Gastrointestinal tract colonization with Candida albicans and mucosal immune response in mice treated with Artemisinin and Fluconazole orally

Abstract

The results showed that after treating <italic>C. albicans</italic> with Artemisinin (0.104 mg/ml) for 3 hours the number of <italic>C. albicans</italic> decreased and affected the morphology hyphae and growth of <italic>Candida</italic> compared with Fluconazole (150 mg/ml) and control (infected mice). The biomarker levels in the tongue showed high levels of MDA and TGFβ (623±1.5pg/ml, 586.1±0.13pg/ml respectively) in (infected mice with fungi). IL-37 was recorded high level (49.21±0.21pg/ml) in (Neoral +Fungi + artemisinin + fluconazole) compared with negative control. The finding biomarker levels in stomach showed high levels of MDA and IL-37 (533.8±1.9, 69.76±0.39pg/ml) in (Neoral + Fungi + artemisinin + fluconazole) compared with control. TGF β was recorded high level (1002±0.32pg/ml) in (Neoral + Fungi + Artemisinin) compared with negative control. The biomarker levels in intestine showed high levels of MDA and TGFβ (1149±0.34pg/ml, 1089±0.3pg/ml respectively) in (mice infected with fungi). IL-37 was recorded high level (74.14±0.14pg/ml) in (Neoral + Fungi + artemisinin + fluconazole) compared with negative control (normal mice). The biomarker levels in serum showed high levels of MDA and TGFβ (1668±0.12 pg/ml, 1629±0.05 pg/ml respectively) in (mice infected with fungi) at (P<0.05). IL-37 was recorded high level (135.1±0.8pg/ml) in (negative control). In conclusion, artemisinin has a role as antifungal and <italic>C. albicans</italic> infection causes the imbalance in immunity.

Key words
Candida albicans; Cyclosporin; MDA; L-37; TGFβ

INTRODUCTION

Candida albicans represents one of the common species of fungi from the mycobiome in humans (Belvoncikova et al. 2022), which colonize the multiple sites of the body, like the oral cavity, gastrointestinal, vaginal tract, and skin (Jassim et al. 2009, Alsharifi 2018, Othman et al. 2018, Kassid & Hamied 2022, Lopes & Lionakis 2022). Fungal infection diseases occurring by C. albicans have increased over years and also represent one of the main reasons for death in intense care among the immunocompromised individuals with HIV, and cancer after chemotherapy and radiation, transplantation organs (Hassan et al. 2019, Mahalingam et al. 2022, Saftien et al. 2023). C. albicans stimulate superficial infections in the oral cavity or skin, but they can occasion systematic infections, like candidemia (Al-Khazraji et al. 2018, Al-Barrak & Al-Kawaz 2011, Muhsen et al. 2020, Talapko et al. 2021). Oxidative stress has an important role in the inflammation of skin disease and other systematic illnesses (Cordiano et al. 2023, Semenova et al. 2024). Dysfunctional immunity response to prompts causes overproduction of proinflammatory cytokine and reactive oxidative stress, which lead to tissue harm and clinical symptoms (Bertino et al. 2020).

Oxidative stress contributes to the inter-regulation of the macroorganism and microbiome and its regulation mechanisms (Sun et al. 2024). The main mechanism of oxidative stress tissue destruction is oxidant-stimulated necrosis and apoptosis occur via an elevated permeability of mitochondria membrane and the liberation of cell-damaging factors (Filomeni et al. 2015).

Malondialdehyde is a composition derived through polyunsaturated fatty acids peroxidation. It was used as a biomarker to estimate oxidative stress in different biological specimens for patients suffering from a broad range of illnesses (Cordiano et al. 2023).

Consequently, increased levels of MDA point to increase oxidative stress during the lipid peroxidation process (Davey et al. 2005). Elevated products of lipid peroxidation are related to many human infections, such as Candida albicans and malaria (Abdel-Megeed et al. 2019, Mueangson et al. 2023).

IL-37 is a new cytokine belonging to the family of IL-1. Several research has illustrated that IL-37 has immunosuppressive impacts against innate and adaptive immunity responses by inhibition of many inflammation mediators (Dinarello et al. 2016). Therefore, IL-37 has anti-inflammatory effects in illnesses involving cancer, autoimmune, cardiovascular disease, and infectious disease. Recent studies demonstrated the role of IL-37 in immunity against bacterial, fungal, and viral infections. IL-37 prevents unsuitable immunity activation and inhibition the inflammation stimulated by the infectious factors. Consequently , IL-37 plays an important role in preventing host tissue from damage during infection via inhibition of excessive inflammation reaction (Allam et al. 2020). TGF (Transforming growth factor) β is one of the multifunctional cytokines produced by tissue and cell types. TGF-β signal transduction can induce varied cellular reactions and is important to embryonic growth, wound recovery, homeostasis tissue, and immunological homeostasis. TGF-β dysfunction has a key role in different illnesses, and many targeted treatments have been improved to correct its pathogenic actions (Deng et al. 2024).

During the process of Candida infection, the hyphal of C. albicans development is considered the most significant virulence factor (Chen et al. 2020). The hyphal of C. albicans can induce oral epithelial cell adhesion by producing adhesins, and an invasion of oral epithelial cells by the mechanical injury and the fungal toxin secreted (Naglik et al. 2017). In addition, the hyphae induce C. albicans resistance to antifungal immune by stimulating the immunity escape from the immunity cells like neutrophils and macrophages (Austermeier et al. 2020). Several studies showed natural productions have a significant source for the original antimicrobial factors resistance discovered due to their chemical structures, varied objectives, and features in averting cross-resistances (Silva et al. 2023). Artemisinin has a different other activity such as antimicrobial, antiviral, and anticancer properties. Artemisinin has a role against different parasite infections (Toxoplasma, Leishmania, and Schistosoma) (Pratama et al. 2019). Therefore, the current study aimed to investigate the effect of C. albicans in activation of immune response and the role of artemisinin as an antifungal agent.

