HMOX1 x KEAP1 x MAPK

The aim of this study is the evaluation of an Azomethine derivative, BCS2, for its antioxidant and anti-tumor activities against mammary carcinoma through the Nrf2- Keap1-HO-1 pathway.

The global prevalence of breast cancer is rising at an alarming rate. The facilitation of abnormal cell proliferation in mammary carcinoma occurs due to the disruption of signaling pathways that balance pro- and antioxidant status, thereby producing oxidative stress that disrupts genomic stability. Therefore, introducing a potent antioxidant molecule with antitumor activity is of paramount importance for treating breast cancer.

Synthesis, characterization, and in-vitro, in-vivo, and in-silico evaluation of an Azomethine derivative, BCS2, for its antioxidant and anti-tumor activities against chemical carcinogen- induced mammary carcinogenesis in Sprague-Dawley rats.

An azomethine derivative, 1-(4-nitrophenyl)-N-phenylmethanimine (BCS2), was synthesized and characterized based on its spectral data. The cytotoxic potential was observed on breast cancer cells, MCF-7, MDA-MB-231, and MDA-MB-468. The in vivo chemotherapeutic potential of BCS2 was established on 7,12-dimethylbenz(a)anthracene (DMBA) induced breast cancer in Sprague-Dawley (SD) rats. The effect of BCS2 on kelch-like ECH-associated protein- 1 (Keap1), Nrf2, heme oxygenase-1 (HO-1), mitogen-activated protein kinase (MAPK), and nuclear factor kappa-light-chain-enhancer of activated-B (NF-κB) was evaluated through ELISA and qPCR techniques. Furthermore, the binding potential and stability of BCS2 with Keap-1, HO-1, and MAPK were predicted using in silico molecular docking and dynamics studies. Additionally, drug-likeness properties of BCS2 were evaluated using in silico ADMET tools.

BCS2 showed remarkable cytotoxic activity on MCF-7 cells followed by MDA-MB- 231 and MDA-MB-468 cells having an IC50 of 2.368μM, 4.843μM and 6.472μM respectively, without affecting normal breast cells, MCF-10A. In the DMBA-induced animal model, BCS2 showed potent antitumor potential and showed protective action on endogenous-enzymatic and non-enzymatic antioxidants in cancer-bearing animals. Marked improvement in cellular architecture and ultrastructure of breast/tumor tissues excised from experimental animals was noted through histopathological and field emission scanning electron microscopy (FESEM) analyses. Significant upregulation of antioxidant proteins, Keap1 and HO-1, and downregulation of inflammatory proteins, MAPK, and NF-κB was observed after BCS2 treatment. The in silico computational studies predicted the potent binding of BCS2 with the active pockets of Keap1, HO-1, and MAPK proteins that validated the biological findings.

The study revealed BCS2's potent antioxidant and antitumor potential against mammary carcinoma through the Nrf2-Keap1-HO-1 signaling pathway.

Alcoholic liver disease is regarded as a leading reason for liver cirrhosis. This study aimed to investigate the protective effect of tetracera asiatica flavonoids (TAF) on alcoholic liver injury (ALI) and explore the associated mechanisms. An ALI rat model was established and then divided into four groups, including ALI group, low-dose TAF (l-TAF) group, medium-dose TAF (m-TAF) group, and high-dose TAF (h-TAF) group. Levels of ALT, AST, ALB, SOD, MDA, NO, CAT, TG, TNF-α, IL-1β, Nrf2, Keap1, HO-1, NQO-1, and GSH-Px were measured in ALI rats in different groups. Pathological changes and inflammatory infiltration were examined using HE staining. Western blot was used to detect expressions of Nrf2, MAPK p38, PERK, NF-κB, ERK1/2 and anti-JNK1/2/3. The results showed that TAF protected against alcoholic liver injury in ALI rats by decreasing ALT and AST levels and inhibiting inflammatory response. TAF significantly reversed alcohol-induced increase in NO (P < 0.05), and remarkably decreased levels of TNF-α (P < 0.001) and IL-1β (P < 0.01), compared with the ALI group. TAF significantly increased the transcription of Nrf2, Keap1, HO-1, NQO-1 and GSH-Px gene (all P < 0.05) and inhibited the alcohol-induced upregulation of MAPK p38 expression (P < 0.001), p-NF-κB/NF-κB ratio (P < 0.001), p-ERK/1/2/ERK1/2 ratio (P < 0.05), and p-JNK1/2/3/JNK1/2/2 ratio (P < 0.05), compared with the ALI group (all P < 0.001). TAF obviously reversed effects of ALI modeling, and remarkably downregulated the expression of PERK and upregulated Nrf2 (all P < 0.001) compared with the ALI rats. In conclusion, TAF attenuates alcohol-induced livery injury through suppressing Keap-1/Nrf2/HO-1, NF-κB/MAPK and PERK/Nrf2 signaling pathways mediated oxidative stress and inflammation in ALI rats.

Betulinic acid (BA) is a pentacyclic triterpenoid with broad pharmacological potential and widely recognized for its neuroprotective effects. This study investigated the potential protective effects of this compound on in vitro differentiated human neuroblastoma SH-SY5Y cells against LPS and FeSO₄-induced ferroptosis, apoptosis, neuroinflammation, and dopaminergic cell death, and explored the underlying mechanisms. Differentiated human neuroblastoma SH-SY5Y cells were exposed to LPS and FeSO₄, and the cellular viability was evaluated using the MTT assay. Flow cytometry was performed to assess apoptotic cell death. Additionally, the expression levels of key markers associated with ferroptosis, apoptosis, and other relevant signaling proteins were analyzed through western blotting and Immunocytochemical staining techniques. However, co-exposure with LPS and FeSO₄ resulted in a dose-dependent reduction in cell viability, which was significantly reversed by pretreatment with BA (0.3-30μM). Exposure to LPS and FeSO₄ increased the DMT1, Bax, caspase-3, and alpha-synuclein, and decreased the GPX4, FTH1, SLC7A11, Nrf2, Keap1, HO-1, PARK7, Bcl-2, NeuN, and TH levels, resulting in cell ferroptosis, apoptosis, and dopaminergic cell death. Furthermore, LPS and FeSO₄ significantly increased the expression of IL-6, TNF-α, and phosphorylation of p38, pMAPK, and pNFkB in the cells. Pretreatment with BA markedly suppressed LPS and FeSO₄-induced upregulation of pro-inflammatory cytokines, ferroptosis, apoptosis, and dopaminergic cell death markers. These findings suggest that BA exerts neuroprotection by modulating the GPX4/Nrf2/Keap-1/HO-1 antioxidant defense and p38MAPK/NF-κB inflammatory signaling pathways, highlighting its potential as a therapeutic agent for oxidative stress-related neurodegenerative conditions, such as Parkinson's disease (PD).