健脾化湿解毒方对急性肝损伤小鼠肠屏障功能的影响

    姜婷婷 江宇泳 叶永安

    

    

    摘要 目的:探讨健脾解毒凉血方治疗肝损伤肠屏障功能障碍的作用靶点。方法:选取C57BL/6雄性小鼠32只,随机分为正常组、模型组、中药组、培菲康组,腹腔注射硫代乙酰胺100 mg/kg造模;中药组予以健脾解毒凉血方灌胃,培菲康组给予培菲康溶液灌胃;16 h后采集小鼠血清、肝组织、小肠组织,观察小鼠肝功能、肠道组织HE染色变化,免疫组织化学染色方法观察小肠闭锁小带蛋白-1表达。结果:模型组、中药组、培菲康组小鼠的ALT、AST均显著高于正常组,模型组、中药组、培菲康组比较差异无统计学意义(P>0.05)。小肠组织在光镜和电镜下均可观察到中药组病变轻于模型组和培菲康组,免疫组织化学染色在小肠上皮细胞上可见闭锁小带蛋白-1棕黄色阳性标记,模型组、中药组、培菲康组阳性标记均显著少于正常组,中药组阳性标记显著多于模型组(P<0.05)。结论:健脾解毒凉血方治疗硫代乙酰胺诱导急性肝损伤小鼠,可维护肠上皮细胞紧密连接部闭锁小带蛋白-1表达,修复肠屏障功能。

    关键词 健脾解毒凉血方;硫代乙酰胺,肝损伤;肠屏障功能;闭锁小带蛋白-1;紧密连接蛋白;小鼠;肠道

    Abstract Objective:To explore the target of Jianpi Jiedu Liangxue Formula in the treatment of intestinal barrier dysfunction in liver injury.Methods:A total of 32 male C57BL/6 mice were randomly divided into a normal group,a model group,a Chinese medicine group and a Peifikang group.Mouse liver injury model by intraperitoneal inject of thioacetamide 100 mg/kg was established.The mice in the traditional Chinese medicine group were given Jianpi Jiedu Liangxue Formula for gavage.The mice in the Peficon Kang group were given Pefikang solution.After16 hours,mice serum,liver tissue,and small intestinal tissue were collected.Liver function,intestinal HE histochemical changes,immunohistochemical staining method were observed the expression of ZO-1 protein in the small intestine.Results:The ALT and AST of the model group,the Chinese medicine group and the Peifikang group were significantly higher than those of the normal group(P>0.05).There was no significant difference between the model group,the Chinese medicine group and the Peifikang group.Small intestinal tissue can be observed under light and electron microscopy.Immunohistochemical staining in the cell membrane of intestinal villus epithelial cells were visible on the zonula occludens protein-1 Brown positive marker.The positive markers of model group,Chinese medicine group and Peifikang group were significantly less than which of normal group.The positive marker of Chinese medicine group were significantly more than that of model group(P<0.05).Conclusion:Jianpi Jiedu Liangxue Formula in the treatment of acute liver injury induced by thioacetamide can maintain the expression of zonula occludens protein-1 in the tight junction of intestinal epithelial cells and repair intestinal barrier function.

    Keywords Jianpi Huashi Jiedu Formula; Thioacetamide; Liver injury; Small intestinal barrier function; Zonula occludens protein-1; Tight junction protein; Mice; Intestinal

    中圖分类号:R289.5文献标识码:Adoi:10.3969/j.issn.1673-7202.2021.07.012

    重症肝病时肠道缺血、缺氧、循环障碍,肠黏膜淤血、水肿、糜烂,通透性增加;肠屏障功能受损导致大量内毒素经门静脉进入肝脏,其诱导的一系列炎症反应加剧了肝损伤,并与自发性细菌性腹膜炎等并发症密切相关[1-2]。在“见肝之病,知肝传脾,当先实脾”的中医经典理论指导下,我们以健脾化湿解毒法治疗重症肝病合并肠功能障碍取得较好的临床效果。为探索中医药修复肝损伤并发肠屏障功能的作用靶点,以硫代乙酰胺(Thioacetamide,TAA)誘导的急性肝损伤小鼠模型,观察健脾化湿解毒方对小鼠小肠组织、小肠上皮细胞闭锁小带蛋白-1(Zonula Occludens Proteins-1,ZO-1)表达的影响。

