Reprogramme Macrophages Phenotype Suppress Liver Fibrosis

Liver fibrosis is the typical response to chronic liver disease and characterized by the massive excess of extracellular matrix deposition.The traditional view of liver fibrosis is an irreversible process.However,increasing evidence shows that that liver fibrosis is an early stage of cirrhosis and the fibrosis regression is rapidly initiated after termination of chronic injury.Macrophages,as one part of the innate immune system,provide the first line of defense against external or internal danger signals in the body.They express a broad array of sensing molecules,which allow them to monitor tissue microenvironments and act as sentinel cells for infection and tissue damage.Besides,they exhibit important homeostatic activity in nearly all organ systems.Traditionally,macrophage function has been assigned as inflammatory or‘‘M1”and anti-inflammatory or‘‘M2”.However,the M1/M2 model is by far too simplistic to describe the polarization of macrophages including in the liver.It has been recently proposed a new nomenclature linked to the activation standards,i.e.,M?IL-4?,M?IFN-g?,M?LPS?.Increasing evidence shows that monocytes/macrophages can be reprogrammed in response to the microenvironment signals,terming heterogeneity.Moreover,the heterogeneity of macrophages was a consequence of interaction with different immunological pathways,rather than attributing them to distinct macrophages subsets.Increasing evidence shows that the hepatic macrophages,Kupffer cells and infiltrated bone marrow-derived monocytes/macrophchenages,fulfill diverse functions in the pathogenesis of liver fibrosis.These central functions include the perpetuation of inflammation and hepatocyte injury,activation of hepatic stellate cells with subsequent fibrogenesis,production of matrix metalloproteinases?MMPs?,the most important degrading effectors of excessive extracellular matrix.Hence,hepatic macrophages can actually exert dual functions in liver fibrosis by either promoting or abrogating the excessive deposition of extracellular matrix.If liver injury ceases,specific molecular signals trigger hepatic macrophages to switch their phenotype towards reparative phagocytes that promote tissue repair and regression of fibrosis.Therefore,novel strategies to treat liver disease aim at targeting macrophages.Resveratrol?trans-3,4,5-trihydroxystilebene?is a natural polyphenolic flavonoid found in grapes,peanuts and berries and nuts,possesses many activities effects.Resveratrol has been shown to attenuate liver injury and fibrosis in many experimental animal models.However,whether its anti-fibrogenic function is associated with the phenotype changes of liver macrophages is poorly understood.Therefore,in order to further understand the mechanism of liver fibrosis and investigate whether resveratrol may exert its anti-fibrogenic effect by facilitating macrophage reprogramming towards M?IL-4?-like phenotype,in this study,the RAW264.7 macrophages and liver fibrosis model were undertook the intervention of resveratrol,and the related molecular will be detect,our results will provide a new clue for the treatment of liver fibrosis.This study will be conducted from the following three aspects:Part one Resveratrol suppress liver fibrosis by facilitating macrophagereprogrammingObjective:Resveratrol has been suggested to mediate liver fibrosis.The switch from classically M?LPS?to alternatively activated M?IL-4?macrophages shows to protect organs from fibrosis.However,the mechanisms remain unclear.The study aimed to investigate whether resveratrol inhibited liver fibrosis by promoting the macrophage polarization in vivo.Methods:Liver fibrosis was induced by carbon