Exploring the effect of Zuojin Pill on inflammatory factors in gastroesophageal reflux disease model rats based on bitter taste receptors and transient receptor potential vanilloid 1
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摘要: 目的 基于苦味受体(TAS2R38)和热敏受体(TRPV1)探讨左金丸对胃食管反流病(gastroesophageal reflux disease，GERD)模型大鼠炎性因子的影响。方法 将40只SPF级雄性SD大鼠随机分为假手术组、模型组及左金丸低、中、高剂量组，每组各8只。除假手术组大鼠外，其余各组采用手术造模方式构建GERD模型大鼠，造模成功4周后给予不同剂量左金丸干预治疗4周后，苏木精-伊红染色观察病理改变，酶联免疫吸附试验(ELISA)检测血清中白细胞介素-1β(interleukin-1β，IL-1β)、IL-6和肿瘤坏死因子(tumor necrosis factor-α，TNF-α)的含量，Western blot法检测食管组织中TAS2R38和TRPV1表达水平。利用分子对接技术将左金丸中黄连和吴茱萸主要药效成分分别与TAS2R38、TRPV1进行分子对接模拟。为了进一步验证活性成分与靶点的关系，将48只SPF级雄性SD大鼠随机分为假手术组、模型组、小檗碱组、小檗碱+U73122组、吴茱萸碱组及吴茱萸碱+RTX组，每组各8只。除假手术组大鼠外，其余各组大鼠进行模型制备，4周后分别给予相应药物治疗，检测上述一致的指标。结果 与模型组大鼠比较，左金丸各剂量组大鼠食管组织病理明显改善，左金丸低、中、高剂量组大鼠血清中IL-1β、TNF-α含量显著降低，左金丸高剂量组大鼠血清中IL-6含量显著降低，左金丸中、高剂量组大鼠食管组织中TAS2R38的表达水平升高(P < 0.05，P < 0.01)，但各组大鼠食管组织TRPV1表达无显著变化(P>0.05)。分子对接结果表明左金丸中黄连和吴茱萸分别作用于TAS2R38、TRPV1的有效成分可能是小檗碱和吴茱萸碱。验证实验进一步证实，与模型组比较，小檗碱组与吴茱萸碱组大鼠食管组织病理明显改善，血清中炎性因子IL-1β、IL-6和TNF-α含量降低(P < 0.05)；与小檗碱组比较，小檗碱+U73122组大鼠血清中IL-6、TNF-α的含量升高，食管组织中TAS2R38表达显著降低(P < 0.05)；与吴茱萸碱组比较，吴茱萸碱+RTX组大鼠血清中IL-1β、IL-6的含量升高，食管组织中TRPV1表达显著降低(P < 0.05)。结论 左金丸能显著下调GERD大鼠炎性因子的含量，其作用机制可能是其有效成分小檗碱和吴茱萸碱分别靶向TAS2R38、TRPV1从而发挥作用。Abstract: Objective To investigate the effect of Zuojin Pill on inflammatory factors in gastroesophageal reflux disease(GERD) model rats based on the bitter taste receptor TAS2R38 and transient receptor potential vanilloid 1(TRPV1).Methods Forty SPF-grade male SD rats were randomly divided into sham surgery group, model group, and low, medium, and high dose groups of Zuojin Pill, with 8 rats in each group.Except for the sham group, surgical modeling was used to construct GERD model for all other groups. After 4 weeks of successful modeling, different doses of Zuojin Pills were administered for intervention treatment. After 4 weeks, hematoxylin-eosin staining was used to observe pathological changes, and enzyme-linked immunosorbent assay(ELISA) was used to detect the content of interleukin-1β(IL-1β), IL-6, and tumor necrosis factor-α(TNF-α) in serum. Western blot method was used to detect the expression levels of TAS2R38 and TRPV1 in esophageal tissue. Based on molecular docking technology, the main pharmacological components of Coptidis Rhizoma and Evodiae Fructus in Zuojin Pill were simulated with TAS2R38 and TRPV1, respectively. In order to further verify the relationship between active components and targets, 48 SPF grade male SD rats were randomly divided into sham surgery group, model group, berberine group, berberine+U73122 group, evodiamine group, and evodiamine+RTX group, with 8 rats in each group. Except for the sham group rats, all other groups of rats were subjected to model preparation, and after 4 weeks, corresponding medication treatment was given and the consistent indicators mentioned above were detected.Results Compared