Abstract: BACKGROUND: Apoptosis is involved in the formation of degenerative joint diseases. Therefore, anti-chondrocyte apoptosis may be an effective way to treat osteoarthritis. Neferine has a wide range of pharmacological activities including anti-inflammatory, anti-tumor and anti-apoptotic activities, and its effect on chondrocyte apoptosis is not clear.
OBJECTIVE: To investigate the effect of neferine on chondrocyte apoptosis induced by interleukin-1β and elucidate its possible mechanism. 
METHODS: (1) The rat chondrocytes at logarithmical stage were taken and intervened in five groups. The control group was cultured routinely. The model group was routinely cultured for 24 hours after treatment with interleukin-1β for 2 hours. The low-, medium-, and high-dose groups were treated with interleukin-1β for 2 hours and then cultured with 5, 10, and 20 μmol/L neferine for 24 hours, respectively. At the end of culture, cell apoptosis, chondroglycoprotein 39 and type II collagen levels in cell supernatants, mRNA and protein expression of apoptosis-related proteins, and mRNA and protein expression of proteins related to the protein kinase R-like endoplasmic reticulum kinase (PERK)/activating transcription factor 4 (ATF4) signaling pathway were detected. (2) Rat chondrocytes at logarithmic growth period were taken and divided into four groups: control group and model group were treated with the same intervention as above, drug group was treated with interleukin-1β for 2 hours and then cultured with 20 μmol/L neferine for 24 hours, and activator group was treated with interleukin-1β for 2 hours and then cultured with 20 μmol/L neferine and CCT020312, an activator of PERK/ATF4 signaling pathway, for 24 hours. At the end of culture, cell proliferation was detected by cell counting kit-8 assay, apoptosis was detected by flow cytometry, and mRNA and protein expressions of apoptosis-related proteins and PERK/ATF4 signaling pathway-related proteins were detected. 
RESULTS AND CONCLUSION: (1) Compared with the control group, the model group showed increased apoptosis (P < 0.05), decreased proliferative activity (P < 0.05), increased level of chondroglycoprotein 39 (P < 0.05), decreased level of type II collagen (P < 0.05), decreased mRNA and protein expression of Bcl-2 protein (P < 0.05), increased mRNA and protein expression of Bax protein, increased caspase-3 mRNA expression, increased Cleaved-caspase-3 protein expression (P < 0.05), increased mRNA expression of PERK, ATF4, and C/EBP homologous protein (P < 0.05), and increased protein expression of p-PERK, ATF4, and C/EBP homologous protein (P < 0.05). Compared with the model group, neferine reversed the above effects of interleukin-1β on chondrocytes in a concentration-dependent manner. (2) Compared with the drug group, the activator group showed increased apoptosis (P < 0.05), decreased proliferative activity (P < 0.05), elevated mRNA expression of caspase-3, ATF4, and C/EBP homologous protein (P < 0.05), and elevated protein expression of Cleaved-caspase-3, ATF4, and C/EBP homologous protein (P < 0.05). (3) To conclude, neferine inhibits interleukin-1β-induced chondrocyte apoptosis and enhances cell proliferation activity, and the mechanism of action may be related to blocking the PERK/ATF4 signaling pathway in the endoplasmic reticulum.

Key words: neferine, interleukin-1β, chondrocyte, apoptosis, endoplasmic reticulum, PERK/ATF4 signaling pathway