ML385 generally inhibits the expression of HO-1, NF-κB (including nuclear NF-κB, cytoplasmic NF-κB, and phosphorylated NF-κB), NQO1, and TNF-α at both protein and gene levels. However, its effects are cell-type dependent, as some cells show no response, indicating that ML385's impact on these genes varies across different cell types. Relevant literature for reference:

ML385 reduces the expression of both protein and mRNA levels of HO-1 and NRF2.

PMID: 31011401

PMID: 30115814

PMD: 32830144

PMD: 32576270

ML385 alone has no effect on NQO1 expression in HaCaT cells but can reduce NQO1 expression induced by H2O2 treatment.

PMID: 38131276

The answer to this question is provided by MedChemExpress Technical Support. [email protected]

HO-1 and NQO1: ML385 routinely suppresses these canonical NRF2-dependent targets (mRNA and protein) in many cell types and in vivo models — so expect HO-1 and NQO1 to go down when ML385 is effective.

NF-κB & p-NF-κB (p65): blocking NRF2 with ML385 often relieves an anti-inflammatory brake, leading to increased NF-κB activation / nuclear p65 in inflammatory or injury models — but this is context dependent (depends on stimulus, timepoint, cell type).

TNF-α: ML385 commonly increases pro-inflammatory cytokines (including TNF-α) in models where oxidative stress / inflammatory signaling are active (e.g., LPS, pyroptosis models). However, in some cancer cell studies ML385’s net effect on cytokines can be variable.

Below I expand on the evidence, caveats, and concrete experimental suggestions you can use for your oxidized fish-oil / herbal medicine / ML385 experiment.

1. Evidence summary (what the literature shows)

HO-1 & NQO1 (NRF2 targets): Multiple papers report that ML385 reduces NRF2 protein/activity and consequently decreases HO-1 and NQO1 expression (both mRNA and protein levels) across cell lines (epithelial, hepatic, renal, cancer lines) and in animal models. These are reliable readouts that ML385 is inhibiting NRF2 signaling.

NF-κB activation (nuclear translocation and phosphorylation): Several injury/inflammation models show that ML385 treatment increases NF-κB pathway activity — e.g., enhanced TAK1-NF-κB signaling, increased p-p65/nuclear p65 and downstream inflammatory signaling when NRF2 is blocked. This is a commonly observed indirect effect (NRF2 normally restrains oxidative stress and some pro-inflammatory cascades). But note: the magnitude/timing differs by cell type and stimulus.

TNF-α and other cytokines: In inflammasome / macrophage / pyroptosis or LPS-like contexts, ML385 pretreatment raises TNF-α, IL-1β, IL-18 in the supernatant / tissue. In cancer cell lines the cytokine response is less consistent and may not increase. So expect TNF-α upregulation when you combine ML385 with an inflammatory/challenge model (oxidized oil, LPS-mimic), but validate empirically.

2. Important caveats & reasons for variability

Stimulus matters. If cells are not exposed to an inflammatory/oxidative stimulus, downstream cytokine changes after NRF2 inhibition may be minimal. In your design oxidized fish oil is a chronic stressor — that increases the likelihood you’ll see NF-κB/TNF changes when NRF2 is blocked.

Cell type matters. Immune cells (macrophages, microglia) and epithelial cells show stronger NF-κB/TNF responses when NRF2 is inhibited, whereas some cancer lines primarily show changes in proliferation/chemosensitivity with smaller cytokine changes.

Compensatory / off-target effects. ML385 was developed as a selective NRF2 inhibitor, but studies have shown it can affect other pathways (e.g., PI3K-mTOR / RagD regulation) at higher doses or in vivo; this can confound interpretation if you only rely on one readout. Always pair ML385 results with genetic perturbation (siRNA/CRISPR) or ARE-reporter readout.

3. Practical experimental suggestions for your groups and endpoints

Concentrations / dosing commonly used:

In vitro: Many studies use 5–10 µM ML385 (pre-treat 1 h before challenge) for epithelial, hepatic, renal and immune cell lines. Confirm with your cell-type viability assay.

In vivo: Reported systemic doses vary (example: 30 mg/kg in some rodent MCAO/brain injury studies given i.p. or oral vehicle). Use published in vivo protocol for route and solvent and consider pharmacokinetics/toxicity.

Timepoints: NRF2 target suppression (HO-1, NQO1) often appears within hours (mRNA) and by 6–24 h at the protein level. NF-κB phosphorylation and cytokine release can be rapid (1–6 h) after the inflammatory stimulus — run a short early time course (1, 6, 24 h) plus a later timepoint for chronic effects.

Assays to run (recommended):

NRF2 activity: ARE-luciferase reporter (if feasible) or nuclear NRF2 by fractionation + Western.

Downstream targets: HO-1 & NQO1 mRNA (qPCR) and protein (WB). These are the most direct readouts of ML385’s on-target effect.

NF-κB: cytoplasmic vs nuclear p65 (subcellular fractionation + Western), p-p65 (WB), and immunofluorescence for nuclear translocation. Measure both basal and stimulus-induced p-p65.

TNF-α: ELISA in supernatant / serum and TNF mRNA by qPCR.

Controls: include KEAP1/Nrf2 siRNA or CRISPR knockout (if possible) to confirm ML385’s on-target effects. Also include vehicle controls for ML385 solvent.

Interpretation rules of thumb:

If HO-1 & NQO1 fall after ML385, the compound is blocking NRF2 signaling (on-target).

If that is accompanied by increased nuclear p65 / p-p65 and higher TNF-α, that supports the model that NRF2 inhibition removes anti-inflammatory restraint.

If HO-1/NQO1 do not change but NF-κB/TNF change, suspect off-target ML385 effects or independent pathway activation — validate with genetic NRF2 knockdown.

4. Example papers you can cite / check for methods & doses

IEC-6 H/R injury study showing ML385 lowers HO-1 and NQO1 (Western blot evidence).

Studies on renal IRI and ML385 pre-injection showing increased inflammation when NRF2 is inhibited.

Papers showing ML385 augments TAK1→NF-κB in SAH/microglia models (useful for neuroinflammation parallels).

A pyroptosis / macrophage paper where ML385 increases TNF-α, IL-1β and IL-18 after challenge.

A mechanistic caveat paper showing ML385 also affects PI3K-mTOR signaling (RagD), useful to justify genetic controls.

5. Specific notes for your oxidized fish-oil experiment

Oxidized oil likely produces ROS / lipid peroxidation and triggers innate inflammatory pathways → this context favors seeing the classic pattern: ML385 ↓ HO-1/NQO1 and ↑ NF-κB activation & ↑ TNF-α versus the herbal-medicine group (if the herb is NRF2-activating). If the herbal medicine is protective through NRF2 activation, adding ML385 should blunt the herb’s induction of HO-1/NQO1 and unmask higher NF-κB/TNF vs herb alone.

6. Final recommended experimental checklist (quick)

ML385 dose-finding + viability for each cell type (5, 10, 20 µM).

3 timepoints (1 h, 6 h, 24 h) after oxidized oil exposure ± herb ± ML385.

Assays: ARE-reporter or nuclear NRF2, HO-1 & NQO1 (qPCR + WB), p-p65 and nuclear p65 (WB and IF), TNF-α ELISA.

Genetic NRF2 knockdown control (siRNA/CRISPR) to confirm on-target effects.

Consider measuring ROS (DCF) to link redox change → NF-κB effects.