| Objective: To investigate the role of the interaction of complement fragment3a (C3a) and itsreceptor C3aR in uric acid or lipopolysaccharide-induced expression of monocye chemoattractantprotein-1(CCL2) and regulated upon activation, normal T cell expressed and secreted factor(CCL5) in human renal proximal tubular epithelial cell line HK-2.Methods: HK-2cells were cultured in vitro, stimulated by uric acid (UA) of differentconcentrations (0,75,150,300,600umol/L) with different periods (6,12,24,48h). Reverse trans-cription polymerase chain reaction (RT-PCR) was used to analyze the transcription of C3, C3aR,CCL2, CCL5. The most appropriate UA concentration and interfering period for inducing C3orCCL2or CCL5were selected according to the results of RT-PCR. Three methods were used tochange the C3a and its receptor’s interaction under the stimulation of lipopolysaccharide(LPS) orUA, including silencing C3expression with small interfering RNA (siRNA) targeted C3, blockingC3aR with small molecule antagonist sb290157and activating the C3aR with synthetic C3a.Realtime quantitative PCR and enzyme-linked immunosorbent assay (ELISA) were used toanalyze the expression of CCL2and CCL5under these conditions.Results:150umol/L uric acid stimulating for12h significantly up-regulated HK-2cells’ CCL2mRNA transcription. In our experiment, CCL5was not successfully induced by uric acid, it wasthen replaced by LPS in CCL5induction. The over-expression of CCL2and CCL5induced by UAor LPS could be inhibited by C3siRNA transfection or C3aR blockage. C3a stimulation alonesignificantly up-regulated the HK-2cells’ CCL5production but barely raise CCL2expression.however, when it stimulated HK-2cells together with UA or LPS, it could further elevate UA orLPS-induced CCL2expression and UA-induced CCL5production.Conclusion: The activation of HK-2cells’ C3aR provides the important stimulating signals forCCL2and CCL5productions, blocking the interaction of C3a-C3aR will significantly inhibit UAor LPS induced CCL2and CCL5productions. |
