The anti-liver fibrosis effect of Kangxian Ruangan prescription: A cell study based on hepatic sinusoidal capillarization

Xu Jie; Liu Yan; Lou WangZhouYang; Xu Jun; Qi Lu; Cheng LiangBin

doi:10.3969/j.issn.1001-5256.2020.02.017

Volume 36 Issue 2

Feb. 2020

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Abstract

References

Article Navigation > Journal of Clinical Hepatology > 2020 > 36(2): 319-323

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The anti-liver fibrosis effect of Kangxian Ruangan prescription: A cell study based on hepatic sinusoidal capillarization

DOI: 10.3969/j.issn.1001-5256.2020.02.017

Clinical College of Traditional Chinese Medicine,Hubei University of Chinese Medicine

Research funding:

Received Date: 2019-09-15
Published Date: 2020-02-20

Abstract

Abstract

Objective To investigate the anti-liver fibrosis effect and mechanism of action of Kangxian Ruangan prescription based on hepatic sinusoidal capillarization. Methods A total of 16 male Wistar rats were divided into Kangxian Ruangan prescription group and control group,with 8 rats in each group. The rats in the Kangxian Ruangan prescription group were given crude drug 40 g/kg body weight by gavage,and those in the control group were given normal saline by gavage. Serum containing Kangxian Ruangan prescription was prepared,and then primary liver sinusoidal endothelial cells were treated with the serum containing Kangxian Ruangan prescription or the control serum. Western blot was used to measure the protein expression of the vascular endothelial markers cluster of differentiation 31( CD31) and von Willebrand factor( vWF),and immunocytochemistry was used to measure the expression of CD31 and vWF in cells. Scanning electron microscopy was used to observe the change in the fenestrae of liver sinusoidal endothelial cells. The t-test was used for comparison of continuous data between two groups. Results Kangxian Ruangan prescription had no marked influence on the morphology of liver sinusoidal endothelial cells. The liver sinusoidal endothelial cells in the control group had a small number of narrow fenestrae with an occluded structure,while those in the Kangxian Ruangan prescription group had an increased number of large fenestrae with a clear structure. The results of immunocytochemistry showed that compared with the control group,the Kangxian Ruangan prescription group had significant reductions in the expression of CD31( 0. 069 ± 0. 015 vs 0. 115 ± 0. 011,t = 4. 257,P < 0. 05) and vWF( 0. 092 ± 0. 009 vs 0. 132 ± 0. 014,t = 4. 274,P < 0. 05). The results of Western blot showed that compared with the control group,the Kangxian Ruangan prescription group had significant reductions in the protein expression of CD31( 0. 334 ± 0. 029 vs 0. 448 ± 0. 013,t = 6. 245,P < 0. 01) and vWF( 0. 560 ± 0. 047 vs0. 709 ± 0. 045,t = 3. 966,P < 0. 05). Conclusion Kangxian Ruangan prescription can significantly resist hepatic sinusoid capillarization and thus prevent the progression of liver fibrosis.
- liver cirrhosis,
- Kangxian ruangan decoction,
- antigens,CD31,
- von Willebrand factor,
- rats,Wistar

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第1例腹腔镜胆囊切除术(laparoscopic cholecystectomy，LC)于20世纪80年代末被报道，其具有手术创伤小、术后恢复快、疗效好等优点，目前在世界范围内得到了广泛应用；而胆总管损伤是LC的常见并发症，发生率为0.4%~0.6%^[1-6]，可导致严重后果。如胆管壁损伤需要胆管缝合或T管引流，而胆总管横断伤则需要胆总管空肠吻合术或Roux-en-Y吻合术。而这些补救措施往往需要将LC中转为开放手术，极大地增加患者的经济负担和身体疼痛。考虑到胆总管损伤的严重性和普遍性，有效预防胆总管损伤的方法亟待应用。

引起胆总管损伤的原因多样，包括胆道解剖变异、胆囊炎反复发作引起胆总管变形、操作人员经验不足及器械不当等^[7-12]。可以认为，准确识别胆总管是预防胆总管损伤的有效方法。因而从20世纪80年代至今，人们为准确识别胆总管和胆囊管做出了巨大努力；尝试过的方法如术中X线造影，但不易操作，且X线会给患者和操作者带来辐射风险^[13]；而术中超声成像也不易用于胆总管的识别。

