Neferine inhibits interleukin-1beta-induced chondrocyte apoptosis / 中国组织工程研究

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.