Volume 16 - 2025 | https://doi.org/10.3389/fimmu.2025.1503355
This article is part of the Research Topic Interplay of Helminths and the Female Reproductive Tract View all articles anti tnfsf13b antibody
Background: Human papillomavirus (HPV) infection is a worldwide reproductive system disease. Baofukang suppository, a traditional herbal preparation that includes curdione and borneol, has been reported to treat bacterial vaginosis (BV) and HPV infection in China. However, the therapeutic mechanism is still unknown. This study aims to explore the molecular mechanisms of curdione and borneol in treating HPV infection.
Methods: We conducted a retrospective cohort analysis of medical records from a single-center study involving 205 HPV patients, focusing on the correlation between HPV clearance and co-infection with other pathogens, confirming the efficacy of Baofukang suppository. Bioinformatics and network pharmacology approaches were employed to identify therapeutic targets of Baofukang suppository for BV/HPV co-infections. qRT-PCR, Western blot, immunofluorescence staining, and flow cytometry were utilized to validate the therapeutic targets of curdione and borneol, along with the associated immune molecular changes. Finally, the molecular mechanisms and therapeutic efficacy of curdione and borneol were confirmed in vivo using an LPS/TC-1 cervical orthotopic injection model.
Results: Curdione and borneol selectively inhibit the secretion of interleukin-6 (IL-6) and interleukin-1β (IL-1β) by macrophages. The reduction in IL-6 and IL-1β levels effectively inhibits the expression of CD274 (Programmed death ligand 1, PD-L1) in infected epithelial cells by inhibiting STAT3 phosphorylation, thereby suppressing their immune evasion capabilities. Furthermore, curdione and borneol enhance the expression of tumor necrosis factor α (TNF-α) and caspase 1 (CASP1) in macrophages, as well as the expression of interleukin 12 (IL-12) and interleukin 23 (IL-23) in dendritic cells (DCs). The expression of these inflammatory factors effectively promotes the migration and differentiation of T cells to the site of infection, completing the clearance of infected epithelial cells.
Conclusion: The main components of Baofukang suppository, curdione and borneol, inhibit the progression of HPV infection and the occurrence of cervical cancer by modulating the communication between innate and adaptive immunity, promoting the recruitment and recognition of CD8+ T cells to eliminate HPV-infected epithelial cells.
HPV infection is one of the most common sexually transmitted infections (STIs) in the world, and it primarily causes genital warts and cervical cancer (1). Although most female patients can clear HPV infection through spontaneous immunity within 6-18 months, the occurrence of mixed infections with other pathogens can lead to persistent HPV infection (2–4). The correlation between BV and HPV infection has been reported in several clinical cohort studies worldwide, and BV infection disrupts the balance of the vaginal flora thereby enhancing HPV susceptibility (3, 5–7). However, current research suggests that beyond the vaginal microbial environment, both the vaginal epithelial barrier and the mucosal immune response play pivotal roles in HPV infection (8–10).
Innate and adaptive immune responses, constituting the primary defenses against pathogens residing on the vaginal mucosal surface, are indispensable for the elimination of HPV infection (10, 11). Upon activation by pathogen-associated molecular patterns, innate immune cells secrete diverse cytokines to control the progression of HPV infection (8, 12). Adaptive immunity activates cytotoxic T-lymphocytes (CTLs) in response to antigen presentation by antigen-presenting cells (APCs) and cytokine signaling to eliminate HPV-infected epithelial cells (13). However, in contrast to other viruses, HPV does not induce epithelial cell lysis, resulting in a significant reduction in antigen presentation by macrophages and DCs (14). Furthermore, HPV has been reported to inhibit DC maturation and downregulate the expression of major histocompatibility complex (MHC) molecules through mechanisms involving interleukin-10 (IL-10), transforming growth factor-β (TGF-β), arginase 1 (Arg-1), or indoleamine 2, 3-dioxygenase (IDO-1), thereby preventing the clearance of infected cells by CTLs (15–18).
