Published online Nov 21, 2026. doi: 10.3748/wjg.121477
Revised: May 11, 2026
Accepted: June 26, 2026
Published online: November 21, 2026
Processing time: 166 Days and 23.2 Hours
Helicobacter pylori (H. pylori) infection remains a major etiological factor in chronic gastritis, peptic ulcer disease, and gastric cancer. The increasing prevalence of antibiotic resistance underscores the urgent need for alternative anti-H. pylori agents with clearly characterised molecular targets.
To investigate whether deoxyshikonin inhibits H. pylori adhesion by modulating urease-associated pathways.
Anti-adhesion activity of deoxyshikonin was evaluated in GES-1 and BGC823 cells using Gram staining, fluores
Deoxyshikonin reduced cell-associated H. pylori in a concentration-dependent manner. Molecular docking sugges
Deoxyshikonin inhibits H. pylori adhesion. The current evidence indicates that the urease subunits UreA and UreB are involved as molecular targets in this effect.
Core Tip: Deoxyshikonin exerted dose-responsive anti-adhesion activity against Helicobacter pylori in gastric epithelial cell models. Using an integrated approach combining drug affinity-responsive target stability-mass spectrometry, molecular docking, reverse transcription-quantitative polymerase chain reaction, western blotting, surface plasmon resonance, and urease activity assays, this study identifies urease, particularly UreB, as a key molecular target of deoxyshikonin. The findings further suggest that interference with urease is accompanied by concomitant changes in SabA and BabA expression; however, the precise regulatory mechanism underlying this coordinated response requires further investigation.
- Citation: Pang GF, Xu JY, Guan AX, Yang SX, Duangsonk K, Huang YQ, Zhou QH, Zhou WT. Study on the adhesive effect of deoxyshikonin on Helicobacter pylori. World J Gastroenterol 2026; 32(43): 121477
- URL: https://www.wjgnet.com/1007-9327/full/v32/i43/121477.htm
- DOI: https://dx.doi.org/10.3748/wjg.121477
A substantial proportion of the global population is infected with Helicobacter pylori (H. pylori). This organism is a major cause of chronic gastritis, peptic ulcers, and gastric cancer and has been linked to a variety of extragastric diseases. From 1980 to 2022, the global prevalence of H. pylori infection decreased from 58.2% to 43.1%; however, it remains an important clinical and public health concern[1-3]. Therapeutic strategies for H. pylori infection vary across regions. In the United States, where the infection rate is approximately 36%, bismuth-containing quadruple therapy is recommended as a first-line treatment. However, rising clarithromycin resistance complicates regimen selection and may compromise eradication success[4].
Shikonin is a bioactive naphthoquinone derived from Zicao, a traditional Chinese medicinal herb, and has a long history of medicinal use. Shikonin and its derivatives have been reported to possess broad pharmacological activities, including anti-inflammatory, anti-tumour, and antimicrobial effects[5,6]. Recent studies have also shown that deoxyshikonin inhibits H. pylori, particularly under acidic conditions; however, its direct molecular targets remain incompletely defined[7]. Shikonin has also been evaluated for anti-H. pylori activity. For example, Kuo et al[8] de
The drug affinity responsive target stability (DARTS) method is a label-free strategy for target discovery that can identify small-molecule targets without chemically modifying the compound[21]. In the present study, we combined DARTS-based screening with analyses using a low-urease-expression mutant, molecular interaction assays, and functional activity assays to assess whether deoxyshikonin inhibits H. pylori adhesion by directly targeting urease and whether parallel changes in adhesin-related gene expression are involved.
H. pylori strains (G27, 26695, and 26695Δcfa) were provided by Professor Hongkai Bi (Nanjing Medical University). All strains used in this study were laboratory strains; no clinical isolates, patient specimens, or human-derived materials were involved. Bacteria were cultured and routinely handled following previously described laboratory procedures[22].
