Palmatine (ChemShuttle, Trust Way, USA), Doxorubicin, Trypan blue (Sigma Aldrich, St. Louis, USA). Roswell Park Memorial Institute 1640 medium, Trypsin/EDTA, l-glutamine (Pan Biotech, Aidenbach, Germany), Fetal bovine serum, Penicillin-streptomycin (Capricorn scientific, Ebsdorfergrund, Germany), MTA1 (Cusabio, Houston, USA) and p53 Antibodies (Sigma Aldrich, St. Louis, USA).

The 4T1 triple negative breast cancer cell line (ATCC CRL-2539) was purchased from AddexBio (San Diego, CA 92117, USA). The 4T1 cells were cultured in Roswell Park Memorial Institute 1640 medium with 10% fetal bovine serum, penicillin 100 U/mL, streptomycin 100 g/ml and l-glutamine 2 mM. The cells were kept at 37°C in a 5% CO₂ humidified atmosphere. Cell viability was determined with a trypan blue assay before the experiment.

Female balb/c mice weighing 20–30 g and aged 6–8 weeks were purchased from the Noguchi Memorial Institute for Medical Research at the University of Ghana, Legon and kept at the animal house of the Department of Pharmacology, Kwame Nkrumah University of Science and Technology (KNUST), Kumasi. The mice were housed in stainless steel cages with delicate wood shavings as bedding, with free access to chow (GAFCO, Tema), and water. The room was maintained at a temperature of 24–28 C with a relative humidity of 60–70%, and a light-dark interval of 12 h. Before the experiment, the animals were acclimatized for a week.

A fixed number of viable 4T1 triple negative breast cancer cells (2×10⁶) were transplanted via the lymphatic thoracic duct into each recipient mouse (Minn et al., 2005) after an anesthetisia of 50 mg/kg pentobarbitone i.p. was given. Following 48 h of inoculation, the animals were randomized into treatment groups;

Animals were administered Palmatine (1, 5, 10 mg/kg i.p.), Doxorubicin (5 mg/kg, i.p. per week), Lung metastasis (4T1 triple negative breast cancer cells only, 2 × 10⁶) and Negative control (normal saline 1 ml/kg). After 28 days, the following assessments were made;

Arterial blood was collected into heparinized tubes through a 23-gauge needle inserted into the aorta. Blood gas analysis was performed immediately after collection with a Blood gas and electrolyte analyzer BGE800 (Biobase, China).

The lung tissues were excised and embedded in paraffin after being fixed in 10% neutral-buffered formalin. 4 µm thick deparaffinized sections were stained with hematoxylin and eosin (H&E) and analyzed at 10 × magnification. The histology sections were evaluated for metastatic lesions, morphological changes and necroinflammation according to a modified histology activity index (Gibson-Corley et al., 2013; Passmore et al., 2018). The sections were scored by a cumulative quantitative score of 0–4 representing; 0—Normal histological alterations, 1–3—Mild/moderate/severe and 4- Extreme histological alterations (Tables 1 and 2).

The immunoreactions of MTA1 and p53 were determined in the lung tissues with mouse polyclonal antibodies. In brief, 5 µm paraffin-embedded tissue sections were deparaffinized with xylene. The endogenous peroxidase activity was then quenched for 30 min in the dark with 3 percent H₂O₂ in methanol. Tissue sections were dehydrated in graded alcohols before performing heat-induced epitope antigen retrieval with 10 mM sodium citrate. Sections were washed with tris borate saline tween-20 (TBST) and then blocked for 1 h with 5 percent bovine serum albumin. The sections were incubated with the mouse polyclonal primary antibody. After washing for 5 min in TBST, the sections were incubated for 1 h with a horseradish peroxidase-conjugated rabbit anti-mouse IgG secondary antibody mixed with TBS in a 1:200 ratio. The sections were incubated with 3,3′-diaminobenzidine and counterstained with hematoxylin. Images of the slides were captured under a 10 × light microscope Leica DM2500 M (Leica Microsystems, Wentzler, Germany). The manifestation of a dark, brown-colored, intracytoplasmic precipitate is indicative of the presence of MTA1 and p53. The antibody stain intensity of the images obtained was analyzed with an IHC Profiler plugin in ImageJ. The Immunohistochemistry optical density score (IODS) was calculated (Seyed Jafari and Hunger, 2017). IHC optical density score = Percentage   contribution   of   high   positive   × 4 + Percentage   contribution   of   positive   × 3 +   Percentage   contribution   of   low   positive   × 2 +   Percentage   contribution   of   negative   × 1 100

