Table 1.
Primer sequence.
Figure 1.
Leptin inhibited aggrecan expression.
(A) Leptin (10 ng/ml) significantly inhibited aggrecan transcription in a time-dependent manner, with a maximal response both at 48 h. Dose-dependent studies demonstrated a maximal response to leptin (1–1000 ng/ml) was at the concentration of 1000 ng/ml at the 48 h time point. (B) The effect of leptin on aggrecan protein expression was detected with western blotting ananlysis using GAPDH as an internal control. Error bars represent standard deviration.
Figure 2.
Leptin promoted ADAMTS-4 and ADAMTS-5 expression.
(A) The effect of leptin on ADAMTS-4 and ADAMTS-5 mRNA expression assessed using Real-time RT-PCR. For the dose-dependent studies, NP cells were treated with either medium only or varying concentrations of leptin (1–1000 ng/ml) for 24 h. For the time-depent studies, NP cells were treated with either medium only or leptin (10 ng/ml) for varing time intervals (0–72 h). (B) The effect of leptin on ADAMTS-4 and ADAMTS-5 protein expression was detected with western blotting ananlysis using GAPDH as an internal control. Error bars represent standard deviration. The medium was changed every day. *p<0.05, ** p<0.01, and ***p<0.001.
Figure 3.
Pharmacological inhibition of p38 MAPK pathways prevented the regulation of aggrecan, ADAMTS-4 and ADAMTS-5 by leptin.
(A) The protein amouts of phosphorylated forms of p38 (P- p38 were detected with western blotting analysis. GAPDH was also detected for a loading control. (B) NP cells were treated with vehicle (contorl) (-), 10 ng/ml leptin (+), 10 µM SB203580 for 48 h. The amouts of aggrecan, ADAMTS-4 and ADAMTS-5 protein were detected with western blotting analysis using GAPDH as an internal control. (C) Aggrecan, ADAMTS-4 and ADAMTS-5 mRNA expression were detected with Real-time RT-PCR analysis using GAPDH as an internal control. Error bars represent standard deviration. The medium was changed every day.***p<0.001.
Figure 4.
ADAMTS-4 and ADAMTS-5 silencing attenuated leptin-induced aggrecan degradation in NP cells.
(A) The protein expression of ADAMTS-4 and -5 were detected with western blotting analysis using GAPDH as an internal control. (B) NP cells were treated with control SiRNA (SiRNA-con), 10 ng/ml leptin, SiRNA-ADAMTS-4 or SiRNA-ADAMTS-5 for 48 h. The amouts of aggrecan protein were detected with western blotting analysis using GAPDH as an internal control. The signal in each lane was quantified using ImageJ software and the ratio of aggrecan to GAPDH was determined. (C) Aggrecan mRNA expression were detected with Real-time RT-PCR analysis using GAPDH as an internal control. Error bars represent standard deviration.