Shaoyao Decoction Ameliorates Colitis-associated Colorectal Cancer By Regulating Inflammatory Cytokines And Lowering Epithelial-mesenchymal Transition

Background and ObjectThe data of American Cancer forecast in 2014 showed that account for new cases and deaths of colorectal cancer would be 136,830 and 50,310,8% of new cancer cases and deaths, respectively. CRC is the third most common cancer in men and women. Ulcerative colitis, familial adenomatous polyposis, hereditary nonpolyposis colorectal cancer syndrome are the risk factors of colorectal cancer. A study found that about 20 percent of the causes of cancer was related with long-term repeated chronic inflammation. Colitis-associated colorectal cancer was one of the causes of death for patients with ulcerative colitis.Currently, surgery is preferred treatment for colorectal cancer, with radiotherapy and chemotherapy treatment. But radiotherapy and chemotherapy treatment cause serious side effects. Recent studies have found that traditional medicine has a very good effect in the prevention of gastrointestinal cancer, mainly in improving the quality of life of cancer patients and prolong its survival and so on. So it is important to find a Chinese herbal compound with effect of anti-tumor which can reduce the toxicity of radiotherapy and chemotherapy treatment, improve the quality of life of cancer patients. Further more clarifying the mechanism of its anti-tumor is very important for clinical medication guide.Shaoyao decoction (SYD) is a canonical Chinese medicine prescription used for the treatment of hot and humid dysentery. It is effective in clearing heat and damp, reconciling qi and blood. Shaoyao decoction was commonly used for treatment of inflammatory bowel disease, dysentery, diarrhea caused by chemotherapy in clinical. It has been reported that SYD has pharmacological effects of anti-inflammatory and antibacterial. Paeonol, the primary phenolic component in Radix Paeoniae Alba (Shaoyao), inhibited the proliferation of colon cancer cells. Currently, if SYD can prevent colitis-associated colon cancer and its mechanism remains unclear. To study the mechanism of how SYD prevent colitis-associated colon cancer is important to exert the anti-tumor effect of traditional Chinese medicine.Epithelial-mesenchymal transition (EMT) is a process in which epithelial cells lose their cell polarity, and the connection between cell adhesion and tight connection is lost, and epithelial cells obtain the ability of invasion and migration, have morphology and characteristics of mesenchymal cells. EMT is recognized to play an important role in the pathogenesis of inflammatory bowel disease. its function in tumor development are lead to more and more attention. At present, the mechanism for the regulation of EMT is not very clear. Studies suggested that it could be regulated by several transcription factors. Snail, a transcription factor having a zinc finger-like structures, not only participate in the start of EMT, but also can regulate EMT. NF-κB is an inflammatory-associated tumor initiation factor which is closely related with the development of tumor. NF-κB can be activated by inflammatory factors, and can induce releasing of inflammatory cytokines. TNF-α, IL-1β and IL-6 are multifunctional cytokine regulated by NF-κB. These cytokine not only involved in the development of inflammation, but also played a crucial role in the startup process of EMT.Chingish in whether SYD can prevent colitis-associated colon cancer, and whether it can prevent colitis-associated colon cancer through lowering EMT and regulating inflammatory factors. Therefore, this study was to provide an experimental basis for clinical treatment of the effect of anti-tumor of SYD. At the same time, to further explain the effect of inflammatory cytokines and EMT in preventing colitis-associated colon cancer.Method1. (High Performance Liquid Chromatography, HPLC) control the quality of SYDEach herb was weighed according to prescription, using traditional decoction method, added water to 10 and 6 times of the dose, boiled twice, filtered and merged the two boiling liquid. Then concentrated to a paste by rotary evaporation, after dry, weighed and recorded. Then, added the appropriate amount of methanol solution to dissolved in ultrasound, used 0.45μm microporous filter to filter. Appropriate amount of berberine and baicalcein was weighed, added methanol solution to make the dose to lmg/mL, used 0.45 μm microporous filter to filter. According to the following chromatographic conditions:Column:Shimadzu C18 VP ODS (250×4.6 mm,5μm); mobile