Xiaotan Sanjie Fang prevents colonic inflammation‐related tumorigenesis by inhibiting COX‐2/VEGF expression cancer

Colitis‐associated bowel cancer (CAC) is one of the most common malignancies associated with inflammation. The aim of this study was to observe a new herbal formula “Xiaotan Sanjie Fang” (XTSJF) derived from the addition and subtraction theory of traditional medicine as an alternative to CAC treatment by “Daotan Decoction” and “Xiaojianzhong Decoction”, which are famous traditional Chinese medicine prescriptions for the treatment of inflammatory diseases of the digestive tract. We constructed a DMH/DSS inflammation‐associated colorectal cancer rat model and treated CAC rats with sulfasalazine and different doses of XTSJF. The results showed that the body weight of rats treated with different doses of XTSJF increased, which was still lower than that of normal rats; AFC decreased significantly compared with the model group and the positive control group, and the final dose was superior to the low dose; histological observation revealed that it could maintain the normal structure of colon tissue, while it could inhibit the secretion of VEGF, COX2, and AQP1 and the expression of pro‐inflammatory cytokines IL‐6, IL‐1β, and TNF‐α, promote the expression of caspase‐3 and BAX and inhibit the expression of Bcl‐2. Taken together, these data suggest that XTSJF can inhibit COX‐2/VEGF expression to prevent the development of inflammation‐associated colorectal cancer.

increases the risk of CRC by approximately 2-3 fold. 10Evidence suggests that screening average-risk individuals can reduce mortality from early cancer detection and cancer prevention by detecting and removing cancer precursor lesions. 11Studies on inhibiting the growth of colorectal cancer precursor lesions have aroused much attention.
First, a rat colitis-associated colorectal cancer model was established, and then the effect of Xiaotan Sanjie Fang on the development of colitis-associated colorectal cancer was examined.

| Animal model
Sixty adult male Sprague-Dawley rats (SD) weighing 140200 g were studied.The animals were purchased from the Shanghai Sippr-BK laboratory animal Co. Ltd. (Shanghai, China).The male rats were housed in metal cages with wire-grid floors.The animals were kept at standard housing facilities (25 ± 2 C, 45% ± 5% humidity and 12 h light/dark cycles).After 2 days of adaptive feeding, all animals were randomly divided into six groups: normal group, model group, low, medium, and high dose groups and positive sulfasalazine tablet group, with 10 animals in each group.All rats except the normal group had free access to 2% DSS in water and started drinking tap water and chow diet in the third week.
1-2dimethylhydrazine (DMH) was dissolved in 1 mM ethylenediaminetetraacetic acid-containing 1 mM sodium bicarbonate (pH 6.5).Animals were given a weekly subcutaneous injection of DMH in the groin at a dose of 40 mg/kg body weight for 8 weeks.They were then fed with low, medium, and high doses of Xiaotan Sanjie Fang and positive sulfasalazine tablets for another 5 weeks.

| Histological analysis
Tissues were carefully excised, rinsed in PBS, and then fixed in 4% Paraformaldehyde (PFA).Samples were dehydrated in a graded ethanol series (70%-100%) and embedded in paraffin.Five-micrometer sections were prepared.According to the standard procedures, samples were stained with either Hematoxylin and Eosin (HE) and immunohistochemistry including VEGF (1:300, Abcam), COX-2 (1:500, Abcam).Immunohistochemistry mean optical density values were analyzed using imagej and SPSS 13.0 was applied for statistical analysis.

| TEM characterization
For preparation of transmission electron microscopy (TEM) samples, rats were perfused with 4% PFA and 0.4% glutaraldehyde under anesthesia.Tissues were post-fixed with osmic acid for 1 h.After dehydration in an acetone gradient, samples were impregnated with a mixture of acetone/resin (1:1, 1 h, room temperature; 1:2, 2 h, room temperature) and incubated with resin at room temperature overnight.Sections were stained and images were obtained under an H-7500 TEM (Hitachi Science Systems, Ltd, Japan).

