The Effect of Rutin and Extracts of Uncaria guianensis (Aubl.) J. F. Gmeland on Primary Endometriotic Cells: A 2D and 3D Study

Abstract

There is increasing interest in the potential of natural compounds to treat diseases, such as endometriosis, a gynecological disorder that affects 10-15% of women of reproductive age, and it is related to severe pelvic pain and infertility. We have evaluated the in vitro effects of rutin and the aqueous bark, roots, and leaf extracts (ABE, ARE, and ALE, respectively) and isolated components of Uncaria guianensis on stromal cells from eutopic endometrium and lesions of patients with endometriosis. Two- and three-dimensional cultures were used to assess the cell death and production of reactive oxygen species (ROS), cytokines and growth factors of cells following exposure to these natural products. The applied treatments did not reduce cellular viability, but ROS production did increase. In addition, significant increases in the levels of interleukin (IL)-15, IL-17A, IL-4, IL-6, tumor necrosis factor-α, and vascular endothelium growth factor were observed when 2D-cells from endometrium of patients with endometriosis were treated with ABE, while exposure to ALE induced significant increases in epidermal growth factor in lesion cells.

Keywords: ROS production; anti-inflammatory; anti-oxidant; cytokines; endometriosis; growth factors; medicinal plants.

Figure 1

Chemical structures of the active principles of Uncaria guianensis extracts: (A) mitraphylline, (B) isomitraphylline, (C) quinic acid, and (D) chlorogenic acid.

Figure 2

Within-group comparisons of viabilities of stromal cells isolated from eutopic endometrium of control patients without endometriosis (EEC group), eutopic endometrium of patients with endometriosis/endometrioma (EEE group), and lesions of patients with endometriosis/endometrioma (LEE group) after treatment with rutin, aqueous bark extract (ABE), aqueous leaf extract (ALE), or aqueous root extract (ARE) from Uncaria guianensis at 1000 µg mL⁻¹, or with mitraphylline, isomitraphylline, quinic acid, or chlorogenic acid at 5 µg mL⁻¹. Assays were carried out using a 2D culture technique and the MTT method. Each bar represents mean percentage viability ± standard deviation (n = 3) in relation to untreated cells (100% viability). Data were analyzed by one-way ANOVA and Tukey test with no statistical significance set at p > 0.05.

Figure 3

Within-group comparisons of the susceptibilities of stromal spheroids isolated from eutopic endometrium of control patients without endometriosis (EEC group), eutopic endometrium of patients with endometriosis/endometrioma (EEE group), and lesions of patients with endometriosis/endometrioma (LEE group) to treatment with rutin, aqueous bark extract (ABE), aqueous leaf extract (ALE), or aqueous root extract (ARE) from Uncaria guianensis at 100 µg mL⁻¹, or with combinations of rutin + ALE, rutin + ABE, and rutin + ARE containing 100 μg mL⁻¹ of each component. Assays were carried out using a 3D cell technique and SYTOX™ Green nucleic acid stain, while untreated spheroids served as control. Each bar represents the mean fluorescence in pixels ± standard deviation (n = 3). Data were analyzed by one-way ANOVA and Tukey test with statistical significance set at p < 0.05.

Figure 4

Within-group comparisons of reactive oxygen species (ROS) production by stromal cells isolated from eutopic endometrium of control patients without endometriosis (EEC group), eutopic endometrium of patients with endometriosis/endometrioma (EEE group), and lesions of patients with endometriosis/endometrioma (LEE group) following treatment with rutin, aqueous bark extract (ABE), aqueous leaf extract (ALE), or aqueous root extract (ARE) from Uncaria guianensis at 100 µg mL⁻¹, or with combinations of rutin + ALE, rutin + ABE, and rutin + ARE containing 100 μg mL⁻¹ of each component. Assays were carried out using a 2D cell technique with dichlorodihydrofluorescein diacetate as probe, while untreated cells served as control. Each bar represents the mean number of pixels ± standard deviation (n = 3). Data were analyzed by one-way ANOVA and Tukey test with statistical significance set at p < 0.05. (* p < 0.05; **p < 0.001).

Figure 5

Fluorescent signals indicating the production of reactive oxygen species (ROS) by (A) eutopic endometrium-derived stromal cells and (B) lesion-derived stromal cells collected from an individual patient with retrocervical endometriosis (RE subgroup). Cells were treated with Rutin, aqueous bark extract (ABE), aqueous leaf extract (ALE), or aqueous root extract (ARE) from Uncaria guianensis at 100 µg mL⁻¹, or with combinations of Rutin + ALE, Rutin + ABE, and Rutin + ARE containing 100 μg mL⁻¹ of each component. Assays were carried out using a 2D cell technique with dichlorodihydrofluorescein diacetate as probe, while untreated cells served as control. Images were acquired and analyzed at 20× magnification with a Carl Zeiss Axio Vert.A1 microscope running Zen 2010 software.

Figure 6

Within-group comparisons of reactive oxygen species (ROS) production by stromal spheroids isolated from eutopic endometrium of control patients without endometriosis (EEC group), eutopic endometrium of patients with endometriosis/endometrioma (EEE group), and lesions of patients with endometriosis/endometrioma (LEE group) following treatment with rutin, aqueous bark extract (ABE), aqueous leaf extract (ALE), or aqueous root extract (ARE) from Uncaria guianensis at 100 µg mL⁻¹, or with combinations of rutin + ALE, rutin + ABE, and rutin + ARE containing 100 μg mL⁻¹ of each component. Assays were carried out using a 3D cell technique with the dichlorodihydrofluorescein diacetate as probe, while untreated spheroids served as control. Each bar represents the mean number of pixels ± standard deviation (n = 3). Data were analyzed by one-way ANOVA and Tukey test with statistical significance set at * p < 0.0001.

Figure 7

Fluorescent signals indicating the production of reactive oxygen species (ROS) by (A) eutopic endometrium-derived stromal spheroids and (B) lesion-derived stromal spheroids collected from an individual patient with retrocervical endometriosis (RE subgroup). Spheroids were treated with rutin, aqueous bark extract (ABE), aqueous leaf extract (ALE), or aqueous root extract (ARE) from Uncaria guianensis at 100 µg mL⁻¹, or with combinations of rutin + ALE, rutin + ABE, and rutin + ARE containing 100 μg mL⁻¹ of each component. Assays were carried out using a 3D cell technique with dichlorodihydrofluorescein diacetate as probe, while untreated spheroids served as control. Images were acquired and analyzed at 20× magnification with a Carl Zeiss Axio Vert.A1 microscope running Zen 2010 software.