Objective: To study the effect of SREBP-1c silencing on lipid metabolism and expression of inflammatory chemokines in a NAFLD model with endoplasmic reticulum stress.
Method: NAFLD model was established in L02 cells treated with oleic acid. SREBP-1c expression was inhibited using RNA interference with a p Silencer-1.0-U6-4476 vector. After transfection with p Silencer-1.0-U6-4476 or control vector for 0 h, 24 h, 48 h and 72 h, the extent of fatty degeneration was shown by Oil Red O staining. The mRNA and protein expression of inflammatory chemokine CCL2 and basic fibroblast growth factor-21 (FGF21) were determined by real time PCR and Western blot respectively.
Results: SREBP-1c silenced L02 cells showed fat droplets with smaller diameter and attenuated fatty deposition, as compared with control cells. The relative CCL2 mRNA levels in SREBP-1c silencing vector transfected L02 cells were 1.03+/-0.11 for 0 h, 1.11+/-0.21 for 24 h, 0.88+/-0.16 for 48 h, and 1.05+/-0.15 for 72 h, which showed no significant difference as compared with control cells (P>0.05, respectively). In addition, no difference was found between the different time points within the same group (P>0.05). However, CCL2 protein levels in SREBP-1c silenced cells were 1.19+/-0.15, 1.07+/-0.18, 0.48+/-0.14, and 0.05+/-0.24 after transfection for 0 h, 24 h, 48 h, and 72 h respectively, which were significantly downregulated as compared to the control group (P<0.01). And CCL2 protein levels between different time points in SREBP-1c silenced cells were also distinct (P<0.01). The relative FGF21 mRNA levels in SREBP-1c silenced L-02 cells were 1.01+/-0.08, 0.91+/-0.22, 0.98+/-0.20, and 1.02+/-0.12 for 0 h, 24 h, 48 h, and 72 h respectively, which were not statistically different as compared with the corresponding control cells. Statistic difference of FGF21 mRNA levels in SREBP-1c knockdown cells of different time points was not found (P>0.05). In striking contrast, robust down regulation of FGF21 protein in SREBP-1c silenced cells was observed, with 0.81+/-0.05, 0.66+/-0.12, 0.58+/-0.08 and 0.19+/-0.13 after transfection for 0 h, 24 h, 48 h and 72 h respectively, as compared to control group (P<0.01). And differences in FGF21 protein level between different time points in SREBP-1c silenced cells were also demonstrated (P<0.01).
Conclusion: SREBP-1c knockdown attenuated fatty deposition in oleic acid treated L02 cells. In addition, silencing of SREBP-1c expression reduced expressions of CCL2 and FGF21 proteins posttranscriptionally, which may play a role in endoplasmic reticulum stress induced inflammatory response in NAFLD.