    硫代乙酰胺是一种具有很强肝毒性的物质,其活性代谢产物如磺胺-5-甲嘧啶、TAA硫二氧化物等共价结合于肝脏大分子,通过氧化应激和脂质过氧化反应导致肝细胞损伤,诱导其坏死和凋亡。TAA诱导的急性肝损伤模型在形态学和致肝损伤机制方面与人类肝损伤相似,TAA 350 mg/kg腹腔注射制备大鼠急性肝衰竭模型,发现大鼠在出现急性肝衰竭变化的同时,还伴有肠壁黏膜上皮细胞坏死脱落,黏膜下层充血水肿,黏膜层炎症细胞浸润明显[3];TAA诱导的肝损伤伴胃肠道损伤及肠源性内毒素血症[4-5]。这是一种典型的伴有胃肠道生物化学、组织结构和功能改变的肝-肠损伤模型。

    1 材料与方法

    1.1 材料

    1.1.1 动物 清洁级野生型C57BL/6小鼠,雄性,8周龄,体质量(20±1)g,购自中国医学科学院动物研究所,所有实验动物均按照动物保护相关法规饲养于中国医学科学院动物所无特定病原体(SPF)级动物室(许可证号:SCXK京2009-0007)。造模前饲养观察室内观察1 d,各组分笼饲养,标准配方饲料喂养,自由进水,室温10~20 ℃,相对湿度为30%~50%,光暗周期为12 h。

    1.1.2 药物 中药购自北京卫仁制药厂,批号20140416,健脾解毒凉血方:党参15 g、黄芪30 g、茵陈30 g、栀子15 g、生大黄10 g、黄芩15 g、黄连10 g、赤芍30 g、生地黄15 g,水煎浓缩至87 mL,浓度为1.9 g/mL,造模同时中药灌胃1次。

    1.1.3 试剂与仪器 硫代乙酰胺试剂(Sigma公司,德国,批号:20140107);兔抗人ZO-1多克隆抗体(Merck-Millipore公司,美国,批号:AB2272)。

    1.2 方法

    1.2.1 分组与模型制备 将小鼠分4组:正常组,模型组,中药组,培菲康组,每组8只。造模前禁食12 h,以TAA 100 mg/kg单次腹腔注射造模,正常组予以0.9%氯化钠注射液单次腹腔注射。造模及治疗后16 h处死,观察肝功能、肝肠组织形态学和肠上皮细胞ZO-1表达。

    1.2.2 干预方法 模型组小鼠造模后立即给予去离子水灌胃;中药组小鼠造模后立即给予健脾解毒凉血方灌胃,培菲康组小鼠造模后立即给予培菲康溶液灌胃,灌胃后小鼠自由进水饮食。灌胃体积依小鼠体质量而定,体质量>22 g予以270 μL灌胃、体质量<18 g予以230 μL灌胃、体质量18~22 g予以250 μL灌胃。

    1.2.3 检测指标与方法 造模后16 h摘取小鼠眼球取外周血,离心后进行血浆谷丙转氨酶(ALT)和谷草转氨酶(AST)检测。取血后立即断颈处死小鼠,迅速取肝左叶0.8 cm×0.5 cm×0.3 cm大小肝组织、十二指肠下约2 cm处约2 cm长的空肠段,置10%中性甲醛溶液固定,24 h后逐级乙醇脱水,二甲苯透明,56 ℃石蜡包埋,4 μm厚切片,用于HE染色和ZO-1免疫组织化学染色。在每组8只小鼠中随机选取3只留取小肠标本,每只取l块长约l cm的小肠组织用2.5%戊二醛固定2 h,二甲砷酸钠缓冲液漂洗3~4遍,1%锇酸固定2 h,丙酮梯度脱水,Epon812环氧树脂浸透包埋。聚合72 h(35 ℃、45 ℃,60 ℃各24 h),LKB-V超薄切片机连续切片,厚度为60~80 nm,采用醋酸铀、柠檬酸铅双重染色,在透射电镜下观察。

