SMMC-7721 liver cancer cells (Shanghai Xinyu Biotechnology Co., Ltd., Shanghai, China); spleen-invigorating and blood stasis-removing recipe (12 g Curcuma, 20 g Atractylodes, 20 g Poria, 12 g Sophora, 12 g Bergamot, and 15 g Hedyotis diffusa) was provided by the Chinese Pharmacy of Guangdong Provincial People's Hospital (Guangzhou, China); fetal liver serum (Gibco, Shanghai, China); RPMI-1640 medium (Hyclone, Beijing, China); streptomycin, PBS buffer, and trypsin (Genview, Shanghai, China); Cell Counting Kit-8 (CCK-8) (Dojindo China Co., Ltd., Shanghai, China); Corning Matrigel Basement Membrane Matrix (Corning, Shanghai, China); DAPT inhibitor (AbMole BioScience, Shanghai, China); Tris-base, glycine, and sodium dodecyl sulfate (SDS) (Sangon Biotech Co., Ltd., Shanghai, China); acrylamide, BIS-acrylamide, PMSF, and Bromphenol Blue (Shanghai Genebase Gene-Tech Co., Ltd., Shanghai, China); BCA protein concentration measurement reagent (Thermo Fisher Scientific, Inc., Waltham, MA, USA); ammonium persulphate (Genview); TRIzol (Takara, Dalian, China); RT-PCR reagents and reverse transcription kit (DBI Bioscience, Shanghai, China); DEPC (Sigma-Aldrich, San Francisco, CA, USA); RNasin (Promega, Madison, WI, USA).

Superclean bench (Suzhou Purification Equipment Co., Ltd., Suzhou, China); low speed centrifuge (Shenzhen Anke High-Tech Co., Ltd., Shenzhen, China); inverted microscope (Motic, Xiamen, China); CO₂ cell incubator (Memmert Trading Co., Ltd., Shanghai, China); enzyme-linked immunosorbent detector (Thermo Fisher Scientific, Inc.); desktop high-speed centrifuge (Shanghai Anting Science Instrument Factory, Shanghai, China); constant pressure constant current electrophoresis instrument (Beijing Liuyi Instrument Factory, Beijing, China); frozen high-speed centrifuge (Zhuhai Heima Medical Instrument Co., Ltd., Zhuhai, China); magnetic stirrer (Changzhou Guohua Electric Appliance Co., Ltd., Changzhou, China); multi-purpose decolorization shaker (Jiangsu Xinkang Medical Devices Co., Ltd., Taizhou, China); ultrasonic crusher (Ningbo Xinzhi Biotechnology Co., Ltd., Ningbo, China); Swirl Oscillator (Shanghai Jingke Industrial Co., Ltd., Shanghai, China); SDS-PAGE vertical electrophoresis tank (Atobo, Beijing, China); scanner (Shanghai Microtek Technology Co., Ltd., Shanghai, China); spectrophotometer (Shimadzu Co., Ltd., Beijing, China); gel scanning system (Zhongshan Golden Bridge, Beijing, China); air thermostat oscillator (Taicang Longway Medical Instrument Co., Ltd., Taicang, China); ABI 9700 PCR Amplifier (Applied Biosystems, Shanghai, China); Stratagene Mx3000P Real-time PCR instrument (Agilent Technologies, Palo Alto, CA, USA).

The composition of the spleen-invigorating and blood stasis-removing recipe (formerly known as 960 mixture) was as described above. The total weight of each prescription was 96 g.

The traditional Chinese medicines were collected according to the prescription ratio. A volatile oil extractor was used to extract volatile oil from curcuma, Atractylodes, and bergamot. Next, the residues of curcuma, Atractylodes, and bergamot were mixed with poria, Sophora flavescens, and Hedyotis diffusa, and were soaked in pure water (10 times volume) for 30 min, and extracted three times at 90–100°C, for 1.5 h each time. The extracts were combined, filtered, and concentrated, and finally mixed with the volatile oil. The ratio of drug to extract was calculated. The resulting extract was stored at −20°C after disinfection by radioactive isotope, cobalt-60.

According to previous studies (11), the equivalent dose ratio calculated based on the body surface area (humans, 70 kg; mice, 0.020 kg) was 0.0026. According to the conversion formula, the equivalent dose for mice was 12.48 g/kg (96 g × 0.0026/0.020 kg = 12.48 g/kg). Therefore, we set the low, middle, and high dose of the spleen-invigorating and blood stasis-removing recipe as 6.24, 12.48, and 24.96 g/kg, respectively.

