Name: establishment of coloproctitis cancer model in mice and evaluation of therapeutic effect of chinese medicine
Uploaded: 2023-10-13T12:50:00.000+00:00
Duration: 6 min 24 s
Description: Watch this Scientific Journal Video about Author Spotlight: Liujunzi Decoction as a Traditional Chinese Treatment for Coloproctitis Cancer at JoVE.com

This work was supported by the Jilin Provincial Department of Science and Technology (YDZJ202201ZYTS181).

The animal procedure has been approved by the Ethics Committee of Changchun University of Chinese Medicine (Record number: 2021214). Specific pathogen-free C57BL/6J mice (8-10 weeks, weight 18-22 g), male and female, were housed in independently ventilated cages at 22 &#176;C and 65% relative humidity. The mice began the experiment after 7 d of adaptive feeding, during which they had free access to water and diet.
1. Drug preparation
<ol>
	<li>Preparation of LJZD 
	&#8203;NOTE: The Chinese medicine used was purchased from the Affiliated Hospital of Changchun University of Chinese Medicine and was identified as genuine Chinese medicine (see Table 1).
	<ol>
		<li>Put Dangshen (12 g), Baizhu (12 g), Gancao (6 g), Chenpi (12 g), Jiangbanxia (9 g) into a specialized ceramic pot (see Table of Materials). Add 1000 mL of distilled water and soak at room temperature for 1 h (see Figure 1A).</li>
		<li>Powder 12 g Fuling into fine powder with a grinder and soak in 300 mL of distilled water in another container for 1 h at room temperature.</li>
		<li>Mix the above Chinese medicine in the specialized ceramic pot. Boil the mixture and then keep on medium flame until only 300 mL of decoction remains. Use medical gauze for filtering and preserve the filtrate at room temperature.</li>
		<li>Add 1000 mL of distilled water and repeat the above decoction operation once more.Filter again using medical gauze. Combine the filtrate and boil it until only 150 mL remains.</li>
		<li>Centrifuge the concentrated liquid at 10,000 x g for 5 min, and further concentrate the obtained supernatant to 30 mL on medium heat. Transfer the final concentrate to a dish and dry it using an electric drying oven until only the solute remains as a powder.</li>
		<li>Weigh the above solid and dissolve it in sterile distilled water to obtain a solution containing 22.85 mg of drug per 0.2 mL (114.16 mg/mL), which is the daily dosage of mice.</li>
	</ol>
	</li>
	<li>Preparation of 5-amino salicylic acid 
	&#8203;NOTE: 5-amino salicylic acid (5-ASA; see Table of Materials) has a good preventive effect on UC-CRC, and it was used as a positive drug in this study23.
	<ol>
		<li>Dissolve 64 mg 5-ASA powder in 200 mL of sterile distilled water to obtain 1.82 mg/mL 5-ASA solution. The daily dosage for a single mouse was 0.2 mL.</li>
	</ol>
	</li>
	<li>Preparation of AOM injection solution
	<ol>
		<li>Add 2.5 mL of sterile distilled water into 25 mg AOM powder (see Table of Materials), and mix by vortex mixer (see Table of Materials) to make 10 mg/mL AOM stock solution, store at -20 &#176;C until use.</li>
		<li>Prepare the AOM injection solution by diluting the AOM stock solution with sterile distilled water at 10:1 (1 mg/mL).</li>
	</ol>
	</li>
</ol>
2. Establishment of UC-CRC model
NOTE: The experiment was divided into 4 groups: control, model, LJZD, and 5-ASA group, 10 mice in each group. Except the control group, the other groups were treated with AOM and DSS.
<ol>
	<li>Intraperitoneal injection of AOM injection solution 
	NOTE: After adaptive feeding for 7 d, mice were given AOM injection solution (1 mg/mL) by intraperitoneal injection (see Figure 1B).
	<ol>
		<li>Hold the mouse with its belly up and head slightly down. Grip the back skin to tighten the abdominal skin and pierce the skin approximately 1 cm to the right of the intersection of the root line of both thighs on the mid-abdomen line with a 1 mL syringe (see Table of Materials).</li>
		<li>Push the 1 mL syringe needle to a distance of 3-5 mm under the skin, keeping it parallel to the abdominal midline, and insert the needle 0.3-0.5 mm into the abdominal cavity at a 45&#176;.</li>
		<li>After the tip passes through the abdominal muscle, the operator senses a sudden loss of resistance. Subsequently, pull the syringe outward and backward to observe whether liquid seepage occurs. If not, push the AOM injection solution slowly into the mice at 0.1 mL/10 g.</li>
	</ol>
	</li>
	<li>Cyclic stimulation of 2% DSS solution 
	&#8203;NOTE: Each AOM-induced mouse was given 500 mL of DSS solution at weeks 3, 6, and 9, and the mice drank freely during this period.
	<ol>
		<li>Prepare 2% DSS solution by adding 500 mL of sterile distilled water to 10 g DSS (see Table of Materials). Mix with a vortex mixer and store at 4 &#176;C until use.</li>
		<li>Each AOM-induced mouse drinks 500 mL of 2% DSS solution freely for 7 d on weeks 3, 6, and 9 after AOM induction.</li>
	</ol>
	</li>
</ol>
3. Drug treatment
NOTE: Adult humans need 63 g LJZD per day. According to the conversion formula of experimental mouse and human drug dose, equivalent experimental dose for mice (mg/kg) = human dose (mg/kg)/body weight (60 kg) x 9.1, the daily dose of mice was about 9.6 g/kg.
<ol>
	<li>Treat the LJZD and 5-ASA group with 0.1 mL/10 g of the prepared LJZD and 5-ASA solution by gastric gavage at week 7 and week 15, respectively.
	<ol>
		<li>For intragastric administration in mice, perform the following procedure. Hold the mouse in the left hand, and in the right hand, hold the stomach perfusion apparatus. Insert the syringe needle in the mouth and slide it down the back wall of the pharynx of the mice. Slide down the pharynx as the mice swallow and continue to move ahead. When there was a sense of resistance, and the syringe could be pushed into the pharynx, pull the needle out and complete the injection.</li>
	</ol>
	</li>
	<li>Treat the control and model group with the same amount of saline (see Table of Materials).</li>
