To investigate the mechanism by which reduced degradation of Bcl-2/adenovirus E1B-19-kDa interacting protein 3 (BNIP3) mediated by neural precursor cell expression developmental downregulation protein 4 (NEDD4) and proteasome β5 subunit (PSMB5) contributes to impaired autophagy flux and cellular damage in arsenic-induced human hepatocellular carcinoma cells (HepG2).

The cytotoxic effects of different doses of sodium arsenite (NaAsO₂) treated HepG2 cells over varying time periods were evaluated by analyzing cell morphology, ultrastructure, cell viability, and hepatic injury enzyme levels (ALT, AST). Immunoprecipitation blotting (Western blot) was used to detect the expression levels of NEDD4, PSMB5, BNIP3, and autophagy-related proteins. Co-immunoprecipitation (Co-IP) experiments were conducted to analyze the interactions among NEDD4, PSMB5, and BNIP3. Specific inhibitors including cycloheximide (CHX), chloroquine (CQ), and proteasome inhibitor (MG132) were employed to inhibit protein synthesis, lysosomal function, and the ubiquitin-proteasome pathway, respectively, to investigate the roles of the ubiquitin-proteasome pathway and autophagy in arsenic-induced HepG2 cell damage.

As the concentration and duration of NaAsO₂ exposure increased, the viability of HepG2 cells decreased and morphogenesis exhibited significant changes. Additionally, autophagy flux was impaired, and cellular damage worsened in a dose- and time-dependent manner. Different concentrations and durations of NaAsO₂ exposure down - regulated NEDD4 and PSMB5 protein expression while up - regulating BNIP3, Beclin-1, P62, and LC3II/LC3I protein expression, showing time - effect and dose - effect relationships (P<0.05). CQ intervention exacerbated NaAsO₂ - induced autophagy flux obstruction and cellular damage (P<0.05). Co-IP and CHX experiments demonstrated that NaAsO₂ exposure reduced the interaction between NEDD4 and PSMB5 with BNIP3, thereby decreasing BNIP3 degradation (P<0.05). MG132 intervention downregulated PSMB5 protein expression and up - regulated BNIP3, P62, Beclin-1, and LC3II/LC3I protein expression, with all differences being statistically significant (P<0.05).

NaAsO₂ exposure can reduce the expression of NEDD4 and PSMB5 in HepG2 cells in a time - and dose - dependent manner, pregulate BNIP3 protein levels, and impair autophagy flux, ultimately leading to cellular damage. The mechanism may involve reduced binding of NEDD4 and PSMB5 to BNIP3, leading to decreased BNIP3 degradation and consequent cumulative accumulation.