Study On The Effect And Mechanism Of Centipede Small Molecule Peptides On Activating TRAIL Receptor And Inducing Apoptosis Of Liver Cancer Cell

Objective: Scolopentide was synthesized to verify its anti-liver cancer effect in vivo and in vitro,and explore its anti-liver cancer mechanism.Methods:1.Biosynthesis and detection of scolopentide small molecule peptides:scolopentide was biosynthesized according to the peptide sequence and molecular formula matched by Mascot Science in the previous period.The purity of Scolopentide was detected by high performance liquid chromatography and the molecular weight was detected by mass spectrometry to ensure that the synthesized Scolopentide met the requirements.2.Anti-tumor effects of biosynthetic Centipede small molecule peptides: In vitro experiments,CCK-8 method was used to detect the effect of scolopentide synthesis on the activity of hepatocellular carcinoma Hep G2 cells.Different concentrations of synthetic peptides(50,100,150,200 μg/m L)were treated on hepatocellular carcinoma Hep G2 cells for 6h,12 h,24h,48 h,and the cell inhibition rate was observed and calculated.Morphological effects of scolopentide on Hep G2 cells were observed by Hoechst staining.Hep G2 cells were treated with scolopentide(100 μg/m L,about 0.098 μmol/m L)for 6h,12 h and 24 h,and morphological changes were observed respectively.In vivo experiment,subcutaneous tumor model of liver cancer was constructed and divided into two groups: scolopentide group(500mg/kg/d)and vehicle group(normal saline of equal volume).The patients were injected intraperitoneally once a day for 14 consecutive days.The subcutaneous tumor volume of transplantation was measured every other day,and the tumor weight was measured after death.3.Antitumor mechanism of biosynthetic centipede small molecule polypeptides : The effect of scolopentide on hepatoma cell apoptosis was detected by flow cytometry.Scolopentide and death receptors DR4 and DR5 were computer simulated for molecular docking,and the binding tightness was analyzed.RT-PCR and WB experiments were performed on the tumor in vivo to detect the expression of DR4,DR5,FADD,caspase-8 and caspase-3 of the key indicators of TRAIL pathway.The expressions of TRAIL pathway inhibitory proteins c-FLIP and XIAP were detected.The expression of Cyto-c and Bax/Bcl-2,the key indexes of mitochondria-dependent apoptosis pathway,and the content of ROS in tumor were detected,and the relationship between scolopentide synthesis and the two apoptosis pathways was analyzed.Results:1.The purity of scolopentide synthesized was 98.014% by HPLC,and its molecular weight was 1018.8Da by mass spectrometry,both of which were similar to the extract.2.In vitro experiments,CCK8 experiment suggested that scolopentide with different concentrations could inhibit proliferation,and the inhibition rate showed concentration-dependent characteristics(P ≤0.001).The inhibitory effect was strongest when the treatment time was12 h,and there was no significant difference between 24 h and 48 h.Hoechst 33342 staining indicated that cytoplasmic bright staining and nuclear pyretosis were observed in Hep G2 cells treated with scolopentide for 12 h and 24 h,the most obvious at 12 h.Synthetic peptides may induce cell apoptosis.In vivo experiments,the tumor volume of subcutaneous liver cancer transplantation in scolopentide group and vehicle group both increased gradually(P=0.000 3),but the growth rate was slower in scolopentide group(P=0.000 1).The average tumor weight in scolopentide group was smaller than that in vehicle group(P=0.0 022).3.Flow cytometry indicated that scolopentide extracted could induce apoptosis of Hep G2 cells in a dose-dependent manner.Computer simu lated molecular docking experiment indicated that the binding energy of DR4 and scolopentide is-10.4 kcal/mol,and the protein binding sites are LYS145,ALA201,PRO219,CYS190,etc.The binding energy of DR5 to scolopentide is-7.1 kcal/mol,and the protein binding sites are TRP74,SER73,ER149,TYR148.It is known that scolopentide is closely bound to DR4 and DR5,and is more closely bound to DR4 than to DR5.RT-PCR and WB experiments indicated that the m RNA and protein expressions of key indexes DR4,DR5,FADD,caspase-8 and caspase-3 of TRAIL pathway were significantly up-regulated under scolopentide synthesis,but gradually decreased.The m RNA expression of TRAIL pathway inhibitory protein c-FLIP was slightly inhibited(P=0.0272),but XIAP was slightly increased without statistical significance(P=0.1125).Even when ROS was upregulated statistically(P=0.0286),m RNA and protein expressions of Cyto-c,a key indicator of mitochondrial apoptosis pathway,were only slightly upregulated statistically(P=0.2574;P=0.1000),m RNA expression of Bax/Bcl-2 was also insignifican tly up-regulated(P=0.1674).Conclusion:1.The purity and molecular weight of biosynthetic scolopentide meet the requirements.2.Scolopentide has anti-liver cancer effect in vivo and in vitro.3.Scolopentide can affect the TRAIL pathway and promote the caspase cascade expression to induce tumor cell apoptosis by activating the death receptors DR4 and DR5,which is more related to the independent mitochondrial apoptosis pathway.