Differentially Expressed Proteins and Their Regulation between Liver-gallbladder Damp-heat Syndrome and Spleen-stomach Damp-heat Syndrome in Chronic Hepatitis B

doi:10.12114/j.issn.1007-9572.2020.00.572

Abstract

Abstract: Background　Liver-gallbladder damp-heat and spleen-stomach damp-heat syndromes，two most common syndromes in chronic hepatitis B（CHB），are difficult to be distinguished due to a similar manifestation of damp-heat.So finding the essence of difference and distinguishable biomarkers of them are of great significance to enrich the modern scientific connotation of TCM syndromes and improve the clinical precision differentiation and treatment.Objective　To investigate the differentially expressed proteins and their regulation as well as potentially distinguishable objective markers between liver-gallbladder damp-heat and spleen-stomach damp-heat syndromes in CHB.Methods　From the General Hospital of Western Theater Command PLA，30 hepatology and gastroenterology outpatients with CHB〔15 with liver-gallbladder damp-heat syndrome（subgroup A）and 15 with spleen-stomach damp-heat syndrome（subgroup B）〕，and 10 heath examinees（control group）were recruited during January 2018 to January 2019.ELISA was used to detect the target proteins and bioinformatics analysis was conducted，involving CEACAM1，MCP-1，SDF-1，ORM2，MSN，FCN2，VCAM-1，IGF-1，NOTCH2，β-APP，ACO1，SORD and ALDH1A1.ROC analysis was conducted to explore the value of differentially expressed proteins in distinguishing liver-gallbladder damp-heat and spleen-stomach damp-heat syndromes in CHB.Results　The mean expression levels of CEACAM1，MCP-1，SDF-1，ORM2，MSN，FCN2，VCAM-1，IGF-1，NOTCH2，β-APP，ACO1，SORD and ALDH1A1 in subgroups A and B were significantly lower than those in control group（P<0.05）.Subgroup B had higher mean expression levels of CEACAM1，MCP-1，MSN and VCAM-1，and lower mean expression levels of SDF-1，ORM2，FCN2，IGF-1，NOTCH2 and β-APP than subgroup A（P<0.05）.Taking inter-subgroup differences reaching a significance level of P<0.01 as a criterion to further screen the differentially expressed proteins，only CEACAM1，SDF-1，MSN and IGF-1 of above-mentioned 10 proteins were enrolled，specifically，subgroup B had higher mean expression levels of CEACAM1 and MSN and lower mean expression levels of SDF-1 and IGF-1（P<0.01）.Bioinformatics analysis results showed that，there were relatively close associations of CEACAM1，MCP-1，SDF-1，ORM2，MSN，FCN2，VCAM-1，IGF-1，NOTCH2 and β-APP between liver-gallbladder damp-heat and spleen-stomach damp-heat syndromes in CHB，moreover，the above-mentioned 10 differentially expressed proteins participated in multiple signaling pathways together.ROC curve analysis results showed that，in distinguishing the two syndromes，the AUC of CEACAM1 was 0.73〔95%CI（0.54，0.87）〕with sensitivity of 86.67% and specificity of 56.25%，the AUC of SDF-1 was 0.70〔95%CI（0.51，0.85）〕with sensitivity of 86.67% and specificity of 56.25%，and the AUC of MSN was 0.72〔95%CI（0.53，0.86）〕with sensitivity of 80.00% and specificity of 56.25%.Conclusion　Liver-gallbladder damp-heat and spleen-stomach damp-heat syndromes in CHB have objective differences，with CEACAM1，SDF-1，MSN and IGF-1 as major differentially expressed proteins.The biological mechanism for the differences may be correlated with oxidative stress response mediated by NF-κB and AGE-RAGE signaling pathways.Moreover，CEACAM1，SDF-1 and MSN may be used as potential biomarkers for distinguishing liver-gallbladder damp-heat and spleen-stomach damp-heat syndromes in CHB.