It is puzzling that while HPV achieves immune evasion by inhibiting APC cells, an overly and persistently activated immune system in co-infection with BV fails to effectively eliminate HPV (19–21). In hepatoma cells, CD274 expression is stimulated by high levels of inflammatory cytokines IL-6 and IL-1β via the STAT3 signaling pathway to enable immune evasion (22). Elevated CD274 expression and impaired T-cell function have also been reported in HPV-associated head and neck squamous cell carcinomas (HNSCCs) and cervical cancer (23–25). Similarly, increased CD274 expression is found in HPV-infected epithelial cells (26). These studies suggest that in the bacterial mixed HPV infection, the persistent HPV infection is not solely due to the damage of vaginal epithelial barrier and microbial environment, but rather involves more complex immune evasion mechanisms.
Baofukang suppository, as a traditional Chinese medicine preparation, is widely used in the clinical treatment of BV in China. In recent years, several researchers have reported that it has also demonstrated satisfactory therapeutic effects in HPV infection (27–29). Baofukang suppositories consist of natural borneol and zedoary oil, which are extracted from the branches and leaves of Cinnamomum camphora (Linn.) Presl and the rhizomes of Curcuma zedoaria (Christm.) Rosc, respectively (30). Borneol and Dl-isoborneol, the primary active components of natural borneol, exhibit diverse biological activities, including anti-inflammatory, antioxidant, anti-apoptotic, and anticoagulant properties (31). Previous literature has reported that borneol ameliorates neuroinflammation in post-stroke mice by modulating immune cell polarization (32). The major active constituents of zedoary oil are terpenes, such as epicurzerenone, curdione, and 1,8-cineole, which are being researched in cancer treatment (30, 33). Curdione has been reported to influence tumor immunity and promote tumor cell apoptosis through IDO1 (34). Although the bioactivities of the primary active components in Baofukang suppository are significant, the molecular mechanisms underlying their therapeutic effects in HPV and BV remain unknown. Furthermore, whether they exhibit synergistic effects in immune regulation warrants further investigation.
In this study, we found that Baofukang suppositories exhibit promising therapeutic effects against bacterial-mixed HPV infections. Employing systems pharmacology, we identified the key active ingredients, curdione and borneol, along with their specific targets of action. Furthermore, we validated that curdione and borneol facilitate HPV clearance by modulating the interplay between innate and adaptive immune cells. Curdione and borneol blocked the IL-6/IL-1β-STAT3-CD274 axis-mediated HPV immune escape by inhibiting excessive immune activation. Meanwhile, they promoted the secretion of adaptive immune-related cytokines by macrophages and DCs, accelerating the maturation and focal infiltration of CD3+ CD8+ T cells. This study proposes an HPV immune escape mechanism under BV/HPV co-infections and explains that curdione combined with borneol achieves HPV clearance through the crosstalk among immune cells. This study aims to establish a theoretical basis for the formal adoption of Baofukang suppositories as an efficacious therapeutic modality for HPV infection.
Curdione (C418576) and borneol (B119291) were purchased from Aladdin (China). Baofukang suppositories (Z46020058) were purchased from Bikai Pharmaceutical (China). LPS (L2880) was purchased from Sigma-Aldrich (USA). CpG (GC68897), and HPV E6 protein (GP25441) were purchased from GLPBIO (USA).
Antibodies: iNOS (22226-1-AP), TLR4 (66350-1-Ig), NF-κB p65 (10745-1-AP), CD274 (66248-1-Ig), ERK1/2 (66192-1-Ig), AKT (60203-2-IG) and STAT3 (60199-1-IG) were purchased from Proteintech (China). Antibodies: IL-6 (DF6087), p-STAT3 (AF3293), p-ERK1/2 (AF1014) and p-AKT (AF0016) were purchased from Affinity (USA). TLR9 (13674) and CD8a (98941) antibodies were purchased from CST (USA). Flow cytometry antibody: FITC-CD3 (E-AB-F1013C), APC-CD8a (E-AB-F1104E), PE-CD8a (E-AB-F1104D), APC-CD4 (E-AB-F1097E) and PE-CD4 (E-AB-F1097D) were purchased from Elabscience (China).
A retrospective cohort analysis was conducted to assess the therapeutic efficacy of Baofukang suppository in patients with bacterial mixed HPV infection. With the informed consent of the participants and ensuring their privacy, we collected the medical information and test results of female patients with HPV vaginal infection who visited the Gynecological Clinic of Wuhu Maternity and Child Healthcare Hospital in Anhui Province from June 2022 to June 2023. Patients were grouped based on their medical information, and underwent follow-up visits, with reassessments of vaginosis and HPV infection status at 3 and 5 months after the consultation. Two authors independently assessed patients for inclusion and risk of bias, extracted data and checked them for accuracy. All patients received basic treatment according to the 2021 Sexually Transmitted Infections Treatment Guidelines and were subsequently administered Baofukang suppositories in combination therapy based on their individual preferences (21). As a traditional Chinese medicine preparation, Baofukang suppository, while clinically proven beneficial for patients with HPV or BV infections, is not considered a necessary treatment option. Some patients are unwilling to undergo the treatment due to its vaginal administration route.
