Abstract
Purpose
Vibrio vulnificus (V. Vulnificus) infection is characterized by rapid onset, aggressive progression, and challenging treatment. Bacterial resistance poses a significant challenge for clinical anti-infection treatment and is thus the subject of research. Enhancing host infection tolerance represents a novel infection prevention strategy to improve patient survival. Our team initially identified cytochrome P4501A1 (CYP1A1) as an important target owing to its negative modulation of the body's infection tolerance. This study explored the superior effects of the CYP1A1 inhibitor bergamottin compared to antibiotic combination therapy on the survival of mice infected with multidrug-resistant V. Vulnificus and the protection of their vital organs.
Methods
An increasing concentration gradient method was used to induce multidrug-resistant V. Vulnificus development. We established a lethal infection model in C57BL/6J male mice and evaluated the effect of bergamottin on mouse survival. A mild infection model was established in C57BL/6J male mice, and the serum levels of creatinine, urea nitrogen, aspartate aminotransferase, and alanine aminotransferase were determined using enzyme-linked immunosorbent assay to evaluate the effect of bergamottin on liver and kidney function. The morphological changes induced in the presence of bergamottin in mouse organs were evaluated by hematoxylin and eosin staining of liver and kidney tissues. The bacterial growth curve and organ load determination were used to evaluate whether bergamottin has a direct antibacterial effect on multidrug-resistant V. Vulnificus. Quantification of inflammatory factors in serum by enzyme-linked immunosorbent assay and the expression levels of inflammatory factors in liver and kidney tissues by real-time quantitative polymerase chain reaction were performed to evaluate the effect of bergamottin on inflammatory factor levels. Western blot analysis of IκBα, phosphorylated IκBα, p65, and phosphorylated p65 protein expression in liver and kidney tissues and in human hepatocellular carcinomas-2 and human kidney-2 cell lines was used to evaluate the effect of bergamottin on the nuclear factor kappa-B signaling pathway. One-way ANOVA and Kaplan-Meier analysis were used for statistical analysis.
Results
In mice infected with multidrug-resistant V. Vulnificus, bergamottin prolonged survival (p = 0.014), reduced the serum creatinine (p = 0.002), urea nitrogen (p = 0.030), aspartate aminotransferase (p = 0.029), and alanine aminotransferase (p = 0.003) levels, and protected the cellular morphology of liver and kidney tissues. Bergamottin inhibited interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α expression in serum (IL-1β: p = 0.010, IL-6: p = 0.029, TNF-α: p = 0.025) and inhibited the protein expression of the inflammatory factors IL-1β, IL-6, TNF-α in liver (IL-1β: p = 0.010, IL-6: p = 0.011, TNF-α: p = 0.037) and kidney (IL-1β: p = 0.016, IL-6: p = 0.011, TNF-α: p = 0.008) tissues. Bergamottin did not affect the proliferation of multidrug-resistant V. Vulnificus or the bacterial load in the mouse peritoneal lavage fluid (p = 0.225), liver (p = 0.186), or kidney (p = 0.637).
Conclusion
Bergamottin enhances the tolerance of mice to multidrug-resistant V. Vulnificus infection. This study can serve as a reference and guide the development of novel clinical treatment strategies for V. Vulnificus.
Keywords: Multidrug-resistant Vibrio vulnificus, Bergamottin, CYP1A1 inhibitors, Infection tolerance
1. Introduction
Vibrio vulnificus (V. Vulnificus) is a gram-negative bacterium commonly found in estuarine waters and is prevalent in seafood, such as molluscan shellfish, especially those that live in warm waters. With the highest case fatality rate among food-borne pathogens, reported cases of V. Vulnificus infection in humans are rising annually, accompanied by increasing rates of associated morbidity and mortality.1 In China, V. Vulnificus is found mainly in regions such as Hong Kong, Taiwan, and the southeastern coast of the mainland.2 Due to its marked negative impact on infected patients, V. Vulnificus has been categorized as one of the most dangerous bacteria.1,3 The clinical manifestations of V. Vulnificus infection include high fever, vomiting, diarrhea, shock, necrotizing fasciitis, and sepsis, leading to septic shock and multiple organ failure within a remarkably short timeframe of 48 h in 50% – 70% of patients.4 At present, third-generation cephalosporins and quinolone antibiotics are commonly employed in the clinical treatment of V. Vulnificus infection. However, the extensive misuse of antibiotics in the aquaculture industry to combat V. Vulnificus has exacerbated the issue of drug resistance in clinical infectious strains, necessitating the development of alternative clinical treatment strategies beyond antibiotic use.
