Longzhu Xiaoliu Fang Reduces Intestinal Adenoma Formation In APC^Min/+ Mice Through Suppressing HIF-1α Induced Tregs

BackgroundColorectal carcinoma （CRC） is a common malignant tumor of the digestive system, accounting for the third leading cause of cancer deaths in western countries. CRC belongs to Fuliang,and ChangQin in traditional Chinese medicine （TCM）.It has been recorded in Suwen that CRC can be defined as Fuliang, Cold Qi hits the intestine and fights with WeiQi. Qi is out of nourishing and pathogenic Qi onsets in the intestine to form colorectal polyp. The balance Hanqi and Weiqi, that means CRC is associated with terrible diet, invasion of Waixie, Imbalance of nutrition and damaged organs. Due to changes of diet and lifestyle for Chinese, CRC becomes one of the fastest ascendant malignants tours with a rasing the mortality. Surgery is the first choice for CRC treatment while giving systemic chemotherapy. Although advanced diagnosis and treatment, the prognosis is poor yet. Except for the fact that the overall survival rates of patients depend on tumor stage, Immune system modulation therapy has been becoming more and more popular for treating CRC. Therefore, it is necessary to use adjuvant therapy and palliative care to improving efficacy and lives. However, there are few available chemotherapeutic drugs, especially the effective, low toxicity and cost one. Molecular targeted drugs provide novel choices for CRC treatment, while it cost too much and its application and effect are not yet clear. The effect of chemotherapy drugs declines obviously because of drugs resistance. In conclusion, it is essential for us to exploit low price, low toxicity and good efficiency natural drugs.Shenling Baizhu San is a canonical Chinese medicine formula recorded in "The Prescriptions of the Bureau of Taiping People’s Welfare Pharmacy" from Song-dynasty. It can effectively extenuate the symptom of postoperative CRC patients like fatigue, yellow skin, anorexia, nausea, diarrhea, and abdominal distension. Also, it is able to largely improve the Karnofsky performance scale （KPS） and increase the number of CD4+, CD8+cells and natural killer cells （NK cells） for postoperative CRC patients.Tregs, Myeloid suppressor cells and various immunosuppressive factors Including interleukin-10 （IL-10）, transforming growth factor-β （TGF-β）, vascular endothelial growth factor, prostaglandin E2 have been found in CRC patients. To promoting immune evasion, they usually enriched in the tumor microenvironment. Thus it is first to fight against the immunosuppressive mechanisms of the tumor microenvironment for wonderful anti-tumor efficacy. Tregs plays an important role in maintaining self-tolerance, protection and response in intestinal inflammation. Meanwhile suppressing the immune surveillance for tumor may be horrible. Foxp3 is key transcription factors to control development and function of CD4+ CD25+ Tregs. It is proved that Tregs reduces the mainly anti-tumor response, which is commonly found in serum in CRC patients with poor prognosis. However, more functions of Tregs in the diagnosis of tumor-associated CRC patients are still unclear.In this study, we have improved the Shenling Baizhu San form Longzhu Xiaoliu Fang （LXF）, It comprises:Atractylodes Humulus scandens,Poria cocos, Polygonum chinense,Coix seed,Cistanche,Ginseng, Tsubaki root tree, Wikstroemia indica, Paridis, Zedoary, Radix paeoniae alba. Moreover, Shenling Baizhu San reduces the tumor weight from heterotopic transplantation mice and increase IL-2、 IFN-y and TNF-a in serum, So we speculated that LXF may through regulating immune and inflammatory intervention in CRC, and the mutant mice by influencing the state of inflammation and immune cell function involved in the occurrence of CRC. Therefore, we proposed to choose ApcMin/+ mice to observe whether LXF can inhibit the incidence of colorectal adenomas in ApcMin/+ mice and to explore whether the regulation in