Investigation of the role of rosmarinic acid treatment in regulating inflammation, cell damage, and angiogenesis in rat ovarian torsion and detorsion models

Abstract

Purpose: To investigate the protective effect of rosmarinic acid (RA) in ovarian ischemia/reperfusion injury using biochemical, histopathological, and immunohistochemical methods.

Methods: Wistar female rats (n = 32) were randomly divided into four groups: control, ischemia, ischemia-reperfusion, and ischemia-reperfusion with RA. Rosmarinic acid was given at a dose of 50 mg/kg by oral gavage three hours after reperfusion. Malondialdehyde (MDA) levels and glutathione peroxidase (GSH-Px) activities were determined in the ovary tissue homogenates for each rat.

Results: In the ischemia-reperfusion with RA group, the epithelial cells are regularly regulated at the periphery, and the degenerative changes in preantral and antral follicle cells are reduced. Follicle cells and cells in the corpus luteum showed a decrease in vascular endothelial growth factor (VEGF) expression, while VEGF demonstrated a positive reaction in vascular endothelial cells and stromal cells. The TNF-α expression due to the decreased degenerative effect and inflammation was positive in the macrophage cells. The expression of caspase-3 as an apoptosis change was negative in antral follicle cells and granular cells around the antral follicle.

Conclusion: Different doses of RA may be useful in preventing ischemic damage after vascularization, inflammation, and apoptotic development after ischemia/reperfusion.

Figure 1. a. Masson’s Trichrome staining (control group). The germinal epithelium was regular and lined throughout the periphery. The cortical preantral and antral follicles were oval, and the luteal cells of the corpus luteum were full of secretory contents. Irregular connective tissue fibers, solitary-localized cells, regular blood vessels, and small hemorrhages were observed in the stromal area. b. Masson’s Trichrome staining (ischemia group). Degeneration of germinal epithelial cells, deterioration of preantral and antral follicles, and increased inflammation in the cortical area were observed (yellow arrow). Hyper-dilated and congestion in blood vessels (red arrow), edema around blood vessels, degenerative changes in collagen fibers were seen. c. Masson’s Trichrome staining (ischemia-reperfusion group). Degenerated preantral and antral follicles with decreased size, apoptotic follicle cells with pyknotic nuclei, increased inflammation outside the follicles (yellow arrow), and intensive congestion in blood vessels were observed. d. Masson’s Trichrome staining (Ischemia-reperfusion + RA group). Epithelial cells were regularly organized on the periphery, and decreased degenerative changes in the preantral and antral follicle cells were observed. Granular cells were small and regular, and some of them had dense secretory granules (yellow arrow). Around the follicles, the collagen fibers were arranged in parallel, and the connective tissue cells were solitarily distributed. Scale bar = 50 μm.

Figure 2. a. VEGF immunostaining (control group). The control group results showed negative expression of VEGF in vascular endothelial cells and stromal macrophage cells outside the preantral and antral follicles. b. VEGF immunostaining (ischemia group). Positive VEGF expression was seen in degenerative preantral and antral follicle cells (red arrow), dilated vascular endothelial cells, and dense inflammatory cells (yellow arrow). c. VEGF immunostaining (ischemia-reperfusion group). The VEGF expression was increased in the luteal cells of the corpus luteum and the vascular endothelial and inflammatory cells (red arrow). d. VEGF immunostaining (ischemia-reperfusion + RA group). Follicle cells and cells in the corpus luteum showed a decrease in VEGF expression (yellow arrow), while the VEGF expression was positive in vascular endothelial and stromal cells. Scale bar = 50 μm.

Figure 3. a. TNF-α immunostaining (control group). Preantral and antral follicle cells and stromal macrophage cells showed a negative TNF-α expression, whereas the TNF-α expression was positive in corpus albicans cells. b. TNF-α immunostaining (ischemia group). The TNF-α was positively expressed in the degenerative follicular cells and numerous inflammatory cells around the stromal blood vessels in the ischemia group (red arrow). c. TNF-α immunostaining (ischemia-reperfusion group). The TNF-α expression was positive in the granular cells of the antral follicles, vascular endothelial cells, and inflammatory cells (red arrows). d. TNF-α immunostaining (ischemia-reperfusion + RA group). The TNF-α expression was positive in granular cells, blood-vessel endothelial cells, and inflammatory cells in the antral follicles (red arrow). The TNF-α expression was positive in some macrophage cells distant from the preantral and antral follicles, while the TNF-α expression was negative in other areas. Scale bar = 50 μm.

Figure 4. a. Caspase-3 immunostaining (control group). The control group caspase-3 results showed that caspase-3 expression was negative in germinal epithelial cells, granular cells of preantral and antral follicles, and endothelial cells. However, it was positive in the stromal cells around the follicle. b. Caspase-3 immunostaining (ischemia group). The positive expression of caspase-3 was observed in degenerated granular cells of preantral and antral follicles, luteal cells of the corpus luteum, and many inflammatory cells in the stromal region (red arrow). c. Caspase-3 immunostaining (ischemia-reperfusion group). Caspase-3 was positively expressed in granular cells in mature antral follicles and inflammatory cells in the stromal region (red arrow). d. Caspase-3 immunostaining (ischemia-reperfusion + RA group). Caspase-3 expression was negative in the preantral follicle cells and granular cells around the antral follicle (red arrow), whereas it was positive in some stromal cells and corpus luteum cells. Scale bar = 50 μm.