Boosting the host's infection tolerance represents a novel strategy for combating infections, which is distinct from conventional methods such as antibiotics, immunomodulators, and vaccines.5 Infection tolerance refers to a host's anti-infection response to pathogens, which aims to preserving the body's homeostasis by mitigating pathological damage caused by the host's immune reaction, thereby facilitating the body's ability to withstand microbial infections.5 Improved infection tolerance is manifested by the ability to maintain pathogen levels without significantly compromising vital host organs or by markedly extending survival duration.
Our team previously reported that cytochrome P4501A1 (CYP1A1) negatively regulates the body's ability to resist infections. Knocking out the gene encoding this molecule significantly prolonged the survival of mice subjected to lethal methicillin-resistant Staphylococcus aureus challenge, although it did not reduce the bacterial load.6 Research has shown that bergamottin is a specific small molecule inhibitor of CYP1A17 that is abundant in citrus fruits such as bergamot and pomelo, as well as present in traditional Chinese medicines such as Qianghuo. Notably, it has the advantages of being inexpensive, readily available, and safe for in vivo applications.8,9 This study revealed that the combined use of gentamicin (Gen), cefotaxime (CTX) sodium (a third-generation cephalosporin antibiotic), and levofloxacin (LVX) (a quinolone antibiotic) had limited protective effects in mice during lethal infections caused by multidrug-resistant V. Vulnificus. However, the administration of bergamottin significantly prolonged the survival of the mice and mitigated damage to vital organs such as the liver and kidney. This mechanism is similar to that of bergamottin-mediated inhibition of the nuclear factor Kappa B (NF-κB) signaling pathway, which alleviates the excessive elevation of inflammatory factor levels that is correlated with infection in mice. This study confirms the potential of bergamottin for the treatment of drug-resistant V. Vulnificus infections, offering valuable insights for the development of novel anti-infective drugs and clinical treatment strategies.
2. Methods
2.1. Materials and reagents
V. Vulnificus was obtained from the Laboratory Department of Daping Hospital. The CYP1A1 inhibitors bergamottin (CAS#HY-N2194), Gen (CAS#HY-A0276A), CTX sodium (CAS#HY-A0088), and LVX (CAS#HY-B0330) were all purchased from MCE Company. The SYBR® Prime Ex Taq™ II assay kit was purchased from TAKARA Corporation. Antibodies against IκBα (CAS#4812), phosphorylated IκBα (p-IκBα) (CAS#2859), p65 (CAS#8242), phosphorylated p65 (p-p65) (CAS#3033), and β-actin (CAS#8457) and secondary antibodies were all purchased from CST (Beverly, MA, USA). Mouse interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α enzyme-linked immunosorbent assay (ELISA) kits were purchased from BOSTER Biological Technology Co. ltd. (Wuhan, China). Modified eagle medium (MEM) (CAS#PM150478) was purchased from Pricella Biotechnology Co., Ltd. (Wuhan, China). Radio-immunoprecipitation assay cell lysis buffer and protease inhibitors (CAS#P0013B), phosphate-buffered saline (CAS#C0221B), and a bicinchoninic acid assay protein assay kit (CAS#ST2222) were purchased from Beyotime (Shanghai, China). Trypsin-ethylenediaminetetraacetic acid solution (0.25%, CAS#T4049), fetal bovine serum (FBS) (CAS#12103C), dulbeccos MEM/nutrient mixture F-12 (CAS#DF-041), total RNA extraction reagent lysis solution (CAS#T9424), and dimethyl sulfoxide (CAS#67-68-5) were purchased from Sigma-Aldrich.
2.2. Animals
Male C57BL/6 mice (20 – 22 g, 8 – 10 weeks) were purchased from Sibeifu Biotechnology Co., Ltd. (Beijing, China). Feeding was performed at the Experimental Animal Center of Army Medical University (Chongqing, China). The license number of the laboratory was SYXK (Yu) 20170002. In this study, the animal experiments performed were approved by the Ethics Committee of Army Medical University (AMUWEC20211429) and were carried out in accordance with the Guidelines for Care and Use of Laboratory Animals of the National Institutes of Health.