MATERIALS AND METHODS

Approval ethics

Approval was obtained from patients, approval for ethics was obtained from Baghdad University/College of Education for Pure Science (Ibn Al-Haitham) for conducting this study. Besides, the Ministry of Health was approved to collect specimens from the hospital (Ref.30014 in 3/9/2024) and to get animals from the Iraqi Center for Genetics and Cancer Research, Al-Mustansiriya University (Ref.2480 in 7/11/2024).

Collection of specimens

One-hundred twenty oral swabs were obtained from people. They admitted to Ghazi Al-Hariri, Baghdad teaching, Al-Alawiya Maternity, Al-Imamain Al-Kadhimain, hospitals Iraq. The study was conducted from July - October 2024.

Preparation of Fluconazole and Artemisinin for in vivo experiment

Fluconazole, weighing 0.263 gm, was prepared and then dissolved in 10 ml of distilled water for mice weighing 20-25 gm, and a dose was 0.15 ml per mouse at the stock concentration of 150 ml/mg. Artemisinin was prepared with a weight of 0.104 mg/10ml and then dissolved in 10 ml of distilled water for mice weighing 20-25 gm and a dose was 0.15 ml per mouse at stock concentration 60 ml/mg.

Detection and isolation C. albicans

The saliva swabs were cultured on SDA (Sabouraud dextrose agar) plates and then incubated at (37°C/48 h). The chromogenic Candida differential agar was applied to detect Candida spp. Also, it was cultured C. albicans on Sabouraud broth at 37°C for 18 h for inoculation animals for animals experiment Further, Viteck compact 2 was applied to investigate Candida albicans (BioMerieux, France) (Salih & Khalil 2019).

Determine of fungal stock solution

Candida albicans were re-cultured on SDA agar plates and incubated at (37°C/48 h). Then C. albicans were cultured in 5 ml of PBS solution to obtain a concentration of ~1x108 CFU. One ml of (~1x108 CFU) solution was added to 9 ml PBS with shaking to obtain ~1x107 CFU. One ml of (~1x107 CFU) solution was added to 9 ml of PBS with shaking to obtain ~1x106 CFU. These solutions compared with MacFarlane’s solution.

Artemisinin inhibits the development of C. albicans hyphae

The effect of Artemisinin on C. albicans development was determined according to (Bortolus et al. 2019) by induction of hyphae growth through suspended of (~1x106) of C. albicans with a concentration of artemisinin (60mg/mL) in 200 µL (RPMI media) with 10% phosphate buffer saline then incubated (37°C/3hour). Moreover this experiment applied with a concentration of Fluconazole (150mg/ml). Also, Candida albicans was monitored at (zero, 2, and 3 hours) for Fluconazole and artemisinin. Germ tubes or hyphae formation was determined using a microscope examination.

Animals experiment in vitro

The experiment was carried out in Iraqi Center for Cancer Research and Medical Genetics / Al-Mustansiriya University according to Asfoor & Hamied (2024). Mice groups involved nine groups: Group 1 animals were as a control (without treated); Group 2 was treated by immunosuppressant Neoral (0.1mg/ml) in oral for one days; Group 3: infected with C. albicans (~1x106CFU) orally for four days; Group 4 were treated by immunosuppressant Neoral (0.1mg/ml) left for three days and infected with Candida albicans (~1x106CFU) for four days and treated with fluconazole 150mg/ml for 5 days; Group 5 were treated with Neoral (0.1mg/ml) left for three days and then infected with Candida albicans (~1x106CFU) for four days and treated with Artemisinin 60mg/ml for 5 days; Group 6 included animals with Neoral (0.1mg/ml) left for three days and then infected with Candida albicans (~1x106CFU) orally for four days, then treated with Artemisinin 60mg/ml and fluconazole 150mg/ml for 5 days. Group 7 included animals included animals infected with C. albicans (~1x106CFU) for four days and treated with fluconazole 150mg/ml for 5 days. Group 8 included infected with Candida albicans (~1x106CFU) for four days and treated with Artemisinin 60mg/ml for 5 days. Group 9 were infected with C. albicans (~1x106CFU) orally for four days, then treated with Artemisinin 60mg/ml and fluconazole 150mg/ml for 5 days.

Impact of Artemisinin, Fluconazole, and fungi infection on the fungal content for Candida and survival rate of mice

To estimate a load of fungi, oral swabs, and feces were collected from mice groups to determine the fungal content after (1,3,5,8) days after administration of immunosuppressant, fungal infection, antifungal, artemisinin. It was dissolving fecal pellets (gut content) with distilled water and 0.1ml from the supernatant was put on SDA. It was also cultured swab on SDA and incubated for 48 hours at 37°C. Candida albicans colonies were confirmed by culturing on chromogenic Candida differential agar was incubated for 24 hours/37°C. Besides, it was used salt mannitol agar to estimate the growth of Staphylococcus aureus with the presence of Candida albicans in gut content. The survival rate of mice groups was determined in the experiment period by monitoring survival mice groups.

Detection levels of MDA, IL-37, and TGF β in tongue, stomach, intestine and serum

To examine the immune response, it was measured the serum levels of IL-37, MDA, and TGF β to detect effect Artemisinin. The experiment was conducted according to (Das et al. 2023). The tongue, stomach, intestine, and serum were used to determine the levels of IL-37, MDA, and TGF β. Blood was collected by the retroorbital route to estimate IL-37, MDA, and TGF β levels. After mice groups were euthanized the stomach, intestine, and tongue were resuspended in PBS (phosphate buffer saline) to get the supernatant and then homogenized by tissues homogenate with vortex for 1 minute and centrifuged at 10000g/10 minutes to obtain supernatant. ELISA applied according to Elabscience/USA to detection levels of IL-37, MDA, and TGF β.

