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Context: Pulmonary fibrosis is a devastating disease without effective treatment. Rosemary is appreciated since ancient times for its medicinal properties, while biomolecules originated from the plant have an antioxidant and antifibrotic effect. Objective: The effects of Rosmarinus officinalis L. (Lamiaceae) leaves extract (RO) on bleomycin-induced lung fibrosis were investigated. Materials and methods: Male Wistar rats were given a single dose of bleomycin (BLM, 4 mg/kg, intratracheal), while RO (75 mg/kg, intraperitoneal) was administered 3 days later and continued for 4 weeks (BLM/RO1-curative group). Alternatively, RO was administered 2 weeks before BLM and continued 15 days thereafter (BLM/RO2-prophylactic group). Antioxidant activities of RO and lung tissues were studied by standard methods. Histological staining revealed lung architecture and collagen deposition. RO was characterized for its polyphenol content and by high-performance liquid chromatography. Results: RO polyphenol content was 60.52 mg/g of dry weight, carnosic and rosmarinic acids being major components (6.886 and 2.351 mg/g). Antioxidant effect of RO (DPPH and FRAP assay) expressed as IC50 values were 2.23 μg/mL and 0.074 μg/mL, respectively. In BLM/RO1 and BLM/RO2 lung architecture was less compromised compared to BLM, which was reflected in lower fibrosis score (2.33 ± 0.33 and 1.8 ± 0.32 vs 3.7 ± 0.3). Malondialdehyde levels were attenuated (141% and 108% vs 258% of normal value). Catalase and glutathione-S-transferase activities were normalized (103% and 117% vs 59%, 85% and 69% vs 23%, respectively). Discussion and conclusion: RO has a protective effect against BLM-induced oxidative stress and lung fibrosis due to its phenolic compounds.

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Pulmonary fibrosis is characterized by over-population and excessive activation of fibroblasts and myofibroblasts disrupting normal lung structure and functioning. Rosemary extract rich in carnosic acid (CA) and rosmarinic acid (RA) was reported to cure bleomycin-(BLM)-induced pulmonary fibrosis. We demonstrate that CA decreased human lung fibroblast (HLF) viability with IC50 value of 17.13±1.06 μM, while RA had no cytotoxic effect. In the presence of 50 μM of RA, dose-response for CA shifted to IC50 value of 11.70±1.46 μM, indicating synergic action. TGFβ-transformed HLF, rat lung fibroblasts and L929 cells presented similar sensitivity to CA and CA+RA (20μM+100μM, respectively) treatment. Rat alveolar epithelial cells died only under CA+RA treatment, while A549 cells were not affected. Annexin V staining and DNA quantification suggested that HLF are arrested in G0/G1 cell cycle phase and undergo apoptosis. CA caused sustained activation of phospho-Akt and phospho-p38 expression and inhibition of p21 protein.Addition of RA potentiated these effects, while RA added alone had no action.Only triple combination of inhibitors (MAPK-p38, pan-caspase, PI3K/Akt/autophagy) partially attenuated apoptosis; this suggests that cytotoxicity of CA+RA treatment has a complex mechanism involving several parallel signaling pathways. The in vivo antifibrotic effect of CA and RA was compared with that of Vitamine-E in BLM-induced fibrosis model in rats. We found comparable reduction in fibrosis score by CA, RA and CA+RA, attenuation of collagen deposition and normalization of oxidative stress markers. In conclusion, antifibrotic effect of CA+RA is due to synergistic pro-apoptotic action on lung fibroblasts and myofibroblasts.

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We aimed to investigate in the present work, using metabonomics approaches, the scalability of lung fibrosis-biomarkers, in bleomycin (BLM) model of pulmonary fibrosis in rats. Sixty male Wistar rats, weighing 250 ± 10 g, were randomly divided into three groups: a negative control group receiving normal saline treatment (G), an intratracheal BLM instilled group (G), and an aerosol BLM instilled group (G). Rats were investigated at various times after BLM instillation. Metabolic changes observed in different biofluids have been integrated into the results of the histological examination (increase in inflammation, fibrosis score, and TGF-β immunostaining) which provide a novel pathway of biomarkers in pulmonary fibrosis. These two BLM-models showed an efficacy in the production of pulmonary fibrosis in rats, accompanied by an oxidative stress in lung tissue as assessed by the increase of lipid peroxidation and the depletion in the level of antioxidant enzymes such as superoxide dismutase and catalase. The aerosol model was more advantageous showing fibrotic foci occupying the majority of the lung in contrast to intratracheal instillation characterized by a non-homogeneous distribution of the fibroblastic foci.

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