Experimental Study Of Ursolic Acid In Prunella Vulgaris Extract Inhibiting The Proliferation Of Thyroid Papillary Carcinoma Cell TPC-1

Objective:Thyroid cancer?TC?is a malignant tumor that occurs in the thyroid and is the most common malignant tumor of the endocrine system,Papillary Thyroid Carcinoma?PTC?originates from the differentiated thyroid cancer of the thyroid parenchyma and is the most common,The occurrence and development of PTC is a complicated pathophysiological process.The previous research results of the research group showed that Ursolic Acid?UA?in Prunella vulgaris extract can down-regulate the expression of oncogene PI3K,AKT and mTOR,and induce the apoptosis of human thyroid papillary carcinoma cells?TPC-1?.This experiment will explore the inhibitory effect of UA on TPC-1 cell proliferation based on the p53/MAPK signaling pathway,and provide experimental basis and reference for the clinical application of UA and the development of new drugs.Method:1 Effect of UA in Prunella vulgaris extract on TPC-1 cell proliferation1.1 Effect of Prunella vulgaris water,ethanol and ethyl acetate extracts on TPC-1 cell proliferationAfter crushing the ears of Prunella vulgaris,add purified water,95%ethanol to soak,boil,filter,concentrate,and sterilize,and take a part of the ethanol concentrate to extract with ethyl acetate,then concentrate and sterilize to obtain Prunella vulgaris water,ethanol,acetic acid Ethyl acetate extract,the three extract concentrations were prepared as 75,37.5,18.75,9.375,4.6875,2.34375 mg/mL.The tetramethylazo blue method?MTT method?was used to observe the proliferation of the three extracts of Prunella vulgaris after different concentrations of TPC-1 cells for 48 h,and the corresponding IC₅₀0 values were calculated.1.2 Determination of UA,oleanolic acid?OA?,corosolic acid?CA?,maslinic acid?MA?,betulinic acid?BA?in the ethyl acetate extract of Prunella vulgarisA C18-column?4.6 mm×250 mm,5?m?,flow rate:1.0 mL/min,column temperature:25?,UV detection wavelength:210 nm,mobile phase:methanol-acetonitrile-0.1%phosphoric acid?80:5:15?Chromatographic conditions for content determination.1.3 Extraction and content determination of UA in Prunella vulgaris extractPrunella vulgaris was crushed,soaked with 95%ethanol,heated,filtered,and concentrated to an extract,dispersed with water,extracted with ethyl acetate,extracted three times,combined ethyl acetate liquid,and concentrated under reduced pressure to a paste.The paste was dissolved in methanol,and the purity was detected by HPLC-ELSD.The detection conditions were mobile phase:methanol:water=98:2,flow rate:1 mL/min,detector:ELSD,chromatography column:C18?4.6 mm×250mm,5?m?.The sample was loaded,separated and purified by preparative liquid chromatography,collected,and concentrated to a powder to obtain a UA sample.Preparative liquid chromatography conditions:chromatograph:varian SD1,column:C18?250 mm×50 mm,10 um?,Flow rate:40 mL/min,mobile phase:methanol:water=95:5.1.4 The effect of UA in Prunella vulgaris extract on the proliferation of TPC-1 and Nthy-ori3-1 cellsDifferent concentrations of UA were used to intervene in TPC-1 and Nthy-ori3-1cells?control group 0?M,experimental group 1.5?M,3?M,6?M,12?M,24?M?,and MTT method was used to observe the effect of UA on TPC-1 and Nthy-Effect of ori3-1 cell proliferation.2 RNA sequencing analysis of PTC in the extract of Prunella vulgarisUsing RNA-Seq sequencing technology for human normal thyroid epithelial cell?Nthy-ori3-1?group,TPC-1 cell group,?UA 3?M+TPC-1,UA 6?M+TPC-1,UA 12?M+TPC-1?Perform transcriptome analysis on the cell group,select differentially expressed genes based on the criteria of Fold Change?1.5 and P value<0.05,and then perform GO and KEGG functional enrichment analysis on these differentially expressed genes.Real-time fluorescent quantitative PCR?QRT-PCR?was used to verify the expression levels of p53/MAPK-related genes p53,Ras,MEK and ERK,TSHR,BRAF,DDR1,PAK4,PKACA,c-Myc,PIK3Ca mRNA in enrichment pathways The accuracy of routing.3 Study on the effect and mechanism of UA in Prunella vulgaris extract on TPC-13.1 UA induces apoptosis in TPC-1 cellsTPC-1 cells in the logarithmic growth phase were selected for the experiment.TPC-1 cells were treated with different concentrations of UA for 48 hours.The flow cytometer Annexin V-FITC/PI double staining method was used to detect the changes in apoptosis.3.2 The effect of UA on TPC-1 cell cycleTPC-1 cells in the logarithmic growth phase were selected for the experiment.TPC-1 cells were treated with different concentrations of UA for 48 h,and flow cytometry?FCM?was used to detect changes in the proportion of cells in each phase of the cell cycle.3.3 The effect of UA in Prunella vulgaris