Lentiviral-mediated RNAi targeting caspase-3 inhibits apoptosis induced by serum deprivation in rat endplate chondrocytes in vitro

Abstract

Current studies find that degenerated cartilage endplates (CEP) of vertebrae, with fewer diffusion areas, decrease nutrient supply and accelerate intervertebral disc degeneration. Many more apoptotic cells have been identified in degenerated than in normal endplates, and may be responsible for the degenerated grade. Previous findings suggest that inhibition of apoptosis is one possible approach to improve disc regeneration. It is postulated that inhibition of CEP cell apoptosis may be responsible for the regeneration of endplates. Caspase-3, involved in the execution phase of apoptosis, is a candidate for regulating the apoptotic process. In the present study, CEP cells were incubated in 1% fetal bovine serum. Activated caspases were detected to identify the apoptotic pathway, and apoptosis was quantified by flow cytometry. Lentiviral caspase-3 short hairpin RNA (shRNA) was employed to study its protective effects against serum deprivation. Silencing of caspase-3 expression was quantified by reverse transcription-polymerase chain reaction and Western blots, and inhibition of apoptosis was quantified by flow cytometry. Serum deprivation increased apoptosis of rat CEP cells through activation of a caspase cascade. Lentiviral caspase-3 shRNA was successfully transduced into CEP cells, and specifically silenced endogenous caspase-3 expression. Surviving cells were protected by the downregulation of caspase-3 expression and activation. Thus, lentiviral caspase-3 shRNA-mediated RNAi successfully silenced endogenous caspase-3 expression, preventing inappropriate or premature apoptosis.

Autophagy: A double-edged sword in intervertebral disk degeneration

“…As a major cell type in the CEPs, chondrocytes play an essential role in maintenance of disc functions and balance between ECM catabolism and anabolism [76]. It is well established that chondrocyte loss because of apoptosis is closely correlated with the development of disc degeneration [77][78][79]. Similar to…”

GanDouLing combined with Penicillamine improves cerebrovascular injury via PERK/eIF2α/CHOP endoplasmic reticulum stress pathway in the mouse model of Wilson’s disease

“…GRP78, an ER chaperone protein, regulates protein translocation, folding, and degradation of the misfolded protein [ 40 ] to restore the homeostasis of ER. Caspase-3 is an executioner in caspase cascades, which are of much concern on apoptosis-induced cell death [ 41 ]. Caspase-12 is the initial factor of dependent cell apoptosis pathway.…”

Effects of CCN3 on rat cartilage endplate chondrocytes cultured under serum deprivation in vitro

“…To quantify apoptosis, the cells were washed with cold PBS and suspended in binding buffer. The cells were then stained with 5 µ l annexin V-FITC and 5 µ l PI at 4°C for 15 min, and were analyzed using FACScan flow cytometry (BD Biosciences, San Jose, CA, USA) ( 22 ). The cells were quantified as follows: i) Annexin V negative/PI negative (viable cells); ii) annexin V positive/PI negative (cells in the initial stages of apoptosis); iii) annexin V positive/PI positive (cells in the advanced stages of apoptosis); and iv) annexin V negative/PI positive (necrotic cells).…”

“…PCR products were subjected to amplification curve analysis and were quantified using SYBR Green (Invitrogen; Thermo Fisher Scientific, Inc.). The data were normalized to GAPDH and were presented as ΔΔCt ( 22 ).…”

“…In the present study, a serum deprivation-induced experimental model of apoptosis was used to explore the biological effects of CCN3 on CEP cells ( 22 ), which mimicked the low nutrient conditions in IVDs.…”