Alpha-lipoic acid inhibits TNF-alpha-induced apoptosis in human bone marrow stromal cells

Abstract

TNF-alpha is an important mediator of bone loss. In the HS-5 hBMSC, TNF-alpha and H2O2 increased intracellular ROS levels and induced cell apoptosis through activation of caspases, JNK and NF-kappaB. alpha-Lipoic acid prevented these changes induced by TNF-alpha and H2O2, suggesting its potential therapeutic applications in attenuating bone loss.

Introduction: Oxidative stress is an important mediator of bone loss. TNF-alpha, which plays a critical role in the bone loss after menopause, has been shown to increase intracellular oxidative stress. Because oxidative stress is associated with cell death, we analyzed the apoptotic effects of TNF-alpha and H2O2 on human bone marrow stromal cells (hBMSCs). We also examined the protective effects of an important biological thiol antioxidant, alpha-lipoic acid (alpha-LA), against TNF-alpha- and H2O2-induced apoptosis.

Materials and methods: Using the HS-5 hBMSC cell line, we tested whether TNF-alpha-induced apoptosis was mediated by the generation of excessive reactive oxygen species (ROS). Apoptosis was determined by 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) assay, trypan blue exclusion assay, quantitation of histone-associated DNA fragments in cytosol, and the activation of caspases. The mechanisms mediating these apoptotic effects were determined by Western blotting and enzyme immunoassay.

Results: Both TNF-alpha and H2O2 increased intracellular ROS levels, reduced total cellular glutathione levels, activated caspases-3, -9, and -8, and enhanced hBMSC apoptosis. The activation of c-jun N-terminal kinase (JNK) and NF-kappaB mediated these apoptotic effects. Pretreatment of cells with alpha-LA prevented these changes induced by TNF-alpha and H2O2.

Conclusions: Our data show that TNF-alpha increases intracellular ROS in hBMSC and that TNF-alpha and H2O2 induce apoptosis in hBMSC through the activation of JNK and NF-kappaB. Our findings also suggest that alpha-LA may have therapeutic applications in halting or attenuating bone loss associated with increased oxidative stress.