The Anticancer Activity Of Xinghuayu Injection In Vivo And Its Mechanism

Xinghuayu injection was a new Ginkgo biloba extract injection. The content of flavonoid glycoside was 49.79%, and terpene was 4.4% in the Ginkgo biloba extract. Many literatures reported that flavanoid had goodish anticancer activity. Xinghuayu injection maybe has latent anticancer activity for it containing affluent flavonoids. The present study mainly explored the inhibitory effect of Xinghuayu injection on tumor growth of H22 tumor-bearing mice and the mechanism of anticancer activity to provide foundation for the clinical application of Xinghuayu injection.Objective: To investigate the anticancer effect of Xinghuayu injection in tumor-bearing mice and study the possible mecha nism.Methods:⑴Study of inhibitory effect on tumor growth: The H22 tumor-bearing mouse model was adopted. The mouse which had beared H22 for seven days was killed by the cervical vertebra luxation. Thereafter, the ascites was taken immediately and was diluted by stroke-physiological saline solution. Then neoplastic cell suspension was mixed uniformity and the concentration of tumor cell was adjusted in sterile saline to 107 cells per ml. Kun ming mice were used in this experiment and one mouse was subcutaneously inoculated with 0.2ml cell suspension at right armpit. Then the mice were divided randomly according to weight into 5 groups with 10 in each group: model group, Xinghuayu injection 17.5mg/kg, 35mg/kg, and 70mg/kg groups, and cyclophosphamide 20mg/kg group. Drugs were given once daily for 7 days. The mice were killed by the cervical vertebra luxation. The tumors were extirpated and weighed to calculate tumor-inhibition rate.⑵Study of synergistic effect: H22 cell suspension was subcutaneously transplanted to right armpit of mice, and the mice were divided randomly according to weight into 4 groups with 10 in each group: model group, Xinghuayu injection 17.5mg/kg group, cyclophosphamide 5mg/kg group and combined group. Drugs were given once daily for 7 days. The combined group was given both Xinghuayu injection and cyclophosphamide. The mice were killed by the cervical vertebra luxation. The tumors were extirpated and weighed to calculate tumor-inhibition rate.⑶Study of mechanism: The colorimetric MTT reduction assay was established to measure NK activity of spleen cells in H22 tumor-bearing mice, ELISA methods were used to examine content of IL-2 and TNF-αin serum of H22 tumor-bearing mice, and the expression of MVD and VEGF were measured with immunohistochemistry staining.Results:⑴Inhibitory effect on tumor growth: Xinghuayu injection was able to inhibit tumor growth of H22-bearing mice apparently. The tumor-inhibitory rate was 22.60%, 48.84% and 40.22% in Xinghuayu injection 17.5mg/kg, 35mg/kg, and 70mg/kg groups (P<0.01),while the tumor inhibitory rate was 57.12% in cyclophosphamide 20mg/kg group (P<0.01).⑵Synergistic effect: The tumor inhibitory rate in Xinghuayu injection 17.5mg/kg group, cyclophosphamide 5mg/kg group and combined treatment group was 18.07%,23.88% and 34.59% (P<0.01),respectively. Combined treatment had better antitumor effect than either of them.⑶The effect on activity of spleen NK cells: Xinghuayu injection could increase the activity of spleen NK cells in tumor-bearing mice apparently, and the kill rate was (43.58±6.95)%, (67.21±3.91)%, (75.06±4.70)%, (72.25±6.94)% in model group and Xinghuayu injection 17.5mg/kg, 35mg/kg, 70mg/kg groups, which in Xinghuayu injection groups was higher than that in model group (P<0.01).⑷The effect on content of serum IL-2 and TNF-αin tumor-bearing mice: Xinghuayu injection could increase the content of serum IL-2 and TNF-αin tumor-bearing mice apparently. The content of serum IL-2 in tumor-bearing mice was 34.23±4.55pg/ml, 38.57±8.33pg/ml, 42.34±7.41pg/ml, 41.60±7.76 pg/ml and 25.35±7.70pg/ml in model group, Xinghuayu injection 17.5mg/kg, 35mg/kg, and 70mg/kg groups and cyclophosphamide 20mg/kg group respectively, which in Xinghuayu injection 35mg/kg group was higher than that in model group (P<0.05); The content of serum TNF-αwas 24.00±6.93pg/ml, 30.77±6.07pg/ml, 53.25±10.69pg/ml, 51.61±9.10pg/ml and 14.35±2.95pg/ml in above five groups, which in Xinghuayu injection 35mg/kg and 70mg/kg groups was higher than that in model group (P<0.01). While the content of serum IL-2 and TNF-αin cyclophosphamide 20mg/kg group was lower than that in model group (P<0.05).⑸The effect on tumor angiogenesis: Xinghuayu injection could significantly inhibit tumor angiogenesis. The microvessel density was 35.48±5.36, 30.40±2.43, 22.72±2.18, 29.74±3.53 and 18.78±1.73 in model group, Xinghuayu injection 17.5mg/kg, 35mg/kg, and 70mg/kg groups and cyclophosphamide 20mg/kg group, respectively. The intensity of VEGF expression was 2.06±0.33, 1.62±0.27, 1.58±0.26, 1.60±0.32 and 1.54±0.35 in above five groups. Otherwise Xinghuayu injection 35mg/kg group and cyclophosphamide 20mg/kg group comparing with model group had significant difference (P<0.05).Conclusion:⑴Xinghuayu injection could significantly inhibit tumor growth of tumor-bearing mice and enhance the tumor- inhibitory effect of cyclophosphamide.⑵Its anticancer mechanism in vivo was concerned with improving immune functions of tumor-bearing mice, and decreasing VEGF content and MVD in H22 cancer.