Statistics data analysis

Graph Pad Prism was applied to the data analysis using Tukey test in one-way ANOVA . P-value less than 0.05 considered a significances difference. The power of size sample was carried out by G power (version 3.1.9.4).

RESULTS

Power of size sample

The power of size sample was estimated at a P value of 0.05 with two-tailed, and the actual power represented 0.91 for the total size sample in G power, which was 89. The power accepted for the sample size was 0.80 (Serdar et al. 2021), which supported the current sample size of 120 for patient samples.

Candida albicans detection

One-hundred twenty samples were detected by chromogenic Candida differential agar and 34 samples were diagnosed as Candida isolates. Candida albicans appeared as a green pale color on agar (Figure 1). The Vitek2 system results exhibited a probability (97-99%) for Candida albicans isolates 12 isolates, C. glabrata 6 isolates, C. krusi 5 isolates, C. tropicalis 4 isolates, C. parapsilosis 4 isolates, C. dubliniensis 3 isolates, C. spherica 2 isolates, C. kefyer 2 isolates, C. lusitaniae 2 isolates (Figure 2).

Figure 1
Figures S1-S14.

Figure 2
Appearance of different species of candida growing on CHROMagar. Candida spp. isolated from oral swap sample after 48 h at 37°C.

Artemisinin inhibit the development of C. albicans hyphae

The results showed after treating C. albicans with Artemisinin (60mg/ml) for 3 hours the number of C. albicans decreased and affected the morphology hyphae and growth of Candida compared with Fluconazole (150mg/ml) and control (infected mice) as shown in Figure 3.

Figure 3
Number of yeast species isolated from oral which characterization with vitek-2 system. Majority of common isolates was C. albicans, and the least number was two for each C. spherica, C.kefyer, C. lusitaniae isolates.

Artemisinin and Fluconazole treatments decrease the GIT colonization of C. albicans in mice

The CFU )colony forming unit) of fungi assay was utilized to estimate the effect of fungi infection, Neoral (Immunosuppressant), fluconazole, and artemisinin on normal flora. It was observed that the fungi distribution in fecal and saliva can increase based on the immunity of host. The fecal has been appeared to have a higher colony forming unit (320 CFU/gram) for the group of mice ((Immunosuppressant( Neoral+Fungi+fluconazole) after 2 day post infection (dpi) and the lowest colony forming unit in group of mice (Fungi+fluconazole +artemisinin) (170.33 CFU/gram) after 2 dpi. Also, it was observed in the fecal a high fungal distribution after 4 dpi in mice group immunosuppressant with Neoral (286 CFU/gram) and the lowest in mice group (Fungi+fluconazole+artemisinin) (79.7 CFU/gram). Besides, the colony forming unit in fecal after 6 and 8 dpi was higher in immunosuppressant group with Neoral (279.3 and 269.3 CFU/gram respectively) and the lowest in mice group (Fungi+fluconazole +artemisinin) (75 and 57 CFU/gram respectively) as shown in Figure 4. Regarding the results of colony forming unit in the saliva was showed highest fungal distribution (14.7 CFU) in mice group ((Immunosuppressant (Neoral+Fungi+ fluconazole) after 2 day post infection (dpi) and the lowest colony forming unit in group of mice (Immunosuppressant) (1.3 CFU) after 2 dpi. Also, it was observed in the saliva a high fungal distribution after 4 dpi in mice group ((Immunosuppressant (Neoral+Fungi+ fluconazole)) (12.9 CFU) and the lowest in mice group (Immunosuppressant) (1.1 CFU/gram). Besides, the colony forming unit in saliva after 6 and 8 dpi was higher in mice group infected with fungi (22 CFU) and the lowest in mice group (Fungi+fluconazole +artemisinin) (0.3 CFU) as shown in Figure 5.

Figure 4
Artemisinin and Fluconazole inhibited the hyphal formation of C. albicans. The DIC images of the germ tube and hyphal formation of C. albicans treated by artemisinin and Fluconazole for 0, 1 and 3 h.

Figure 5
Fungal burden in the feces of mice groups. The rate of Candida albicans growth in mice’s feces were checked at different treatments groups. Mice were administered orally with 1x108 cells/ml and treated with Fluconazole and Artemisinine. Feces were collected at 2, 4, 6, 8 days post infection. IMS=Immunosuppression.

In addition, the results showed that the infection with Candida albicans in mice activates bacterial infections such as Staphylococcus aureus, as shown in Figure S1.

Survival rate in mice

To examine the immunity system of mice C. albicans was infected to mice orally for 4 days and post-treatment . In models of infection for groups , the mice survival was monitored daily as shown in Figure S2. The mice group had the lowest survival rate in (Neoral+ Fungi+ artemisinin+fluconazole). While the highest survival was registered in mice groups treated with (Neoral); (Fungi); (Neoral+Fungi+fluconazole); (Neoral+Fungi+Artemisinin); (Fungi+ fluconazole); (Fungi+artemisinin); (Fungi+fluconazole+artemisinin). These group recorded less mortality.

Detection levels of MDA, IL-37, and TGF β in tongue, stomach, intestine, and serum

The finding biomarker levels in the tongue showed high levels of MDA and TGF β (623±1.5pg/ml, 586.1±0.13pg/ml respectively) in group 3 (mice infected with fungi) at (P<0.05). Whereas IL-37 was recorded high level (49.21±0.21pg/ml) in group 6 (Neoral+Fungi+artemisinin+fluconazole) with significance differences (P<0.05) compared with negative control (normal mice). However, the lowest levels of MDA was (104.4±0.2) and TGF β was (94.4±0.04 ) in group 5 (Neoral+ Fungi+Artemisinin) at P value <0.05. The lowest level of IL-37 was (3.3±0.3) in group 2 (mice with fungi) as shown in Table I and Figures S3, S4 and S5.