近红外造影术于近年来兴起，其中吲哚菁绿(indocyanine green, ICG)造影术最为简便^[14]，被广泛应用于各类外科手术，包括肿瘤边界识别、甲状旁腺保护、肝段切除、乳腺癌前哨淋巴结活检等^[15]。与其他方法相比，ICG造影安全性高。据报道^[16]，仅0.001 67%的患者对ICG发生过敏反应。且ICG使用方便，可于术前经静脉注射，如于术前24 h予ICG 25 mg以显影肝癌边界；术前50 s给予7.5 mg ICG以鉴别甲状旁腺；术前10 min予ICG 1 mg协助乳腺癌前哨淋巴结活检^[15]；离断操作前3 min予ICG 5 mg行肠血运灌注评估等^[17]。

此前已有研究^[18-19]对术中ICG胆道造影鉴别胆总管和胆囊管方法的有效性和安全性进行了评估，但此法的长期效果仍需进一步确认。本研究首次应用倾向评分匹配法比较了ICG胆道造影组和常规LC组患者中胆总管损伤的发生率，探讨ICG胆道造影的应用效果。

1. 资料和方法

1.1 研究对象

选取珠海市人民医院2021年4月—6月行LC患者。纳入标准：经超声、CT或MRI检查并结合病史确诊为胆囊良性疾病需行LC的患者。排除标准：急性胆囊炎、胰腺炎、血管性胆囊炎和胆总管结石；胆囊穿刺引流，引流管未拔出者；有心脏、肺部等基础疾病，不宜手术者；美国麻醉师学会评分≥2分。

将同意接受ICG胆道造影的患者纳入ICG胆道造影组，其余纳入常规LC对照组。常规LC组患者用腹腔镜白光，ICG胆道造影组用近红外光检查胆总管、胆囊管和胆囊。采用倾向评分匹配法对两组患者进行基线指标的平衡。

1.2 术前评估

所有患者术前均行心电图、胸部X线检查、超声、CT、血型、血常规、凝血功能、感染性疾病筛查、肝肾功能检查等。所有检查结果正常的患者可安排手术。有其他基础疾病的患者，需经相关专科和麻醉科会诊确认可耐受全麻。对所有患者术前均进行LC围手术期注意事项的宣教；另对ICG胆道造影组患者进行ICG胆道造影知识宣教；其他术前准备按上腹部手术常规进行。

1.3 手术方式

两组患者均行LC。患者头部抬高30°，身体左倾30°。全麻后建立静脉通路，用插入式Veress针头向腹腔内注入CO₂。于脐下行一1 cm切口。经脐下套管针导入腹腔镜。在胆囊的体表投影处行一0.5 cm切口，于剑突下行一1 cm切口。将腹腔镜器械置入腹腔，解剖Calot三角。夹闭切断胆囊管和胆囊动脉。取胆囊送病理检查。关闭皮肤切口，视情况放置腹腔引流管(图 1)。

图 1 ICG造影引导下腹腔镜胆囊切除术过程

注：a、b，荧光及白光下分离胆囊三角周围粘连的过程；c、d，荧光及白光下分离胆囊床及游离胆囊过程；e、f，荧光及白光下结扎胆囊管过程。

下载: 全尺寸图片幻灯片

1.4 ICG的注射

对ICG胆道造影组患者于术前30 min(切皮前60 min)静脉注射ICG (瑞度，丹东医药)0.4 mg·kg^-1·mL^-1(基于ICG在血液中浓度于2 h达到最大值的原理确定此给药时间)。

1.5 荧光成像系统

荧光成像系统由近红外光源、控制手柄、腹腔镜、脚踏开关、设备摄像机和显示屏组成。该系统可作为常规腹腔镜手术系统呈现全彩色图像，并可在操作者踩下脚踏开关后发出波长约800 nm的近红外光用于ICG胆道造影；专门设计的腹腔镜可传输近红外光；通过操作控制手柄，术者可拍摄术中ICG胆道造影的照片或视频。

1.6 ICG胆道造影中胆管的识别

ICG胆道造影组在夹闭胆囊管前后，应用ICG胆道造影排除异位胆管的存在，并区分胆总管和胆囊管。所有的手术操作完成后，2名术者需用ICG胆道造影2次检查胆囊管残端，确保胆总管完好。比较2组术中出血量、手术时间、术后住院时间及胆总管损伤发生率。

