Effects Of Total Glucosides Of Paeony On Diethylnitrosamine-induced Hepatocellular Carcinoma And Its Mechanisms

Hepatocellular carcinoma (HCC) is one of the most common malignancies and the third cause of cancer-related death in the world. It accounts for approximately 70%-80% of all primary liver cancer cases. Due to the growing incidence, high malignance, poor prognosis and hidden development of HCC, the therapy for HCC is still in challenging. Nowadays some research has reported that immunological factors were contributed to the tumor remission, indicating the important role of the immune system in the control of HCC. However, it has been reported that HCC could induce a suppressive network to evade the host immune response. Regulatory B cells (Bregs), functionally defined as aB cells subset have been shown to play a pivotal role in autoimmune diseases and allograft tolerance. Bregs orchestrate the immune system and maintain the suppressive function of B cells that was mainly restricted to their ability to produce’inhibitory’ cytokines such as IL-10. Many studies have confirmed the important role of Bregs in the development of HCC. Although Bregs have been shown to possess a critical role in HCC, microenvironmental factors or cytokines involved in the induction of Bregs in HCC remain largely uncharacterized. B cell-activating factor (BAFF), a member of TNF family cytokines, is a key regulator for B cell maturation and function. The early literatures have shown that BAFF has been generally considered as a driving factor for its pro-inflammatory function and demonstrated that BAFF induces MZ B cell differentiation into IL-10-producing B cells with a regulatory function both in vitro and in vivo. These findings support that BAFF plays a dual role in modulating B cell maturation and function. Total glucoside of paeony (TGP) is an effective composition extracted from the root of Paeonia Lactiflora, which contains many effective constituents including hydroxyl-paeoniflorin, paeonin, albiflorin and benzoylpaeoniflorin, and exhibits wide biological properties including anti-inflammatory, anti-oxidative, hepatoprotective and immuno-regulatory activities. TGP has been well known for a long time for the treatment of autoimmune diseases, which exhibited good curative effects and without evident toxicity or side effects. TGP can suppress inflammatory mediator production and regulate immune cells responses. More recently, it has been reported that TGP has protective effects on liver diseases, such as immunological hepatic injury and fibrosis in rats. These findings provide valuable clues for us theoretically to speculate whether TGP can inhibit the escaping immuno-surveillance of HCC or not. Based on the above data, the present study aimed to investigate whether TGP could attenuate the development of HCC and to reveal the possible underlying mechanisms of its anti-tumor effect against DEN-induced HCC in rats.ObjectiveBased on the above data, the present study aimed to investigate whether TGP could attenuate the development of HCC and to reveal the possible underlying mechanisms of its anti-tumor effect against DEN-induced HCC in rats.MethodsMale SD rats, weighing 130-150 g, after acclimating for 7 days, rats were randomly divided into five groups, including (ⅰ) Normal control group. Rats were intragastrically administrated with CMC-Na. (ⅱ) Model group. Rats were intragastrically administrated with DEN (8 mg/kg) for 6 days every week and persisting 16 weeks. (ⅲ) TGP treated groups. Rats were intragastrically administrated with the same DEN as model group and administrated TGP (30 mg/kg,60 mg/kg,120 mg/kg) at the twelfth week and persisting 4 weeks. Rats were weighed and sacrificed at the fourth, twelfth and sixteenth week respectively. Liver samples were surgically removed and weighed. Visible nodules (greater than approximately 1 mm in diameter) on the liver surface were recorded. Then the right lobe of each liver was fixed in 10%　formalin solution for histopathologic examination. The activities of ALT, AST, AFP, ALP, IL-10, IL-4, IL-7 and BAFF in serum were evaluated by commercially available kits. Bregs were measured by flow cytometry and data were analyzed by FlowJo. The protein expressions of BCMA、TACI and BAFF-R were measured by western blot.The lymphocytes of SD rats were culture under different concentration of BAFF and BAFF, Pae in vitro. Bregs were measured by flow cytometry and data were analyzed by FlowJo. The activities of IL-10 in normal lymphocytes supernatant was evaluated by commercially available kits.Results1. Therapeutic effects of TGP on DEN-induced HCC rats1.1 Effects of TGP on hepatocarcinogenesis in DEN-treated ratsTo determine the anti-tumor effects of TGP against hepatocarcinogenesis in rats, the mortality, the number of nodules and index of liver and spleen of experimental rats were observed. Results showed that the mortality in each group was 0%,33%,27%, 20% and 7% respectively. The number of nodules and index of liver and spleen inmodel group was increased significantly compared with normal group. TGP (60 mg/kg,120 mg/kg) significantly decreased the number of nodules and index of liver and spleen compared with model group.1.2 Effect of TGP on histopathology change in