Excluding invalid data, a comprehensive dataset of 205 cases was successfully amassed for analysis. The valid data were categorized into two groups: a control group (n = 104) and a Baofukang suppository-treated group (n = 101), based on the administration status of Baofukang suppositories. This clinical cohort study revealed no statistically significant differences between the two groups of participants in terms of baseline characteristics and vaginal pathogenic microorganism examination indicators, including mean age, pregnancy history, delivery history, age at first pregnancy, sexual intercourse frequency, microbial diversity, microbial density, Nugent score, and Amsel (AV) criteria level (Supplementary Table 1, P > 0.05). The study protocol was approved by the Ethics Committee of Wuhu Maternal and Child Health Center (Approval Number: 20230006).
Inclusion criteria: 1. Female individuals with a history of sexual intercourse; 2. Meeting the relevant diagnostic criteria for infection with high-risk HPV types, with positive results in high-risk HPV tests; 3. No sexual intercourse, vaginal lavage, or intravaginal medication use within 3 days before the examination; 4. No receipt of any anti-HPV treatment within 4 weeks before consultation; 5. Patients with high-risk HPV positivity, undergoing colposcopy and biopsy, with pathological findings indicating inflammation or low-grade lesions; 6. Fulfilling the medication indications of this study; 7. Having signed the informed consent form.
Exclusion Criteria: 1. Presence of malignant tumors or precancerous lesions confirmed by pathological examination; 2. Existence of immune system or hematological disorders; 3. Concurrent dysfunction of other vital organs; 4. Cognitive or communication impairments; 5. Failure to adhere to medication instructions or discontinuation of treatment; 6. Pregnancy or lactation; 7. Receipt of other traditional Chinese medicine treatments within 7 days before the intervention; 8. Loss to follow-up.
Patients should administer one Baofukang suppository into the posterior fornix of the vagina after cleansing the external genitalia before bedtime, during non-menstrual periods. The administration should be done every other day, with a total of 10 administrations comprising one treatment course. Continuous treatment for three courses is recommended. During the treatment period, patients are advised to avoid bathtub baths and sexual intercourse, refrain from overwork, and ensure proper scheduling of rest and moderate exercise to enhance their immune resistance.
To evaluate the correlation between patients’ baseline characteristics (including mean age, pregnancy history, delivery history, age at first pregnancy, and sexual intercourse frequency) and indicators related to vaginosis (including microbial diversity, microbial density, Nugent level, AV level, and bacterial infection type) with HPV clearance rate, a Cox analysis is conducted. The univariate and multivariate Cox analysis was conducted by the survival coxph function of the R package.
Least Absolute Shrinkage and Selection Operator (LASSO) is a regression analysis method that performs gene selection and classification. Separate LASSO regression analyses were performed on the set of disease targets for BV and HPV vaginal infections to identify signature genes, using the R package “glmnet” (34). Characterization genes are detailed in Supplementary Table 4.
CIBERSORT can use deconvolution with a collection of 22 immune cell signature genes to derive the proportion of various immune cells from tissue RNAseq data (35). For identifying potential immune cell targets related to BV and HPV vaginal infections, immune infiltration analyses were performed on co-DEGs, utilizing the R package “CIBERSORT”.
Lung epithelial cells (TC-1) expressing mouse HPV E6/E7 proteins, macrophages (Raw 264.7) and DCs (DC 2.4) were cultured in RPMI 1640 medium (Gibco, USA) supplemented with 10% FBS (Gibco, USA) in a humidified incubator containing 5% CO2. The co-culture model was based on Transwell 12 well plates (3401, Corning, USA), where DCs were mixed with macrophages in a 1:3 ratio in the upper chamber, and TC-1 cells were cultured in the lower chamber and synchronized with splenic lymphocytes in the stimulated addition chamber. Splenic lymphocytes were added to the lower chamber at the time of administration of the stimulus. Under unspecified conditions, Baofukang suppository, curdione and Borneol were all stimulated at 100 μg/mL for 24 hours. LPS stimulation at 2 μg/mL was employed to establish a model of bacterial infection-associated inflammation. To mimic a viral infection-associated inflammatory response, cells were stimulated with 1 μg/mL CpG in combination with 10 μg/mL HPV E6 protein. In the co-culture model, 20 ng/mL of IL-6 and IL-1β were used to induce CD274 expression in TC-1 cells.