	<li>Treat the mice in each group with appropriate drug once a day at the same time during the administration period.</li>
</ol>
4. Evaluation of UC-CRC model and efficacy of LJZD
<ol>
	<li>Disease activity index score 
	NOTE: According to Table 2, the disease activity index (DAI) score was evaluated by combining weight loss, fecal viscosity, and stool bleeding of the mice.
	<ol>
		<li>Record the weight of the mice daily from the beginning of adaptive feeding until the end of drug treatment.</li>
		<li>Observe the fecal consistency of each experimental mouse carefully and record the bowel movements as one of the three conditions: normal, loose stools, and watery diarrhea.</li>
		<li>Record the fecal bleeding of experimental animals as one of the three conditions: no bleeding, little bleeding, and visible blood in stool.</li>
	</ol>
	</li>
	<li>Detection of IL-6 level in serum
	<ol>
		<li>Treat mice with LJZD or 5-ASA for 9 weeks. Fix the mice by grasping the neck skin of the mouse with the left hand and gently pressing on the experimental table to take the lateral decubitus position. Cut off the whiskers of the mice with scissors. Cut off the whiskers of the mice with scissors carefully(see Table of Materials) to prevent blood contamination. 
		NOTE: Mice that could not wiggle freely were considered to be properly fixed. If this does not happen, the mice should be fixed.</li>
		<li>Anesthetize the mouse by inhalation of 2% isoflurane. Sterilize the skin around the eyeball with ethanol (see Table of Materials). Press the eye skin on the side gently to make the eyeball congested and protruding.</li>
		<li>Clamp the eyeball with an elbow tweezer and remove the eyeballs accurately and quickly. Allow the blood to drip into the centrifugal tube (see Table of Materials). In the process, tap the mouse&#39;s heart to speed up blood collection.</li>
		<li>Keep the collected blood at room temperature for 30 min and centrifuge it at 3,500 x g for 10 min. Collect the supernatant and detect the IL-6 level according to the IL-6 content detection kit instructions (see Table of Materials).</li>
	</ol>
	</li>
	<li>Separating colorectal tissue
	<ol>
		<li>Euthanize the mice after blood sampling by inhaling 5% overdosed isoflurane and cervical dislocation (see Table of Materials) in accordance with animal ethics.</li>
		<li>Keep the mice in a cryogenic anatomical environment. Immobilize the mice in a supine position. Cut the lower abdomen hair with scissors and sterilize it with ethanol.</li>
		<li>Pinch the intersection point between the two thigh roots and the abdominal midline with eyelid forceps (see Table of Materials). Cut a transverse incision about 1-1.5 cm with scissors.</li>
		<li>Clip a longitudinal incision along the median line of the abdomen from the midpoint of the transverse incision towards the xiphoid process.</li>
		<li>Remove the peri-colorectal tissues in the direction of the anus to separate the colorectum from the surrounding tissue. Be careful not to damage the colorectum.</li>
		<li>Push the skin of the abdomen to the sides to fully expose the colorectum. Remove the colorectum from the abdominal cavity with eyelid forceps and cut off the segments from the anus to the cecum (excluding); the total length is about 10 cm. Store the obtained colorectal tissues in saline at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Evaluation of the length and weight of the rectum
	<ol>
		<li>Extract the saline at 4 &#176;C with a 5 mL needle (see Table of Materials) to flush the inside of the colorectum. Then, place the colorectum on absorbent paper to absorb the moisture of the tissue.</li>
		<li>Weigh the colorectal tissues and then place them on A4 paper to measure their length.</li>
	</ol>
	</li>
	<li>Measure the number of tumors in the colorectum
	<ol>
		<li>Cut the colorectum lengthwise to fully unfold it and observe the number and size of the tumors in the colorectum.</li>
	</ol>
	</li>
	<li>Pathological analysis of the colorectum
	<ol>
		<li>Fix the colorectum in 4% paraformaldehyde for 24 h. Embed the fixed rectal tissue in melted paraffin and section continuously with a thickness of 5 &#181;m by a tissue freezing microtome.</li>
		<li>According to the procedure of Hou et al.24, dewax the sections in xylene and then dehydrate it with serial concentrations of ethanol. After staining with hematoxylin solution for 5 min, rinse the sections with pure water. After that, stain with 0.5% eosin solution for 1 min(see Table of Materials).</li>
		<li>Perform gradient dehydration and xylene transparent treatment again. Seal the sections and observe them under the optical microscope (see Table of Materials) and photograph, as described by Xie et al.25.</li>
	</ol>
	</li>
	<li>Immunohistochemical analysis of colorectum
	<ol>
		<li>Dewax and dehydrate the sections according to the above method. Repair the antigen in the sections with high-pressure thermal repair technique, as described by Gok et al.26.</li>
		<li>Soak the sections in endogenous peroxidase blockers at room temperature for 15 min. Seal the sections with goat serum (see Table of Materials).</li>
		<li>Add the primary antibody ZO-1 (1:1000), Occludin (1:1000) and KI67 (1:500; see Table of Materials) to the sections and incubate overnight at 4 &#176;C. Rinse the sections with PBS buffer (see Table of Materials), add general purpose secondary antibody (1:5000; see Table of Materials) to it and incubate at 37 &#176;C for 30 min.</li>
		<li>Add the DAB solution (see Table of Materials) to the sections for color development. Counterstain the sections with hematoxylin solution.</li>
		<li>Dehydrate, transparentize, and seal the sections again. Observe the expression of protein by the optical microscope.</li>
	</ol>
	</li>
</ol>

Liujunzi Decoction: An Effective Treatment for UC-CRC Prevention

The authors have nothing to disclose.