Human gastric epithelial GES-1 cells and human gastric carcinoma BGC823 cells were maintained according to the proto
For microscopic adhesion assessment, GES-1 cells (3 × 105 cells/well) were seeded in 6-well plates (WHB-6, lot no. 2022120701) and cultured for 24 hours. H. pylori G27 was added at a multiplicity of infection of 100:1 and treated with deoxyshikonin (Chengdu Ruifensi Biotechnology Co., Ltd, CAS: 43043-74-9, 99.53% by HPLC) at 2, 8, and 16 μg/mL for 2 hours. After Gram staining, bacterial adhesion was evaluated by light microscopy.
In fluorescence-based assays, BGC823 cells (5 × 104 cells/well) were seeded in 24-well plates (WHB-24, lot no. 2022111601) and cultured for 24 hours. H. pylori G27, 26695, or 26695Δcfa was labeled with SYTO9 (Thermo Fisher, L-7012, United States), added to the cells under the same above-mentioned treatment conditions, and observed using a fluorescence microscope (Olympus, IX73+DP80, Japan). Non-adherent bacteria were removed by washing with phosphate-buffered saline (PBS). Cells were then lysed with 5 g/L saponin (McLean, CAS: 8047-15-2, United States), and adherent bacteria were quantified by Alamar Blue fluorescence assay and dilution plating for colony enumeration. Amoxicillin was used as a positive control.
Logarithmic-phase H. pylori G27 cultures were adjusted to 1 × 108 CFU/mL and treated with deoxyshikonin at 4 μg/mL, which corresponded to the half-maximal inhibitory concentration (IC50) value determined in this study. Bacterial proteins were extracted using a lysis buffer containing Triton X-100, and the protein concentration was determined with a bicinchoninic acid protein assay kit (Beyotime, P0010, China). Deoxyshikonin (4 mg/mL) was prepared in TNC buffer (Tris, NaCl, and CaCl2) and incubated with the protein lysate at 25 °C for 30 minutes, followed by limited proteolysis with protease from Streptomyces griseus (Yuanye Bio-Technology Co., Ltd, CAS: 9036-06-0, China) at 37 °C for 5 minutes. The reaction was stopped by adding 5 × sodium-dodecyl sulfate loading buffer (Solarbio, P1040, China), and the samples were immediately boiled for 10 minutes. Proteins were separated by sodium-dodecyl sulfate gel electrophoresis at 100 V for 90 minutes and stained with Coomassie Brilliant Blue. Bands showing differential staining intensity were excised and sent to Wuhan GeneCreate Biological Engineering Co., Ltd. for mass spectrometry-based protein identification and downstream bioinformatics analysis.
Logarithmic-phase H. pylori G27 cultures were adjusted to 1 × 108 CFU/mL and treated with deoxyshikonin (4 μg/mL) for 8 hours. Total RNA was extracted using a Vazyme FastPure Cell/Tissue Total RNA Isolation Kit V2 following the instructions provided by the manufacturer and stored at -80 °C until use. The RNA was subsequently reverse-transcribed into complementary DNA using a reverse transcription kit (Monad, MR05101M, China). Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed on a LightCycler instrument following the kit instructions (Monad, MQ10301S, China): Pre-denaturation at 95 °C for 30 seconds, denaturation at 95 °C for 10 seconds, and extension at 60 °C for 30 seconds, for a total of 40 cycles. A dissociation curve analysis was performed to confirm amplification specificity. The 16S ribosomal RNA gene served as an internal control for data normalisation, and relative transcription levels were calculated using the 2-∆∆CT method. Each group was prepared in triplicate.
Logarithmic-phase H. pylori G27 cultures were adjusted to 1 × 108 CFU/mL and incubated with deoxyshikonin at 0, 4, and 16 μg/mL for 8 hours. Bacterial pellets were collected, washed twice with sterile PBS (Sangon, B040100-0005, China), and lysed in radioimmunoprecipitation assay buffer containing a protease inhibitor cocktail (Beyotime, P1005, China). Total protein was quantified using a bicinchoninic acid protein assay kit. Equal amounts of protein were separated by sodium-dodecyl sulfate gel electrophoresis and transferred onto polyvinylidene fluoride membranes. The membranes were incubated with an anti-UreB antibody (GeneTex, GTX627292; 1:10000, United States), followed by incubation with an appropriate horseradish peroxidase-conjugated secondary antibody. UreB band intensity was quantified by densitometry and normalised to total protein loading. All experiments were independently repeated three times.