extract on p53/MAPK signaling pathway-related proteins in TPC-1 cellsTPC-1 cells in the logarithmic growth phase were selected for the experiment,using different concentrations of UA intervention cells?negative control group,vehicle control group,UA 3?M,UA 6?M,UA 12?M?,Western blot method detected by UA intervention The expression of TSHR,BRAF,PAK4,P-PAK4,PKAPA,p53,c-Myc protein in TPC-1 cells after 48 h.Result:1 Effect of UA in Prunella vulgaris extract on TPC-1 cell proliferation1.1 Effect of Prunella vulgaris water,ethanol and ethyl acetate extracts on TPC-1 cell proliferationAfter different concentrations of Prunella vulgaris water,ethanol,and ethyl acetate extracts acted on TPC-1 cells,the IC50 values??obtained were:30.473,47.617,and 18.433 mg/mL,that is,Prunella vulgaris ethyl acetate partially inhibited the proliferation of TPC-1 cells.The effect is stronger.1.2 Determination of UA,OA,CA,MA,BA in the ethyl acetate extract of Prunella vulgarisThe UA,OA,CA,MA,BA chromatographic peaks and impurity peaks in the ethyl acetate extract of Prunella vulgaris were well separated.There was no tailing in the chromatographic peaks,and the UA peak area was the largest.1.3 Extraction and content determination of UA in Prunella vulgaris extractThe prepared UA sample was detected by HPLC-ELSD,and the purity was relatively pure.In the linear range,the UA content increased with the extension of ultrasound time,and it was the highest at 60 min.1.4 The effect of UA in Prunella vulgaris extract on the proliferation of TPC-1 and Nthy-ori3-1 cellsThe IC₅₀0 value of TPC-1 cells treated by UA was 9.19±0.35?M by MTT method.The low,medium and high concentrations of UA in subsequent experiments were 3,6,12?M.2 RNA sequencing analysis of PTC in the extract of Prunella vulgarisFrom the KEGG pathway enriched in differentially expressed genes:Compared with the Nthy-ori3-1 cell group,the TPC-1 cell group has a down-regulated pathway including the p53 signaling pathway,and the up-regulated pathway has a MAPK signaling pathway.In comparison,?UA 3?M+TPC-1,6?M+TPC-1,12?M+TPC-1?cell group up-regulated pathways have p53 signaling pathways,down-regulated pathways have MAPK signaling pathways,etc.,the enrichment degree increases with concentration Large and enlarged.QRT-PCR verification of genes in the p53 signaling pathway and MAPK signaling pathway showed that:compared with the negative control group,the expression level of p53 mRNA in the cells of the UA 3,6,12?M administration group was up-regulated?P<0.05?,Ras,MEK,and ERK mRNA and related genes TSHR,BRAF,DDR1,PAK4,PKACa,c-Myc,PIK3Ca mRNA expression levels were down-regulated?P<0.05?,and the p53/MAPK signaling pathway was used as the future research direction.3 The effect and mechanism of UA on TPC-1 in Prunella vulgaris extract3.1 UA induces apoptosis in TPC-1 cellsFlow cytometry results showed that after 48 hours of UA treatment of TPC-1cells,compared with the negative control group,the difference in early apoptosis rate,late apoptosis rate and total apoptosis rate of UA groups with different concentrations were statistically significant?P<0.05?,the rate of apoptosis increases with increasing concentration.3.2 The effect of UA on TPC-1 cell cycleAfter 48 h of different concentrations of UA on TPC-1 cells,the proportion of cells in each phase of the cell cycle changed.Compared with the negative control group,the proportion of S-phase cells increased significantly with the increase of UA concentration,and the difference was statistically significant?P<0.05?.3.3 The effect of UA in Prunella vulgaris extracts on p53/MAPK signaling pathway-related proteins in TPC-1 cellsWestern blot results showed that compared with the negative control group,the vehicle control group p53,TSHR,BRAF,c-Myc,PKACa,PAK4,P-PAK4 and other proteins were not significantly different?P>0.05?;different concentrations of UA administration The p53,TSHR,BRAF,c-Myc,PKACa,PAK4,P-PAK4 and other proteins were all expressed in the group of cells.After 48 hours of UA acting on TPC-1 cells,the expression of p53 protein was up-regulated with the increase of UA concentration.The difference was statistically significant?P<0.05?,the expressions of TSHR,BRAF,PKACa,PAK4 protein were all down-regulated,and the difference was statistically significant?P<0.05?,the expression of c-Myc and P-PAK4 protein were all down-regulated in UA When the concentration was 6,12?M,the difference was statistically significant?P<0.05?.Conclusion:The effect of UA in Prunella vulgaris extract on inhibiting the proliferation of human thyroid papillary carcinoma cell TPC-1 is related to promoting apoptosis,blocking cell cycle in S phase,promoting p53 signaling pathway and inhibiting MAPK signaling pathway.