Table I
Levels of MDA, IL-37 and TGF β in tongue.

Parameters	MDA	IL-37	TGF β
Group 1	231.8±0.9A	33.2±1.5A	239.1±0.12A
Group 2	354.7±2.3B	3.3±0.3B	356.3±0.2B
Group 3	623±1.5C	9.2±0.2C	586.1±0.13C
Group 4	592.4 ±1.2D	15.04±0.04D	547.2±0.2D
Group 5	104.4±0.2E	16.16±0.15E	94.4±0.04E
Group 6	196.5±3.2F	49.21±0.21F	140.1±0.09F
Group 7	126.4±3.2G	37.25±0.3G	113±0.01G
Group 8	244.2±2.1H	28.2±0.2H	227±0.047H
Group 9	142.5±1.2K	33.3±0.3A	140.1±0.09F
Different letters in the column mean a significant difference of <0.01; Similar letters in the column mean nonsignificant difference of >0.05.

• Negative control (group1); Neoral (group2); Fungi (group3); Neoral+Fungi+ fluconazole (group4); Neoral+ Fungi+ Artemisinin (group5); Neoral+Fungi+ artemisinin+ fluconazole (group6); Fungi+ fluconazole (group7); Fungi+artemisinin (group8); Fungi+fluconazole +artemisinin (group9). MDA=Malondialdehyde.

The finding biomarker levels in stomach showed high levels of MDA and IL-37 (533.8±1.9, 69.76±0.39pg/ml) in group 6 (Neoral+Fungi+artemisinin+fluconazole) at (P<0.05) comparing with control. Whereas TGF β was recorded high level (1002±0.32pg/ml) in group 5 (Neoral+Fungi+Artemisinin) with significance differences (P<0.05) compared with negative control (normal mice). However, the lowest levels of MDA and TGF β were (173.5±1, 179.3±0.28) in group 2 (mice with Neoral) at P value <0.05. While the lowest levels of IL-37 was (20.22±0.22) in group 3 (mice with fungi) as shown in Table II and Figures S6, S7, and S8.

Table II
Levels of MDA, IL-37 and TGF β in stomach.

Parameters	MDA	IL-37	TGF β
Group 1	330.5±0.3A	64.27±0.3A	327.3±0.3A
Group 2	173.5±1.8B	24.03±0.03B	179.3±0.28B
Group 3	1551±0.7C	20.22±0.22C	1519±0.26C
Group 4	974.6±2.3D	29.22±0.22D	901.1±0.09D
Group 5	1027±1.02E	26.01±0.01E	1002±0.32E
Group 6	533.8±1.9F	69.76±0.39F	579.1±0.07F
Group 7	305.1±2.5G	35.33±0.33G	325.1±0.05G
Group 8	270.2±1.2H	37.02±0.02H	246.1±0.14H
Group 9	350.6±0.3K	69.1±0.011F	354.3±0.3K
Different letters in the column mean a significant difference of <0.01; Similar letters in the column mean a nonsignificant difference of >0.05.

• Negative control (group1); Neoral (group2); Fungi (group3); Neoral+Fungi+ fluconazole (group4); Neoral+ Fungi+ Artemisinin (group5); Neoral+Fungi+ artemisinin+ fluconazole (group6); Fungi+ fluconazole (group7); Fungi+artemisinin (group8); Fungi+fluconazole +artemisinin (group9). MDA=Malondialdehyde.

The finding biomarker levels in intestine showed high levels of MDA and TGFβ (1149±0.34pg/ml, 1089±0.3pg/ml respectively) in group 3 (mice infected with fungi) at (P<0.05). Whereas IL-37 was recorded high level (74.14±0.14pg/ml) in group 6 (Neoral+Fungi+ artemisinin+fluconazole) with significance differences (P<0.05) compared with negative control (normal mice). However, the lowest levels of MDA was (108.7±0.4) in group 8 (Fungi+artemisinin) at P value <0.05. While the lowest levels of TGF β was (96.04±0.4) in group 5 (Neoral+ Fungi+Artemisinin) at P value <0.05. While the lowest levels of IL-37 was (9.041±0.04) in group 2 (mice with Neoral) as shown in Table III and Figures S9, S10, and S11.

Table III
Levels of MDA, IL-37 and TGF β in intestine.

Parameters	MDA	IL-37	TGF β
Group 1	239.7±0.4A	48.17±0.2A	274.1±0.11A
Group 2	113.8±0.43B	9.041±0.04B	119±0.034B
Group 3	1149±0.34C	9.1±0.2C	1089±0.3C
Group 4	718.7±0.34D	23.29±0.3D	659.1±0.07D
Group 5	477.8±0.4E	31.12±0.12E	488.3±0.3E
Group 6	318.0±0.6F	74.14±0.14F	305.1±0.1F
Group 7	203±0.6G	37.29±0.3G	149.1±0.09G
Group 8	108.7±0.4H	29.06±0.05H	96.04±0.4H
Group 9	296.3±0.3K	31.16±0.2E	280.3±0.3K
Different letters in the column mean a significant difference of <0.01; Similar letters in the column mean a nonsignificant difference of >0.05.

• Negative control (group1); Neoral (group2); Fungi (group3); Neoral+Fungi+ fluconazole (group4); Neoral+ Fungi+ Artemisinin (group5); Neoral+Fungi+ artemisinin+ fluconazole (group6); Fungi+ fluconazole (group7); Fungi+artemisinin (group8); Fungi+fluconazole +artemisinin (group9). MDA=Malondialdehyde.

