The Inhibition Of HepG2 Proliferation And Immune Regulation Mechanism On Anti-hepatoma Of LEA

1 Purpose and SignificanceThis study aims to extract and separate the effective components in litchi seeds ethyl acetates(LEA), and to screen out the LEA-sensitized Hep G2 cells by comparing the inhibition of Hep G2 and MCF-7 in the in-vitro experiment based on previous results, then to investigate the inhibition mechanism of Hep G2 and related apoptosis induction mechanism. Another purpose is to provide the immune mechanism in vivo on inhibiting H22 of LEA, which can work as the theory basis for further research of the pharmacological efficacy.This work will implement a preliminary screening of litchi active ingredients with the inhibiting tumor cell activity, and further confirm their function pathway of inducing apoptosis. It supposes that the possible efficacy components are the total flavonoids. This study can has practical application value on the development and utilization of antitumor ingredients of litchi seeds.2 Method2.1 Extraction, separation and identification of effective components in LEAThe total compounds were dissolved in 70% ethanol with assistance of ultrasound method. After extraction and drying, LEA were reserved and marked as the raw material for antitumor experiments. HCl-Mg reaction and UV spectrophotometry were used to identify the flavonoids and determine their content in litchi seed extracts.2.2 Inhibition on Hep G2 and MCF-7 of LEAHep G2 and MCF-7 cells were selected as experimental targets in this part. After cultured in different concentration of LEA for 24 h and 48 h, respectively, we used the MTT-test to measure the proliferation inhibition rate of tumor. The viable tumor cells were detected bythe crystal violet staining method. According to the inhibition efficacy, the significantly-suppressed tumor type was filtered out for the cell apoptosis and mechanism research.2.3 Proliferative effect on mouselymphocyt cells of LEATo study the effect of LEA on normal lymphocyte, mouselymphocyt spleen lymphocytes, thymus lymphocytes, and peritoneal macrophages were taken as experimental subjects. After cultured in different concentration of LEA for 24 h and 48 h, respectively, MTT-test was used to evaluate thelymphocyt cell proliferation rate. As a result, we obtained the optimal non-toxic concentration of normal lymphocyte and selected thelymphocyt cells with high lymphatic activity after using LEA.2.4 Effect on co-culture system analysis of LEAThe co-culture system comprises of mouselymphocyt spleen lymphocytes and Hep G2 with ratio of 3:1. After incubated after 24 h using LEA, the suspended lymphocytes and drug-containing nutrient solution were sucked out. Then the tumor cell proliferation inhibition rate was determined by MTT method, and drawn in the growth-inhibitory curves. The part is to study the efficacy changes of LEA in coexistence of tumor and lymphocyte cells, and to provide the basis of in-vivo experiments.2.5 Apoptosis mechanism of Hep G2 cells induced by LEAThe flow cytometry and DNA Ladder were used to investigate the apoptosis mechanism of Hep G2 cell. After treated with different concentration of LEA within 24 h, the Annexin V-FITC/PI Apoptosis Detection Kit was employed to detect the total apoptosis rate. Then we measured the obvious ladder banding in DNA Ladder test to estimate the apoptosis of Hep G2 induced by LEA, and used the Rhodamine 123 to detect the ?Ψm change in mitochondrial during the induced apoptosis.2.6 Effect of LEA on tumor-burdened miceIn order to establish the liver solid tumor model, the BALB/c mice were inoculated with H22 cells. These tumor-burdened mice that were divided in different groups(model group, positive control, and drug groups with different doses) were injected with different concentration of LEA and 5-Fu, respectively, drenched once a day and 10 days in a row. After cultivated for 10 days, the ocular blood was collected and centrifuged to obtain the standby serum. The tumor tissue, thymus and spleen were completely stripped and weighed, as the basis of the inhibitory rate, thymus