Establishment of Hepatocarcinoma in BALB/c-nu Mice and Investigation of the Therapeutic Effect of the Sanleng Jiashen Formula

Here, we present the step-by-step protocol to investigate the therapeutic and vascular inhibitory effects of the Sanleng Jiashen formula on BALB/c-nu mice with orthotopic hepatocellular carcinoma.

The lethality of liver cancer and the resistance to chemical drugs have forced the search for effective prescriptions of traditional herbs for liver cancer. Animal models that are repeatable, easy to manipulate, and highly mimic the pathophysiological processes of liver cancer are the prerequisite for the successful screening of effective drug candidates. Meanwhile, reliable drug efficacy evaluation indicators and means are also the guarantee of anti-liver cancer drug research and development.

Sanleng Jiashen formula, a representative prescription of traditional Chinese medicine containing Sparganium stoloni erum, Buch. -Ham. (Sanleng), Panax ginseng C. A. Mey. (ginseng), Rheum officinale Baill. (rhubarb), and Ligusticum chuanxiong Hort. (Chuanxiong), is prescribed to nourish the liver and clear heat, remove toxins, and promote blood circulation to treat liver cancer. This experimental protocol describes the preparation of lyophilized Sanleng Jiashen formula and the establishment process of in-situ liver cancer in BALB/c-nu mice. Histopathological staining, immunohistochemical detection of cancer markers, in vivo imaging of mice, and chick embryo chorioallantoic membrane test were used to explore the inhibition and anti-angiogenesis effect of Sanleng Jiashen formula on malignant proliferation of liver cancer tissue. The data show that the Sanleng Jiashen formula can effectively resist the malignant proliferation of liver cancer tissue, which is manifested by reduced tumor mass volume, improved pathological damage, and lower levels of the cancer marker ki67.

The superior inhibition of angiogenesis also suggests that the Sanleng Jiashen formula may have the potential to treat and prevent the progression and deterioration of liver cancer. The whole experimental scheme shows a comprehensive process of traditional Chinese medicine components in the treatment of mouse liver cancer, which provides a reference for the establishment and optimization of a liver cancer model, as well as the research and development of drugs to prevent and treat liver cancer.

Liver cancer is a deadly cancer that originates in the liver and is the second most common cause of cancer-related death worldwide¹. Liver disease rates in the United States and some developing countries are showing high levels, and there could be more than 1 million liver cancer cases worldwide by 2030². Due to unhealthy living habits such as smoking, obesity, and drinking, and the ravages of hepatitis B, hepatitis C, and other viruses, the incidence and mortality of liver cancer have increased significantly year by year³. As a heterogeneous malignant tumor with different histological features and poor prognosis, liver cancer has a very wide range of incidence, including hepatocellular carcinoma, intrahepatic cholangiocarcinoma, and fibrolamellar liver cancer⁴. Liver cancer is often diagnosed at an advanced stage, and many advanced patients are not candidates for radiation therapy⁵. The multikinase inhibitor sorafenib is a highly accepted drug for patients with advanced liver cancer, but patients often develop significant resistance within a short period⁶. At present, immune checkpoint inhibitors PD-1 and PD-L1 combined with sorafenib have made some progress in controlling the development of liver cancer, but the therapeutic effect is still controversial⁷.

Orthotopic liver cancer is an early type of liver cancer: cancer cells have not spread around the liver tissue or occur interorgan metastasis, and the function of the liver is not greatly affected⁸. Because orthotopic liver cancer generally does not appear obvious discomfort symptoms, some patients may have mild liver pain, fatigue, and other symptoms⁹. In clinical practice, color Doppler ultrasonography of the liver and enhanced computerized tomography examination can diagnose substantial space-occupying lesions in the liver¹⁰. Although the local liver cancer that has not spread and metastasized can be removed by surgery, there are certain surgical risks and the inevitable psychological burden for the patients¹¹. Chemoradiotherapy can kill all or part of liver cancer cells, and long-term immunosuppression in patients with medication may affect the effectiveness of treatment and increase the risk of other diseases¹². Therefore, seeking safe and effective drug therapy for early liver cancer prevention and treatment is the key to curbing the progression and deterioration of liver cancer.

