Frankincense ameliorates endometriosis via inducing apoptosis and reducing adhesion

Abstract

Background: Frankincense, a resin derived from trees of the Boswellia genus, has been used as an incense and a type of herbal medicine for treating inflammatory diseases such arthritis, chronic bowel illness, and asthma. While endometriosis is a well-known inflammatory gynecological illness caused by the ectopic attachment and development of uterine tissue over the menstrual cycle, the impact of frankincense on this illness is poorly understood. The purpose of this study was to explore the effects of frankincense on endometriosis.

Methods: We used a network pharmacological assessment, in vitro and in vivo investigations with a human endometriotic cell line as well as a syngeneic uterine transfer mouse model. High-performance liquid chromatographic analysis was used to compare water-extracted frankincense (Fr) to its reference compounds and validate the sample.

Results: A network pharmacological analysis suggested a positive effect of Fr on endometriosis. Fr relieved endometriosis by reducing ectopic endometrial adherence and development, according to both in vivo and in vitro models. We suggested that the ER stress/p53-apoptosis and chemokine-migration/adhesion pathways underlie Fr's anti-endometriotic action using RNA sequencing and bioinformatic analysis.

Conclusion: This study revealed the potential effect of Fr on endometriosis using an experimental investigation. Fr may have the potential to be an effective and safe treatment for endometriosis.

Keywords: Adhesion; Apoptosis; Endometriosis; Frankincense.

Fig. 1

Fr was validated by high performance liquid chromatography. Fr was subjected to 20 min of ultrasound sonication before being passed through a 0.45 μm syringe filter. Beta-boswellic acid, 3-acetyl-11-keto-beta-boswellic acid, and 11-keto-beta-boswellic acid were employed as reference materials. The samples were injected with 20 μL and examined using an ultraviolet detector at 205 nm for β-boswellic acid and 3-acetyl-11-keto-beta-boswellic acid (A), and at 250 nm for 11-keto-beta-boswellic acid and (B).

Fig. 2

The potential therapeutic effect of Fr on endometriosis is presented via network pharmacological analysis. The DisGeNet database was used to compile the list of endometriosis-related genes. A Venn diagram analysis was performed utilizing endometriosis-related genes and Fr targets. 38 common genes were listed (A). Cytoscape with the STRING plugin was used to show the protein-protein interaction network of the 38 common genes (B).

Fig. 3

Fr reduced human endometriotic cell proliferation. Fr was dissolved in DMSO and diluted in DMEM-F12 medium. Indicated concentrations of Fr was treated to human endometriotic epithelial (12Z) cells. After 24 h (A) and 48 h (B), the cell viability was analyzed by measuring at a wavelength of 450 nm using MTT. Data shown as mean ± SEM of quintuplicated and are representative of three independent experiment. Statistical analysis was conducted using a Dunnet one-way ANOVA (*; p < 0.05, **; p < 0.01, ***; p < 0.001).

Fig. 4

Fr reduces the size and number of murine ectopic endometrial lesions. The pictures of the ectopic endometrial tissues and uterus. Red arrows indicate endometrial tissues (A). The weight and number of ectopic endometrial tissues (B-C). Data are expressed as mean ± SEM. Statistical analysis was conducted using a t-test (*; p < 0.05, ***; p < 0.001).

Fig. 5

Fr activates the apoptosis-death signaling pathway in endometriotic cells. For analysis of frequency of apoptotic or dead cell populations, 12Z cells were incubated with the increasing doses of Fr for 24 h, and then the percentage of Annexin⁺ or PI⁺ cells were measured at excitation (Ex) 494/emission (Em) 525 nm for Annexin V and Ex 535/Em 617 nm for PI (A, B). Mitochondria-relative apoptosis molecules, Bax and Bcl-2 (C) and the cleavage forms of caspase −3 and −9, and PARP, main intracellular apoptosis signaling proteins (D), were detected 24 hrs after Fr treatment. GAPDH was used as control, and the specific molecular weight is represented on the left.

Fig. 6

Fr decreased the ability of endometrial cells to adhere to mesothelial cells. 12Z cells were stained with CMFDA after 24 h of Fr treatment and then gently transferred onto a red fluorescent Met-5A cell monolayer. After 90 min with gentle shaking at 20 rpm, unbound 12Z cells were removed, and cells were then visualized using a fluorescent microscope (200 × magnification) (A). The number of 12Z cells bound to Met-5A cells was manually counted in four randomly chosen areas for statistical analysis (B). Data are representative of one of five independent experiments and values are expressed in mean ± SEM. Statistical analysis was conducted using a Dunnet one-way ANOVA (***; p < 0.001).

Fig. 7

RNA-sequencing analysis suggests possible mode of action (MoA) underlying anti-endometriosis effect of Fr. The 12Z cells were treated with Fr (70 μg/ml) for 12 h. The RNA-sequencing dataset supplied by commercial service was applied to GSEA. The results from hallmark and KEGG analysis were visualized by bubble plot. The major pathways related with endometriosis was indicated by red (upregulated) or blue (downregulated) characters (A, B). The data from GO analysis was described by Cytoscape with enrichment map visualization. The cutoff value was set as p > 0.001 and q > 0.5 (C). The DEG analysis visualized as log2 fold change and -log10 p-value. Genes that were noticeably up- or down-regulated were denoted by red or blue characters, respectively. The genes participating in endometriosis-related pathways, as shown in the A and B panels, were indicated by a symbol for each gene (D). Heatmap analysis was used to display the relative expression levels of genes connected to endometriosis-related pathways (E). A graphical scheme was used to outline the potential mechanisms of action (MoA) underlying the relieving effects of Fr on endometriosis (F).