Chito-oligosaccharides and macrophages have synergistic effects on improving ovarian stem cells function by regulating inflammatory factors

Abstract

Background: Chronic low-grade inflammation and ovarian germline stem cells (OGSCs) aging are important reasons for the decline of ovarian reserve function, resulting in ovarian aging and infertility. Regulation of chronic inflammation is expected to promote the proliferation and differentiation of OGSCs, which will become a key means for maintaining and remodeling ovarian function. Our previous study demonstrated that Chitosan Oligosaccharides (Cos) promoted the OGSCs proliferation and remodelled the ovarian function through improving the secretion of immune related factors,but the mechanism remains unclear, and the role of macrophages, the important source of various inflammatory mediators in the ovary needs to be further studied. In this study, we used the method of macrophages and OGSCs co-culture to observe the effect and mechanism of Cos on OGSCs, and explore what contribution macrophages give during this process. Our finding provides new drug treatment options and methods for the prevention and treatment of premature ovarian failure and infertility.

Methods: We used the method of macrophages and OGSCs co-culture to observe the effect and mechanism of Cos on OGSCs, and explore the important contribution of macrophages in it. The immunohistochemical staining was used to locate the OGSCs in the mouse ovary. Immunofluorescent staining, RT-qPCR and ALP staining were used to identify the OGSCs. CCK-8 and western blot were used to evaluate the OGSCs proliferation. β-galactosidase(SA-β-Gal) staining and western blot were used to detect the changing of cyclin-dependent kinase inhibitor 1A(P21), P53, Recombinant Sirtuin 1(SIRT1) and Recombinant Sirtuin 3(SIRT3). The levels of immune factors IL-2, IL-10, TNF-α and TGF-β were explored by using Western blot and ELISA.

Results: We found that Cos promoted OGSCs proliferation in a dose-and time-dependent manner, accompanied by IL-2, TNF-α increase and IL-10, TGF-β decrease. Mouse monocyte-macrophages Leukemia cells(RAW) can also produce the same effect as Cos. When combined with Cos, it can enhance the proliferative effect of Cos in OGSCs, and further increase IL-2, TNF-α and further decrease IL-10, TGF-β. The macrophages can enhance the proliferative effect of Cos in OGSCs is also associated with the further increase in IL-2, TNF-α and the further decrease in IL-10, TGF-β. In this study, we determined that the anti-aging genes SIRT-1 and SIRT-3 protein levels were increased by Cos and RAW respectively, whereas the senescence-associated SA-β-Gal and aging genes P21 and P53 were decreased. Cos and RAW had a protective effect on OGSCs delaying aging. Furthermore, RAW can further decrease the SA-β-Gal and aging genes P21 and P53 by Cos, and further increase SIRT1 and SIRT3 protein levels in OGSCs by Cos.

Conclusion: In conclusion, Cos and macrophages have synergistic effects on improving OGSCs function and delaying ovarian aging by regulating inflammatory factors.

Keywords: Chitosan oligosaccharides; Macrophages; Ovarian aging; Ovarian germline stem cells.

Fig. 1

Localization of Mouse OGSCs in the Ovary. A Immunohistochemistry of MVH in in OGSCs cells and oocytes of all stages in ovarian cortex; B Immunohistochemistry of OCT-4 in OGSCs cells in ovarian cortex. Scales bar: 100 μm

Fig. 2

Localization of Mouse Ovarian Germline Stem Cells in the Ovary. A Single-cell cloning of OGSCs derived from a two-step enzymatic method; B Immunofluorescence of OCT-4 and EDU on OGSCs; C RT-qPCR identification (M: mark; O: ovary; OG: OGSCs; N: negtive)and D ALP staining of OGSCs. Scales bar: 10 μm

Fig. 3

RAW Enhanced the Proliferation Effect, Anti-aging and Anti-inflammation of Cos on OGSCs. A The relative protein levels; B The morphology of OGSCs at 72 h; C The SA-β-Gal expression of OGSCs. D The proliferation vitality curves of OGSCs; D, E, F and I The statistics histogram of western blotting were expressed as band density normalized versus GAPDH; G and H The levels of immune factors in supernatant, *p < 0.05, **p < 0.01, ***p < 0.001, * compared with the Control group; #p < 0.05, ##p < 0.01, ###p < 0.001, # compared with the RAW group. Scale bar: 50 μm

Fig. 4

Cos Improved Ovarian Function and Delayed Ovarian Aging via Modulating Macrophage’s Function