The finding biomarker levels in serum showed high levels of MDA and TGFβ (1668±0.12 pg/ml, 1629±0.05 pg/ml respectively) in group 3 (mice infected with fungi) at (P<0.05). Whereas IL-37 was recorded high level (135.1±0.8pg/ml) in group 1 (negative normal control). However, the lowest levels MDA and TGFβ (86.11±0.11pg/ml, 69.67±0.33pg/ml respectively) in group 9 (Fungi+fluconazole+artemisinin) at (P<0.05). While the lowest levels of IL-37 was (9.041±0.04) in group 3 (mice with Neoral) as shown in Table IV and Figures S12, S13, and S14.

Table IV
Levels of MDA, IL-37 and TGF β in serum.

Parameters	MDA	IL-37	TGF β
Group 1	541.2±0.2A	135.1±0.8A	542.3±0.3A
Group 2	104.9±0.5B	26.22±0.2B	100.3±0.3B
Group 3	1668±0.12C	16.02±0.02C	1629±0.05C
Group 4	914.2±0.15D	27.20±0.019D	796.3±0.3D
Group 5	627.3±0.26E	45.24±0.24E	616.3±0.3E
Group 6	308.3±0.3F	72.22±0.2F	294.1±0.15F
Group 7	170.2±0.2G	38.22±0.23G	136±0.04G
Group 8	148.1±0.07H	29.03±0.03H	119.2±0.2H
Group 9	86.11±0.11K	123.1±0.12K	69.67±0.33K
Different letters in the column mean a significant difference of <0.01; Similar letters in the column mean nonsignificant difference of >0.05.

• Negative control (group1); Neoral (group2); Fungi (group3); Neoral+Fungi+ fluconazole (group4); Neoral+ Fungi+ Artemisinin (group5); Neoral+Fungi+ artemisinin+ fluconazole (group6); Fungi+ fluconazole (group7); Fungi+artemisinin (group8); Fungi+fluconazole +artemisinin (group9). MDA=Malondialdehyde.

DISCUSSION

C. albicans is a microorganism found in 50% of asymptomatic people. Nevertheless, oropharyngeal candidiasis (thrush) occurs by the commensal of C. albicans that attaches irreversibly and colonizes oral cavities. Many autoimmune illnesses and drugs facilitated the development and passing of C. albicans to mucosal surface (Alsalleeh 2019).

The present study showed that the infection of Candida albicans in oral can activate bacterial infection. This results agreed with other study. Van Dyck et al. (2020) showed in their study Candidalysin peptide secretion by C. albicans has play role in the activation of the immune and damage of cells, and is demand for colonization of C. albicans and later dissemination of bacteria. The physical interplay with C. albicans induced the uptake of bacteria via phagocytes, that way can be a dissemination opportunity.

According to Montelongo-Jauregui & Lopez-Ribot (2018), Candida albicans frequently co-isolates with other infections in oral cavities and is known to be associated with other commensal bacterium species. Gram-positive commensal bacteria such as S. aureus mostly grow in the nares and skin, but it is also thought to be a part of the oral microbial flora (Thomer et al. 2016). Despite having many different virulence factors, S. aureus does not invade and cannot produce systemic infections without a portal of entry (DeLeo et al. 2010, Kong et al. 2016). It is commonly known that S. aureus and Candida albicans interact synergistically, with significant clinical and therapeutic ramifications, including increased mortality. Also, it was confirmed the significance Candida proteins in adherence of Staphylococcus such as the Als3 protein in the interaction with S. aureus is demonstrated by the decreased adherence of S. aureus to Candida albicans hyphae in strains with ALS3 deletion (Peters et al. 2012).

Another study revealed the impact of immunosuppression in the oral co-infection paradigm was studied to look at a potential function for the host immune response in bacterial dispersion following C. albicans colonization. Since mice that are not immunosuppressed will not develop Oropharyngeal candidiasis, cortisone administration is essential for C. albicans colonization of the oral cavities (Solis & Filler 2012).

Concerning the results of the role of Neoral in immunosuppression. One study revealed that Cyclosporin A (NeoralTM) has a role as an immunosuppressive medicine it was used widely in mice with dose (10 - 200 mg/kg). The experiments were administrated (75 mg/kg) from Neoral to mice (BALB/cJ) via oral gavage to verruca formation in mice, to suppress immune mice for susceptible papillomavirus infection (Ticar et al. 2023).

Cyclosporine A has long suppressed response of T cell by preventing IL-2 production which induce proliferation of T cell and, therefore used as a therapeutic for autoimmunity and transplantation. Nevertheless, cyclosporine A affects innate immunity cells involving dendritic cell, macrophages, and neutrophils. Cyclosporine A can modify cytokine release and the expression of surface molecules that interact with T cells in dendritic cells, which are crucial for T cell primed. This results in a different activation of T cell responses (Liddicoat & Lavelle 2019).

Regarding the results of effect Artemisinin on Candida albicans showed the Artemisinin inhibits the development of C. albicans hyphae more than Fluconazole.

The infection by C. albicans is treated by three commonly antifungal classes: polyenes, echinocandins, and azoles. Fluconazole in azoles is applied for preventive and therapeutic aims due to its suitable characteristics like high activity, bio-availability, decreasing toxicity, and inexpensive (Lohse et al. 2020). Nevertheless, the common and periodic administration of Fluconazole in clinical treatment decreases its activity by inducing the development of resistance strains to Fluconazole (Li et al. 2019, Perfect & Ghannoum 2020). This impedance can belong to different mechanisms, involving mutations in enzyme targets, transcription agents , transporters, and stimulated efflux pump action (Perfect & Ghannoum 2020).

