A Study On The Herb Pair Of Hedyotis Diffusa And Lobelia Inhibiting Colon Cancer By Regulating Wnt/β-catenin Signaling Pathway Through Spalt-like Transcription Factor 4

Background:As the third largest cancer in China,the incidence rate and mortality rate of colon cancer are increasing year by year.Traditional treatment is surgery combined with chemotherapy.Therefore,the side effects,including loss of appetite,anemia and decreased immunity caused by chemotherapy are still troubling the patients.Traditional Chinese medicine has achieved good results in alleviating side effects and enhancing treatment.The herb pair of Hedyotis diffusa and Lobelia chinensis is the primal medicine of Xiaoai jiedu prescription.It is also a common herb pair in clinical antitumor therapy.Xiaoai jiedu prescription is created by Master of national physician Zhou Zhongying.This prescription is based on "Ai Du" theory which is the guidance of cancer treatment.Zhou insisted that cancer is a special poison,which is called "Du" in traditional Chinese medicine,if we want to restore patients to health,detoxication is needed.Xiaoai jiedu prescription is used to treat colon cancer patients after surgery.Clinical practice and previous literature studies have confirmed that the composition of Hedyotis diffusa and Lobelia lobelis can have synergistic effect and excellent therapeutic effect.With the development of modern medicine,the research on the mechanism of drugs acting on diseases is more and more complete.However,it mainly focuses on single component,there are few studies on herb pairs.Spalt-like transcription factor 4(SALL4)is a zinc finger transcription factor,which regulates the self-renewal of embryonic stem cells.SALL4 is also involved in tumorigenesis and tumor progression.Wnt/β-catenin signaling pathway is a classical and significant signaling pathway.It has been proved to be abnormally activated in a variety of tumors,including colon cancer.Objectives:The aim of the study was to explore the mechanism of Hedyotis diffusa and Lobelia chinensis herb pair on inhibiting colon cancer cells.It provides scientific basis for anti-tumor therapy of traditional Chinese medicine,and also provides ideas for clinical treatment of other tumors.This will provide major benefits to the advantages of traditional Chinese medicine.Methods:1.Experiment 1:The colon cancer cell lines were divided into negative control group and herb pair group(S1:B1、S1:B2、S2:B1).The herb pair group was treated with corresponding drug-containing serum,and the negative control group was treated with PBS solution.The following step was analyzing cell functions of colon cancer cell lines by using CCK-8 assay,colony formation assay,Transwell assay and flow cytometric analysis.2.Network pharmacologic analysis:Forecasting potential target gene and signaling pathway of Hedyotis diffusa and Lobelia chinensis herb pair.3.Experiment 2:①Based on the results of network pharmacologic analysis,qRT-PCR and Western blot were used to detect the expression of SALL4 in colon cancer samples and adjacent normal tissue samples.The methods were also used to detect the expression of SALL4 in colon cancer cells and normal intestinal mucosa cells.Correlation analysis was performed on clinicopathological characteristics of the patients and the expression level of SALL4.②The colon cancer cell lines were divided into negative control group and herb pair group.qRT-PCR and Western blot was used to detect the expression of S ALL4 in colon cancer cell lines treated with drug-containing serum.③The colon cancer cell lines were divided into negative control group,herb pair group and herb pair plus SALL4 overexpression group.Rescue experiments,including CCK-8 assay,colony formation assay,Transwell assay and flow cytometric analysis,were performed to confirm the rescue effect of SALL4 overexpression on Hedyotis diffusa and Lobelia chinensis herb pair.4.Experiment 3:The colon cancer cell lines were divided into negative control group,herb pair group and herb pair plus SALL4 overexpression group.Western blot was used to detect the expression level of proteins of Wnt/β-catenin signaling pathway in each group.5.Experiment 4:Ki67 staining,TUNEL staining and immunohistochemical staining were performed on subcutaneous tumor of nude mice.The metastasis ability of colon cancer cell lines was assessed by in vivo imaging.Hematoxylin and eosin staining were performed on the harvested lungs from nude mice.The expression of SALL4 and Wnt/β-catenin signaling pathway proteins in subcutaneous tumor were detected by Western blot.Conclusions:1.The herb pair of Hedyotis diffusa and Lobelia chinensis can inhibit proliferation,migration,invasion and induce apoptosis of colon cancer cell lines.The best proportion of Hedyotis diffusa and Lobelia chinensis herb pair was 2:1.2.SALL4 was highly expressed in colon cancer tissues compared with normal adjacent tissues,and was positively correlated with tumor size,T stage and lymph node metastasis.The herb pair of Hedyotis diffusa and Lobelia chinensis inhibits proliferation,migration,invasion and promotes apoptosis of colon cancer cells by down-regulating SALL4 expression.3.The herb pair of Hedyotis diffusa and Lobelia chinensis inhibits colon cancer by inactivating Wnt/β-catenin signaling pathway via targeting SALL4.