Study On The Anti Colorectal Cancer Mechanism Of Fufangchangtai Based On Gut Microbiota

Objective:This study summarized the clinical experience of medical experts in the treatment of colorectal cancer(CRC),and provides background support for the mechanism research of Fufangchangtai(FFCT).The anti CRC mechanism of FFCT was discussed from the perspective of gut microbiota(GM).The effect of FFCT on host GM,and the effect of GM on FFCT metabolism were discussed respectively.Based on the interaction between FFCT and GM,the active monomer of FFCT was excavated.A further study on clarifying the anti-tumor mechanism of active monomer was performed.The study provided an experimental support for adjusting the GM of patients with CRC,optimizing the prescription of FFCT and exerting far greater anti-tumor effect of FFCT.Methods:(1)The clinical experience of 18 famous medical experts in the treatment of CRC was screened from the literature database.Based on data mining technology,the prescriptions,pathogenesis and treatments of CRC were summarized by using the methods of frequency analysis,cluster analysis and association rule analysis,so as to find the general rules and methods of CRC in clinic.A common analysis was made to discuss the prescription idea of FFCT.(2)CT26-luc colon cancer cells were cultured and injected subcutaneously into mice.Tumor bearing mice were treated with different concentrations(0.65 mg/g,1.3 mg/g,2.6 mg/g)of FFCT.The weight and tumor volume of mice were recorded;the T lymphocytes(CD3+CD4+,CD3+CD8+,MAIT)in blood,spleen and tumor of mice were determined using flow cytometry;To find out significant landmark GM and applicable drug dose,the GM of mice treated with different concentrations of FFCT were determined by the 16S rRNA gene sequencing.(3)Mouse models with none,normal or CRC GM were established by fecal microbiota transplantation(FMT)before intervented with FFCT.The T lymphocytes(CD3+CD4+,CD3+CD8+)in blood and spleen of mice were detected by flow cytometry to evaluate the immune status;metabolites in mouse serum were detected by Liquid chromatography-mass spectrometry(LC-MS),so as to evaluate the effect of different GM on immunity and drug metabolism.(4)The tumor bearing mice were intervented by FFCT,and the tumor volume was detected by Small living animal imaging technology;metabolites in mouse serum were detected by LCMS;the active monomers of FFCT were explorebased on the differential metabolites between FFCT-treated group and FFCT-treated tumor group.(5)CT26-luc colon cell lines were cultured and treated with different doses of Matrine(0μg/mL,200 μg/mL,400 μg/mL and 800 μg/mL).The cell morphology was observed under microscope,and the cell proliferation was detected using CCK8 and colony formation assay;the cell migration and invasion was assessed using transwell;the cell apoptosis was determined by Hoechst/PI assay.To explore the anti-tumor mechanism of Matrine,PI3K/Akt/mTOR signal pathway were further verified using RT-qPCR and Western blot were used to verify the effect of Matrine on based on KEGG pathway analysis.Results:(1)The clinical experience of famous medical experts in the treatment of CRC was summarized.It was found that the commonly used drugs were Coix seed,Atractylodes macrocephala,Poria cocos,Licorice,Tangerine peel,Radix Paeoniae Alba,Astragalus membranaceus,Pinellia ternata,Codonopsis pilosula and Hedyotis diffusa,most of which were tonic drugs.Warm drugs were used to avoid severe damage on Qi.Drugs were mostly attributed to the spleen,liver and stomach meridians,so as to strengthen the spleen Qi,regulate the liver Qi and protect the stomach Qi.It was a pervasive notion that deficiency in origin and excess in superficiality was the basic pathogenesis of CRC.Deficiency in origin mainly referred to asthenia of both the spleen and the Qi,and excess in superficiality mainly referred to pathogenic blood stasis,dampness and poison.Attention on treatments should be paid to strengthening the spleen and kidney,consolidating the foundation,supplemented by detoxification,removing blood stasis,regulating Qi and others.The summarized clinical experience is consistent with the formula idea of FFCT,which reflects the universal applicability of the experience.(2)It was found that compared with the control group(CT26 group),high-dose FFCT could significantly increase the expression of CD3+CD4+T lymphocytes in blood and CD3+CD8+T lymphocytes in spleen(P<0.05).Compared with the none tumor-bearing state(PBS group),the tumor-bearing state will cause GM disorder in mice,which showed the significantly increased Bacteroides abundance(55.45%vs 32.23%,P<0.05)and decreased Firmicutes abundance(61.32%vs 39.32%,P<0.05).After intervented with FFCT,Bacteroides abundance decreased and Firmicutes abundance increased,suggesting that FFCT could remedy the disorder of GM.(3)It was found that CD3+CD8+T cells in the spleen of GM clearance mice and CRC GM colonized mice were lower than that of healthy GM colonized mice,suggesting that the GM influenced the immune level.Serum metabolome detection showed that there was 8hydroxyguanine,a marker metabolite of oxidative damage in CRC GM colonized mice.After intervented with FFCT,the efficacy components of FFCT in serum such as Ganosporeic acid A,m-Coumaric acid and Jasmonic acid were affected,and these differential metabolites were mainly enriched in the primary bile acid biosynthesis pathway.(4)There was no significant difference in tumor volume with and whiout FFCT treatment.The serum metabolites of mice with or without tumor were detected followed FFCT treated.With the presence of tumor,the efficacy components of FFCT in serum such as Matrine,Isogingerenone B and Armillaripin were affected,and Matrine is the clear active monomer of FFCT among these differential metabolites.(5)400 μg/mL and 800 μg/mL Matrine solution could significantly inhibit the proliferation of CT26-luc colon cancer cells and induce late apoptosis of tumor cells.When the concentration of Matrine solution was greater than or equal to 200 μg/mL,it could significantly inhibit the migration and invasion of tumor cells.The expressions of PI3K and Akt at mRNA level were both significantly increased at concentration of 400 μg/mL and 800 μg/mL.But,the expressions of proteins(PI3K,p-PI3K,Akt,p-Akt,mTOR,p-mTOR)in PI3K/Akt/mTOR signaling pathway in CT26-luc cells were significantly decreased(P<0.05).Conclusion:The anti-tumor mechanism of FFCT may be related to remedying the GM dysbiosis,as the corrected GM was conducive to the absorption of Matrine into the blood.Matrine was the active monomer of FFCT,which could inhibit the progress of CRC cells by down-regulating the expression of PI3K/Akt/mTOR signal pathway.