Rhein inhibits Chlamydia trachomatis infection by regulating pathogen-host cell

The global incidence of genital Chlamydia trachomatis infection increased rapidly as the primary available treatment of C. trachomatis infection being the use of antibiotics. However, the development of antibiotics resistant stain and other treatment failures are often observed in patients. Consequently, novel therapeutics are urgently required. Rhein is a monomer derivative of anthraquinone compounds with an anti-infection activity. This study investigated the effects of rhein on treating C. trachomatis infection. Rhein showed significant inhibitory effects on the growth of C. trachomatis in multiple serovars of C. trachomatis, including D, E, F and L1, and in various host cells, including HeLa, McCoy and Vero. Rhein could not directly inactivate C. trachomatis but could inhibit the growth of C. trachomatis by regulating pathogen-host cell interactions. Combined with azithromycin, the inhibitory effect of rehin was synergistic both in vitro and in vivo. Together these findings suggest that rhein could be developed for the treatment of C. trachomatis infections.


Introduction
Chlamydia trachomatis (C. trachomatis), a Gram-negative bacterial pathogen, is a causative agent of sexually transmitted infections in humans. There are an estimated 127 million new cases of C. trachomatis genital infection annually worldwide (1). C. trachomatis is classified into 15 serovars, with serovars D to K causing urinary or genital tract infections and serovars L1 to L3 being associated with lymphogranuloma venereum (2,3). The developmental cycle of C. trachomatis is unique and biphasic, featuring an infective, metabolically inactive, elementary body (EB) and a metabolically active, intracellular, reproductive reticular body (RB) (4). Many individuals infected with C. trachomatis are asymptomatic, but chlamydia . /fpubh. .
infections can have serious consequences. Untreated or recurrent chlamydial urogenital infections can lead to severe complications such as pelvic inflammatory disease and infertility (5). Genital infection with C. trachomatis also facilitates other sexually transmitted infections such as HIV (6) and human papillomavirus (7,8). Thus, the potential risk chlamydia poses to human health should not be underestimated. Currently, 1 g azithromycin (AZM) or 100 mg doxycycline twice a day for 7 days is the unanimously recommended, firstline treatment for uncomplicated C. trachomatis infections of the urogenital tract in the United States (9), China (10, 11), Europe (12), and Australia (13). Nevertheless, antibiotic resistance or treatment failure is not uncommon in C. trachomatis infections (10, 14,15). For example, AZM treatment failure has been reported in 5% to 23% of Chlamydia-positive men with non-gonococcal urethritis and women with cervicitis not at risk of reinfection (16). A partner-treatment study reported that 8% of women had persistent chlamydial infection despite stating they had no sexual contact after treatment (17). These treatment failures may be due to resistance in members of the genus C. trachomatis and persistent infection. The tet (M) gene confers resistance to tetracycline antibiotics, while mutations in the 23S rRNA gene confer resistance to macrolides (18)(19)(20). The rates of 23S rRNA gene mutations and the abundance of tet (M) in C. trachomatis were higher in a treatment-failure group than in a treatment-success group (21). Furthermore, following exposure to antimicrobial drugs at sub-inhibitory concentrations, C. trachomatis can transform into a surviving reticulate with an almost persistently quiescent metabolism, which further increases the resistance to antibiotics (22). The emergence of antibiotic resistance and treatment failure indicated the need to identify novel antichlamydial agents.
Phytochemicals have attracted attention in recent years because of their therapeutic potential against a wide variety of pathogenic microorganisms (23). Compounds extracted from biomaterials and phytochemicals include flavones (24), terpenoids (25), alkaloids (26), and essential oils (27), and many of these compounds have antimicrobial properties. In a previous study, rhubarb inhibited C. trachomatis infection (28). Rhein (4, 5-dihydroxyanthraquinone-2-carboxylic acid; Figure 1A) is a monomer primarily extracted from rhubarb (29,30). This lipophilic, naturally occurring compound has numerous pharmacological properties, including antitumor, antioxidant, anti-inflammatory, antimicrobial, hepatoprotective, and nephroprotective activities (31). As an antimicrobial, rhein has effects against Staphylococcus aureus (32, 33), Pseudomonas aeruginosa (34), Porphyromonas gingivalis (35), and influenza virus (36), among others. In the current study, the effects of rhein on C. trachomatis replication in cell culture and in mice were investigated with the aim of determining whether rhein had potential as a novel therapeutic against C. trachomatis infections.
To obtain sufficient quantity of C. trachomatis, confluent HeLa cells were infected with C. trachomatis and centrifuged for 60 min at 1,000 ×g. After centrifugation, the supernatants were replaced with 1 ml maintenance medium supplemented with 1.0 µg/ml cycloheximide (MedChemExpress, Monmouth Junction, NJ, USA). At 48 hpi, infected cells were sonicated and resuspended in sucrose-phosphate-glutamate. Stocks were divided into small aliquots and stored frozen at −70 • C.

