| Retinoblastoma(RB)is a highly aggressive ocular tumor,most commonly seen in children under 5 years of age.The effectiveness of retinoblastoma treatment depends on early detection and early treatment,but due to socioeconomic and medical constraints,many children are diagnosed and treated in the metaphase and advanced clinical stages,resulting in high rates of blindness and disability.Chemotherapy,radiotherapy,surgery and immunotherapy are currently available for the treatment of retinoblastoma,among which chemotherapy-based combination therapy is the most commonly used regimen for children with intermediate to advanced disease.In clinical treatment,the existence of resistance and toxic side effects of chemotherapy drugs makes it difficult for some children to receive timely and effective treatment.Therefore,less toxic and affordable treatments or drugs need to be sought.Panax Notoginseng Saponins(PNS)is a biologically active ingredient extracted from the traditional Chinese medicine Panax ginseng,which is widely used in the treatment of diabetic retinopathy,retinal vein obstruction and other ophthalmic diseases.The clear antitumor activity of PNS has been confirmed in recent studies on liver cancer,breast cancer,osteosarcoma,lymphoma,pancreatic cancer and other tumor cells,However,its use in the treatment of ocular tumors has not yet been reported.Objectives:To investigate the effects of PNS on the proliferation,apoptosis and PI3K/AKT signaling pathway of retinoblastoma Y79 cells in order to explore the mechanism of action of PNS in inhibiting retinoblastoma.Methods:1.The CCK-8 assay was used to detect the effects of PNS and carboplatin on the proliferation of Y79 cells,respectively,and then the IC50 values of the two drugs were calculated.Referring to the results of CCK-8 experiment,Y79 cells were divided into negative control group,different concentrations(200,400,600ug/ml)of PNS group and carboplatin positive control group.The effect of PNS on apoptosis was detected using flow cytometry,and the m RNA and protein expression of apoptosis-related genes were analyzed by Western blot and real-time fluorescence quantitative PCR.2.In the mechanism experiment,the effect of PI3 K inhibitor LY294002 on the proliferation of Y79 cells was analyzed by CCK-8 assay and IC50 was calculated.PI3K/AKT signaling pathway proteins were detected in the negative control group and in the PNS group with concentration gradients of 200ug/ml,400ug/ml and 600ug/ml of PNS.Then Y79 cells were grouped and treated according to the negative control group,PNS group,combined drug group and PI3 K inhibitor group.The apoptosis and the expression of PI3K/AKT pathway proteins and apoptosis-related proteins in the cells were detected in Y79 cells of each group.3.The cell cycle of Y79 cells in the negative control group,PNS group(200ug/ml group,400ug/ml group and 600ug/ml group)and carboplatin positive control group were examined using flow cytometry.Western blot and real-time fluorescence quantitative PCR were applied to detect the m RNA and protein expression of cell cycle-related genes.Results:1.PNS could effectively inhibit the proliferation of Y79 cells compared with the negative control group(P<0.05).The IC50 of PNS on Y79 cells at 24 h,48h and 72 h were calculated as 429.2ug/ml,343.8ug/ml,271.5ug/ml after the analysis of the results of CCK-8 experiments.The 24 h IC50 value of carboplatin on Y79 cells was 63.60ug/ml.The apoptosis rate of Y79 cells treated with PNS increased with increasing drug concentration(P<0.05),and 600ug/ml PNS had the most obvious effect of promoting apoptosis in Y79 cells,with an apoptosis rate of 22.30±1.08%.The results of Western blot and real-time fluorescence quantitative PCR showed that the m RNA and protein expression of apoptosis-related genes Bax,Caspase-3,Caspase-8,and Caspase-9 were significantly higher in Y79 cells treated with PNS compared with the negative group(P<0.05),and the activation proteins of apoptosis-promoting proteins Cleaved caspase-3,Cleaved caspase-8,and Cleaved caspase-9 were also significantly increased in expression(P<0.05),whereas the m RNA and protein expression of the apoptosis suppressor gene Bcl-2 were suppressed(P<0.05).The results of comparison between groups showed that 600ug/ml PNS had the strongest induction of m RNA and protein expression of apoptosis-promoting genes(P<0.05),and this group also had the most significant m RNA and protein inhibition of Bcl-2(P<0.05).Bax/Bcl-2 values were significantly higher in Y79 cells treated with PNS compared with the negative group(P<0.01),and the Bax/Bcl-2 values of the 600ug/ml PNS group were higher than those in the 200ug/ml(P<0.001)and 400ug/ml groups(P<0.005).2.We found that PI3 K inhibitor LY294002 could effectively inhibit the proliferation of Y79 cells by CCK8 cell proliferation assay,and its 24-hour IC50 value of Y79 cells was 26.25umol/L.The results of apoptosis assay showed that the apoptosis rate of PNS group,LY294002 group and combined drug group were higher than that of the negative control group(P<0.001),and the highest apoptosis rate of Y79 cells was in the combined drug group,which was 35.59±0.85%.The expression of total PI3 K and AKT1 proteins in Y79 cells treated with different concentrations of PNS did not show differences compared with the negative group(P>0.05),but the expression of phosphorylated proteins p-PI3 K,p-AKT(Thr308),p-AKT(Ser473)and p-m TOR in the PI3K/AKT pathway was significantly lower than that in the negative control group(P<0.05).The expression of phosphorylated proteins in the PNS treated group decreased in a gradient with increasing drug concentration(P<0.05).Compared with the negative group,200ug/ml PNS had no significant effect on the expression of the PI3K/AKT pathway antagonist protein PTEN(P>0.05),but the expression of PTEN protein was significantly higher in both 400ug/ml and 600ug/ml PNS groups(P<0.01).The expression of apoptosis promoting proteins Bax,Caspase-3,Caspase-8,Caspase-9 and activation proteins Cleaved caspase-3,Cleaved caspase-8,Cleaved caspase-9 in the PNS group,the combination drug group and the PI3 K inhibitor group were significantly increased(P<0.05),and the expression of apoptosis promoting proteins in the combined drug group was higher than that in the PNS group and the LY294002 group(P<0.05).The expression of Bcl-2 protein in the cells of each drug group was lower than that of the negative group(P<0.01),and the comparison between groups showed that the combined drug group had the strongest inhibitory effect on Bcl-2 protein(P<0.005).The Bax/Bcl-2 values of the drug-treated cells were all significantly higher than those of the negative group(P<0.005),and the highest Bax/Bcl-2 values were found in the combined drug group,which were 2.62±0.16 times higher than those of the negative group.3.Compared with the negative group,the percentage of Y79 cells in G0/G1 phase significantly increased(P<0.005),and the percentage of cells in S and G2/M phases decreased(P<0.05)after treatment with PNS.The cycle reports showed that the highest percentage of Y79 cells in G0/G1 phase was 72.42±0.65% in the group with PNS at a concentration of 600ug/ml.The expression of Cyclin D1,CDK4,CDK6,m RNA and protein in G0/G1 phase related genes were significantly decreased after PNS treatment of Y79 cells compared with the negative group(P<0.05),and it was concentration dependent,with the most significant decrease in the 600ug/ml PNS group(P<0.05).Conclusion:1.PNS can inhibit the proliferation and promote the apoptosis of retinoblastoma Y79 cells.2.The apoptosis-promoting effect of PNS on Y79 cells may be achieved by inhibiting the PI3K/AKT signaling pathway and subsequently regulating the expression of apoptosis-related genes.3.PNS could induce G0/G1 phase block in Y79 cells by inhibiting the m RNA and protein expression of Cyclin D1,CDK4 and CDK6. |
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