The Study On The Mechanism Of Apoptosis Of Human Papillary Thyroid Cancer Cell TPC-1 Induced By Ursolic Acid In Prunella Vulgaris L.

Objective:The occurrence and development of papillary thyroid cancer(PTC)is a complex pathophysiological process.The results of the previous research group suggest that ursolic acid(UA)in Prunella vulgaris can upregulate the expression of PTEN in human papillary thyroid cancer cells(TPC-1).This study will further investigate the inhibitory effect of UA on PTC and its related mechanism of action,providing experimental basis for the development of new drugs and clinical application of UA.Method:1.Optimization of Ultrasonic Extraction of UA in Prunella vulgarisBased on the experiments of ultrasonic extraction time,material-liquid ratio,and ethanol volume fraction as single factors,the best technique of ultrasonic extraction of UA in Prunella vulgaris was optimized by response surface methodology.2.Using the maximum dose method(5g/kg.bw),using SPF grade ICR mice,male and female,according to 40mL/kg dosing volume within 24 hours of a single oral acute toxicity test,monitoring Acute toxicity and death of UA in mice.3.RNA sequencing(RNA-Seq)analysis of PTC treated by Ursolic Acid in Prunella vulgarisThe experiments were divided into three groups:human normal thyroid epithelial cells(Nthy-ori3-1)group,TPC-1 cell group,(UA+TPC-1)cell group,three samples in each group.The total RNA of each group was extracted and subjected to RNA-Seq,selection of differentially expressed genes according to a relative expression fold(Fold Change)?1.5,P<0.05.Then,the G0 and KEGG functions of these differentially expressed genes were analyzed.Real-time fluorescence quantitative PCR(QRT-PCR)was used to verify the PI3K,AKT1,mTOR,PTEN and NIS genes in the enrichment pathway PI3K and bcl-2,bax,caspase-3,caspase-8 and caspase-9 in mitochondrial apoptotic pathways.The level of expression further confirms the accuracy of selected pathways.4.Effect and mechanism of UA on TPC-1 cells in Prunella vulgaris4.1 Effect of UA on proliferation of TPC-1 cells in Prunella vulgarisThe Nthy-ori3-1 cells and TPC-1 cells in the logarithmic growth period were used for the experiment.First,the growth curves of these two cells were drawn by four methazo azazolus(MTT)method,and the optimum inoculation density of the two cells was obtained.The two cells were treated with different concentrations of UA(the control group 0?M;the experimental group UA 3?M,UA 6?M,UA 12?M).MTT assay was used to observe the effects of different concentrations of UA and UA on the proliferation of Nthy-ori3-1 cells and TPC-1 cells at different time(24h,48h,and 72h).4.2 Apoptosis of TPC-1 cells induced by UA in Prunella vulgarisTPC-1 cells in logarithmic growth phase were selected for experiment.The cells were treated with different concentrations of UA(negative control group,vehicle control group,UA 3?M,UA 6?M,UA 12?M).The morphologic changes of TPC-1 cells after 48h intervention were observed by inverted microscope;Hoechst 33342-PI double staining fluorescence microscopy was used to observe the changes of apoptosis of TPC-1 cells after UA intervention for 48h;Flow cytometry Annexin V-FITC/PI double staining method was used to detect the apoptosis of TPC-1 cells after UA intervention for 24h,48h and 72h;The expression of bcl-2,bax,caspase-3,caspase-8 and caspase-9 in TPC-1 cells was detected by Western blot.QRT-PCR test was used to detect the expression of bcl-2,bax,caspase-3,caspase-8 and caspase-9 m RNA in TPC-1 cells after UA intervention 48h.4.3 UA causes TPC-1 cell cycle arrest in S phase in Prunella vulgarisTPC-1 cells in the logarithmic growth phase were selected for experiments.Different concentrations of UA were used to intervene cells(negative control group,vehicle control group,UA 3?M,UA 6?M,UA 12?M).Flow cytometry(FCM)was used to detect UA.Intervention at different time(24h,48h,72h)changes in the proportion of cells in each phase of TPC-1 cell cycle.4.4 Effects of UA in Prunella vulgaris on PI3K/PTEN/NIS/AKT/mTOR pathway in TPC-1 cellsTPC-1 cells in logarithmic growth phase were selected for experiments.Different concentrations of UA were used to intervene cells(negative control group,vehicle control group,UA 3?M,UA 6?M,UA 12?M).Western blot was used to detect the expression of p85-PI3K,p110-PI3K,AKT1,p-AKT1(Thr308),p-AKT1(Ser473),mTOR,PTEN and NIS proteins in TPC-1 cells 48 h after UA intervention;QRT-PCR test was used to detect the expression of PI3K,AKT1,mTOR,PTEN and NIS mRNA in TPC-1 cells after UA intervention 48h.5.Effect of UA in Prunella vulgaris on the transplanted tumor model of TPC-1 cells in nude miceThe healthy BALB/c-nu/nu female nude mice were selected and transplanted into right axillary TPC-1 