CRC is one of the most common cancers worldwide, with approximately 1,148,000 new cases and more than 576,000 deaths each year. CRC can be divided into three types according to different causes, including hereditary, sporadic and UC-CRC31. The incidence of CRC in patients with inflammatory bowel diseases such as UC is significantly higher than that in the general population. UC stimulates the development of CRC through the inflammatory-cancer pathway, which differs from the typical adenoma-adenocarcinoma pathway6. At present, the cause of UC-CRC is unknown, mainly caused by long-term recurrent chronic inflammation, with a mortality rate of up to 60%32,33. There is no effective treatment for UC-CRC, which has been the hotspot of research in recent years. Understanding the molecular mechanism of UC-CRC is crucial for early detection and precise treatment of UC-CRC.
Currently, there are many methods to construct animal models of UC-CRC, and the formation mechanisms are different. IL-10 or Muc2/4 gene knockout, resulting in epithelial barrier defects, can induce spontaneous colitis in mice, so it is often used to construct animal CRC models based on genetic defects associated with UC34,35,36. Nevertheless, the animal model of UC-CRC induced by gene knockout cannot simulate the complete pathogenesis of the disease, and its operation method is complex, the experiment is expensive, and it has obvious limitations. Chemical induction is the classical and common method to construct UC-CRC animal models37,38. DSS, AOM, and dimethylhydrazine are all commonly used chemical inducers for the construction of UC-CRC. However, using these chemical reagents alone to establish animal models takes a long time and has a low success rate39. Studies have shown that the incidence of UC-CRC induced by the combination of AOM and DSS can reach almost 100%40. Compared with other methods, the combination of AOM/DSS has the advantages of simple operation, strong controllability, short cycle, and high replication rate, and can better simulate human UC-CRC in terms of pathology and molecular mechanism40,41,42. Although the use of AOM/DSS cycle stimulation to construct UC-CRC model has many advantages, it also has the disadvantages of long modeling time, expensive modeling reagents, and still cannot fully simulate the pathogenesis of human UC-CRC.
In order to seek a potential therapeutic target for UC-CRC, we used AOM, a chemical carcinogen, in combination with DSS-induced mice to establish a UC-CRC model. In this study, mice exposed to AOM and cyclic stimulation of DSS showed varying degrees of UC-CRC-related symptoms. Compared with the control group, the DAI score of the model group was significantly increased, which was manifested as weight loss, blood stool, loose stool, or watery diarrhea. Whereas LJZD significantly reduced the DAI score. Shortened colorectum length is an important marker of inflammatory response in UC-CRC43,44. In this study, the colorectum length of the model mice was significantly shorter than that of the control group, and LJZD could improve the shortened colorectum. The colorectal mucosa develops chronic inflammation, which gradually evolves into atypical hyperplasia of colorectal tissue and eventually causes cancer45. In this study, the number of tumors in the colorectal tissue of mice in the model group increased significantly compared with the control group, and correspondingly, the colorectal tissue weight also increased. After LJZD treatment, the number of tumors decreased significantly, and the colorectal weight decreased. It is suggested that LJZD has a significant inhibitory effect on the formation of CRC induced by UC. Previous studies have found that LJZD can alleviate inflammation and reduce the expression levels of TNF-&#945;, IL-6, and IL-1&#946; in vivo16,28,46. In this study, the serum IL-6 level of mice in the model group was increased, but the production of serum IL-6 was significantly inhibited after LJZD treatment.
Microscopic observation showed inflammatory infiltration of rectal tissue, destruction of the mucosal barrier, reduction of mucus secretion, irregular or disappearance of goblet cells, distortion or atrophy of intestinal crypts, and obvious disorder of gland structure in AOM/DSS-treated mice. After LJZD treatment, the morphology of goblet cells in colorectal tissue was normal, the structure of recess and gland was regular, and the damage to the mucosal barrier was improved. Epithelial tight junction (TJ) is an indispensable mechanical barrier to maintain cell integrity and permeability, mainly including ZO-1 and Occludin29. It has been reported that the expression of TJ proteins such as ZO-1 in colorectal tissue of UC-CRC patients is significantly reduced47,48. This study showed that the colorectal mucosal tissue structure of UC-CRC mice was destroyed, and the expression of ZO-1 and Occludin in the tissue decreased, while LJZD could reverse this decrease, suggesting that LJZD may protect the integrity of the colorectal mucosal barrier by increasing the expression of ZO-1 and Occludin. Nuclear-associated antigen (KI67) is an essential component of cell cycle regulation and is widely used as an indicator of proliferative activity of tumor cells30,49. In this study, KI67 protein expression was increased in the model group compared with the control group, indicating significant tumor cell proliferation in colorectal tissue. After LJZD treatment, the expression of KI67 protein was decreased, which showed that LJZD had a good effect on UC-CRC.
In summary, AOM intraperitoneal injection and DSS circulation stimulation can effectively establish a UC-CRC model in a short period and are similar to human UC-CRC in terms of histopathology and molecular mechanisms of formation. As a classical prescription, LJZD can reduce the DAI score, colorectal tissue weight, and inflammatory factor levels, effectively inhibit the shortening of colorectal length, and play a role in the stage of UC in UC-CRC mice. Simultaneously, it can increase the expression of TJ proteins, significantly improve the pathological damage of colorectal tissue, reduce the expression of KI67 protein, significantly reduce the incidence of tissue tumors, and inhibit the transformation process of UC to CRC. This study provides a new idea for the treatment of UC-CRC.

