Relationship between senescence in macaques and bone marrow mesenchymal stem cells and the molecular mechanism

Morphological characteristics of BMSCs

With prolonged in vitro culture, some cells in the aging group became broad, and the number of polygonal and irregularly shaped cells began to increase. The nuclear-to-cytoplasmic ratio of the cells decreased. In addition, there were small particles or small vacuoles observed in the cytoplasm. The shape of the cells in the young group was relatively uniform, with a strong refractive index and optimal growth (Figure 1).

Figure 1. Cell morphology of young and aged macaque BMSCs (×100). (A, B) show the primary BMSCs of young and aged macaques, respectively; (C, D) show the P3 BMSCs of young and aged macaques, respectively. With prolonged in vitro culture, some cells in the aging group became broad, and the number of polygonal and irregularly shaped cells began to increase.

Immunophenotype of macaque BMSCs

Flow cytometry was performed on P3 BMSCs, and the cell surface antigens CD29, CD105, CD73, CD184, CD90, CD45 and HLA-DR were detected. There were several differences in the surface antigens of the BMSCs in the young and aged groups, but these differences were not statistically significant (Table 1). Only CD90 showed a significant difference (P < 0.05). The percentage of CD90-positive cells (99.43 ± 0.45%) of young macaques was significantly higher than that of aged macaques (24.37 ± 17.35%).

Ability to induce differentiation of macaque BMSCs

P3 BMSCs in the young and aged groups underwent osteogenic induction. After staining with alizarin red, the dark-red calcium nodules were quantified (Figure 2A, 2B). The number of calcium nodules in the aged group was fewer than that in the young group. In the young group and the aged group, the macaque BMSCs underwent adipogenesis. After adding the oil red O stain, the dye was dissolved in the intracellular lipids, and the lipid droplets in the cells were red (Figure 2C, 2D). Fewer lipid droplets were produced in the aged group than in the young group. In the young group and the aged group, the macaque BMSCs underwent cartilage induction, and alcian blue staining showed the proteoglycans synthesized by the blue chondrocytes (Figure 2E, 2F). The aged group had less proteoglycans than the young group. We then used ImageJ 1.37c software to quantitate the osteogenic, adipogenic and chondrogenic results, which are shown in Figure 2G. Overall, in the aged group, the osteogenic, adipogenic and chondrogenic markers produced were less than those in the young group (^*p<0.05, n=3).

Figure 2. Macaque BMSCs differentiated into multiple cells. (A, B) show the osteogenic induction of young and aged macaque BMSCs, respectively (×40); (C, D) show the adipogenic induction of young and aged macaque BMSCs, respectively (×200); (E, F) show the cartilage induction of young and aged macaque BMSCs, respectively. (×100); (G) We used ImageJ 1.37c software to quantitate the osteogenic, adipogenic and chondrogenic results. In the aged group, less osteogenic, adipogenic and chondrogenic markers were produced than in the young group (^*p<0.05, n=3).

Proliferative ability of macaque P3 BMSCs

It was observed that compared with that in the young group, the expansion rate of P3 BMSCs in the aged group was significantly lower, and the proliferative ability was significantly reduced (Figure 3, Table 2, P < 0.05).

Figure 3. Proliferation curve of BMSCs from aged and young macaques as determined by the CCK-8 method. It was observed that compared with that in the young group, the expansion rate of P3 BMSCs in the aged group was significantly lower, and the proliferative ability was significantly reduced.

Detection of senescence-related SA-β-gal in macaque BMSCs

P3 BMSCs from young and aged macaques were stained with SA-β-gal. The results showed that the percentage of positive BMSC staining in the young group was 1.33 ± 1.51%, which was significantly lower than that in the aged group (35.33 ± 3.88%, p<0.01, Figure 4), suggesting that the BMSCs in aged macaques exhibit cell senescence earlier than those in young macaques.

Figure 4. Macaque P3 BMSCs stained with senescence-associated SA-β-gal (A) and (B) show young macaque and aged macaque P3 BMSCs, respectively (×100). The results showed that the percentage of positive BMSC staining in the young group was 1.33 ± 1.51%, which was significantly lower than that in the aged group (35.33 ± 3.88%, p<0.01), suggesting that the BMSCs in aged macaques exhibit cell senescence earlier than those in young macaques.