2.3. Preparation of multidrug-resistant V. Vulnificus
The method for preparing multidrug-resistant bacteria was borrowed from the previously published work.10 First, 20 mL of Luria-Bertani liquid culture medium was added to 5 sterile centrifuge tubes (50 mL). The concentrations of Gen added to each tube were 10 μg/mL, 8 μg/mL, 6 μg/mL, 4 μg/mL, and 2 μg/mL. A total of 200 μL (1 × 109 colony forming units (CFU)/mL) of V. Vulnificus culture was inoculated into each tube and incubated at 37 °C and 200 rpm for 14 h, after which the absorbance of each bacterial culture was measured at a wavelength of 570 nm using a microplate reader. A tube of bacterial culture with a relatively high concentration of Gen and a relatively high absorbance was used to obtain a low-resistance V. Vulnificus strain. The above experiment was repeated to ultimately obtain a Gen-resistant V. Vulnificus strain. The concentration of Gen was maintained to continue induction, and a gradient concentration pressure-induced screening method was used to identify the dominant bacteria resistant to Gen and CTX sodium. The concentrations of Gen and CTX sodium were maintained to continue induction, and the dominant bacteria resistant to Gen, CTX sodium, and LVX were screened through a gradient concentration pressure-induced screen for LVX to obtain multidrug-resistant V. Vulnificus strains resistant to Gen, CTX sodium, and LVX.
2.4. Identification of multidrug-resistant V. Vulnificus
Sensitive strains of V. Vulnificus and multidrug-resistant strains of V. Vulnificus were inoculated using a plate-coating method on LB solid culture medium (60 mm × 15 mm) supplemented with or without Gen, CTX sodium, and LVX. The extent of bacterial proliferation was used to determine whether multidrug-resistant V. Vulnificus strains were resistant to Gen, CTX sodium, and LVX.
2.5. Mouse survival experiments
C57BL/6 mice were divided into a control group, an antibiotic group, and a bergamottin group using a random number table method. Twelve hours before establishing the infection model, the bergamottin group mice were intraperitoneally injected with 200 μL of bergamottin solution (25 mg/kg body weight; the solvent was 10% dimethyl sulfoxide), and the antibiotic group and control group mice were injected with an equal volume of 10% dimethyl sulfoxide. To establish the infection model, sensitive or multidrug-resistant V. Vulnificus strains were intraperitoneally injected into mice (1 × 108 CFU/mouse). After the infection model was established, the antibiotic group mice were injected with 100 μL of Gen, CTX sodium, and LVX (Gen: 16 mg/kg, CTX: 10 mg/kg, LVX: 8 mg/kg body weight; the solvent was physiological saline). The survival status of each group of mice was observed at 48 h.
2.6. Growth curve determination for multidrug-resistant V. Vulnificus
Twenty microliters of multidrug-resistant V. Vulnificus were inoculated into a centrifuge tube containing 20 mL of LB liquid medium (1:1000). Then, different concentrations (50 μM, 25 μM, 12.5 μM, 6.25 μM, and 0 μM) of bergamottin were added to the cocultured multidrug-resistant V. Vulnificus. The mixture was continuously oscillated (200 rpm) at 37 °C for 4 h (early index) or 8 h (late index). The centrifuge tubes were removed at 4 h, 8 h, 12 h, and 24 h, and the optical density (OD) was measured at a wavelength of 570 nm (OD570nm). With time as the x-axis and OD570nm as the y-axis, a growth curve of multidrug-resistant V. Vulnificus was created.
2.7. Determination of liver and kidney function in mice
The grouping and experimental methods for establishing experimental models are described in section 2.5 above, but sensitive or multidrug-resistant V. Vulnificus strains were intraperitoneally injected into mice (1 × 107 CFU/mouse). After 6 h, 1.5 mL of whole blood was collected via the orbital blood collection method, allowed to stand for 1 h, and then centrifuged (3000 rpm, 5 min) to obtain the serum. The levels of creatinine, urea nitrogen, aspartate aminotransferase, and alanine aminotransferase in the serum were detected according to the instructions of the reagent kit.