For the qRT-PCR assay, total RNA was extracted from treated cells using Trizol reagent (Invitrogen, USA). 3 μg of mRNA was reverse transcribed to cDNA and qRT-PCR was performed to detect mRNA expression of specific genes (Vazyme, China). The primer sequences are shown in Supplementary Table 5.
For the WB assays, cells were treated with RAPI lysate (Biosharp, China) containing protease and phosphatase inhibitors. The protein concentration was determined by the BCA Protein Assay Reagent (Pierce, USA). 45 μg of protein from each sample was separated by electrophoresis (10%, w/v) and transferred to the NC membranes (Amersham, USA). Membranes were blocked with 5% BSA (Biosharp, China) for 1 hour and then incubated with the corresponding antibodies overnight. After washing with TBST, they were incubated with secondary antibodies (Proteintech, USA) for 2 hours. Following another wash, the membrane was visualized by Universal Hood II (BIO-RAD, USA). The gray value of the bands was quantified using ImageJ.
Immunofluorescence staining was performed to detect the expression level and spatial distribution of specific proteins. The treated cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature. After permeabilization with 0.1% Triton X-100 for 10 minutes, cells were blocked with 3% BSA for 30 minutes. After 3 washes with PBS, the cells were incubated overnight at 4°C with the indicated primary antibody and then with the fluorescent secondary antibody (Proteintech, USA) for 1 hour at room temperature.
Gardnerella vaginalis (ATCC 14018) was obtained from the American Type Culture Collection (ATCC). Colonies were inoculated onto blood agar plates and cultured in a 5% CO2 atmosphere. Gradient dilutions were performed to ensure that Gardnerella vaginalis grew as individual colonies in the antimicrobial assay. Bacterial solutions were mixed with 100 μg/mL of Baofukang suppositories or Curdione + borneol, respectively, and then inoculated. The colonies were counted after 24-48 hours of incubation.
Female C57BL/6 mice (aged 6-8 weeks, weighing 18-20 g) were purchased from the Liaoning Changsheng Laboratory Animal Center (China) and maintained in an SPF-grade environment. Mice were anesthetized by intraperitoneal injection of 0.4ml/20g of 0.3% pentobarbital sodium. BV/HPV co-infections were mimicked by injecting 20 μL of TC-1 cell suspension (5 × 105 cells/mL) mixed with 5 μg of LPS into the cervical wall tissue of mice. After the LPS/TC-1 cervical in situ injection model had been established for 4 days, vaginal lavage treatment was administered according to the group assignments. The vaginal lavage treatment consisted of a single dose of 0.5 mL, administered once every two days, for a total of seven treatments. Animal procedures and care were guided by the Animal Research: Reporting In Vivo Experiments (ARRIVE) guidelines. All protocols related to animal experiments in this study were approved by the Ethics Committee of Wuhu Maternal and Child Health Center (Approval Number: 2024009).
To extract mesenchymal cells from the vagina and cervix, the corresponding tissues were collected and minced. The tissues were digested with 0.125% trypsin and 0.8 mg/mL collagenase I at 37°C for 1 hour. After digestion, the tissues were passed through a 200-mesh strainer to obtain a single-cell suspension, which was subsequently cultured in RPMI 1640 medium containing 10% FBS for biocompatibility experiments.
For lymphocyte extraction, the appropriate tissues were collected and crushed in a mortar. The mixture was passed through a 200-mesh strainer. Cells were then collected by centrifugation, and excess erythrocytes were removed using an erythrocyte lysate solution. The single-cell suspensions were washed with PBS to obtain a pure single-cell suspension for flow cytometry.
The treated cells were collected and stained with appropriate concentrations of fluorescently labeled antibodies for 30 minutes, protected from light, at 37°C. After washing with PBS, the stained cells were stored in 2% paraformaldehyde awaiting detection. Flow cytometry was performed by LSRFortessa X-20 (BD, USA) and the data were analyzed by FlowJo.