Colorectal cancer (CRC) is a common gastrointestinal malignancy, the third most common malignancy, and the second most common cause of death in the world, accounting for 10% of the global cancer incidence and 9.4% of the total cancer-related death1,2. Genetic factors, chronic inflammation, high-fat diet, diabetes, and abnormal intestinal flora are risk factors for CRC3,4. Among them, inflammatory bowel disease, especially ulcerative coloproctitis (UC), is a clear risk factor for CRC5,6. UC-associated CRC (UC-CRC) is a transition process of inflammation, atypical hyperplasia, and cancer based on chronic inflammation of the colorectum, which is different from the typical adenoma-adenocarcinoma development model of CRC7,8. Compared with the general population, the risk of CRC is approximately 10-40 times higher in patients with inflammatory bowel disease9.
Currently, surgery is still the standard treatment for CRC, and depending on the location and stage of the tumor, radiation therapy, systemic drug therapy, or a combination of both are possible10. Although these traditional treatment modalities have made great progress, due to the high heterogeneity and recurrence rate of CRC, the prognosis is poor, and the treatment effect is not ideal11,12. Therefore, early detection, early diagnosis, and comprehensive treatment are key to improving the survival rate of CRC patients, and it is particularly important to pay attention to the transformation of UC to CRC. Over the years, traditional Chinese medicine (TCM) has attracted much attention in the treatment of UC-CRC or chronic gastritis due to its limited side effects and significant efficacy. Based on dialectical treatment, famous Chinese medicine practitioners of various generations have created a large number of classic prescriptions, such as Huangqi Jianzhong decoction13, Sijunzi decoction14, and Sishen pill15.
Liujunzi decoction (LJZD) originated from the works of Yi Xue Zheng Zhuan compiled in the Ming Dynasty and is a classic prescription in TCM16. As shown in Table 1, LJZD consists of six traditional herbs, including Codonopsis pilosula (Franch.) Nannf. (Dangshen), Poria cocos (Schw.) Wolf (Fuling), Atractylodes macrocephala Koidz. (Baizhu), Glycyrrhiza uralensis Fisch. (Gancao), Citrus reticulata Blanco (Chenpi) and Pinellia ternata (Thunb.) Breit (Banxia), which has the effect of replenishing qi and strengthening the spleen, drying dampness, and resolving phlegm. In modern clinical practice, it is often used to treat chronic gastritis, gastric ulcers, and duodenal ulcers. Modern pharmacological research has shown that LJZD and modified LJZD have high application value in the adjuvant treatment of UC and digestive tract cancer17,18,19.
At present, there are many ways to construct UC-CRC mouse models, but the azoxymethane (AOM)/dextran sulfate sodium (DSS) induced mouse model is the most widely used UC-CRC model; the clinical symptoms, morphological, and pathological observations have proved that the model is very similar to human UC-CRC20,21. The basic principle is to first induce carcinogenesis with chemical carcinogen AOM and then continuously expose mice to the inflammatory stimulation environment of DSS to simulate the continuous damage and repair of intestinal mucosal epithelium, thereby constructing a UC-CRC mouse model22. The aim of this study is to establish a mouse model of UC-CRC by intraperitoneal injection of AOM and cyclic stimulation of DSS in the short term and to evaluate the effect of the drug and the molecular mechanism of LJZD on UC-CRC in order to provide a scientific basis for the treatment of UC-CRC.

<table><tbody><tr><td>Azoxymethane</td><td>Sigma</td><td>A5486</td><td></td></tr><tr><td>5-amino salicylic acid</td><td>Kuihua Pharmaceuticals Group Jiamusi Luling Pharmaceutical Co., Ltd</td><td>3819413</td><td></td></tr><tr><td>C57BL/6J mice</td><td>Liaoning Changsheng Biotechnology Co., Ltd</td><td>NO 210726210100853716</td><td></td></tr><tr><td>Cover slip</td><td>Jiangsu Shitai Experimental Equipment Co., Ltd</td><td>10212432C</td><td></td></tr><tr><td>DAB color development kit</td><td>Jiangsu Shitai Experimental Equipment Co., Ltd</td><td>2005289</td><td></td></tr><tr><td>Dewatering machine&amp;nbsp;</td><td>Wuhan Junjie Electronics Co., Ltd</td><td>JJ-12J</td><td></td></tr><tr><td>Dextran sulfate sodium</td><td>Dalian Meilun Biotechnology Co., Ltd</td><td>MB5535</td><td></td></tr><tr><td>Embedding machine</td><td>Wuhan Junjie Electronics Co., Ltd</td><td>JB-P5</td><td></td></tr><tr><td>Hematoxylin-eosin dye</td><td>Wuhan Hundred Degree Biotechnology Co., Ltd</td><td>B1000</td><td></td></tr><tr><td>IL-6</td><td>Jiangsu Meimian Industrial Co., Ltd</td><td>MM-0163M2</td><td></td></tr><tr><td>Isoflurane</td><td>RWD Life Science Co., Ltd</td><td>R510-22-10</td><td></td></tr><tr><td>KI67 primary antibody</td><td>Google Biotechnology Inc</td><td>GB121141</td><td></td></tr><tr><td>Neutral gum</td><td>Wuhan Hundred Degree Biotechnology Co., Ltd</td><td>10004160</td><td></td></tr><tr><td>Object slide</td><td>Jiangsu Shitai Experimental Equipment Co., Ltd</td><td>10212432A</td><td></td></tr><tr><td>Occludin primary antibody</td><td>Affnity</td><td>DF7504</td><td></td></tr><tr><td>Orthostatic optical microscope</td><td>Nikon</td><td>Nikon Eclipse CI</td><td></td></tr><tr><td>Pathological microtome</td><td>Shanghai Leica Instrument Co., Ltd</td><td>RM2016</td><td></td></tr><tr><td>ZO-1 primary antibody</td><td>Abcam</td><td>ab221547</td><td></td></tr></tbody></table>

establishment of coloproctitis cancer model in mice and evaluation of therapeutic effect of chinese medicine

Colorectal cancer (CRC) is a common malignancy of the digestive system and has become the third most common malignancy worldwide and the second leading cause of malignancy-related death. Ulcerative coloproctitis (UC) is a precancerous lesion, and UC-associated CRC (UC-CRC) is the most common subtype of CRC. Therefore, a reasonable UC-CRC model is the cornerstone and guarantee of new drug development. Traditional Chinese medicine (TCM) has been widely used in the treatment of UC-CRC due to its good efficacy. As a classic tonic prescription of TCM, Liujunzi decoction (LJZD) has been widely used in the treatment of UC-CRC. In this study, a UC-CRC model was established by combining azomethane and dextran sulfate sodium, and the LJZD was administered. The data confirmed that LJZD can effectively inhibit cancer transition in UC-CRC by using mouse body weight, colorectal length, pathological and inflammatory factors, colorectal barrier function, and cancer markers. This protocol provides a system for evaluating the efficacy of TCM in the prevention and treatment of UC-CRC.

