Effect Of Marrow Mesenchymal Stem Cells On Regulatory B Cells In Acute On Chronic Liver Failure Mice

[Background]Liver failure is a clinical syndrome that caused by multiple factors, including hepatitis viruses, drugs, toxics and so on, leading to hepatic function decompensation, such as detoxification, biosynthesis, excretion and biotransformation disorder. The mortality of liver failure is above 60%. Liver failure in Asian-Pacific region is mostly attributed to acute on chronic liver failure(ACLF). Liver transplantation is so far the most effective therapy for ACLF, but it is limited by several factors such as insufficient donors, transplant reaction and tremendous economical burden. Marrow mesenchymal stem cells(MSCs) transplantation is a new promising method for ACLF. MSC is reported to have potent multi-potency capacity and immunoregulatory effects on both innate and adaptive immune cells. Its favorable therapeutic effects in ACLF are complex and not yet fully understood. Regulatory B cells(Breg) are a specific subsets of B cells that are identified to down-regulate adaptive and innate immunity, inflammation, and autoimmunity, and they play an important role in immunoregulation, However, Breg is rarely researched in ACLF, and how MSCs to treat ACLF is unknown, too.[Objective]1. To observe the immunoregulatory role of regulatory B cells(Breg) in Acute on Chronic Liver Failure(ACLF) mice.2. To survey the immunoregulatory role of regulatory B cells during MSC treating acute on chronic liver failure mice.[Materials and Methods]1.Hepatic fibrosis model was prepared by intraperitoneal injection of carbon tetrachloride(10%) twice per week for eight consecutive weeks,after that a sublethal dose of carbon tetrachloride(50%) was administered through intraperitoneal injection, thus the acute on chronic mice model was established. 24 hours accumulative survival rate of mice in ACLF group and control group were investigated.2.Marrow mesenchymal stem cells were isolated and cultivated,then identified by flow cytometry. Mice in MSC group were treated with MSCs transplantation via caudal vein injection, whereas the control ACLF mice were treated with saline.3. Hepatic, splenic and peripheral blood lymphocytes were separated from each mice, and then the percent of CD19+IL-10+ B cells were detected by flow cytometry(FCM), IL-10 concentration in the lymphocytes culture supernatant was tested by ELISA.[Results]1. Compared to the control group, the 24 hours accumulate survival rate of mice in the ACLF group was reduced(20% VS 100%, P<0.001).2. The high mortality of ACLF mice was accompanied by a significantly higher.3. percent of IL-10-producing CD19+ B cells in liver, spleen and peripheral blood((2.97±0.33)% Vs(0.75±0.11)%,(4.13±0.67)% Vs(0.79±0.11)%,(5.87±1.00)% Vs(0.68±0.09)%,P<0.001, each respectively).4. The concentration of IL-10 in the supernatant of cultured lymphocytes, from liver, spleen and peripheral blood, separately, was also elevated in ACLF mice((16.76±1.73) Vs(6.29±0.70)pg/ml,(21.21±1.62) Vs(8.23±1.52)pg/ml,(31.32±2.95) Vs(7.49±1.22)pg/ml,all P values were less than 0.001).5. 24 hours accumulate survival rate of mice in the MSCs treating ACLF group was higher than that in the control ACLF group(60% VS 10%, P<0.05).6. IL-10-producing CD19+ B cells in liver, spleen and peripheral blood of mice with ACLF in the MSC group were decreased compared to the control group((2.27±0.17)% Vs(3.67±0.21)%,P<0.001,(2.57±0.25)% Vs(5.14±0.59)%,P<0.05,(3.00±0.24)% Vs(5.93±0.99)%,P<0.05).7. After MSCs transplantation, the concentration of IL-10 in the supernatant of cultured lymphocytes, from liver, spleen and peripheral blood, separately, were also reduced in MSC group mice((14.08±2.04) Vs(19.97±1.06)pg/ml, t =2.458, P <0.05;(16.30±2.22) Vs(23.41±1.41)pg/ml, t =2.77,P <0.05;(18.05±2.39) Vs(33.21±3.31)pg/ml, t =3.53,P <0.01).[Conclusions]1. The 24 hours accumulate survival rate of mice with ACLF was lower than that in control group, along with increased number of CD19+IL-10+B cells and higher concentration of IL-10 produced by lymphocytes, which presented that regulatory B cells might play an important role in the progression of ACLF.2. The MSCs transplantation can improve the accumulate survive rate of mice with ACLF, and both of the CD19+IL-10+B cells and the concentration of IL-10 produced by lymphocytes were declinsing. These observation reminded us that MSCs might modulate the immune responses of ALCF mice through down-regulate Breg cells and IL-10 until the balance.