WO2022068077A1 - 石胆酸用于制备缓解肝纤维化的药物中的应用 - Google Patents
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Abstract
胆汁酸中的石胆酸(LCA)通过与肠道菌群相互作用,维持肝脏免疫稳态抑制炎症缓解肝脏纤维化的用途,即石胆酸用于制备缓解肝纤维化的药物中的应用。具体的,用CCl4造的小鼠药物性肝炎模型中,LCA具有减少肝脏中NKT细胞数目,降低TNF‐a浓度,抑制肝脏炎症,最终缓解肝脏纤维化的进展的作用。当用抗生素杀死小鼠肠道中共生菌后,LCA的抑炎症反应反而减弱。LCA在肝脏炎症中的作用是由肠道微生物介导的。胆汁酸‐微生态‐肝脏疾病三角将可能有助于炎性肝脏疾病的研究和治疗。
Description
本发明涉及胆汁酸中的石胆酸(LCA)通过与肠道菌群相互作用,维持肝脏免疫稳态抑制炎症缓解肝脏纤维化的用途,即石胆酸用于制备缓解肝纤维化的药物中的应用。
胆汁酸在肝脏中经由细胞色素P450介导的胆固醇的氧化而合成,包括经典和替代途径。在肝脏中,胆汁酸主要通过甘氨酸和牛磺酸耦合,然后再通过胆盐输出泵(bile salt export pump,BSEP)运送到胆囊中储存。同时,肝脏也能通过磺基转移酶和UDP‐葡糖醛酸转移酶作用生成磺酸化和葡糖醛酸化的胆汁酸,然后再与甘氨酸和牛磺酸耦合,通过多药耐药相关蛋白2(MRP2)转运并分泌到胆囊中。在小肠中,耦合形式的胆汁酸被胆酸盐水解酶BSH水解变成游离形式的胆汁酸,紧接着又通过7‐α脱羟基作用生成次级胆汁酸。胆酸(CA)和鹅脱氧胆酸(CDCA)分别转化为脱氧胆酸(DCA)和LCA。参与胆汁酸代谢的主要肠道菌群包括具有解耦合作用的拟杆菌,梭菌,乳酸杆菌,双歧杆菌和李斯特菌;Bacteroides、Eubacterium、Clostridium、Escherichia、Eggerthella、Eubacterium、Peptostreptococcus和Ruminococcus能使C3,C7和C12的羟基氧化和差向异构化;梭菌和真细菌具有7‐脱羟基作用;拟杆菌,真细菌和乳酸杆菌具有酯化作用;梭菌,梭杆菌,球菌和假单胞菌具有脱硫作用。游离胆汁酸可以消除细胞膜内外的酸碱度差异。最终导致质子泵驱动的生物能量消失,从而对细胞膜造成直接损害。通过这种方式,胆汁酸抑制某些细菌的生长,参与肠道微生物的组成。
代谢系统和免疫细胞之间的相互作用在炎性疾病中发挥重要作用。胆汁酸主要通过激活FXR和TGR5受体发挥生物学作用,改善代谢综合征和控制炎症,而LCA是TGR5的最强天然激动剂。已有数据证明胆汁酸可以通过TGR5‐cAMP‐PKA轴抑制NLRP3炎症小体的激活。TGR5胆汁酸受体诱导的PKA激酶活化导致NLRP3泛素化,而NLRP3泛素化与pka诱导NLRP3在一个单独的残基Ser291磷酸化。更重要的是,这种PKA诱导的NLRP3的磷酸化是NLRP3炎症小体激活的关键制动点。此外,活体实验结果显示胆汁酸和TGR5激活可以阻断NLRP3炎症小体依赖性炎症,包括脂多糖诱导的全身炎症、alum诱导的腹膜炎症和2型糖尿病相关炎症。
肝脏纤维化是继发于肝脏炎症或损伤后组织修复过程中的代偿反应,最终可发展为肝硬化甚至肝癌。有研究表明,抑制肝脏炎症肝脏纤维化可发生逆转,而LCA具有抑制肝脏炎症的功能。鉴于肝脏‐微生态轴在多种炎症疾病中的重要作用以及LCA与肠道菌群的密切关系。通过肠道‐胆汁酸‐肝脏轴调节肝脏炎症性疾病是缓解肝脏纤维化的一个重要治疗靶点。
发明内容
本发明解决的技术问题是,提供石胆酸的新用途,即一种调节肠道微生态和石胆酸肝脏浓度来抑制肝脏炎症及缓解肝脏纤维化的方法。
石胆酸用于制备缓解肝纤维化的药物中的应用。
石胆酸用于制备抑制肝脏炎症的药物中的应用。
本发明的技术方案是,利用肠道微生态‐胆汁酸‐肝脏三角,通过口服石胆酸并与肠道共生菌相互作用,调节肝脏免疫细胞及炎症因子分泌降低肝脏炎症水平,缓解肝脏纤维化的作用。
本发明的有益效果是,LCA能够调节肝纤维化小鼠的肠道微生态,降低杜氏杆菌的相对丰度,增加益生菌乳酸杆菌及拟杆菌的肠道相对丰度。同时LCA抑制肝脏中NKT细胞活化,减少TNF‐a分泌,缓解肝脏纤维化。
本发明涉及肠道菌群通过石胆酸抑制炎症缓解肝脏纤维化的用途。具体的,用CCl4造的小鼠药物性肝炎模型中,LCA具有减少肝脏中NKT细胞数目,降低TNF‐a浓度,抑制肝脏炎症,最终缓解肝脏纤维化的进展的作用。当用抗生素杀死小鼠肠道中共生菌后,LCA的抑炎症反应反而减弱。LCA在肝脏炎症中的作用是由肠道微生物介导的。胆汁酸‐微生态‐肝脏疾病三角将可能有助于炎性肝脏疾病的研究和治疗。
