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. 2023 Mar 1:14:1086232.
doi: 10.3389/fimmu.2023.1086232. eCollection 2023.

Chitosan oligosaccharide improves ovarian granulosa cells inflammation and oxidative stress in patients with polycystic ovary syndrome

Qi Xie  1   2   3 , Wenli Hong  1   4 , Yuan Li  1 , Shuyi Ling  1 , Ziqiong Zhou  1 , Yuqing Dai  1 , Wenbo Wu  1 , Ruoxin Weng  1 , Zhisheng Zhong  1 , Jun Tan  5 , Yuehui Zheng  1
Affiliations

Chitosan oligosaccharide improves ovarian granulosa cells inflammation and oxidative stress in patients with polycystic ovary syndrome

Qi Xie et al. Front Immunol. .

Abstract

Introduction: Polycystic Ovary Syndrome (PCOS) is the most common reproductive endocrine disorder among women of reproductive age, which is one of the main causes of anovulatory infertility. Even though the rapidly developed assisted reproductive technology (ART) could effectively solve fertility problems, some PCOS patients still have not obtained satisfactory clinical outcomes. The poor quality of oocytes caused by the abnormal follicular development of PCOS may directly contribute to the failure of ART treatment. Ovarian granulosa cells (GCs) are the most closely related cells to oocytes, and changes in their functional status have a direct impact on oocyte formation. Previous studies have shown that changes in the ovarian microenvironment, like oxidative stress and inflammation, may cause PCOS-related aberrant follicular development by impairing the physiological state of the GCs. Therefore, optimizing the ovarian microenvironment is a feasible method for enhancing the development potential of PCOS oocytes.

Methods: In this study, we first detected the expression of inflammatory-related factors (TGF-β1, IL-10, TNFα, IL-6) and oxidative stress-related factors (HIF-1α and VEGFA), as well as the proliferation ability and apoptosis level of GCs, which were collected from control patients (non-PCOS) and PCOS patients, respectively. Subsequently, human ovarian granulosa cell line (KGN) cells were used to verify the anti-inflammatory and anti-oxidative stress effects of chitosan oligosaccharide (COS) on GCs, as well as to investigate the optimal culture time and concentration of COS. The optimal culture conditions were then used to culture GCs from PCOS patients and control patients.

Results: The results showed that GCs from PCOS patients exhibited obvious inflammation and oxidative stress and significantly reduced proliferation and increased apoptosis. Furthermore, COS can increase the expression of anti-inflammatory factors (TGF-β1 and IL-10) and decrease the expression of pro-inflammatory factors (TNFα and IL-6), as well as promote the proliferation of GCs. Moreover, we found that COS can reduce the level of reactive oxygen species in GCs under oxidative stress by inhibiting the expression of HIF-1α and VEGFA and by suppressing the apoptosis of GCs induced by oxidative stress.

Conclusion: We find that inflammation and oxidative stress exist in the GCs of PCOS patients, and COS can reduce these factors, thereby improving the function of GCs.

Keywords: chitosan oligosaccharide; granulosa cells; inflammation; oxidative stress; polycystic ovary syndrome.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Expression of factors associated with inflammation and oxidative stress in GCs from PCOS (P) and control (C) group. Compared to the control group, the expression of IL10 and TGF-β1, which were related to anti-inflammatory, was significantly decreased in GCs from PCOS group (n=25 to 50 in each group). Meanwhile, the expression of pro-inflammatory cytokines (TNFα and IL-6) and oxidative stress-related factors (HIF-1α and VEGFA) was remarkably increased in GCs from PCOS group when compared with control group (n=25 to 46 in each group). (*P<0.05, **P<0.01, ***P<0.001).
Figure 2
Figure 2
Proliferation and apoptosis of GCs from PCOS and control group. The proliferation ability of granulosa cells in PCOS group was significantly lower than that in control group (A, B), n=5 in each group. The apoptosis level of granulosa cells in PCOS group was significantly higher than that in control group (C–F), n=5 in each group. *P<0.05, **P<0.01 vs Control group.
Figure 3
Figure 3
COS improves inflammatory and oxidative stress in KGN cells. COS promotes the expression of anti-inflammatory factors TGF-β1 and IL-10 protein in KGN cells in a dose and time-effect relationship. (Protein expression of TGF- β1 and IL-10 after COS culture for 24 hours (A); Protein expression of TGF- β1 and IL-10 after COS culture for 36 hours (B). COS inhibits the expression of pro-inflammatory factors TNF-α and IL-6 in KGN cells in a dose and time-dependent relationship. (Protein expression of TNF-α and IL-6 after COS culture for 24 hours (C); Protein expression of TNF-α and IL-6 after COS culture for 36 hours (D). Determination of IC50 Value of Hydrogen Peroxide Damage to KGN Cells (E). COS reduces H2O2-induced reactive oxygen generation in KGN cells (F). COS inhibits the protein expression of HIF-1α and VEGFA in KGN cells in a dose-dependent manner (G). *P<0.05, **P<0.01, ***P<0.001 vs Control group; # P<0.05, ## P<0.01 vs Control group; # P<0.05, ## P<0.01 vs H2O2 group.
Figure 4
Figure 4
COS promoted KGN cell proliferation and inhibits apoptosis. The cell proliferation curve shows that the COS promotes the proliferation of KGN cells in a dose and time-dependent relationship (A). COS reverse H2O2-induced apoptosis of KGN cells (B). *P <0.05, **P <0.01, ***P <0.001 vs Control group; # P<0.05, ## P<0.01 vs H2O2 group.
Figure 5
Figure 5
COS improves ovarian GCs inflammation and oxidative stress in PCOS patients. Effect of COS on the expression of inflammatory factors in ovarian GCs in patients with PCOS (A). COS inhibits the expression of oxidative stress related factors in ovarian GCs of patients with PCOS (B). COS decreases reactive oxygen species in GCs of patients with PCOS (C). *P<0.05, **P<0.01, ***P<0.001 vs Control group; # P<0.05, ## P<0.01 vs PCOS+300 μg/mL group.
Figure 6
Figure 6
The microenvironment of GCs in PCOS patients is characterized by inflammation and oxidative stress. COS improves the inflammation and oxidative stress of GCs and thus enhances the proliferative capacity of GCs, reduces the apoptotic level of GCs, and ultimately improves abnormal follicular development in PCOS patients.
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Grants and funding

This research was supported by the Natural Science Foundation of Jiangxi Province (No. 20181ACB20018), Basic Research Project of Shenzhen Science, Technology and Innovation Commission in 2020 (JCYJ20190812161405275), and the National Natural Science Foundation of China (Nos. 81671455 and 81960271).