phase:acetonitrile -0.5‰ aqueous acetic acid; flow rate:1.0mL/min; column temperature:25℃; PDA detection:254nm. Analyzed the repeatability of 6 different batches of SYD and detected the content of berberine and baicalcein of SYD by Shimadzu HPLC, recorded the chromatograms, and analyzed.2. To induce colitis-associated colon cancer in mice by AOM/DSS65 SPF male C57BL/6 J mice were randomly divided into three groups,15 mice in control group,30 in model group and 20 in SYD group. Before the experiment recorded body weight and number of mice. Established animal models of colitis-related colon cancer according to literature, given a single intraperitoneal injection of a small dose of AOM, combined with three cycles of DSS feeding. Mice in SYD group was administered SYD (7.12g/kg/d) by gavage excep giving the same administration of model group. Mice were monitored for changes of body weight and food intake during the experiment. Observed the general well-being of mice, such as the general state of the hair, activity, blood stool, rectal prolapse and recorded cases of death of mice. At the end of week 15 of the experiment, mice were weighed and recorded the weight of mice. Obtained blood by excising eyeball, separated the serum; removed spleen and colon from mouse, measured its weight; colon tumor site was observed in mice, the size, the number and the weight of tumor was measured; the length and weight of the colon was measured. Tissues were saved in aliquot to be used in subsequent experiments.3. HE staining observe changes of histology of colon in miceColon tissue of mice was made to paraffin sections. To observe changes of histology of colon in mice under a microscope by hematoxylin and eosin (HE), photograph and analyze the level of differentiation of colon tumor tissue and the degree of invasion.4. Immunohistochemistry observe the expression of oncogenic protein, EMT-related protein and NF-κB, F4/80Colon tissue of mice was made to paraffin sections. To observe the expression and distribution site of oncogenic protein PCNA, p53, COX-2, β-catenin and EMT-related protein E-cadherin, Vimentin, Fibronectin, N-cadherin and NF-κB, F4/80 under the microscope by immunohistochemistry SP method. To analyze the effect of SYD in the expression of these proteins.5. Western blot analyze the effect of SYD in the expression of oncogenic protein, EMT-related proteinExtract the total protein of colon tissue. Western blot detected the expression of oncogenic protein PCNA, p53, COX-2, β-catenin and EMT-related protein E-cadherin, Vimentin, N-cadherin, Snail, Claudin-1. To analyze the effect of SYD in the expression of these proteins.6. MTT assay detect the effect of SYD on the colon cancer cells vitalityTwo kinds of colon cancer cells SW480, HCT116 were cultured in 37℃ 5% CO2 incubator.The cells in logarithmic growth phase were seeded on 96-well plates with 1×104/well Cells in a 100 μl volume. After the cells were adherent, a concentration gradient of SYD (2mg/mL、4mg/mL、6mg/mL、8mg/mL) was added to each well,100μl/well. Each concentration set six wells, and set blank well. Respectively, intervene 12h,24h, added 20μL 5mg/mL MTT to each well, cultured 4h. Discard the old solution, added 150μL dimethyl sulfoxide to each well, shaker 10min, to completely dissolved purple crystals. Microplate reader at 490nm absorbance measured. Cell survival rate= absorbance values in test group/ absorbance values in control. The experiment was repeated three times.7. MTT assay detect the effect of Paeonol on the colon cancer cells vitalityTwo kinds of colon cancer cells SW480, HCT116 were cultured in 37℃ 5% CO2 incubator.The cells in logarithmic growth phase were seeded on 96-well plates with 1×104/ well Cells in a 100μl volume. After the cells were adherent, a concentration gradient of Paeonol (0.2mM/L、0.4mM/L、0.6mM/L、 0.8mM/L) was added to each well, 100μl/well. Each concentration set six wells, and set blank well. Respectively, intervene 24h,48h, added 20μL 5mg/mL MTT to each well, cultured 4h. Discard the old solution, added 150μL dimethyl sulfoxide to each well, shaker 10min, to completely dissolved purple crystals. Microplate reader at 490nm absorbance measured. Cell survival rate= absorbance values in test group/ absorbance values in control. The experiment was repeated three times.8. Western blot analyze the effect of SYD and Paeonol on the expression of oncogenic proteinColon cancer cells SW480, HCT116 were seeded in 6-well plates with 1 × 105/ well cells. After the cells were adherent, a concentration gradient of SYD (2mg/mL、 4mg/mL) were added to each