The principle of Chinese medicine treating liver cancer is based on the concept of balance and harmony within the body. Chinese medicine views liver cancer as an imbalance or disharmony in the body's energy, known as Qi, which affects the liver¹³. Chinese medicine treatment for liver cancer involves various approaches, including herbal medicine, acupuncture, dietary therapy, and lifestyle recommendations. The principle is to restore the balance of Qi, strengthen the body's defense mechanisms, and promote the body's ability to heal itself^14,15. Liver cancer in the traditional Chinese medicine belongs to the "tympanites" and "liver accumulation" category. As early as the "Inner Canon of Huangdi" has a detailed record of its symptoms, liver cancer is caused by blood stasis and Zhengqi deficiency and is treated by removing blood stasis, eliminating tympanites, and strengthening the body resistance to consolidate the constitution¹³. Sanleng pill originated from the book "Weisheng Baojian" written by Luo Tianyi, a famous doctor in the Yuan dynasty. It is composed of three kinds of drugs: Sparganium stoloni erum, Buch. -Ham. (Sanleng), Ligusticum chuanxiong Hort. (Chuanxiong), and Rheum officinale Baill. (rhubarb). These three kinds of drugs can enter the liver meridian and have therapeutic value for the treatment of liver cancer¹⁴. Sanleng Jiashen formula is composed of 16 g of Sanleng, 16 g of Panax ginseng C.A.Mey. (ginseng), 8 g of rhubarb, and 2 g of Chuanxiong (Figure 1), which is prescribed to tonify the liver, clear heat, eliminate toxins, and promote blood circulation. These herbs are believed to have anti-cancer properties and help reduce tumor growth, alleviate symptoms, and improve overall well-being. In the previous in vitro experiments, the Sanleng Jiashen formula can inhibit the proliferation of liver cancer cells¹⁵. However, how to evaluate the inhibitory effect of Chinese herbal compounds on liver cancer efficiently and reasonably? By establishing a BALB/c-nu mouse in situ liver cancer model, the inhibitory effect of the Sanleng Jiashen formula on liver cancer and angiogenesis was investigated mainly through small animal live imaging and chick embryo chorioallantoic membrane test.

BALB/c-nu male nude mice that were specific pathogen-free, 5 weeks of age, and weighing 18-22 g were fed in the Animal Experimental Center of Transformation Medical College of Jilin University; the feeding conditions were 22 °C-24 °C with 40%-60% relative humidity. The experimental animal license number is SYXK(Ji) 2018-0006, and the experimental process complies with the rules and regulations of the Ethics Committee of Jilin University, and the animal ethics approval number is 20221228-01.

1. Preparation of freeze-dried Sanleng Jiashen formula¹⁵

1. Place 16 g of Sanleng, 16 g of ginseng, 8 g of rhubarb, and 2 g Chuanxiong in an intelligent decocting pot (see Table of Materials) and soak in 294 mL of deionized water at room temperature for 30 min. After heating and boiling, continue to cook for 30 min at 100 °C, and filter the liquid through double medical gauze (see Table of Materials). Add 252 mL of deionized water to the residue, cook for 30 min at 100 °C, and filter the liquid with double gauze.
2. Collect the filtrate from step 1.1 in a clean stainless steel dish and freeze for 12 h at -80 °C (see Table of Materials).
3. Place the Sanleng Jiashen formula from step 1.2 in a vacuum freeze dryer (see Table of Materials) (cold trap temperature: -55 °C) and dry for 48 h to obtain the freeze-dried powder of the Sanleng Jiashen formula.