    ZO-1免疫组织化学染色阳性细胞计数判定在200倍显微镜下分别对每张组织切片的肠上皮细胞内的ZO-1阳性信号进行半定量评分:A为阳性细胞数分级:0级,75%阳性信号。B为阳性细胞显色强度分级:0(阴性),1(弱阳性),2(阳性),3(强阳性)。每张切片的染色积分(HIS)以这二者相乘的乘积数表示,即HIS=A×B。

    1.3 统计学方法

    采用SPSS 21.0统计软件进行数据分析,计量资料用单因素方差分析,方差齐性检验,分别采用LSD进行两两比较。等级资料采用非参数检验中的Krukal Wallis检验,以P<0.05为差异有统计学意义。

    2 结果

    2.1 小鼠血浆ALT和AST比较 与正常组比较,各造模小鼠均表现出进食减少,行动缓慢,活动减少。模型组、中药组、培菲康组的ALT、AST均显著高于正常组(P0.05)。见表1。

    2.2 小鼠肝组织病理结果

    肉眼观察:正常组小鼠肝脏色泽红润,大小适中,表面光滑边缘锐利,质软而富有弹性。与正常组比较,模型组小鼠肝脏体积增大,呈深红色,质脆,表面淤血呈片状。中药观察组肝脏颜色接近正常组小鼠,表面散在坏死灶,面积较模型组小,无明显淤血。培菲康组肝脏体积增大,深红色,表面淤血,质地松脆。

    肝组织切片HE染色光镜下观察:正常肝脏组织可见肝细胞排列整齐,肝小叶结构完整,无变性、坏死及炎症细胞浸润。模型组肝组织可见显著的肝细胞水肿,气球样变性,肝小叶中心坏死,伴大面积出血性改变和炎症细胞浸润。中药组肝细胞水肿,气球样变性较轻,出血坏死面积较模型组小,汇管区少量炎症细胞浸润;培菲康组可见肝细胞凝固性坏死和空泡形成,肝小叶中心出血坏死,面积较大。

    2.3 小鼠小肠组织光镜下病理结果 正常空肠组织可见黏膜结构完整,绒毛上皮无缺损,未见淋巴滤泡增生及炎症细胞浸润。模型组空肠组织可见黏膜上皮糜烂,绒毛样结构减少,绒毛倒伏,顶端大片坏死并脱落于肠腔内,绒毛水肿,间隙明显增宽,固有层大量炎症细胞浸润。中药组可见肠黏膜绒毛略有水肿,绒毛间隙略宽,顶端有少量绒毛脱落,较少淋巴滤泡增生,较少炎症细胞浸润;培菲康组与模型组相比小肠结构未见明显改善。

    2.4 小鼠小腸组织电镜下超微结构结果 在透射电镜下观察,正常组小鼠小肠上皮细胞的微绒毛排列整齐,上皮细胞顶端微绒毛下可见显色清晰、染色浓聚的双层膜样的紧密连接蛋白。模型组小肠上皮细胞微绒毛下的紧密连接蛋白范围变小,纤细色淡。中药组小肠微绒毛下细胞间紧密连接显色清晰,范围较模型组大。培菲康组小肠微绒毛下细胞间紧密连接显色较淡,范围较小。见图1。