Logarithmic-phase H. pylori G27 cultures were adjusted to 1 × 108 CFU/mL and treated with deoxyshikonin at 4 or 16 μg/mL for 4 hours. Acetohydroxamic acid (75 μg/mL; McLean, CAS: 546-88-3, United States) served as the positive control. Urease activity was then measured using a Urease Activity Assay Kit (Boxbio, China) following the ma
H. pylori G27 cultures were suspended in normal saline and adjusted to 1 × 107 CFU/mL. The bacterial suspension was mixed with a urease reaction mixture containing 15 g/L urea and phenol red indicator, and the absorbance at 590 nm was measured after 30 minutes using a microplate reader. Normal saline was used as the blank control, and acetohydroxamic acid was used as the positive control. The volume ratio of bacterial suspension to working solution was set at 5:200. Each group was measured in triplicate. This assay was performed with reference to the method reported by Lee et al[23].
H. pylori strains were cultured as described above, and the H. pylori 26695Δcfa strain was constructed as previously described[24]. Bacterial suspensions (OD600 nm = 0.3 and 3) were evenly spread on Columbia agar plates containing 100 mL/L calf serum using sterile cotton swabs. Filter paper discs (6 mm) were loaded with deoxyshikonin (50 or 100 μg) and placed on the inoculated plates. After incubation for 5 days under microaerobic conditions, the plates were pho
A Biacore T200 instrument (Cytiva, United States) was used to determine the binding affinity of deoxyshikonin for UreB by surface plasmon resonance (SPR). After the NTA chip (Cytiva, United States) was conditioned with EDTA (350 mmol/L) and NaOH (50 mmol/L), UreB at a concentration of 25 μg/mL was immobilised on its surface for 420 seconds at a flow rate of 10 μL/minutes in PBS containing 0.5 mL/L Tween-20. Deoxyshikonin was injected at a series of concentrations: 0, 0.7812, 1.5625, 3.125, 6.25, 12.5, and 25 μM, and the binding responses were recorded. All binding analyses were conducted in PBS containing 0.5 mL/L Tween-20 at pH 7.4 and 25 °C. The association and dissociation phases were set to 120 seconds and 180 seconds, respectively. The chip surface was regenerated after each cycle using EDTA (350 mmol/L) and NaOH (50 mmol/L). Before analysis, multiple reference subtractions were conducted to remove bulk refractive index variations, injection noise, and data drift. Binding affinity was calculated by global fitting to a Langmuir 1:1 model using Biacore Evaluation software (Cytiva, United States). The reagents used included NTA chips (BR100532) and NTA kits containing Ni (28995043).
Nucleotide sequences of ureA, ureB, SabA, and BabA were downloaded from the NCBI Nucleotide database. Pairwise RNA-RNA interaction prediction was performed using IntaRNA via the Freiburg RNA Tools web server under default settings. As this was an exploratory and prediction-based analysis, the results were used only to assess potential transcript-level interactions and were not considered evidence of a direct regulatory relationship.
To examine the functional relationship between urease activity and bacterial adhesion, wild-type H. pylori 26695, the low-urease-expressing mutant H. pylori 26695Δcfa, and Staphylococcus aureus (S. aureus) Newman was grown to the logarithmic phase and adjusted to 1 × 108 CFU/mL. Urea was added to each bacterial suspension at a final concentration of 1.5%, and exogenous urease was added at final concentrations of 0.25, 1, or 4 μg/mL. Bacteria were labelled with SYTO9, washed thrice with PBS, and resuspended in antibiotic-free RPMI 1640 medium. BGC823 cells were seeded in 24-well plates at 1 × 105 cells/well and cultured for 24 hours. The labelled bacteria were then added to BGC823 cells at a multiplicity of infection of 100:1 and co-incubated for 2 hours in the presence or absence of deoxyshikonin (16 μg/mL). After washing, bacterial adhesion was examined using an inverted fluorescence microscope.
