Protective Effect of L-carnitine on the Oxidative Stress Injury of Human Ovarian Granulosa Cells

The protective effect of L-carnitine (LC) on the oxidative stress (OS) injury and the effect of L-carnitine on follicular stimulating hormone receptor (FSHR) of ovarian granulosa cells (GCs) were investigated. OS was induced by treatment with H 2 O 2 . We cultured KGN cells in four groups: the blank group, OS group and two L-carnitine pretreatment group (low, high). In the OS group, cell nuclear pyknosis was observed, mitochondria swelled irregularly and their cristae were fractured. Meanwhile, the cell viability, superoxide dismutase (SOD) and glutathione (GSH) contents, mitochondrial membrane potential (ΔΨm) and the level of FSHR expression were signicantly decreased in the OS group. However, malonaldehyde (MDA) content, reactive oxygen species (ROS) level and apoptosis rate were signicantly increased. Compared with the OS group, the morphology of cells and mitochondria in the L-carnitine pretreatment group were improved, the cell viability and the expression of FSHR was signicantly increased, and the OS level was decreased. These results indicated that L-carnitine can protect the cells from OS damage induced by H 2 O 2 , enhance the antioxidant and anti-apoptotic ability of GCs, and alleviate the decrease of FSHR expression on GCs caused by OS. Therefore, L-carnitine may help prevent the ovarian aging and improve the quality of follicles.


Introduction
As the basic unit of female reproduction, follicle consists of oocyte and granulosa cells (GCs) [1]. GCs play an important role in supporting and nourishing oocytes during follicular development and maturation [2] [3]. OS caused by the increase of reactive oxygen species has an important effect on oocytes and embryo quality [4] [5]. Current studies have found that the reactive oxygen species in follicular uid of patients with polycystic ovary syndrome (PCOS) and endometriosis (EMT) are signi cantly increased, while in the healthy population, the total oxidation state (TOS) and total antioxidant state (TOA) in the follicular uid also tend to be abnormal with the aging [6] [7][8]. And some studies have con rmed that OS can regulate ovarian FSH reactivity by changing the expression of FSH regulatory gene P450 aromatase [9]. Therefore, it is of great signi cance to protect GC from OS injury to improve the quality of follicles and embryos and protect female reproductive function.
L-carnitine (LC) is a kind of water-soluble vitamin occurring in human body naturally. As an important substance of energy metabolism and transformation, LC shows high antioxidant effect. Previous studies have indicated that LC is effective in the treatment of male asthenospermia and the improvement of male reproductive ability. Although the protective role of LC in female reproduction have been investigated [10] [11] [12], the protective role LC on GCs is rarely reported. This study used H 2

Measurement of ΔΨm and cell apoptosis
ΔΨm was detected by JC-1 uorescent probe under a uorescence microscope. Green uorescence was used to detect JC-1 monomer and red uorescence was used to detect JC-1 polymer. Annexin V-FITC assay was used to detect cell apoptosis by ow cytometry.

Detection of FSHR protein levels in granulosa cells
The total protein of KGN cells was extracted after culturing, and the expression of FSHR protein in granulosa cells was detected by Western blotting.
Statistical analyses SPSS and GraphPad Prism 8.0 software were used for statistical analysis. Data were expressed as (mean ± SD). Independent sample t-test was used for comparison between two groups, and one-way ANOVA and Dunnett's T3 were used for comparison among multiple groups. All experiments were repeated for 3 times or more, and P < 0.05 was considered statistically signi cant.

Determination of the hydrogen peroxide concentration to induce OS
We cultured KGN cells at different H 2 O 2 concentration gradients and time. Through CCK8 assay, we observed that the cell viability of each group decreased compared with the blank group. Among which, the IC 50 of KGN cells cultured with H 2 O 2 for 24 hours was 109.12µmol/L. Therefore, we chose to use 100 µmol/L H 2 O 2 to treat KGN cells for 24h to induce oxidative stress in human ovarian granular cells ( Figure   1).

Determination of the optimal L-carnitine concentration
We found that L-carnitine had no signi cant effect on cell viability ( Figure 2A). ELISA results showed that when the concentration of L-carnitine in the medium was greater than 80µmol/L, the increasing trend of intracellular L-carnitine concentration slowed down. Therefore, we chose 80µmol/L L-carnitine concentration for subsequent experiments ( Figure 2B).

Effect of L-carnitine pretreatment on cell viability in the presence of H 2 O 2 -induced OS
Compared with the blank group, the cell viability in the OS group was signi cantly decreased, and the difference was statistically signi cant (P < 0.01). Compared with the OS group, the cell viability of Lcarnitine pretreated group increased in a dose-dependent manner ( Figure 3).