Antiaging role of macaque BMSCs in cocultures

Results of the induction of senescence in 293T cells

After induction by different concentrations of t-BHP, cells showed different apoptosis rates, and some cells showed senescence after induction (Figure 5A–5G). Induction with 200 μmol/L t-BHP for 6 h was performed to establish an aging model with macaque BMSCs.

Figure 5. 293T cells treated with different concentrations of t-BHP and stained with SA-β-gal (×100). BMSCs were treated with t-BHP at concentrations of 0 μmol/L (A), 100 μmol/L (B), 200 μmol/L (C), 300 μmol/L (D), 400 μmol/L (E), and 500 μmol/L (F). We have quantified the apoptosis rate in (G). Induction with 200 μmol/L t-BHP for 6 h was performed to establish the aging macaque BMSC model.

Flow cytometry analysis of 293T cells before and after the induction of senescence showed that the detection rates of the surface antigens CD29, CD73, CD105 and HLA-ABC in senescent 293T cells were significantly decreased (Table 3, P < 0.05).

Table 3. Flow cytometry analysis of surface antigens in 293T cells before and after senescence induction ($\overline{X}\pm \text{s}$, n = 3)

Surface antigens	Groups	Positive cell rate(%)	P-value
CD14	Normal group	78.8 ± 0.8	0.089
	Induction group	76.3 ± 1.7
CD29	Normal group	88.3 ± 1.1	0.009
	Induction group**	84.5 ± 0.9
CD90	Normal group	75.5 ± 1.0	0.078
	Induction group	72.7 ± 1.8
CD73	Normal group	4.55 ± 0.53	0.010
	Induction group*	2.77 ± 0.42
CD105	Normal group	83.2 ± 2.1	0.008
	Induction group**	68.4 ± 4.8
HLA-ABC	Normal group	1.12 ± 0.04	<0.001
	Induction group**	0.19 ± 0.02

Telomerase and TCAB1 mRNA expression levels in 293T cells

After the 293T cells were cocultured with BMSCs for 5 d, the mRNA levels of telomeres in the control group and the induced senescence group were significantly higher than those measured before coculture (P < 0.05, Figure 6A), and the mRNA levels of TCAB1 were also significantly increased (P < 0.05, Figure 6B).

Figure 6. Relative expression levels of telomeres and TCAB1 in 293T cells by RT-PCR. (A) shows the senescence-induced 293T cells cocultured with BMSCs. (B) shows the control 293T cells cocultured with BMSCs. (C) shows the senescence-induced 293T cells. (D) shows the control 293T cells. After the 293T cells were cocultured with BMSCs for 5 d, the mRNA levels of telomeres in the control group and the induced senescence group were significantly higher than those measured before coculture (P < 0.05, Figure 6A), and the mRNA levels of TCAB1 were also significantly increased (P < 0.05, Figure 6B).

TCAB1 protein expression levels in 293T cells

TCAB1 is expressed in 293T cells. After coculture with BMSCs, the expression level of TCAB1 was significantly higher than that before coculture. The TCAB1 expression level was weakly positive before induction and positive after induction (Figure 7).

Figure 7. Relative expression of TCAB1 in 293T cells. (A) shows TCAB1 expression in senescence-induced 293T cells cocultured with BMSCs. (B) shows TCAB1 expression in control 293T cells cocultured with BMSCs. (C) shows TCAB1 expression in senescence-induced 293T cells, and (D) shows TCAB1 expression in control 293T cells. (E) shows the Western blots with the quantitated results. TCAB1 expression was weakly positive before induction and positive after induction.

Results of induced aging in macaque BMSCs

After induction with different concentrations of t-BHP for 6 h, SA-β-gal staining showed that some cells were senescent at a concentration of 200 μmol/L; the rate of positive staining was 34.0 ± 4.0%, and the number of SA-β-gal-positive cells was significantly higher than that in the other groups (P < 0.05). No cell death was observed. Therefore, the aging macaque BMSC model was established by exposure to 200 μmol/L t-BHP for 6 h (Figure 8).

Figure 8. BMSCs treated with different concentrations of t-BHP and stained with SA-β-gal (×100). The concentration of t-BHP used to treat BMSCs is 100 μmol/L (A), 200 μmol/L (B), 300 μmol/L (C), 400 μmol/L (D), and 500 μmol/L (E), and 600 μmol/L (F). The aging macaque BMSC model was established by inducing cells with 200 μmol/L t-BHP for 6 h.

