summary
Ovarian fibrosis is a pathological disorder associated with aging and is associated
Brief introduction
Aging is a multifactorial process characterized by a series of progressive changes such as mitochondrial dysfunction, oxidative stress, DNA damage, and chronic inflammation throughout the body (1
Tissue fibrosis can be attributed to excessive deposition of extracellular matrix (ECM) components, often caused by chronic inflammation (7).
In our previous study, we reported that postmenopausal women with type 2 diabetes (T2D) using metformin had normal ovarian fibrosis and decreased M2 macrophage polarization compared to postmenopausal non-diabetic women (11).
Given the possible effects of metformin on age-related ovarian fibrosis, we sought to investigate whether metformin can prevent and/or reverse ovarian fibrosis
outcome
Metformin prevents but does not reverse age-related ovarian fibrosis
Mice were acquired at 14 and 18 months of age, respectively, and aged indoors for 6 months with or without metformin addition to drinking water as a preventive and reversal treatment for ovarian fibrosis (Figure 1).
Light is not specific to collagen, but in combination with polarized light, and fiber collagen undergoes a special birefringence enhancement that visualizes and quantifies fiber thickness (22, 23).
Using second harmonic microscopy and imaging techniques, high-resolution quantitative images
The establishment of ovarian fibrosis in immunocompromised mice accelerates
Although the initial study was conducted with C57BL/6 (the most commonly used mouse strain in the study), we sought to investigate whether other immunocompetent or immunocompromised mouse strains showed age-related developments of
Comprehensive atlas of ovarian cell types after aging and metformin treatment
To study the changes in the cell population that make up the senescent ovaries and the effects of metformin treatment, we collected and isolated the entire ovaries
Single-cell sequencing shows changes in fibroblast subsets in aging ovaries treated with metformin
Because the ECM components produced by tissue fibrosis are primarily produced and maintained by fibroblasts (25) Next, we attempted to study fibroblast subtypes and gene expression
To determine how aging and metformin affect the composition of fibroblast populations, we used a Monte Carlo/permutation test to calculate the cell ratio of each cluster between aging (young vs.
To reveal the overall structure of the fibroblast lineage in our data, we performed a trajectory inference analysis using the "Slingshot" software package (27) which characterizes the trajectories of fibroblast clusters into different lineages in a semi-supervised manner (black dot and black line Fig.
The expression of fibroblast genes is specific and specific between clusters
To study cluster characteristics and functional pathways associated with each cluster, we identified DEGs (Table S2)
Two different populations of myofibroblasts (Acta2+) were also identified and DEGs were measured (Figure 6D).
To identify biological processes associated with each population, we used PANTHER to assess pathway enrichment of DEGs per cluster (Table S3).
The effect of metformin on the ovaries of mice may be indirect
Metformin is known to function in the liver by targeting complex 1 of the mitochondrial electron transport chain and activating AMPK [(19)].
Because of its structure, metformin relies on membrane transporters that are absorbed and secreted by cells, such as 22 members of the solute carrier family 1, 2, 3, and 4 (Slc22a1, Slc22a2, Slc22a3, and Slc22a4).
), 47 members of the solute carrier family 1 and 2 (Slc47a1 and Slc47a2), and 29 members of the solute carrier family 4 (Slc29a4) (18) .
To determine whether metformin may be transported into ovarian cells, we studied gene expression of all known metformin transporters reported in our scRNA sequence analysis (Figure S3A) and found no expression of any known transporters, except for Slc22a1 in myofibroblasts, which are reported to be less than 6%
of cells in this cluster.
According to the human protein atlas SLC22A type 1 gene (also known as OCT1) is not expressed in human ovarian tissue [(35), humanproteinatlas.
org website] suggests that metformin's effect on the ovaries is not mediated by direct action on ovarian fibroblasts
。
Because metformin primarily targets inhibitory mitochondrial complexes I (NADH: ubiquinone oxidase) and oxidative stress response pathways (19, 36), we visited a common list of genes for these pathways to assess their expression in our fibroblasts (Figure S3B).
We found no significant reduction in gene scores for the metformin complex I gene module and no increase in oxidation reaction gene scores if metformin directly targeted fibroblasts
, as we expected.
The metformin group had a slight increase in mitochondrial gene module scores compared to older mice, possibly due to alterations in SASP-associated fibroblasts, which had lower mitochondrial activity (37, 38) and a higher
proportion of fibroblasts activated in the metformin group.
Lack of metformin transporter expression or alterations in cell bioavailability suggest that metformin may not have a direct effect
on ovarian tissue.