2.8. ELISA for quantifying various inflammatory markers in mouse serum
Twelve C57BL/6 mice were divided into a control group, an antibiotic group, and a bergamottin group using a random number table method. The model establishment and serum separation methods are described in section 2.7. ELISAs were used to measure the levels of secreted IL-1β, IL-6, and TNF-α in the serum.
2.9. Measurement of the in vivo bacterial load in mice
After blood collection in the experiment described in section 2.7, the mice were euthanized via cervical dislocation. After intraperitoneal lavage with 4 mL of precooled phosphate-buffered saline, the irrigation solution was collected. The liver and kidney homogenates were homogenized on ice and then diluted 100 times. After overnight cultivation at 37 °C, the bacteria were inoculated on LB solid culture medium via the plate-coating method. Images of the resultant plates were acquired, and the cells were counted.
2.10. Real-time quantitative polymerase chain reaction (RT-qPCR)
Total RNA from liver and kidney tissues was extracted using TRIzol reagent and reverse transcribed into complementary DNA. RT-qPCR was used to determine the expression levels of the inflammatory factors IL-1β, IL-6, and TNF-α in the tissues. The RT-qPCR primers used were as follows:
| IL-1β | 5′-CAACTGTTCCTGAACTCAACT-3′ |
| 3′-ATCTTTTGGGGTCCGTCAACT-5′ | |
| IL-6 | 5′-ACCACGGCCTTCCCTACTTC-3′ |
| 3′-CTCATTTCCACGATTTCCCAG-5′ | |
| TNF-α | 5′-GAGTCCGGGCAGGTCTACTTT-3′ |
| 3′-CAGGTCACTGTCCCAGCATCT-5′ | |
| β-actin | 5′-AGCCATGTACGTAGCCATCC-3′ |
| 3′-CTCTCAGCTGTGGTG-5′ |
2.11. Cell culture and treatment
The human hepatocellular carcinomas-2 (HepG2) cell culture medium consisted of MEM + 10% FBS, and the human kidney-2 (HK2) cell culture medium consisted of dulbeccos MEM/nutrient mixture F-12 + 10% FBS. When the cells reached approximately 90% confluence in the culture bottle, 0.25% trypsin was added for digestion for 2 min. After digestion was completed, fresh medium was added to the cells to terminate the digestion, the cells were transferred to a centrifuge tube, and the solution was centrifuged at 1000 rpm for 3 min. The supernatant was discarded, and the cells were resuspended in fresh medium and then evenly divided into culture bottles for culture. The cells were divided into a control group, an lipopolysaccharide group, and a bergamottin group. Bergamottin-treated HepG2 and HK2 cells were pretreated with 20 μM bergamottin and stimulated with 100 μM or 50 μM lipopolysaccharide, respectively. The cells were collected 12 h later for the experiment.
2.12. Western blotting
Mouse liver and kidney tissues were homogenized after being placed in RIPA cell lysis buffer supplemented with protease inhibitors and centrifuged at 12,000 g for 10 min at 4 °C, after which the supernatant was discarded. The protein concentrations in the cell lysates were determined using a bicinchoninic acid protein assay kit. Loading buffer was added, and the mixture was boiled at 100 °C for 10 min to denature the protein. The protein samples were subjected to 80 V sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‒PAGE) for 30 min and then subjected to 120 V SDS‒PAGE for 50 min. The proteins were subsequently transferred to polyvinylidene fluoride membranes at 260 mA for 120 min. After being blocked with 5% bovine serum albumin for 2 h, the membranes were incubated with the primary antibody overnight at 4 °C. The next day, the membranes were washed 3 times with tris buffered saline with Tween-20 solution. After incubation with the secondary antibody for 2 h, the membranes were washed 3 more times with tris buffered saline with Tween-20 solution. Immunoreactive bands were detected using an enhanced chemiluminescence detection system (Bio-Rad).
2.13. Histopathology
Liver and kidney tissues were collected and fixed with 4% paraformaldehyde after the mice were euthanized. After they were dehydrated, made transparent, treated with paraffin, and embedded, they were sectioned and stained with hematoxylin and eosin. The sections were observed and photographed under an optical microscope.