Data were analysed using GraphPad Prism 7.0. Results are expressed as the mean ± SD from three independent experiments. Two-group comparisons were performed using Student’s t test, whereas multiple-group comparisons were performed using one-way analysis of variance followed by an appropriate post hoc multiple-comparison test. Statistical significance was defined as P < 0.05.
Gram staining showed that H. pylori attached abundantly to GES-1 cells in the PBS control group, and the extent of adhesion decreased with increasing deoxyshikonin concentrations (Figure 1A). SYTO9 labelling showed that BGC823 cells exhibited red fluorescence, whereas bacteria exhibited green fluorescence. Consistently, H. pylori adhesion showed a concentration-dependent decrease, in agreement with the Gram staining results (Figure 1B).
Quantitative analysis further showed that cell-associated H. pylori progressively decreased with increasing deoxyshikonin concentrations (Figure 1C). Colony counting also confirmed that the number of recovered H. pylori colonies decreased in a concentration-dependent manner (Figure 1D). Together, these data indicate that deoxyshikonin reduces H. pylori cell association and colony recovery in a concentration-dependent manner.
The IC50 of deoxyshikonin against H. pylori strain G27 was determined to be 4 μg/mL (Figure 2A). DARTS performed at this concentration revealed differential protein bands (Figure 2B). These bands were excised and sent to Wuhan GeneCreate Biological Engineering Co., Ltd. for protein identification. The data were analysed using the ProteinPilot software with the AB SCIEX TripleTOF® 5600+ mass spectrometer. The Venn diagram showed that 91 proteins were identified, including 21 and 25 unique proteins in the control and experimental groups, respectively (Figure 2C). Gene Ontology (GO) enrichment analysis indicated that the identified proteins were mainly associated with cellular and metabolic processes (Figure 2D). Cluster of Orthologous Groups (COG) annotation primarily associated these proteins with translation, ribosomal structure and biogenesis, as well as posttranslational modification, protein turnover, and chaperones (Figure 2E). Paralog analysis revealed enrichment of P-loop-containing nucleoside triphosphate hydrolases (Figure 2F). Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis highlighted metabolic pathways, biosynthesis of secondary metabolites, and microbial metabolism in diverse environments (Figure 2G). Subcellular localization prediction using WoLFPSORT and PSORTb indicated that most of the identified proteins were localised to the cytoplasm, whereas a smaller proportion was predicted to reside in the cytoplasmic membrane (Figure 2H).