index, and spleen index. Meanwhile, MTT andELISA method were used to stimulate the proliferation index and detect the secretion of IL-1β, TNF-α and INF-γ in serum, respectively.3 Results3.1 Extraction and identification of LEALEA is obtained after alcohol extraction of dry litchi seeds. The total content(rutin meter) of flavonoids in LEA is 0.91 mg/ml and the crude extract flavonoids content is 0.21 mg/ml by using UV spectrophotometry. Thus the flavonoids content of LEA is obviously higher than that of the crude extract content in litchi.3.2 Effect of LEA on Hep G2 and MCF-7 cellsLEA can inhibit the proliferation of Hep G2 and MCF-7 cells after cultured for 24 h than 48 h, with the IC50 up to 59.52 g/ml and 301.7 g/ml, respectively. The crystal violet staining results indicate that in range of 30 g/ml-240 g/ml the higher drug concentrations make the less adherent cells and the lighter crystal violet staining. Therefore, the LEA shows the optimal inhibitory effect on Hep G2 cell and can serve as the subject for later experiment.3.3 Effect of LEA on spleen lymphocytesLEA at 60 g/ml show a proliferation rate of 321.33% on mouse spleen lymphocyte with P < 0.01, compared to the control group, which proves LEA to be non-toxic to normal lymphocyte in 30 g/ml-240 g/ml. Once over 240 g/ml, cells change in abnormal accumulation and no clear oval shape. It indicates that high levels LEA enables to be toxic. Therefore, the experimental concentration of LEA is recommended to 30 g/ml-240 g/ml.3.4 Effect of LEA on co-culture systemThe inhibition rate of Hep G2 cells is 35.28% when pure LEA is used. In co-culture system of mouse spleen lymphocytes and Hep G2 cell, the inhibition is significantly enhanced to 72.34% when LEA is added. It shows that LEA can inhibit the tumor growth by activating the lymphocyte.3.5 Detection of Hep G2 apoptosis induced by LEAAnnexin V-FITC/PI results show that the total apoptosis rate reaches to 57.78% when 60 g/ml LEA was imported in the co-culture system of spleen and Hep G2 cells for 24 h.3.6 Effect of LEA on tumor-burdened miceMouse IL-1β ELISA kit obtains the serum IL-1β of 49.79 pg/ml in a tumor-burdened mice dose group(30g/kg). The serum IL-1β of mice spleen supernatant in middle tumor-burdened dose group fluid content is 46.32 pg/ml, which shows that LEA not only inhibit the tumor growth, but also promote the secretion of IL-1β. These results suggest that LEA may reinforce the immune function and induce the tumor apoptosis by increasing the secretion level of IL-1β coupling with the antitumor immune response of effector T cells suppressed.4 Conclusions4.1.This work extracted and separated LEA by alcohol extraction method, and the total content of flavonoids in LEA was detected to 0.91 mg/ml via a UV spectrophotometry.4.2.The IC50 of LEA for inhibiting Hep G2 cells is 59.52 g/ml, coupling with a inhibition rate of 57.26% as the LEA concentration was set in range of 30-240 g/ml, where it shows nontoxicity to normal lymphocyte and enhanced immune activity to some extent.4.3.In the co-culture system of mouse spleen lymphocyte and Hep G2 cells, 60 g/ml LEA can inhibit 72.34% of tumor cells, suggesting that assembling LEA with co-culture system can play a better role on the suppression of Hep G2.4.4.Annexin-V/PI test presented that the total apoptosis rate reached 57.78% when 60 g/ml LEA was used in the co-culture system of mouse splenocyte and Hep G2 cells. It proved that LEA not only can inhibit the growth of Hep G2 cells alone, also activate the spleen lymphocyte to facilitate the migration of immune cells to the tumor tissue.4.5.H22 solid tumor-bearing mice model in vivo revealed that the inhibitory rate in middle dose group can reach 50.15% with the increase of IL-1β secretion at same time. It is possible that LEA strengthens the immune activity and induce the tumor apoptosis by increasing the secretion of IL-1β.However, whether LEA can be used in the clinical treatment at soon still needs great patience and carefulness. And the exact antitumor molecular mechanism and molecular targets in the cell level remains to be further researched.