João Vitor Geisteira Oliveira da Silva¹, Fernanda da Silva Marinho¹, Guilherme Ferreira da Motta Resende², Fábio da Silva de Azevedo Fortes³, Carlos Vinicius Ferreira⁴, Eidy de Oliveira Santos¹

Non-Alcoholic Fatty Liver Disease (NAFLD) is one of the leading global causes of morbidity and mortality, with the potential to progress to Non-Alcoholic Steatohepatitis (NASH), fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). Around 30% of the global population is estimated to have NASH, with half progressing to severe forms. Cirrhosis, characterized by replacement of functional liver tissue with fibrosis, is the main risk factor for HCC and is present in up to 50% of cases at diagnosis. In the absence of approved therapies, complications pose a significant burden on public health systems. Understanding NASH progression mechanisms is essential for early diagnosis and effective intervention. The pathophysiology of NAFLD involves metabolic, inflammatory, and molecular changes, often modulated by the gut and oral microbiota. Dysbiosis promotes the translocation of LPS and other microbial molecules, activating receptors such as TLRs and triggering chronic liver inflammation. Characterizing these signatures may reveal clinically relevant biomarkers. This study aimed to identify biochemical, proteomic, and microbiological candidates associated with NASH progression, using blood, saliva, and urine samples from individuals aged 40–70 years with varying degrees of fibrosis (3 controls, 4 with absent/mild fibrosis, and 4 with advanced fibrosis/cirrhosis). Biochemical alterations in fibrotic patients included elevated CRP (3.72 mg/L; ref: 0–1.0), VLDL (34.3 mg/dL; ref: 0–30), glucose (109.2 mg/dL; ref: 74–99), and ALT/AST (41.5/46.5 U/L; ref: 10–35). All NASH patients had comorbidities such as diabetes, hypertension, hypercholesterolemia, and dyslipidemia. Among 230 proteins identified by LC-MS in saliva and urine, targets with p<0.001, high fold change (fc), and clinical relevance were prioritized. In saliva, DMBT1 (fc: 112.65), S100A9 (fc: 26.28), and S100A8 (fc: 23.19) stood out, linked to systemic inflammation and neoplasms. Carbonic anhydrase VI (CA6; fc: 15.89) showed potential as a cancer and periodontal inflammation marker. Albumin (fc: 3.77), most abundant in urine, reflected proteinuria, impaired hepatic synthesis, and chronic inflammation. In urine, UMOD (fc: 42.56) and AZGP1 (fc: 5.87) were related to tubular integrity and renal fibrosis; AMBP (fc: 65.10) to tubular proteinuria and liver fibrosis. CD59 (fc: 53.41), a complement pathway regulator, was altered in renal/bladder cancers and glomerulopathies. Salivary microbiota, analyzed by MALDI-TOF MS, included 188 bacterial isolates identified by genus (score 1.70–1.99) and species (score 2.00–3.00): 77 from controls, 56 with mild fibrosis, and 55 with advanced fibrosis. Only 18 were not identified (score <1.70). Streptococcus was common to all groups. The control group showed K. pneumoniae, Enterococcus, and Microbacterium. In absent/mild fibrosis, Weissella, Staphylococcus, Klebsiella, and Limosilactobacillus were observed. In advanced fibrosis, Staphylococcus, R. mucilaginosa, Priestia, Levilactobacillus, Klebsiella, and C. koseri predominated — mostly linked to infections, hepatic abscesses, endocarditis, and cholangitis. In conclusion, saliva is a promising matrix for the non-invasive detection of proteomic and microbial profiles associated with NASH progression. This study supports the relevance of clinical, omics, and microbiological approaches in identifying biomarkers with potential impact on personalized medicine and public health.

Agradecimentos: Acknowledgments: The authors thank CAPES, CNPq, and FAPERJ for financial support. We also acknowledge the Laboratory of Technology in Biochemistry and Microbiology, UERJ – RJ/Brazil and Department of Internal Medicine (HUCFF) – UFRJ/Brazil, for providing infrastructure and technical assistance for the development of this project.