Chitosan Oligosaccharides Alleviate H2O2-stimulated Granulosa Cell Damage via HIF-1α Signaling Pathway

Oocyte maturation disorder and decreased quality are the main causes of infertility in women, and granulosa cells (GCs) provide the only microenvironment for oocyte maturation through autocrine and paracrine signaling by steroid hormones and growth factors. However, chronic inflammation and oxidative stress caused by ovarian hypoxia are the largest contributors to ovarian aging and GC dysfunction. Therefore, the amelioration of chronic inflammation and oxidative stress is expected to be a pivotal method to improve GC function and oocyte quality. In this study, we detected the protective effect of chitosan oligosaccharides (COS), on hydrogen peroxide- (H2O2-) stimulated oxidative damage in a human ovarian granulosa cell line (KGN). COS significantly increased cell viability, mitochondrial function, and the cellular glutathione (GSH) content and reduced apoptosis, reactive oxygen species (ROS) content, and the levels of 8-hydroxy-2′-deoxyguanosine (8-OHdG), 4-hydroxynonenal (4-HNE), hypoxia-inducible factor-1α (HIF-1α), and vascular endothelial-derived growth factor (VEGF) in H2O2-stimulated KGN cells. COS treatment significantly increased levels of the TGF-β1 and IL-10 proteins and decreased levels of the IL-6 protein. Compared with H2O2-stimulated KGN cells, COS significantly increased the levels of E2 and P4 and decreased SA-β-gal protein expression. Furthermore, COS caused significant inactivation of the HIF-1α-VEGF pathway in H2O2-stimulated KGN cells. Moreover, inhibition of this pathway enhanced the inhibitory effects of COS on H2O2-stimulated oxidative injury and apoptosis in GCs. Thus, COS protected GCs from H2O2-stimulated oxidative damage and apoptosis by inactivating the HIF-1α-VEGF signaling pathway. In the future, COS might represent a therapeutic approach for ameliorating disrupted follicle development.