Detection of cytokine content in the culture supernatant of macaque BMSCs

The BMSC culture supernatants from the juvenile cell group, old cell group and induced aging group were analyzed by ELISA. IL-6, IL-11 and GM-CSF levels were significantly decreased in the elderly group and the induced aging group compared with the juvenile group (P < 0.01). IL-6, IL-11 and GM-CSF levels were also significantly lower in the induced aging group than in the elderly group (P < 0.01, Figure 9).

Figure 9. Cytokine levels in the culture supernatant of the young, aged and induced aged macaque BMSCs. IL-6, IL-11 and GM-CSF levels were significantly decreased in the elderly group and the induced aging group compared with the juvenile group (P < 0.01). IL-6, IL-11 and GM-CSF levels were also significantly lower in the induced aging group than in the elderly group (P < 0.01).

RT-PCR detection of senescence-associated mRNA expression levels in BMSCs

RT-PCR was used to detect the expression levels of senescence-associated mRNA in the juvenile cell group, old cell group and induced senescence group (Table 4). The results showed that TERT, SIRT1 and SIRT6 levels were significantly lower in the aged cell group than in the juvenile cell group (P < 0.01, Figure 10A, 10D, 10E). The level of P21 was significantly increased (P < 0.01, Figure 10C), and the level of TCAB1 was significantly decreased (P < 0.05, Figure 10B). Compared with those in the juvenile cell group, TERT and SIRT6 levels were significantly decreased (P < 0.01, Figure 10A) and P21 levels were significantly increased (P < 0.01, Figure 10C) in the induced senescence group. The level of TERT was significantly decreased in the induced aging group compared with the aged cell group (P < 0.01, Figure 10A), and that of SIRT6 was significantly decreased (P < 0.05, Figure 10E).

Figure 10. RT-PCR detection of the relative expression of TERT, TCAB1, P21, SIRT1 and SIRT6 in BMSCs. The results showed that TERT, SIRT1 and SIRT6 expression levels were significantly lower in the aged cell group than in the juvenile cell group (P < 0.01). P21 expression was significantly increased (P < 0.01), and TCAB1 expression was significantly decreased (P < 0.05). Compared with those in the juvenile cell group, TERT and SIRT6 levels were significantly decreased (P < 0.01) and P21 levels were significantly increased (P < 0.01) in the induced senescence group. TERT expression was significantly decreased in the induced aging group compared with the aged cell group (P < 0.01), and SIRT6 expression was significantly decreased (P < 0.05).

Western blot analysis of aging BMSC-related protein expression

Western blotting was used to detect the expression of senescence-associated proteins in juvenile cells, aged cells, and senescence-induced BMSCs. The levels of TCAB1 and SIRT6 were relatively low in the aged and induced aging groups compared with those in the young group. p21 and p53 levels were relatively high (Figure 11) in the aged and induced aging groups. ^*p<0.05 compared to the other two groups.

Figure 11. Expression of aging-associated proteins in the young cell group, aged cell group and induced aged BMSC group. The levels of TCAB1 and SIRT6 were relatively low in the aged and induced aging groups compared with the young group. p21 and p53 expression levels were relatively high in the aged and induced aging groups. ^*p<0.05 compared to the other two groups.

Angiogenic factors in macaques

There were significant differences in angiogenesis-related factors between the juvenile group and the old group of macaques (P < 0.05). The levels of HB-EGF and PIGF in the juvenile group were lower than those in the old group, while the levels of VEGF in the juvenile group were higher than those in the old group (Table 5).

Peripheral blood cell analysis

The number of white blood cells and lymphocytes isolated from the peripheral blood of old macaques was significantly increased (P < 0.05) and higher than the normal reference values. The other indicators showed no significant changes (Table 6). The results suggest that elderly macaques may be in a state of chronic inflammation.

Transcriptome sequencing results of macaque peripheral blood mononuclear cells

Differentially expressed mRNA was analyzed by sequencing peripheral blood mononuclear cells from the juvenile and the aged groups. The clustered genes, which may have similar biological functions, are displayed in a heat map (Figure 12). According to the analysis, a total of 5,711 differentially expressed GO terms were detected, of which 2,636 were downregulated and 3,075 were upregulated. The GO terms of the three groups with corresponding gene numbers greater than two were screened. Each of the 10 items was ranked by the associated -log10 ^(P-value) value. The top 30 bar graphs of the GO enrichment analysis are shown in Figure 13A and 13B. The top 30 downregulated and upregulated GO terms are listed in Table 7 according to their P-value and sorted from small to large. The gene functions involved in the downregulated GO terms mainly included regulation of the IFN-γ response, defense against viruses, the type I IFN signaling pathway, replication of the negative regulatory viral genome, response to viruses, innate immune response, the IFN-γ signaling pathway, the response of IFN-α, the response of IFN-β, and the negative regulation of type I IFN product. The gene functions involved in the upregulated GO terms mainly included the transport of mevalonate and the behavioral response to nutrition, spectrin-related cytoskeleton, organic cyclic compound binding, directed red blood cell differentiation, hemidesmosome, regulation of DNA replication, hemidesmosome assembly, other microorganisms that kill cells, and regulation of cell migration.

