| Liver injury occurs as a common pathological response to acute liver disease, and is characterized by increased inflammation. Kupffer cells (KCs), crucial resident macrophage populations in the pathogenesis of acute liver disease, are driver of both initiation and progression of hepatic inflammation. Protein interacting with C kinase 1 (PICKl) is a scaffolding protein mainly implicated in neurological diseases. Emerging evidence has recognized PICK1 as a critical anti-inflammatory driver in oxidative stress activated microglia during neurological disorders, however, the function of PICK1 in acute liver injury (ALI) remains unknown. Due to the specific plasticity, the current study focuses on the research of the regulatory role PICK1 in macrophage polarization during ALI and its regulatory mechanism, which is used to develop novel targets for the prevention and treatment of ALI.The main contents are divided into four sections, as follows:1. Expressions of PICK1 in the progression of LPS-induced murine acute liver injuryC57 mouseswere intraperitoneally (i.p.) injected with 10 mg/kg of LPS for 24h to induce acute liver injury.Real-time PCR, Western blot were used to demonstrate that the model of acute liver injury was successfully established after 24h, confirming by increased TNF-a, IL-6, CD86 expressions and decreased PICK1 expression time-dependently as compared to that in control group.2. To evaluate the modulation of FSC-231 on inflammation and PICK1 expression in ALI modelWe addressed the function of PICK1 in pathogenesis of ALI by administrating FSC-231, a pharmacological inhibitor of PICK1,2h prior to the LPS injection. C57BL/6J mice were divided randomly into 5 groups (n= 5-8) including control group, LPS group, LPS+FSC-231 groups, which were pretreated with different concentrations of FSC-231 (5μmol/L,25μmol/L,50μmol/L) 2h prior to the LPS injection. Thereafter 24h, both centrifugated serum, liver tissues and isolated KCs were harvested for the further analysis. The results of Serology, Histopathology, Real-time PCR, Western blotindicated that deterioration of liver function and augmentative inflammation were largely enhanced, which was further evident by increased ALT and AST activity in serum after administrating different concentrations of FSC-231 (5 μmol/L,25 μmol/L, 50 μmol/L). Moreover, rather lower PICK1 expression, as well as extensive inflammatory indicators occurred in liver tissues and KCs from FSC-231 pretreated groups than that in the LPS-treated group and control group.These observations indicated that down-regulation of PICK1 might be involved in Ml phenotype switch dutring the progression of ALI, a procedure induced originally by macrophages.3. PICK1 expression in LPS activated macrophages and M1/M2 macrophagesThe murine RAW 264.7 cell lines were frequently applied to demonstrate the interplay between PICK1 and macrophage polarization in vitro. RAW 264.7 cells without treatment were considered as the normal control (MO), the cells were incubated in medium supplemented with LPS (1 μg/ml) alone or LPS combined with interferon gamma (IFN-y,20 ng/ml) for 24h to orient towards M1 macrophages, while macrophages were cultured in medium supplemented with IL-4 (20 ng/mL) for 48h to generate M2 macrophages.To identify the expression of PICK1 in M1/M2 macroohages, we performed Real-time PCR and Western blot. The results showed that the expression of PICK1 in both LPS inducedactivated macrophages and LPS+IFN-y induced Ml macrophages were significantly decreased as compared to untreated macrophages. Nevertheless, IL-4 elevated PICK1 expression in vitro. These observations were in consistence with the decreased PICK1 expression in murine tissures and KCs of ALI, which implied that PICK1 might be associated with the pathogenesis of ALI.4. Modulation of macrophage polarization by PICK1In vitro, using the transfection of GV143-PICK1 or PICK1-siRNA on the M1 and M2 macrophages to over-express or knockdown PICK1, respectively, the results showed that over-expression of PICK1 reduced the mRNA levels of M1 biomarkers (IL-6, IL-1β, IL-12, MCP-1, iNOS, CD86), protein levels of TNF-α, IL-6, iNOS, CD86 and IL-6 production as compared to that in control Ml cells via suppressing NF-κB activity, while enhanced expression of M2 bio marker Arg-1 and IL-10 level in supernatant via promoting STAT6 and p38/MAPK activity. In contrast, PICK1-knockdown or FSC-231 promoted M1, but suppressed M2 macrophage polarization. Additionally, PDTC (inhibitor of NF-κB signaling) at different concentrations could decrease the expression of phospho-p65 and increase PICK1 expression in M1 macrophages. Caveolin-1-knockdown could abrogated the modulation of PICK1 on the M2 biomarkers, which identified caveolin-1 as a potential target gene controlled by PICK1 in macrophages. The mechanistic investigation revealed a dual role of PICK1 in regulating macrophage polarization and implied PICK1 as a potential therapeutic target in ALI. |