The decoction of LJZD was prepared according to the composition ratio of drugs in Table 1 and the decoction method of TCM in Figure 1A. According to the time point indicated in Figure 1B, mice were intraperitoneally injected with 1 mg/mL AOM on the 7th day, and mice were given free access to drinking water containing 2% DSS in the 3rd, 6th, and 9th weeks. The UC-CRC mouse model was successfully established in the 15th week. Meanwhile, mice were treated with LJZD by gavage from week 7 to week 15. The data showed that compared with the control group, the UC-CRC model group had a significant weight loss, which was alleviated by LJZD treatment (Figure 2A, P &#60; 0.01). At the end of the trial, LJZD treatment improved the DAI score compared with the UC-CRC model group (Figure 2B). Compared with the control group, the UC-CRC model group had a shorter colorectal length, which was increased by LJZD treatment (Figure 2C,D, P &#60; 0.01). The ratio of colorectal weight to body weight reflects the development of CRC in mice, and a higher ratio indicates acute tumor development27. Compared with the control group, the colorectal organ index of the model group was significantly increased, and LJZD treatment greatly reduced the colorectal organ index (Figure 2E, P &#60; 0.05). In addition, LJZD treatment also inhibited the formation of colorectal tumors (Figure 2F, P &#60; 0.01) and the level of serum pro-inflammatory factor IL-628 (Figure 2G, P &#60; 0.05).
Pathological results confirmed that compared with the control group, the mice in the UC-CRC model group had larger colorectal tumors and had formed adenocarcinoma, while LJZD treatment reduced the size and grade of the tumors (Figure 3). Immunohistochemical data demonstrated that LJZD treatment enhanced colorectal barrier function in UC-CRC model mice, as indicated by elevated protein expression of ZO-1 and Occludin29 (Figure 4). In parallel, LJZD treatment suppressed the protein expression levels of the cancer marker KI6730 (Figure 4).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66045/66045fig01.jpg" /> 
Figure 1: Preparation of LJZD and establishment of a mouse model of coloproctitis cancer.(A) The mixture of Dangshen (12 g), Baizhu (12 g), Gancao (6 g), Chenpi (12 g) and ginger-processed Ban Xia (9 g) was immersed in 1000 mL of distilled water at room temperature for 1 h. The 12 g Fuling powder was soaked in 300 mL of distilled water at room temperature for 1 h. The mixture of the above six herbs was decocted at 100 &#176;C for 40 min. (B) On day 7, C57BL/6J mice were intraperitoneally injected with 1 mg/mL AOM. Mice were given water containing 2% DSS ad libitum in the 3rd, 6th, and 9th weeks. From 7 to 15 weeks, mice were given LJZD by gavage. Abbreviations: AOM, azomethane; DAI, disease activity index; DSS, dextran sulfate sodium; LJZD, Liujunzi Decoction; w, week. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66045/66045fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66045/66045fig02.jpg" /> 
Figure 2: Evaluation of UC-CRC model and efficacy of LJZD. (A)&#160;LJZD improved body weight in UC-CRC model mice. (B) LJZD alleviated DAI scores in UC-CRC model mice. (C, D) LJZD increased the length of the colorectum in UC-CRC model mice. (E) LJZD reduced the organ index of the colorectum in UC-CRC model mice. (F) LJZD inhibited colorectal tumorigenesis in coloproctitis cancer model mice. (G) LJZD suppressed serum IL-6 level in UC-CRC model mice. #P&#60; 0.05 and ##P&#60; 0.01, compared with the control group; *P &#60; 0.05 and **P&#60; 0.01, compared with the model group. Data were expressed as the mean &#177; standard deviations (n=10) and were analyzed by one-way analysis of variance (ANOVA) followed by Tukey&#39;s test. P &#60; 0.05 indicated a statistically significant difference. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66045/66045fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66045/66045fig03.jpg" /> 
Figure 3: Evaluation of the pathological characteristics of colorectal tissues in mice by hematoxylin-eosin staining. There was no pathological damage in the control group. The tumor tissue of the UC-CRC model group was large and had formed adenocarcinoma and high-grade neoplasia, while the LJZD treatment group had reduced tumor tissue accompanied by a small amount of local adenoma and low-grade neoplasia. The black arrow represents the precancerous dysplastic tumor gland. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66045/66045fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66045/66045fig04.jpg" /> 
Figure 4: Effect of LJZD on colorectal barrier function and cancer marker in UC-CRC model mice. (A) Immunohistochemical images of ZO-1, Occludin and KI67. (B) Statistical results of protein expression of ZO-1, Occludin and KI67. LJZD treatment increased the expression of colorectal barrier functional proteins ZO-1 and Occludin, while decreased the level of cancer marker KI67 in UC-CRC model mice. ## P&#160;&#60; 0.01, compared with the control group; ** P &#60; 0.01, compared with the model group. Data were expressed as the mean &#177; standard deviations (n=10) and were analyzed by one-way analysis of variance (ANOVA) followed by Tukey&#39;s test. P &#60; 0.05 indicated a statistically significant difference. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66045/66045fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Components</td>
			<td>Pinyin</td>
			<td>Weight (g)</td>
		</tr>
		<tr>
			<td>Codonopsis Radix</td>
			<td>Dangshen</td>
			<td>12</td>
		</tr>
		<tr>
			<td>Poria cocos</td>
			<td>Fuling</td>
			<td>12</td>
		</tr>
		<tr>
			<td>Atractylodis Macrocephalae Rhizoma</td>
			<td>Baizhu</td>
			<td>12</td>
		</tr>
		<tr>
			<td>Licorice</td>
			<td>Gancao</td>
			<td>6</td>
		</tr>
		<tr>
			<td>Dried orange peel</td>
			<td>Chenpi</td>
			<td>12</td>
		</tr>
		<tr>
			<td>Rhizome Pinelliae Preparata</td>
			<td>Jiangbanxia</td>
			<td>9</td>
		</tr>
	</tbody>
</table>
Table 1: Composition and proportion of drugs in LJZD.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Items</td>
			<td>Grade or symptoms</td>
			<td>Scores</td>
		</tr>
		<tr>
			<td>Weight loss</td>
			<td>&#60; 1%</td>
			<td>0</td>
		</tr>
		<tr>
			<td></td>
			<td>1%-5%</td>
			<td>1</td>
		</tr>
		<tr>
			<td></td>
			<td>5%-10%</td>
			<td>2</td>
		</tr>
		<tr>
			<td></td>
			<td>10%-15%</td>
			<td>3</td>
		</tr>
		<tr>
			<td></td>
			<td>&#8805;15%</td>
			<td>4</td>
		</tr>
		<tr>
			<td>Faecal occult blood testing</td>
			<td>negative</td>
			<td>0</td>
		</tr>
		<tr>
			<td></td>
			<td>positive</td>
			<td>2</td>
		</tr>
		<tr>
			<td></td>
			<td>visible blood in the stool by the naked eye</td>
			<td>4</td>
		</tr>
		<tr>
			<td>Fecal consistency</td>
			<td>normal</td>
			<td>0</td>
		</tr>
		<tr>
			<td></td>
			<td>loose stool</td>
			<td>2</td>
		</tr>
		<tr>
			<td></td>
			<td>watery diarrhea</td>
			<td>4</td>
		</tr>
	</tbody>
</table>
Table 2: Disease activity index score for UC-CRC model in mice.