cells.After the tumor model was successfully transplanted,the nude mice were divided into two groups(model group and UA group).Group UA was given 250mg/kg weight,and the model group was treated with the same amount of solvent(sodium carboxymethyl cellulose),and every day was given to the stomach for 14 days.The weight and size of nude mice were measured every 2 days from the beginning of self medication to the end of the experiment.Finally,the inhibition rate of UA on the proliferation of TPC-1cells in nude mice was calculated.Results:1.Optimization of Ultrasonic Extraction of UA in Prunella vulgarisThe optimal ultrasonic extraction process of UA in Prunella vulgaris was as follows:the ratio of material to solution was 1:5,the ultrasonic extraction time was 1.5 h,and the volume fraction of ethanol was 100%.2.The maximum dose of the acute toxicity test of UA mice in 24 hours was 5g/kg.bw.After the mice were given UA,there was no obvious toxicity and death,indicating that the toxicity of UA was small.3.RNA-seq Analysis of PTC treated by Ursolic Acid in Prunella vulgarisAccording to the set criteria,the differential genes between the groups were obtained and the KEGG pathway enriched by these differentially expressed genes was obtained.Compared with the Nthy-ori3-1 cell group,the upregulation pathway of the TPC-1 cell group was:mitochondrial withering Pathway of death and PI3K signaling pathways;compared with TPC-1 cell group,the down-regulation pathways of(UA+TPC-1)cell group were mitochondrial apoptosis pathway and PI3K signaling pathway.We performed QRT-PCR validation on the PI3K pathway and mitochondrial apoptotic pathway genes in the screening results.The results showed that bcl-2,PI3K,and AKT1 were present in the TPC-1 cell group compared with the Nthy-ori3-1 cell group.mTOR mRNA expression increased(P<0.01),bax,caspase-3,caspase-8,caspase-9,PTEN,NIS m RNA expression decreased(P<0.01);compared with TPC-1 cell group,(UA+TPC-1)The expression of bcl-2,PI3K,AKT1 and m TOR mRNA was decreased in the cell group(P<0.05),and the expression levels of bax,caspase-3,caspase-8,caspase-9,PTEN and NIS mRNA were increased(P<0.05).The seq results are consistent,and the PI3K signaling pathway and the mitochondrial apoptotic pathway are further confirmed as the future research directions.4.Effect and mechanism of UA on TPC-1 cells in Prunella vulgaris4.1 Effect of UA on proliferation of TPC-1 cells in Prunella vulgarisThe results of MTT assay showed that the optimal inoculum density of Nthy-ori3-1 cells and TPC-1 cells was 6.67×10~4/mL;UA had no inhibitory effect on the proliferation of Nthy-ori3-1 cells;The inhibition of proliferation of TPC-1 cells was statistically significant compared with the control group(P<0.05),but the inhibitory effect of UA on the proliferation of TPC-1 cells was not time-dependent.4.2 Apoptosis of TPC-1 cells induced by UA in Prunella vulgarisInverted microscopy showed that compared with the negative control group,there was no significant change in cells in the vehicle control group,but the cells in the UA group became round.As the concentration of UA increased,there were more and more dead cells,and the adherent cells became sparse.The amount of particulate matter in the slurry increases.Observed by Hoechst 33342-PI double staining method,there were no obvious apoptotic cells in the negative control group and the vehicle control group.The nuclei were normal and light blue fluorescence.Compared with the negative control group,early apoptotic cells were seen in the UA group,and nuclear condensation or Round-strained,bright blue fluorescence,UA concentration of 12?M group showed late apoptotic cells,nuclear chromatin aggregation,red fluorescence,indicating that with the increase of UA concentration,apoptosis gradually changes from early apoptosis to late apoptosis.The results of FCM showed that after 24,48and 72 hours of UA treatment of TPC-1 cells,the apoptosis rate and early apoptotic rate in different concentrations were statistically different(P<0.05);with the increase of the concentration,the late withered.The mortality rate increased,but only at UA=12?M,there was a statistical difference(P<0.05),suggesting that the inhibitory effect of UA on the proliferation of TPC-1 cells may be achieved through induction of apoptosis,and that UA induces TPC-1 cells.Apoptosis does not show time dependence.Western blot results showed that bcl-2,bax,and caspase-9 proteins were expressed in the negative control group,the vehicle control group,and the UA group.There was no significant difference between the vehicle control group and the negative control group(P>0.05).With the increase of UA concentration,the expression of bcl-2 was decreased.On the contrary,the expression of