Study On The Substantial Basis And Mechanism Of Caulis Spatholobi Concentracted Decoction In Treating Colorectal Cancer

Study Objective:At present,many studies have confirmed that Caulis Spatholobi or Spatholobus Composite has an inhibitory effect on multiple tumors or enhance the effect of adjuvant chemotherapy drugs.Therefore,this experiment intends to study the effects of Caulis Spatholobi concentrated decoction drug serum?the main active ingredient glycyrrhizin liquiritigenin and isoliquiritin and its chemically synthesized derivatives in vitro on the proliferation and apoptosis of colorectal cancer,and of ILQG-2d on colon cancer HCT116 cells in vivo and in vitro on the proliferation and apoptosis of HCT116 cells.And further discuss its related mechanisms to provide more theoretical basis for better treatment of colon cancer through flow cytometry,Western Blot,Real Time PCR,Transwell invasion and migration,plasmid transfection and other experimental methods.Research Method:in vitro1.To investigate the effects of Caulis Spatholobi concentrated decoction drug serum on the proliferation of colorectal cancer through the CCK8experiment.Screen the appropriate serum concentration of Caulis Spatholobi concentrated decoction drug serum,and observe the effect of different concentration of Caulis Caulis Spatholobi concentrated decoction drug serum on the morphological changes of HCT116 cells under an inverted phase-contrast microscope.Use flow cytometry to observe the effect of Caulis Spatholobi concentrated decoction drug serum on the apoptosis and cell cycle of HCT116cells.Use Western Blot to observe the Caulis Spatholobi concentrated decoction drug serum on the apoptosis-related proteins of HCT116 cells.2.Use MTT to detect the inhibitory effect on cell proliferation against HCT116,HT29,DLD-1 and SW480 cells at different time under different concentrations of liquiritigenin and isoliquiritin.Choose HCT116 as sensitive cells,and detect the proliferation inhibitory effect of various isoliquiritin chemically synthesized derivatives on HCT116 by MTT.Screened out ILQG-2d as the most toxic drug against HCT11 cells,and it was chosen for the further mechanism study.3.Observe the effect of ILQG-2d on the morphological changes of HCT116cells with an inverted phase contrast microscope.Observe the effect of ILQG-2d on the apoptosis and cell cycle of HCT116 cells by flow cytometry.Wound healing assay and transwell invasion and migration was used to detect the effect of ILQG-2d on the metastasis of HCT116 cells.Western Blot(WB)was used to detect the effect of ILQG-2d on protein expression related with HCT116migration,apoptosis,cell cycle and the expression of related proteins such as PI3K/AKT signaling pathway.Real Time-PCR(RT-PCR)was used to detect the effect of ILQG-2d on the expression of apoptosis-related genes in HCT116 cells.Use the p53 inhibitor PFT-?and sh RNA p53 to block the p53 signaling pathway,and observe the effect of ILQG-2d on the apoptosis of HCT116 cells with down-regulated or inhibited expression of p53 protein.In vivoUse human colon cancer HCT116 cells to establish colon cancer nude mice transplanted model,which is divided into Intraperitoneal injection of positive drug group(5-Fu group)and model group,solvent control,ILQG-2d high,medium,and low groups,by intragastric administration and observed the inhibitory effect on the growth of subcutaneously transplanted tumors in nude mice.Use the HE staining method to observe the effect of ILQG-2d on the morphological changes of the transplanted tumor in nude mice and TUNEL staining method to observe the effect of ILQG-2d on the apoptosis of transplanted tumor cells in nude mice.Western Blot experiment and immunohistochemistry experiment were used to detect the expression of apoptosis-related proteins.Research Result:in vitro1.The results of CCK8 experiment showed that the 5%,10%and 20%of the high-dose serum of the Caulis Spatholobi concentrated decoction had most obviously inhibitory effect on HCT116 cells.The results of the Annexin-V FITC/PI staining experiment showed that the serum of Caulis Spatholobi concentrated decoction could induce cell apoptosis,and as the concentration