Cytotoxicity assays with rhein
Cytotoxicity of rhein in HeLa cells was assessed using a Cell Counting Kit-8 (CCK-8) (Dojindo, Tokyo, Japan) according to the manufacturer's instructions. Briefly, HeLa cells were seeded at 1 × 10 4 cells per well in 96-well plates and incubated overnight. Cell monolayers were exposed to various concentrations of rhein (0, 5, 10, 20, 40, 80, and 160 µM) for 48 h, then the CCK-8 kit was utilized, measuring the absorbance of the cells, and the results were expressed as percent viable cells.

Immunofluorescent staining
Confluent HeLa cells were centrifuged at 1,000 ×g with C. trachomatis for 1 h and then placed at 37 • C in an incubator supplied with 5% CO 2 for 1 h. The medium was then changed to maintenance medium in the presence of 40 µM rhein or DMSO. Infected HeLa cells were cultured on cell slides for 48 h and fixed with 4% paraformaldehyde for 20 min at room

Confocal microscopy
Samples were examined under a confocal microscopy at ×200 magnification (Nikon, Tokyo, Japan) and the number of inclusions and nuclei were counted in 15 random fields from triplicate samples in each experiment. The number of inclusions and nuclei were used to assess infectivity by calculating the inclusion/nuclei percent with Nikon AR NIS 5.02.00 software. The software was also used to measure the area of inclusion bodies.

Electron microscopy
Infected HeLa cells were centrifuged at 1,000 ×g for 1 h and then placed at 37 • C in an incubator supplied with 5% CO 2 for 1 h. The medium was then changed to maintenance medium in the presence of 40 µM rhein or DMSO. At 48 hpi, cells treated with rhein or DMSO were collected, pelleted by centrifugation at 1,000 ×g for 5 min, and were then embedded and examined by transmission electron microscopy (Japan Electron Optics Laboratory, Tokyo, Japan).

Titer of infectious progeny assay
C. trachomatis-infected cells were collected and sonicated. EBs were released from the cells and used to infect fresh cell monolayers. Total inclusions were counted, and numbers of inclusion-forming units (IFU/ml) were calculated at 48 hpi.

Direct interaction assay
EBs of C. trachomatis were co-incubated with 40 µM rhein for 12, 24, 36, or 48 h at 4 • C before infection (37); DMSO was used as a positive control. C. trachomatis was washed with PBS to remove the residual rhein and was then used to infect HeLa cells in 24-well plates. At 48 hpi, cells were fixed with paraformaldehyde and observed by confocal microscopy.

Influence-on-cell assay
HeLa cells were seeded in 24-well plates at 1 × 10 5 cells/well and 40 µM rhein was added to the culture medium and incubated for 24 h. Cell monolayers were washed three times with PBS, then the pretreated cells were infected with C.
trachomatis; DMSO treatment served as a positive control. At 48 hpi, cells were stained with MOMP antibody and observed using confocal microscopy.

Influence-on-adsorption assay
HeLa cells were infected with C. trachomatis and simultaneously exposed to 40 µM rhein in the culture medium; a control group received the equivalent amount of DMSO. The culture plate was centrifuged at 1,500 ×g for 1 h and then placed at 37 • C in an incubator supplied with 5% CO 2 for 1 h. The medium containing rhein was then discarded, and cells were washed with PBS three times before addition of the maintenance medium. Immunofluorescence staining was conducted at 48 hpi.