Based on protein identification and quantitative results, 11 candidate target proteins were selected through integrated analysis of protein identification confidence (unused ProtScore ≥ 1.3, corresponding to ≥ 95% confidence); quantitative differences between the deoxyshikonin-treated (S2) and dimethyl sulfoxide control (D1) groups; GO and COG functional annotations; KEGG pathway enrichment; and subcellular localisation predictions (WoLF PSORT/PSORTb). GO analysis indicated that the 91 overlapping proteins were predominantly enriched in cellular and metabolic processes; COG annotation revealed that these proteins were associated with translation, ribosomal biogenesis, and post-translational modification; KEGG enrichment analysis further highlighted their involvement in metabolic pathways and the biosynthesis of secondary metabolites. Subcellular localisation prediction indicated that most candidates were cytoplasmic, with a subset predicted to localise to the cytoplasmic membrane. Based on these combined criteria, 11 proteins with potential relevance to H. pylori virulence, colonisation, or metabolic fitness were selected for STRING protein-protein interaction network analysis. A complete list of these 11 proteins, together with their mass spectrometry identification parameters, is provided in Supplementary Table 1. It should be noted that DARTS detects changes in protease sensitivity upon ligand binding; therefore, the resulting candidate list may include indirect interactors or spatially proximate proteins. Accordingly, these hits were considered preliminary candidates requiring orthogonal validation. Notably, the outer-membrane adhesins SabA and BabA were not identified among the DARTS candidates, suggesting that deoxyshikonin does not directly bind to these proteins. Protein network interaction analysis was performed using the STRING database (Figure 3A). The network revealed a high-confidence interaction between UreA and UreB. The three-dimensional structures of the corresponding proteins were retrieved from UniProt (https://www.uniprot.org/). Deoxyshikonin was docked with each target protein using AutoDockTools 1.5.6 software, and the lowest-energy binding conformation was selected for visualisation in PyMOL. Deoxyshikonin could occupy the binding pockets of UreA (Figure 3B) and UreB (Figure 3C), with binding energies of -4.9 and -6.1 kcal/mol, respectively. The docking analysis indicated that deoxyshikonin formed hydrogen bonds, van der Waals interactions, and π- Sigma/alkyl interactions within the protein binding pockets. For UreA, deoxyshikonin formed hydrogen bond interactions with Leu65, along with additional van der Waals interactions involving Gln59, Thr63, Leu64, Pro99, and Ile100. Meanwhile, the naphthoquinone ring of deoxyshikonin formed π-alkyl interactions with Arg62, Lys66, and Pro67. For UreB, a hydrogen bond was formed with Gly279, and van der Waals interactions were observed with Asn168, Gly280, His248, His274, and His322. The naphthoquinone ring of deoxyshikonin further formed π-Cation, π-π stacking, and π-Sigma interactions with Arg348, His221, and Ala169 of UreB. These contacts support the formation of stable deoxyshikonin-protein complexes within the predicted pockets. AutoDock Vina was used for semi-flexible docking, and the binding energy data are summarised in Table 1. As previously reported, an AutoDock Vina docking score lower than -7.0 kcal/mol indicates strong binding affinity, a score between -5.0 and -7.0 kcal/mol indicates favourable binding capability, while a score between -5.0 and -4.25 kcal/mol suggests detectable interaction. The docking scores suggest that deoxyshikonin can interact with both UreA and UreB, with a preferential affinity for UreB.
| UniProt accession | Target protein | PDB ID | Ligand | Binding energy (kcal/mol) | Grid center (x, y, z) | Grid size (x, y, z) |
| P69996 | UreB | 1E9Y | Deoxyshikonin | -6.1 | 127.68, 126.29, 87.37 | 20, 20, 20 |
| P14916 | UreA | 1E9Z | Deoxyshikonin | -4.9 | 146.94, 115.36, 52.21 | 20, 20, 20 |
After identifying urease as a candidate target through DARTS and molecular docking, we examined whether deoxyshikonin treatment affected urease expression and enzymatic activity. In the phenol red assay, yellow colouration inhibits urease activity (maintenance of an acidic pH), whereas red colouration indicates active urease (alkaline pH shift). Deoxyshikonin (128 μg/mL) significantly inhibited H. pylori urease activity, as evidenced by the yellow colouration (Figure 4A and B). To further assess urease involvement in the antibacterial activity of deoxyshikonin to urease, the H. pylori 26695Δcfa strain, which exhibits reduced urease expression, was used. RT-qPCR analysis showed (Figure 4C) that the expression levels of ureA and ureB were significantly reduced in this mutant. The antibacterial activity of deoxy