| ELISA
Tissues were excised, and cleaned with PBS to remove blood, then homogenated with 0.9% normal saline in the ratio of 100 μL normal saline per 10 μg tissue by using homogenizer.After centrifugation (3000 rpm) for 10 min at 4 C, the supernatant was collected.The secretion level of VEGF(ml064281), COX-2(ml058808), and AQP1 (ml024219) was detected by using a commercially available enzymelinked immunosorbent assay (ELISA) system (Mlbio, Shanghai, China) following the instructions.

| Statistical analysis
Data are presented as the mean ± S.D., and statistical analysis was performed using SPSS 13.0 software (IBM Corporation, Armonk, NY, USA).Differences among groups were assessed using one-way ANOVA followed by post hoc tests.The values of p < .05were considered statistically significant.

| Aberrant crypt foci counting
In this study, we used 1-2dimethylhydrazine (DMH) to introduce a colorectal cancer model in rats.We examined the body weight of each rat during the whole process.Before establishing the cancer model, there was no difference on body weight among these groups.While, the body weights of normal rats were significant higher than the other groups from 2 to 8 weeks.After 8 weeks, different dose of Xiaotan Sanjie Fang was given to those rat for another 5 weeks.However, the body weight of these rats still lower than normal rats (Figure 1A).Finally, we counted the aberrant crypt foci (ACF).As shown in Figure 1B, the ACF of low, medi Xiaotan Sanjie Fangum and high dose of Xiaotan Sanjie Fang treated rats were obviously decreased compared to the model group and anti-cancer drug control group.And, the ACF of medium and high dose of treated rats were less than low dose Xiaotan Sanjie Fang treated rats.

| Xiaotan Sanjie Fang maintains normal structure of colon tissue
Following, the general histological observations were performed.As shown in Figure 2, in the model group, the colon tissue showed severe dysplasia, the gland was disordered, the cell layer was increased and crowded, the mucus secretion was significantly reduced, the mucosal epithelium was shed, the erosion was heavy, the ulcer was large and large, and the mucosa was severely congested and edema.The above phenomenon was improved in each Xiaotan Sanjie Fang treated group, but different degrees of cell crowding, nuclear enlargement and stratification occurred.Moreover, precise structure of colon tissue of each group were obtained by TEM.In the normal group, normal intestinal epithelial structure was observed.In the model group, the chromatin of the small intestine of the small intestine was aggregated, and there were many vacuoles in the cytoplasm with different sizes, mitochondrial vacuoles, and the cell epidermis was disorderly F I G U R E 1 Aberrant crypt foci counting after low, medium, and high dose of Xiaotan Sanjie Fang treated rats.(A) The body weight of normal rats, model rats, anti-cancer drug, low, medium, and high dose of Xiaotan Sanjie Fang treated rats.(B) The aberrant crypt foci counting of normal rats, model rats, anti-cancer drug, low, medium, and high dose of Xiaotan Sanjie Fang treated 5 weeks rats.arranged and partially broken.In the control group, the mucosal epithelial brush element was closely arranged, and a small amount of vacuoles were present in the cytoplasm.In the high dose of Xiaotan Sanjie Fang treated group, the intestine villi were sparsely arranged, but no break was observed, and there was a small amount of vacuoles in the cytoplasm.In the Medium dose of Xiaotan Sanjie Fang treated group, the small intestine villi are arranged neatly, and some cells have a small amount of vacuole structure in the cytoplasm.In the low dose of Xiaotan Sanjie Fang treated group, there is cytoplasmic shedding in the low dose (Low) part, vacuoles in the cytoplasm, and the brush elements are arranged neatly.