Ultrastructure of KGN cells
KGN cells in the blank group showed intact nucleolus, clear nuclear membrane and normal mitochondrial morphology. But in the OS group and the 40 and 80µmol/L L-carnitine pretreatment groups, different degrees of early and late apoptosis appeared, such as increased nucleolar lobulation, blurred nuclear membrane, mitochondrial swelling, mitochondrial cristae rupture, and increased intracellular phagocytes.

OS biomarkers of KGN cells
After treatment, the contents of ROS, MDA, GSH and the activity of SOD in KGN cells were detected ( Figure 5). In blank group, the levels of ROS and MDA in cells were lower, while the GSH and SOD activities were higher. After 100µmol/L H 2 O 2 induction, ROS and MDA levels in OS group were signi cantly increased, while GSH content and SOD activity were signi cantly decreased (P < 0.01).
These results indicated that 100µmol/L H 2 O 2 could effectively induce OS injury in KGN cells. Compared with OS group, OS levels in KGN cells were signi cantly alleviated in 40µmol/L and 80µmol/L L-carnitine pretreatment groups (P < 0.05, P < 0.01), and the effect of 80µmol/L L-carnitine pretreatment was better than 40µmol/L pretreatment.

ΔΨm and cell apoptosis of KGN cells
Compared with the blank group, the ΔΨm of KGN cells induced by 100µmol/L H 2 O 2 decreased signi cantly, and the cell apoptosis rate increased. Compared with OS group, 40 and 80µmol/L Lcarnitine treatments could up-regulate ΔΨm and reduce cell apoptosis rate. These results indicated that Lcarnitine in both groups could prevent the early apoptosis of KGN cells after oxidative stress injury and reduce the apoptosis after injury, and the effect of L-carnitine at 80µmol/L was more obvious.

Expression level of FSHR protein in KGN cells
Compared with the blank group, the expression of FSHR in KGN cells was signi cantly decreased in the OS group. Compared with OS group, 40 and 80µmol/L L-carnitine pretreatment could effectively upregulate FSHR protein expression, and there was no signi cant difference in FSHR expression between 80µmol/L L-carnitine pretreatment and normal culture KGN.

Discussion
As one of the important components of follicles, ovarian granulosa cells play an important role in signal transduction and nutritional support for oocytes. The decrease of the number of GCs and their function damage may lead to the decline of oocyte quality, and eventually the decline of ovarian function, which could cause female infertility.
The oocytes show different sensitivity to oxidative stress at different stages of follicle development [13].
It is known that when the dominant follicle enters the rst meiosis, a certain degree of OS is present to maintain a high metabolic level, while the subsequent process requires low OS to avoid cell damage [14].
Compared with somatic cells, GCs are always at a high metabolic level during the entire process of follicular development, while the oxidation and antioxidant systems in the follicular microenvironment maintain a dynamic balance to meet the needs of oocytes. When this system is disrupted by stress stimuli, OS damage and reduced local repair capability will damage the various biomolecules in the follicular microenvironment. A large number of follicle atresia causes an irreversible decline in ovarian function and eventually leads to premature ovarian failure. Some studies have also found that the high level of ROS in follicular uid is associated with poor follicular quality, dysontogenesis, and failure of in vitro fertilization-embryo transfer (IVF-ET)[16] [17]. Moreover, the increase of ROS in follicular uid was also found in common female reproductive diseases such as PCOS and EMT, indicating that ROS may be correlated with the occurrence and development of ovarian disorders. Therefore, it is important to protect ovarian granulosa cells from OS damage for female reproductive health.
L-carnitine is an amino acid analogue naturally occurring in the human body. It can promote lipid metabolism and protect plasma and mitochondrial membrane from damage by lipid peroxidation. LC has been widely used as an antioxidant and studies have found that the addition of L-carnitine in sperm culture improves sperm motility [18]. Addition of LC in the oocyte culture can improve oocyte quality, which may be related to the reduction of GCs apoptosis and improvement of mitochondrial function [19]. It is believed that the supplement of LC is bene cial to alleviate the delayed embryonic development, high DNA fragmentation and abnormal blastocyst development after long-term culture mediated by the increased ROS [20]. Therefore, treatment with LC may have the potential to improve the quality of follicles and the pregnancy outcome of IVF-ET.
Oxidative stress injury of granulosa cells can lead to imbalanced follicular microenvironment, reduced function of granulosa cells, and ovarian aging [21] [22]. In this study, we found that LC can effectively alleviate the generation of excess oxygen free radicals induced by H 2 O 2 and maintain the redox balance.
We also found the level of MDA, the end-product of lipid peroxidation, was signi cantly reduced in GCs after LC treatment, indicating that LC alleviated the cell damage caused by OS and mitochondrial oxidative respiratory chain. The SOD activity and GSH content were measured and the results indicated that LC pretreatment improved the antioxidant activity and reduced the consumption of antioxidant, which is helpful to maintain the dynamic balance of redox in the follicular microenvironment.
Damaging of macromolecules such as proteins and DNA by OS will eventually lead to apoptosis of GCs [23]. We observed that LC pretreatment can effectively prevent the formation of apoptotic bodies, mitochondrial swelling and vacuolation, nuclear membrane blurring and other phenomena induced by H 2 O 2 . The protective effect of LC was con rmed by uorescence detection of ΔΨml and cell apoptosis study. Moreover, it is well-known that there is a signi cant correlation between the FSHR expression on ovarian GCs and ovarian activity [24] [25]. Some studies have proposed that OS can change FSH activity by regulating the expression of P450 aromatase [26]. Our results con rmed that the expression level of FSHR in the OS group was signi cantly decreased compared with the control group. We found that the expression of FSHR on GCs in the presence of H 2 O 2 was signi cantly increased after being pretreated with LC, further demonstrating the protective effect of LC.
In conclusion, LC was shown to have high protective effect from the injury of ovarian GCs induced by H 2 O 2 . Therefore, LC treatment has the potential to reduce OS injury of GCs, decrease GCs apoptosis, and provide better nutritional support for oocytes to improve the quality of follicles.