2. Construction of HCCLM3 cell line expressing luciferase stably¹⁶

1. Add 1.0 mL of HCCLM3 cell suspension in the 24-well plate (see Table of Materials) with 5 × 10⁵ cells/well and culture in a complete medium (10% fetal bovine serum + 1% penicillin-streptomycin solution + DMEM) (see Table of Materials) containing 3 µg/mL puromycin for 72 h in a cell incubator (37 °C, 5% CO₂, see Table of Materials).
2. Discard the cell supernatant in step 2.1, and add 100 µL of 0.25% trypsin solution to digest the cells in a cell incubator for 1 min (see Table of Materials). Add 300 µL of complete medium and use a pipette to free the adherent cells and terminate digestion. Collect all cell suspensions and place them in a 15 mL centrifuge tube at room temperature at 1,000 × g for 5 min. After discarding the supernatant, add 1 mL of complete medium and mix by pipetting.
3. Add 100 µL of cell suspension of 4 × 10³ HCCLM3 cells/well from step 2.2 into a 96-well plate and culture in a cell incubator for 24 h (37 °C, 5% CO₂, see Table of Materials).
4. Add 0.27 µL of GFP lentivirus to the 96-well plates in step 2.3 and culture in a cell incubator for 15 h (37 °C, 5% CO₂, see Table of Materials). Discard the cell supernatant, add 100 µL of complete culture medium, and acquire images using a fluorescence microscope at the excitation wavelength of 488 nm and the emission wavelength of 507 nm(see Table of Materials).
5. Replace 2 mL of GFP lentivirus and continue to culture for 24 h. Replace 2 mL of complete culture medium without GFP lentivirus for another 24 h.
   NOTE: The transfection efficiency of GFP lentivirus into HCCLM3 cells was 80% as seen under an inverted fluorescence microscope.
6. Add 5 mL of 2 × 10⁵ cell suspension from step 2.1 into a 60 mm Petri dish and culture in a cell incubator for 24 h. Add 64.5 µL of luciferase, and continue to culture in a cell incubator for 15 h (37 °C, 5% CO₂, see Table of Materials).
7. Discard the supernatant and add 5 mL of complete medium to continue to culture until the cells have become 90% confluent in the Petri dish. Discard the cell supernatant, wash with 2 x 1 mL of PBS, and add 1 mL of 0.25% trypsin solution to digest the cells in a cell incubator for 1 min (37 °C, 5% CO₂, see Table of Materials). Add 3 mL of complete medium and pipette up and down to free adherent cells and terminate digestion. Collect all cell suspensions for subsequent mouse injection.
   NOTE: To obtain a sufficient number of cells, the cells were continuously passaged and cultured.

3. Establishment of BALB/c-nu nude mice model of liver cancer in situ and treatment¹⁵

1. Add 100 µL of 1 × 10⁷ HCCLM3 cells transfected with luciferase in step 2.7 and 100 µL of matrix glue (see Table of Materials) into a 1.5 mL centrifuge tube (see Table of Materials), and lightly mix with a 1 mL pipette (see Table of Materials).
   NOTE: The whole process of solution preparation must be carried out on ice to prevent the matrix glue from curing.
2. Disinfect the left and right armpits of BALB/c-nu nude mice with iodopine (see Table of Materials) and deiodize with alcohol (see Table of Materials).
3. Slowly inject 100 µL of the mixture of HCCLM3 cells and matrix glue from step 3.1 into the left and right armpits of BALB/c-nu nude mice with a 1 mL disposable syringe.
   NOTE: After approximately 10 days, BALB/c-nu nude mice developed a tumor mass of approximately 0.5 cm³ at the bilateral underarm injection site.
4. Anesthetize BALB/c-nu nude mice in step 3.3 by intraperitoneal injection of 1%, 50 mg/kg sodium pentobarbital. Cut the skin of both armpits with an ophthalmic scissor and tweezer (see Table of Materials), and gently peel the subcutaneous tumor mass to place it in a cell culture dish (see Table of Materials) containing pre-cooled PBS buffer (see Table of Materials), and cut it into 1 mm³ pieces with a scalpel (see Table of Materials).
   NOTE: The underarm tumor mass of BALB/c-nu nude mice must be harvested on ice within 0.5 h to maintain tumor cell activity and increase the model's success rate. At the end of sampling, the mice were killed by cervical vertebrae removal. If there is a necrotic part of the tumor, it needs to be removed, leaving only the fish white tumor to prevent infection during the orthotopic cancer transplantation.
5. Anesthetize BALB/c-nu nude mice with 1%, 50 mg/kg sodium pentobarbital intraperitoneally (see Table of Materials), disinfect the abdomen with iodine (see Table of Materials), and deiodize with alcohol (see Table of Materials).
6. Use an ophthalmic scissor and tweezer (see Table of Materials) to cut a 45° surgical incision about 1 cm at the position of 0.5 cm below the xiphoid process to expose the liver, gently press the liver to squeeze out liver tissue, and fix the liver with weighing paper.
   NOTE: Cotton swabs or sticks can be placed under the ribs of mice to effectively expose the liver.
7. Take a small piece of tumor tissue in step 3.4 with a 10 mL disposable injection needle, and implant the tumor mass into the liver about 0.5 cm parallel to the liver to fix it under the liver capsule and slowly withdraw the needle.
   NOTE: Use a 1 mL disposable syringe to drop 2 drops of the melted matrix glue, observe the tumor mass without any accidental slips and no bleeding, and then suture the surgical incision.
8. Close the surgical incision with a 12 cm needle holding forceps and 6-0 surgical sutures (see Table of Materials). Apply erythromycin eye ointment (see Table of Materials) to the surgical site to prevent infection. Place the mice on a heated pad (see Table of Materials) and keep them in separate cages after awakening.
9. Starting from the first day after modeling, administer 7.5 mg/kg sorafenib and Sanleng Jiashen formula (7.8 g/kg, 15.6 g/kg, and 31.2 g/kg) via gavage to the mice from step 3.8 for 14 days, once a day. Administer an equal volume of normal saline to the mice in the model group.