Figure 12. Heat map of the mRNA clustering analysis of macaque mononuclear cells from juvenile and old groups. Samples_1, 2, and 3 correspond to the elderly group, and samples_7, 8, and 9 correspond to the young group. According to the analysis, a total of 5,711 differentially expressed GO terms were identified, of which 2,636 were downregulated and 3,075 were upregulated.

Figure 13. GO enrichment analysis of differentially expressed mRNA in macaque mononuclear cells from the juvenile and old age groups. (A), Downregulated GO terms. (B), Upregulated GO terms. Red indicates the biological process-related GO term; green indicates the cell component-related GO term; and blue indicates the molecular function-related GO term. The top 30 bar graphs of the GO enrichment analysis are shown in Figure 13A and 13B. The top 30 downregulated and upregulated GO terms are listed in Table 7 according to their P-value and sorted from small to large.

KEGG analysis of the mRNA target genes mainly showed enrichment in 322 pathways, of which 170 were upregulated and 152 were downregulated, showing a top-enriched KEGG pathway map (Figure 14, Table 8). The enriched downregulated KEGG pathways mainly included the NOD-like receptor signaling pathway, the RIG-I-like receptor signaling pathway, the cytosolic DNA-sensing pathway, the Toll-like receptor signaling pathway, nucleotide metabolism-related pyrimidine metabolism, glycosphingolipid biosynthesis-ganglio series related to polysaccharide biosynthesis and metabolism, nicotinate and nicotinamide metabolism associated with cofactor and vitamin metabolism, one carbon pool by folate, growth-related osteoclast differentiation, environmental information processing, and the signal transduction-related NF-kappa B signaling pathways. The enriched upregulated KEGG pathways mainly included the carbon fixation pathways in prokaryotes related to energy metabolism, the mTOR signaling pathway related to environmental information processing and signal transduction, cell growth and apoptosis, antigen processing and presentation related to the immune system, hematopoietic cell lineage, the Fanconi anemia pathway, homologous recombination, growth-related axon guidance, digestion-related digestion and absorption, bile secretion associated with genetic information processing, and replication and repair.

Figure 14. Enrichment analysis of KEGG pathways in differentially expressed mRNA in macaque mononuclear cells from the juvenile and old age groups. The enrichment analysis showed that the target gene was mainly enriched in 322 pathways, of which 170 were upregulated and 152 were downregulated, resulting in a top-enriched KEGG pathway map (A), downregulated KEGG pathways; and (B), upregulated KEGG pathways.

Impact of aging on BMSCs

The life expectancy of macaques is approximately 25-30 years and may be longer under artificial breeding conditions. Female macaques are approximately 3 years old and males are 4 years old when they begin to mature. Therefore, macaques are considered young when they are within 3 years of age and considered of old age when they exceed 13 years of age; these guidelines were applied for macaque grouping.

Senescence-associated SA-β-gal is a hydrolase that catalyzes the hydrolysis of β-galactoside to monosaccharides in senescent cells. This hydrolase is therefore considered a biomarker of senescent cells, as is p16^INK4A [20, 21]. In this study, SA-β-gal staining was used to quantify the number of senescent cells; and the conditions suitable for inducing cell senescence were evaluated. The aging macaque BMSC model was constructed by treating BMSC with 200 μmol/L t-BHP for 6 h. The overall deterioration of cell stemness and the decrease in MSC activity leading to insufficient functional cell renewal in tissues and organs may be an important mechanism of aging.

BMSCs can secrete a variety of biologically active molecules, including cytokines, chemokines and growth factors, which can promote the repair and regeneration of damaged tissue and organs and maintain many physiological functions, such as homeostasis. In this study, 293T cells were cocultured with BMSCs for 5 d, and the expression of telomeres and the telomerase core protein component TCAB1 was detected. The expression of telomeres and TCAB1 mRNA was significantly increased after coculture of 293T cells with BMSCs. After coculture with BMSCs, it was possible to reverse telomere shortening and improve telomerase activity due to the secretion of various biologically active substances, thereby improving the aging state of the cells.

