HMGB1 mediated autophagy and apoptosis in human nucleus pulposus cells; chloroquine amplified apoptosis by inhibiting autophagy

Investigate the cellular response of human nucleus pulposus (HNP) cells to serum deprivation, focusing on the role of high mobility group box1(HMGB1) in regulating autophagy and apoptosis, and elucidate the time-dependent activation of autophagy shifting toward apoptosis under nutrient stress. Additionally, the study evaluated the impact of autophagy inhibition by chloroquine (CQ) on apoptosis progression. HNP samples were obtained from the human biobank with exemption from IRB screening (IRB number DC25SASI0012) to evaluate the impact of nutritional deprivation. Comprehensive analyses encompassed detailed evaluations of cellular morphology, viability, DNA integrity, and metabolic function, providing an integrated view of cellular status. Western blotting (WB), fluorescence-activated cell sorting (FACS), and immunofluorescence (IF) were used to detect LC3, P62, HMGB1, and cleaved caspase-3. Real-time quantitative polymerase chain reaction (RT-qPCR) further revealed changes in gene expression related to autophagy (LC3, P62) and apoptosis (caspase-3), highlighting cellular stress responses. Serum deprivation markedly reduced HNP cell viability, altered morphology, and suppressed metabolic activity, while inducing a time-dependent increase in autophagy, peaking at 48 h. Furthermore, elevated LC3-II, decreased P62, and increased cytoplasmic translocation of HMGB1 indicate activation of HMGB1-mediated autophagy. Simultaneously, cleaved caspase-3 levels rose, suggesting HMGB1's involvement in shifting the balance toward apoptosis. IF and RT-qPCR confirmed enhanced LC3 and cleaved caspase-3 expression, while FACS analysis revealed increased apoptotic cell populations with declining serum levels. These findings highlight a crucial interplay between autophagy and apoptosis regulated by HMGB1 under nutrient-deprived conditions. Eventually, CQ treatment inhibited autophagic flux by blocking LC3-II degradation, thereby amplifying apoptosis. Serum deprivation potently induced HMGB1-mediated autophagy-apoptosis interplay in HNP cells, with CQ enhancing apoptosis by inhibiting autophagy.