得到兰坪被毛孢种子液;
将兰坪被毛孢种子液以8 %的接种量接种至RSP培养基(大米80%、土豆粉5%、葡萄糖1%、蛋白胨2%、纯水11.2%,KH 2PO4 0.5%,MgSO4 0.3%,自然pH值,121 ℃灭菌30 min)中,置于18℃培养箱内培养15 d后,转入20 ℃光照培养箱中(每天光照12 h)培养15 d,最后在22 ℃培养箱内弱光培养至兰坪被毛孢子实体成熟,用小刀将RSP中培养的兰坪被毛孢菌丝体在尽可能去除培养基的情况下进行收集,将收集兰坪被毛孢菌丝体40 ℃烘箱干燥至恒重,研磨成粉后过200 目筛备用;
2、在兰坪被毛孢菌粉中添加90 ℃水浸提30 min,过滤后收集滤液,滤渣加蒸馏水重复提取4次,收集合并滤液,置于旋转蒸发仪90 ℃浓缩,在浓缩液中加入无水乙醇,搅拌混匀,4℃静置过夜,过滤收集滤液,滤液中再加入无水乙醇4℃静置过夜,重复至液体中不产生沉淀为止,将液体90 ℃旋干,用蒸馏水溶解得到兰坪被毛孢核苷提取物,HPLC检测核苷提取物中核苷种类及含量,结果如表1所示;
表1兰坪被毛孢核苷核苷提取物成分(μg/g)
实施例2:动物模型的构建、分组及采样
40只SPF级雄性C57BL/6小鼠,7周龄,体重18~22 g,将其饲养于温度为24±2 ℃,湿度为60±5%,光照时间为12 h/天的动物房中,适应环境一周后,随机分为4组(每组10只):空白对照组(Control)、模型组(Model)、兰坪被毛孢核苷提取物低剂量治疗组OLPL(200 mg/kg)、兰坪被毛孢核苷提取物高剂量治疗组OLPH(600 mg/kg);空白组和模型组灌胃生理盐水,治疗组每天分别灌胃兰坪被毛孢核苷提取物200 mg/kg和600 mg/kg,连续给药28 天,除空白组外,OLP给药后,间隔4 h,以10 mL/kg的50%乙醇溶液灌胃,建立小鼠酒精性肝损伤模型;
实验期间,每天记录小鼠的体重变化及健康状况,连续给药28 天后进行实验小鼠样本采集,称量小鼠体重,采集小鼠的血样和肝脏,观察小鼠肝部形态学变化,模型组小鼠较空白组小鼠有严重的肝肿大现象,并且与空白组对比,模型组小鼠肝脏表面白色弥漫、粗糙、边缘变厚、质地变硬;与模型组比较,经过兰坪被毛孢核苷提取物给药后,小鼠肝脏病变减弱,兰坪被毛孢核苷提取物高剂量组小鼠肝脏色泽红润,质地柔软,边缘形态已恢复至接近空白组。以上研究证明了兰坪被毛孢核苷提取物可以有效改善酒精诱导的肝损伤小鼠肝脏病变,根据小鼠体重和肝重计算小鼠肝指数,肝指数(mg/g)=肝重(mg)/小鼠体重(g),结果如图1所示,模型组小鼠肝指数远高于各治疗组,说明治疗组效果明显,预测肝脏病变较轻。
实施例3:小鼠肝组织病理学检测
组织病理学检查:不同实验组取肝左叶相同部位,用10%中性甲醛固定24 h后,按照常规操作制备小鼠肝组织切片,具体操作步骤如下:
肝组织病理切片的制备:将肝组织置于PBS缓冲液浸泡24 h后,进行乙醇梯度脱水,60%乙醇脱水10 h后,70%乙醇脱水8 h,80%乙醇脱水6 h,95%乙醇脱水4 h,最后使用无水乙醇脱水1 h,将脱水完成后的组织置于二甲苯浸泡2 h至其透明后使用石蜡包埋。石蜡包埋的步骤如下:将石蜡置于65℃烘箱中放入透明的肝组织,浸蜡2 h,每隔1 h换一次蜡,重复三次。浸蜡完成后置于室温中冷却凝固,完成组织包埋。将包埋好的组织进行组织切片:切片机对组织蜡块进行连续切片,切片厚度为4.0 μm,然后挑选合适的切片进行展片,使用30%的乙醇进行第一次展片,完成后将其转移至40 ℃的恒温水浴锅中进行二次展片直至组织薄片完全贴附于载玻片上。最后一个步骤为烘片:将载玻片放置于支架上,37 ℃烘箱烘干后即可用于组织染色。制备好的切片烘干后用于H&E染色,按照试剂盒说明对组织切片染色处理后,置于显微镜下观察;
结果如图2所示,与空白组比较,模型组小鼠肝脏中肝小叶结构破坏,汇管区扩大变形,肝细胞和肝血窦排列紊乱无规则,肝细胞存在大量变性坏死,炎性细胞浸润。经过兰坪被毛孢核苷提取物给药后,肝脏受损情况随剂量升高而逐渐改善,OLPH组小鼠肝脏几乎无肝细胞变性坏死情况,炎性细胞浸润情况明显改善,汇管区扩大变形的现象消失,肝脏恢复至接近空白组。
实施例4:小鼠血清生物标志物检测
谷草转氨酶(AST)和谷丙转氨酶(ALT)主要存在于肝细胞中,当肝细胞发生损伤时,AST和ALT会从肝细胞中流出并进入血液,因此血清中AST和ALT活性的增加是作为判断肝损伤的重要指标,采用AST检测试剂盒和ALT检测试剂盒检测实施例2采集的血样中的AST和ALT,结果见图3,小鼠血清AST、ALT检测结果表明,与空白组比较,模型组小鼠血清中的AST、ALT含量显著升高;给予兰坪被毛孢核苷提取物后,小鼠血清中的AST、ALT含量剂量呈依赖性降低,与模型组相比,兰坪被毛孢核苷提取物高剂量组中的AST、ALT含量降低至接近空白组;结果说明兰坪被毛孢核苷核苷提取物可以有效降低酒精诱导的肝损伤小鼠血清中AST、ALT的含量。