DEN-induced HCC ratsMicroscopically, the livers of normal rats looked smooth and shiny, typical hepatic lobules and small uniform nuclei. The liver tissue samples from model group exhibited disordered architecture with a large number of pseudolobules, infiltration of inflammatory cells and abnormal cells with irregular-shaped cytoplasm which generally tended to be poorly differentiated. In TGP treatment groups, the hepatic pathological lesions including pseudolobules, fibrosis, inflammatory cell infiltration and cellular atypia were markedly improved, and the tumors in TGP treatment groups were histologically well-differentiated.1.3 Effect of TGP on the levels of serum AST、ALT and ALP in DEN-induced HCC ratsActivities of serum ALT, AST and ALP indicating hepatic cell damage and precancerous lesions, were significantly higher in the HCC model group than in the normal control group. But the AST and ALP were significantly lower in the TGP (60mg/kg,120mg/kg) treatment group than in the HCC model group. The ALT was significantly lower in the TGP (120mg/kg) treatment group than in the HCC model group.1.4 Effect of TGP on the levels of serum AFP in DEN-induced HCC ratsSerum AFP level is the most common predictor of HCC; Compared with normal control group, DEN has been effective in increasing the levels of AFP. AFP was significantly lower in the TGP (120mg/kg) treatment group than in the HCC model group.1.5 Effect of TGP on the the levels of serum IL-7、IL-4 and BAFF in DEN-induced HCC ratsResults showed that the expression levels of serum BAFF in HCC model were obviously higher compared with normal group, the rats in twelfth stage were significantly higher than in eighth stage, and the rats in sixteenth stage were significantly higher than in twelfth stage. BAFF was significantly elevated in model group. While the expression levels of serum IL-7 and IL-4 were no obvious change. TGP (60mg/kg,120mg/kg) could reduce serum BAFF expression significantly. TGP (30mg/kg) had no obvious effect on the level of serum BAFF levels. While IL-7 and IL-4 were no obvious change.1.6 Effect of TGP on the the levels of serum IL-10 in DEN-induced HCC ratsThe levels of serum IL-10 was significantly higher in the HCC model group than in the normal control group. But the IL-10 was significantly lower in the TGP (60mg/kg, 120mg/kg) treatment group than in the HCC model group.1.7 Effects of TGP on the proportion of Bregs in HCC ratsThe frequency of IL-10-producing Bregs (CD19+ IL-10+ B cells) was analyzed in spleen of HCC rats by flow cytometry. Results showed that following 4,12 and 16 weeks of DEN treatment, the frequency of IL-10-producing Bregs in the model group was significantly increased compared with that in normal control group. While the TGP (60,120mg/kg) treatment group obviously decreased the proportion of Bregs cells, TGP (30 mg/kg) treatment group had no significant changes on the proportion of Bregs.1.8 Effect of TGP on the expression of BCMA、TACI and BAFF-R in spleen tissues in DEN-induced HCC ratsBAFF-R protein was more obviously upregulated in model group than those in normal group, while BCMA and TACI proteins were no obvious change. BAFF-R protein was more obviously downregulated in TGP-treated(60、120 mg·kg-1)rats than those in model rats, while BCMA and TACI proteins were no obvious change. 1.9 The correlation analysis between BAFF and Bregs after TGP treatmentTo understand the relationship between BAFF and IL-10-producing Bregs in promoting the HCC development, the correlation analysis was performed. Results showed that the increase of BAFF in model group was positively correlated with the change of IL-10-producing Bregs in spleen of model group throughout the course of the disease, indicating the increase of BAFF up-regulated the proportion of IL-10-producing Bregs and promoted the progress of HCC. To further confirm the mechanism of TGP in attenuating the rat HCC, correlation Analysis was performed. Results showed that the level of BAFF reduced with TGP treatment was positively correlated with the change of Bregs with TGP treatment. These indicated TGP might down-regulate the proportion of IL-10-producing Bregs by inhibiting BAFF level, which resulted in the anti-tumor effect of TGP on rat HCC.1.10 Effect of BAFF on the proportion of Bregs in normal lymphocytesThe frequency of IL-10-producing Bregs (CD19+IL-10+ B cells) was analyzed by flow cytometry. Results showed that the frequency of IL-10-producing Bregs in the BAFF (20 ng/ml) group was significantly increased compared with that in normal control group. While the Pae (10-6mol/L) group obviously decreased the proportion of Bregs cells.1.11 Effect of BAFF on the the levels of serum IL-10 in normal lymphocytes supernatantThe levels of serum IL-10 was significantly higher in BAFF (20 ng/ml) group than in the normal control group. But the IL-10 was significantly lower in the Pae (10-6mol/L) treatment group than in BAFF (20 ng/ml) group.ConclusionTGP obviously prevented DEN-induced HCC in terms of nodule incidence, number of nodules, serum biochemical parameters and histopathological changes.TGP had a good therapeutic action on DEN-induced HCC rats, which might be related to the down-regulation of IL-10-producing Bregs by reducing the level of BAFF.