| Strains (amount of bacteria) | 50 μg/disk deoxyshikonin | 100 μg/disk deoxyshikonin |
| H. pylori 26695 (109 CFU/mL) | 7.225 | 9.25 |
| H. pylori 26695Δcfa (109 CFU/mL) | 7.175 | 8.4 |
| H. pylori 26695 (108 CFU/mL) | 8.275 | 10.3 |
| H. pylori 26695Δcfa (108 CFU/mL) | 8.05 | 9.3 |
Urease subunits UreA and UreB are mainly synthesised in the cytosol, with a fraction associated with the cell membrane. SabA and BabA are outer-membrane adhesins. Exploratory computational RNA-RNA interaction analysis using IntaRNA predicted potential transcript-level interactions among the mRNAs encoding these genes (Supplementary Figure 1), although these predictions require further experimental validation. To experimentally assess the effects of deoxyshikonin on these transcripts, RT-qPCR was performed. After treatment with deoxyshikonin (4 μg/mL) for 8 hours, the transcript levels of SabA, BabA, ureA, and ureB were significantly downregulated (Figure 5A). Western blotting was used to evaluate UreB protein expression in H. pylori treated with deoxyshikonin (0, 4, and 16 μg/mL). The results showed that de
| Number | Primer name | Sequence (5’-3’) |
| 1 | UreA-F | GCCAATGGTAAATTAGTT |
| 2 | UreA-R | CTCCTTAATTGTTTTTAC |
| 3 | UreB-F | TCTATCCCTACCCCACAACC |
| 4 | UreB-R | CCATCCACGAACACATGGTA |
| 5 | SabA-F | AAAGCATTCAAAACGCCAAC |
| 6 | SabA-R | CCCGCATAAAGACTCCAAAA |
| 7 | BabA-F | ATCGATCCACTTCCATCACT |
| 8 | BabA-R | GTTACGCTTTTGCCGTCTAT |
To further evaluate the relationship between urease and adhesion, adhesion of wild-type H. pylori 26695 and the low-urease-expression mutant H. pylori 26695Δcfa to BGC823 cells was compared following deoxyshikonin treatment. Fluorescence microscopy showed that under the same deoxyshikonin treatment, the green fluorescence signal, which indicates bacterial adhesion, was weaker for the Δcfa mutant than for the wild-type strain, indicating a reduced adhesion capacity (Figure 6).
To further test the functional link between urease and adhesion, exogenous urease supplementation experiments were performed (Figure 7). Wild-type H. pylori 26695, the Δcfa mutant, and S. aureus Newman, which served as a urease-negative control, were co-incubated with BGC823 cells in the presence of increasing concentrations of exogenous urease (0.25, 1, and 4 μg/mL), urea, and deoxyshikonin. BGC823 cells exhibited endogenous Rhodamine red fluorescence, whereas bacteria were labelled with SYTO9 and visualised in green. The adhesion capacity of all three strains increased with increasing concentrations of exogenous urease, including the urease-negative S. aureus, suggesting that urease may facilitate bacterial-cell interaction, at least in part, through a non-species-specific mechanism. In the presence of deoxyshikonin, adhesion was attenuated but not completely abolished at higher urease concentrations, supporting the notion that deoxyshikonin exerts its anti-adhesion effect, at least partially, by interfering with urease function. The differential adhesion observed among strains with varying endogenous urease levels further supports a functional association between urease activity and bacterial adhesion.
Although many natural products have been reported to exhibit anti-H. pylori activity, defining their specific molecular targets remains challenging. In this study, we provide evidence that deoxyshikonin may inhibit bacterial adhesion partly through interaction with urease, particularly UreB. Our previous study demonstrated that deoxyshikonin exhibits potent inhibitory activity against H. pylori, with a minimum inhibitory concentration (MIC) of 0.5-1 μg/mL[22]. Evaluation of bacterial morphology and phenotypic changes can provide a direct approach for preliminary target identification[25,26]. Adhesion is a critical initial step in H. pylori pathogenesis. Our previous work showed that deoxyshikonin inhibits H. pylori adhesion to gastric epithelial cells, prompting us to investigate adhesion-associated proteins as potential drug targets. Adhesion and colonisation by H. pylori are required for disease initiation[27,28], and multiple factors contribute to adhesion, including the adhesins SabA and BabA, flagella, pili, biofilm, and urease.