1.7 伦理学审查

本研究方案经由珠海市人民医院伦理委员会审批，批号：2019-02；并在中国临床试验注册中心注册，注册号：ChiCTR1900024594。

1.8 统计学方法

采用SPSS 25.0进行统计分析。正态分布的计量资料采用x±s表示，2组间比较采用t检验；计数资料2组间比较采用χ²检验。多元逻辑回归模型评估每位受试者的倾向性得分。协变量包括以下基线特征：年龄、性别、复杂性胆结石等。P＜0.05为差异有统计学意义。

2. 结果

2.1 基线特征

共纳入68例行LC患者，其中ICG胆道造影组12例，常规LC组56例，患者基线特征如表 1所示。行倾向评分匹配后，获得12对患者。匹配后，两组患者的基线变量差异被有效校正，两组患者特征比较，差异均无统计学意义(P值均＞0.05)。

表 1 ICG胆道造影组和常规LC组患者匹配前后基线特征比较

项目	匹配前				匹配后
项目	常规LC组(n=56)	ICG胆道造影组(n=12)	标准化差异(%)	P值	常规LC组(n=12)	ICG胆道造影组(n=12)	标准化差异(%)	P值
年龄(岁)	53.2±6.5	63.7±5.1	19.7	0.001	57.1±8.3	57.2±9.6	0.18	0.372
男/女(例)	39/27	8/4	28.3	0.001	8/4	8/4	0	＞0.05
复杂性胆结石(例)	21	3	33.3	0.006	3	3	0	＞0.05

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| 显示表格

2.2 治疗结果

ICG胆道造影组和常规LC组治疗结果见表 2，ICG胆道造影组术中出血量、手术时间、术后住院时间、胆总管损伤发生率与常规LC组比较，差异均有统计学意义(P值均＜0.05)。常规组出现胆总管周围组织出血1例(8.3%)，患者进行了止血药保守处理后于术后1周内康复。所有切除的胆囊均显示良性病理结果。

表 2 ICG胆道造影组和常规LC组患者的手术效果比较

项目	ICG胆道造影组(n=12)	常规LC组(n=12)	统计值	P值
手术时间(min)	20.2±1.6	48.3±5.1	t=-19.856	＜0.01
术中出血量(mL)	3.1±0.9	10.8±2.3	t=-22.709	＜0.01
胆总管相关损伤[例(%)]	0	1(8.3)	χ²=1.287	0.013
住院时间(d)	1.2±0.3	2.3±0.8	t=-19.507	＜0.01

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| 显示表格

3. 讨论

常规LC在手术过程中有发生胆总管损伤的风险，而胆总管的医源性损伤主要与外科医师对胆道的认识不准确有关。ICG胆道造影可通过显示胆囊管和胆总管的结构来预防术中胆总管损伤；本项研究表明，在LC中，ICG胆道造影可确保对胆总管和胆囊管的准确识别，有效区分胆总管与胆囊管的结构，准确解剖Calot三角，从而有效保护胆总管。

ICG过敏反应是最严重的并发症，但发生率极低；Perry等^[16]研究显示，ICG过敏反应仅为1/80 000，并且术前碘过敏实验可预测及预防ICG过敏的发生。

本研究结果显示，ICG胆道造影组中无患者发生胆总管损伤；常规组出现胆总管周围组织出血1例，患者进行了止血保守处理后于术后1周内康复。且两组在术中手术时间、术中出血量、术后住院时间等方面均有显著性差异。究其原因，常规LC常需剥离胆管周围组织以显示其形态及结构，此过程将不可避免造成失血，并延长手术时间；而ICG胆道造影可直接显示胆总管及胆囊管结构，无需剥离周围组织，从而避免术中出血，并缩短手术时间，显示出ICG胆道造影在LC中的显著优势。

本研究为ICG胆道造影预防LC术中胆总管损伤的有效性和安全性提供了依据。由于ICG胆道造影具有效果好、精密性和准确性高、安全性强、可对狭窄胆道进行显影等优点，有望成为LC治疗胆囊结石时重要的辅助方法。然而本研究的样本量较少，需在未来招募更多患者、增加病例数量以更进一步的深入研究。

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The anti-liver fibrosis effect of Kangxian Ruangan prescription: A cell study based on hepatic sinusoidal capillarization

DOI: 10.3969/j.issn.1001-5256.2020.02.017