Watch this Scientific Journal Video about Author Spotlight: Liujunzi Decoction as a Traditional Chinese Treatment for Coloproctitis Cancer at JoVE.com

Author Spotlight: Liujunzi Decoction as a Traditional Chinese Treatment for Coloproctitis Cancer

This protocol provides a mouse model of ulcerative coloproctitis-associated colorectal cancer induced by azomethane combined with dextran sulfate sodium. The model was used to evaluate the efficacy of traditional Chinese medicine compounds in the prevention and treatment of colorectal cancer.

Establishment of Coloproctitis Cancer Model in Mice and Evaluation of Therapeutic Effect of Chinese Medicine

Colorectal cancer (CRC) is a common malignancy of the digestive system and has become the third most common malignancy worldwide and the second leading ...

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Nanyang Technological University

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Medicine

784 Views.  Changchun University of Chinese Medicine. Deze studie ging over de preventie en behandeling van coloproctitiskanker met traditionele Chinese geneeskunde. We streven ernaar een muismodel voor coloproctitiskanker op te stellen en de indicatoren van de traditionele Chinese geneeskunde te evalueren. We hebben bevestigd dat het Chinese medicijn Liujunzi-afkooksel een effectieve behandeling is voor de preventie van coloproctitis kanker.In de toekomst zullen we werken aan het identificeren van gevoeligere biomarkers voor darm- en end...

Video: Auteur in de schijnwerpers: Liujunzi-afkooksel als een traditionele Chinese behandeling voor Coloproctitis-kanker

Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Orthotopic mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in hollow organs such as the large bowel. In order to produce uniform tumors which reliably grow and metastasize, standardized techniques of tumor cell preparation and injection are critical. 
We have developed an orthotopic mouse model of colorectal cancer (CRC) which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor cells from either primary tumors, 2-dimensional (2D) cell lines or 3-dimensional (3D) organoids are injected into the cecum and, depending on the metastatic potential of the injected tumor cells, form highly metastatic tumors. In addition, CTCs can be found regularly. We here describe the technique of tumor cell preparation from both 2D cell lines and 3D organoids as well as primary tumor tissue, the surgical and injection techniques as well as the isolation of CTCs from the tumor-bearing mice, and present tips for troubleshooting.

We describe the establishment of orthotopic colorectal tumors via injection of tumor cells or organoids into the cecum of mice and the subsequent isolation of circulating tumor cells (CTCs) from this model.

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate ...

Cancer Research

A Genetically Engineered Mouse Model of Sporadic Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, colorectal cancer mouse models based on tumor cell injection have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Genetically engineered mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in large organs such as the colon in which only a single tumor is desired.
As a result, an immunocompetent, genetically engineered mouse model of colorectal cancer was developed which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor development is initiated by surgical, segmental infection of the distal colon with adeno-cre virus in compound conditionally mutant mice. The tumors can be easily detected and monitored via colonoscopy. We here describe the surgical technique of segmental adeno-cre infection of the colon, the surveillance of the tumor via high-resolution colonoscopy and present the resulting colorectal tumors.

A protocol for the establishment of a genetically engineered mouse model of colorectal cancer by segmental adeno-cre infection and its surveillance via high-resolution colonoscopy is presented.

Despite the advantages of easy applicability and cost-effectiveness, colorectal cancer mouse models based on tumor cell injection have severe limitations ...

Orthotopic Mouse Model of Colorectal Cancer

The traditional subcutaneous tumor model is less than ideal for studying colorectal cancer. Orthotopic mouse models of colorectal cancer, which feature cancer cells growing in their natural location, replicate human disease with high fidelity. Two techniques can be used to establish this model. Both techniques are similar and require mouse anesthesia and laparotomy for exposure of the cecum. One technique involves injection of a colorectal cancer cell suspension into the cecal wall. Cancer cells are first grown in culture, harvested when subconfluent and prepared as a single cell suspension. A small volume of cells is injected slowly to avoid leakage. The other technique involves transplantation of a piece of subcutaneous tumor onto the cecum. A mouse with a previously established subcutaneous colorectal tumor is euthanized and the tumor is removed using sterile technique. The tumor piece is divided into small pieces for transplantation to another mouse. Prior to transplantation, the cecal wall is lightly damaged to facilitate tumor cell infiltration. The time to developing primary tumors and liver metastases will vary depending on the technique, cell line, and mouse species used. This orthotopic mouse model is useful for studying the natural progression of colorectal cancer and testing new therapeutic agents against colorectal cancer.