图1各组小鼠ALT和AST表达差异。*为与一组比较p<0.05,#为与四组比较p<0.05;ALT谷丙转氨酶、AST谷草转氨酶;一组空白对照组、二组四氯化碳模型组、三组抗生素处理模型干预组、四组模型干预组。
图2为各组小鼠肝脏组织的Masson染色结果。*为与一组比较p<0.05,#为与四组比较p<0.05;CVF胶原容积分数;一组空白对照组、二组四氯化碳模型组、三组抗生素处理模型干预组、四组模型干预组。
图3各组肝脏纤维化指标RNA表达水平。*为与一组比较p<0.05,#为与二组比较p<0.05; CollagenI I型胶原酶、CollagenIII III型胶原酶、a‐SMA平滑肌肌动蛋白、Timp‐1组织金属蛋白酶1、MMP‐2基质金属蛋白酶2;一组空白对照组、二组四氯化碳模型组、三组抗生素处理模型干预组、四组模型干预组。
图4为各组小鼠肝脏中NKT细胞的百分比及各组小鼠血液中TNF‐a浓度差异。*为与一组比较p<0.05,#为与四组比较p<0.05;NKT自然杀伤T细胞、TNF‐a肿瘤坏死因子;一组空白对照组、二组四氯化碳模型组、三组抗生素处理模型干预组、四组模型干预组。
图5各组小鼠不同菌群相对丰度。与一组比较p<0.05,#为与四组比较p<0.05;con空白对照组、CCl4四氯化碳模型组、CCl4+LCA+VA抗生素处理模型干预组、CCl4+LCA模型干预组。
通过下面具体的实施例可以更容易的了解本发明的内容,本发明的保护范围不局限于下述特定的具体实施方案。
实施例1 LCA可以抑制肝脏炎症缓解肝脏纤维化
实验方法
取小鼠40只,随机分为4组,每组10只。
1组:每周生理盐水腹腔注射两次,去离子水灌胃两次;
2组:四氯化碳模型组(CCl4):橄榄油1:4,1ul/g剂量每给小鼠每周腹腔注射两次。去离子水每周灌胃两次;
3组:抗生素处理模型干预组(CCl4+LCA+VA):橄榄油1:4,1ul/g剂量每给小鼠每周腹腔注射两次,LCA以1mg/15g剂量每周给小鼠灌胃2次。用抗生素(万古霉素0.5g/L+氨苄西林1g/L即VA)杀灭小鼠肠道菌群;
4组:模型干预组(CCl4+LCA):橄榄油1:4,1ul/g剂量每给小鼠每周腹腔注射两次,LCA以1mg/15g剂量每周给小鼠灌胃2次。
以上各组,持续干预8周。处理前24h收集各组小鼠的新鲜粪便,‐80℃冻存。末次给药12h后采血,用酶比色法测定血清中的ATL和AST。留取肝脏组织,做HE染色和Masson染色。
实验结果:
小鼠长期石胆酸灌胃,其血液中的ALT和AST与正常组小鼠无明显差异,当用抗生素杀灭肠道菌群,小鼠的ALT和AST明显增高(图1)。肝脏组织经Masson染色后,各组间的肝脏纤维化程度差异也显示LCA具有抗纤维化的作用,但是当消除肠道菌群的协同作用后,LCA的抗纤维化作用减弱(图2)。用ELISA检测肝脏组织中的纤维化指标CollagenI、CollagenIII、 Timp‐1、MMP‐2和a‐SMA的RNA表达量(图3),结果与上述一致。
实施例2 LCA与肠道菌群相互作用
实验方法:
以上各组,处理前24h收集各组小鼠的新鲜粪便,‐80℃冻存。用16sRNA技术检测各组小鼠间的肠道菌群,并比较各组间的菌群差异。
实验结果:
发现LCA灌胃组小鼠肠道中有害菌杜氏杆菌(Dubosiella)相对丰度降低,益生菌乳酸杆菌和拟杆菌总的相对丰度增加(图5)。用抗生杀灭肠道共生菌,LCA的抑制肝脏炎症及缓解纤维化的作用消失。证明肠道微生态‐胆汁酸‐肝脏三角的相互作用是LCA作用的基石。
实施例3 LCA通过抑制肝脏NKT细胞激活剂降低TNF‐a浓度达到抗纤维化作用
实验方法:
留取小鼠新鲜肝脏,制备肝脏单细胞悬液,用流式仪检测肝脏内的免疫细胞:NKT细胞、NK细胞和巨噬细胞,及小鼠血液中的巨噬细胞。对小鼠的血清进行流式CBA检测。
实验结果:
发现LCA具有抑制NKT细胞活化的作用,而NKT细胞活化表现为肝脏组织中NKT细胞下调,这与NKT细胞激活后特异性抗原丢失有关。抗生素杀灭肠道菌群后,NKT细胞百分比增加。血清中的TNF‐a浓度在各组中的变化,与上述结果一致(图4)。
Claims (4)
- 石胆酸用于制备缓解肝纤维化的药物中的应用。
- 石胆酸用于制备抑制肝脏炎症的药物中的应用。
- 石胆酸用于制备抑制肝脏中NKT细胞活化的药物中的应用。
- 石胆酸用于制备降低杜氏杆菌的相对丰度、增加乳酸杆菌或增加拟杆菌的肠道相对丰度的药物中的应用。
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