Animals
Female BALB/c mice (4-6-week-old) were purchased from the Southern Medical University (Guangzhou, China). At 10 and 7 days before infection, all mice were injected subcutaneously with 2.5 mg medroxyprogesterone acetate (Bayunshan Pharmaceutical Company, Guangzhou, China) to synchronize estrus (38). After treatment, the mice were vaginally infected with 1 × 10 7 C. trachomatis IFU or an equal volume of sucrose-phosphate-glutamate. Experiments were conducted in the Experimental Animal Center of South China Agricultural University and in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All procedures performed in studies involving experiments on animals were approved by the Ethics Committee of South

Drug treatment in vivo
Mice were divided into five groups: negative control, positive control, rhein treatment, AZM treatment and rhein + AZM combined treatment. Rhein was dissolved in DMSO at 10 mg/ml, and AZM was dissolved in ethanol at 0.084 mg/ml. Mice were treated with 120 mg/kg rhein, 1.0 mg/kg AZM or a combination of 120 mg/kg rhein and 1.0 mg/kg AZM in 0.5% carboxymethylcellulose sodium (CMC-Na) once daily by gavage from day 4 to day 10. Control mice were gavaged with 0.5% CMC-Na. Vaginal swabs were taken for cell culture on day 4 (before gavage), day 7 and day 10 after infection, and the number of inclusions were measured.

Statistical analyses
GraphPad Prism 8 (GraphPad Software, La Jolla, CA, USA) was used to generate graphs, and statistical analyses were conducted using SPSS 15.0 (SPSS Inc., Chicago, IL, USA). Quantitative data are presented as mean ± standard deviation. The Shapiro-Wilk test was used to test the normality of quantitative data. Fisher's exact test and Bonferroni's multiple comparisons were used to assess infectivity. Kruskal-Wallis and Dunn's multiple comparisons tests were used to evaluate the area of inclusions. An unpaired t-test was used to analyze the difference in EB titer between groups. P-values were calculated using one-way ANOVA followed by Bonferroni correction for multiple comparisons. A nonparametric Wilcoxon test was used for mouse model statistics. Differences were considered significant at P <0.05 ( * ), P <0.01 ( * * ) and P <0.001 ( * * * ).

Rhein e ectively inhibited C. trachomatis replication
Cell viability was approximately 95% in samples exposed to 40 µM rhein ( Figure 1B). The anti-chlamydial effects of rhein were investigated in HeLa cells infected with the more prevalent serovars of C. trachomatis (serovars D, E and F) and the L1 serovar that can lead to venereal lymphogranuloma (39). A few aberrant RBs were observed by transmission electron microscopy in HeLa cells infected with C. trachomatis serovar D and treated with 40 µM rhein, compared with many small, mature EB particles within the inclusion of DMSO-treated cells at 48 hpi ( Figure 1C). Immunofluorescent staining revealed that the inclusion bodies became smaller and the infectivity, inclusion size and infectious progeny decreased in the presence of 40 µM rhein (Figures 1D-G). These results demonstrated that rhein effectively inhibited the growth and reproduction of different serovars of C. trachomatis in HeLa cells.
The e ect of rhein on C. trachomatis was dose-and time-dependent The effect of different concentrations of rhein (0, 5, 10, 20, 40, and 80 µM) on HeLa cells infected with C. trachomatis was examined, and the infectivity, inclusion area and infectious progeny are decreased in the presence of rhein in a dosedependent manner (Figure 2A). HeLa cells infected with C. trachomatis were also exposed to 40 µM rhein at various time points (0, 6, 12, 18, and 24 h) after infection. C. trachomatis inclusions were larger and more numerous with rhein exposure at 24 hpi compared with rhein exposure at 0 hpi ( Figure 2B). The titer of infectious progeny also increased with the delay in exposure to rhein ( Figure 2B). These findings indicated that rhein inhibited the replication of C. trachomatis in a dose-and time-dependent manner, and suggested that the earlier cells are treated with rhein, the better the inhibition of C. trachomatis.

Rhein did not directly inactivate C. trachomatis elementary bodies
Rhein and other anthraquinone drugs, including emodin and aloe-emodin, have been extracted from rhubarb. Emodin and aloe-emodin have antibacterial or virucidal activity by destroying the envelope of bacteria or viruses (40)(41)(42)(43). Rhein was previously demonstrated to directly inhibit the growth of S. aureus (33). To determine whether rhein could directly impair C. trachomatis activity, 40 µM rhein was co-incubated with C. trachomatis serovar D for 12, 24, 36, and 48 h, respectively (36, 40). The infectivity and inclusion area of C. trachomatis exposed to rhein were not significantly different from those of the corresponding DMSO control (P > 0.05; Figure 3). This suggested that rhein did not directly inactivate C. trachomatis EBs.