Evaluation of the aging macaque BMSC model

In this study, expression of the senescence-associated gene p21 in BMSCs was detected by RT-PCR. The aged group and the induced senescence group had significantly increased levels of p21 compared with the young group. Western blot analysis showed that both p21 and p53 were highly expressed in the aged group and the induced aging group. The results indicated that both groups of cells had entered the aging state.

Sirtuin (SIRT) is a family of proteins homologous to silent information regulator 2 (Sir2), which are also known as antiaging enzymes. The positive role of SIRT1 in combating aging in MSCs has been confirmed. SIRT1 protects cells from aging-related DNA damage, induces the expression of TERT, and enhances telomerase activity [22, 23]. There is a close correlation among SIRT1, telomere length and human life [24]. SIRT1 plays an important role in maintaining the young state of MSCs [25].

SIRT6 is another member of the Sirtuin family with antiaging effects. In this study, SIRT6 was detected by RT-PCR and Western blotting. The expression of SIRT6 in the aged and the induced senescence BMSC groups was significantly lower than that in the juvenile cell group. BMSCs in the aged cell group and the induced senescence group became aged, and the ability of cells to become activated decreased, while accelerated senescence was observed.

TERT is an important substance for stabilizing the length and structure of telomeres. In this study, the expression of TERT and TCAB1 in the aged cell group was significantly lower than that in the juvenile cell group, as observed by RT-PCR analysis of TERT and TCAB1. The telomerase activity of BMSCs was decreased, the telomere length was shortened, and the proliferation potential of cells in vitro was reduced.

BMSCs can secrete a variety of cytokines and play an immunomodulatory role. In this study, the cytokine levels in the cell culture supernatants of each group were detected by ELISA. The cytokine levels in the aged cell group and the induced aging group were significantly lower than those in the juvenile cell group. The results indicated that the aging of cells is accompanied by a decline in paracrine ability, which affects the immune regulation function.

Some authors [26] have found that bone marrow-derived multipotent MSCs are the most frequently investigated cell type for potential regenerative strategies because they are relatively easy to isolate and are able to differentiate into several mesenchymal lineages. These authors concluded that antioxidant supplementation during MSC expansion reduces the DNA damage load and increases the MSC yield.

Effects of aging on macaques

Increased risk of bacterial infection and chronic inflammation often accompany aging. The proportion of white blood cells and lymphocytes in the peripheral blood of macaques in the elderly group was elevated, suggesting that the old macaques may be in a chronic inflammatory state. The bone marrow supernatant of macaques was analyzed using an angiogenic protein-antibody microarray. The levels of HB-EGF and PIGF in aged macaques were significantly increased, suggesting that the risk of tumors in elderly macaques is increased or that tumors may be present in aged macaques.

Some authors [27] have found that the regeneration potential of MSCs diminishes with advanced age, and this diminished potential is associated with changes in cellular functions. These changes represent novel insights into the aging process and could have implications regarding the potential for autologous stem cell therapy in older patients. In our current manuscript, an aging macaque BMSC model was successfully developed. Both the aged cell group and the induced aging group showed aging morphology; in addition, the expression of aging genes was increased, and the expression of aging-related proteins was increased. The expression trends of the aged cell group and the induced aging group were largely similar. Transcriptome sequencing of macaque mononuclear cells showed that senescence in macaques was related to multiple biological processes, multiple cellular components and multiple molecular functions. Therefore, the novelty of the current manuscript is straightforward.

Macaque mononuclear cell transcriptome sequencing analysis

Regarding the genomic sequence, the similarity between macaque and human is approximately 93%, and the miRNAs are relatively conserved during evolution; these miRNAs can inhibit gene expression and can have biological significance for tumorigenesis, development, metabolism and viral diseases [28].

Peripheral blood mononuclear cells were sequenced to analyze the differentially expressed mRNA. Through GO enrichment analyses, it was found that the GO terms of the functions of downregulated genes mainly included regulation of the IFN-γ response, defense against virus, the type I interferon signaling pathway, negative regulation of viral genome replication, the innate immune response IFN-γ mediated signaling pathway, IFN-α response, IFN-β response, and negative regulation of type I IFN products. The gene functions involved in the downregulated GO terms are primarily related to the immune system. The immune system is an important defense mechanism of the body against the stress of pathogens. Age-related immune system dysfunction is also called immune aging, which is characterized by increased susceptibility to infectious diseases, risk of developing autoimmune diseases, and risk of tumor occurrence [29]. In general, the downregulated GO terms are closely related to the immune system. Immunosuppression leads to decreased secretion of related cytokines and weakened immune function. The data suggest that the ability of the old macaques to defend against viruses is reduced.