Introduction
Due to environmental pollution, life pressure, radiation and chemotherapy administered to patients with cancer, and other factors that damage female ovarian function and shorten human life, infertility, premature ovarian failure (POF), and aging have become major medical problems and social problems threatening human health. The speed of ovarian aging is significantly faster than that of other organs of the body. Some scholars have considered ovarian aging as pacemakers of female body aging. Ovarian aging is the initial factor triggering cardiovascular and cerebrovascular diseases, neurodegenerative diseases, and other chronic diseases [1]. Unfortunately, few methods are available to protect the ovary and delay ovarian aging in healthy aged women. In pathological conditions, treatments include administration of gonadotrophin-releasing hormone (GnRH) analogs, transposition of the ovaries outside of the fields of radiation in cancer patients, and cryopreservation of the ovarian cortical tissue. All these methods are either less effective or are still being investigated [2]. The fundamental reason is the mechanism of ovarian aging. Chemicals that potentially protect the oocyte and its feeder cells from injury would be highly useful.
Ovogenesis has a close relationship with the follicle growth and its maturity. Primary oocytes are originated from primordial germ cells [3,4]. Primary oocytes interact with the surrounding monolayer of follicular cells and gradually developed into primitive follicles. The primitive follicle terminates the dormancy in certain condition; next, it enters the growth state [5]. During the normal follicle development process, oocytes gradually develop and mature, while granulosa cells (GCs) surrounding oocytes also proliferate and differentiate continuously [4]. Nevertheless, more than 99% of mammalian follicles evolve into atresia state in physiological phenomenon, they degenerate in a period of growth and development, and only a few follicles have the ability to accomplish the development process [6][7][8]. Follicular atresia is attributed to the programmed cell death of ovarian GCs. GCs communicate with and form a protective barrier between the oocyte and follicle microenvironment by transmitting nutrients, energy, and signals to oocytes and play roles in the androgen to estrogen conversion and progesterone synthesis; thus, a decrease in GC quantity and function leads to decreased production of estradiol (E 2 ) and progesterone (P 4 ) and may trigger follicular atresia [9,10]. The morphology and quantity of GCs have been used as biomarkers of developmental ability and pregnancy outcomes [11]. Therefore, the disorder of GCs caused by various factors is the key cause of disrupted oocyte maturation.
The mechanisms underlying decreased GC function and subsequent ovarian aging are not wholly clear. Previous studies have shown that chronic inflammation and oxidative stress (OS) caused by ovarian hypoxia are the main causes of ovarian aging and GC dysfunction [12,13]. Hypoxia, via a hypoxia-inducible factor-1α-(HIF-1α-) dependent manner, promotes GC expression of more than 70 downstream genes (including nuclear factor kappa-B (NF-κB), nitric oxide synthase (NOS), and VEGF), resulting in the overproduction of IL-6 and ROS [14][15][16][17][18]. ROS promote the formation of DNA adducts such as 8-hydroxy-2′-deoxyguanosine  and lipid peroxidation such as 4-hydroxynonenal (4-HNE). Both of these adducts and inflammatory factors jointly cause the accumulation of DNA damage, epigenetic changes, abnormal gene expression and altered cell signaling pathways in GCs, leading to cell and organ aging [19][20][21][22]. At present, our group and others' studies have shown that ovarian aging is delayed by improving mild inflammatory and OS environment in the ovary [23,24]. Therefore, the search for natural materials and chemicals to improve the inflammatory and OS environment of GCs is expected to provide a novel method to rejuvenate the ovary via GC function restoration. COS might be the target chemical meeting these properties. COS is a derivative of chitin that is mainly derived from crustaceans, fungi, insects, and algae cell membranes [25]. COS, water-soluble alkaline polysaccharide with positively charged, has good biocompatibility and is widely used in food, medicine, the chemical industry, environmen-tal protection, cosmetics, agriculture, and other applications [26]. It is known as the "sixth life element" after protein, fat, sugar, vitamin, and mineral in the biomedical field. COS can be used as a functional food and is known as "soft gold." COS is an oligomer composed of β-(1 ➔ 4)-linked d-glucosamine, and it is an oligosaccharide obtained by the hydrolysis of chitosan (CTS) [27]. COS has distinct characteristics, such as a low molecular weight, noncytotoxicity, good water solubility, and easy absorption in the intestine. Meanwhile, COS possesses various diverse biological features, including anti-inflammatory, radical-scavenging, antioxidant, and antidiabetic effects [28]. Currently, products of health care and skincare and other applications have been applied COS as a supplement.
The role of reactive oxygen species (ROS) concentrations in follicular fluid in gynecological diseases and assisted reproduction is relevant to its role in reproductive outcomes [29,30]. Patients with premature ovarian failure, polycystic ovarian syndrome (PCOS), and physiologically aging ovaries experience a decrease in follicle quality and disrupted oocyte maturation. These tissues are all in chronic low-grade inflammation state caused by OS [31][32][33]. Tiwari and Chaube found that a gentle increase in ROS levels in the follicle is advantageous for meiotic resumption and first polar body extrusion [34]. In contrast, the excess accumulation of ROS such as H 2 O 2 and superoxide anion radicals leads to OS, and progressive OS generally induces an inflammatory state [35,36]. High levels of ROS in the follicle microenvironment prevent the recovery of meiosis of diploid terminated oocyte and GC function, causing poor communication between GCs and oocytes by reducing the supply of nutrition, and eventually negatively affect oocyte quality and maturation [37][38][39][40].
Hypoxia-inducible factor (HIF) expression is induced by hypoxia and by other pathological environments associated with inflammation, aging, infectious microorganisms, and tumors [41][42][43][44]. It is composed of three subunits: 1α, 2α, and 1β. HIF-1α is the main subunit that is widely expressed and regulates a variety of target genes, such as glucose transporters, and VEGF [45]. Recent research has reported that inflammation may lead to cumulate HIF-1α protein in macrophages via a mechanism relative to ROS [46]. In addition, KC7F2 is an HIF-1α protein inhibitor but does not affect the protein degradation rate or mRNA transcription. TGF-β (transforming growth factor-β), along with its superfamily number anti-Müllerian hormone (AMH), bone morphogenetic protein-15 (BMP-15), and growth differentiation factor-9 (GDF-9), plays important roles in influencing the developing follicle microenvironment through autocrine and paracrine manner [47]. In ovary and other organs, hypoxia significantly inhibited GSH activities and superoxide dismutase (SOD) and enhanced the production of malondialdehyde (MDA) [48][49][50].
However, it's not clear whether COS has a role in GC development and function by controlling chronic low-grade inflammation and OS. We studied the direct effects of different COS concentrations on a human ovarian granulosa cell line (KGN) as a first step to examine the potential protective effect of COS on H 2 O 2 -stimulated GC dysfunction. Also, we 2 Oxidative Medicine and Cellular Longevity studied the effects of COS on KGN cells mediated by the HIF-1α signaling pathway. Phase C: the optimal concentration of COS was studied to explore the mechanism underlying the protective effect.