Rhein inhibited C. trachomatis through regulation of host cells
C. trachomatis is an obligate intracellular parasitic pathogen that needs to combine with host surface receptors to enter a cell. The pathogen then uses host cell nutrients to replicate and reproduce by regulating the interaction with the host cell (44). Rhein did not have a direct inactivation effect on C. trachomatis. To elucidate the potential inhibitory mechanism .

FIGURE
Inhibitory e ects of rhein on C. trachomatis infection were dose-and time-dependent. (A) HeLa cells were infected with C. trachomatis serovar D at multiplicity of infection (MOI) and were exposed to various concentrations of rhein ( , , , , and µM) or DMSO for h before fixation and immunostaining. (B) HeLa cells infected with C. trachomatis were exposed to rhein ( µM) at , , , , and hpi. Cells were fixed, and C.trachomatis were stained with a FITC-conjugated anti-MOMP antibody (green), while host cell nuclei were counterstained with DAPI (blue). Scale bars, µm. Data bars in the graphs represent the mean ± standard deviation. NS, not significant; *p< . , **p < . , ***p< . . of rhein, a set of influence-on-cell, influence-on-adsorption, and influence-on-post-adsorption assays were designed (37, 45) ( Figure 4A). The first two assays were used to determine whether rhein affected the adhesion and binding of EB particles to cell membranes, while the third assay was used to determine whether rhein inhibited C. trachomatis during its replication stage. The influence-on-post-adsorption assay showed a significant inhibitory effect of rhein ( Figures 4B-D  h. Images were captured at × magnification. Scale bars, µm. Data represent the mean ± standard deviation of triplicate samples. NS, not significant. *p< . , **p < . , ***p< . . primate (Vero) origin, the antibacterial activity of rhein was also exerted during the replication stage of C.trachomatis (47) (Supplementary Figure S1). These observations suggest that the inhibitory activity of rhein may not be host cell-specific and that rhein may regulate host cells and change the environment to inhibit C. trachomatis replication.

Rhein and AZM had a synergistic inhibitory e ect against C. trachomatis
AZM is a first-line drug for treating C. trachomatis infections, but treatment failure has been reported (15, 17).
Although rhein alone impaired growth of C. trachomatis, an experiment was conducted to investigate whether rhein and AZM had a synergistic suppressive effect on C. trachomatis infection. Sub-inhibitory concentrations of 20 µM rhein and 0.005 µg/ml AZM were tested. The infectivity, the area of inclusions and infectious progeny of C. trachomatis were reduced by the two individual treatments (rhein alone and AZM alone) ( Figure 5). However, a greater inhibitory effect on C. trachomatis replication was observed when rhein was combined with AZM compared with rhein alone and AZM alone ( Figure 5). Thus, the combination of rhein and AZM potentially has great clinical value.

Rhein combined with AZM inhibited C. trachomatis infection in mouse models
The in vitro experiments demonstrated that rhein effectively inhibited C. trachomatis infection, and when combined with AZM, there was a synergistic inhibitory effect. The inhibitory effect of rhein on C. trachomatis was therefore tested in vivo in a mouse model. Six-week-old female BALB/c mice were infected with C. trachomatis serovar D, then DMSO, AZM, rhein and AZM + rhein were administered orally from day 4 to day 10 post-infection. Vaginal swabs were taken on days 4, 7 and 10 for cell culture and to determine the number of infectious progenies. The number of infectious progenies in the DMSO control and rhein-treated group was not significantly different between days 4, 7, and 10 ( Figures 6A,B). However, the number of infectious progenies in the AZM-treated group decreased significantly from day 4 to day 10 ( Figure 6C), and the number of infectious progenies in the AZM + rhein treatment group decreased significantly from day 4 to day 7 ( Figure 6D). Murine tissues were examined by hematoxylin and eosin (H&E) staining on day 22 after C. trachomatis infection. Edema and hypertrophy were observed in the uterus of infected mice ( Figure 6E), but the uterine edema was relieved in the rhein and/or AZM treatment groups. There were no pathological changes in the heart, liver, spleen, or kidney of mice in any treatment group as revealed by H&E staining (Supplementary Figure S2).

