Experiment Study On The Inhibition Of Thyroid Follicular Carcinoma Proliferation By Polyphyllin Ⅰ

Objective:Follicular thyroid carcinoma（FTC）has a high lethality rate,and there are few studies on FTC at home and abroad,and the clinical treatment is relatively simple.This experiment is based on RNA sequencing results,related pathway proteins and m RNA detection,and investigates the mechanism of inhibition of FTC proliferation by Polyphyllin I,and provides an experimental basis and theoretical basis for the clinical treatment of FTC.Methods:1.Extraction and quality control of mixtures such as PolyphyllinⅠ.In this study,the mixture of PolyphyllinⅠwas extracted by reflux add ultrasound assisted extraction with Rhizoma Paridis Yunnanensis as the raw materials,and the quality control of PolyphyllinⅠwas carried out by RP-HPLC method.2.Effect of the PolyphyllinⅠon the proliferation of FTC-133 cellsThe growth curve of FTC-133 cells was plotted using the tetramethylazole blue（MTT）method,and the optimal inoculum density was selected.Six different concentrations（0,0.25,0.5,1,2 and 4μM）of the main rebaudiosides（Polyphyllin I,Polyphyllin II,Polyphyllin VI and Polyphyllin VII）were set.And the effect of the four compounds on the proliferation of FTC-133 cells was detected using the MTT method,and the better inhibitory effect of Polyphyllin I.The MTT method was used to detect the effect of the four compounds on the proliferation of FTC-133 cells,and the one with the better inhibitory effect was selected as PolyphyllinⅠ.The MTT method was used to detect the effect of different concentrations of PolyphyllinⅠ（0,0.25,0.5,1,2,4μM）and different times（24h,45h,72h）of the same administration concentration on the proliferation of FTC-133 cells.3.RNA sequencing analysis of the effect of Polyphyllin I on FTC-133Three experimental groups were set up:FTC-133 cell group（F1,F2,F3）,FTC-133+Polyphyllin I（1μM）group（FTC-133+Polyphyllin I 1,FTC-133+Polyphyllin I 2,FTC-133+Polyphyllin I 3）,Nthy-ori3-1 cell group（N1,N2,N3）,total RNA was extracted from each sample.After the samples were tested RNA sequencing analysis was performed,and differential genes were screened for GO and KEGG analysis according to the criteria（Fold Change）>1.5,P<0.05),and QRT-PCR was used to verify the expression of genes related to the enrichment pathway.4.Inhibitory effect of Polyphyllin I on the proliferation of FTC-133 cellsFTC-133 cells at logarithmic growth stage were taken and given complete medium containing different concentrations（0.25μM,0.5μM and 1μM）of Polyphyllin I 24 h after inoculation,lysate control group was given DMSO complete medium solution at a concentration of 1μM and negative control group was given normal complete medium.After48h of administration,cells were collected for total protein extraction,protein concentration was detected and split protein solution was prepared.The effect of different concentrations of PolyphyllinⅠon protein expression related to the enrichment pathway was investigated by sampling,electrophoresis,membrane transfer,incubation of primary and secondary antibodies,and protein bands were calculated and analyzed after development.5.Acute toxicity study of Polyphyllin I administered intraperitoneally to miceSeven experimental groups were set up with different concentrations of PolyphyllinⅠ（0 mg/kg,3.13 mg/kg,6.25 mg/kg,12.5 mg/kg,25 mg/kg,50 mg/kg,100 mg/kg）,and the maximum tolerated dose（LD₀）and absolute lethal dose(LD₁₀₀)were calculated by the number of mouse deaths.);in between the two values,different dosing concentrations（13.1mg/kg,16.4 mg/kg,20.5 mg/kg,25.6 mg/kg,32 mg/kg,40 mg/kg）were set in a gradient（0.8）and the drug was administered intraperitoneally in a single dose,and the mice were closely observed for 14 days.The LD₅₀ was calculated according to the formula.A dosing group and a solvent control group were set up according to the dosing concentration at which the mice started to show toxic reactions during the previous experiments,with half of the males and half of the females administering the drug intraperitoneally once a day for 5 days.6.Effect of Polyphyllin I on FTC-133 cell transplantation tumor model in nude miceHealthy SPF-grade Balb/c female nude mice were inoculated with FTC-133 cells at a density of 1×10⁷ cells/m L in the right axilla for the establishment of a transplantation tumor model.The animals were grouped