Two techniques can be used to establish this model: injection of a cancer cell suspension into the cecal wall or transplantation of a piece of subcutaneous tumor onto the cecum. This model is useful for studying the natural progression of colorectal cancer and testing new therapeutic agents against colorectal cancer.

The traditional subcutaneous tumor model is less than ideal for studying colorectal cancer. Orthotopic mouse models of colorectal cancer, which feature ...

Biology

Modeling Colitis-Associated Cancer with Azoxymethane (AOM) and Dextran Sulfate Sodium (DSS)

Individuals with inflammatory bowel disease (IBD), such as Crohn's disease (CD) or ulcerative colitis (UC) are at increased risk of developing colorectal cancer (CRC) over healthy individuals. This risk is proportional to the duration and extent of disease, with a cumulative incidence as high as 30% in individuals with longstanding UC with widespread colonic involvement.1 Colonic dysplasia in IBD and colitis associated cancer (CAC) are believed to develop as a result of repeated cycles of epithelial cell injury and repair while these cells are bathed in a chronic inflammatory cytokine milieu.2 While spontaneous and colitis-associated cancers share the quality of being adenocarcinomas, the sequence of underlying molecular events is believed to be different.3 This distinction argues the need for specific animal models of CAC.
Several mouse models currently exist for the study of CAC. Dextran sulfate sodium (DSS), an agent with direct toxic effects on the colonic epithelium, can be administered in drinking water to mice in multiple cycles to create a chronic inflammatory state. With sufficient duration, some of these mice will develop tumors.4 Tumor development is hastened in this model if administered in a pro-carcinogenic setting. These include mice with genetic mutations in tumorigenesis pathways (APC, p53, Msh2), as well as mice pre-treated with genotoxic agents (azoxymethane [AOM], 1,2-dimethylhydrazine [DMH]).5 
The combination of DSS with AOM as a model for colitis associated cancer has gained popularity for its reproducibility, potency, low price, and ease of use. Though they have a shared mechanism, AOM has been found to be more potent and stable in solution than DMH. While tumor development in other models generally requires several months, mice injected with AOM and subsequently treated with DSS develop adequate tumors in as little as 7-10 weeks.6, 7 Finally, AOM and DSS can be administered to mice of any genetic background (knock out, transgenic, etc.) without cross-breeding to a specific tumorigenic strain. Here, we demonstrate a protocol for inflammation-driven colonic tumorigenesis in mice utilizing a single injection of AOM followed by three seven-day cycles of DSS over a 10 week period. This model induces tumors with histological and molecular changes closely resembling those occurring in human CAC and provides a highly valuable model for the study of oncogenesis and chemoprevention in this disease.8

Modeling Colitis-Associated Cancer with Azoxymethane AOM and Dextran Sulfate Sodium DSS

We demonstrate a protocol in which administration of the genotoxic agent azoxymethane (AOM) followed by three cycles of the pro-inflammatory agent dextran sulfate sodium (DSS) rapidly and consistently generates colon tumors in mice with morphologic and molecular similarities to those seen in human colitis-associated cancer.

Individuals with inflammatory bowel disease (IBD), such as Crohn's disease (CD) or ulcerative colitis (UC) are at increased risk of developing colorectal ...

Flexible Colonoscopy in Mice to Evaluate the Severity of Colitis and Colorectal Tumors Using a Validated Endoscopic Scoring System

The use of modern endoscopy for research purposes has greatly facilitated our understanding of gastrointestinal pathologies. In particular, experimental endoscopy has been highly useful for studies that require repeated assessments in a single laboratory animal, such as those evaluating mechanisms of chronic inflammatory bowel disease and the progression of colorectal cancer. However, the methods used across studies are highly variable. At least three endoscopic scoring systems have been published for murine colitis and published protocols for the assessment of colorectal tumors fail to address the presence of concomitant colonic inflammation. This study develops and validates a reproducible endoscopic scoring system that integrates evaluation of both inflammation and tumors simultaneously. This novel scoring system has three major components: 1) assessment of the extent and severity of colorectal inflammation (based on perianal findings, transparency of the wall, mucosal bleeding, and focal lesions), 2) quantitative recording of tumor lesions (grid map and bar graph), and 3) numerical sorting of clinical cases by their pathological and research relevance based on decimal units with assigned categories of observed lesions and endoscopic complications (decimal identifiers). The video and manuscript presented herein were prepared, following IACUC-approved protocols, to allow investigators to score their own experimental mice using a well-validated and highly reproducible endoscopic methodology, with the system option to differentiate distal from proximal endoscopic colitis (D-PECS).

Over the past few years, new generation endoscopes have emerged as important diagnostic research aids for evaluating murine colitis and colorectal tumors. We present herein a detailed protocol for endoscopic assessment of inflammation and colorectal tumors in mice, as well as a novel scoring system that uses decimal identifiers to document the endoscopic severity of colitis and colorectal tumors.

The use of modern endoscopy for research purposes has greatly facilitated our understanding of gastrointestinal pathologies. In particular, experimental ...