Discussion
Rhein significantly inhibited C. trachomatis replication across various serovars and in HeLa, McCoy and Vero host cells. In combination with AZM, rhein exerted a synergistic suppressive effect on C. trachomatis infection, both in vitro and in vivo. In addition, rhein may regulate host cells and change the environment to inhibit C. trachomatis replication. Taken together, the findings of this study suggest that rhein may be a potential treatment for C. trachomatis infection.
Rhein was previously reported to have effective antibacterial and antiviral activity against S. aureus, Helicobacter pylori, influenza A virus, and hepatitis B virus (HBV) (33,36,46). The mechanism of action of rhein was shown to involve direct impairment of pathogens or regulation of host cell signaling pathways. Rhein increased the transcription of genes encoding the iron-regulated surface determinants system and genes involved in the ribonucleotide reductase systems of S. aureus (33). In addition, rhein exerted its antimicrobial activity against S. aureus by reducing the transcription of genes responsible for .
/fpubh. .   (49). Rhein has multiple targets and consequently regulates multiple pathways at the molecular level, including the MAPK signaling pathway, the PI3K-AKT signaling pathway, and the Wnt signaling pathway (31). Among these pathways involved in the pharmacological activity of rhein, the MAPK signaling pathway can be considered one of the most interactive pathways and rhein can regulate the Ras/Raf/MEK/ERK pathway to inhibit the phosphorylation of ERK1/2 (50, 51). The ERK pathway is considered crucial in cell proliferation and migration and RSK is an important downstream effector of the Ras/Raf/MEK/ERK signaling pathway (52, 53). Phosphorylated substrates of RSK are involved in diverse cellular processes including gene transcription, protein synthesis, cell cycle regulation, and cell survival (54,55). ERK signaling pathways are the most prominent kinase signaling network utilized by C. trachomatis and have been characterized as being instrumental in nutrient acquisition, host cell apoptosis resistance, immune responses, and even pathology associated with chlamydial infections (56)(57)(58). Moreover, our previous study suggested that ERK/RSK may be a novel target for C. trachomatis therapeutics (46). In this study, phosphorylated ERK/RSK was reduced upon exposure to rhein, suggesting that rhein may inhibit C. trachomatis infection by regulating the ERK/RSK pathway.
In the process of infectious disease treatment and drug development, host-directed therapy (HDT) is a novel strategy for treating bacterial and viral infections. Biological products or small molecules are used to interfere with replication or persistence of the pathogen by regulating host factors (59). Currently, small-molecule drugs have been proposed for the management of tuberculosis, HBV and HIV by HDT (60-62). C. trachomatis development requires host cell energy and nutrients and may therefore be a suitable pathogen for the development of HDT (63)(64)(65). The small molecule mycophenolate mofetil was recently demonstrated to effectively inhibit C. trachomatis growth by targeting the rate-limiting enzyme inosine-5 ′monophosphate dehydrogenase in the biosynthesis of guanine nucleotides in host cells (66). In addition, our research team reported that inhibitors targeting ERK/RSK had potential in the treatment of C. trachomatis infection (46). Findings from the current study indicated that rhein may regulate host cells and change the environment to inhibit C. trachomatis replication. Moreover, rhein and AZM had a synergistic inhibitory effect on C. trachomatis in vitro and in vivo. Rhein may therefore be a potential drug for a HDT strategy of managing chlamydial infections. Although rhein was demonstrated to inhibit C. trachomatis infection, the precise molecular mechanism of rhein on C. trachomatis has not yet been elucidated. Current research suggests that rhein inhibits C. trachomatis survival most likely through targeting host factors. Future work will explore the molecular mechanism by which rhein affects C. trachomatis replication.
In summary, this study provided evidence that rhein reduced C. trachomatis replication in vitro and in vivo and indicated that rhein may have potential in drug development for the treatment of C. trachomatis.

Data availability statement
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Ethics statement
The animal study was reviewed and approved by the Ethics Committee of South China Agricultural University (SCAU, Guangzhou, China and approval number: 2020c035).

Author contributions
XY and QX performed most of the experiments in this study and jointly wrote the draft manuscript. WC and ZM were responsible for the initial data analysis. XS and LM compiled figure preparation and statistical analysis. JO and YL provided experimental assistance and constructive comments to this study. HZ and YX had the leading contribution to the design of studies and interpretation of the whole dataset. All authors read and approved the manuscript.