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. 2024 Feb 19:15:1355460.
doi: 10.3389/fmicb.2024.1355460. eCollection 2024.

In vitro anti- Helicobacter pylori activity and the underlining mechanism of an empirical herbal formula - Hezi Qingyou

Zhong Feng #  1   2   3 , Hui Li #  2   3 , Yajie Hao  2   3 , Chang Peng  1 , Ling Ou  1 , Junwei Jia  2   3 , Mingjin Xun  2   3 , Yuanjing Zou  1 , Meiyun Chen  1 , Guimin Zhang  2   3 , Meicun Yao  1   4
Affiliations

In vitro anti- Helicobacter pylori activity and the underlining mechanism of an empirical herbal formula - Hezi Qingyou

Zhong Feng et al. Front Microbiol. .

Abstract

Background: Helicobacter pylori (H. pylori) is thought to primarily colonize the human stomach and lead to various gastrointestinal disorders, such as gastritis and gastric cancer. Currently, main eradication treatment is triple or quadruple therapy centered on antibiotics. Due to antibiotic resistance, the eradication rate of H. pylori is decreasing gradually. Therefore, searching for anti-H. pylori drugs from herbal sources has become a strategy for the treatment. Our team proposed a Hezi Qingyou Formula (HZQYF), composed of Chebulae Fructus, Ficus hirta Vahl and Cloves, and studied its anti-H. pylori activity and mechanism.

Methods: Chemical components of HZQYF were studied using UHPLC-MS/MS and HPLC. Broth microdilution method and agar dilution method were used to evaluate HZQYF's antibacterial activity. The effects of HZQYF on expression of adhesion genes (alpA, alpB, babA), urease genes (ureE, ureF), and flagellar genes (flaA, flaB) were explored using Reverse Transcription-quantitative Polymerase Chain Reaction (RT-qPCR) technology. Effects on morphology and permeability of the extracellular membrane were studied using scanning electron microscopy (SEM) and N-phenylnaphthalen-1-amine (NPN) uptake. Effect on urease activity was studied using a urease kinetics analysis in vitro. Immunofluorescence staining method was used to examine the effect on adhesion. Western blot was used to examine the effect on cagA protein.

Results: Minimum inhibitory concentration (MIC) values of the formula against H. pylori clinical strains and standard strains were 80-160 μg/mL, and minimum bactericidal concentration (MBC) values were 160-320 μg/mL. The formula could down-regulate the expression of adhesion genes (alpA, alpB, babA), urease genes (ureE, ureF) and flagellar genes (flaA, flaB), change the morphology of H. pylori, increase its extracellular membrane permeability, and decrease its urease activity.

Conclusion: Present studies confirmed that HZQYF had promising in vitro anti-H. pylori activities and demonstrated its possible mechanism of action by down-regulating the bacterial adhesion, urease, and flagellar gene expression, which provided scientific bases for further clinical investigations.

Keywords: Helicobacter pylori; Hezi Qingyou Formula; antibacterial activity; in vitro; mechanism of action.

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Conflict of interest statement

ZF, HL, YH, JJ, MX, and GZ were employed by Lunan Pharmaceutical Group Co., Ltd., and Shandong New Time Pharmaceutical Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Total ion chromatogram of HZQYF.
Figure 2
Figure 2
HPLC fingerprint chromatogram of HZQYF.
Figure 3
Figure 3
(A) Inhibiting kinetics curves of HZQYF on ATCC 43504. Concentration of CLR was 0.016 μg/mL. (B) Inhibiting kinetics curves of HZQYF on ATCC 700392. Concentration of CLR was 0.004 μg/mL. (C) Killing kinetics curves of HZQYF on ATCC 43504. (D) Killing kinetics curves of HZQYF on ATCC 700392.
Figure 4
Figure 4
RT-qPCR analysis results showed the effects of HZQYF on the mRNA levels of H. pylori virulence genes. The bacteria strain was ATCC 700392, and the concentration of HZQYF were 160 μg/mL (A) and 80 μg/mL (B). And the incubation time of HZQYF was all 24 h. p < 0.05, vs. control group. **Stands for p < 0.01, and ****Stands for p < 0.0001, compared with control group.
Figure 5
Figure 5
SEM images of ATCC 700392. Morphological images of H. pylori cells on SEM (magnification of 20.0, 40.0, and 50.0 kx) of control (A–C) and HZQYF treatment (D–F) at MIC (160 μg/mL) for 24 h.
Figure 6
Figure 6
(A) The impact of cell membrane permeability of HZQYF on ATCC 43504. (B) The impact of cell membrane permeability of HZQYF on ATCC 700392. (C) The change in urease activity of ATCC 43504. (D) The change in urease activity of ATCC 700392. **Stands for p < 0.01, ***Stands for p < 0.001, and ****Stands for p < 0.0001, compared with control group.
Figure 7
Figure 7
CagA protein expression in H. pylori ATCC 43504 and ATCC 700392 in different groups. *Stands for p < 0.05, and ****Stands for p < 0.0001, compared with control group.
Figure 8
Figure 8
Effect of HZQYF (80,160, 320 μg/mL) on adhesion of ATCC 700392. The first row was marked with 1% FITC for H. pylori, and the second row was marked with DAPI, and all groups except cell group were added to H. pylori at MOI = 100:1.
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