2.14. Statistical analyses
Statistical analysis was conducted using GraphPad Prism 8.0. Quantitative data are expressed as the mean ± SD. One-way ANOVA or Kaplan-Meier survival analysis was used. Differences were considered statistically significant at p < 0.05.
3. Results
3.1. Preparation of multidrug-resistant V. Vulnificus
We cultured antibiotic-sensitive V. Vulnificus in marine bacterium-specific LB medium supplemented with Gen, CTX sodium, and LVX. Multidrug-resistant V. Vulnificus was screened using a pressure-induced screening method with increasing concentration gradients as described in detail in the Method (Fig. 1A). The obtained strain was firstly authenticated by growth on V. Vulnificus identification medium (Fig. 1B). Then we conducted plate culture-based validation of the drug resistance of the obtained V. Vulnificus strains. At 48 h after coating on a composite antibiotics plate (containing 3 kinds of antibiotics: Gen, CTX sodium, and LVX), sensitive strains could not grow on the composite antibiotics plate, while multidrug-resistant strains could still grow well (Fig. 1C). The above results confirmed that we obtained multidrug-resistant V. Vulnificus through a pressure-induced screening method.
Fig. 1.
Preparation of multidrug-resistant V. Vulnificus. (A) Multidrug-resistant strains obtained by pressure-induced screening; (B) V. Vulnificus was identified by growth in selective culture medium; (C) Validation of drug resistance in V. Vulnificus.
Gen: gentamicin; CTX: cefotaxime; V. Vulnificus: Vibrio vulnificus.
3.2. Bergamottin can prolong the survival of mice infected with drug-resistant V. Vulnificus
We administered sensitive or multidrug-resistant strains of V. Vulnificus to mice via intraperitoneal injection (1 × 108 CFU/mouse), and antibiotics (Gen: 16 mg/kg body weight, CTX sodium: 10 mg/kg body weight, and LVX: 8 mg/kg body weight) or bergamottin (25 mg/kg body weight) were administered. The combination of antibiotics effectively prolonged the survival time of mice infected with sensitive strains, but there is no significant protective effect on survival when the mice were infected with multidrug-resistant V. Vulnificus (Fig. 2A). When the mice were infected with drug-resistant strains, bergamottin effectively protected them, even when combination antibiotics treatment was ineffective (Fig. 2B). The above results confirmed that bergamottin has a protective effect in mice infected with multidrug-resistant V. Vulnificus. Moreover, we also provided evidence to show the therapeutic effects of bergamottin + antibiotics on treating V. Vulnificus infection in mice. As demonstrated in Fig. 2C, when treating the mice with sensitive V. Vulnificus infection, bergamottin + antibiotics showed slightly better protective effects than the use of antibiotics or bergamottin alone, though there was no significant difference (Fig. 2C). But this suggested the different mechanisms involved in the anti-infection effects of antibiotics and bergamottin. However, as shown in Fig. 2D, bergamottin + antibiotics treatment showed similar effects with bergamottin alone on the multidrug-resistance V. Vulnificus infected mice, which might be attributed to the useless of antibiotics to treat this strain (Fig. 2D).
Fig. 2.
Bergamottin can effectively prolong the survival of multidrug-resistant V. Vulnificus infected mice. (A) The survival rate of mice infected with V. Vulnificus in the sensitive V. Vulnificus + saline, sensitive V. Vulnificus + antibiotics, multidrug-resistant V. Vulnificus + saline and multidrug-resistant V. Vulnificus + antibiotics groups. (B) The survival rate of mice infected with multidrug-resistant V. Vulnificus for in the saline, antibiotics and bergamottin groups. The survival rate of mice infected with (C) V. Vulnificus, and (D) multidrug-resistant V. Vulnificus in the saline, antibiotics, bergamottin and antibiotics + bergamottin groups. The data are presented as the mean ± SD.
∗∗p < 0.01, ∗∗∗∗p < 0.0001.
