基于抑制细菌RNA聚合酶探索杨梅素的抗结核作用及机制

doi:10.3877/cma.j.issn.1674-6880.2025.06.001

目的

从抑制细菌RNA聚合酶（RNAP）角度探讨杨梅素的抗结核作用及机制。

方法

选取大肠杆菌RNAP（EcoRNAP）、结核分枝杆菌RNAP（MtbRNAP）为靶标蛋白，通过体外转录实验筛选出细菌RNAP抑制剂，杨梅素的活性验证分为A、B两组，每个大组设置4个浓度梯度，A组先加浓度为0、12.5、25、50 μmol/L杨梅素后加20 nmol/L启动子DNA；B组先加20 nmol/L启动子DNA后加浓度为0、12.5、25、50 μmol/L杨梅素。凝胶电泳迁移实验（EMSA）探索抑制机制，实验分为两组，不加杨梅素的样品为对照组，加杨梅素的样品为实验组。最后，药敏试验验证抑制剂对结核分枝杆菌敏感株和耐药株是否具有抑菌作用。

结果

体外转录实验表明，杨梅素对EcoRNAP（F = 13.290，P = 0.015）及MtbRNAP（F = 9.431，P = 0.028）均有抑制活性。EMSA提示，杨梅素在转录起始阶段通过阻碍EcoRNAP（t = 22.692，P < 0.001）、MtbRNAP（t = 21.581，P < 0.001）与启动子DNA的相互作用从而抑制RNAP的转录活性。药敏试验证实杨梅素对结核分枝杆菌敏感株和耐药株均具有抑菌作用。

结论

杨梅素通过抑制细菌RNAP与启动子DNA结合抑制细菌转录，对利福平和异烟肼耐药的结核分枝杆菌有抑菌作用，有望成为新型抗结核先导化合物。

关键词: 杨梅素, 细菌RNA聚合酶, 抑制剂, 抗生素耐药, 抗结核

Objective

To investigate the antituberculosis activity and mechanism of myricetin from the perspective of inhibiting bacterial RNA polymerase (RNAP).

Methods

The Escherichia coli RNAP (EcoRNAP) and Mycobacterium tuberculosis RNAP (MtbRNAP) were selected as target proteins. Bacterial RNAP inhibitors were identified using in vitro transcription experiments. The activity verification of myricetin was conducted in groups A and B, with four concentration gradients for each group. In the group A, myricetin at concentrations of 0, 12.5, 25, and 50 μmol/L was added first, followed by 20 nmol/L promoter DNA; the group B first added 20 nmol/L promoter DNA, followed by myricetin at concentrations of 0, 12.5, 25, and 50 μmol/L. The inhibition mechanism was studied using electrophoretic mobility shift assay (EMSA) in two groups: the control group (samples without myricetin) and the experimental group (samples with myricetin). Finally, the antituberculosis activity was tested against both antibiotic sensitive and resistant Mycobacterium tuberculosis strains.

Results

In vitro transcription experiments demonstrated that myricetin had inhibitory activity against both EcoRNAP (F = 13.290, P = 0.015) and MtbRNAP (F = 9.431, P = 0.028). EMSA indicated that myricetin disrupted RNAP-promoter DNA interactions during transcription initiation, especially EcoRNAP (t = 22.692, P < 0.001) and MtbRNAP (t = 21.581, P < 0.001). Drug sensitivity experiments confirmed that myricetin had antimicrobial activity against both antibiotic sensitive and resistant Mycobacterium tuberculosis strains.

Conclusions

Myricetin inhibits bacterial transcription by disrupting RNAP-promoter DNA interactions and has antimicrobial activity against rifampin and isoniazid resistant Mycobacterium tuberculosis. Myricetin may serve as a novel antituberculosis lead compound.

Key words: Myricetin, Bacterial RNA polymerase, Inhibitor, Antibiotic resistance, Antituberculosis

表1 EcoRNAP和MtbRNAP的表达载体与菌株信息汇总表

蛋白名称	表达载体	抗性	表达菌株
EcoRNAP核心酶	PIA900	氨苄青霉素	BL21（DE3）
大肠杆菌σ⁷⁰	pGEMD	氨苄青霉素	BL21（DE3）
MtbRNAP核心酶	pETDuet-Mtb-rpoBC，pACYCDuet-Mtb-rpoAZ	氨苄青霉素，氯霉素	BL21（DE3）
结核分枝杆菌σ^A	pET28a-Mtb-σ^A	卡那霉素	BL21（DE3）

表1 EcoRNAP和MtbRNAP的表达载体与菌株信息汇总表

蛋白名称	表达载体	抗性	表达菌株
EcoRNAP核心酶	PIA900	氨苄青霉素	BL21（DE3）
大肠杆菌σ⁷⁰	pGEMD	氨苄青霉素	BL21（DE3）
MtbRNAP核心酶	pETDuet-Mtb-rpoBC，pACYCDuet-Mtb-rpoAZ	氨苄青霉素，氯霉素	BL21（DE3）
结核分枝杆菌σ^A	pET28a-Mtb-σ^A	卡那霉素	BL21（DE3）

图1 杨梅素抑制细菌RNAP的体外转录活性注：RNAP. RNA聚合酶；EcoRNAP.大肠杆菌RNAP；MtbRNAP.结核分枝杆菌RNAP；a图为基于荧光的体外高通量筛选方法；b图为杨梅素抑制EcoRNAP的体外转录活性（n = 4）；c图为杨梅素抑制MtbRNAP的体外转录活性（n = 4）；A组先加杨梅素后加DNA，B组先加DNA后加杨梅素；与同组0 μmol/L比较，^aP < 0.05；与A组同浓度比较，^bP < 0.05

图2 杨梅素阻碍RNAP与启动子DNA的相互作用注：RNAP. RNA聚合酶；EcoRNAP.大肠杆菌RNAP；MtbRNAP.结核分枝杆菌RNAP；a图为200 μmol/L杨梅素对RNAP-DNA复合物形成的影响分析；b图为对a图中复合物浓度的统计分析（n = 3），与EcoRNAP + DNA比较，^aP < 0.05；与MtbRNAP + DNA比较，^bP < 0.05

表2 杨梅素的MIC

菌株	最小抑菌浓度（mg/L）
结核分枝杆菌2 572（利福平耐药、异烟肼耐药）	10
结核分枝杆菌2 878（利福平耐药、异烟肼耐药）	10
结核分枝杆菌2 321（利福平耐药、异烟肼耐药）	20
结核分枝杆菌2 586（利福平敏感、异烟肼敏感）	20
结核分枝杆菌2 590（利福平敏感、异烟肼敏感）	20
结核分枝杆菌2 581（利福平敏感、异烟肼敏感）	10
结核分枝杆菌2 585（利福平敏感、异烟肼敏感）	10

表2 杨梅素的MIC

菌株	最小抑菌浓度（mg/L）
结核分枝杆菌2 572（利福平耐药、异烟肼耐药）	10
结核分枝杆菌2 878（利福平耐药、异烟肼耐药）	10
结核分枝杆菌2 321（利福平耐药、异烟肼耐药）	20
结核分枝杆菌2 586（利福平敏感、异烟肼敏感）	20
结核分枝杆菌2 590（利福平敏感、异烟肼敏感）	20
结核分枝杆菌2 581（利福平敏感、异烟肼敏感）	10
结核分枝杆菌2 585（利福平敏感、异烟肼敏感）	10

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Antituberculosis activity and mechanism of myricetin based on inhibition of bacterial RNA polymerase

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Antituberculosis activity and mechanism of myricetin based on inhibition of bacterial RNA polymerase