The extract was harvested according to the ratio of drug to extract, and was mixed with saline to prepare an administration dose of 20 ml/kg. Nude mice received intragastric administrations of saline containing the extract, and the administration volume was 20 ml/kg. Mice were treated for 15 consecutive days. The mice in the normal control group were given equal volumes of saline.

The same batch of SPF nude mice was randomly divided into the normal control group, spleen-invigorating and blood stasis-removing recipe high dose group, spleen-invigorating and blood stasis-removing recipe middle dose group, and spleen-invigorating and blood stasis-removing recipe low dose group, with 10 mice per group. The clinical dose was calculated according to a previous study (6). Saline or traditional Chinese medicine compounds were administered intragastrically for 15 days. Blood from the abdominal aorta was collected on day 16, and serum was separated, filtered, and divided into smaller volumes for storage at −80°C.

Based on our study, different concentrations of the spleen-invigorating and blood stasis-removing recipe had different inhibitory effects on the in vitro proliferation of hepatocarcinoma cells. The half-maximal inhibitory concentration (IC₅₀) and the inhibitory effects of the high dose of the spleen-invigorating and blood stasis-removing recipe (24.96 g/kg) were both optimal among the four groups (P<0.05). Therefore, drug serum derived from the high dose treatment was used in this study.

Complete medium preparation for the normal serum group: RPMI-1640 medium + 10% normal nude mouse serum (from the normal control group) + 1% double antibiotics (100 U/ml penicillin and 100 U/ml streptomycin). The preparation was performed at 4°C, and the medium was used after being warmed in a 37°C water bath.

Complete medium preparation for the drug serum group: RPMI-1640 medium + 10% dosing nude mouse serum (from the spleen-invigorating and blood stasis-removing recipe high dose group) + 1% double antibiotics (100 U/ml penicillin and 100 U/ml streptomycin). The preparation was performed at 4°C, and the medium was used after being warmed in a 37°C water bath.

Complete medium preparation for the inhibitor group: RPMI-1640 medium + 10% normal nude mouse serum (from the normal control group) + 1% double antibiotics (100 U/ml penicillin and 100 U/ml streptomycin) + 1 µM of DAPT. The preparation was performed at 4°C, and the medium was used after being warmed in a 37°C water bath.

Liquid phase conditions were used according to a previous study (12). The fingerprint of the drugs of the spleen-invigorating and blood stasis-removing recipe and drug serum were identified by HPLC at 323 nm.

The water bath was preheated to 37°C; the frozen tube of cells was quickly placed in the heated water for rapid thawing; cells were transferred to a centrifuge tube, and RPMI-1640 complete medium was added, followed by centrifugation in a low-speed centrifuge. After centrifugation, cells were resuspended with medium, cell suspensions were added to cell culture flasks, and incubated in the incubator with 5% CO₂ at 37°C.

When approximately 80% of cells adhered to the wall of culture flasks, the culture medium was replaced under aseptic conditions. The culture conditions were 5% CO₂, saturated humidity, and 37°C.

The experimental grouping was the same as described above. The cultured SMMC-7721 cells were collected to prepare single cell suspensions under aseptic conditions, and the cell suspensions were adjusted to a density of 1×10⁵/ml. The cells were seeded in 96-well plates, with 100 µl per well. Complete medium was added and the cells were cultured in an incubator (37°C, 5% CO₂) for 0, 24, 48 and 72 h. At the end of cell treatment, the culture solution was aspirated and 100 µl of CCK-8 was added to each well without generation of air bubbles. The plates were incubated in the incubator for 4 h. After, the medium was transferred to ELISA plates. The OD value at 450 nm was measured by an ELISA detector, and the data were analyzed.

The experimental grouping was the same as described above. Matrigel stock solution was maintained at room temperature for melting overnight. Matrigel stock solution and serum-free RPMI-1640 medium were used to prepare a gel solution, and the solution was placed in 96-well plates, followed by incubation for 2 h for it to solidify. The cultured SMMC-7721 cells were collected and digested to prepare single cell suspensions under aseptic conditions, and the cell suspensions were adjusted to a density of 1×10⁵/ml. The cells were seeded in 96-well plates with 100 µl per well. The plates were incubated in an incubator (5% CO₂, 37°C) for 6–8 h, and a Motic inverted microscope was used to observe the cells and take pictures.