4. In vivo imaging evaluation

NOTE: On day 14, live imaging was performed 1 h after the end of administration.

1. Power on the computer and in vivo imaging instrument (see Table of Materials). Double-click on the Living Image icon on the desktop to launch the program. Click on initialize IVIS system in the control panel to start the entire IVIS system.
   NOTE: After the machine completes the self-test, the temperature status light in the control panel is red. Wait about 5-10 min after the temperature drops and the light turns green, image acquisition can be performed.
2. Disinfect the abdomen of mice with alcohol and intraperitoneally inject 10 µL D-luciferin per g of body weight (see Table of Materials) with a disposable syringe. Anesthetize mice by intraperitoneal injection of 1%, 50 mg/kg sodium pentobarbital.
   NOTE: D-luciferin was diluted with sterile PBS to 15 mg/mL.
3. Put the anesthetized mice in step 4.3 into the observation box and close the door, set the exposure time as 30 s in the control panel: Mode selection: select luminenscence according to the sample type, select automatic program by default, and press acquire to acquire images.
4. Click on Save Living Image Data, select All Living Image Data Files, and save it on the computer desktop.
5. Click on Exit in Living image on the main screen. Turn off camera power, imaging instrument power, and computer power in turn.
6. After imaging, euthanize the anesthetized mice (intraperitoneal injection of 1%, 50 mg/kg sodium pentobarbital) by cervical vertebrae dislocation.

5. Hematoxylin-eosin staining¹⁷

1. Fix the fresh liver tissue of mice from step 4.7 in 4% paraformaldehyde for 48 h, and then rinse with running water 3 times for 5 min each time to wash away the paraformaldehyde (see Table of Materials).
2. Dehydrate the liver tissues in step 5.1 in 30% ethanol, 50% ethanol, 70% ethanol, 95% ethanol , 95% ethanol , and anhydrous ethanol for 1 h in turn (see Table of Materials).
3. Put the dehydrated liver tissues in step 5.2 to 50% ethanol, 50% xylene, xylene , xylene , and xylene sequentially to make them transparent for 1 h (see Table of Materials).
   NOTE: Xylene is volatile and toxic, this step is carried out in the fume hood.
4. Melt the paraffin wax and keep the temperature at about 55 °C. Place the liver tissue blocks in step 5.3 into a paraffin embedding machine (see Table of Materials) filled with wax solution for embedding.
5. Fix the embedded wax block of mice liver tissue in step 5.4 on the paraffin slicing machine (see Table of Materials), cut it into 5 µm thick slices, lay the slices completely flat in 50 °C water, and scoop them up with a slide. Finally put them in a biological tissue baking machine for drying 20 min at 70 °C (see Table of Materials).
6. Soak the 5 µm sections in step 5.5 into xylene , xylene , anhydrous ethanol , and anhydrous ethanol for 10 min successively, then soak in 95% ethanol, 90% ethanol, 80% ethanol, and 70% ethanol for 5 min respectively, and finally wash with distilled water for dewaxing treatment (see Table of Materials).
7. Dye the dehydrated, 5 µm thick, parrafin wax liver tissue sections with hematoxylin for 8 min, and then rinse with running water.
8. Differentiate in 1% hydrochloric acid solution for 10 s, and rinse with running water for 30 min (see Table of Materials).
9. Dye with 0.5% eosin solution for 1 min (see Table of Materials).
10. Put the slices into 95% alcohol , 95% alcohol , anhydrous ethanol , anhydrous ethanol , xylene , and xylene for 5 min each to dehydrate (see Table of Materials).
11. Seal with neutral gum, and take photos under an inverted light microscope (see Table of Materials).

