Curcumin Protects Against Liver Fibrosis In Mice By Upregulating Regulatory B Cells

BACKGROUNDLiver cirrhosis(cirrhosis,LC)is a serious public health problem that can lead to clinical problems such as portal hypertension,liver failure,and liver cancer,and is the most important cause of liver transplant demand.Cirrhosis is result from necroinflammation and fibrosis due to liver damage with varying causes.Liver fibrosis is a complex,closely regulated process,a balance between the production and degradation of extracellular matrix,which reveals the pathogenesis of liver fibrosis.The prevention of LC is essential for the prevention and treatment of cirrhosis.Inflammation is an important factor to drive and maintain fibrosis,and the immune response within the host is the key to the development of liver fibrosis.Overall,liver fibrosis is the result of the interaction between stromal cells,extracellular matrix and leukocytes.Although myofibroblasts and macrophages are considered to be the center in the pathogenesis of hepatic fibrosis,many unsolved problems still exist.It is necessary to define the role of different types of cells during hepatic fibrosisRecent studies have shown that,in addition to antibody secretion,B cells also have important immune regulatory functions.B cells can play a role in the pathogenesis of liver fibrosis by secreting cytokines in a non-antibody-dependent manner,revealed in the animal model.B cells are divided into effector B cells(Be)and regulatory B cells(Breg)based on the cytokine profiles and functions.It has been demonstrated that Breg cells produce strong anti-inflammatory and negative immunomodulatory effects mainly by secreting IL-10 in animal models of infection,immunity,tumors,and humans.IL-10 is also an important anti-fibrotic cytokine,and Breg cells serve as a major source of IL-10 in B cells.Recent studies have shown that it is also involved in the pathogenesis of liver fibrosis,revealing that the proportion of peripheral blood Breg cells increases with the degree of liver fibrosis in chronic hepatitis B patients.Animal models also show that infusion of exogenous Breg cells can reduce the degree of fibrosis.However,evidence of the role of Breg in liver fibrosis is still scarce.Therefore,this study attempt to further clarify the role of Breg cells in the pathogenesis of liver fibrosis in both human and animal models.It is well established that liver fibrosis is a reversible process,so finding antifibrotic targets and drugs is essential for reversing liver fibrosis.Curcumin is a natural polyphenol compound extracted from rhizomes of Curcuma turmeric,turmeric,curcuma and other traditional Chinese medicines.Due to its anti-inflammatory,antioxidant,anti-mutagenic and anti-tumor properties,it has been used as a traditional medicine in some countries for the last century.At the same time,many animal experiments have also shown that curcumin has good anti-fibroic effects.One of the mechanisms is that curcumin can regulate the function of immune cells and promote the secretion of IL-10,but it is not clear if curcumin can regulate Breg cells.In a GVHD(graft-versus-host disease)mouse model,curcumin promotes an increment of immature B cells in the spleen,therefore we speculate that curcumin can exert anti-inflammatory and anti-fibrotic functions by IL-10 secretion by promoting the upregulation of Breg cells.In our study,peripheral blood,liver of patients with chronic hepatitis B and hepatitis B-related cirrhosis were collected.The proportion of peripheral blood B cells and Breg cells were analyzed by flow cytometry.The number of B cells,Breg cells,and IL-10+ cells in the liver were analyzed using immunohistochemistry.Further,the mouse model of acute liver injury and liver fibrosis induced by CCl4 were further established.The proportions and numbers of B cells and Breg cells in peripheral blood,liver,and spleen were analyzed by flow cytometry,immunofluorescence,and ELISA.The role of B cells,Breg cells,and IL-10 in liver injury and liver fibrosis were examined.The effects of curcumin on liver injury and fibrosis and the regulatory mechanism of upregulating Breg cells and promoting IL-10 secretion were explored.Our data provide a theoretical basis for the clinical application of curcumin and provide new ideas for reversing liver fibrosis.MethodPart I Characteristics of Breg Cells in Patients with Hepatitis B Virus Infection Related Liver Cirrhosis(HBV-LC)1.Characteristics of Breg cells in peripheral blood of patients with HBV-LCIn our cohort,there are 32 cases of chronic hepatitis B(CHB)patients,among which 24 patients underwent liver biopsy,16 patients with HBV-LC and 10 healthy individuals.The distribution of CD19+ B cells and their subsets were used by flow cytometry.The proportion of peripheral blood B-cell subsets in patients and healthy individuals was compared to analyze the correlation of CD19+CD24hiCD38hi Breg cell ratios with other clinical indicators,liver inflammation grades and fibrosis stages.2.Characteristics of Breg cells in the liver of patients with CHB and HBV-LCParaffin-embedded specimens from the liver of 24 patients with CHB and HBV-LC,10 liver grafts from normal donor were collected.The distribution of CD20+B cells and IL-10+PAX5+Breg cells,IL-10+cells in liver tissue were detected by immunochemistry.The B cells,Breg cells,and IL-10+cell counts were compared between patients and healthy individuals,and between patients with different inflammation grades and fibrosis stages.The correlations