V. Vulnificus: Vibrio vulnificus.
3.3. Bergamottin reduced liver and kidney damage in mice infected with multidrug-resistant V. Vulnificus
Then, to further clarify whether bergamottin could alleviate injuries on major organs during infection, we used saline, combination antibiotics, and bergamottin to treat drug-resistant V. Vulnificus-infected mice after infection for 12 h, and collected the peripheral blood serum, liver, and kidney samples for further detection. Liver and kidney injuries were evaluated by determining the levels of creatinine, blood urea nitrogen, aspartate aminotransferase, and alanine aminotransferase in serum by ELISA as well as performing histopathology by hematoxylin-eosin staining. The results showed that the combination of antibiotics did not effectively reduce creatinine (44.78 ± 6.95 vs. 49.23 ± 9.92, p = 0.759), blood urea nitrogen (11.48 ± 0.94 vs. 14.10 ± 1.77, p = 0.332), aspartate aminotransferase (89.52 ± 13.92 vs. 95.43 ± 11.89, p = 0.893), or alanine aminotransferase (74.12 ± 34.56 vs. 128.10 ± 26.89, p = 0.062) levels, while bergamottin significantly reduced the levels of all the indicators (creatinine: 25.93 ± 7.89 vs. 49.23 ± 9.92, p = 0.002, blood urea nitrogen: 8.82 ± 2.53 vs. 14.10 ± 1.77, p = 0.030, aspartate aminotransferase: 64.05 ± 3.48 vs. 95.43 ± 11.89, p = 0.029), alanine aminotransferase: 33.18 ± 7.20 vs. 128.10 ± 26.89, p = 0.003) (Fig. 3A). The hematoxylin-eosin staining results of the liver and kidney also showed that in the absence of combined antibiotics, bergamottin significantly reduced liver and kidney damage (Fig. 3B). These results indicated the protective effects of bergamottin on relieving injuries of important organs during drug-resistant V. Vulnificus infection.
Fig. 3.
Bergamottin effectively alleviates liver and kidney damage caused by drug-resistant V. Vulnificus infection. (A) Changes in the serum creatinine, blood urea nitrogen, aspartate aminotransferase, and alanine aminotransferase levels in the control, saline, antibiotics and bergamottin groups; (B) Morphological changes in liver and kidney tissues from mice in the control, saline, antibiotics and bergamottin groups.
Scale bar = 100 μm.
The data are presented as the mean ± SD.
∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.
V. Vulnificus: Vibrio vulnificus.
3.4. Bergamottin has no direct killing effects on multidrug-resistant V. Vulnificus
We investigated whether bergamottin had a protective effect against multidrug-resistant V. Vulnificus infections in mice through direct antibacterial functions. To this end, we added gradient concentrations (0 – 50 μM) of bergamottin to the culture medium of sensitive and multidrug-resistant V. Vulnificus and constructed the growth curves, respectively. The results showed that different concentrations of bergamottin did not significantly affect the proliferation of the 2 strains (Fig. 4A). Then we detected the bacterial load in vivo and it was reflected that compared with the no treatment groups, the bergamottin treatment group did not significantly reduce the bacterial load in the abdominal lavage fluid (420750.0 ± 190002.0 vs. 703333.0 ± 86217.0, p = 0.225), liver (66140.0 ± 28619.0 vs. 107667.0 ± 29918.0, p = 0.186) or kidney (16633.0 ± 11824.0 vs. 22333.0 ± 4500.0, p = 0.637) of the multidrug-resistant V. Vulnificus-infected mice (Fig. 4B). The above results indicated that bergamottin did not have direct antibacterial effects. Based on the above evidence that bergamottin protected the multidrug-resistant V. Vulnificus -infected mice without directly cleaning the invasive pathogens, we considered this compound actually improved the tolerance of host to multidrug-resistant V. Vulnificus infection.
Fig. 4.
Bergamottin had no direct killing effect on multidrug-resistant V. Vulnificus. (A) Growth curve of multidrug-resistant V. Vulnificus strains at different concentrations of bergamottin at the early stage (left) or late stage (right) of the exponential growth. (B) Bacterial load of the peritoneal lavage fluid, liver, and kidney of mice in the saline, antibiotics and bergamottin groups.
The data are presented as the mean ± SD.