A responsive macrophage population was found in the metformin treatment group
Because our observations in NSG mice show that immune cells play an important role in the establishment of ovarian fibrosis, we attempted to further investigate immune cells in single-cell RNA expression data and cell population proportions
associated with senescence and metformin treatment.
From the original dataset (Figure 4B), we sorted 12 clusters identified as CD45 + and then reclassified these immune cells, identifying a total of 16 different CD45+ clusters (Figure 7A ).
As expected, aging alters the immune structure of the ovaries, which is associated with an increase in the proportion of B cells, T cells, natural killer T cells, native lymphocytes (ILC) and mast cells, and a decrease in macrophages and dendritic cells (Figure 7B).
Compared with elderly mice, metformin prophylaxis in elderly mice led to an increase in B cells and T cells, as well as the recruitment of metformin-reactive macrophage populations
.
Therefore, we identified a population of macrophages specific to the metformin group, called "macrophage III" (Figure 7C).
From the original CD45+ data, we divided macrophages into four categories: macrophages I, II, III, and monocytes, and reclassified these populations as shown in UMAP (Figure 7D).
We then performed trajectory inference with the Slingshot software package to determine whether the population came from a monocyte line in the same tissue, or whether it was recruited into the ovaries (black dot and haddock figure 7D).
One possible explanation for the separation between macrophage I and macrophage III subpopules and the lack of phylogenetic links between monocytes and macrophage III clusters is that these macrophages may differentiate
in different tissues before they are recruited to the ovaries and infiltrated into the ovaries.
Further work is needed to determine the origin of
macrophage subsets in the treatment of metformin in the ovaries.
To determine the unique characteristics of the metformin-induced macrophage population, we compared its gene expression profiles with all other macrophages (see Table S4 for a complete list of Figure 7E).
Based on statistical enrichment analysis of DEGs and PANTHER pathway annotations, we found that macrophage III clusters were enriched into activation of B cells and T cells, phosphatidylinositol 3 kinase (PI3K) kinase activity, and activity of integrins and apoptotic signaling pathways (Figure 7F).
Since we found that cytokine- and chemokine-mediated inflammation, T cell activation, and IFNg signaling in fibroblasts are associated with senescence and ovarian fibrosis (Figure 6E), we further investigated the cellular communication networks of this unique macrophage population by using LIANDA R packets for ligand receptor analysis of single-cell data (Table S5) (39).
We limited the analysis to the communication of homologous receptors expressed by 7 fibroblast clusters as ligand sources with immune cells as the ligand source and 7 fibroblast clusters as recipient populations to obtain a list
of possible signaling ligands secreted by immune cells and acting on fibroblasts.
Notably, of all immune cells, macrophages appear to interact the most with fibroblasts (Figure 8A).
To determine the specific ligands secreted by macrophage III cells, we further screened all macrophages for ligands uniquely expressed and compared the list of ligands in three groups of macrophages (Figure 8B).
We found 8 macrophage swarm III-specific ligands and their target receptors in fibroblast clusters (Figure 8C
).
Taken together, our findings suggest that age-related ovarian fibrosis is associated
with a higher proportion of immune aging and fibroblasts with SASP-like phenotypes and expression of non-classical MHC molecules.
As a limitation of this study, it is unclear whether SASP accounts for a higher proportion of Cd74Hi fibroblasts as a result of ovarian fibrosis or the cause thereof
.
Nonetheless, we demonstrate that metformin prevents ovarian fibrosis by modulating immune cells, which is associated with a decrease in the proportion of SASP associated with an increased
proportion of Cd74 Hi fibroblasts and positive remodeling myofibroblasts.
Future work should focus on the involvement of the immune system in the process of ovarian fibrosis, as well as the mechanisms
by which metformin regulates the role of these cells to prevent ovarian fibrosis.
Finally, we know that young and healthy ovaries continue to be extensively reshaped by ECM in their reproductive lives (77).
Thus, the ovarian interstitium is constantly remodeled to accommodate follicle generation, ovulation, luteal formation, and tissue repair, all depending on the estrous cycle (41).
During menopause, the ovaries stop circulating, which limits the pressure of matrix remodeling in the estrus cycle and may promote permanent collagen deposition, leading to ovarian fibrosis
.
As another contributing factor, immune aging blocks the clearance of SASP-producing fibroblasts, further promoting a favorable microenvironment
.
Our findings suggest that metformin intake can prevent the persistence of SASP-produced fibroblasts and maintain healthy tissue homeostasis
by recruiting immune cells into aging ovaries.