6. Immunohistochemical staining¹⁸

1. Drop 1.5% H₂O₂ onto the 5 µm sections of mouse liver in step 5.6 and incubate at room temperature for 10 min, then wash with PBS for 2 x 5 min (see Table of Materials).
2. Repair tissue with a citric acid solution (see Table of Materials) for 5 min and wash with PBS for 3 x 5 min.
3. Incubate with 0.2% triton X-100 (see Table of Materials) at room temperature for 4 min, then incubate with 0.5% triton X-100 at room temperature for 4 min, and wash with PBS.
4. Add goat serum (see Table of Materials) to seal for 15 min, then add the ki67 primary antibody (see Table of Materials) diluted with PBS (1:300) to incubate at 4 °C overnight. Wash with PBS for 3 x 5 min.
5. Incubate with secondary antibody diluted with PBS (1:200) at room temperature for 1 h, and wash with PBS for 3 x 5 min (see Table of Materials).
6. Stain with the developing solution for 5 min and rinse with double distilled water (see Table of Materials).
7. Re-stain with hematoxylin for 2 min, differentiate in 1% hydrochloric acid solution for 10 s, and rinse with running water for 30 min (see Table of Materials).
8. Put the slices into 95% alcohol , 95% alcohol , anhydrous ethanol , anhydrous ethanol , xylene , and xylene for 5 min each to dehydrate (see Table of Materials).
9. Seal with neutral gum and take photos under an inverted light microscope (see Table of Materials).

7. Chick embryo chorioallantoic membrane test¹⁹

1. Leave the white feather chicken eggs (see Table of Materials) at room temperature for 2 h and spray with alcohol to disinfect. Drill a hole in the air chamber with a skull drill (see Table of Materials), and incubate the eggs with the air chamber up for 7 days (Incubation temperature 37.8-38.2 °C and relative humidity 65%).
   NOTE: During the first 3 days of incubation, the automatic egg turning button is turned on to prevent the chick embryo chorioallantoic membrane from adhering to the shell and creating a false air chamber.
2. Discard the weak/unfertilized eggs in the egg illuminator (see Table of Materials). Peel out a 1 x 1 cm window carefully with eye tweezers (see Table of Materials) after drilling a hole in the fake air chamber with a skull drill (see Table of Materials).
   NOTE: Please note that the eggshell should not fall into the chick embryo chorioallantoic membrane.
3. On day 8 of incubation, add 100 µL of tumor cell suspension (1 × 10⁶ cells) to each egg from the fake air chamber except for the control group. In the drug groups, add 100 µL of sorafenib solution and Sanleng Jiashen formula. In the control and model groups, add 100 µL of PBS.
   NOTE: The tumor cell suspension is composed of HCCLM3 cells and matrix glue in equal volume. The drug concentration is set as follows: 7.5 mg/kg sorafenib and Sanleng Jiashen formula (7.8 g/kg, 15.6 g/kg, and 31.2 g/kg).
4. Cover the window with transparent dressing (see Table of Materials) and disinfect with alcohol spray (see Table of Materials). After 7 days of incubation, remove the transparent dressing, and add 1 mL of fixative (methanol:acetone = 1:1) (see Table of Materials) from the fake air chamber for a 30 min fixation.
5. Discard the fixative with a 1 mL syringe. Cut ~5 x 5 cm chorioallantoic membrane in the fake air chamber with ophthalmic scissors and place in a 60 mm Petri dish.
   NOTE: For chick embryo chorioallantoic membrane with blood, rinse with PBS. For chick embryo chorioallantoic membrane attached to the tissue, separate with ophthalmic scissors.
6. Put the chorioallantoic membrane from step 7.5 under an optical microscope and photograph the vessels (see Table of Materials).

