Risk-of-bias assessment of preclinical animal studies (murine and rat models) investigating antibiotics and vascular ageing.
| Ref # | Study type | Drug/Class | Model/Population | Tools used | Key bias domains (summary) | Overall risk | Ageing effects (biomarkers) |
|---|---|---|---|---|---|---|---|
| [63] | Animal | Doxycycline | ApoE−/−, ovariectomized mice | SYRCLE | Randomization not reported/unclear; no blinding; attrition not described | Some concerns | ↓ Aortic lesion area; ↓ MMP-2 activity; ↓ macrophage infiltration; modest ↑ eNOS (endothelial function) |
| [64] | Animal | Doxycycline | DOCA-salt hypertensive rats | SYRCLE | Allocation concealment not reported; selective reporting; no blinding | High | ↑ Systolic BP; ↓ endothelium-dependent relaxation; microbiota shift linked to ↑ vascular inflammation |
| [61] | Animal | Vitamin C + Doxy + Azithro | Murine MSC xenograft context | SYRCLE | Small sample; randomization not reported; blinding absent | High | Modulation of senescence-related genes (↓ p16/p21 signals reported); effect size uncertain |
| [91] | Animal | Chloroquine | Middle-aged male mice | SYRCLE | Lifespan assay only; randomization not reported; attrition not described | Some concerns | ↑ Survival; signals of ↑ autophagy/↓ proteasome activity; indirect ↓ inflammaging |
| [38] | Animal | Antibiotic protocols | Germ-free/ABX-treated mouse models | SYRCLE | Randomization not reported; blinding absent | Some concerns | Microbiota depletion → endothelial dysfunction phenotype; ↑ systemic inflammation |
| [51] | Animal | Aged microbiota transfer | Germ-free mice receiving aged donors | SYRCLE | Allocation concealment not reported; blinding absent | Some concerns | ↑ Inflammaging; ↑ circulating IL-6/TNF-α; ↓ endothelial function; ↑ oxidative stress |
| [56] | Animal | Dysbiosis (age-associated) | Aged mouse microbiome models | SYRCLE | Randomization not reported; attrition bias unclear; no blinding | High | ↑ Vascular inflammation; ↑ ROS; impaired NO bioavailability; ↑ arterial stiffness |
| [57] | Animal | Ageing microbiome | Murine gut microbiome alterations | SYRCLE | Randomization not reported; selective reporting; small cohorts | Some concerns | ↑ Senescence/SASP signaling; altered immune tone; endothelial dysfunction |
| [71] | Animal | Fluconazole | Murine gut barrier/immune model | SYRCLE | High dose; allocation concealment unclear; blinding absent | High | Altered gut barrier → ↑ systemic inflammation; indirect ↑ vascular oxidative stress |
| [74] | Animal | Amoxicillin-clavulanate | Murine mycobiota development | SYRCLE | Randomization not reported; blinding absent | Some concerns | Perturbed mycobiome → ↑ bacterial/fungal imbalance; signals of ↑ inflammaging |
| [75] | Animal | CKD-microbiota transfer/vancomycin, neomycin, metronidazole, and ampicillin. | CKD murine models | SYRCLE | Dietary control limited; blinding absent | Some concerns | ↑ TMAO and pro-inflammatory milieu; ↓ endothelial function; ↑ arterial stiffness |
| [77] | Animal | Antifungal immunity (CX3CR1+) | Murine intestinal immunity | SYRCLE | Randomization not reported; no blinding | Some concerns | Better fungal control → ↓ systemic inflammation; potential ↓ endothelial activation |
| [78] | Animal | Mycobiome manipulation | Murine co-colonization models | SYRCLE | Small n; allocation concealment not described; no blinding | High | Fungal shifts tied to ↑ inflammatory markers; indirect ↑ vascular aging signals |
| [140] | Animal | Tenofovir disoproxil fumarate (antiviral; NRTI) | Adult Wistar rats (13-week oral exposure) | SYRCLE | Randomization unclear; baseline similarity adequate; blinding not reported; incomplete outcome data low risk; selective reporting low risk | Moderate | ↑ Protein carbonyls (+50%); ↓ GSH (–50%); ↓ SOD (–57%); ↓ GPx (–45%); ↓ GR (–150%); ↓ carbonic anhydrase (–45%); ↓ SDH (–29%); marked mitochondrial swelling and loss of cristae |
| [141] | Animal | Praziquantel (antiparasitic) | Bleomycin-induced PF-mice | SYRCLE | No randomization/blinding was fully described• No sample-size justification• Lung injury model (not vascular) reduces external validity• Short-term exposure period• Induced injury rather than natural ageing | Some high risk (indirect for vascular aging) | ↓ Collagen deposition; ↓ hydroxyproline; ↓ TGF-β; ↓ MMP-12; ↓ fibroblast proliferation; ↓ inflammatory infiltration; altered M2/M1 macrophage ratio (anti-fibrotic/remodeling suppression) |
| [142] | Animal | Ribavirin | Caenorhabditis elegans (nematode) | SYRCLE | The model is an invertebrate (nematode), not mammalian or vascular• Drug pharmacokinetics/metabolism in worms may not mirror mammal/human• Longevity endpoints (whole-organism)—no tissue-specific data (e.g., vascular)• Relevance to vascular aging (arterial stiffness, endothelial senescence) is indirect/speculative | High risk/indirect relevance | ↑ Median and maximal lifespan; improved healthspan; ↑ AMPK activation; ↓ mTOR signaling; ↓ cellular ATP levels |