Experimental data were expressed as Mean ± standard error of the mean. Analysis of data was done by Graph Prism software version 8.0.1- using One-way analysis of variance (ANOVA) followed by Dunnett’s post hoc tests. The criterion for significant difference between groups was set at p ≤ 0.05.

Palmatine (1–10 mg/kg) demonstrated a dose dependent increase in the partial oxygen pressure (P_aO₂) 190.70 ± 2.19, 207.0 ± 3.51, 218.5 ± 3.75 and decrease in the partial carbon dioxide pressure (P_aCO₂) 40.75 ± 0.58, 35.10 ± 0.92, 30.00 ± 0.68; bicarbonate (HCO₃) 16.30 ± 0.17, 14.00 ± 0.20, 11.75 ± 0.20; base excess (BE) −1.45 ± 0.02, −0.50 ± 0.03, −0.37 ± 0.01 and anion gap 20.46 ± 0.27, 16.41 ± 0.70, 14.19 ± 0.32 respectively compared to a decreased PaO₂ 185.70 ± 1.89, increased PaCO₂ 43.83 ± 0.51; HCO₃ 17.23 ± 0.20, BE −1.47 ± 0.02 and anion gap 21.98 ± 0.78 in the untreated lung metastasis group consistent with hypoxemia and respiratory distress. This resulted in reduced activity levels and labored breathing. There were no significant changes in values recorded for potential of hydrogen (pH) and chloride (Cl⁻) for all groups in the experiment (Figure 1A–J)

After transplantation of the 4T1 breast cancer cells, metastatic colonization resulted in a remodeling of the lung parenchyma, a large metastatic growth of breast cancer cells marked by peribronchial and perivascular inflammation with the formation of lymphoid nodules. A histology score of 3.67 ± 0.33 (Figures 2B, 1K) was obtained for the untreated lung metastasis group. Palmatine 1 mg/kg showed diffused interstitial fibrotic changes, thickening of alveoli epithelial cells typical of metastatic progression. Administering palmatine 5 and 10 mg/kg dose dependently preserved lung morphology with increased interstitial spaces, thin-walled alveoli and attenuated metastasis-associated endothelial injury; a histology score of 3.33 ± 0.33, 1.67 ± 0.33, 1.33 ± 0.33 (Figures 2C–E, 1K) was obtained for palmatine 1–10 mg/kg respectively.

The expression of MTA1 indicated by a brown stain was evaluated by a semi-quantitative method using the Immunohistochemistry Optical density score (IODS). Tissue sections from the untreated lung metastasis group showed positive immunoreactivity in the cytoplasm for the MTA1 protein; IODS 3.11 ± 0.07 (Figures 3B, 1L) compared to the low expression in palmatine treated groups 1–10 mg/kg; IODS 2.19 ± 0.12, 1.83 ± 0.04, 1.84 ± 0.05 respectively (Figures 3C–E, 1L).

Tissue sections from palmatine treated groups 1–10 mg/kg showed positive immunoreactivity in the cytoplasm for the p53 protein; IODS 1.99 ± 0.06, 2.27 ± 0.12, 2.34 ± 0.12 respectively (Figures 4C–E, 1M) compared to the low expression in the untreated lung metastasis group; IODS 1.64 ± 0.06 (Figures 4B, 1M).