Key words: Chronic hepatitis B, Symptom complex, Liver-gallbladder damp-heat, Spleen-stomach damp-heat, Proteins, Bioinformatics

摘要： 背景　湿热证是慢性乙型肝炎（CHB）的常见证候，而作为CHB常见证型的肝胆湿热证与脾胃湿热证有湿热共性，辨证分型较易混淆，探讨二者证候差异的本质及区分二者的生物学标志物对丰富中医证候现代科学内涵、提高精准化辨证论治等具有重要意义。目的　从差异表达蛋白及其调控层面探讨CHB肝胆湿热证与脾胃湿热证的证候差异，并探寻区分二者的潜在客观标志物。方法　选取2018年1月—2019年1月中国人民解放军西部战区总医院肝病科门诊、消化科门诊收治的CHB患者30例，其中辨证分型为肝胆湿热证者15例（肝胆湿热组）、脾胃湿热证者15例（脾胃湿热组）；另选取同期在中国人民解放军西部战区总医院体检健康者10例作为对照组。采用酶联免疫吸附试验（ELISA）检测目标蛋白〔包括人癌胚抗原相关细胞黏附分子1（CEACAM1）、单核细胞趋化蛋白1（MCP-1）、基质细胞衍生因子1（SDF-1）、人类黏蛋白2（ORM2）、人膜突蛋白（MSN）、纤维胶凝蛋白2（FCN2）、血管细胞黏附分子1（VCAM-1）、胰岛素样生长因子1（IGF-1）、人神经源性基因座同源缺口蛋白2（NOTCH2）、人β-淀粉样前体蛋白（β-APP）、人乌头酸酶（ACO1）、人山梨醇脱氢酶（SORD）、人乙醛脱氢酶1家族成员A1（ALDH1A1）〕，并进行生物信息学分析；绘制受试者工作特征（ROC）曲线以分析差异表达蛋白对CHB肝胆湿热证与脾胃湿热证的鉴别诊断价值。结果　肝胆湿热组、脾胃湿热组患者CEACAM1、MCP-1、SDF-1、ORM2、MSN、FCN2、VCAM-1、IGF-1、NOTCH2、β-APP、ACO1、SORD、ALDH1A1蛋白表达量均低于对照组（P<0.05）；脾胃湿热组患者CEACAM1、MCP-1、MSN、VCAM-1蛋白表达量高于肝胆湿热组，SDF-1、ORM2、FCN2、IGF-1、NOTCH2、β-APP蛋白表达量低于肝胆湿热组（P<0.05）。以P<0.01为标准进一步筛选肝胆湿热证与脾胃湿热证差异表达蛋白，则上述10种差异表达蛋白仅CEACAM1、SDF-1、MSN、IGF-1入选，其中脾胃湿热组患者CEACAM1、MSN蛋白表达量高于肝胆湿热组，SDF-1、IGF-1蛋白表达量低于肝胆湿热组（P<0.01）。生物信息学分析结果显示，CHB肝胆湿热证与脾胃湿热证之间CEACAM1、MCP-1、SDF-1、ORM2、MSN、FCN2、VCAM-1、IGF-1、NOTCH2、β-APP存在较紧密的关联，且这10种差异表达蛋白共同参与多条信号通路。ROC曲线分析结果显示，CEACAM1、SDF-1、MSN鉴别诊断CHB肝胆湿热证与脾胃湿热证的ROC曲线下面积分别为0.73〔95%CI（0.54，0.87）〕、0.70〔95%CI（0.51，0.85）〕、0.72〔95%CI（0.53，0.86）〕，灵敏度分别为86.67%、86.67%、80.00%，特异度均为56.25%。结论　CHB肝胆湿热证与脾胃湿热证之间存在客观差异，CEACAM1、SDF-1、MSN、IGF-1是二者主要差异表达蛋白；CHB肝胆湿热证与脾胃湿热证证候差异的生物学机制可能与核因子（NF）-κB信号通路、晚期糖基化终末产物（AGE）-晚期糖基化终末产物受体（RAGE）信号通路介导的氧化应激反应等有关，而CEACAM1、SDF-1、MSN可作为区分CHB肝胆湿热证与脾胃湿热证的潜在客观标志物。

关键词: 乙型肝炎, 证候, 肝胆湿热, 脾胃湿热, 蛋白质类, 生物信息学

DAN Lijuan,CHAI Shaozhu,LI Guiyu, et al. Differentially Expressed Proteins and Their Regulation between Liver-gallbladder Damp-heat Syndrome and Spleen-stomach Damp-heat Syndrome in Chronic Hepatitis B　[J]. Chinese General Practice, 2021, 24(3): 348-354. DOI: 10.12114/j.issn.1007-9572.2020.00.572.
淡丽娟,柴少竹,黎桂玉等. 从差异表达蛋白及其调控层面探讨慢性乙型肝炎肝胆湿热证与脾胃湿热证的证候差异[J]. 中国全科医学, 2021, 24(3): 348-354. DOI: 10.12114/j.issn.1007-9572.2020.00.572.

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