The Study On Separated Recipes Of Tianma Granules And The Effedt Of The Optimized Recipes On PI3K/Akt/mTOR Singaling Pathway In Colorectal Cancer

Objective:In order to Optimizate of formula composition of Tianma granules（TMG）,the study on separated recipes of Tianma granules was carried out by orthogonal experimental design.To observe the antitumor effects of Tianma granules optimized recipes（OR）in vivo and vitro,and to explore the role on PI3K/Akt/mTOR signaling pathway.Method:1.Experimental study on Separated Recipes of TMG:The 17 traditional Chinese medicines of TMG and 2 doses（original dose and 0 dose）were established L20(2¹⁷)orthogonal table.And prepared 20 groups medicated serum.The optical density（OD）of colorectal cancer cell HCT8,HCT116,HT29,CoLo320,SW480,SW620,which were intervened with 5%,10%,20%medicated serum for 24h,48h,72h,were detected by CCK-8 method.The proliferation inhibition rates of the 6 kinds colorectal cancer cell were analyzed by variance analysis.2.Experimental study on OR in vitro:SD rats were prepared medicated serum by continuing intragastric administration for 7 days with using 2.5 times,5 times,10times of routine dose of OR and 10 times of routine dose of the original.The OD of colorectal cancer cell HCT8,HCT116,HT29,CoLo320,SW480,SW620,which were treated with 5%,10%,20%medicated serum for 24h,48h,72h,were detected by CCK-8 method.So The sensitive cell lines,medicated serum concentration and action time of the best proliferation inhibition were screened.The HCT116 cells,which were affected with 20%medicated serum for 24h,were used to be observed cells morphological changes,and detected apoptosis and cell cycle with flow cytometry.3.Experimental study on OR in vivo:We established xenografts tumor nude mice model of colorectal cancer cell HCT-116.And these xenografts tumor nude mice were respectively given intragastric administration with the original,low dose,medium dose and high dose of OR,and purified water for 4 weeks.From the day of given intragastric administration,the long and short diameter of tumor in nude mice were measured by vernier caliper every 4 days and the tumor volume was calculated to draw the tumor growth curve;The xenografts tumor nude mices were killed after28 days.The xenografts tumor volume and weight were measured.The expression of PI3K/Akt/mTOR signaling pathway protein of tumor tissues was detected by Western blot method;Tumor tissues HE staining were used to observe and compare the inhibition of tumor growth of the original,OR and purified water in vivo.Result:1.Experimental study on Separated Recipes of TMG:The centipede,scorpion,lobelia,amur cork-tree,burreed,rhubarb,bile arisaema,seaweed,milkvetch root and common yam rhizome played major roles in TMG on the proliferation inhibition of 6colorectal cancer cells（all P<0.05）,and the centipede was the most important in TMG（P=0.001）.However,the paris root,zedoary,Prunella,angelica,codonopsis,plantain seed and hemp seed have no significant effect in TMG on the proliferation inhibition of colorectal cancer cells（all P>0.05）.2.Experimental study on OR in vitro:The proliferation inhibition rate of HCT116 cell was significantly higher than that of HCT8,HT29,Colo320,SW480 and SW620（P<0.05）.HCT116cell,which was affected with 20%medicated serum for24h,could gotten the best proliferation inhibition rate.Compared with the control group,the HCT116 cells were affected with 20%medicated serum became round,light transmittance difference,some cells irregular morphology,and some died cells floated in culture medium;With increasing dose of OR,the HCT116 cells morphology was more and more irregular,transparency was getting worse,the died cells gradually increased,and adherent cells more and more sparse.There were significant differences in the apoptosis rate between all the affected groups and the control group（all P<0.01）;There was no significant difference of the apoptosis rate between the high dose group of OR and the original group（P>0.05）,but those were significantly higher than that of low and medium dose group of OR（P<0.01）;There were no significant differences of the apoptosis rate between low,medium and high dose group of OR（P<0.01）.The number of HCT116 cells in G0/G1 phase of the high dose group of OR and the original group had no significant difference（P>0.05）,and were significantly more than other groups（P<0.05）;The number of cells in S phase was no significant difference between the high dose group of OR and the original group（P>0.05）,and were significantly less than the other groups（all P<0.05）.The cells number in G2 phase were no significant differences between all groups（P>0.05）.3.Experimental study on OR in vivo:The xenografts tumor volume were compared between each group.The control group and the low dose group of OR had no significant difference（P>0.05）,but significantly more than other groups（all P<0.05）.The low dose group and middle dose group of OR had no significant difference（P>0.05）,but significantly more than other administration groups（all P<0.05）;The differences between the 3 groups of the original group,medium dose group and high dose group of OR were no significant（all P>0.05）.There was statistical significance of the drug group and dosing time interaction（F=77.659,P<0.01）;（2）The tumor weight were compared between each group.The tumor weight of each administration group was significantly lower than the control group（all P<0.01）;The tumor weight of the high dose group of OR had no significant difference with the original group（P>0.05）,but those were significantly lower than those of other groups（P<0.01）.The tumor relative growth rates were compared between each treated group.The differences between the low dose group of OR and the others group were statistically significant（all P<0.05）;The differences between the 3 groups of the original group,medium dose group and high dose group of OR were no significant（all P>0.05）;There was statistical significance of the drug group and dosing time interaction（F=2.038,P<0.05）.The expression of PI3K/Akt/mTOR signaling pathway protein in tumor tissue showed:the differences between each treated group and control group were statistically significant（all P<0.01）;There was no significant difference between the high dose group of OR and the original group（P>0.05）;The differences between the 3 groups of OR were significant（all P<0.01）.（5）The xenografts Tumor tissue HE staining showed:the necrotic area in all the treated groups;With the dose increased of OR,more and more apoptotic cells.The xenografts Tumor tissue of the high dose group of OR and the original included a large number of vacuoles.And there were a large number of inflammatory cell infiltration in the border areas of necrosis and tumor tissue.Conclutions:The OR included centipede,scorpion,lobelia,amur cork-tree,burreed,bile arisaema,seaweed,milkvetch root,common yam rhizome and rhubarb.OR inhibited the proliferation of colorectal cancer cells,induced the apoptosis of HCT-116 cells and blocked the cell cycle in G0/G1 phase。OR inhibited the growth of xenografts tumor of HCT116 cell in nude mice.It may be related to down regulating the expression of PI3K/Akt/mTOR signaling pathway protein.There was no significant difference in the antitumor effect between OR and the original in vivo and vitro.