tetrachloride?CCL₄?.The male BALB/C mice were divided into three groups:?1?Group 1?n=6?control animals were received the vehicle only?olive oil?;?2?Group 2?n=6?,micwere injected intraperitoneally with CCL₄?Sigma??0.1 ml/20 g of CCL₄ dissolved in a1:10 ratio olive oil?three times per week for 4 weeks to induce the liver fibrosis;?3?Group 3?n=6?mice were injected intraperitoneally with resveratrol?TCI,Europe??400mg/kg/d?plus CCL₄?0.1 ml/20g of CCL₄ dissolved in a 1:10 ratio olive oil?three times per week for 4 weeks.All the animals were killed under light ether anesthesia 72h after the last dose of CCL₄ or olive oil.Blood was collected by cardiac puncture and the liver was rapidly removed.All samples were kept on ice until analysis.Liver fibrosis was assessed by histology,Masson's trichrome staining,and the measurement of hydroxyproline.RNA levels were assessed by quantitative real time polymerase chain reaction?RT-PCR?.Protein expressions were determined by immunohistochemical staining,immunohistofluorescence and TUNEL.Results:?1?Treatment with resveratrol was remarkably alleviated liver fibrosis as assessed by METAVIR and ISHAK fibrosis scoring systems and significantly inhibited the secretion of hydroxyproline.?2?A prominent enlargement and aggregation of Kupffer cells in periportal area was observed in both CCL₄ group and CCL₄+Resveratrol group.Moreover,resveratrol treatment significantly elevated the intrahepatic mRNA expression of F4/80?P<0.05?,which indicated that resveratrol's anti-fibrogenic function may be associated with up-regulation of the numbers of the Kupffer cells.?3?Treatment with resveratrol markedly increased the expression of intrahepatic mRNA of the M?IL-4?related genes Arg1,CD163,Mrc1 and Mrc2,while slightly suppressed the M?LPS?related genes iNOS,TNF-?and MCP1expression and up-regulated IL-1?mRNA expression.Similarly,treatment with resveratrol significantly increased the number of CD206 positive cells and slightly decreased the number of iNOS positive cells.These results suggested that resveratrol promotes M?LPS?to M?IL-4?macrophages polarization in the CCL₄-induced liver fibrosis of mice.?4?Treatment with resveratrol significantly upregulated the intrahepatic IL-10 mRNA expression in CCL₄-induced liver fibrosis mice?P<0.05?.?5?Treatment with resveratrol significantly upregulated the apoptosis rate of the M?LPS?in CCL₄-induced liver fibrosis mice?P<0.05?.Conclusions:Our in vivo results demonstrate that resveratrol was effective in protecting the liver from CCL₄-induced liver fibrosis of mice.This is attributed to the effects of resveratrol by inducing more production of endogenous IL-10 and promoting the apoptosis of M?LPS?cells to change the microenvironment,which promoted the polarization of M?LPS?macrophages to M?IL-4?-like macrophages.Part two Resveratrol Facilitating M?LPS?Macrophages Transformation In vitro Objective:The study aimed to verify whether resveratrol inhibited liver fibrosis by delivering Interleukin-10?IL-10?to reprogramme the M?LPS?macrophages phenotype in vitro.Methods:RAW264.7 cells were serum-starved for 24h and pretreated with LPS 1ug/ml for 24h to be swiched into M?LPS?cells.After gently washed with serum-free DMEM medium for three times,the M?LPS?cells were stimulated with?1?M?LPS?+medium;?2?M?LPS?+LPS?1ug/ml?;?3?M?LPS?+Resveratrol?30?M?;?4?M?LPS?+IL-4?5ng/ml?;?5?M?LPS?+cont CM;?6?M?LPS?+LPS CM;?7?M?LPS?+Resveratrol CM;?8?M?LPS?