As shown in Figure 2A, we established an in-situ liver cancer model of BALB/c-nu mice and evaluated the antitumor effect of the Sanleng Jiashen formula. Figure 2B presents that the Sanleng Jiashen formula can significantly suppress the growth of liver tumors. The pathological results indicated that, compared with the control group, the tumor foci in the model group had clear boundaries with the surrounding tissue, larger tumor foci, nodular expansion, and compression growth. Meanwhile, the arrangement of tumor cells in tumor tissue is disordered with no liver lobular structure. Conformably, the tumor cells are less heterogeneous, and there are large blood vessels around the tumor body. Compared with the model group, the tumor area of different concentrations of the Sanleng Jiashen formula was reduced, indicating that the tumor size of mice in situ cancer was inhibited after drug intervention (Figure 2C). Further immunohistochemical results of ki67, a tumor proliferation marker in Figure 2D, also demonstrated that the Sanleng Jiashen formula can greatly inhibit tumor proliferation. To more intuitively and accurately explore the effect of the Sanleng Jiashen formula on the growth of liver tumors, small animal imaging was used to visualize the tumor mass size of in-situ liver cancer. As shown in Figure 3, the Sanleng Jiashen formula signally reduced the volume of liver tumors. To further confirm whether the Sanleng Jiashen formula inhibits the growth of liver cancer by inhibiting angiogenesis phenotype, the chick embryo chorioallantoic membrane test was conducted in this study according to Figure 4A. Figure 4B,C demonstrate that compared with the control group, the number of new blood vessels in the model group was distinctly increased with chaotic distribution. Compared with the model group, the small blood vessels in different concentrations of Sanleng Jiashen formula were markedly reduced, and some large blood vessels were broken with insufficient blood circulation, manifesting that angiogenesis was reduced after drug intervention.

Figure 1: Drug composition and proportion of Sanleng Jiashen formula. (A) Sanleng (16 g). (B) ginseng (16 g). (C) rhubarb (8 g). (D) Chuanxiong (2 g). Please click here to view a larger version of this figure.

Figure 2: Establishment and tissue staining of in situ liver cancer model in BALB/c-nu mice. (A) Schematic diagram of an in situ liver cancer model. (B) Naked eye view of the volume of liver cancer tissue. The blue arrows indicate tumor areas of the liver. (C) Pathological staining of liver cancer tissue. The blue dotted circles indicate the tumor area in the liver histopathologic sections. Scale bars = 200 µm. (D) ki67 immunohistochemical staining of hepatocellular carcinoma. Scale bars = 200 µm. Please click here to view a larger version of this figure.

Figure 3: In vivo imaging of orthotopic liver cancer in BALB/c-nu mice. Compared with the model group, Sanleng Jiashen formula groups showed a concentration-dependent decrease in the volume of liver tumors in BALB/c-nu mice; n = 5. Please click here to view a larger version of this figure.

Figure 4: Schematic diagram and results of chick embryo chorioallantoic membrane test. (A) The specific process of chicken embryo chorioallantoic membrane test. (B) Imaging the number of blood vessels. Scale bars = 0.2 mm. (C) Statistical results of blood vessel area of chick embryo chorioallantoic membrane. Compared with the control group, the vascular area of the model group was significantly increased. In comparison with the model group, Sanleng Jiashen formula groups showed a concentration-dependent decrease in vascular proliferation; n = 3. Compared with the control group, ^###p < 0.001; Compared with the model group, ^***p < 0.001. The data were analyzed by One-way ANOVA followed by Tukey's test. Data are presented as mean ± SD. Please click here to view a larger version of this figure.

A large amount of evidence has confirmed that Sanleng, ginseng, rhubarb, and Chuanxiong in the Sanleng Jiashen formula have pharmacological effects on the treatment of liver cancer. Studies have shown that Sanleng can significantly inhibit the proliferation and growth of tumor cells to prevent tumor cell invasion and metastasis, induce cell apoptosis, and regulate the cell cycle and tumor microenvironment²⁰. It has been proved that kaempferol, the active ingredient in Sanleng, can inhibit the occurrence and development of gastric cancer²¹, prostate cancer²², and liver cancer²³ by inhibiting proliferation, promoting autophagy, and regulating oxidative stress. Myricetin is a key compound of ginseng that possesses antitumor activity. It is known that myricetin can confine the growth of bladder cancer by inducing apoptosis²⁴ and intercept the proliferation of lung adenocarcinoma by inducing cell cycle arrest²⁴. More importantly, myricetin can restrict the growth of liver cancer cells by inhibiting angiogenesis²⁵. Currently, myricetin has been marketed as a healthcare product in Europe for liver protection and its drug value is worth further exploration.

