MiodesinTM Positively Modulates the Immune Response in Endometrial and Vaginal Cells

Endometriosis presents high prevalence and its physiopathology involves hyperactivation of endometrial and vaginal cells, especially by bacteria. The disease has no cure and therapies aiming to inhibit its development are highly desirable. Therefore, this study investigated whether MiodesinTM (10 µg/mL = IC80; 200 µg/mL = IC50), a natural compound constituted by Uncaria tomentosa, Endopleura uchi, and astaxanthin, could exert anti-inflammatory and anti-proliferative effects against Lipopolysaccharides (LPS) stimulation in endometrial and Candida albicans vaginal cell lines. VK2 E6/E7 (vaginal) and KLE (epithelial) cell lines were stimulated with Candida albicans (1 × 107 to 5 × 107/mL) and LPS (1 μg/mL), respectively. MiodesinTM inhibited mRNA expression for Nuclear factor kappa B (NF-κB), ciclo-oxigenase 1 (COX-1), and phospholipase A2 (PLA2), beyond the C–C motif chemokine ligand 2 (CCL2), CCL3, and CCL5 in VK2 E6/E7 cells (p < 0.05). In addition, the inhibitory effects of both doses of MiodesinTM (10 µg/mL and 200 µg/mL) resulted in reduced secretion of interleukin-1β (IL-1β), IL-6, IL-8, tumor necrosis factor α (TNF-α) (24 h, 48 h, and 72 h) and CCL2, CCL3, and CLL5 (p < 0.05) by VK2 E6/E7 cells. In the same way, COX-1 MiodesinTM inhibited LPS-induced hyperactivation of KLE cells, as demonstrated by reduced secretion of IL-1β, IL-6, IL-8, TNF-α (24 h, 48 h, and 72 h) and CCL2, CCL3, and CLL5 (p < 0.05). Furthermore, MiodesinTM also inhibited mRNA expression and secretion of matrix metalloproteinase-2 (MMP-2), MMP-9, and vascular endothelial growth factor (VEGF), which are key regulators of invasion of endometrial cells. Thus, the study concludes that MiodesinTM presents beneficial effects in the context of endometriosis, positively affecting the inflammatory and proliferative response.


Introduction
Endometriosis is estimated to affect around 10% of the female population in reproductive age, reaching 20-90% in those with pelvic pain and/or infertility. It currently has no cure, and it is not possible to predict whether it will have relapses. Many patients report cations of the stem bark of E. uchi are diverse and include anti-inflammatory, antioxidant, antibacterial, and antifungal activities [17][18][19][20]. H. pluvialis (Chlorophyceae, Volvocales) is a unicellular freshwater microalga found in temperate regions around the world and considered to be the main source of astaxanthin (3,3 -dihydroxy-β-carotene-4,4 -dione) [21]. Astaxanthin is often referred as a "super-antioxidant" molecule, because of its capacity to reduce free radicals and oxidative stress: 65 times greater than vitamin C, 54 times more than β-carotene, and 100 times more than α-tocopherol [22]. Additionally, the molecule also presents immunomodulating activity [23][24][25].
Miodesin TM has previously been shown to exert anti-inflammatory effects in reducing pelvic pain in patients with endometriosis and leiomyoma, supporting the role of naturally occurring anti-inflammatory medications in the treatment of this disease [12,26,27]. However, the underlying mechanisms involved in such beneficial effects remains unexplored.
Therefore, the present study investigated the effects of Miodesin™ on activation, proliferation, and cytotoxicity of key cells (vaginal and endometrial) involved in endometriosis and leiomyoma.

Effects of Miodesin TM on Cell Line Viability
Different concentrations of Miodesin TM were tested (0-1000 µg/mL) and the IC 50 value (200 µg/mL) and IC 80 value (10 µg/mL) were defined as the study dose in endometrial ( Figure 1A) and vaginal ( Figure 1B) cell lines.
Molecules 2022, 27, x FOR PEER REVIEW 3 of 1 unicellular freshwater microalga found in temperate regions around the world and con sidered to be the main source of astaxanthin (3,3′-dihydroxy-β-carotene-4,4′-dione) [21 Astaxanthin is often referred as a "super-antioxidant" molecule, because of its capacity t reduce free radicals and oxidative stress: 65 times greater than vitamin C, 54 times mor than β-carotene, and 100 times more than α-tocopherol [22]. Additionally, the molecul also presents immunomodulating activity [23][24][25]. Miodesin TM has previously been shown to exert anti-inflammatory effects in reducin pelvic pain in patients with endometriosis and leiomyoma, supporting the role of natu rally occurring anti-inflammatory medications in the treatment of this disease [12,26,27 However, the underlying mechanisms involved in such beneficial effects remains unex plored.
Therefore, the present study investigated the effects of Miodesin™ on activation, pro liferation, and cytotoxicity of key cells (vaginal and endometrial) involved in endometri osis and leiomyoma.

