Characterization of Gastric Mucosal Flora and Inflammation Level in Hp-Related Gastropathy with Spleen-Stomach Damp-Heat and Spleen-Qi Deficiency Syndromes
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摘要:
目的 探讨幽门螺杆菌(Helicobacter pylori, Hp)相关性胃病脾胃湿热证、脾气虚证形成的生物学机制。 方法 收集107例Hp相关性胃病患者的胃黏膜样本, 其中脾胃湿热证70例，脾气虚证37例, 同时招募平和体质健康受试者10名作为正常对照组。采用16S rDNA技术检测胃黏膜样本菌群组成, 免疫组织化学法检测胃黏膜NLRP3和白介素-1β(Interleukin-1β, IL-1β)蛋白表达水平。 结果 与正常对照组比较, 脾胃湿热证患者胃黏膜菌群放线菌门、拟杆菌门的相对丰度显著降低(P < 0.05), 脾气虚证组患者胃黏膜菌群的门水平物种丰度无明显差异；线性判别分析显示, 与正常对照组比较, 幽门螺杆菌属为Hp相关性胃病脾胃湿热证、脾气虚证患者的胃黏膜标志菌属。与正常对照组比较, 脾胃湿热证、脾气虚证患者胃黏膜NLRP3和IL-1β蛋白表达水平均显著升高(P < 0.05)。 结论 胃黏膜菌群结构差异、炎症水平升高可能是Hp相关性胃病脾胃湿热证、脾气虚证形成的内在生物学机理。 Abstract:OBJECTIVE To investigate the biological mechanisms underlying the formation of spleen-stomach damp-heat and spleen-qi deficiency syndromes in helicobacter pylori (Hp)-related gastropathy. METHODS Gastric mucosa samples were collected from 107 patients with Hp-related gastropathy, including 70 cases with spleen-stomach damp-heat syndrome and 37 cases with spleen-qi deficiency syndrome, while 10 healthy subjects with moderate constitution were recruited as normal control group. Besides, the gastric mucosa samples were examined by 16S rDNA technique, and the expression levels of NLRP3 and interleukin-1β (IL-1β) in the gastric mucosa were measured by immunohistochemistry. RESULTS Compared with the normal control group, the relative abundance of the gastric mucosal flora of Actinobacteria and Bacteroidetes phylum was significantly lower in patients with spleen-stomach damp-heat syndrome (P < 0.05), while the abundance of gastric mucosal flora in patients with spleen-qi deficiency syndrome was not obviously different. In addition, the linear discriminant analysis showed that Hp was the marker genus of gastric mucosa in Hp-related gastropathy patients with spleen-stomach damp-heat and spleen-qi deficiency syndromes. What is more, the expression levels of NLRP3 and IL-1β proteins in the gastric mucosa of patients with spleen-stomach damp-heat and spleen-qi deficiency syndromes were significantly higher compared with those of normal controls (P < 0.05). CONCLUSION The structural differences in gastric mucosal flora and elevated inflammation levels may be the underlying biological mechanism for the formation of Hp-related gastropathy with spleen-stomach damp-heat and spleen-qi deficiency syndromes. -
图 2 脾胃湿热证组、脾气虚证组和正常对照组胃黏膜菌群的多样性PCoA图
Figure 2. PCoA diagram of diversity of gastric mucosa microflora in spleen and stomach damp-heat syndrome group, spleen-qi deficiency syndrome group and normal control group
图 3 脾胃湿热证组、脾气虚证组和正常对照组在门(A)、属(B)水平的物种丰度组成
Figure 3. Species abundance at phylum (A) and genus (B) level in spleen and stomach damp-heat syndrome group, spleen-qi deficiency group and normal control group
图 4 Hp相关性胃病脾胃湿热证优势菌群
Figure 4. The dominant flora of Hp-related gastropathy with spleen-stomach damp-heat syndrome
图 5 Hp相关性胃病脾气虚证组优势菌群
Figure 5. The dominant flora of Hp-related gastropathy with spleen-qi deficiency syndrome
图 6 胃黏膜组织中NLRP3蛋白表达水平(×200)
注: A.脾胃湿热证组; B.脾气虚证组; C.正常对照组
