| Objective:To explore the change and the underline mechanism of phenotype of M1 macrophage after the application of TDZD-8 or LiCl, well-known inhibitors for GSK3β,and SiRNA to block GSK3β activity in vitro. And to investigate the effect of TDZD-8 on macrophage phenotype conversion and its role on preventing kidney injury induced by folic acid in a mouse model.Methods:Bone marrow derived macrophage (BMDM) was polarized to Ml phenotype by LPS and IFNγ. Then they were cultured with different concentrations of TDZD-8 or manipulated by SiRNA to knockdown GSK3β gene for 48 hours. The phenotype of cultured cell was determined by detecting the biomarkers for M1 such as iNOS and IL-1β or M2 such as arginase I or MR through immunocytochemistry, flow cytometric analysis and Western blot. And the expression of STAT1, pSTAT1, STAT3, pSTAT3, STAT6, pSTAT6, PPARγ, pPPARγ were also evaluated. STAT3 inhibitor and PPARγ inhibitor were applied to demonstrate by which pathway TDZD-8 regulates the phenotype conversion of M1 macrophage. For in vivo experiments, C57BL/6 mice were randomly assigned to four groups as Group Ctrl; Group FA treated with folic acid:Group TDZD-pre treated with folic acid and TDZD-8 was given 0.5 hour before folic acid exposure; Group TDZD-post treated with folic acid and TDZD-8 was given 5 days after folic acid exposure. Mice were sacrificed at day 7,14 and 28 to get kidney tissues to investigate the changes of histology. Blood samples were collected at days0,5,7,14 and 28 to assess the renal function.Results:In vitro, mouse BMDMs underwent a shift from M1 to M2 with the presence of TDZD-8 or specific knockdown of GSK3β gene. Among canonical signal transducers and activators of transcription (STAT) signaling pathways, STAT3 inhibition was observed. However, the inhibition of STAT3 was not the main reason for the phenotype conversion of M1 macrophage induced by TDZD-8 for its specific inhibitor did not polarize M1 to M2 in vitro. On the other way, we found that the PPARγ activity was upregulated after GSK3β inhibition. And the inhibitor of PPARγ could largely abrogate the effect of TDZD-8 on converting M1 to M2 macrophage. For in vivo experiments, both pre- and post- treatment with TDZD-8 lowered levels of serum creatinine. More rapid recovery of renal function was observed in TDZD-post mice after day 5 as compared with TDZD-pre mice. Both pre- and post- treatment with TDZD-8 attenuated injury on the tubular cells, prompted cell proliferation and regeneration at day 7, as well as ameliorated the long-term renal fibrosis at day 28. Most macrophages infiltrated to the kidney were MR negative in group FA. However, there was an evident increase in MR positive macrophages in the kidney in TDZD-post mice, but not in TDZD-pre mice, by immunocytochemistry. Immunoblots of MR and arginase I in cortical kidney tissue and flow cytometry detection of MR positive macrophage in the kidney supported the findings in immunostaining. PPARγ activity was found significantly upregulated in TDZD-post mice.Conclusions:TDZD-8 can induce M1 macrophage polarization to M2 through activating PPARγ pathway. Post treatment with TDZD-8 can attenuate renal injury and fibrosis after acute kidney injury. This protective effect of TDZD-8 is partially related to its ability on switching macrophage phenotype to M2 phenotype through activating PPARγ signaling pathway. Our results suggest that TDZD-8 might be a novel method for preventing the progress of kidney injury after acute insult. |
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