V. Vulnificus: Vibrio vulnificus; OD: optical density.
3.5. Bergamottin effectively ameliorated the excessive inflammatory response in mice infected with multidrug-resistant V. Vulnificus
During the infection process, an excessive inflammatory response leading to organ damage is an important cause of host death.11 Therefore, we tested the changes in inflammatory factor levels in the serum, liver, and kidneys of mice infected with multidrug-resistant V. Vulnificus after receiving bergamottin treatment. In the peripheral blood of infected mice, ELISA showed that bergamottin reduced the levels of the inflammatory factors IL-1β (62.26 ± 2.72 vs. 253.50 ± 108.00, p = 0.010), IL-6 (288.60 ± 32.21 vs. 485.80 ± 51.55, p = 0.029), and TNF-α (267.70 ± 111.00 vs. 620.60 ± 153.20, p = 0.025), while the combination of antibiotics did not affect their levels (IL-1β: 242.20 ± 48.24 vs. 253.50 ± 108.00, p = 0.994, IL-6: 498.20 ± 104.50 vs. 485.80 ± 51.55, p = 0.995, and TNF-α: 519.30 ± 138.30 vs. 620.60 ± 153.20, p = 0.723) (Fig. 5A). We found that the liver and kidneys were significantly protected by bergamottin (Fig. 3B). Therefore, we further determined the expression of IL-1β, IL-6 and TNF-α in the liver and kidney and it turned out that bergamottin treatment significantly reduced the mRNA levels of the 3 inflammatory factors when infected with multidrug-resistant V. Vulnificus (liver: IL-1β: 1.01 ± 0.83 vs. 7.50 ± 3.44, p = 0.010, IL-6: 4.44 ± 2.84 vs. 16.86 ± 10.44, p = 0.011, and TNF-α: 5.11 ± 1.15 vs. 9.60 ± 1.92, p = 0.037, kidney: IL-1β: 5.98 ± 0.07 vs. 10.21 ± 2.09, p = 0.016, IL-6: 17.81 ± 18.85 vs. 98.25 ± 47.15, p = 0.011, and TNF-α: 5.60 ± 4.17 vs. 27.34 ± 14.85, p = 0.008) (Fig. 5B&C). The above results indicate that bergamottin treatment can reduce the excessive inflammatory response in mice infected with multidrug-resistant V. Vulnificus.
Fig. 5.
Bergamottin effectively alleviated the excessive inflammatory response in mice infected with multidrug-resistant Vibrio vulnificus (V. Vulnificus). (A) Changes in the levels of the inflammatory cytokines IL-1β, IL-6, and TNF-α in the serum of mice in the control, saline, antibiotics, and bergamottin groups. (B) Changes in the levels of the inflammatory cytokines IL-1β, IL-6, and TNF-α in the livers of mice in the control, saline, antibiotics, and bergamottin groups. (C) Changes in the levels of the inflammatory cytokines IL-1β, IL-6, and TNF-α in the kidneys of mice in the control, saline, antibiotics, and bergamottin groups.
The data are presented as the mean ± SD.
∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.
V. Vulnificus: Vibrio vulnificus; IL: interleukin; TNF: tumor necrosis factor.
3.6. Bergamottin inhibits the inflammatory NF-κB signaling pathway
NF-κB is a classic inflammation-related signaling pathway. During the infection process, activation of the NF-κB signaling pathway promotes the secretion of various inflammatory factors, making the NF-κB signaling pathway an important target for inhibiting inflammation.12,13 Therefore, we investigated whether bergamottin inhibited the NF-κB signaling pathway in liver and kidney tissues. In our research, the levels of p-IκBα and p-p65 were significantly increased in the liver and kidney from the mice infected with multidrug-resistant V. Vulnificus. Bergamottin treatment reduced the expression levels of p-IκBα and p-p65, but antibiotics did not have any influence (Fig. 6A&B). Additionally, the same phenomenon was observed in HepG2 and HK2 cells (Fig. 6C&D). The above results indicate that bergamottin inhibited the activation of the NF-κB signaling pathway in mice infected with multidrug-resistant V. Vulnificus.
Fig. 6.
Bergamottin inhibits the inflammatory nuclear factor kappa-B signaling pathway. (A) The protein expression levels of IκBα, p-IκBα, p65, and p-p65 in the livers of mice in the control, saline, antibiotics, and bergamottin groups. (B) The protein expression levels of IκBα, P-IκBα, p65, and p-p65 in the kidneys of mice in the control, saline, antibiotics and bergamottin groups. (C) The protein expression levels of IκBα, P-IκBα, p65, and p-p65 in HepG2 cells in the control, lipopolysaccharide (100 μM) and bergamottin (20 μM) groups. (D) The protein expression levels of IκBα, P-IκBα, p65, and p-p65 in HK2 cells in the control, ipopolysaccharide (50 μM) and bergamottin (20 μM) groups.
p-IκBα: phosphorylated IκBα; p-p65: phosphorylated p65; HepG2: human hepatocellular carcinomas-2; HK2: human kidney-2.