At present, ginsenosides Rh2 and Rg3 are commonly used in clinical antitumor drugs, which can not only inhibit tumor growth, tumor angiogenesis, invasion, and metastasis but also improve the immunity of the body²⁶. Shenyi capsule with ginsenoside Rg3 as its main component has been marketed for improving the symptoms of Qi deficiency in tumor patients. Clinically, it is believed that the Shenyi capsule can regulate the immunity of patients with primary lung cancer²⁷ and liver cancer, improve the toxic and side effects of chemotherapy in patients with advanced liver cancer, and serve as adjuvant therapy for patients with sorafenib resistance²⁸. In addition, ginsenoside Rg3 can inhibit angiogenesis and promote anti-tumor immunity to reshape the tumor microenvironment and effectively block the pathway of tumor absorption of nutrients, thereby inhibiting its growth and metastasis²⁹.

As two effective components of rhubarb, emodin, and aloe emodin are the key compounds for rhubarb to exert antitumor activity. Evidence shows that emodin can induce apoptosis of colon cancer, liver cancer, and laryngeal cancer cells in vitro and in vivo through PI3K/AKT and MAPK signaling pathways³⁰. In vivo experiments, emodin improved liver and kidney function to inhibit tumor growth, possibly by inhibiting proliferation and inducing apoptosis³¹. Meanwhile, through co-culture of breast cancer cells and vascular endothelial cells, it was indicated that after the intervention of aloe emodin, the proliferation of human umbilical vein endothelial cells in the co-culture system decreased observably, manifesting that aloe emodin can inhibit the proliferation of breast cancer cells by inhibiting angiogenesis pathway³². Tetramethylpyrazine can reverse multidrug resistance of liver cancer, and its mechanism is related to inhibiting the expression of P-gp³³. Ligusticum chuanxiong polysaccharide has an obvious inhibitory effect on HepG2 cells, which is related to cell cycle arrest, induction of tumor cell apoptosis, and inhibition of tumor angiogenesis^34,35. To sum up, the Sanleng Jiashen formula has great potential application value in the treatment of liver cancer. This protocol also preliminarily confirmed that the Sanleng Jiashen formula could inhibit the proliferation of in-situ liver cancer in BALB/c-nu mice, and showed superior pharmacological activity to inhibit angiogenesis. These data provide a reference for further exploring the potential molecular mechanism of Sanleng Jiashen prescription in the treatment of liver cancer.

Tumor vascular dysplasia is a significant feature of cancer occurrence and development. In this study, the chick embryo chorioallantoic membrane test was selected to evaluate whether the Sanleng Jiashen formula can inhibit tumor angiogenesis. First, experiments with the chicken embryo do not require additional animal ethical approval and are inexpensive and easy to perform. Second, the immune system of chicken embryos does not develop within 15 days of incubation, which is a relatively perfect tumor drug efficacy evaluation model, and the price is lower than that of nude mice. Third, it has been reported that the chicken embryo allantoic membrane test can also detect the results in real time by combining optical coherence tomography, confocal microscopy, magnetic resonance imaging (MRI), and fluorescence imaging. In in vivo imaging of small animals, whether it is bioluminescence or fluorescence imaging, the luminescence results are affected by many factors. For example, in the process of bioluminescence, the intensity of fluorescence is significantly related to the quality of the injected cells, the time of luciferase injection, and the depth of the model site. Therefore, MRI technology is also required for the diagnosis of liver cancer and the evaluation of drug efficacy.

In summary, this article established a BALB/c-nu mouse model of in-situ liver cancer and confirmed that the traditional Chinese medicine Sanleng Jiashen formula can effectively inhibit the proliferation of in-situ liver cancer through small animal imaging and chick embryo chorioallantoic membrane experiments, which may be related to the inhibition of angiogenesis. These established methods provide a reference for the rapid and efficient screening of traditional Chinese medicines for the treatment of liver cancer.However, the material basis and molecular mechanism of the Sanleng Jiashen formula containing complex components in the treatment of liver cancer still need to be further clarified by combining chemical analysis techniques¹⁸ and molecular interaction research methods, such as high-performance liquid chromatography and surface plasmon resonance^36,37.

The authors have nothing to disclose.

This work was supported by the Philosophy and Social Science Foundation of China (20VYJ070), the Project of Jilin Provincial Education Department (JJKH20230963KJ), and the Second Batch of Xinglin Scholars Engineering Project in Changchun University of Chinese Medicine (QNKXJ2-2021+ZR25).