increases,the apoptosis rate gradually increased,showing a good dose-effect relationship.The results of cycle analysis by flow cytometry showed that the medicated serum of Caulis Spatholobi concentrated decoction can induce cell cycle arrest in G2/M phase.The results of Western Blot experiment showed that the expression of apoptosis protein Bax in the serum group of Caulis Spatholobi was increased compared with the control group,and the expression of anti-apoptosis protein Bcl-2 was decreased compared with the control group.2.The results of MTT showed that liquiritigenin and isoliquiritin were the most toxic to HCT116 cells,showing a good concentration and time dependence,but the IC₅₀ value is relatively high.Through the MTT assay,the IC₅₀ value of the inhibitory effect of various chemically synthesized derivatives of isoliquiritin on HCT116 was analyzed,and it was found that the IC₅₀ value of the chemically synthesized derivatives of ILQG-2d on HCT116 cells was the lowest,the 24 h and 48 h IC50 values were 9.429?M and 2.567?M,respectively.ILQG-2d and HCT116 were selected as drugs and cells for subsequent experimental mechanism research.3.It was found that compared with the blank control group,the cell morphology of HCT116 gradually changed,the gaps between the administered cells increased,the arrangement was disordered,the antennae decreased,and the morphology gradually constricted and became rounded,Some cells basically lost their original shape and floated.The results of wound healing assay and transwell invasion and migration experiments showed that compared with the control group,ILQG-2d could significantly inhibit the migration and invasion of HCT116 cells.The results of the Annexin-V FITC/PI staining experiment showed compared with the control group,different concentrations of ILQG-2d(6.25,12.5,25?M)against HCT116 cells for 24 hours could induce cell apoptosis,and as the dose of ILQG-2d increased,the rate of apoptosis gradually increased,showing a good dose-effect relationship.The results of cell cycle analysis by flow cytometry showed that the effect of ILQG-2d on the cell cycle of HCT116 was manifested in the G2/M phase.Western Blot(WB)experiment results showed:ILQG-2d could increase the expression of E-Cadherin,p53,p21,Cleaved-PARP,Bax,BAK,Caspase-9,Cleaved-caspase-9,and Caspase-3,and decrease MMP-3,MMP-9,Bcl-2,Cyclin-D1,Cyclin-B1,p-PI3K,p-AKT expression.Real Time-PCR(RT-PCR)results showed that:ILQG-2d up-regulated the pro-apoptotic Bax m RNA expression level,down-regulated the Bcl-2 m RNA expression level to decrease.It was observed that the inhibitory effect of ILQG-2d against HCT116 cells was decreased by the blocking of p53 signaling pathway.in vivoIt was found that compared with the model group,the 5-Fu group and ILQG-2d treatment group has significantly reduced both tumor volume and tumor weight,indicating that the positive drug group and the ILQG-2d treatment groups could inhibit the growth of subcutaneous xenograft tumors in nude mice.The HE and TUNEL staining showed that ILQG-2d could inhibit the growth of tumor cells in nude mice and increase the rate of apoptosis in tumor tissues.The immunohistochemical(IHC)staining showed that the expression of p53,PTEN,and Bax in the middle-dose group,high-dose group,5-Fu group was significantly increased compared with the model group.Western Blot showed that the expressions of Caspase 3,Bax,p53,and p21 were higher than those in the model group,further verifying that the drug could induce apoptosis in vivo.Conclusion:1.The Caulis Spatholobi concentrated decoction and its active ingredients,liquiritigenin,isoliquiritin,can inhibit the proliferation of colorectal cancer in a concentration-time dependent manner,and the anti-tumor mechanism may be related to induce apoptosis of cancer cells and G2/M cell cycle arrest.Among those isoliquiritin chemically synthesized derivatives,ILQG-2d has a significant inhibitory effect on the proliferation of HCT116 cells in vivo and in vitro by inducing tumor cell apoptosis,blocking the G2/M phase of the cell cycle,and inhibiting tumor cell metastasis.Its mechanism of action may be achieved by regulating PI3K/AKT or activating the p53 pathway.