Through DARTS-based screening, we identified UreA and UreB as the primary deoxyshikonin-binding proteins. Subsequent molecular docking, RT-qPCR, western blotting, and SPR analyses collectively support urease as a key target of deoxyshikonin. UreB emerges as the most robustly validated binding partner, while UreA remains a candidate interacting subunit. Experiments using the H. pylori 26695Δcfa low-urease-expression mutant provided supportive, though not definitive, genetic evidence; the generation of a direct ureB-deletion mutant will be necessary for conclusive proof. Molecular docking identified key binding residues, including Gln59, Thr63, Leu64, Pro99, and Ile100 for UreA and Asn168, Gly280, His248, His274, and His322 for UreB. Urease is a major virulence factor of H. pylori, playing essential roles in acid neutralisation, adhesion, colonisation, and metabolism. It facilitates the hydrolysis of urea, producing ammonia and carbamate, which subsequently decomposes into an additional molecule of ammonia and carbonate. Carbonic acid then dissociates into carbon dioxide and water, while ammonia neutralises gastric acid, maintaining the peribacterial pH at approximately 6.1 and ensuring the survival of H. pylori in the stomach[29]. By binding to and functionally inhibiting the UreA and UreB subunits, deoxyshikonin disrupts acid neutralisation, thereby reducing bacterial adhesion and colonisation.
BabA and SabA are key outer-membrane adhesins of H. pylori that recognise host glycan receptors in the gastroduo
Overall, the data support a model in which urease represents a principal, though likely not the sole, molecular target of deoxyshikonin, particularly UreB, resulting in reduced urease activity, impaired local acid acclimation, a diminished capacity of H. pylori to maintain a favourable peribacterial microenvironment, and consequently decreased bacterial adhesion and colonisation. The downregulation of SabA and BabA may reflect an indirect consequence of urease inhibition, potentially mediated through regulatory networks such as ArsRS, rather than a direct pharmacological effect. The current data do not exclude the possibility that deoxyshikonin interacts with additional molecular targets beyond urease.
Several limitations should be considered. First, the mechanistic findings were obtained in vitro and require validation in animal or in vivo colonisation studies. Second, the H. pylori 26695Δcfa strain was used as a low-urease-expression model rather than as a direct urease knockout, and therefore the genetic evidence remains supportive but not definitive; the construction of a targeted ureB-deletion mutant is needed for conclusive genetic verification. Third, the RNA interaction analysis was exploratory and computational and did not experimentally prove a regulatory mechanism. Fourth, the adhesion assays in this study quantified cell-associated bacteria after washing and host-cell lysis and did not specifically differentiate surface-adherent from intracellular bacteria. Fifth, certain experiments employed deoxyshikonin at concentrations substantially exceeding the IC50 (4 μg/mL) and MIC (0.5-1 μg/mL); at such doses, contributions from non-specific cytotoxicity or growth inhibition cannot be fully excluded. Sixth, the exogenous urease supplementation assay showed that urease also augmented adhesion of the urease-negative S. aureus strain, suggesting that urease may promote bacterial-cell interactions through non-species-specific mechanisms in addition to any H. pylori-specific effects. Future studies should further validate the direct binding interface between deoxyshikonin and urease using a targeted ureB-deletion mutant; conduct dose-response mechanistic experiments within a concentration range encompassing the IC50/MIC; determine whether UreB serves as the dominant functional target in vivo; and elucidate how urease inhibition secondarily affects adhesin-related pathways during colonisation.
Deoxyshikonin inhibits H. pylori adhesion and colonisation-related phenotypes in vitro. The available evidence supports urease, particularly UreA and UreB, as the primary molecular targets of deoxyshikonin, accompanied by coordinated changes in adhesin gene expression. Further studies are needed to determine whether additional targets contribute to the overall pharmacological effect. These findings provide mechanistic support for further development of deoxyshikonin as a nontraditional anti-H. pylori lead compound.
The authors thank Professor Bi Hong-Kai of Nanjing Medical University for providing the Helicobacter pylori strains used in this study.
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