The Role Of PTEN In Regulation Of Hepatic Macrophages Activation And Function In Progression And Reversal Of Liver Fibrosis

Background: Hepatic fibrosis,manifested by extensive deposition of extracellular matrix(ECM),is a morbid consequence of chronic liver injury of any etiology.Advanced liver fibrosis results in cirrhosis or even liver cancer.Resident macrophages in liver tissue called Kupffer cells(KCs).Activation of KCs plays a pivotal role in the pathogenesis of liver fibrosis.Accumulating data have indicated that,dual functionally distinct types of hepatic macrophages are activated in the fibrotic liver,which defined as classical activated macrophages(M1)and alternative activated macrophages(M2),participating in formation and reversal of fibrosis.Besides,M2 macrophages participate in Th2-type responses and resolution of inflammation,and promoting progression,tissue repair,and remodeling by secreting fibroblast factors such as IL-10 and TGF-β.It is one of the important way to cure liver fibrosis that the regulation of KC polarization is investigated during hepatic fibrosis.Phosphatase and tensin homolog deleted on chromosome 10(PTEN)is known as a tumor suppressor,which encodes a protein with dual phosphatase activity.PTEN involves in the regulation of innate immunity by negatively regulating PI3K/Akt signaling pathways.Some studies have indicated that PTEN plays an important role in the polarization of tumor-associated macrophages and adipose tissue-associated macrophages.However,the effect of myeloid PTEN in liver fibrosis has not beeninvestigated yet.This study aimed to investigate the regulator role and Mechanisms of PTEN in hepatic macrophages activation and function in progression and reversal of liver fibrosis.Methods: Fibrosis groups were administrated with biweekly subcutaneous injection of a 10% solution of CCl4 in olive oil for 2 to 8 weeks.After 8 weeks,the reversal model terminated CCl4 administration and recovered spontaneously for 2 to 8 weeks.The liver tissues and primary hepatic macrophages(KCs)in each point were collected and liver pathological changes and index of liver fibrosis were measured to confirm that the model of liver fibrosis and reversal was established successfully.RT-q PCR and Western Blot were used to detect the polarization phenotype of KCs in progression and reversal of liver fibrosis.Besides,the expression of PTEN in KCs was detected by immunohistochemistry,RT-q PCR and Western Blot.LPS and IL-4 induced polarization of RAW264.7 macrophages,and the expression of PTEN was detected by RT-q PCR,Western Blot and immunofluorescence staining.Then we transfected si PTEN or p EGFP-N1-PTEN into cells.RT-q PCR,Western Blot,ELISA,immunofluorescence staining and flow cytometry were used to detect the changes of macrophage polarization markers.Akt,p-Akt,STAT6 and p-STAT6 were also detected to determine the relationship between PTEN and PI3 K / Akt / STAT6 signaling pathway.LY294002 is a specific inhibitor of PI3 K / Akt.RT-q PCR,Western Blot and ELISA were used to detect the effect of LY294002 on the expression of polarization markers in macrophages.Results: In vivo,the results indicated that CCl4 treatment triggered a mixed M1/M2 macrophage phenotype in progression and regression of liver fibrosis.In addition,the levels of PTEN in KCs were stikingly decreased in liver fibrosis mice,but it was up-regulated in reversed mice,respectively.Lower expression of PTEN was found in murine M2 macrophages in vitro.M2 macrophages biomarkers were remarkably elevated when si PTEN was transfected into RAW264.7 cells while over-expression of PTEN clearly led the murine macrophage cell to reducing the level of M2 macrophage markers.Blockage of PTEN with si RNA enhanced the protein phosphorylation and expression of p-Akt and p-STAT6,while over-expression of PTEN diminished the expression levels of p-Akt and p-STAT6 in M2 macrophages.It proved that PTEN is a major negative regulator of the PI3K/Akt/STAT6 signaling pathway in M2 macrophages.LY294002 could significantly suppress the expression of M2 macrophage markers.Conclusion: In progression and regression of liver fibrosis,hepatic macrophages are polarized to M1 and M2 phenotypes.Meanwhile,the reduction of PTEN expression in macrophages promotes enhanced-M2 macrophages.These results suggest that PTEN may regulate macrophage polarization via activation of PI3K/Akt/STAT6 signaling to participate in hepatic fibrosis.