colorectal tumor infiltrating Tregs and control the growth of CRC.ObjectiveThe purpose of this study was to investigate the influence of LXF on colorectal adenomas in ApcMin/+ mice, and to explore the molecular mechanism of LXF for the antitumor activation. It may find a theoretical basis on molecular biology of the antitumor activation and provide more reliable basis for clinical application and new targets for the prevention and treatment of CRC.Mterial and Methods1 Preparation of LXFAqueous extracts of LXF were extracted at 80℃ by stirring it for 1h using 4 volumes of distilled water （v/m）. Then, we centrifuged the extract at 1,500 ×g at room temperature. To obtain the semisolid LXF solution, the supernatant was collected and subjected to condensation under reduced pressure at 70℃. Quality control of LXF was performed by high-performance liquid chromatographic （HPLC） analysis. LXF was suspended again in 0.9% saline at a final concentration of 2 g/mL. The solution was stored in aliquots at-20℃.2 Animals model for analysis of the influence of LXF on CRC in vivoAPC^Min/+ Male Mice on a C57BL/6 background were bred with female C57BL/6mice. Offspring were genotyped as heterozygotes by real-time polymerase chain reaction （RT-PCR） for the Ape gene by taking tail snips at weaning. C57BL/6 mice were used as a wild-type control. ApcMin/+ mice were randomly assigned to either LXF or placebo treatment. LXF （11.21 g/kg） or equivalent normal saline was administered by gavage using a tube twice a day. LXF feedings began at 4 weeks of age and continued for 14 weeks, until animals reached 18 weeks of age, at which time they were sacrificed. Each group included 6 mice. The physiological state and weight including the operation of ApcMin/+ mice were recorded. The weight of solid tumors, the length of the colons, the size and weight of spleen from ApcMin/+ mice were measured finally.3 Flow cytometric analysis for the influence of LXF on Tregs in peripheral blood and spleen in ApcMin/+ micePeripheral blood were collected by taking out an eye from mice, stewing at room temperature for 2 to 4 h, and obtaining the serum at 1,500×g for 15 minutes. After spleen cells stained with a FITC-conjugated anti-mouse CD4 mAb, anti-mouse CD25 mAb, and a PE-Cy5-conjugated anti-mouse/rat Foxp3 mAb were separated as usual, Tregs of every group were analyzed by using a FACSCalibur flow cytometer and CellQuest software （Blank, administration, control）.4. Immunohistochemistry for the influence of LXF on related protein in the tissue of CRCParaffin-embedded colon sections were dewaxed, rehydrated, and pre-treated with hydrogen peroxidase in PBS buffer. Heat-induced antigen retrieval was performed. Sections were incubated with anti-Proliferating cell nuclear antigen （PCNA）, anti-β-catenin, anti-p53, or anti-COX-2, anti-HIF-1α. After incubation with HRP-conjugated secondary antibody and tyramide amplification followed by streptavidin-HRP, positive signals were visualized by DAB kit. Section were examined at a magnification of 400X and analyzed using NIS-Elements. Double staining of the section with CD4+ and CD25+ was carried out as above.5. Western blot analysis for the influence of LXF on related protein in the tissue of CRCTotal protein of each group was collected respectively, with quantification in Bradford. After mixing 5 times Loading buffer,50μg of total protein was separated on a 10% SDS-polyacrylamide get and transferred eletrophoretically to a PVDF membrane,which were blocked with 5% non-fat dry milk for 1h and incubated sequentially with the primary antibodies anti-PCNA, anti-β-catenin, anti-p53, or anti-COX-2, anti-Foxp3, anti-HIF-1α, at 4℃ overnight. The HRP-conjugated secondary antibody was added to the preparation at 37℃ for 1h.The protein bands were captured and documented by KODAK Image Station and 2000MM Digital Imaging System.6. Cell culture for the association between HIF-la and Foxp3Jurkat T cells was cultured for 0,6,12,24h