Expression of Cytokine and Inflammatory Mediators at mRNA Levels in Vaginal Cell Line (VK2 E6/E7)
We also investigated the effects of Miodesin TM (10 µg/mL and 200 µg/mL) on the mRNA expression of transcription factor, inflammatory mediators, and chemokines in VK2 E6/E7 cells. Similar results were found for both doses of Miodesin TM (Figure 2A,B, 200 µg/mL; Figure 2C,D, 10 µg/mL), resulting in reduced expression of total NF-κβ transcription factor, inflammatory mediators, and chemokines at mRNA levels. These results indicate that Miodesin TM significantly suppressed the expression of NF-κβ transcription factor and also that of enzymes that participate in the arachidonic acid cascade (inflammatory mediators), such as COX-1 and PLA2, as well as reducing chemokine expression, CCL2/MCP-1, CCL3/MIP-1α, and CCL5/RANTES.

Miodesin TM Regulates MMP-2, MMP-9, and VEGF in Endometrial Cells
To investigate whether Miodesin TM regulates MMP-2, MMP-9, and VEGF expression at transcriptional level, reverse transcription polymerase chain reaction (RT-PCR) was performed. As shown in Figure Table 3 shows the results obtained with endometrial cells (KLE) stimulated with LPS which were pre-treated with 200 µg/mL and 10 µg/mL of Miodesin TM . Miodesin TM significantly reduced the chemokine release (CCL2/MCP-1, p < 0.05; CCL3/MIP-1α, p < 0.05; CCL5/RANTES, p < 0.05). Again, such results clearly show the anti-inflammatory effects of Miodesin™ on endometrial cells, not only by inhibiting the release of pro-inflammatory cytokines, but also by inhibiting the release of chemokines.       Real-time RT-PCR was performed to measure the mRNA levels of MMP-2, MMP-9, and VEGF in KLE cells. Glyceraldehyde-3-phosphate dehydrogenase (GADPH) was used as an internal control. Data are presented as the means ± SEM of three independent experiments. (*) p < 0.05.
Endometriosis is still a disease with uncertain pathophysiology. However, the inflammatory character associated with the role of cytokines, hormones, and soluble factors seems to play an important role in its pathophysiology [28]. Indeed, the inflammatory character in endometriosis has been studied in the peritoneal fluid and serum, in addition to the eutopic and ectopic endometrium [29][30][31][32]. In this way, increased levels of TNF-α, IL-6, IL-8, IL-10, VEGF, and MCP-1/CCL2 [29,32,33], as well as different metalloproteinases (MMPs), are associated with the establishment and progression of endometriosis [34]. In fact, the present study revealed that vaginal mucosa and endometriotic cells responded to increased levels of IL-6, IL-8, IL-1β, and TNF-α when challenged by Candida albicans or
Endometriosis is still a disease with uncertain pathophysiology. However, the inflammatory character associated with the role of cytokines, hormones, and soluble factors seems to play an important role in its pathophysiology [28]. Indeed, the inflammatory character in endometriosis has been studied in the peritoneal fluid and serum, in addition to the eutopic and ectopic endometrium [29][30][31][32]. In this way, increased levels of TNF-α, IL-6, IL-8, IL-10, VEGF, and MCP-1/CCL2 [29,32,33], as well as different metalloproteinases (MMPs), are associated with the establishment and progression of endometriosis [34]. In fact, the present study revealed that vaginal mucosa and endometriotic cells responded to increased levels of IL-6, IL-8, IL-1β, and TNF-α when challenged by Candida albicans or by LPS, and these cells present a pivotal role in the inflammatory process in endometriosis and leiomyoma.
On the other hand, in 2018 and 2019, Maia et al. published the first manuscripts showing the role of Miodesin TM in reducing pelvic pain in patients with endometriosis and leiomyoma, supporting the role of naturally occurring anti-inflammatory medications in the treatment of this disease [8,26,27]. However, no analysis concerning the anti-inflammatory effects of Miodesin TM was addressed in such studies. So, the present study shows for the first time that Miodesin TM (10 µg/mL and 200 µg/mL) inhibited the hyperactivation of vaginal mucosa and endometriotic cells driving an inflammatory response, as demonstrated by reduced release of cytokines (IL-1β, IL-6, IL-8 and TNF-α; p < 0.01) and chemokines (CCL2, CCL3 and CCL5; p < 0.01)-in addition to the reduction on the expression at mRNA levels of transcription factor (NF-kβ; p < 0.01), inflammatory enzymes (COX-1, COX-2, PLA2, iNOS; p < 0.01), and chemokines (CCL2, CCL3 and CCL5; p < 0.01). Of note, the cytokines inhibited by Miodesin TM , such as IL-8 [32,35], IL-6 [36,37], IL-1β [38,39], and TNF-α [40,41] are thought to have a key role in the inflammatory process in endometriosis, becoming an interesting tool as an adjuvant for therapeutic schemes in the treatment of endometriosis. Thus, the inhibitory effects of Miodesin TM on epithelial cells in the human lower female genital tract clearly showed its anti-inflammatory potential, ensuring further randomized clinical trials.
