Inhibitory Effect And Underlying Mechanisms Of Galanin On Murine Macrophage Cell Lines

Background and Aims:Nonalcoholic fatty liver disease（NAFLD）,whose disease spectrum including simple fatty liver（SFL）,nonalcoholic steatohepatitis（NASH）and its related fibrosis and cirrhosis,is a metabolic stress induced liver injury raletad to insulin resistance and genetic susceptibility.It is well accepted that inflammatory response is the key rate-limiting step.Plenty of evidence shows that Kupffer cell（KC）play a crucial role in the disease progress to NAFLD.KC can be activated by LPS or endotoxin.When hepatic steatosis occurs,the KCs can be actived with the combination of the Toll-like receptor 4 through the Damage-associated molecular patterns.The activated KCs release a large number proinflammatory factors including TNF-αand IL-6,which can aggravate hepatic steatosis and liver cell damage.The TNF-αcan also lead to self-activated KCs through an autocrine manner.KCs play a predominant role in liver steatosis and progression to NASH,as well as sepatic fibrosis.Therefore,it is crucial to look for the possible endogenous regulatory molecules of KCs.Only with that can we know the pathogenesis of metabolic inflammation and the reason that how simple fatty liver develop to NASH,then to find the possible therapeutic targets.Galanin serves as a regulatory factor of appetite and energy metabolism.Galanin is a sensory neuropeptide with a length of 29 amino acids.Some researchers showed that the local inflammation might be involved in the regulation of galanin expression in liver,while there was few research on the physiological function of galanin in the liver.What we found in our earlier studies was that the dynamic changes of circulating levels of galanin was associated to the high fat diet induced hepatic steatosis in rats and the progress of NASH.What’s more,the level of serum galanin was related to the proinflammatory factors such as MCP-1 as well.Further animal studies have demonstrated that exogenous galanin injection could inhibit the CD68 positive cells aggregation in the mouse model of NASH,while it failed to relieve hepatic steatosis and ballooning.The above mentioned research falied to illustrate the underlying mechanism,here we hypothesized that galanin might directly affect the function of liver macrophages or KCs.There is no report of galanin in regulating KCs function in NASH.Based on the foundation that galanin is an important regulatory factor of energy metabolism,we hypothesized that galanin might function through a certain key signaling pathway or protein to affect metabolism in inflammation,inhibiting the inflammatory cells and inflammatory reaction.Insight into the role of metabolism in inflammation is supported by recent findings about the role of AMPK in inflammation and immunity:inhibition by AMPK of lipopolysaccharide-induced NF-κB phosphorylation and TNF-αproduction in macrophages.Galanin may affect the energy metabolism,then activate AMPK signaling pathway,effect the macrophage function,severs as a suppressor of inflammation.Our study will test the above mentioned hypothesis.Methods:RAW264.7 and J774A.1 mouse macrophage cells were cultured in DMEM（100 U/ml of penicillin and 100 g/ml of streptomycin）containing 10%FBS at37℃,in a humidified 5%CO₂ atmosphere.Cells were divided into three groups,namely the control group（vehicle only）,lipopolysaccharides（LPS）group（LPS only）and galanin treatment group（LPS plus galanin）.Cells were serum-starved for 4 h followed by LPS（1μg/ml）or vehicle incubation for 2 h;Cell were then incubated with or without galanin（250 nmol/L,500 nmol/L,1000 nmol/L）for 24 h.Cell morphology was observed under the microscope;Cell proliferation assays were performed by using Cell Counting Kit-8;mRNA levels of tumor necrosis factor-α（TNF-α）,interleukin-6（IL-6）and interleukin-1β（IL-1β）were measured by real-time RT-PCR and Western blotting analysis were performed to determine the inducible nitric oxide synthase（iNOS）and arginase 1（Arg1）protein levels;The phagocytotic activity of RAW264.7 or J774A.1 cells was assessed by the latex beads;luorescent measurement of intracellular reactive oxygen species（ROS）was assessed by the fluorescentprobe DCFH-DA;Western blotting analysis were performed to determine the AMPKα,pAMPKα,ACC and pACC protein levels;Semi quantitative RT-PCR was performed to determine the expression of galanin receptors（GalR1,GalR2 and GalR3）.Results:In the RAW264.7 cell line,the mRNA levels of IL-6,IL-1βand TNF-αin LPS group were increased by（7083±432.5）,（3106±104.3）and（108.0±47.58）folds of that in the control group,respectively（P<0.05）;and the mRNA levels of IL-6,IL-1βand TNF-αin cells treated with galanin decreased by 55.16%,23.71%and95.10%,respectively,as compared with LPS group（P<0.05）;Relative level of iNOS protein in LPS group increased by（19.5±1.964）folds compared with that of the control group,and relative level of Arg1 protein in LPS group was decreased by62.18%compared with that of the control group;After galanin treatment,iNOS protein level decreased by 41.63%and Arg1 protein increased by（2.31±0.12）folds lower or greater than that treated with LPS alone（P<0.05）.The phagocytotic activity in cells treated with galanin（250nmol/L,500nmol/L and 1000nmol/L）decreased by35.73%,38.63%and 50.36%,respectively,as compared with LPS group（P<0.05）;The ROS levels in cells treated with galanin decreased by 18.22%,19.17%and21.35%compared with LPS group（P<0.05）.GalR2 and GalR3 are expressed in the control group,the LPS group,and the galanin treatment group.In the J774A.1 cell line,relative level of iNOS protein in LPS group increased by（1.7±0.13）folds compared with that of the control group;After galanin treatment,iNOS protein level decreased by 49.17%and 55.36%and Arg1 protein increased by（1.49±0.12）folds lower or greater than that treated with LPS alone（P<0.05）.The phagocytotic activity in cells treated with galanin（250nmol/L,500nmol/L and1000nmol/L）decreased by 35.73%,38.63%and 50.36%,respectively,as compared with LPS group（P<0.05）;The ROS levels in cells treated with galanin（250nmol/L,500nmol/L and 1000nmol/L）decreased by 18.22%,19.17%and 21.35%compared with LPS group（P<0.05）.The relative level of pAMPK protein was increased after galanin treatment as compared with LPS challenge alone or control group in both the RAW264.7 and J774A.1 cell lines（P<0.05）;The relative level of pACC protein was also increased after galanin treatment as compared with LPS challenge alone or control group in both the RAW264.7 and J774A.1 cell lines（P<0.05）.Conclusions:Galanin exhibits a significant inhibitory effect on proinflammatory response induced by LPS in mouse Raw264.7 and J774A.1 macrophage in vitro;the inhibitory effect of galanin on the proinflammatory response may relate to AMPK signaling pathway.