Study Of Anti-hepatoma Effects And Mechanisms Of Rosmarinic Acid

Objective:The aim of this study was to observe the inhibitory effects of rosmarinic acid（RA）in human hepatocellular carcinoma cell lines（SMMC-7721 and HepG-2）and H22 tumor-bearing mice,and explore the anti-hepatomamechanismsofRAthroughregulationoftumor microenvironment and influence on tumor apoptosis so as to lay foundation for further clinical application of RA in liver cancer treatment.Methods:Chapter 1.The human hepatocellular carcinoma cell line SMMC-7721 and HepG-2 were cultured and treated with different concentrations of RA for 48h.Cell growth inhibitory rates were determined by tetrazolium salt（MTT）assay.The H22 tumor-bearing mice were randomly divided into five groups:the non-tumor group,model group,cyclophosphamide（CTX）group,the low-,middle-and high-dose RA groups（75,150,300 mg/kg）.After the 10 days treatment,the inhibitory rates of tumor weights,the liver,spleen and thymus indexes in mice were tested.Blood routine examination was measured by automatic hematology analyzer.Serum levels of alanine aminotransferase（ALT）,aspartate aminotransferase（AST）,blood urea nitrogen（BUN）,uric acid（UA）and creatinine（CRE）were determined by automatic biochemical analyzer.HE staining was performed to evaluate the histopathologic changes of tumor tissues.Levels of interleukin-1β（IL-1β）,tumor necrosis factor-α（TNF-α）,vascular endothelial growth factor（VEGF）,transforming growth factor-β1（TGF-β1）,interleukin-2（IL-2）,interferon-γ（IFN-γ）,interleukin-6（IL-6）,interleukin-10（IL-10）and immune globulin G（IgG）were measured by enzyme linked immunosorbent assays（ELISA）.Expressions of NF-κB p65,phosphorylated p65,STAT3,p38 MAPK and phosphorylated p38were detected by western blot.Protein levels of NF-κB p65,STAT3,Bax,Bcl-2and Caspase-3 were detected by immunohistochemistry.mRNA levels of NF-κB p65,STAT3,Bax,Bcl-2 and Caspase-3 was analyzed by qRT-PCR.The percentages of CD3⁺,CD4⁺and CD8⁺lymphocyte cell were counted by flow cytometry.Results:RA displayed significant growth inhibitory effects on SMMC-7721 and Hep G-2（P<0.05）,and the IC₅₀0 were 316.5μM and 230.7μM,respectively.Compared with the model group,RA could effectively inhibit the tumor growth in H22 tumor-bearing mice.The inhibitory rate of tumor weights of high-dose RA group was 48.24%.RA had little impacts on thymus,spleen and liver indexes.The levels of WBC and PLT were unchanged whereas RBC and HGB were decreased.The levels of the renal function parameters,including ALT,BUN,UA,and CRE,remained unchanged after the treatment of RA.HE staining showed the tumor cells of RA groups were decreased in varying degrees.Compared with the model group,RA treatment could increase the levels of IL-2and IFN-γ,and decrease the levels of IL-1β,TNF-α,VEGF,TGF-β1,IL-6,IL-10 and IgG（P<0.05）.CTX group could increase the level of IL-2 and decrease the levels of IL-1β,TNF-α,VEGF,TGF-β1,IL-6,IL-10 and IgG compared with model group.Western blot analyses showed that the middle-and high-dose RA decreased expressions of NF-κB p65 and phosphorylated p65.Three dosages RA decreased protein expression of STAT3.RA had no effect on the protein expression of p38 MAPK,but middle-dose RA treatment could increasetheexpressionsofphosphorylatedp38（P<0.05）.Immunohistochemistry analyses showed that the middle-and high-dose RA decreased protein expression of p65.Three dosages RA decreased protein expression of STAT3.High-dose RA treatment could increase the protein expressions of Bax and Caspase-3,and decrease the protein expression of Bcl-2（P<0.05）.q RT-PCR result showed that the mRNA level of p65 was decreased after middle-dose RA treatment compared with model group.mRNA level of STAT3 was decreased after middle-and high-dose RA treatment.High-dose RA treatment could increase the mRNA expressions of Bax and Caspase-3,and decrease the mRNA expression of Bcl-2（P<0.05）.In the flow cytometry analysis,RA had no effect on the percentage of CD3⁺lymphocyte cell（P>0.05）.Compared with the model group,low-and middle-dose RA treatments could decrease the percentage of CD8⁺whereas CTX could increase it.High-dose RA could increase the ratio of CD4⁺/CD8⁺（P<0.05）.Conclusions:RA can inhibit the growth of liver cancer in vitro and in vivo,and showed little toxic effects in H22 tumor-bearing mice.The possible anti-hepatoma mechanisms of RA may be related to the inhibition of inflammationfactorsandNF-κBp65pathwayininflammatory microenvironment,regulation of immunocytes,immune factors and STAT3pathway in immunosuppression microenvironment,and influence on the expressions of tumor apoptosis factors and p38 MAPK pathway.