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 an....

Protocol

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.

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.......

Materials

Name	Company	Catalog Number	Comments
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