For H&E staining, paraffin tissue sections were first deparaffinized, and then stained with hematoxylin for 5 minutes. After washing with ddH2O, they were stained with eosin for 1 minute. After transparentization with alcohol and xylene, neutral resin was used to seal the slides.
For immunohistochemical staining, antigen retrieval was performed in citrate buffer solution (0.01 M) after deparaffinization of paraffin tissue sections. After treatment with 3% H2O2, the sections were incubated with the indicated antibodies overnight at 4°C. The following day, the sections were washed with PBS and incubated with the secondary antibodies for 30 minutes at room temperature. Subsequently, 3,3’-Diaminobenzidine (DAB) and hematoxylin were used for staining.
One-way analysis of variance (ANOVA) was used to compare data between multiple groups, while Student’s t-test was used to compare data between two groups. GraphPad Prism 8.0.2 is utilized for graphing and statistical analysis. Non-quantitative data from the clinical cohort information were compared using the nonparametric Mann-Whitney U test by SPSS 25.0. The threshold for statistical significance was set at P < 0.05.
We collected the medical information from patients diagnosed with vaginal HPV infection who visited a single center within one year from June 2022 and followed up continuously, with a total of 205 complete cases collected. Patients were categorized into control and Baofukang groups based on whether they were treated with Baofukang suppositories or not, and all patients with concurrent vaginosis were routinely treated. There were no group differences in age, sexual frequency, and number of pregnancies between the control and treatment groups (Supplementary Table 1).
We investigated the impact of HPV-negative conversion in patients receiving conventional treatment. Multivariate Cox analysis showed only the type of initial bacterial infection was an independent influencing factor in the control group (Figure 1A). Although HPV will gradually be cured over time, the type of initial bacterial infection will affect the difficulty of HPV curing (Figure 1B). Introducing the Baofukan group and conducting a multivariate Cox analysis, the results showed that both the treatment with Baofukang suppository and the type of initial bacterial infection were independent influencing factors (Figure 1C). The complexity of initial bacterial infection still affected HPV cure in patients, but Baofukang suppository was effective in promoting cure of bacterial mixed HPV infections (Figure 1D). Concurrently, Baofukang suppository also mitigated the complexity of bacterial infection (Figure 1E, F). Nevertheless, we found that at therapeutic concentrations, Baofukang suppositories exhibited no significant bactericidal impact against Gardnerella vaginalis, a common pathogen associated with bacterial vaginosis (Figure 1G, H). Baofukang suppository can effectively promote the cure of HPV/BV co-infections, but not by directly killing the mixed infection pathogens and the mechanisms remain to be further investigated.
Figure 1. Baofukang suppositories facilitate HPV-negative conversion in cases of bacterial mixed HPV infection and restore the vaginal microbial environment. (A) Multifactorial Cox analysis of a cohort of 104 HPV patients who were not given pharmacological interventions for HPV treatment (control group); (B) Analysis of additional vaginal pathogen infections versus HPV-negative conversion in the control group at the third versus fifth month after consultation (No-infection: Someone infected with HPV only; Simple-infection: Someone infected with HPV and an additional pathogen; Mixed-infection: Someone infected with HPV and multiple additional pathogens.); (C) Multifactorial Cox analysis on a cohort consisting of control group and 101 HPV patients who were administered Baofukang suppositories (Baofukang group); (D) Statistical graph of additional vaginal pathogen infections versus HPV negative conversion in the control group versus the Baofukang group at the third versus fifth month after consultation; (E) The control and Baofukang groups with additional pathogen infections in the vagina at the time of consultation, and at the third and fifth month after consultation; (F) HPV- negative conversion in the control and Baofukang groups versus time; (G) Bactericidal properties of Baofukang suppositories against Gardnerella vaginalis at no-cytotoxic concentration (cytocompatibility, Supplementary Figure 2) and (H) the quantification of colonization (n = 3). Data are shown as mean ± SD; P values were calculated using Student’s t-test.
Bacterial infections in this cohort of HPV patients were predominantly BV alone, or BV combined with aerobic vaginitis (AV) or vulvovaginal candidiasis (VVC) infections, with mixed BV infections being the most prevalent (106/161). Consequently, BV was selected as a representative case of additional pathogen infection for further investigation.