Study On Mechanism Of Guzheng Xiaoai Decoction On The Glucose Metabolism Of Colorectal Cancer

Background:The continuous high-speed proliferation,non-stop metastasis,invasion of tumor cells,and the tenacious resistance to chemotherapeutic drugs are largely dependent on the acid-hypoxic microenvironment created by the body.This kind of microenvironment will undoubtedly kill normal body cells and provide the result of energy metabolism that violates the law.As early as 1924,the well-known medical scientist Otto Herinrich Warburg proposed the "Warburg Effect" theory for this abnormal metabolic form.This new metabolic coding program is considered in modern research to initiate local hypoxia and acidic environment of tumors.And the key to infinite growth of deformities.From the perspective of glucose metabolism to study anti-tumor drugs has become a new hope for a breakthrough in the cure of colon cancer.Professor Zhou Zhongying,a master of traditional Chinese medicine,has refined the "cancer toxicity theory"that has the basis of traditional Chinese medicine,scientificity and practicability in the long-term clinical work.Combining the advantages of traditional Chinese medicine prescriptions under the treatment concept of"Removing toxins means strengthening the body",a prescription for eliminating cancer and detoxifying is formed,which integrates heat-clearing and detoxification,strengthening vitality and qi,and promoting blood circulation.Whether it is in basic research or clinical practice,this prescription can prove that it can achieve the purpose of coexisting with cancer.In the clinical application of Zhou Zhongying’s Xiaoai Jiedu Decoction in outpatient treatment of colon cancer patients,the research team with the help of Zhou Zhongying’s team modified Xiaoai Jiedu Decoction and evolved into Guzheng Xiaoai Decoction.Practical observations believe that it is more targeted and applicable to colon cancer patients,and greatly improves the quality of life of colon cancer patients.Objective:From the perspective of glucose metabolism and tumor microenvironment,to explore the specific mechanism of Guzheng Xiaoai Decoction in inhibiting colon cancer cells,so as to further consolidate the "cancer toxicity theory" and give play to the advantages of Chinese medicine in anti-tumor.Method:1.Cell experiment1.1 Select HT-29 human colon cancer cell lines and divide the cell lines into four groups randomly.Namely① Blank control group(Control):cells were inoculated in normal rat serum and RPMI-1640 medium for culture;② Low dose group(Low):cultured in RPMI-1640 medium containing 10%Guzheng Xiaoai Decoction serum;③Medium dose group(Medium):Cultured in RPMI-1640 medium containing 20%Guzheng Xiaoaifang medicated serum;④High-dose group(High):Cultured with RPMI-1640 containing 30%Guzheng Xiaoai’s medicated serum Base culture.Observe the Transwell test of HT-29 cells cultured in different groups for 12h and 24h;determine the glucose content,lactate level,lactate dehydrogenase content(LDH),and the influence of intracellular and extracellular pH,the concentration of ATP in each group after 72h;Western Blot was used to detect hypoxia inducible factor 1α(HIF-1α),cell energy receptor AMPK α1,pHospHorylated AMPK α1(p-AMPK α1),hexokinase(HK2);AnnexinV-FITC/PI double labeling flow cytometry was used to detect the apoptosis rate of HT-29 cells.1.2 Select HT-29 human colon cancer cell lines and divide the cell lines into four groups randomly.Namely① Blank control group(Control):HT-29 cells were inoculated in normal rat serum and RPMI-1640 medium and cultured;②High dose group(High):Using RPMI-1640 medium containing 30%Guzheng Xiaoai Decoction serum HT-29 cells;③High-dose combination and small molecule interference AMPK(High+Si-AMPK)group:The RPMI-1640 medium containing 30%Guzheng Xiaoai Decoction-containing serum and specific silencing AMPK can work together on HT-29 cells.④High-dose combination and AMPK activator(High+AMPK activator)group:Use RPMI-1640 containing 30%Guzheng Xiaoai Decoction medicated serum and AMPK agonist AICAR to act on HT-29 cells.Calculate the OD value of the cells after 24h,48h,72h culture in different groups;scratch and Transwell test on HT-29 cells under 4 groups to observe the migration and invasion ability of 12h and 24h;use Western Blot to detect different groups HIF-1α,the expression of glucose transporter 4(Glut4),apoptosis-related proteins Bax,Bcl-2 and p53.2.Animal experimentSelect HT-29 human colon cancer cell line to construct tumor-bearing nude mice models,and randomly divide them into 5 groups,each with 8 mice,half male and half female.Namely,the blank control group,model group,low-dose Guzheng Xiaoai Decoction group(5g/kg),medium-dose Guzheng Xiaoai Decoction group(10g/kg),and high-dose Guzheng Xiaoai Decoction group(20g/kg).Drug intervention was performed for 14 days,after sacrifice,nude mice were subjected to HE staining of liver,kidney,and spleen;tumor tissue was used to detect the expression of Cleaved-caspase-3,HIF-1α,HK2,Ki67,and p-AMPK α1;Western Blot was used to detect AMPKα1,p-AMPKα1,HIF-1α,HK2,and Glut4 protein in tumor;the last administration to nude mice in model group,low-dose group and high-dose group After 2h,the serum was collected for glucose metabolomics Liquid ChromatograpH Mass Sepctrometer(LC-MS)analysis.Result:1.Cell experimentIn the cell in vitro experiment,it was found that after the intervention of different groups of drugs,it was confirmed that the Guzheng Xiaoai