    2.5 小鼠小肠绒毛ZO-1免疫组织化学染色比较

    免疫组织化学染色显示ZO-1阳性细胞呈柱状,阳性信号位于细胞膜下,呈棕黄色或褐色颗粒状。正常组小鼠小肠绒毛上皮细胞胞膜下可见线性棕黄色阳性染色,表达充分,阳性细胞数占肠绒毛上皮细胞总数的50%以上,大多数细胞阳性信号着色清晰连续。模型组小鼠小肠绒毛上皮细胞ZO-1阳性染色明显减少,着色较淡,不连贯。

    小鼠小肠组织内的ZO-1半定量评分显示:正常组ZO-1的HIS(9.38±1.92)分,中药组为(5.63±1.96)分,模型组为(3.00±0.76)分,在小肠绒毛上,模型组、中药组、培菲康组小鼠ZO-1表达均明显弱于正常组,差异有统计学意义(P0.05)(图2)。

    3 讨论

    既往研究表明,以TAA200 mg/kg腹腔注射建立急性肝衰竭小鼠模型,健脾解毒凉血方灌胃较模型组的存活率提高了37.5%,2组的存活率差异有统计学意义[6]。TAA诱导的肝衰竭小鼠模型有多脏器损伤,健脾解毒凉血方未能改善小鼠肝功能,可能是通过其他途径提高肝衰竭小鼠的生存率。本研究进一步探讨了健脾化湿解毒方对急性肝损伤小鼠的肠道屏障功能的影响。

    肠黏膜上皮细胞的完整性及上皮细胞间的紧密连接在维护肠道屏障功能中发挥重要作用,通过研究ZO-1基因敲除的小鼠,发现ZO-1在紧密连接的形成过程中起关键作用[7];在实验性多发伤和失血性休克的小鼠模型中,肠上皮细胞中ZO-1的表达显著降低,被认为与肠道通透性增加,细菌成分导致肠上皮细胞产生炎症反应和趋化介质有关[8];肠上皮细胞的ZO-1的表达受到膜型基质金属蛋白酶-2及E-钙黏蛋白的调节[9]。肝衰竭时肠道ZO-1的表达也是减少的,TNF-α使急性肝衰竭小鼠结肠紧密连接ZO-1mRNA及ZO-1蛋白的表达显著降低[10]。酒精性肝损伤时肠道ZO-1,Claudin-1等蛋白表达下降,肠屏障损伤,毒素吸收增加,激活肝内毒素-TLR4-核因子κB通路加重肝损伤[11-12]。非酒精性脂肪性肝炎患者肠ZO-1和Occludin的表达水平与转氨酶水平呈负相关[13]。改善肠道屏障,可以同时改善肠道炎症反应、内毒素血症、肝脏脂肪变性和肝损伤[14-15]。多数情况下紧密连接功能的损伤伴随ZO-1的表达减少,故ZO-1常被用于判断肠紧密连接功能和肠机械屏障通透性的指标。有许多研究报道了中药、西药通过增强肠黏膜ZO-1等紧密连接蛋白的表达,而发挥改善肠屏障功能的作用,同时能改善乙醇、代谢紊乱引起的肝损伤。枳椇子提取物显著抑制乙醇引发的肝损伤大鼠TLR4通路及其下游炎症介质,并上调ZO-1和Occludin在肠道中的表达,通过调节肠-肝轴的异常发挥其保肝作用[16]。石斛多糖可上调ZO-1等紧密连接蛋白的表达,下调Caspase-3蛋白的表达,维持肠平衡,抑制LPS-TLR4-核因子κB信号通路的激活,减轻肝纤维化[17];山楂总黄酮可调控核因子κB P65介导的MLCK-MLC信号通路减轻TNF-α诱导的肠上皮屏障缺损[18];芒果仁提取物治疗恢复ZO-1和Claudin-1的表达,从而防止高脂饮食引起的肠通透性增加[19]。复方银杏叶上调紧密连接蛋白ZO-1的表达,降低慢性酒精性肝损伤大鼠的转氨酶和二胺氧化酶水平[20]。体外实验表明金银花提取液与RAW 264.7细胞共培养,降低了ZO-1和Claudin-1的基因表达,通过调节肠道菌群分布和肠道通透性来改善肥胖和相关的代谢性内毒素血症[21]。黄精多糖增加了肠道ZO-1蛋白的表达,降低了高脂饮食喂养的大鼠血清内毒素[22];口服姜黄素可促进肠道ZO-1和Claudin-1的表达,降低血浆内毒素水平,改善高脂饮食引起的代谢紊乱[23]。龙眼多糖可以增加肠上皮Caco-2细胞中ZO-1紧密连接蛋白的表达[24],广藿香醇改善肠黏膜炎大鼠肠上皮细胞ZO-1、Claudin-1等蛋白表达,预防肠黏膜炎的发展[25]。逍遥散能有效改善抑郁大鼠结肠病理及超微结构改变,上调结肠ZO-1、Occlutdin的表达,提高下丘脑和结肠黏膜5-HT水平[26]。栀子苷通过增加紧密连接蛋白ZO-1表达和减少炎症反应,氧化应激和细胞凋亡来防止缺氧/再灌注诱导的血脑屏障损伤[27];参麦注射液通过调节脂筏中Occludin的表达和运输,维持局灶性脑缺血后的血脑屏障完整性[28],中医药通过调节上皮细胞ZO-1的表达不仅影响着肠屏障功能,也可以防止血脑屏障的损伤。

