Turtle nail decoction pill is from the book “Jin Kui Yao” written by Zhang Zhong Jing, a doctor in the Eastern Han Dynasty. It has been reported to be effective in treating liver cancer, esophageal cancer, leukemia, uterine fibroids and so on. The drug composition of Turtle Nail Decoction Pills is composed of products that break blood, resolve blood stasis and disperse knots. The effect of these blood-breaking, blood-stasis-dispersing and nodule-dispersing drugs on tumors and whether they promote tumor angiogenesis and tumor metastasis are questions that deserve to be seriously discussed. We have performed in vivo herbal interventions to observe the expression of microvascular count (MDC), vascular endothelial growth factor (VEGF) and proliferating cell nuclear antigen (PCNA) in tumor masses of tumor-bearing mice by establishing an animal model of tumor-bearing mice to illustrate the effects of turtle shell decoction pill on tumor vasculature. 1. Materials and methods 1.1 Experimental animals: 60 Kunming breed mice, 4-6 weeks old, weighing 18-22 g, males, purchased from the Animal Center of Guangzhou University of Chinese Medicine. The mice were housed in separate cages, fed and watered freely, and the mouse room was kept ventilated. 1.2 Cell line: The cell line is mouse ascites-type hepatocellular carcinoma cell line H22 cells, which can generate sarcoma by subcutaneous inoculation. The cells were purchased from the Institute of Oncology, School of Medicine, Sun Yat-sen University, and were routinely resuscitated and cultured. 1.3 Main reagents: PCNA primary antibody, VEGF primary antibody and kits, all of which are products of Wuhan PhD Biological Engineering Co. 1.4 Drug preparation: turtle shell decoction, manufactured by Hangzhou Hu Qingyutang Co., Ltd, batch number 020602. The daily dose for mice was calculated according to the formula for converting human and mouse drug doses. The suspensions of turtle shell decoction pills were prepared with distilled water to contain 0.048g and 0.024g of raw drug per ml for the high and low dose groups respectively, stored at 4℃ in the refrigerator and shaken well when used. Cyclophosphamide, produced by Jiangsu Hengrui Pharmaceutical Co., Ltd, lot No. 02071121. 2mg/ml of cyclophosphamide was diluted with sterile saline and stored at 4℃ in the refrigerator. 1.5 Establishment of tumor-bearing mouse model [1]: in vitro resuscitation of cultured H22 cells: made into cell suspension as usual. Adjusted to 2-3×106/ml tumor cells, 10 healthy Kunming breed mice were randomly selected and each was injected intraperitoneally with 0.4 ml of the above cell suspension, and the abdomen of the inoculated mice was obviously enlarged and protruded after about one week. The cervical vertebrae were dislocated and executed, fixed on wax plates, and the milky white ascites fluid was aseptically extracted, and after dilution and staining, the concentration of the tumor cell suspension was adjusted to 6×106/ml tumor cells, and each mouse was inoculated subcutaneously with 0.2ml of the right hind limb buttocks. 44 mice were inoculated in total. 24 hours later, the drug was administered. 1.6 Experimental grouping and drug administration: 44 mice with successful modeling were randomly divided into 4 groups of 11 mice each and numbered within the group. 4 groups were: turtle shell decoction pill high-dose group, turtle shell decoction pill low-dose group, negative control group and cyclophosphamide group. All treatments were started from the second day of modeling: 12g?kg-1?d-1 and 6g?kg-1?d-1 gavage for the high-dose and low-dose groups of turtle shell decoction, respectively; the same dose of distilled water gavage for the negative control group; and 20mg?kg-1?d-1 intraperitoneal injection for the cyclophosphamide group [2 -1 intraperitoneal injection [2]. The drug was discontinued after 15 days of continuous administration, and the tumor masses were stripped after execution of the mice and fixed in 10% formaldehyde. 1.7 Processing of the extracted material: including fixation, dehydration, wax dipping, wax wrapping, and sectioning. 1.8 Judgment of results: microvessel counting (MDC) within the tumor: the material was taken from 5 different parts within the tumor of each tumor-bearing mouse, each block was 5 mm×5 mm×2 mm, excluding the tumor hemorrhage area and marginal reaction area. In order to avoid the interference of larger vessels, vessels with thick smooth muscle envelope or lumen of 8 erythrocytes area were not counted. The mean value is the MDC value of the tumor in the case [3]. The positive criteria of PCNA, the nucleus stained brown or tan is positive, 10 high magnification fields of each specimen were randomly observed, and the number of positive cells among 100 tumor cells in each high magnification field was calculated, and the average value was taken as the number of positive cells of PCNA [4]. The percentage of positive area to the total area of tumor cells was calculated. 