4. Discussion
The core protective mechanism of infection tolerance is control of tissue damage, such as anti-stress, antioxidant, and anti-damage responses.14 Tolerance-related molecules uniformly promote tolerance to infection. The reported tolerance-related molecules include the ferritin heavy chain in liver parenchymal cells15, autophagy-associated protein 7 or 16L in the lungs16,17, insulin-like growth factor in white adipose tissue18, aromatic hydrocarbon receptors in splenic dendritic cells19, amphiregulin produced by natural lymphocytes 2 or regulatory T cells in the lungs20,21, nuclear factor erythroid 2-related factor 222, nicotinamide adenine dinucleotide phosphate oxidase subunit Cybb23 and leukotriene B4-I type interferon axis components24. Although some researchers have suggested employing infection tolerance strategies for sepsis treatment25, there is a lack of published reports on the application of infection tolerance regulators in clinical treatment. Our team's preliminary research revealed that CYP1A1 is a negative regulator of infection tolerance. This study aimed to explore the mechanism by which the CYP1A1 inhibitor bergamottin protects the host against drug-resistant V. Vulnificus infection. Here, we investigated the protective mechanism of CYP1A1 inhibitor bergamottin-mediated protection in host defense during multidrug-resistant V. Vulnificus infection, and we found that bergamottin inhibits the activation of the NF-κB signaling pathway and mitigates excessive inflammatory responses, thereby reducing liver and kidney tissue damage and prolonging the survival of mice.
V. Vulnificus has a wide distribution area and diverse infection pathways, and it proliferates rapidly upon invasion of the host. Due to the lack of typical infection symptoms in the early stages, susceptible individuals can quickly progress to critical illnesses such as sepsis. Current challenges in the clinical treatment of V. Vulnificus infection include (1) increasing resistance rates of V. Vulnificus due to excessive antibiotic use in aquaculture and clinical practice, as well as (2) incomplete understanding of the synergistic pathogenic mechanism of the multiple virulence factors of V. Vulnificus.26 Moreover, (3) slow progress has been made in the development of antibacterial drugs or vaccines targeting V. Vulnificus. This study revealed that bergamottin enhances host tolerance to multidrug-resistant V. Vulnificus infection. Based on the recently proposed "host-directed therapy"27 anti-infection strategy, bergamottin can prevent drug resistance issues and exhibits the advantages of stability, efficiency, and targeting. These findings offer insights into the clinical treatment of V. Vulnificus infections.
Funding
This research was supported by the National Natural Science Foundation of China (grant number: 82104247); Chongqing Talent Innovation Leading Plan (grant number: cstc2021ycjh-bgzxm0340); Chongqing Doctor Express Project (grant number: CSTB2022BSXM-JCX0024); Hainan Clinical Medical Research Center Project (grant number: LCYX202205); and Hainan Province Health Industry Research Project (grant number: 22A200082).
Ethical statement
The animal experiments performed were approved by the Ethics Committee of Army Medical University (AMUWEC20211429) and were carried out in accordance with the Guidelines for Care and Use of Laboratory Animals of the National Institutes of Health.
Declaration of competing interest
The authors declare that they have no competing interests.
Author contributions
Ruo-Bai Qiao and Wei-Hong Dai conducted most of the experiments and wrote the manuscript; they contributed equally to the study; Wei Li, Xue Yang, Dong-Mei He, Rui Gao and Yin-Qin Cui helped culture Vibrio vulnificus and perform the animal experiments; Ri-Xing Wang and Xiao-Yuan Ma helped with the statistical analyses; Fang-Jie Wang and Hua-Ping Liang designed the study, revised the manuscript and supervised the entire project.
Footnotes
Peer review under responsibility of Chinese Medical Association.
Contributor Information
Fang-Jie Wang, Email: wangfj10086@163.com.
Hua-Ping Liang, Email: 13638356728@163.com.
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