well,2ml/well, intervene 24h and a concentration gradient of Paeonol (0.2mM/L、0.4mM/L) were added to each well,2ml/well, intervene 48h. Set blank well. Extract the total protein of cells and protein quantify, taking 50μg protein samples each and adding five-times sample buffer to carry samples on 10% SDS-PAGE. Use the wet transfer method to transfer protein to a PVDF membrane.5% skim milk closed 1h at room temperature. Incubating overnight at 4℃ by primary antibody, next day the membrane was incubated by HRP-conjugated appropriate secondary antibody (1:2000 dilution) for 1h at room temperature. Then use chemiluminescence assay to obtain Image.9. WB analyze the effect of SYD and Paeonol on Snail-induced EMTSW480 cells in logarithmic growth phase were seeded in 6-well plates with 1× 105/well cells. After cells were adherent, transfect Snail 1 plasmid into SW480 cell according to the manufacture’s protocol. A concentration gradient of SYD (2mg/mL、 4mg/mL) were added to transfected cells,2ml/well, intervene 24h and a concentration gradient of Paeonol (0.2mM/L、0.4mM/L) were added to transfected cells,2ml/well, intervene 48h. Set blank well. Extract the total protein of cells, detected the expression of EMT-related protein and analyze the effect of SYD and Paeonol in the expression of these proteins.10. Luminex assay detected the content of serum IL-1β, IL-6, TNF-αLuminex assay detected the content of serum IL-1β, IL-6, TNF-α using a mouse multi-factor assay kit. To analyze the effect of SYD in inflammatory factors.11. Statistical MethodsAll data were analyzed using SPSS 13.0 statistical software, experimental data were presented as mean ± standard deviation (x± SD). For quantitative data, data between two groups were compared with 2-independent samples tests, Mean values of data from more than 3 groups were analyzed by one-way analysis of variance (ANOVA), homogeneity of variance using LSD method, heterogeneity of variance using Dunnett T3 method. Ranked data were analyzed by nonparametric test (K Independent Samples Test). Survival rate was analyzed with Kaplan-Meier survival analysis. A value of P< 0.05 was considered as statistically significant.Result1. The repeatability of SYD is stableEvaluate the similarities of the 6 batches of SYD by HPLC found that the peaks in the spectra were matched automatically. The similarities of repeatability were from 0.937 to 0.977. SYD content berberine and baicalein.2. Shaoyao decoction ameliorated the general well-being of mice effectively and reducing the mortality of miceAfter intervening by AOM/DSS, mice became significantly less dynamic and burnout, dark hair, listlessness in model group. Some severe cases may appear loose stools, bloody stools. Body weight of mice lossed significantly. After being treated with SYD, the general well-being of mice effectively improved. The degree of loss of weight was lighter than model group (P< 0.01).Mice in control group all survived, survival rate of mice in model group was only 23.30% while was 60.00% in SYD group. Compared with model group, SYD significantly prolonged the survival time of mice, improved survival rate of mice after intervening by AOM/DSS, the value of P is 0.032.3. Shaoyao decoction reduced the number and size of tumor induced by AOM/DSSThe formation rate of colonic neoplasm was 100% in model group and SYD group. Neoplasms were principally distributed in the middle and distal colon. Compared with model group, the total multiplicity of colorectal neoplasms decreased by 42.80% after treating with SYD (P< 0.01). Shaoyao decoction significantly retarded the development of large neoplasms (diameter> 3mm) by 59.20% (P< 0.01), and significantly reduced tumor weight and volume. Compared with control group, the weight of colon was increased and the length of colon was shortened significantly in model group (P< 0.01). Shaoyao decoction significantly ameliorated the degrees of shorten of colon length (P< 0.05) and increased of colon weight (P< 0.01).4. Shaoyao decoction ameliorated level of differentiation and invasion of neoplasmsThe result of HE staining showed that the neoplasms in AOM/DSS-induced colitis-associated colon cancer were mostly tubular adenoma or adenocarcinoma. The neoplasms was mostly high-differentiation with invasion in model group. Shaoyao decoction ameliorated the level of differentiation and invasion of neoplasms, P= 0.041.5. Shaoyao decoction reduced the expression of oncogenic proteinImmunohistochemistry results showed that the expression of oncogenic protein PCNA and COX-2 increased in model group. p53 majorly express in the neoplastic epithelium and in nondysplastic crypts, implicating the involvement of p53 mutation in our AOM/DSS-induced caCRC model.