Declarations Ethics approval and consent to participate
This study does not address ethical issues.

Consent for publication
There is no con ict of interest among the authors in this study, and we all agree to publish.
Availability of data and material SPSS and GraphPad Prism 8.0 software were used for statistical analysis. Data were expressed as (mean ± SD). Independent sample t-test was used for comparison between two groups, and one-way ANOV A and Dunnett's T3 were used for comparison among multiple groups. All experiments were repeated for 3 times or more, and P < 0.05 was considered statistically signi cant.
L-carnitine and hydrogen peroxide were purchased from Sigma-Aldrich (USA). KGN human ovarian granulocyte cells were purchased from Yaji Biotechnology Co. Ltd. (China).

Competing interests
The authors declare that they have no known competing nancial interests or personal relationships that could have appeared to in uence the work reported in this paper.

Funding
This study is funded by Weifang Health Committee (wfwsjk_2019_029).    Effects of L-carnitine pretreatment with concentration of 20 40 80μmol/L on the activity of cells injured by oxidative stress induced by H2O2. note ** indicates that compared with the blank control group, P < 0.01,## indicates that compared with the injury model group, P < 0.05, (n=3, mean± SD). Ultrastructure of KGN cells: A. in the blank control group, the nucleus was complete, the nuclear membrane was clear, the mitochondrial structure was complete and the mitochondrial ridge was clearly visible; B. The concentration is 80μmol/L L-carnitine pretreatment group, the nucleus was complete, the nuclear membrane was basically clear, the mitochondrial structure was basically complete, and a clear mitochondrial ridge could be seen; C. In the pretreatment group with 40μmol/L L-carnitine, the nucleus was heterogeneous, the nuclear membrane in some areas was fuzzy, some mitochondria were visible, the structure in mitochondria was unclear, and apoptotic and phagocytic bodies were visible in the cytoplasm; D. In the oxidative stress injury group, the nucleolus was diffused, the nuclear membrane was blurred, the nuclear membrane in some areas disappeared, it was di cult to nd the mitochondria with complete structure, and there were a large number of apoptosis bodies and phagocytosis bodies in the cytoplasm; note The left gure in the gure shows the transmission electron microscope with magni cation of 2.0k, The eld of vision is the panorama of cells in 5μm, The right gure in the gure shows the transmission electron microscope with magni cation of 5.0k, The eld of vision is the local area of cells in 2μm; In this picture, The M is mitochondria, and the arrow points to the nuclear membrane.

Figure 5
Oxidative stress level of KGN cells: A. ROS content; B. MDA content; C. GSH content; D. SOD content. As shown in the gure, compared with the blank control group, in the oxidative stress injury model induced by H2O2, the content of ROS and MDA increased, the content of GSH decreased, and the activity of SOD decreased. The injury model group was successfully constructed. Compared with the damage model group, the concentration is 40μmol/L and 80μmol/L L-carnitine could improve the oxidative stress in KGN cells in a concentration dependent manner. note In the gure, * * indicates that there is a statistical difference between the injury model group and the blank control group, P < 0.01; # indicates that there is a statistical difference compared with the injury model group, (P < 0.05); ## indicates that there is a statistical difference compared with the injury model group, (P < 0.01). The content unit of each substance is in parentheses, "-" indicates that it is not added.