One study revealed the efficiency of artemisinin on Candida albicans by reducing of biofilm formation and supporting the antibiofilm effect of artemisinin through molecular reactions by interacting artemisinin with proteins that are involved in the formation of biofilm (Sumlu et al. 2024). Another study showed the effect derivatives of artemisinin in colony counts of Candida albicans with 10mg/ml for artesunate in artemisinin (Pratama et al. 2019).

The current study revealed the increased levels of biomarker included MDA, IL-37, and TFGβ in mice infected with Candida albicans and immunosuppressed with Neoral also, increased levels biomarker in mice treated with artemisinin

As is well known, membrane lipid peroxides and MDA production might result from the intracellular generation of ROS (Li et al. 2016). To look at the impacts of oxidative stress, the MDA level was also assessed. The amount of produced MDA elevated steadily in a dose-dependent way. In particular, when C. albicans was treated with Methylaervine alkaloids from natural products (Liu et al. 2024).

Artemisinin and its derivatives have the immunomodulatory effects, it was demonstrated that dihydroartemisinin, an artemisinin derivative, improves psoriatic skin inflammation and its recrudesce through selectively decreasing memory CD8+T cell. Besides the anti-inflammatory effects of artemisinin and derivatives (Qiu et al. 2021).

One study showed the impact of artemisinin as antifungal compound against Candida albicans infection by impacting on the development of hyphae (Liang et al. 2023).

One study showed the role of fungal infections such as Malassezia in increased oxidative stress by MDA level that was higher in patients. Oxidative stress has an important role in the pityriasis versicolor pathogenesis as shown by MDA presence (Sabry et al. 2022).

Another study observed an increase in some levels of cytokines (IL-12p70 and IFN gamma) in the tongue homogenate after 48 hours of inoculation because Candida albicans invade the surface of tongue and cause acute inflammation. This indicates the infected tongue by Candida was affected by neutrophil accumulation and raised levels of some cytokine (Okada et al. 2013). Macrophages and neutrophils represent important cells that are found in tissue and organs to demolish fungi by phagocytosis. Antimicrobial matters liberated by antifungal cells involving Reactive Oxygen Species by liberation of the inflammation signals such as cytokines and chemokines which have a main role in inducting immune cells in the infected places (Hamied 2021).

IL-37 is considered one of the anti-inflammatory cytokine. IL-37 absence in mice complicated studying its role in vulvovaginal candidiasis (VVC) in the mice model. IL-37 triggers a functional pathway, which potently prohibits recruitment of neutrophil in peritoneal C. albicans infections. Also, IL-37 can prevent immunopathology of VVC as anti-inflammatory cytokine (Cheng et al. 2024)

Th1 cell-produced cytokines, like TGF, were demonstrated to possess a regulatory effect on the replication of HIV and to be a strong immune system control and suppressor; consequently, excessive TGF production may lead to immunosuppression in HIV-infected patients, while activating phagocytic cells to combat candidiasis. Additionally, it has been demonstrated that this cytokine is anti-inflammatory, preventing the release of pro-inflammatory cytokines and preventing phagocytes from performing their antifungal effector functions. Also, fungal infections reduce significantly of TGF-β level than nonfungal infections (Izadi et al. 2015).

Subsequently, the studies conducted on the microbiota, their metabolites, and their interact with the pathogenic microbes colonized in intestine are significant to know the ability pathogens in pathogenicity (Kunyeit et al. 2019).

CONCLUSIONS

The present study indicated that artemisinin has a role as an antifungal for Candida albicans during the development of biofilm. Also, it was observed that the fungal load increased in oral cavity and gut during immunosuppression and decreased during treatment with Artemisinin and Fluconazole. Levels of MDA, IL-37, and TGF β increased in tissues (tongue, stomach and intestine) and serum during Candida albicans infection occurs due to an imbalance in immunity resulting from an immune response and oxidative stress.

SUPPLEMENTARY MATERIAL

Figures S1-S14.

Acknowledgements

We thank all patient participants and the staff of Ghazi Al-Hariri, Baghdad Teaching, Al-Alawiya Maternity, Al-Imamain Al-Kadhimain, hospitals for facilitating sample collection.

