Purpose: Systemic administration of young mice plasma (YMP) in aged mice exhibits positive intervention effects on inhibiting microglia activation in the brain^1-4. However, the underlying mechanism remains unclear. The purpose of this study is to investigate the underlying mechanisms of young mice serum (YS, instead of YMP in vivo) on microglia in vitro.

Methods: Primary microglia isolated from postnatal mice were cultured for varying durations of 2, 5, 8, 11, and 16 days in vitro (DIV) to establish a model of chronic senescent microglia that mimics the behavior of microglia in the aged mouse brain⁵. Senescence associated β-galactosidase (SA-β-gal) staining, western blot, and quantitative PCR were performed to evaluate the states of cell senescence. Additionally, primary microglia from DIV2 were exposed to 100 ng/mL LPS to construct an acute inflammatory model. The senescent and inflammatory microglia were then cultured in different conditions: 5% FBS + 5% YS (YS) as a youthful environment, 5% FBS + 5% serum from aged mice (OS) as an aging environment, and 10% FBS as a control (FBS) for a period of 3 days. Bulk RNA sequencing was performed to profile transcriptomic changes under YS treatment. In vitro [¹⁸F]FDG PET imaging and gamma counter were performed to evaluate cellular glucose metabolism in microglia. The capacity of glycolysis and oxidative phosphorylation (OXPHOS) were assessed by seahorse extracellular flux experiments. Microglial phenotypes were analyzed by flow cytometry.

Results: Analysis through senescence associated β-galactosidase (SA-β-gal) staining, western blot (P53, P21), and quantitative PCR analysis (P16^ink4a, P21, IL-1β, IL-6) demonstrated that microglia underwent senescence with prolonged culturing, and exhibiting significant senescence‐like characteristics from DIV8 in vitro (p < 0.05). Therefore, the DIV 8 microglial cells were chosen for investigating the effects of YS in subsequent experiments. Flow cytometry analysis showed that YS treatment decreased CD86 (proinflammatory marker) and increased CD206 (anti-inflammatory maker) expression in microglia. A total of 4468 differentially expressed genes (DEGs) (adjust p ≤ 0.05 and fold change ≥ 2) were identified by RNA sequencing between FBS, YS and OS-treated microglia. Subsequent gene set enrichment analyzes (GSEA) and heatmap analysis revealed a significant upregulation of gene sets associated with OXPHOS and tricarboxylic acid (TCA) cycle in YS-treated microglia. Seahorse extracellular flux analysis further demonstrated an increase in oxygen consumption rate (OCR) and a decrease in extracellular acidification rate (ECAR) in microglia following treatment with YS. Additionally, in vitro [¹⁸F]FDG PET imaging and gamma counter analysis provided further evidence that YS significantly reduced cellular glucose uptake in microglia cells (p < 0.05). These findings suggest that YS induces a shift in energy metabolism from glycolysis to OXPHOS in microglia (p < 0.05).

Conclusions: Our findings indicate that the beneficial effects of YS in inhibiting microglia proinflammatory activation may be attributed to the alteration of glucose metabolism from glycolysis to oxidative phosphorylation. [¹⁸F]FDG PET imaging provides a new avenue for exploring the underlying mechanisms of YS therapy and offers great promise for studying energy metabolism-related diseases.

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Figure. YS regulates microglial glucose metabolism and inhibits microglial pro-inflammatory activation. a, Primary microglia purified from the cerebrum of P0-P3 neonatal mice were cultured in a medium containing 10% FBS and stained with senescence-associated β-galactosidase (SA-β-gal) at several different time points (DIV 2, DIV 5, DIV 8, DIV 11 or DIV16). n=3 individual cultures per group. b, c, The levels of proteins associated with senescence (p53 and p21) were analyzed by Western blot. Red box highlights p53 and p21 were significantly increased at DIV 8. n=3 individual experiments. d-g, mRNA expression levels of senescence and senescence-associated secretory phenotype (SASP) markers (p16inka, p21, IL-1β and IL-6) was significantly elevated in primary microglia starting at DIV 8. DIV, days in vitro. h, An experimental schematic of in vitro studies for testing mitochondrial function. LPS, lipopolysaccharide. i,j, Analysis of the CD86 fluorescence for the detection of M1 activation marker expression in primary microglia. n = 3 independent experiments. k, GSEA shows significantly enriched Oxidative phosphorylation (OXPHOS) and Citrate cycle (TCA cycle) pathways in YS-treated microglia compared with OS-treated microglia and FBS-treated microglia, respectively. The statistical significance (p value) was generated by employing an empirical phenotype-based permutation test procedure. i, Heatmap showing 20 significant DEGs (FDR-corrected p < 0.05) related to OXPHOS and TCA cycle pathway in different in vitro microglial groups. m,n, Oxygen consumption rate (OCR) measurement of primary microglia after different treatment. Olig, oligomycin; FCCP, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone; Rot/AA, antimycin A/rotenone; n = 4-7 independent cultures per group. o,p, Extracellular acidification rate (ECAR) measurement of primary microglia after different treatment. 2-DG, 2-Deoxyglucose; n = 8-12 independent cultures per group. q, In vitro [¹⁸F]FDG PET imaging shows the cellular glucose uptake of primary microglia after different treatment. n = 3 independent cultures per group. r, Comparison of the radioactivity levels (CPM) of primary microglia with a gamma counter. CPM, counts per minute. n = 3 independent cultures per group. Data are expressed as mean ± SEM. one-way ANOVA with Tukey’s test; n.s, no significance; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

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