The gene functions involved in the upregulated GO terms mainly included the transport of mevalonate, the behavioral response to nutrition, the spectrin-associated cytoskeleton, the binding of organic cyclic compounds, and the differentiation of erythrocytes. The relationship between these gene functions and aging is still unclear and warrants further investigation. Overall, the downregulation of immune function-related pathways suggests a decrease in immune function in aged macaques, similar to the results of the GO enrichment analysis.

The upregulated KEGG pathways involved the carbon fixation pathways in prokaryotes, the mTOR signaling pathways, and the cell apoptosis pathways. In this study, sequencing analysis showed that the expression of the mTOR signaling pathway was upregulated, suggesting an increased risk of tumors in elderly macaques. Cell apoptosis involves the removal of damaged or redundant cells by activating cysteine-containing aspartate-specific cysteine proteases, and the results suggest that there is an increase in apoptosis in aged macaques.

Detection of the proliferation, senescence, phenotype and differentiation ability of macaque BMSCs

Three macaques with an average age of 2.7 years (2.7 ± 0.58) and three macaques with an average age of 20.7 years (20.7 ± 0.58) were selected for the young and old groups. Bone marrow was collected using the aseptic technique. BMSCs were isolated and cultured by adherent culture screening. The structure and morphological changes were observed under the microscope. The CCK-8 method was used after 7 d of dynamic monitoring to study the old and young macaque P3 BMSCs. The β-galactosidase reagent detected the proportion of senescent cells. Flow cytometry was used to detect the cell surface antigens CD29, CD45, CD73, CD90, CD105, CD184 and HLA-DR. BMSCs were induced for differentiation into bone, fat and chondrocytes. Experimental protocols were approved by the Experimental Animal Ethics Committee of Kunming General Hospital of The People’s Liberation Army.

Construction and evaluation of the aging macaque BMSC model

Young cynomolgus BMSCs were treated with t-BHP at a concentration of 100-600 μmol/L for 6 h. The senescence ratio of the cells was measured using the β-galactosidase reagent. ELISA was used to detect the levels of IL-6, IL-11 and GM-CSF in young, aged and induced aged BMSC culture supernatants. RT-PCR was used to detect the mRNA expression levels of TERT, TCAB1, P21, SIRT1 and SIRT6 in BMSCs. The expression levels of the aging-associated proteins TCAB1, P21, P53 and SIRT6 in BMSCs were detected by Western blotting.

Anti-293T cell aging effect of young macaque BMSCs

For this experiment, 293T cells were treated with Tert-butyl hydroperoxide (t-BHP) at a concentration of 0-500 μmol/L for 2 h, and the senescence ratio of the cells was measured using the β-galactosidase reagent. The senescent 293T cells were cultured in the Transwell coculture system together with young macaque BMSCs. The relative expression of the telomere- and telomerase-related gene TBAB1 was detected by RT-PCR. The expression changes of the TCAB1 protein were detected by Western blotting.

Detection of cytokines in macaque bone marrow tissue and sequencing of the mononuclear cell transcriptome

A protein-antibody microarray was used to detect the levels of angiogenesis-related factors in the supernatant of bone marrow from macaques in the aging and young groups. The mRNA of mononuclear cells in the peripheral blood from macaques in the old and young groups was sequenced by second-generation gene sequencing technology. The sequencing data were analyzed by bioinformatics methods to reveal the differences in transcriptome levels in the young and old groups.

Statistical analysis

All statistical analyses were performed using SPSS 21.0 statistical software. The measurement results were expressed as the mean ± standard deviation ($\overline{X}\pm \text{s}$). The paired t-test was used for pairwise comparison between groups. Three-group comparisons and the above results were analyzed by one-way analysis of variance (one-way ANOVA). Analysis of the differential expression of transcripts was performed using the negative binomial distribution test for the differential gene significance test of read values. GO analysis and KEGG pathway analysis were performed using the hypergeometric distribution test for significant differential gene enrichment, and P < 0.05 was considered statistically significant.