with the model group rats, the pathological changes of the esophageal tissue in each dose group of Zuojin Pill were significantly improved, and the content of IL-1β, TNF-α in the low, medium, and high dose groups of Zuojin Pill rats were significantly down-regulated. The content of IL-6 in the serum of rats in the high-dose Zuojin Pill group was significantly decreased. The expression level of TAS2R38 in the esophageal tissue of rats in the middle and high-dose groups of Zuojin Pill were increased(P < 0.05, P < 0.01), but there was no significant change in the expression of TRPV1 in the esophageal tissue of rats in each group(P>0.05). The molecular docking results indicate that the effective components of Coptidis Rhizoma and Evodiae Fructus in Zuojin Pill, which act on TAS2R38 and TRPV1 respectively, may be berberine and evodiamine. The validation experiment further confirmed that compared with the model group, the pathological changes in the esophageal tissue of rats in the berberine group and evodiamine groups were significantly improved, and the content of inflammatory factor including IL-1β, IL-6, and TNF-α were decreased.Compared with the berberine group, the levels of IL-6 and TNF-α increased and the expression of TAS2R38 in esophageal tissue significantly decreased in the berberine+U73122 group.Compared with the r evodiamine group, the content of IL-1β and IL-6 increased and the expression of TRPV1 in esophageal tissue significantly decreased, in the evodiamine+RTX group, with statistical significance(P < 0.05).Conclusion Zuojin Pill can significantly down-regulate the content of inflammatory factors in GERD rats, and its mechanism is that berberine and evodiamine are the effective components of Zuojin Pill respectively, exert anti-inflammatory effects by targeting TAS2R38 and TRPV1.
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表 1 各组大鼠血清中IL-1β、IL-6、TNF-α含量
pg/mL，X±S 组别 例数 IL-1β IL-6 TNF-α 假手术组 3 54.86±5.972) 88.99±28.832) 66.85±13.632) 模型组 3 172.94±26.11 255.30±25.38 281.83±47.23 左金丸低剂量组 3 93.58±10.332) 199.50±68.32 176.16±18.582) 左金丸中剂量组 3 90.67±15.552) 161.51±36.46 154.30±13.632) 左金丸高剂量组 3 82.93±29.642) 138.18±14.911) 135.15±17.302) 与模型组比较，1)P < 0.05，2)P < 0.01。 
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表 2 左金丸中关键活性成分与TAS2R38和TRPV1分子对接结果
kJ/mol 化合物 TRPV1-结合能 化合物 TAS2R38-结合能 吴茱萸碱 -10.0 小檗碱 -8.0 吴茱萸次碱 -9.4 表小檗碱 -7.8
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表 3 小檗碱和吴茱萸碱对GERD大鼠血清中IL-1β、IL-6、TNF-α含量的影响
pg/mL，X±S 组别 例数 IL-1β IL-6 TNF-α 假手术组 3 69.38±13.061) 95.65±15.341) 80.35±17.101) 模型组 3 179.50±22.76 247.43±36.23 289.12±35.62 小檗碱组 3 88.20±20.481) 139.31±12.561) 126.70±11.911) 小檗碱+U73122组 3 154.55±13.65 205.85±16.012) 223.99±26.552) 吴茱萸碱组 3 91.10±11.371) 127.92±18.741) 139.73±8.931) 吴茱萸碱+RTX组 3 147.88±12.783) 204.84±16.503) 207.43±15.81 与模型组比较，1)P < 0.01；与小檗碱组比较，2)P < 0.01；与吴茱萸碱组比较，3)P < 0.05。
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