2, Results 2.1 Effect of turtle shell decoction pill on microvessel count (MDC) of tumor tissues As shown in Table 1: compared with the negative group, the MDC of both turtle shell decoction pill high dose group and turtle shell decoction pill low dose group were significantly lower (P0.01). In contrast, there was no significant difference in MDC in the cyclophosphamide group compared with the negative control group (P>0.05). The MDC of the high-dose group of Turtle shell decoction pill was significantly lower than the MDC of the low-dose group of Turtle shell decoction pill (P0.01). The MDC of both the high-dose group and the low-dose group of Turtle shell decoction pill was significantly lower, and there was a significant difference compared with the cyclophosphamide group (P0.01). Table 1 Effect of turtle shell decoction pill on microvascular density (MDC) of tumor tissues ( ) Group n Initial dose (g/kg) MDC (bars) Negative control group 11 11 – 15.7±2.3 Cyclophosphamide group 11 11 20 14.6±2.2 Turtle shell decoction pill high dose group 11 11 12 4.1±1.2*▲△ Turtle shell decoction pill low dose group 11 11 6 7.6±1.4*△ Note: Compared with the negative control group: *P0.01; compared with the turtle shell decoction pill low dose group: ▲P0.01; compared with the cyclophosphamide group: △P0.01 2.2 The effect of turtle shell decoction pill on vascular endothelial growth factor (VEGF) in tumor tissues The expression of VEGF was significantly weaker in both the turtle shell decoction pill high dose group and the turtle shell decoction pill low dose group than the negative control group (P0.01). P0.01). The expression of VEGF was significantly weaker in the turtle shell decoction pill high-dose group and turtle shell decoction pill low-dose group, which was significantly different from the cyclophosphamide group (P0.01, P0.05, respectively). The expression of VEGF was also significantly weakened in the Turtle shell decoction pill high dose group compared with the Turtle shell decoction pill low dose group (P0.01). The results are shown in Table 2: Table 2 Effect of turtle shell decoction pill on vascular endothelial growth factor (VEGF) in tumor tissues ( ) Group n Initial end dose (g/kg) VEGF (%) Negative control group 11 11 – 13.18±1.47 Cyclophosphamide group 11 11 20 9.18±3.40* Turtle shell decoction pill high dose group 11 11 12 2.36± 1.12*△△△ ▲ Turtle shell decoction pill low dose group 11 11 6 5.91±1.38*△ Note: compared with negative control group: *P0.01; compared with cyclophosphamide group: △P0.05, △△△P0.01; compared with Turtle shell decoction pill low dose group: ▲P0.01 2.3 Effect of Turtle shell decoction pill on proliferating cell nuclear antigen (PCNA) of tumor tissue As shown in Table 3, the expression of PCNA expression of turtle shell decoction pill high dose group and turtle shell decoction pill low dose group were significantly weakened compared with the negative control group (P0.01). Compared with the cyclophosphamide group, the expression of PCNA was significantly weakened in both the Turtle shell decoction pill high-dose group and the Turtle shell decoction pill low-dose group (P0.01). The PCNA expression in the high-dose group of Turtle shell decoction pill was significantly weaker than that in the low-dose group of Turtle shell decoction pill (P0.01). Table 3 Effect of turtle shell decoction pill on proliferation nuclear antigen (PCNA) in tumor tissues ( ) Group n Initial dose (g/kg) PCNA (%) Negative control group 11 11 – 57.45±2.16 Cyclophosphamide group 11 11 20 34.18±2.64* Turtle shell decoction pill high dose group 11 11 12 12.90±4.16* △▲ The tumor angiogenesis is a difficult problem to be solved because it is difficult to be effective by a single route of drugs. Turtle shell decoction pill is composed of multiple drugs with multifaceted targets, including regulating the overall functional state, attacking localized pathogenic evil, supporting positive energy and dispersing masses, and has been clinically proven to be an effective meridian formula. This formula contains a large number of blood-breaking and blood-stasis-dispersing drugs, such as rhubarb, zweiwei, tulip worm, dung beetle, rattle, danpi, and peach kernel for breaking blood stasis and dispersing lumps; turtle nail, half asia, scapularia, wu fan, and red nitrate for softening and dispersing lumps; bee balm for detoxifying and dispersing lumps; and scutellaria baicalensis and chai hu for