References

• ABDEL-MEGEED RM, KADRY MO, FAYED DB & ABDEL-HAMID AHZ. 2019. Antimicrobial activity and acetylcholinestrase inhibition of novel synthesized pyrimidine derivatives versus Candida albicans trafficking to brain and kidney. Toxicol Rep 6: 262-266.
• AL-BARRAK AY & AL-KAWAZ IA. 2011. Adherence of Candida albicans to Uroepithelial Cells. Iraq J M S 9: 4.
• AL-KHAZRAJI SI, MOHAMMED BL & AL-SALIHI SS. 2018. Synthesis and Antifungal Activity Against of Candida Species for Some New Heterocyclic Compounds Containing Schiff Base, Oxazepine, Indoline or Imidazolo Units and Their Spectral Characterization. IHJPAS (IHSCICONF) 2017: 273-293.
• ALLAM G, GABER AM, OTHMAN SI & ABDEL-MONEIM A. 2020. The potential role of interleukin-37 in infectious diseases: role of IL-37 in HIV-1, viral myocarditis, HCV, HBV, tuberculosis, leprosy, pneumonia, listeria, aspergillosis, candidiasis, and eumycetoma infection. Int Rev Immunol 39(1): 3-10.
• ALSALLEEH F. 2019. Oral Candidiasis Susceptibility of Mice Lacking Interferon Regulatory Factor 3, A Preliminary Report. Bio Med J Sc Tech Res 14(4): 1-4.
• ALSHARIFI EA. 2018. Epidemiology of vaginal candidiasis among pregnant women attending Tikrit teaching hospital/Iraq. J Fac Med Baghdad 59(4): 321-324.
• ASFOOR HM & HAMIED AS. 2024. Immune response to colonization of Candida albicans in mice treated with Cefoperazone. Cytokine 179: 156611.
• AUSTERMEIER S, KASPER L, WESTMAN J & GRESNIGHT MS. 2020. I want to break free–macrophage strategies to recognize and kill Candida albicans, and fungal counter-strategies to escape. Curr Opin Microbiol 58: 15-23.
• BELVONCIKOVA P, SPLICHALOVA P, VIDENSKA P & GARDLIK R. 2022. The human mycobiome: colonization, composition and the role in health and disease. J Fungi 8(10): 1046.
• BERTINO L, GUARNERI F, CANNAVO SP, CASCIARO M, PIOGGIA G & GANGEMI S. 2020. Oxidative stress and atopic dermatitis. Antioxidants 9(3): 196.
• BORTOLUS C, BILLAMBOZ M, CHARLET R, LECOINTE K, SENDID B, GHINET A & JAWHARA S. 2019. A small aromatic compound has antifungal properties and potential anti-inflammatory effects against intestinal inflammation. Int J Mol Sci 20(2): 321.
• CHEN H, ZHOU X, REN B & CHENG L. 2020. The regulation of hyphae growth in Candida albicans Virulence 11(1): 337-348.
• CHENG KO, MONTANO DE, ZELANTE T, DIETSCHMANN A & GRESNIGT MS. 2024. Inflammatory cytokine signalling in vulvovaginal candidiasis: a hot mess driving immunopathology. Oxf Open Immunol 5(1): iqae010.
• CORDIANO R, DI GIOACCHINO M, MANGIFESTA R, PANZERA C, GANGEMI S & MINCIULLO PL. 2023. Malondialdehyde as a potential oxidative stress marker for allergy-oriented diseases: an update. Molecules 28(16): 5979.
• DAS D, HOGENESCH H & THANGAMANI S. 2023. Intestinal colonization with Candida auris and mucosal immune response in mice treated with cefoperazone oral antibiotic. Front Immunol 14: 1123200.
• DAVEY MW, STALS E, PANIS B, KEULEMANS J & SWENNEN RL. 2005. High-throughput determination of malondialdehyde in plant tissues. Anal Biochem 347(2): 201-207.
• DELEO FR, OTTO M, KREISWIRTH BN & CHAMBERS HF. 2010. Community associated meticillin-resistant Staphylococcus aureus Lancet 375: 1557-1568.
• DENG Z ET AL. 2024. TGF-β signaling in health, disease and therapeutics. Sig Transduct Target Ther 9: 61.
• DINARELLO CA, NOLD-PETRY C, NOLD M, FUJITA M, LI S, KIM S & BUFLER P. 2016. Suppression of innate inflammation and immunity by interleukin-37. Eur J Immunol 46(5): 1067-1081.
• FILOMENI G, DE ZIO D & CECCONI F. 2015. Oxidative stress and autophagy: the clash between damage and metabolic needs. Cell Death Differ 22(3): 377-388.
• HAMIED AS. 2021. Candida and Candidiasis - A review of Virulence Factors. JOBRC 15(2): 13-20.
• HASSAN RA, AL-ABASSI HM & AL-HUSSAIN MA A. 2019. New immunological technique for diagnosis of Candida albicans in different sites. Bioch Cell Arch 19: 2.
• IZADI A, ASADIKARAM G, NAKHAEE N, HADIZADEH S & AYATOLLAHI MOUSAVI A. 2015. Plasma levels of Transforming Growth Factor Beta in HIV-1 patients with oral candidiasis. Curr Med Mycol 1(1): 22-25.
• JASSIM AN, MAJEED HA & HABIB KA. 2009. Estimation of Immunoglobulins and complements and Using Enzyme linked Immuno sorbant Assay in Identification of Vulvovaginal candidiasis. Baghdad Sc J 6(3): 427-431.
• KASSID LM & HAMIED AS. 2022. Infection frequency of Candida SP. in Iraqi patients suffering from UTI. IJHS 6(S2): 2824-2831.
• KONG EF, JOHNSON JK & JABRA-RIZK MA. 2016. Community-Associated Methicillin-Resistant Staphylococcus aureus: An Enemy amidst Us. PLoS PATHOG 12: e1005837.
• KUNYEIT L, KURREY NK, ANU-APPAIAH KA & RAO RP. 2019. Probiotic yeasts inhibit virulence of non-albicans Candida species. MBio 10(5): e02307-19.
• LI L, SUN J, XIA S, TIAN X, CHESEREK MJ & LE G. 2016. Mechanism of antifungal activity of antimicrobial peptide APP, a cell-penetrating peptide derivative, against Candida albicans: intracellular DNA binding and cell cycle arrest. Appl Microbiol Biotechnol 100(7): 3245-3253.
• LI Y, JIAO P, LI Y, GONG Y, CHEN X & SUN S. 2019. The synergistic antifungal effect and potential mechanism of D-penicillamine combined with fluconazole against Candida albicans. Front Microbiol 10: 2853.