β-catenin occurs nuclear translocation in tumor tissue. Compared with control group, four oncogenic protein significantly increased in model group, P< 0.01, the difference was statistically significant. SYD can significantly reduce the expression of four oncogenic proteins (P< 0.05). WB showed that compared with control group, four oncogenic protein are highly expressed in model group, there was a significant difference. Consistented with the immunohistochemistry results, SYD significantly decreased the expression of four oncogenic proteins (P< 0.05), compared with model group, the difference was statistically significant.6. Shaoyao decoction and Paeonol inhibited CRC cell proliferationTwo kinds of colon cancer cells SW480, HCT116were intervened by 2mg/mL, 4mg/mL,6mg/mL,8mg/mL SYD for 12h,24h, the viability of cells were significantly decreased (P< 0.01). Compared with control group, the viability of cells in intervened group were significantly decreased in both two cell lines (12h:P< 0.01; 24h:P<0.05).SW480, HCT116 were intervened by 0.2mM/L,0.4mM/L,0.6mM/L,0.8mM/L Paeonol for 24h,48h, the viability of cells was significantly decreased, the viability of cells between different concentration had significant differences (P< 0.01). Compared with control group, the viability of cells in intervened group were significantly decreased in both two cell lines (P< 0.01).After intervened by different concentration of SYD and Paeonol, cell viability was significantly decreased. While prolonged intervention time, cell viability was decreases. Shaoyao decoction and Paeonol decreased cell viability in a time-and dose-dependent manner.7. Shaoyao decoction and Paeonol decreased oncogenic proteins in CRC cellsThe result of WB showed that oncogenic protein PCNA, p53, COX-2, β-catenin were highly expressing in colon cancer cells. Treated with different concentrations of SYD (2mg/mL,4mg/mL) 24h, Paeonol (0.2mM/L,0.4mM/L) 48h, the expression of PCNA, p53, COX-2, β-catenin were significantly decreased compared with control group (P< 0.05).8. Shaoyao decoction ameliorated AOM/DSS-induced and Snail-induced EMT, Paeonol inhibited Snail-induced EMTThe result of IHC showed that the expression of E-cadherin, the marker protein of EMT was decreased in AOM/DSS-induced colon cancer in mice. Compared with control group, the expression of N-cadherin, Fibronectin, Vimentin were significantly increased (P< 0.01). Compared with model group, SYD significantly increased the expression of E-cadherin and decreased the expression of N-cadherin, Fibronectin, and Vimentin (P< 0.05).The results of WB was consistent with the results of IHC. The expression of E-cadherin was missing in model group, while the expression of N-cadherin, Vimentin, Snail, Claudin-1 were significantly increased, compared with control group, (N-cadherin, Vimentin, E-cadherin:P< 0.05; Snail, Claudin-1:P< 0.01). Shaoyao decoction significantly upregulated the expression of E-cadherin, downregulated the expression of N-cadherin, Vimentin, Snail, Claudin-1, compared with model group (P < 0.05).Overexpression of Snail in SW480 induced upregulation of N-cadherin, Vimentin, and downregulation of E-cadherin. Shaoyao decoction and Paeonol can significantly decreased the expression of N-cadherin, Vimentin and increased the expression of E-cadherin induced by Snail (P< 0.01).9. Shaoyao decoction decreased expression of macrophage cell surface marker F4/80 and NF-κBThe results of IHC showed that compared with control group, the expression of F4/80 and inflammation-associated regulatory factor NF-κB significantly increased in model group (P< 0.01). Shaoyao decoction significantly reduced the expression of them in tumor tissue compared with model group (P< 0.05).10. Shaoyao decoction reduced the level of inflammatory factors in serumThe level of IL-1β, IL-6, TNF-α in serum were significantly increased in model group. After SYD intervention, the level of IL-1β,IL-6, TNF-α in serum decreased significantly compared with model group (IL-1β;P< 0.01; IL-6, TNF-α: P<0.05).Conclusions1. The preparation process of Shaoyao decoction is reasonable.2. Shaoyao decoction inhibit AOM/DSS-induced colitis-associated colorectal cancer.3. Shaoyao decoction ameliorates AOM/DSS-induced colitis-associated colorectal cance by lowering epithelial-mesenchymal transition (EMT).4. Shaoyao decoction ameliorates AOM/DSS-induced colitis-associated colorectal cance by suppressing NF-κB activation and downregulating inflammatory cytokines.