    本研究显示,健脾解毒凉血方灌胃可以减轻小鼠肝组织的出血坏死,减轻小肠绒毛的水肿、炎症细胞浸润,与模型组比较,显著改善了肠上皮细胞的ZO-1蛋白表达。本方的主要药物成分具有改善肠功能的功效。大黄素可通过抑制缺氧诱导因子-1α和核因子κB信号通路来抑制内毒素和缺氧诱导的肠上皮细胞屏障功能障碍,防止紧密连接屏障损伤和ZO-1表达的下降[29]。黄连素通过抑制缺氧诱导因子-1α介导肌球蛋白轻链磷酸化激酶、依赖肌球蛋白轻链磷酸化信号传导通路,来抑制IFN-γ和TNF-γ诱导的肠上皮屏障功能障碍[30];同样黄连素通过肌球蛋白轻链磷酸化依赖途径改善重症急性胰腺炎肠屏障功能障碍[31]。电镜下模型组小肠上皮细胞微绒毛下的紧密连接蛋白范围较正常组变小,纤细色淡。中药组小肠微绒毛下细胞间紧密连接显色清晰,范围较模型组大。免疫组织化学染色也提示健脾化湿解毒方明显改善TAA小鼠的小肠ZO-1的表达。

    肠屏障功能障碍,肠源性内毒素血症是导致肝损伤加重的二次打击的重要因素。肠功能障碍并非局限于肠腑之疾,也并非单纯腑实证。诊治需审证求因,追寻其发病原因。对于脾胃虚弱,升降失常的患者,顾护脾胃不足,通利周身气机,恢复脾胃升降枢纽功能,有助于调节肠功能;同时兼顾疏肝柔肝,调畅气机,对机体脏腑功能的纠正、内环境的调节有益,最终从根本上使得其恢复生理状态,以达培本复元之效。健脾解毒凉血方以党参、黄芪健脾益气,茵陈、栀子清热利湿;大黄、黄芩、黄连清热解毒;赤芍、生地黄凉血解毒,全方共奏顾护中焦,清利肝胆之功效。TAA诱导的急性肝衰竭模型是“肝-肠损伤”模型,健脾解毒凉血方治疗TAA诱导的急性肝损伤小鼠,可减轻小肠绒毛水肿,对于肠上皮细胞的ZO-1蛋白表达有较强的保护作用,可能是其顾护中焦,保护肠黏膜的生物学基础之一。

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    (2019-05-16收稿 責任编辑:杨觉雄)