Orthotopic Implantation of Patient-Derived Cancer Cells in Mice Recapitulates Advanced Colorectal Cancer

Over the last decade, more sophisticated preclinical colorectal cancer (CRC) models have been established using patient-derived cancer cells and 3D tumoroids. Since patient derived tumor organoids can retain the characteristics of the original tumor, these reliable preclinical models enable cancer drug screening and the study of drug resistance mechanisms. However, CRC related death in patients is mostly associated with the presence of metastatic disease. It is therefore essential to evaluate the efficacy of anti-cancer therapies in relevant in vivo models that truly recapitulate the key molecular features of human cancer metastasis. We have established an orthotopic model based on the injection of CRC patient-derived cancer cells directly into the cecum wall of mice. These tumor cells develop primary tumors in the cecum that metastasize to the liver and lungs, which is frequently observed in patients with advanced CRC. This CRC mouse model can be used to evaluate drug responses monitored by microcomputed tomography (&#181;CT), a clinically relevant small-scale imaging method that can easily identify primary tumors or metastases in patients. Here, we describe the surgical procedure and the required methodology to implant patient-derived cancer cells in the cecum wall of immunodeficient mice.

This protocol describes the orthotopic implantation of patient-derived cancer cells in the cecum wall of immunodeficient mice. The model recapitulates advanced colorectal cancer metastatic disease and allows for the evaluation of new therapeutic drugs in a clinically relevant scenario of lung and liver metastases.

Over the last decade, more sophisticated preclinical colorectal cancer (CRC) models have been established using patient-derived cancer cells and 3D ...

Non-invasive Assessment of the Efficacy of New Therapeutics for Intestinal Pathologies Using Serial Endoscopic Imaging of Live Mice

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic mechanisms that contribute to the onset and progression of intestinal diseases. The identification of new molecules that promote these pathologies has led to a flurry of activity focused on the development of potential new therapies to inhibit their function. As a result, various pre-clinical mouse models with an intact immune system and stromal microenvironment are now heavily used. Here we describe three experimental protocols to test the efficacy of new therapeutics in pre-clinical models of (1) acute mucosal damage, (2) chronic colitis and/or colitis-associated colon cancer, and (3) sporadic colorectal cancer. We also outline procedures for serial endoscopic examination that can be used to document the therapeutic response of an individual tumor and to monitor the health of individual mice. These protocols provide complementary experimental platforms to test the effectiveness of therapeutic compounds shown to be well tolerated by mice.

We describe methods for longitudinal monitoring of the efficacy of therapeutics for the treatment of colonic pathologies in mice using a rigid endoscope. This protocol can be readily used for the characterization of the therapeutic response of an individual tumor in live mice and also for monitoring potential disease relapse.

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic ...

Murine Distal Colostomy, A Novel Model of Diversion Colitis in C57BL/6 Mice

Diversion colitis (DC) is a frequent clinical condition occurring in patients with bowel segments excluded from the fecal stream as a result of a diverting enterostomy. The etiology of this disease remains ill-defined but appears to differ from that of classical inflammatory bowel diseases such as Crohn's disease and ulcerative colitis. Research aimed to decipher the pathophysiological mechanisms leading to the development of this disease has been severely hampered by the lack of an appropriate murine model. This protocol generates a murine model of DC that facilitates the study of the immune system's role and its interaction with the microbiome in the development of DC. In this model using C57BL/6 animals, distal parts of the colon are excluded from the fecal stream by creating a distal colostomy, triggering the development of mild to moderate inflammation in the excluded bowel segments and reproducing the hallmark lesions of human DC with a moderate systemic inflammatory response. In contrast to the rat model, a large number of genetically-modified murine models on the C57BL/6 background are available. The combination of these animals with our model allows the potential roles of individual cytokines, chemokines, or receptors of bioactive molecules (e.g., interleukin (IL)-17; IL-10, chemokine CXCL13, chemokine receptors CXCR5 and CCR7, and the sphingosine-1-phosphate receptor 4) to be assessed in the pathogenesis of DC. The availability of congenic mouse strains on the C57BL/6 background largely facilitates transfer experiments to establish the roles of distinct cell types involved in the etiology of DC. Finally, the model offers the opportunity to assess the influences of local interventions (e.g., modification of the local microbiome or local anti-inflammatory therapy) on mucosal immunity in affected and non-affected bowel segments and the on systemic immune homeostasis.

Murine distal colostomy provides a murine model for human diversion colitis, a predominantly lymphocytic colitis in colon segment excluded from the fecal stream.

Diversion colitis (DC) is a frequent clinical condition occurring in patients with bowel segments excluded from the fecal stream as a result of a ...

Murine Appendectomy Model of Chronic Colitis Associated Colorectal Cancer by Precise Localization of Caecal Patch

The human appendix has been recently implicated to play important biological roles in the pathogenesis of various complex diseases, such as colorectal cancer, inflammatory bowel disease, and Parkinson&#8217;s disease. To study the function of the appendix, a gut disease-associated murine appendectomy model has been established and its step-by-step protocol is described here. This report introduces a facile protocol for caecal patch removal in mice followed by the chemical induction of chronic colitis-associated colorectal cancer using a combination of dextran sulfate sodium (DSS) and azoxymethane (AOM). IgA specific cells and IgA concentration were significantly reduced upon removal of the caecal patch in male C57BL/6 mice&#160;compared to those in the sham group. Simultaneously administering 2% DSS and AOM resulted in nearly 80% mice survival in both sham and appendectomy groups without significant body weight loss. Histological results confirmed colonic inflammation and different degrees of adenocarcinoma. This model can be used for the study of the functional role of the appendix in maintaining gut microbiota homeostasis and pathogenesis of gut colitis and malignancies, as well as for the potential development of drug targeting therapies.

The presented protocol describes a facile surgical removal of the appendix (caecal patch) in a mouse followed by the induction of inflammatory bowel disease-associated colorectal cancer. This murine appendectomy model enables investigation of the biological role of the appendix in the pathogenesis of human gastrointestinal disease.

The human appendix has been recently implicated to play important biological roles in the pathogenesis of various complex diseases, such as colorectal ...

Impact of Sanleng Jiashen Formula on Orthotopic Hepatocellular Carcinoma in Mice

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

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.

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 ...

Establishment of Coloproctitis Cancer Model in Mice and Evaluation of Therapeutic Effect of Chinese Medicine

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