Enzyme-Linked Immunosorbent Assay (ELISA).
The centrifuged supernatant of the medium was determined using ELISA kits (Westang, China) for the levels of IL-6, IL-10 (cell lysates), E 2 , and P 4 , according to the manufacturer's recommendations.

Detection of ROS Production. The Reactive Oxygen
Species Assay Kit (Solarbio, China) was used to determine ROS level. After COS treatment, KGN cells were loaded with 10 μM DCFH-DA in FBS-free medium for 20 min at 37°C. Then, the medium containing DCFH-DA was removed, and KGN cells were washed to remove unconjugated DCFH-DA.
All of the fluorescence intensity was examined under an Olympus 1X71 microscope and analyzed with ImageJ. The results are from 3 replications.

Cell
Counting Kit-8. Cell viability was measured using the Cell Counting Kit-8 assay (CCK-8, APExBIO, USA) in 96-well plates at a density of 8 × 10 3 cells/well. The mixture liquid (10 μl CCK − 8 assay reagent + 90 μl FBS − free medium) was added to each well and incubated in the dark for 2 h at 37°C. The optical density (OD) was assessed by a microplate reader (Bio-Rad, USA).

Flow
Cytometry. KGN cells were resuspended in annexin V binding buffer with FITC-conjugated annexin V (Bestbio, China). Then, 10 μl of propidium iodide was added. A flow cytometer was used to analyze the KGN cells (Beckman Coulter, USA).
2.8. SA-β-Gal Assay. The SA-β-gal assay was carried out according to the manufacturer's instructions (Beyotime, China). Freshly collected KGN cells were fixed for 15 min with β-galactosidase fixative at room temperature, washed, and stained with X-gal solution overnight at 37°C. Cells were photographed under an Olympus 1X71 microscope. The percentage of blue SA-β-gal positive cells was determined using ImageJ software.
2.9. GSH. The cellular total GSH were measured using a GSH Assay Kit (Beyotime, China). KGN cells were collected after centrifugation. Then, the supernatant was removed. M solution for protein removal was added, and the volume was three times the cell pellet volume. The samples were rapidly freeze-thawed three times. Afterward, suspension was centrifuged. The supernatant was collected to measure the total GSH content.
2.10. Statistical Analysis. All data (three independent cultures) are presented as the means ± SEM (standard errors of the means). Statistical analyses were performed using Student's t-test and one-way ANOVA with SPSS software. P < 0:05 were considered statistically significant.  (Figure 1). When cells were treated with 100 μM H 2 O 2 for 4 h, the cell survival rate was 53:25 ± 3:76 %, which was closest to the IC50. Therefore, 100 μM and 4 h were selected for subsequent experiments as the optimal concentration and time, respectively.   Oxidative Medicine and Cellular Longevity viability (Figure 2(c), P < 0:001), but the viability of COS treatment groups was higher than that of the H 2 O 2 group, except the 200 COS group, but the differences were not significant (P > 0:05). In Figure 2(a), annexin V and PI assays were conducted to evaluate apoptosis. The percentage of apoptotic cells was calculated from H2 (late stage of apoptosis) and H4 (early stage of apoptosis). In Figure 2(  (c-f) The levels of IL-6, IL-10, E 2 , and P 4 . Data are presented as the means ± SEM (n = 3). * P < 0:05, * * P < 0:01, or * * * P < 0:001 vs. the control group; # P < 0:05, ## P < 0:01 or ### P < 0:001 vs. the H 2 O 2 group. 5 Oxidative Medicine and Cellular Longevity and P 4 were increased by COS treatment at different doses ranging from 100 to 300 μg/ml.