| [137, 138] | Animal | Minocycline | Mice with Ang II-induced hypertension | SYRCLE | • Hypertension model, not aging model • Short-to-medium term drug intervention • Drug used as an anti-inflammatory, not for infection• No vascular-aging specific endpoints (arterial stiffness, senescence, ROS, ECM remodeling)• Confounding by hypertension/neuro-inflammation/gut-brain axis | High risk/indirect relevance | ↓ Blood pressure (attenuated hypertension)Normalization of gut microbiota compositionImprovement in gut wall integrity/reduced gut pathology/inflammation |
| [67] | Animal | Azithromycin (senotherapy) | Murine endometriosis model | SYRCLE | Preventive context; randomization not described; no blinding | High | ↓ Lesion progression; signals of ↓ senescence burden in lesions; systemic vascular effects unclear |
| [72] | Animal | Gut mycobiome & aging/multiple antifungal drugs | Murine ageing models | SYRCLE | Exploratory; small cohorts; randomization not described | Some concerns | Fungal composition shifts ↔ ↑ inflammaging; potential ↑ endothelial activation |
| [42] | Animal | Salinomycin (immune senolysis) | Mouse cancer/ageing models | SYRCLE | Randomization not reported; allocation unclear; attrition bias possible | High | Enhanced immune clearance of senescent cells; ↓ senescence load (p16/SASP) |
| [49] | Animal | Vancomycin and metronidazole | Murine inflammatory disease model | SYRCLE | Allocation concealment unclear; no blinding | Some concerns | ↑ Systemic cytokines; microbiota-driven ↑ oxidative stress; endothelial dysfunction |
| [50] | Animal | Microbiota-sarcopenia | Aged murine muscle function models | SYRCLE | Transfer valid; blinding absent; attrition possible | Some concerns | ↑ Frailty/Sarcopenia indices; systemic inflammaging; potential ↓ endothelial function |
| [58] | Animal | Antibiotics & dysbiosis | Murine dysbiosis models | SYRCLE | Allocation concealment not reported | Some concerns | Broad ABX → microbiome loss; ↑ inflammatory tone; ↓ vascular resilience |
| [59] | Animal | Multiple antivirals/antibiotics | Murine infection/ageing models | SYRCLE | Randomization not reported; no blinding | Some concerns | ↑ Susceptibility to bacterial virulence, ↑ inflammaging, and endothelial activation |
| [73] | Animal | Antiretrovirals | Murine gut microbiome (HIV drug exposure) | SYRCLE | Small number; no blinding | Some concerns | ↑ Mitochondrial toxicity signals; ↑ oxidative stress; ↓ endothelial function |
| [79] | Animal | Multiple antibiotics | Murine ageing gut cohorts | SYRCLE | Observational cohorts; allocation concealment unclear | Some concerns | Age-linked microbiome patterns ↔ ↑ inflammatory cytokines; endothelial dysfunction |
| [101] | Animal | Immunosenescence pathways | Ageing murine models | SYRCLE | Blinding absent; attrition bias possible | Some concerns | Altered innate/adaptive responses; ↑ SASP cytokines; endothelial activation |
| [94] | Animal | Oxidative-stress proteins | Aged murine vascular models | SYRCLE | Randomization not reported; selective reporting | Some concerns | ↑ Protein damage; ↑ ROS; impaired autophagy/proteostasis; endothelial dysfunction |
| [130] | Animal | Miconazole | Murine colitis model | SYRCLE | Confounding (inflammatory model, disease state), lack of vascular context, limited to colon tissue, and no long-term outcome data | Some concerns | ↓ Senescence markers in colon; ↓ NF-κB signaling; improved colitis pathology; altered gut microbiota composition |
| [147] | Animal | Nitroxoline (antimicrobial, 5-nitro-8-hydroxyquinoline) | ApoE−/− mice, 20 weeks old, high-cholesterol diet; nitroxoline 10 mg/kg/day for 8 weeks | SYRCLE | Randomisation not reported; allocation concealment absent; no comment on blinding outcome assessment; incomplete data unlikely (all animals accounted for); no selective reporting observed. | Moderate risk | ↓ LDL-C, ↓ TG, ↓ total cholesterol; ↓ aortic lipid deposition; ↑ LDLR expression; ↓ Ppp2ca; ↓ hepatic steatosis & fibrosis. These changes influence vascular ageing via improved lipid metabolism and reduced plaque formation |
| [23] | In vivo (HGPS accelerated-aging mouse) | Doxycycline (tetracycline antibiotic) | Zmpste24–/– mice (HGPS model) | SYRCLE | Disease-specific model; dosage not equivalent to human; no long-term wild-type validation | Moderate | ↑ Survival +20–25%; ↓ IL-6 50%; normalization of aortic α-tubulin acetylation; ↓ senescence burden; ↑ bone density; ↑ exercise capacity |
The SYRCLE tool was applied to evaluate randomization, allocation concealment, blinding, attrition, and reporting. Ageing effects reported included vascular stiffness, atherosclerotic burden, senescence markers, hypertension, and endothelial dysfunction (n = 31). ↑: increased/upregulated/elevated; ↓: decreased/downregulated/reduced. DOCA: deoxycorticosterone acetate; ECM: extracellular matrix; HGPS: Hutchinson-Gilford progeria syndrome; mTOR: mechanistic target of rapamycin; NO: nitric oxide; ROS: reactive oxygen species; SASP: senescence-associated secretory phenotype; NF-κB: nuclear factor kappa-light-chain-enhancer of activated B cells.