Although inflammation plays a pivotal role in endometriosis, which is regulated by pro-inflammatory cytokines [42,43], it is important to note the modulatory capacity of Miodesin TM reducing the release of IL-8 by vaginal mucosa and endometriotic cells. IL-8 is also considered a chemokine, which is involved in the pathogenesis of endometriosis [44]. Chemokines are proteins with the ability to control mainly immune cell chemoattraction, but also centrally involved in cell proliferation, migration, and apoptosis [45]. In the present study, stimulation with C. albicans induced a significant increase in the secretion of chemokines CCL2, CCL3, and CCL5 by vaginal mucosa cells. Again, treatment with Miodesin TM resulted in significant reduction in CCL2, CCL3, and CCL5 expression at mRNA level and only for CCL2 and CCL3 at protein levels. In addition, chemokines form a complex network and stimulate other cells, such as macrophages, stromal cells, epidermal cells, and smooth muscle cells [46]. Recent studies have shown several types of chemokines produced by the endometrial implant foci, thus affecting the progression of endometriosis [46]. In this sense, both IL-8 and CCL2 are elevated in endometriotic tissues and in peritoneal implants, reinforcing the role of chemokines in the progression of ovarian and peritoneal endometriosis [47]. CCL5 is another example of a chemokine found high in the peritoneal fluid of women with endometriosis and is proportional to the stage of the disease that seems to mediate the chemotactic activity of monocytes in the peritoneal cavity, suggesting that it may contribute to the progression of this disease [48]. Thus, in this study we also evaluated the behavior in the secretion of chemokines CCL2, CCL3, and CCL5 in the KLE endometrial cell line. The results showed, once again, the ability of Miodesin TM to reverse the increase in chemokine secretion, when endometrial cells were stimulated by LPS, reinforcing the therapeutic potential of Miodesin TM in therapeutic endometriosis treatment schemes.
Furthermore, in endometrial cells, we found that the secretion and expression of the mRNA levels of the metalloprotease enzymes MMP-2 and MMP-9 and of growth factor VEGF were reduced. The results showed that treatment with Miodesin TM led to a significant reduction in the secretion of MMP-2, MMP-9, and VEGF by KLE cells, measured at protein levels. Similarly, such inhibitory effects of Miodesin TM were also seen at mRNA levels. Studies suggest that MMPs are associated with the establishment and progression of endometriosis and their levels appear to be increased in ectopic endometriotic tissues [5]. MMP-2 and MMP-9 have been extensively studied in the scope of endometriosis, mainly because they are present at high levels in the peritoneal fluid of patients with endometriosis [49]. In addition, MMP-2 and MMP-9 are found to have high levels and activity in human endometriotic tissues [50]. Downregulation of MMP-2 and MMP-9 by Miodesin TM may involve the inhibition of migration and invasion in human endometriotic cells, considering that MMPs have been implicated in this phenomenon. VEGF is one of the main growth factors in angiogenesis and since endometriosis is characterized by marked vascularization in and around ectopic tissue, VEGF-induced angiogenesis may be a critical aspect of the pathophysiology of endometriosis [46].
Finally, in addition to VEGF, chemokines and cytokines can play important roles in angiogenesis associated with endometriosis. IL-8, for example, a chemoattractant for neutrophils, is an angiogenic agent, inducing proliferation of endothelial cells [51]. TNFα, which induces the growth of new blood vessels and secreted by macrophages, also stimulates the proliferation of endometriotic stromal cells [52].

