Hypothesis

Age‑related loss of intestinal barrier function occurs only when endocannabinoid signaling declines sufficiently to disinhibit the CB1‑miR‑191‑5p‑NF‑κB‑Rock2 cascade, leading to myosin‑II‑driven tight‑junction remodeling that is detectable first in the distal ileum. In healthy elderly humans, compensatory mechanisms preserve endocannabinoid tone, preventing this cascade and maintaining barrier integrity; barrier loss therefore reflects a secondary, phenotype‑specific shift rather than an inevitable aging process.

Mechanistic Basis

CB1 activation normally suppresses miR‑191‑5p transcription through Gαi‑mediated inhibition of adenylyl cyclase and reduced CREB activity. When CB1 signaling wanes, miR‑191‑5p rises and directly targets the 3′‑UTR of RhoGDIα, lowering its protein levels. Reduced RhoGDIα frees RhoA, which activates ROCK2. ROCK2 phosphorylates the myosin light chain (MLC2), increasing actomyosin contractility and pulling ZO‑1 and occludin away from the junctional complex without necessarily decreasing their total expression. This mechanism explains why rodent aging shows tight‑junction protein loss (due to combined transcriptional and contractile effects) while some human studies report preserved protein levels but increased permeability (purely contractile dislocation). The pathway also links to inflammaging: ROCK2‑mediated contractility promotes formation of paracellular pores that favor LPS translocation, triggering TLR4‑MyD88/TRIF signaling and TNF‑α/IL‑6 production, which further inhibit CB1 expression via NF‑κB, creating a feed‑forward loop.

Testable Predictions

1. In aging humans, the ratio of fecal anandamide (AEA) to 2‑arachidonoylglycerol (2‑AG) will inversely correlate with ileal miR‑191‑5p expression and ROCK2 activity, and positively with ZO‑1/occludin junctional localization (measured by immunofluorescence tension assays).
2. Individuals exhibiting a low AEA/2‑AG ratio (<0.3) will show increased serum zonulin and LPS‑binding protein (LBP) within 12 months, preceding measurable rises in plasma IL‑6 and TNF‑α.
3. Pharmacological CB1 activation with a peripherally restricted agonist (e.g., ACEA) in ex‑vivo ileal mounts from low‑ratio donors will reduce p‑MLC2 levels and restore ZO‑1/occludin junctional localization without altering total protein abundance.
4. Longitudinal cohorts with high baseline AEA/2‑AG ratio will maintain barrier integrity (normal lactulose‑mannitol ratio) and inflammaging markers over 3 years, whereas low‑ratio cohorts will develop progressive barrier leak and inflammaging, independent of changes in tight‑junction gene expression.

Experimental Design

• Participant stratification: Recruit 200 adults aged 60‑80, stratify by baseline fecal AEA/2‑AG ratio (high vs. low tertiles).
• Baseline assays: Quantify fecal endocannabinoids (LC‑MS/MS), ileal biopsies (obtained via colonoscopy) for miR‑191‑5p (qPCR), ROCK2 activity (phospho‑MYPT1 ELISA), junctional protein localization (confocal ZO‑1/occludin intensity at apical membranes), and serum zonulin/LBP.
• Follow‑up: Every 6 months for 2 years, collect serum cytokines (IL‑6, TNF‑α, IL‑1β) via Simoa, urine lactulose‑mannitol ratio, and repeat fecal endocannabinoid profiling.
• Intervention substudy: Randomly assign 30 low‑ratio participants to 12‑week ACEA (1 mg/kg/day) or placebo; repeat ileal biopsies and permeability assays pre‑ and post‑treatment.
• Statistical plan: Mixed‑effects models testing interaction between time, AEA/2‑AG ratio, and outcome variables; mediation analysis to assess whether changes in p‑MLC2 mediate the effect of endocannabinoid decline on permeability.

Potential Implications

If validated, this hypothesis repositions the endocannabinoid system as a modifiable checkpoint that determines whether age‑associated tight‑junction remodeling progresses to pathological barrier leak. It suggests that measuring fecal AEA/2‑AG ratio could serve as an early biomarker for inflammaging risk, and that peripherally acting CB1 agonists might prevent barrier‑driven inflammaging without central psychoactive effects. Furthermore, it reconciles species discrepancies by highlighting that rodents exhibit a steeper decline in endocannabinoid tone with age, whereas humans retain compensatory mechanisms until disrupted by disease‑specific triggers (e.g., diet‑induced obesity, antibiotic‑mediated microbiota shifts).