Protective effect of L‑carnitine against oxidative stress injury in human ovarian granulosa cells

Abstract

Granulosa cells (GCs) are important for supporting and nourishing oocytes during follicular development and maturation. Oxidative stress (OS) injury of GCs can lead to decreased responsiveness of follicles to follicular stimulating hormone (FSH), which will accelerate ovarian senescence and adversely affect oocyte and embryo quality. Since L-carnitine has been previously reported to exert strong antioxidant activity, the present study aimed to explore the possible effects of L-carnitine on OS injury and FSH receptor (FSHR) expression in ovarian GCs, results of which may be of significance for GCs protection. In the present study, OS was induced in vitro in KGN cells by treatment with H₂O₂. KGN cells were cultured and divided into the following four groups: Blank, OS, and 40 and 80 µmol/l L-carnitine pre-treatment groups. In the OS group, cells showed nuclear pyknosis, mitochondria swelled irregularly whilst featuring fractured cristae. In addition, cell viability, ROS levels, superoxide dismutase levels, glutathione levels, malondialdehyde levels, the mitochondrial membrane potential and FSHR expression, as determined by Cell Counting Kit-8 (CCK-8), 2,7-dichloro-dihydrofluorescein diacetate, spectrophotometry, ELISA, spectrophotometry, JC-1 and western blot analyses, respectively, were all significantly different in the OS group compared with those in the control group. However, malonaldehyde levels, reactive oxygen species levels and the apoptosis rate according to flow cytometry were all significantly increased compared with those in the control. Compared with those in the OS group, the morphology of cells and mitochondria in the L-carnitine pre-treatment groups were improved, whilst cell viability and the expression of FSHR were significantly increased but oxidative stress injury was decreased. The present results suggest that L-carnitine can protect the cells from OS damage induced by H₂O₂, enhance antioxidant activity whilst suppressing the apoptosis of GCs, in addition to preserving FSHR expression in GCs under OS. Therefore, the present study revealed that the introduction of L-carnitine in clinical medicine or dietary supplement may protect GCs, improve follicular quality and female reproductive function.

Keywords: L-carnitine; follicular stimulating hormone receptor; mitochondria; ovarian granulosa cells; oxidative stress.

Figure 1

Cell viability is decreased in KGN cells challenged with H₂O₂. Effect of several H₂O₂ treatment concentrations and durations on the cell viability of KGN cells. ^*P<0.05 and ^**P<0.01 vs. control (untreated cells), Tukey's test. Data are represented as the mean ± standard deviation (n=3).

Figure 2

L-carnitine concentration. (A) Effects of different concentrations of L-carnitine on the viability of KGN cells. (B) Analysis of the intracellular concentrations of L-carnitine after exposure to different concentrations of L-carnitine. Data are represented as the mean ± standard deviation (n=3).

Figure 3

Effects of L-carnitine pre-treatment on the viability of KGN cells challenged with H₂O₂. ^**P<0.01 vs. control and ^##P<0.01 vs. H₂O₂-only, Tukey's test. Data are represented as the mean ± standard deviation (n=3).

Figure 4

Ultrastructure of untreated KGN cells, those treated with H₂O₂ and those pre-treated with L-carnitine as analyzed using transmission electron microscopy. The arrows indicate the nuclear membrane. (A) In the control group, the nucleus is complete, the nuclear membrane is clear, the mitochondrial structure is complete and the mitochondrial ridge is clearly visible. (B) In the H₂O₂-only group, the nucleolus is diffused, the nuclear membrane is blurred, the nuclear membrane in some areas disappeared. The majority of mitochondria do not present with the complete structure and there is a large number of apoptotic and phagocytic bodies in the cytoplasm. (C) In the low L-carnitine group (40 µmol/l), the nucleus is heterogeneous, the nuclear membrane in some areas is fuzzy, some mitochondria were visible, the structure in the mitochondria is unclear and apoptotic. Phagocytic bodies are visible in the cytoplasm. (D) In the high L-carnitine group (80 µmol/l), the nucleus is complete, the nuclear membrane is basically clear, the mitochondrial structure is basically complete and a clear mitochondrial ridge can be seen. Magnifications: Left, x2,000; right, x5,000. M, mitochondria; P, phagosome; A, apoptotic body.

Figure 5

OS levels in KGN cells. (A) ROS content quantification and corresponding flow cytometry histogram. (B) MDA, (C) GSH and (D) SOD content. Compared with the control group, the KNG cells challenged with H₂O₂ showed an increase in the content of ROS and MDA, whilst the content of GSH and the activity of SOD were decreased. The OS injury model group was successfully constructed. Compared with that in the OS group, 40 and 80 µmol/l L-carnitine pre-treatment ameliorated the effect of H₂O₂ in KGN cells. ^**P<0.01 vs. untreated cells and ^##P<0.01 vs. H₂O₂-only; (A, C and D) Tukey's test; (B) Dunnett's T3 test. Data are presented as the mean ± standard deviation (n=3). OS, oxidative stress; ROS, reactive oxygen species; MDA, malondialdehyde; GSH, reduced glutathione; SOD, superoxide dismutase.

Figure 6

mitochondrial membrane potential of KGN cells. Mitochondrial membrane potential in the (A-a) H₂O₂-only, (A-b) low L-carnitine (40 µmol/l), (A-c) high L-carnitine (80 µmol/l) and (A-d) control groups. Magnification, x200. (B) Percentage of ΔΨm (JC-1 monomer-positive cells). ^**P<0.01 vs. control and ^##P<0.01 vs. H₂O₂-only, Tukey's test. Data are represented as the mean ± standard deviation (n=3).

Figure 7

Cell apoptosis analysis through flow cytometry. (A) Representative flow cytometry dot plots in (A-a) H2O2-only, (A-b) low L-carnitine (40 µmol/l), (A-c) high L-carnitine (80 µmol/l) and (A-d) control groups (B) Percentage of apoptotic cells in each group. ^**P<0.01 vs. control and ^##P<0.01 vs. H₂O₂-only, Tukey's test. Data are represented as the mean ± standard deviation (n=3).

Figure 8

Effect of L-carnitine pretreatment on the expression of FSHR in KGN cells in a H₂O₂-induced oxidative stress injury model. (A) Representative western blotting image. (B) Densitometry data generated from the western blotting images. ^**P<0.01 vs. control and ^##P<0.01 vs. H₂O₂, Dunnett's T3 test. Data are represented as the mean ± standard deviation (n=3). FSHR, follicle stimulating hormone receptor.