实施例5:小鼠肝组织氧化应激指标检测
采用试剂盒检测小鼠肝组织中的丙二醛(MDA)、超氧化物歧化酶(SOD)、谷胱甘肽(GSH)的含量;将小鼠肝组织与生理盐水按质量比1:9的比例进行研磨,研磨后使用低温离心机4000rpm/min,离心15min,取上清后按照试剂盒步骤说明分别检测肝组织中MDA、SOD、GSH的含量。
结果如图4所示,MDA作为过氧化物的产物,在模型组小鼠中的水平明显上升,且远高于正常小鼠;经兰坪被毛孢核苷核苷提取物治疗后小鼠肝组织中MDA水平均有不同程度的下降,其中兰坪被毛孢核苷核苷提取物高剂量组最为显著。SOD和GSH是两种重要的抗氧化酶,可以维持体内的氧化与抗氧化的平衡,模型组中SOD和GSH的含量较正常组均大幅度降低,但与模型组相比,兰坪被毛孢核苷核苷提取物治疗后二者的水平均有不同程度的回升。
实施例7:小鼠肝组织中促炎因子基因表达水平的检测
提取小鼠肝组织中RNA,逆转录后进行荧光定量PCR以检测肝组织中促炎基因表达量的变化,具体步骤如下:
按照试剂盒说明配置好所需试剂,在研钵中加液氮研磨小鼠肝组织至粉末状,加入500μL异硫氰酸胍,40μL巯基乙醇,混匀冰上静置2 min,加入100μL醋酸钠、500μL水饱和酚、100μL氯仿,充分混匀静置离心取上清。将上清液转移至吸附柱中,并加入上清1/2体积的无水乙醇混匀后12000rpm/min离心1min,加入提前配置好的500μLGT Solution,静置后离心。向吸附柱中加入提前配置好的500μL NT Solution,静置后离心。然后,加入30μLDEPC水冰上静置后离心2 min,将这一过程重复两次后获得小鼠肝组织RNA。将获得的RNA通过逆转录试剂盒逆转录为cDNA,根据RNA浓度大小计算逆转录时加入量。向离心管中加入3μL MixⅠ和一定量的RNA,最后添加ddH2O至总体积为15μL,混匀后,42℃孵育2 min,然后加入5μL Mix plus,混匀后按照逆转录程序:25℃反应时间2min,55℃反应时间15min,85℃反应时间5 min,获得小鼠肝组织cDNA。获得的小鼠肝组织cDNA通过qPCR检测其促炎因子基因表达量的变化,扩增引物如下,以稀释后的cDNA为模板,以β-actin为内参基因,在反应条件为95℃,15s;60℃,30s;72℃,30s;的条件下进行42个循环完成荧光定量PCR。测量出的内参基因和目的基因的Ct值后,采用实验组/对照组=2-ΔΔct为基因表达量计算TNF-α、IL-1β、IL-6 和NF-κB的基因表达水平;
IL-1β-F:GTCTCTCTCTCTCTCTCTCTTTCCCCCC;
IL-1β-R:AAGCAACAGCAGAGCCAAACCCTAATA;
IL-6-F:AGAGGATACCACTCCCAACAGACC;
IL-6-R:ACAACTCTTTTCTCATTTCCACGAT;
TNF-α-F:GCCACCACGCTCTTCTGTCTAC;
TNF-α-R:GGGCTACAGGCTTGTCACTCG;
NF-κB-F:GAGATTGACTTTTGTGCCCAGC;
NF-κB-R:GAGAGAGCAGACAGACGGACGG;
β-actin-F:GAAATCGTGCGTGACATCAAAGA;
β-actin-R:CCCAAGAAGGAAGGCTGGAAAA;
实时荧光定量PCR结果如图5所示,与空白组对比,经酒精灌胃处理后,模型组小鼠肝脏中TNF-α、IL-1β、IL-6 和NF-κB mRNA表达水平明显升高;与模型组对比,兰坪被毛孢核苷核苷提取物治疗组小鼠肝脏中TNF-α、IL-1β、IL-6 和NF-κBmRNA表达水平均明显降低。说明兰坪被毛孢核苷核苷提取物可以有效缓解酒精引起的小鼠肝脏TNF-α、IL-1β、IL-6和NF- κBmRNA表达水平升高。因此可以表明,兰坪被毛孢核苷核苷提取物可以有效抑制酒精诱导的肝损伤小鼠肝脏中炎症因子TNF-α、IL-1β、IL-6和NF-κB合成,从而达到缓解小鼠肝损伤的目的。
Claims (1)
1.兰坪被毛孢(Hirsutella lanpingensis)核苷提取物在制备防治酒精性肝损伤药物中的应用。
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