+IL-4 CM respectively for an additional 6h or 24h period.RNA levels were assessed by quantitative real time polymerase chain reaction?QRT-PCR?.Protein expressionsweredeterminedbyimmunohistochemicalstaining,immunohistofluorescence.The apoptosis rate were assessed by AO/PI,HO/PI and TUNEL staining.Results:IL-4 and resveratrol promote the polarization of macrophages to an M?IL-4?phenotype and LPS promotes to an M?LPS?phenotype.RAW264.7cells were exposed to LPS for 24h to be polarized to M?LPS?phenotype,to mimic the inflammatory environment in vitro,Treatment with resveratrol CM treatment significantly increased the CD206 expression as assessed by double immunohistofluorescence staining and decreased the mRNA expression of iNOS and increased the TNF-?,MCP1 and Mrc2 mRNA expression.These results indicated that resveratrol via its indirect effect can facilitate M?LPS?macrophage reprogramming towards M?IL-4?-like phenotype in vitro.Of note,compared to the LPS-treated group?M?LPS?+LPS group?,neither M?IL-4?inducer nor M?IL-4?conditioned media could significantly reduce the TNF-?and MCP1 expression,but up-regulate the IL-1?mRNA expression.Treatment with resveratrol CM and IL-4 significantly increased the expression of IL-10mRNA?P<0.05?.Moreover,the IL-10 mRNA expression was also significantly increased in the M?LPS?+LPS group?P<0.05?.Treatment with IL-4,resveratrol and resveratrol CM significantly upregulated the apoptosis rate of the M?LPS?.Conclusions:In inflammatory context,treatment with IL-4 and resveratrol significantly upregulated endogenic production of IL-10 and promoted the apoptosis of M?LPS?cells,leading to the changes of microenvironment and promoting the polarization of M?LPS?to M?IL-4?-like macrophages in vitro.Part three The M2 inducer targeting NF-kappa B1 to reprogramme M?LPS?Objective:The study aimed to investigate the signaling pathway of transformation of macrophage from M?LPS?to M?IL-4?by M2 inducer.Methods:?1?In vivo,Liver fibrosis was induced by carbon tetrachloride?CCL₄?.The male BALB/C mice were divided into three groups:?1?Group 1?n=6?control animals were received the vehicle only?olive oil?;?2?Group 2?n=6?,mice were injected intraperitoneally with CCL₄?Sigma??0.1 ml/20g of CCL₄ dissolved in a 1:10 ratio olive oil?three times per week for 4 weeks to induce the liver fibrosis;?3?Group 3?n=6?mice were injected intraperitoneally with resveratrol?TCI,Europe??400mg/kg/d?plus CCL₄?0.1 ml/20g of CCL₄dissolved in a 1:10 ratio olive oil?three times per week for 4 weeks.All the animals were killed under light ether anesthesia 72 h after the last dose of CCL₄or olive oil.All samples were kept on ice until analysis.RNA levels were assessed by quantitative real time polymerase chain reaction?QRT-PCR?.?2?In vitro,RAW264.7 cells were serum-starved for 24h and pretreated with LPS 1ug/ml for 24h to be swiched into M?LPS?cells.After gently washed with serum-free DMEM medium for three times,the M?LPS?cells were stimulated with?1?M?LPS?+medium;?2?M?LPS?+LPS?1ug/ml?;?3?M?LPS?+Resveratrol?30?M?;?4?M?LPS?+IL-4?5ng/ml?;?5?M?LPS?+cont CM;?6?M?LPS?+LPS CM;?7?M?LPS?+Resveratrol CM;?8?M?LPS?+IL-4 CM respectively for an additional 6h.RNA levels were assessed by quantitative real time polymerase chain reaction?QRT-PCR?.Results:In vivo,resveratrol improved CCL₄-induced liver fibrosis,increased the mRNA expression of TLR4,TLR2,ERK,NF-?B1,C-maf and IL-10.In vitro,consistent with the in vivo results,resveratrol and IL-4 promote the the mRNA expression of TLR4,TLR2,ERK,NF-?B1,C-maf and IL-10.Conclusions:?1?Resveratrol is able to significantly ameliorate liver fibrosis by activating the transcription factors NF-?B1,which has positive regulation on the TLRs-MYD88-ERK–IL-10 signaling pathway,leading to more production of endogenous IL-10 and promoting the polarization of M?LPS?to M?IL-4?-like macrophages.Hence,NF-?B1 is a key target for M?LPS?macrophage reprogramming and anti-fibrosis therapy.?2?C-maf may be another important target for M?LPS?macrophage reprogramming and anti-fibrosis therapy.