and administered at a concentration of 1/10 of the LD₅₀dose by intraperitoneal injection once a day for 14 days.The animals were administered intraperitoneally once a day for 14 days,and their body weight and tumour volume were measured every two days.The effect of the drug on the volume of transplanted tumours and the body weight of FTC-133 cell nude mice was observed.Results:1.Extraction and quality control of mixtures such as PolyphyllinⅠ.The extraction of the mixture and the determination of the main compounds in the Rhizoma Paridis Yunnanensis showed that the masses of PolyphyllinⅠ,Ⅱ,VI and VII showed good linearity in the range of 0.404-2.828 ug,0.0945-0.6615 ug,0.0788-0.5516 ug and0.406-2.842 ug,respectively,and the correlation coefficients were all greater than The mean recoveries were 101.57%,100.09%,105.96%and 99.53%respectively,and the RSD values were in accordance with the requirements.2.Effect of the Polyphyllin I on the proliferation of FTC-133 cellsThe growth curve was plotted and the inoculation density of FTC-133 cells was determined to be 8.89×10⁴ cells/m L;The proliferation of FTC-133 cells was inhibited by compounds such as Polyphyllin I.etc.According to the experimental results,the tested drug was chosen to be Polyphyllin I.The inhibition rate of the proliferation of FTC-133 cells was calculated by the different concentrations of Polyphyllin I acting on FTC-133 cells,Determine the final administration of the drug at low,medium and high concentrations of0.25,0.5 and 1μM,respectively,based on the IC₅₀.;And the inhibition of FTC-133 cell proliferation by Polyphyllin I at different dosing concentrations did not show a time-dependent effect,so the later dosing time was set to 48h.3.RNA sequencing analysis of Polyphyllin I action on FTC-133Sequencing results showed that:tissue Apelin signaling pathway,AGE-RAGE signaling pathway,TNF signaling pathway,etc.are related to the development of FTC,cancer-related pathways mainly in PI3K-AKT-m TOR signaling pathway,MAPK-ERK signaling pathway,etc.may be related to the treatment of thyroid follicular carcinoma by PolyphyllinⅠ.Applying QRT-PCR to validate the pathways,the results showed that the expression of Bax,Caspase-9 and PTEN were up-regulated,and the expression of Bcl-2,PI3K,AKT1,m TOR,MEK and ERK were down-regulated.4.Inhibitory effect of Polyphyllin I on the proliferation of FTC-133 cellsFrom the protein expression level,all of Polyphyllin I could promote the apoptosis of FTC-133 cells.Polyphyllin I could make the expression of Bax,Caspase-9 and PTEN up-regulated;PI3K,KAT1,p-PI3K,p-KAT,MAPK,MEK and RAS down-regulated in FTC-133 cells.5.Acute toxicity study of Polyphyllin I in mice administered intraperitoneallyThe LD₁₀₀ in mice was 50.0 mg/kg and the LD₀ was 12.5 mg/kg after the intraperitoneal administration of Polyphyllin I.The LD₅₀ was calculated as 21.4 mg/kg according to the formula.6.The effect of PolyphyllinⅠon FTC-133 cell transplantation tumor model in nude miceAccording to the change of body weight,the nude mice in the model group gradually lost weight with the extension of time,while the body weight of the administered drug remained more stable;at the beginning of the administration,there was no significant change in the tumor volume of the nude mice,and after the 10th day,the tumor volume of the model group grew faster than that of the administered drug group;suggesting that Polyphyllin I may have some inhibitory effect on the growth of the tumor of FTC-133 cell nude mice.Conclusion:1.1.Determination of the content of compounds such as Polyphyllin I in Rhizoma Paridis Yunnanensis,which can provide reference for its quality control and research of production process.2.all the compounds such as Polyphyllin I can inhibit the proliferation of FTC-133 cells to different degrees.3.that Polyphyllin I can inhibit the proliferation of FTC-133 cells by a mechanism related to apoptosis,and that Polyphyllin I can affect the proliferation of this cell by inhibiting the PI3K-AKT-m TOR and MAPK-ERK signaling pathways.4.the LD₅₀ of Polyphyllin I for acute toxicity test in mice administered intraperitoneally was 21.4 mg/kg.5.The mechanism of action of Polyphyllin I on FTC-133 cell transplanted tumor in nude mice is related to the inhibition of the proliferation of FTC-133 cells,It may provide some reference for clinical research in the treatment of FTC.