clearing heat and dispersing lumps. Whether these drugs promote or inhibit tumor angiogenesis needs to be proved by experiments. The growth, infiltration and metastasis of tumors require the support of the nascent capillary network. Malignant tumor cells rely on the abundant neovascular network to get the nutrients and oxygen needed for their rapid and unlimited growth in time and to transport the metabolites away. Tumors can secrete substances that erode blood vessels and promote neovascularization during growth. VEGF is a multifunctional factor that acts specifically on endothelial cells and has an important role in the induction and regulation of tumor angiogenesis.VEGF increases the permeability of blood vessels, especially microvessels [5], and provides a fibrous network for the migration of multiple cells during angiogenesis.VEGF is a selective pro-vascular endothelial cell mitogen [6] that directly stimulates endothelial cell proliferation and produces VEGF has been used as a prognostic indicator to reflect tumor angiogenesis and determine the biological heterogeneity of tumors. In this experiment, the expression of VEGF in tumor masses of tumor-bearing mice was detected by immunohistochemistry. The results showed that the positive rate of VEGF in both the high-dose group and the low-dose group of turtle shell decoction pill was significantly lower than that in the negative control group (P0.01). Compared with the cyclophosphamide group, the expression of VEGF was also significantly weaker in the Turtle shell decoction pill high-dose group and the Turtle shell decoction pill low-dose group (P0.01, P0.05, respectively). There was also a significant difference between the high-dose group and the low-dose group of turtle shell decoction pill (P0.01). It indicates that the expression of VEGF in this tumor-bearing mouse was significantly inhibited by Turtle shell decoction pill, and there was a quantitative effect trend. The increased density of blood vessels increases the opportunity for tumor cells to enter the circulation, and tumor cells are more likely to cross these vessels and metastasize to distant sites [7]. In this experiment, we showed that the microvessel count was significantly lower in both the high-dose and low-dose groups of turtle shell decoction pill, which was significantly different from the MDC of the negative control group (P0.01). In contrast, there was no significant difference in MDC in the cyclophosphamide group compared with the negative control group (P>0.05). The MDC of the high-dose group of turtle shell decoction pill was significantly lower than that of the low-dose group of turtle shell decoction pill (P0.01). This indicates that turtle shell decoction pill has an inhibitory effect on tumor vasculature in hormonal mice. PCNA is an intranuclear protein that is expressed along with cell proliferation. As a marker of endogenous proliferating cells, PCNA is closely related to the growth activity, metastasis and prognosis of malignant tumors. The detection of PCNA expression in malignant tumor tissues is important for determining the malignancy of tumors, predicting the trend of lymph node metastasis, guiding clinical treatment plans, and improving patient prognosis [8]. In this experiment, we applied the method of immunohistochemistry to detect the expression of PCNA in tumor tissues of tumor-bearing mice, and explored the effect of Jingfang Turtle shell decoction pill on PCNA in tumor tissues. The results showed that the expression of PCNA in the high-dose and low-dose groups of Turtle shell decoction pill was significantly weakened compared with the negative control group (P0.01), and also significantly weakened compared with the cyclophosphamide group (P0.01). The expression of PCNA in the high-dose group of Turtle shell decoction was significantly weaker than that in the low-dose group of Turtle shell decoction (P0.01). These indicate that the expression of PCNA in this tumor-bearing mice was significantly inhibited by Turtle shell decoction pill, and there was a certain trend of quantitative effect. 4, Conclusion The decoction of turtle shell decoction can significantly reduce the microvessel count of tumor in tumor-bearing mice, which indicates that turtle shell decoction can inhibit the angiogenesis of tumor in tumor-bearing mice to achieve the tumor-suppressive effect. The inhibition of tumor angiogenesis by turtle shell decoction pill may be achieved through the inhibition of tumor VEGF. The inhibition of tumor angiogenesis by turtle shell decoction may also be attributed to the inhibition of tumorigenesis by turtle shell decoction, which significantly inhibited the expression of PCNA in tumor-bearing mice.