after treating with LXF. Western blot was carried out for analyzing the association between HIF-1α and Foxp3. Next, Jurkat T cells was treated with HIF-la siRNA for 48h, followed by exposure to LXF for 24 h. Finally, Western blot was carried out again.Statistical AnalysisEach experiment was repeated at least three times. Data were presented as mean x±S. All data were analyzed using the SPSS statistical package （version 16.0, SPSS Inc, Chicago, USA）. Data between two groups were compared with 2-independent samples tests. Mean values of data from more than 3 groups were compared with one-way analysis of variance （ANOVA） and multi-comparison was performed. A value of P<0.05 was considered as statistically significant.Results1 LXF suppresses colorectal adenomas growth in ApcMin/+ miceDuring the animal experiment, LXF increased body weight and lower systemic toxicity, the size or number of colorectal adenomas significantly in ApcMin/+ mice as compared to wild type mice, besides smaller spleen.2 LXF reduces expression of β-catenin, COX-2, PCNA, and p53 in colon tumorsBased on immunohistochemistry, the expression of β-catenin, COX-2, PCNA, and p53 were highly elevated in intestinal polyps of ApcMin/+ mice. The positive cells of β-catenin, COX-2 and PCNA decreased 91.6%（P<0.01）,62.5%（P<0.01） and 83.3%（P<0.01） by LXF treatment, respectively. Western blotting analysis confirmed the results that LXF significantly decreased P-catenin, COX-2, PCNA, and p53 expression. Therefore, LXF effectively ameliorated CRC through downregulating neoplastic markers.3 LXF downregulates peripheral and spleen CD4+CD25+Foxp3 Tregs in ApcMin+ miceThe percentage of CD4+CD25+Foxp3 Tregs in the peripheral blood and the spleen significantly increased in the ApcMin/+ mice. LXF significantly decreased the ratio of Tregs as compared to ApcMin/+ mice and no significant difference was found as compared to the wild type mice. These results indicated that Tregs play essential role in the carcinogenesis of ApcMin/+ mice, LXF reduced the formation of intestinal adenomas through regulating the percentage of Tregs in ApcMin/+ mice.4. LXF reduces in situ expression of CD4+, CD25+and Foxp3 in intestinal adenomas of ApcMin/+ miceThrough immunohistochemical double staining of CD4+CD25+ cells and western blotting of Foxp3. CD4+CD25+positive cells was increased in ApcMin/+ group and decreased by LXF treatment significantly. This result was consistant with the data from flow cytometric analysis of CD4+CD25+cells in the peripheral blood and the spleen. These results further confirmed that LXF suppressed the accumulation of the tumor infiltrating Tregs.5. LXF depresses Foxp3 expression via inhibition of HIF-1α in ApcMin/+ miceBoth immunohistochemistry and western blotting revealed a remarkable decrease in expression of HIF-1α in the intestinal adenoma treatment with LXF as compared with ApcMin/+groups. The expression of HIF-1a was increased in a time dependent manner by incubation with 1% O2 for Oh,6h,12h, and 24h. The expression of Foxp3 under hypoxia condition was increased correspondingly with the extention of the culturing time. To determine whether the inhibition of HIF-la could suppress Foxp3 expression in hypoxia, Jurkat T cells was cultured with HIF-1a siRNA for 48 h, followed by exposure to LXF for 24 h. The results showed that LXF decreased hypoxia-induced the expression of Foxp3. Expression of Foxp3 and HIF-1α were positively correlated under hypoxia conditions. Thus, the results above indicated that LXF could suppress HIF-1α expression and depress Foxp3 activity in T cells.Conclusions1 LXF suppresses colorectal adenomas growth in ApcMin/+ mice and reduces expression of p-catenin, COX-2, PCNA, and p53 in colon tumors.2 LXF downregulates peripheral and spleen CD4+CD25+Foxp3 Tregs and reduces the expression of CD4+, CD25+and Foxp3 in intestinal adenomas of ApcMin/+ mice.3 LXF depresses Foxp3 expression via inhibition of HIF-1α in ApcMin/+ mice.