Rapamycin Uncovers Neuropeptide Y as a Hidden Driver of Joint Inflammation and Cellular Aging
A murine arthritis study finds rapamycin suppresses neuropeptide Y, linking cellular senescence to joint inflammation via SASP cytokines.
Summary
Researchers used rapamycin treatment in a mouse model of rheumatoid arthritis to map the molecular bridge between cellular senescence and joint inflammation. RNA sequencing of joint tissue revealed neuropeptide Y (NPY) as a key differentially expressed gene connecting these two processes. Rapamycin reduced arthritis severity, lowered NPY protein levels, and modulated senescence markers including beta-galactosidase and autophagy genes like Sirt1 and Sirt6. When NPY was silenced in fibroblast-like synoviocytes in vitro, levels of inflammatory cytokines TNF-alpha, IL-1beta, and IL-6 dropped significantly, while Sirt1 expression increased. These findings position NPY as a novel therapeutic target in rheumatoid arthritis.
Detailed Summary
Rheumatoid arthritis (RA) is a chronic autoimmune disease marked by synovial inflammation, joint destruction, and features of accelerated biological aging. Patients with RA show hallmarks of premature aging including telomere shortening, epigenetic aging, and immunosenescence — particularly the senescence-associated secretory phenotype (SASP), which drives persistent cytokine-mediated inflammation. Despite this recognized overlap, the molecular mechanisms linking cellular senescence to joint-specific inflammation remain poorly defined. This study set out to close that gap using rapamycin, an mTOR inhibitor with established anti-inflammatory and anti-senescence properties, as a pharmacological probe in a well-validated murine arthritis model.
Collagen-induced arthritis (CIA) was established in 26 male DBA/1 mice (aged 8–10 weeks) via intradermal injection of type II bovine collagen with Freund's adjuvant, followed by a booster on day 14. Once arthritis was confirmed (total clinical score >4), mice were randomized into two groups of eight: untreated CIA-control and CIA-rapamycin (44 ppm in drinking water for 40 days). An additional 10 CIA mice were used for fibroblast-like synoviocyte (FLS) isolation. Clinical arthritis scores were assessed twice weekly using a validated 0–4 paw scoring system, and histological evaluation used hematoxylin and eosin staining scored for inflammatory infiltrate, synovial hyperplasia, enthesitis, cartilage damage, and bone erosion.
RNA sequencing of joint tissue was performed using the TruSeq Stranded mRNA Library Prep Kit on an Illumina NextSeq 2000 platform. Differential expression analysis via BioJupies identified genes with p≤0.05 and |log2FC|≥1.0. Rapamycin treatment significantly reduced arthritis incidence and severity, with histological improvements across all scored parameters. Transcriptomic analysis identified neuropeptide Y (Npy) as a prominently differentially expressed gene, with reduced expression in rapamycin-treated joints. Protein-protein interaction network analysis using STRING 11.5 (interaction score >0.4, k-means clustering into three clusters) placed NPY at the intersection of senescence and inflammatory signaling pathways.
Immunohistochemistry and RT-qPCR validated the transcriptomic findings. Rapamycin treatment reduced NPY protein levels in joint tissue, paralleling reductions in TNF-alpha and beta-galactosidase — a canonical marker of cellular senescence. NPY receptor expression (Npy1r and Npy2r) was also downregulated in treated animals. Autophagy-related and senescence-regulatory genes Sirt1, Sirt6, and Lc3b were modulated in vivo, consistent with rapamycin's known role in promoting autophagy and suppressing SASP. The mTOR target gene Rps6 was also reduced, confirming effective pathway inhibition.
In vitro validation using siRNA-mediated Npy silencing in CIA-derived FLS (passage 3, 72-hour transfection with 25 pmol siRNA) produced striking results: knockdown of Npy significantly reduced expression of the SASP cytokines Tnfa, Il1b, and Il6, downregulated both Npy1r and Npy2r, and increased Sirt1 expression — suggesting that NPY actively sustains a pro-inflammatory, pro-senescent state in synoviocytes. Cell viability was confirmed via MTT assay. These findings collectively identify NPY as a functional mediator of the senescence-inflammation axis in arthritis, regulated downstream of mTOR, and capable of driving SASP cytokine production through autocrine/paracrine receptor signaling.
Key Findings
- Rapamycin (44 ppm in drinking water for 40 days) significantly reduced arthritis incidence and clinical severity scores in CIA DBA/1 mice compared to untreated controls
- RNA sequencing identified Npy as a top differentially expressed gene (|log2FC|≥1.0, p≤0.05) in joint tissue, with reduced expression following rapamycin treatment
- Immunohistochemistry confirmed reduced NPY protein levels in rapamycin-treated joints, paralleling decreases in TNF-alpha and beta-galactosidase senescence marker
- NPY receptor genes Npy1r and Npy2r were downregulated in rapamycin-treated joint tissue, suggesting disruption of autocrine/paracrine NPY signaling
- Autophagy and senescence regulators Sirt1, Sirt6, and Lc3b were modulated in vivo by rapamycin, consistent with anti-senescence mechanism of action
- siRNA silencing of Npy in CIA-derived FLS significantly reduced SASP cytokines Tnfa, Il1b, and Il6, and increased Sirt1 expression, confirming NPY's pro-inflammatory role
- STRING network analysis (interaction score >0.4, k-means 3-cluster method) placed NPY at the functional intersection of senescence and inflammatory signaling pathways
Methodology
26 male DBA/1 mice with collagen-induced arthritis were randomized into untreated (n=8) and rapamycin-treated (n=8) groups, with 10 additional mice used for FLS isolation; rapamycin was administered at 44 ppm in drinking water for 40 days. Joint RNA was sequenced on an Illumina NextSeq 2000 platform and analyzed via BioJupies (DEG threshold: p≤0.05, |log2FC|≥1.0); protein-protein interaction networks were built using STRING 11.5 with k-means clustering. In vitro validation used siRNA-mediated Npy knockdown (25 pmol, 72 hours) in passage-3 CIA-derived FLS, with RT-qPCR quantification using the ΔΔCt method and Rpl13 as reference gene.
Study Limitations
The study used a murine CIA model, which may not fully recapitulate human RA immunopathology, and all subjects were male, limiting generalizability across sexes. Sample sizes were small (n=8 per group), and the in vitro FLS experiments were conducted in duplicate rather than triplicate, which may affect statistical robustness. The authors did not report specific p-values or effect sizes for most individual gene expression comparisons, and no conflicts of interest were disclosed in the available text.
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