of B-cells,Breg cells,and IL-10+cell counts with the clinical indicators,liver inflammation grade,and fibrosis stage among patients were analyzed.3.Cytokine profile of HBV-LC patientsThe serum from 32 CHB patients,16 HBV-LC patients and 10 healthy individuals were collected.The expression level of a panel of cytokines,including Th1 cytokines IFN-?,TNF-?,TNF-?,IL-12p40,IL-12p70,and IL-15,Th2 type cytokines IL-5,IL-6,IL-10,as well as Th17 type cytokine IL-17a,chemokine IL-8,IP-10 were detected by Luminex method.The cytokines between patients and healthy individuals were compared,and the characteristics of serum cytokines in HBV-LC patients were analyzed,their correlation with clinical indicators were explored.Part ? Curcumin Upregulates Breg Cells to Promotes IL-10 Secretion to protect against Liver Fibrosis in Mice induced by CCl4To establish a liver fibrosis model,C57BL/6 mice were injected intraperitoneally with CCl4 for 6 weeks.Curcumin was administered by gavage and the serological levels of ALT and AST in mice were measured.HE and Masscon staining of liver and spleen tissue were performed.The effect of curcumin on liver fibrosis were evaluated.The proportion of CD19+B cells and CD19+CD1dhi CD5+ Breg cells in liver and spleen were detected by flow cytometry.IL-10+PAX5+ Breg cells and B cells in spleen,the distribution of IL-10+ cells and.their infiltration in the liver were detected by double labeling immunofluorescence method.IFN-?,IL-2,IL-4,IL-10,IL-17a and TGF-?1 in mice were detected by ELISA.The role of B cells and Breg cells du:ring the pathogenesis of liver fibrosis induced by CCl4 and the potential mechanism of curcumin acting as an anti-fibrotic agent by up-regulating Breg cells and promoting IL-10 secretion were explored.ResultsPart I Characteristics of Breg Cells in CHB-LC Patients1.The Distribution of B Cell Subsets in Peripheral Blood of HBV-LC PatientsThe proportion of CD 19+B cells in the peripheral blood of CHB and HBV-LC patients did not differ from healthy individuals,and the proportion of CD19+CD24hi CD38hi Breg cells was higher than that of healthy individuals.The proportion of CD 19+CD24intCD38int mature B was higher in the HBV-LC patients than in the CHB patients and the healthy individuals,and the proportion of CD19+CD24hiCD38-memory B cells was lower in the HBV-LC group than in the CHB group and the healthy individuals.The proportion of Breg cells in peripheral blood was positively correlated with G grade and S stage.However,there was no correlation between Breg cells with Child-Pugh score.2.Distribution of Breg cells in the liver of patients with CHB and HBV-LCThe amounts of CD20+B cells and IL-10+cells in liver of CHB and HBV-LC patients were higher than that of healthy individuals.The amount of liver B cells was positively correlated with inflammation grade(G),and the amount of Breg cells and IL-10+cells in liver were positively correlated with fibrosis stages(S).3.The profile of serum cytokine in HBV-LC patientsThe serological IL-10,IL-15,IL-6 and TNF-a in patients with HBV-LC were higher than those in healthy individuals.Serum IL-10 expression was positively correlated with ALT and AST,and also positively correlated with IL-6,IL-15,IL-12p70,IL-17a and TNF-? expression,respectively.Part ? Curcumin protects against liver fibrosis in mice induced by CCl4 by upregulating Breg cells and IL-10 secretionRepeated injection of CCl4 can cause liver fibrosis in mice along with significantly higher serological ALT and AST levels than those in the control group.Heavy necrosis of hepatocytes,disordered lobular structure,and infiltrated inflammatory cells were revealed by liver HE staining.Collagen fibers in the tissue were significantly elevated and hepatic lobular fibrous septum increased compared with the control group,demonstrated by Massaon staining.Curcumin treatment can significantly reduce serum ALT,AST levels,liver cell damage and inflammatory cell infiltration and ultimately,reduce liver fibrosis in mice.After repeated intraperitoneal injections of CC1,,the proportion of blood B cells in the model group was lower than that in control group,and the proportion of liver and spleen B cells did not change.The proportion of splenic Breg cells in the model group was lower than that in control group,and the ratio of Breg cells in blood and liver was not significantly changed.Curcumin can upregulate the proportion of B cells in peripheral blood and Breg cells in the spleen of mice.The infiltrations of the B cells,Breg cells,and IL-10+cells into liver increased in the model group.Further,Splenic B cells,Breg cells,and IL-10+cells increased compared with control group.Curcumin can upregulate the number of splenic Breg cells and IL-10+cells in the mice model of liver fibrosis.The liver and splenic Breg cells,IL-10+cells,and liver B cells were positively correlated with ALT and AST.The levels of serum IFN-v,IL-2,IL-17a,IL-10,IL-4 and TGF-B1 in mice increased compared with the control group.Curcumin treatment lead to elevated serum IL-10,and decreased IFN-y,IL-2,IL-17a.The level of serum IL-10 was positively correlated with the serological level of IFN-?,IL-2,IL-4,IL-17a and TGF-?1.Conclusion1.The proportion of Breg cells in peripheral blood of CHB-LC patients increased,and the number of Breg cells in the liver increased.2.Curcumin has protective role against liver fibrosis,and the mechanism of anti-fibrosis may be contributed to the upregulation of splenic Breg cells?IL-10 positive cells and IL-10 secretion.