Culture and Cytotoxicity Evaluation of Miodesin by MTT Assay
Endometrial cell line KLE (epithelial) and vaginal cell line VK2 E6/E7 (epithelial) were cultured in Dulbecco's modified Eagles' medium (DMEM high glucose) supplemented with 10% v/v fetal bovine serum (FBS), 1% L-glutamine, 100 U/mL penicillin and 100 mg/mL streptomycin, and in keratinocyte serum-free medium (Gibco) supplemented with 50 µg of bovine pituitary extract, 0.1 ng of epidermal growth factor, 100 U of penicillin, and 100 µg of streptomycin, respectively and maintained at 37 • C in a humidified atmosphere of 5% CO 2 . The cells were trypsinized every 72 h using 0.01% trypsin and 1 mmol ethylenediamine tetra-acetic acid (EDTA). For all the experiments, the Miodesin™ was dissolved in the culture medium in appropriate concentrations. The cell viability of control and Miodesin TM (1-1.000 µg/mL)-treated cells were measured using a standard MTT assay. Briefly, 5 × 10 4 viable cells were seeded into clear 96-well flat-bottom plates (Corning) in RPMI medium supplemented with 10% fetal bovine serum and incubated with different concentrations of the extract for 24 h. Then, 10 µL/well of MTT (5 mg/mL) was added and the cells were incubated for 4 h. Following incubation, 100 µL of 10% sodium dodecyl sulfate solution in deionized water was added to each well and left overnight. The absorbance was measured at 595 nm in a benchtop multimode reader (Molecular Device). The established cell lines KLE and VK2 E6/E7 were kindly provided by Dr. Andre Luis Lacerda Bachi, from University Santo Amaro, São Paulo, SP, Brazil.

Candida Albicans (Stimulator Cells)
The isolate of Candida albicans was grown on Sabouraud dextrose agar (Becton Dickinson, Cockeysville, MD, USA) at 30 • C, and one colony was used to inoculate 10 mL of Phytone-peptone broth (Becton Dickinson) supplemented with 0.1% glucose. Broth cultures were grown to stationary phase for 18 h at 25 • C in a shaking water bath. The blastoconidia were collected, washed with phosphate-buffered saline, and enumerated on a hemacytometer by using trypan blue dye exclusion.

Coculture Supernatant Collection for Examination of Cytokines and Chemokines
For examination of cytokines and chemokines, epithelial cells (1 × 10 5 to 5 × 10 5 cells/mL) were cocultured with Candida albicans (1 × 10 7 to 5 × 10 7 /mL) at a ratio of 1:100 in separate wells for 0, 24, 48, and 72 h for VK2 E6/E7 cells in a total volume of 2 mL of tissue culture medium in 24-well tissue culture plates (Costar, Corning, NY, USA). Controls included VK2 E6/E7 cells (constitutive production) cultured alone in separate wells, Candida albicans cultured alone (negative control), and cells cultured with tumor necrosis factor alpha (TNFalpha; 10 to 20 ng/mL [quality control]; Pharmingen, San Diego, CA, USA) independently in tissue culture medium alone over the same 96-h period. At each time point, the VK2 E7/E6 cell-Candida coculture and the respective control cultures were aspirated from a new set of individual wells and centrifuged for 5 min at 800× g. Thereafter, the supernatants were collected and stored at −70 • C until assayed.

Reverse Transcription-Quantitative PCR (RT-qPCR)
Total RNA extracted from cells samples was converted to cDNA using a SuperScript ® III RT kit (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's protocol. The concentration of RNA was detected using a NanoDrop 2000 (Thermo Fisher Scientific, Inc., Waltham, MA, USA). GAPDH and 18 S rRNA were used as the internal control. The thermocycling conditions were as follows: 95 • C for 10 min followed by 35 cycles of 95 • C for 15 s and 55 • C for 40 s. The 2 −∆∆Cq method was used to quantify the relative gene expression levels of the target genes. Relative standard curves were generated by serial dilutions and all samples were run in triplicates. The table below indicates the sequences of primers used in qRT-PCR analysis.

Gene
Primer sequences Cell supernatant was centrifuged at 12,000× g for 15 min at 4 • C. MMP2 ELISA kits (RAB0365; Sigma, San Louis, MO, USA) and MMP9 ELISA kits (RAB0372; Sigma, Nanjing, China) were used to measure the levels of protein secretion of MMP2 and MMP9 by glioma cells, respectively, according to the manufacturer's instructions. To detect VEGF concentration in supernatants, ELISA was performed with a commercially available ELISA kit (Abcam, Cambridge, UK). The VEGF concentrations were detected according to the manufacturer's instructions.

Statistical Analysis
The unpaired Student's t test was used to analyze constitutive and Candida-specific cytokine production between primary oral and vaginal epithelial cells. Significant differences were defined as having a p value ≤ 0.05. One-way analysis of variance (ANOVA) was used for multiple comparisons. The obtained results were expressed as the mean ± standard error of mean from at least three independent experiments, unless stated otherwise.

Conclusions
Miodesin TM exerts anti-inflammatory effects against C. albicans and LPS stimulation, inhibiting vaginal mucosa and endometriotic cells hyperactivation. The results show that Miodesin TM is a promising phytotherapeutic candidate for treatment of endometriosis and leiomyoma.

Informed Consent Statement: Not applicable.
Data Availability Statement: All raw data will be available upon a reasonable justification request.