| Xiaotan Sanjie Fang promotes secretion of VEGF, COX2, and AQP1
As shown in Figure 3A, the secretion of VEGF in model rats, anticancer drug, low, medium, and high dose of Xiaotan Sanjie Fang treated rats was higher than normal group.The secretion of COX2 in model rats, anti-cancer drug, low, medium, and high dose of Xiaotan Sanjie Fang treated rats was higher than normal groupV (Figure 3B).
The secretion of AQP1 in model rats, anti-cancer drug, low, medium, and high dose of Xiaotan Sanjie Fang treated rats was higher than normal group (Figure 3C).In order to verify the above ELISA results, the immunohistochemical staining was performed.As shown in Figure 4, the expression of VEGF in model rats, anti-cancer drug, low, medium, and high dose of Xiaotan Sanjie Fang treated rats was higher than normal group.Similarly, the expression of COX2 in model rats, anticancer drug, low, medium, and high dose of Xiaotan Sanjie Fang treated rats was higher than normal group.

| Xiaotan Sanjie Fang inhibits apoptosis
As shown in Figure 5A, the relative expression of caspase-3 and BAX in model rats, anti-cancer drug, low, mediumand high dose of Xiaotan Sanjie Fang treated rats was lower than normal group.On the contrary, the expression of BCL-2 in model rats, anti-cancer drug, low, medium, and high dose of Xiaotan Sanjie Fang treated rats was higher than normal group.Furthermore, the western blot results show a similar tread on the expression of caspase-3 and BCL-2 (Figure 5B).

| DISCUSSION
Research indicates that traditional chinese medicine offers significant treatment for most types of cancers including lung cancer, 17 liver cancer, 18 stomach cancer, 19 breast cancer, 20 esophageal cancer, 21 colorectal cancer, 15 and nasopharyngeal (throat and sinus) cancer. 22ncer patients mostly are given traditional Chinese medicine and conventional medical treatments. 23Traditional chinese medicine treatment resulted in improvement of cancer symptoms with many of those reporting reduced pain.It also shows reduced tumor size, increased survival rates, increased quality, lower relapse rates and reduced complications. 24The Xiaotan Sanjie Fang, a Chinese famous herbal prescription was reported here has ability to inhibit the growth of colorectal cancer precursor lesions.We also explore the potential mechanism of Xiaotan Sanjie Fang inhibits the growth of colorectal cancer precursor lesions.
Angiogenesis is essential for cancer development and growth, because it requires blood vessels to supply nutrients and oxygen. 25e production of VEGF and other growth factors by the tumor results in the "angiogenic switch," where new vasculature is formed in and around the tumor, allowing it to grow exponentially. 26In this study, we found the Xiaotan Sanjie Fang was able to reduce the expression of VEGF.Suggesting the treatment of Xiaotan Sanjie Fang was capable of suppressing angiogenesis in colorectal cancer precursor lesions when compared to model group.
Cyclooxygenase-2 (COX-2), an inducible form of the enzyme that catalyzes the first step in the synthesis of prostanoids, is associated with inflammatory diseases and carcinogenesis, which is suspected to promote angiogenesis and tissue invasion of tumors and resistance to apoptosis. 27In this study, we found Xiaotan Sanjie Fang could reduce COX-2 secretion compared to model group, which may contribute to reduce angiogenesis.Aquaporin 1 (AQP1) is a membrane protein whose main function is to transfer water across cellular membranes.Recent studies have described important roles for AQP1 in epithelial carcinogenesis and tumor behavior. 28Yuzo et al. revealed that AQP1 played a role in suppressing apoptosis in esophageal squamous cell carcinoma cells lines. 29In the presented study, we found Xiaotan Sanjie Fang could reduce AQP1 secretion compared to model group.And the expression of apoptosis gene, caspase-3 and BAX was significantly increased and anti-apoptosis gene, BCL-2, was obviously decreased.These results were consistent with others' report.
In summary, in the presented study, we found Xiaotan Sanjie Fang was able to inhibit the growth of colorectal cancer precursor lesions.The potential mechanism including reduced secretion of VEGF, COX-2 and AQP1, and decreased anti-apoptosis gene expression and increased apoptosis gene expression.