Establishment of Hepatocarcinoma in BALB/c-nu Mice and Investigation of the Therapeutic Effect of the Sanleng Jiashen Formula
Summary
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.
Abstract
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.
Introduction
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 itself14,15. 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.
Protocol
Representative Results
Discussion
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 occu…
Divulgations
The authors have nothing to disclose.
Acknowledgements
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).
Materials
| 0.25% trypsin solution | HyClone, America | SH30042.01B | |
| 1 mL disposable syringe | Mingankang Medical Equipment Co., Ltd., China | RWSB | |
| 1% Penicillin & streptomycin solution | Hyclone, America | SV30010 | |
| 1.5 mL centrifuge tube | Corning, America | MCT-150-C-S | |
| 12 cm needle forceps | Hesdick, China | HKQS-211 | |
| 6-0 surgical sutures | Shanghai Jinhuan Medical Equipment Co., Ltd., Shanghai, China | CR631 | |
| 75% alcohol | Sichuan Youbang Enterprise Co., Ltd., Sichuan, China | 1.00009E+11 | |
| 96-well plates | NEST, China | 701001 | |
| Acetone | Tianjin Dingfu Chemical General Plant, Tianjin, China | GB686-89 | |
| BALB/c-nu male nude mice | Beijing Weitonglihua Experimental Animal Technology Co., Ltd., Beijing, China | SYXK(Ji) 2018-0006 | |
| Biological tissue baking machine | Leica, Germany | Leica HI1220 | |
| Cell culture dish | NEST, China | NEST.706001 | |
| Citric acid solution | Wuhan Canos Technology Co., Ltd., Wuhan, China | sj1074 | |
| D-luciferin | Gold Biotechnology®, Inc., China | LUCK-100 | |
| DMEM | Hyclone, America | SH30243.01 | |
| Egg illuminator | Shandong Weizhen Incubation Equipment Company, Shandong, China | WZSDT | |
| Eosin staining solution | Hunan BKMAM Biotechnology Co., Ltd., Hunan, China | 110703061 | |
| Erythromycin eye ointment | Cisen Pharmaceutical Co., Ltd., Shanghai, China | H37022025 | |
| Fetal bovine serum | Clark, America | FB25015 | |
| Fully automatic intelligent chick incubator | Shandong Weizhen Incubation Equipment Company, Shandong, China | 29538 | |
| GFP lentivirus solution | Suzhou GenePharma Co.,Ltd., Suzhou, China | F22AZ | |
| Goat serum | Shenyang Wanlei Biotechnology Co., Ltd., Shengyang, China | WLA067 | |
| Hand-held mouse skull drill | STRONGWT, China | 190 | |
| HCCLM3 cell line | WheLab, Shanghai, China | C1010 | |
| Heating pad | Zhongke Life Technology Co., Ltd., Hangzhou, China | GEJRD-10W | |
| Hematoxylin | Hunan BKMAM Biotechnology Co., Ltd., Hunan, China | B-YH250-1 | |
| Intelligent decoction pot | Hangzhou Jiuyang living Appliance Co., Ltd., Hangzhou, China | 3003BQ | |
| Inverted fluorescence microscope | Olympus, Japan | IX73 | |
| ki67 primary antibody | Wuhan Servicebio Technology Co., Ltd., Wuhan, China | GB121141-100 | |
| Lodophor disinfectant | Cofoe Medical Technology Co.,Ltd., Hunan, China | 202110073 | |
| Luciferase | Suzhou GenePharma Co.,Ltd., Suzhou, China | E26JZ | |
| Matrix glue | Corning, America | 356234 | |
| Medical gauze | Yunnan Chenye Biotechnology Co., Ltd., Yunnan, China | 71712049971 | |
| Methanol | Guangdong Guanghua Sci-Tech Co., Ltd., Guangdong, China | 1.17001.023 | |
| Ophthalmic scissor | Hesdick, China | HKQS-209 | |
| Ophthalmic tweezer | Hesdick, China | HKCL-20 | |
| Paraffin embedding machine | Leica, Germany | EG1150H | |
| Paraffin slicing machine | Leica, Germany | Leica CM1950 | |
| Paraformaldehyde | Biosharp, China | BL539A | |
| PBS buffer | Wuhan Servicebio Technology Co., Ltd., Wuhan, China | G2156-1L | |
| Puromycin | Beijing Solarbio Science & Technology Co., Ltd., Beijing, China | P8230 | |
| Refrigerator | Thermo Scientific, America | TDE40086FV-ULTS | |
| Scalpel | Hesdick, China | HKCL-93 | |
| Secondary antibody | Wuhan Servicebio Technology Co., Ltd., Wuhan, China | GB23301 | |
| Small animal live imaging system | Caliper Life Sciences, America | IVIS Lumina XR | |
| Sorafenib | Shanghai Aladdin Biochemical Technology Co., Ltd., Shanghai, China | 284461-73-0 | |
| Transparent dressing | 3M, America | 9534HP | |
| Triton X-100 | Beijing Solarbio Science & Technology Co., Ltd., Beijing, China | T8200 | |
| Vacuum freeze dryer | Ningbo Xinzhi Biotechnology Co., Ltd., Ningbo, China | sz-10N | |
| White feather chicken eggs | Shandong Haotai Experimental Animal Breeding Co., Ltd., Shandong, China | SCXK(Lu) 20180004 | |
| Xylene | Sigma-Aldrich, , America | 534056 |
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