• LIANG X, CHEN D, WANG J, LIAO B, SHEN J, YE X & ZHOU X. 2023. Artemisinins inhibit oral candidiasis caused by Candida albicans through the repression on its hyphal development. Int J Oral Sci 15(1): 40.
• LIDDICOAT AM & LAVELLE EC. 2019. Modulation of innate immunity by cyclosporine A. Biochem Pharmacol 163: 472-480.
• LIU J, WANG L, SUN Y, XIONG Y, LI R, SUI M & DAI J. 2024. Antifungal Activity and Multi-Target Mechanism of Action of Methylaervine on Candida albicans Molecules 29(18): 4303.
• LOHSE MB, GULATI M, CRAIK CS, JOHNSON AD & NOBILE CJ. 2020. Combination of antifungal drugs and protease inhibitors prevent Candida albicans biofilm formation and disrupt mature biofilms. Front Microbiol 11: 1027.
• LOPES JP & LIONAKIS MS. 2022. Pathogenesis and virulence of Candida albicans. Virulence 13(1): 89-121.
• MAHALINGAM SS, JAYARAMAN S & PANDIYAN P. 2022. Fungal colonization and infections—interactions with other human diseases. Pathogens 11(2): 212.
• MONTELONGO-JAUREGUI D & LOPEZ-RIBOT JL. 2018. Candida Interactions with the Oral Bacterial Microbiota. J Fungi (Basel) 4(4): 122.
• MUEANGSON O, MAHITTIKORN A, ANAIRE NG, MALA W & KOTEPUI M. 2023. Increased Blood Concentrations of Malondialdehyde in Plasmodium Infection: A Systematic Review and Meta-Analysis. Antioxidants 12(8): 1502.
• MUHSEN TA, HAWAR SN, MAHDI TS & KHALEEL R. 2020. Effect of Eucalyptus and Myrtus extracts identification by gas chromatography-mass spectrometry on some species of Candida as amodel of medical plants. Ann Trop Med and Public Health 23(S10): 1-11.
• NAGLIK JR, KONIG A, HUBE B & GAFFEN SL. 2017. Candida albicans–epithelial interactions and induction of mucosal innate immunity. Curr Opin Microbiol 40: 104-112.
• OKADA M, HISAJIMA T, ISHIBASHI H, MIYASAKA T, ABE S & SATOH T. 2013. Pathological analysis of the Candida albicans-infected tongue tissues of a murine oral candidiasis model in the early infection stage. Arch Oral Biol 58(4): 444-450.
• OTHMAN KI, ABDULLAH SM, ALI B & MAJID M. 2018. Isolation and Identification Candida spp from Urine and Antifungal Susceptibility Test. Iraq J Sc 59(4B): 1981-1988.
• PERFECT JR & GHANNOUM M. 2020. Emerging issues in antifungal resistance. Infect Dis Clin North Am 34(4): 921-943.
• PETERS BM ET AL. 2012. Staphylococcus aureus adherence to Candida albicans hyphae is mediated by the hyphal adhesin Als3p. Microbiology-Sgm 158: 2975-2986.
• PRATAMA MRF, TJAHJONO DH & GUSDINAR T. 2019. The antifungal activity of Artesunate toward Candida albicans: two opposite activities. Microbiology Indonesia 13(3): 5.
• QIU F, LIU J, MO X, LIU H, CHEN Y & DAI Z. 2021. Immunoregulation by Artemisinin and Its Derivatives: A New Role for Old Antimalarial Drugs. Front Immunol 12: 751772.
• SABRY HH, HAMED AM, DEL ELFALLAH AA & WAFEEK SM. 2022. Serum Levels of Total Antioxidant Capacity and Malondialdehyde in Patients with Pityriasis Versicolor. BJAS 7(3): 61-66.
• SAFTIEN A, PUSCHOF J & ELINAV E. 2023. Fungi and cancer. Gut 72(7): 1410-1425.
• SALIH S & KHALILS. 2019. The inhibitory effect of partially purified lipopolysacharide extracted from Pseudomonas aeruginosa bacteria on Candida glabrata yeast. Bioch & Cell Arch 19(2): 4207-4209.
• SEMENOVA N, GARASHCHENKO N, KOLESNIKOV S, DARENSKAYA M & KOLESNIKOVA L. 2024. Gut Microbiome Interactions with Oxidative Stress: Mechanisms and Consequences for Health. Pathophysiology 31(3): 309-330.
• SERDAR CC, CIHAN M, YUCEL D & SERDAR MA. 2021. Sample size, power and effect size revisited: simplified and practical approaches in pre-clinical, clinical and laboratory studies. Biochem Med 31(1): 010502.
• SILVA RR ET AL. 2023. DVL, lectin from Dioclea violacea seeds, has multiples mechanisms of action against Candida spp via carbohydrate recognition domain. Chem Biol Interact 382: 110639.
• SOLIS NV & FILLER SG. 2012. Mouse model of oropharyngeal candidiasis. Nat Protoc 7: 637-642.
• SUMLU E, AYDIN M, KORUCU EN, ALYAR S & NSANGOU AM. 2024. Artemisinin May Disrupt Hyphae Formation by Suppressing Biofilm-Related Genes of Candida albicans: In Vitro and In Silico Approaches. Antibiotics (Basel, Switzerland) 13(4): 310.
• SUN Y, WANG X, LI L, ZHONG C, ZHANG Y, YANG X & YANG C. 2024. The role of gut microbiota in intestinal disease: From an oxidative stress perspective. Front Micr 15: 1328324.
• TALAPKO J, JUZBASIC M, MATIJEVIC T, PUSTIJANAC E, BEKIC S, KOTRIS I & ŠKRLEC I. 2021. Candida albicans—the virulence factors and clinical manifestations of infection. J Fung 7(2): 79.
• THOMER L, SCHNEEWIND O & MISSIAKAS D. 2016. Pathogenesis of Staphylococcus aureus Bloodstream Infections. Annu Rev Pathol 11: 343-364.
• TICAR V, TSCIRLEY A, WILSON M, PLESSIS AD & HIBMA M. 2023. Case report: An adverse response to cyclosporin A treatment in BALB/cJ mice. Lab Anim 57(6): 669-675.
• VAN DYCK KK, VIELA F, MATHELIE-GUINLET M, DEMUYSER L, HAUBEN E & JABRA-RIZK MA. 2020. Adhesion of Staphylococcus aureus to Candida albicans during co-infection promotes bacterial dissemination through the host immune response. Front Cell Infect Microbiol 10: 624839.

Publication Dates

• Publication in this collection
  14 July 2025
• Date of issue
  2025

History

• Received
  21 Feb 2025
• Accepted
  18 Mar 2025