bax,caspase-9 and bax/bcl-2 protein was increased.Compared with the negative control group,P<0.05,the higher the UA concentration,the TPC.Apoptosis anastomosis was more likely to occur in-1,while no expression of caspase-3 and caspase-8 was observed in negative control group,vehicle control group,and UA group.QRT-PCR experiments showed that the expression of bcl-2,bax,caspase-3,caspase-8,and caspase-9 mRNA in the negative control group,vehicle control group,and UA group increased with the increase of UA concentration.There was no significant difference between the vehicle control group and the negative control group(P>0.05).The expression was decreased,whereas the ratios of bax,caspase-3,caspase-8,caspase-9 mRNA and bax/bcl-2 mRNA were increased.Compared with the negative control group,the difference was statistically significant(P<0.05).4.3 UA causes TPC-1 cell cycle arrest in S phase in Prunella vulgarisAfter different concentrations of UA interfered with TPC-1 cells,the proportion of cells in the cell cycle varied significantly.With the increase of UA concentration,the proportion of cells in S phase increased significantly.Compared with negative control group,the difference was statistically significant(P<0.05);The same concentration of UA acting TPC-1 cells at different time(24h,48h,72h),with the increase of time,the proportion of S phase cells increased significantly,compared with the negative control group,the difference was statistically significant(P<0.05),indicating that UA blocks TPC-1 cells in concentration and time dependent at S phase.4.4 Effects of ursolic acid in Prunella vulgaris on PI3K/PTEN/NIS/AKT/mTOR pathway in TPC-1 cellsWestern blot results showed that the expressions of p85-PI3K,p110-PI3K,AKT1,p-AKT1(Thr308),p-AKT1(Ser473),mTOR,PTEN,and NIS proteins were detected in the negative control group,vehicle control group,and UA group.The difference between the vehicle control group and the negative control group was not statistically significant(P>0.05);after UA was treated with TPC-1 cells for 48 hours,the expression of p110-PI3K protein was not significantly changed with the increase of UA concentration(P>0.05);p85-PI3K protein expression only decreased by UA(12?M)(P<0.05);AKT1 protein expression did not change significantly(P>0.05);p-AKT1(Thr308)protein expression was significantly reduced(P<0.05),and the ratio of p-AKT1(Thr308)/AKT1 was significantly decreased(P<0.05);p-AKT1(Ser473)protein was only decreased when UA=12?M(P<0.01),and p The ratio of AKT1(Ser473)/AKT1 decreased significantly(P<0.01).The expression of PTEN and NIS increased only at the concentration of UA(6,12?M)(P<0.01).The expression of mTOR protein decreased significantly(P<0.01).The results of QRT-PCR showed that PI3K,AKT1,m TOR,PTEN,and NIS mRNA were expressed in the negative control group,vehicle control group,and UA group.After UA was treated with TPC-1 cells for 48 hours,PI3K,compared with the negative control group,were detected.The expression levels of AKT1 and mTOR m RNA decreased with the increase of UA concentration(P<0.05),and the expression levels of PTEN and NIS mRNA increased with the increase of UA concentration(P<0.05).5.Effect of UA on the transplanted tumor model of TPC-1 cells in nude mice in Prunella vulgarisDuring the experiment,the body weight of nude mice in UA group increased.After the drug was over,the body weight of the nude mice was(22.05±0.99)g.The weight of the nude mice in the model group gradually decreased during the experiment.After the administration,the body weight was approximately(16.82±0.68)g.Compared with the model group,the UA There was a statistically significant difference in body weight changes in nude mice(P<0.01);at the end of administration,the tumor volume in the model group was(3.14±0.12)cm ~3,and the tumor volume in the UA group was(2.32±0.11)cm ~3 Compared with the model group,the tumor volume in the UA group was decreased,and the difference was statistically significant(P<0.05).The experimental results show that UA can inhibit the growth of transplanted tumors of TPC-1 cells in nude mice,but there is no statistical difference in the inhibition rate of transplanted tumors in UA group(P>0.05).Conclusion:1.UA can inhibit the proliferation of TPC-1 cells and induce apoptosis,which is related to the activation of mitochondrial apoptosis pathway,blocking cell cycle in S phase and inhibiting the PI3K/PTEN/AKT/mTOR signaling pathway;2.UA can enhance the expression of NIS in TPC-1 cells,and may enhance the iodine uptake ability of TPC-1 cells,which needs further verification;3.The toxicity of UA is low,which has a certain inhibitory effect on transplanted tumors of TPC-1 cells in nude mice,which provides a theoretical basis for future experiments.