Decoction can inhibit the invasion and migration of tumor cells in Transwell;the ATP concentration test results showed that the serum concentration of the Guzheng Xiaoai Decoction was inversely proportional to the concentration of ATP.The higher the serum concentration of Guzheng Xiaoai Decoction,the lower the concentration of ATP.When the concentration of Guzheng Xiaoai Decoction contains certain essential substances,it can reduce the acquisition of glucose by HT-29 cells.Slow down the process of glucose metabolism;At the same time,Guzheng Xiaoai Decoction can also correspondingly reduce the lactic acid produced by HT-29 cells and limit the expression of LDH;Guzheng Xiaoai Decoction containing serum can decrease the intracellular pH value,reduces the pH difference between the inside and outside of the cell,and the effect is enhanced with the increase of the intervention concentration of Guzheng Xiaoai Decoction;the serum of Guzheng Xiaoai Decoction can down-regulate the expression of HK2 and HIF-1α protein,Obviously activates the AMPK signal pathway,regulates the metabolism of HT-29 cells;Annexin—FITC/PI flow cytometry also confirmed that Guzheng Xiaoai Decoction can inhibit the proliferation of HT-29 cells in a dose-dependent manner.According to the results of the cell experiment study on the effect of Guzheng Xiaoai Decoction on the AMPK pathway of HT-29 cells,the proliferation of HT-29 cells in the High group was weaker than that in the High+Si-AMPK group,but the HT-29 cells in the High group The proliferation of HT-29 cells is stronger than that of the High+AMPK activator group,indicating that the Guzheng Xiaoai Decoction-containing serum can act on HT-29 cells by activating the AMPK signaling pathway,and the degree of inhibition of tumor cells is affected by the amount of activated AMPK;The serum containing Guzheng Xiaoai Decoction can weaken the migration and invasion ability of HT-29 cells by activating the AMPK pathway;Western Blot results in the High group showed that the expression of Glut4 was significantly inhibited,which was less than that of the blank control group(P<0.05);High+Si-The Glut4 expression of AMPK group was higher than that of High group(P<0.05);the expression of Glut4 of High+AMPK activator group was lower than that of High group.It shows that the serum containing Guzheng Xiaoai Decoction can regulate the expression of Glut4 protein through AMPK pathway,thereby inhibiting the glucose metabolism of HT-29 cells.There was no difference in HIF-1α expression between High group and Control group.There was no difference in HIF-la expression between High+Si-AMPK group and High group;HIF-la expression in High+AMPK activator group was significantly lower than High Group(P<0.05).The expression of p53 protein in the High group was significantly higher than that in the blank control group(P<0.05),and the p53 content in the High+AMPK activator group was also higher than that in High(P<0.05),indicating that p53 depends on activated AMPK to exert anti-tumor effects;inhibiting AMPK in HT-29 cells will reduce the expression of p53 in the High+Si-AMPK group and affect the apoptosis of HT-29 cells.The expression of Bcl-2 in the High+Si-AMPK group was higher than that in the High group(P<0.05).It is speculated that the proliferation of HT-29 cells will increase after AMPK is silenced.The Bax content in the High+AMPK activator group was higher than that in the High group,but there was no statistical difference;however,the Bcl-2 expression in the High+AMPK activator group was lower than that in the High group(P<0.05),which can be considered as the activation of AMPK to a certain extent can promote the apoptosis of HT-29 cells,and the result is that AMPK may affect the downstream Bcl-2 pathway.2.Animal experimentIn vivo experiments on tumor-bearing nude mice showed that Guzheng Xiaoai Decoction did not cause significant damage to liver,spleen,and kidney;immunohistochemical examination of tumor tissue showed that Guzheng Xiaoai Decoction would down-regulate Ki67,HIF-1α,HK2 protein and promote the expression of Cleaved-caspase-3,p-AMPK al protein,in order to control the proliferation of tumor cells and promote their apoptosis by restricting glucose metabolism;Western Blot test shows that Guzheng Xiaoai Decoction also activates the AMPK pathway,restricts the expression of glucose metabolism-related enzymes HIF-la,HK2,and Glut4,pHospHorylates p53,and induces programmed apoptosis of colon cancer cells.Serum LC-MS analysis of nude mice in the model group,low-dose group,and high-dose group:the aggregation of each group was obvious,and five different carbohydrate metabolites were screened out:Mannose,Glucose,Arabinose,Fucose,Fructose and multiple signal pathways such as:ABC transporter pathway,galactose metabolism pathway.Conclusion:1)Guzheng Xiaoai Decoction can reduce the energy source of colonrectal cancer cells from the glucose metabolism pathway,improve the surrounding acidic environment,and down-regulate related proteins to play an anti-tumor effect.2)Under the action of Guzheng Xiaoai Decoction,the activation of AMPK pathway can also interfere with the process of glucose metabolism of colonrectal cancer cells and induce cell apoptosis.3)LC-MS analysis indicated that the different glucose metabolites under the action of Guzheng Xiaoai Decoction were Mannose,Glucose,Arabinose,Fucose,and Fructose.Signal pathways include the ABC transporter pathway,the galactose metabolism pathway and so on.