Figure 6. Expression of NLRP3 in gastric mucosa tissue(×200)
图 7 胃黏膜组织中IL-1β蛋白表达水平(×200)
注: A.脾胃湿热证组; B.脾气虚证组; C.正常对照组
Figure 7. Expression of IL-1β in gastric mucosa tissue(×200)
表 1 Hp相关性胃病不同证候患者性别、年龄比较(x±s)
Table 1. Comparison of sex and age of different evidence of Hp-related gastropathy (x±s)
证型 例数 性别 年龄/岁 男 女 脾胃湿热证组 70 46 24 54.23±12.63 脾气虚证组 37 13 24 51.27±13.03 正常对照组 10 5 5 51.20±10.62 注: 组间比较, χ性别2=9.32, P < 0.05。 
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表 2 Hp相关性胃病不同证候患者病种比较
Table 2. Comparison of disease types between different evidence of Hp-related gastropathy
证型 例数 CNAG CAG GC 脾胃湿热证组 70 34 29 7 脾气虚证组 37 15 16 6
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表 3 Hp相关性胃病脾胃湿热证胃黏膜菌群的α多样性分析[M(P25, P75)]
Table 3. α diversity analysis of gastric mucosa microflora in Hp-related gastropathy with spleen-stomach damp-heat syndrome [M(P25, P75)]
组别 例数 Chao Ace Shannon Simpson 脾胃湿热证组 70 181.98(114.09, 226.39) 182.39(116.12, 225.58) 2.00(0.38, 3.27)** 0.23(0.90, 0.89)** 正常对照组 10 221.02(101.00, 281.27) 218.46(82.01, 272.30) 3.21(2.85, 3.65) 0.08(0.05, 0.11) 注：组间比较，**P < 0.01。
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表 4 Hp相关性胃病脾气虚证胃黏膜菌群的α多样性分析[M(P25, P75)]
Table 4. α diversity analysis of gastric mucosa microflora in Hp-related gastropathy with spleen-qi deficiency syndrome [M(P25, P75)]
组别 例数 Chao Ace Shannon Simpson 脾气虚证组 37 177.16(123.75, 270.21) 180.37(125.54, 277.81) 2.94(1.41, 3.51) 0.14(0.06, 0.53)* 正常对照组 10 221.02(101.00, 281.27) 218.46(82.01, 272.30) 3.21(2.85, 3.65) 0.08(0.05, 0.11) 注：组间比较，*P < 0.05。
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表 5 Hp相关性胃病脾胃湿热证门、属水平差异菌群(x±s)
Table 5. Differential flora in Hp-related gastropathy with spleen and stomach damp-heat syndrome at the phylum and genus level (x±s)
组别 门水平 属水平 Actinobacteria Bacteroidetes Helicobacter Neisseria 脾胃湿热证组 2.547±3.498** 6.963±9.752* 40.904±44.899** 2.141±3.717* 正常对照组 4.851±2.921 12.390±9.970 1.531±4.813 4.281±6.283 注：组间比较，*P < 0.05，**P < 0.01。
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表 6 Hp相关性胃病脾气虚证属水平差异菌群(x±s)
Table 6. Differential flora in Hp-related gastropathy with spleen-qi deficiency syndrome at the genus level (x±s)
组别 Helicobacter 脾气虚证组 28.269±39.237** 正常对照组 1.531±4.812 注：组间比较，**P < 0.01。
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表 7 Hp相关性胃病脾胃湿热证、脾气虚证的NLRP3蛋白表达
Table 7. NLRP3 protein expression in Hp-related gastropathy with spleen-stomach damp-heat syndrome and spleen-qi deficiency syndrome
组别 例数 阴性 轻度 中度 重度 脾胃湿热证组 70 7 18 31 14 脾气虚证组 37 3 10 16 8 正常对照组 10 3 6 0 1 注: 经Kruskal-Wallis H检验，与正常对照组比较, 脾胃湿热证组统计值为30.186，P < 0.05，脾气虚证组统计值为31.432，P < 0.05。
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表 8 Hp相关性胃病脾胃湿热证、脾气虚证的IL-1β蛋白表达
Table 8. IL-1β protein expression in Hp-related gastropathy with spleen-stomach damp-heat syndrome and spleen-qi deficiency syndrome
组别 例数 阴性 轻度 中度 重度 脾胃湿热证组 70 7 29 22 12 脾气虚证组 37 4 11 16 6 正常对照组 10 5 3 2 0 注: 经Kruskal-Wallis H检验，与正常对照组比较, 脾胃湿热证组统计值为28.629，P < 0.05，脾气虚证组统计值为32.639，P < 0.05。
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