RNA extraction: An appropriate amount of cells was collected and ground with liquid nitrogen. Total RNA extraction was performed using appropriate reagents. cDNA synthesis: A total of 1 µg of total RNA was used as template, and the reaction system was prepared according to the instructions of the Bestar qPCR RT kit. Fluorescence quantification: The reaction was performed with a volume of 20 µl (DBI Bestar^® SYBR-Green qPCR Master mix) using real-time PCR. The reaction system was prepared according to Table I. Reaction conditions for qPCR: 94°C for 2 min, followed by 40 cycles of 94°C for 20 sec, 58°C for 20 sec, and 72°C for 20 sec. Melting curve analysis: Temperature between 62°C and 95°C. Fluorescence quantitative PCR was performed using Agilent Stratagene fluorescence quantitative PCR Mx3000P. The housekeeping gene, GAPDH, was used as the endogenous reference. Primer sequences are shown in Table II. The relative expression of target gene mRNA was calculated according to the 2^−ΔΔCq method, where ΔCq = Cq (target gene) - Cq (internal reference gene), ΔΔCq = ΔCq (experimental group) - ΔCq (control group).

Cells were lysed with RIPA buffer, and the concentration of the protein lysate was determined using a BCA protein quantification kit. Total protein (20 µg) from each group was electrophoresed using 10% SDS-PAGE for 2 h at 120 V constant voltage. Total protein was transferred to NC membranes. Membranes were then incubated with rabbit polyclonal Notch1 antibody (dilution, 1:1,000; cat. no. ab27526); rabbit monoclonal VEGF antibody (dilution, 1:1,000; cat. no. ab109536) and rabbit monoclonal Jagged1 antibody (dilution, 1:1,000; cat. no. ab32152), which were all purchased from Abcam (Cambridge, MA, USA) at 4°C overnight. The membranes were washed three times, and the corresponding secondary antibody was added and incubated for 2 h followed by three washes. ECL luminescence solution was added for chemiluminescence, exposure, and development. X-film was used for exposure and scanned, and the net density value was analyzed using a gel image processing system (Image-Pro Plus 6.0; QMgene, Beijing, China).

Cells were collected, protein was extracted, and the concentration was measured using the Co-IP kit. A total of 100 µl (300 µg) of the protein extract and beads that were previously combined with the Notch1 antibody were mixed and incubated at 4°C overnight. The obtained pure protein and antibody complex was washed and eluted for western blot detection.

Data were analyzed using SPSS13.0 statistical software (SPSS, Inc., Chicago, IL, USA). Numerical data are presented as false ± SD. Comparisons between two groups were by two-sample t-test. Comparisons within a group were by paired sample t-test.

HPLC analysis showed that drug serum contained the ingredients of the spleen-invigorating and blood stasis-removing recipe (Fig. 1).

SMMC-7721 cells were cultured in serum, drug serum, and serum containing inhibitor, and cell proliferation was detected using the CCK-8 method at 0, 24, 48 and 72 h, respectively. There was no significant difference in OD value between the three groups at 0 h (P>0.05), although significant differences were found at 24, 48, and 72 h (P<0.01) (Fig. 2).

The normal serum group, drug serum group, and inhibitor group were observed under an inverted microscope (×200). Compared with the drug serum group and inhibitor group, SMMC-7721 cells in the normal serum group formed a larger area of new blood vessels with denser blood vessels (Fig. 3).

qPCR analysis showed that the levels of Jagged1 (Fig. 4A), Notch1 (Fig. 4B), and VEGF (Fig. 4C) in SMMC-7721 cells of the drug serum group and inhibitor group were significantly lower than those of the normal serum group (P<0.01).

The expression of Jagged1, Notch1, and VEGF protein in SMMC-7721 cells was measured by western blot analysis. The levels of Jagged1, Notch1, and VEGF protein in SMMC-7721 cells of the drug serum group were significantly lower than those of the normal serum group (P<0.01). The levels of Jagged1 and Notch1 were decreased after adding the Notch inhibitor, DAPT, indicating that DAPT successfully inhibited the Jagged1/Notch signaling pathway. In addition, the expression of VEGF protein was reduced after adding DAPT (Fig. 5).

The interaction between Jagged1 and Notch1 in SMMC-7721 cells of the drug serum group was significantly lower than that in the normal serum group (P<0.01). The interaction between Jagged1 and Notch1 in SMMC-7721 cells was also significantly reduced after adding DAPT compared with that of the normal serum group (P<0.01) (Fig. 6).