Discussion
Aging, mental stress, endocrine disorders, and chemo-and/ or radiotherapy all exert substantial effects on female fertility [53][54][55]. Currently, few methods are available to preserve ovarian function in human being. Furthermore, these methods are less effective and still under investigated in experimental research. Chemicals that potentially protect the follicle of oocyte and GCs from injury might be the next female fertility-reserving medicine. A vital antioxidant and a forceful free radical scavenger, COS might be this type of agent.
GCs promote oocyte maturation and protect oocytes from OS damage via supplying essential nutrients and maturation-promoting factors; they play crucial roles in folliculogenesis [56]. GCs are susceptible to ROS, which are byproducts produced in the course of the citric acid cycle. The generation of endogenous ROS is through the following: one is various metabolic processes, and the other is the electron transport chain. Mitochondria are intimately involved in cellular respiration and metabolism, and thus, they are the main generator of endogenous ROS and the main target organelle for OS damage. There are two main methods to remove ROS. One is the internal antioxidant system, which consists of enzyme such as SOD and the nonenzymatic antioxidant such as GSH [49,50]. GSH is a major cellular antioxidant, also recognized key indicator to determine the degree of OS [50]. The other is exogenous antioxidant from supplement to improve the antioxidant capacity [57]. Obviously, it leads to a better effect that endogenous and exogenous antioxidant methods simultaneously remove ROS to resist oxidant damage. ROS is not only considered to be the most critical induction factor of cell damage caused by oxidative stress; meanwhile, free radicals mainly attack macromolecules such as biofilms, membrane system, DNA, and protein. Free radicals easily react with unsaturated fatty acids on the membrane then produce lipid radicals, destroying the original membrane structure [58]. The levels of ROS are markedly accumulative after stimulation and induction OS in GCs, leading to GC dysfunction [59]. Hence, ROSinduced injury of OS and GC decline in quantity are deemed the primary etiological factors of ovarian dysfunction. In this study, H 2 O 2 was applied to induce OS and GC apoptosis [60][61][62]. We found that COS-mediated GC protection depends on improved mitochondrial function and a decrease in apoptosis. Therefore, our results suggest that COS inhibits OS-induced damage in GCs by ameliorating mitochondrial damage. Numerous animal studies have shown that OS and chronic inflammation promote each other in a vicious cycle: OS leads to an increase in ROS production through NOD-like receptor 3 (NLRP3) and NF-κB, resulting in a series of inflammatory responses and increased secretion of IL-1β, IL-8, and TNF-α, which in turn promote OS and accelerate the organ aging process [63]. So we evaluated the protective effect of COS on H 2 O 2 -oxidative damage and cytokine production in KGN cells. COS significantly increased cell viability, the cellular GSH content, and mitochondrial function and reduced ROS production; the levels of 8-OHdG, 4-HNE, IL-6, HIF-1α, and VEGF; and cell apoptosis. COS also significantly increased TGF-β1 and IL-10 expression in the H 2 O 2 -stimulated KGN cells, significantly increased E 2 and P 4 levels, and decreased in SA-βgal protein expression, implying that COS attenuated oxidative and inflammatory injury in H 2 O 2 -stimulated KGN cells (Figure 9). Partial results showed that the effect of the  * P < 0:05, * * P < 0:01, or * * * P < 0:001 vs. the control group; # P < 0:05, ## P < 0:01, or ### P < 0:001 vs. the H 2 O 2 group; & P < 0:05, && P < 0:01, or &&& P < 0:001 vs. the H 2 O 2 +100 COS+KC7F2 group. 9 Oxidative Medicine and Cellular Longevity H 2 O 2 +200 COS group was lower than that of the other two groups, but the P values between the most optimal concentration group (the H 2 O 2 +100 COS group) and the H 2 O 2 +200 COS group were not significant except the results in IL-6 and IL-10. It may be related to the higher sensitivity of ELISA than western blotting in quantifying. Meanwhile, it proved that COS was not concentration-dependent in the KGN OS model.
We were very curious about the triggers of inflammation and OS-induced damage. What is the mechanism of action?
We speculated that the mechanism may be related to ovarian hypoxia. Hypoxia, the imbalance between the oxygen supply and demand, is the factor that induces chronic inflammation and OS. Due to the unique tissue construct and large volume of oocyte, the ovary is a hypoxic organ. On the one hand, with the growth and development of follicular oocytes and the proliferation and division of GCs, the oxygen demand gradually increases; on the other hand, continuous ovulation leads to an increase in the amount of fibrous connective tissue and a significant decrease in the    (e, f) Cellular GSH content and cell viability. Data are presented as the means ± SEM (n = 3). * P < 0:05, * * P < 0:01, or * * * P < 0:001 vs. the control group; # P < 0:05, ## P < 0:01, or ### P < 0:001 vs. the H 2 O 2 group; & P < 0:05, && P < 0:01, or &&& P < 0:001 vs. the H 2 O 2 +100 COS+KC7F2 group.