Rab18 Down-regulating PLIN2 Through PPARγ To Reduce Lipid Accumulation In Macrophages

[Background and objective] Previous research from our group has found that high expression of adipose differentiation-related protein PLIN2 can promote lipid accumulation in THP-1 macrophages and accelerate the development of atherosclerosis,but its specific mechanism is not fully understood.Therefore,the object of this study was to clarify the relationship between Rab18,PPARγ and PLIN2,then to explore the mechanism of PLIN2 promoting lipid accumulation in THP-1macrophages.[Methods] Firstly,50 μg / mL Ox-LDL was co-incubated with THP-1 macrophages at different time points.The protein level of Rab18,PPARγ,and PLIN2 were detected by Western blot in the cells.At the same time,oil red O staining was used to observe the lipids accumulation in cells.Then,the plasmids of Rab18(WT),Rab18(Q67L)and Rab18(S22N)were transfected into THP-1 macrophages to prepare wild-type Rab18 cells,high activity type Rab18 cells and low-activity Rab18 cells,and the plasmid transfection was detected by immunofluorescence and Western blot.After the results showed that successful transfection,we incubated 50 μg / mL Ox-LDL with each transfected cell for 24 hours.The protein level of PPARγ and PLIN2 were detected by Western blot.Oil red O staining was used to observe lipid accumulation stuation and cellular immunofluorescence was used to observe whether there was co-localization between Rab18 and PPARγ and the effect of Rab18 on PPARγ nuclear translocation.Finally,the high activity type Rab18 cells were pretreated with PPARγ agonists and inhibitors for 2 hours,and then treated with 50 μg / mL Ox-LDL.The protein changes of PPARγ and PLIN2 were detected by Western blot and immunofluorescence in macrophages.[Results] The protein of Rab18,PPARγ and PLIN2 increased with the prolonging of 50 μg / mL Ox-LDL treatment time within 24 hours,and lipid accumulation also increased with time in THP-1 macrophages.Compared with the control group,Rab18 was highly expressed in each transfection group,but there was no differential expression among the groups.Then,Ox-LDL treatment showed that after lipid loading in wild-type Rab18 cells and high activity type Rab18 cells,the expression of intracellular PPARγ and PLIN2 were significantly down-regulated compared with the control group,and intracellular lipid accumulation was significantly reduced.However,in the low-active Rab18 cells,the contentof these two kinds of protein and the lipid accumulation there were no significant change.Moreover,the co-localization of Rab18 and PPARγwas observed by immunofluorescence,and the expression and nuclear translocation of PPARγ in activated Rab18 cells were significantly reduced.In high activity type Rab18 cells,the addition of PPARγ agonist can significantly increase the expression of PPARγ and PLIN2,reversing the inhibitory effect of Rab18 on PPARγ and PLIN2.The addition of PPARγ inhibitor can significantly decrease the expression of PPARγ and PLIN2,and enhances the inhibitory effect of Rab18 on PPARγ and PLIN2.That indicates that Rab18 does have an inhibitory effect on PPARγ and PLIN2.[Conclusion] Rab18 downregulates PLIN2 expression through PPARγ and inhibits lipid accumulation in macrophages.