| Lung cancer is the most common clinical tumor,and its morbidity and mortality rate rank among the top of all malignant tumors in my country.Among them,non-small cell 1 ung cancer accounts for 80%of all lung cancer patients.At present,the main treatments for non-small cell lung cancer include surgery,radiotherapy,chemotherapy,targeted therapy,and immunotherapy.In the past thirty years,apoptosis has been extensively studied and is considered to be the main mechanism of cell development and programmed death.Disregulation of apoptosis is one of the main causes of tumors.Inducing tumor cell apoptosis is currently one of the main methods of tumor treatment.Therefore,inducing tumor cell apoptosis and restoring the balance between cell proliferation and programmed death in the body is the key to the treatment of tumors.JNKs can induce cell apoptosis through the influence on gene expression and the regulation of endogenous and exogenous apoptotic pathways.Bcl-2 family proteins are key regulators of apoptosis.PUMA is one of the most effective killers in the subgroup of BH3 among the members of the Bcl-2 family.PUMA may be an excellent therapeutic target,because activating PUMA can inhibit tumor growth by restoring the apoptosis of cancer cells."National Famous Chinese Medicine Doctor" Professor Piao Bingkui,based on years of clinical experience and combined with the screening results of modern medical drugs,has developed Feiliuping ointment with definite effects on lung cancer.The Feiliuping ointment is divided into Qi-invigorating drugs,detoxification drugs,blood-stimulating drugs,phlegm-resolving drugs.According to the factorial study of the detoxification group,the detoxification group mainly exerts an anti-tumor effect,and the Qi-invigorating may mainly be adjusted.The body's immune function indirectly exerts an anti-tumor effect,and the combination of Qi-enriching drugs and detoxification drugs can synergistically improve the effect of suppressing tumors.The role of Platycodon grandiflorum,which regulates qi and resolves phlegm,needs to be further studied.Platycodon grandiflorum is an important part of the whole prescription to relieve phlegm.It has the effects of relieving lung and relieving asthma,resolving phlegm and draining pus.According to the Chinese Pharmacopoeia,Platycodin D is the main bioactive component of the Chinese herbal medicine Platycodon grandiflorum.Studies have found that Platycodin D has anti-inflammatory and analgesic,lipid-lowering,hypoglycemic,anti-tumor and other functions.Studies have reported that Platycodin D can inhibit the proliferation of human gastric cancer cells.Therefore,we speculate that Platycodin D may be one of the effective components of the Feiliuping ointment group.ObjectiveTo observe the effects of Fei Liuping(phlegm-resolving group)drug-containing serum on human non-small cell lung cancer cells and its main component Platycodin D on human non-small cell lung cancer cell tumor tissues in a tumor-bearing animal model and non-small cell lung cancer cell lines.The influence of JNK/PUMA protein expression in Chinese medicine reveals the molecular mechanism of Feiliuping(phlegm-resolving group)in preventing and treating lung cancer from the perspective of inducing apoptosisMethod(1)After Feiliuping ointment(phlegm-resolving group)drug-containing serum applied to H1299 cells,CCK8 detects the proliferation of H1299 cells,and Western blot detects the effect of related molecule JNK expression;(2)After Platycodin D is applied to H1299,H2030,A549 cells,the ATK luminescence method is used to detect the cell activity,and the IC50 value of Platycodin D on the above cells is calculated;(3)After Platycodin D is applied to H1299 and H2030 cells,apoptosis is detected by flow cytometry,and the expression of apoptosis-related molecules is detected by western blot;(4)After Platycodin D is applied to H1299 and H2030 cells,Western blot was used to detect the expression of proteins related to the JNK/PUMA signaling pathway in the cells,and the changes in PUMA mRNA were detected by qPCR;(5)Reduce the expression of JNK1 and JNK2 in H1299 cells by synthesizing si-RNA and transfection technology.After the intervention of Platycodin D,the interference efficiency was verified by qPCR,and the protein expression related to the JNK/PUMA signal pathway was detected by Western blot;(6)After Platycodin D is applied to H1299 cells,construct a plasmid to detect the changes of cellular transcription factor AP-1 through the dual luciferase reporter gene;(7)The H1299 cell line was used to inoculate the right axilla of NSG nude mice to establish a H1299 human non-small cell lung cancer tumor-bearing animal model.After 14 days of intervention with physiological saline,low-dose platycodin D,and high-dose platycodin D.The material was collected,tumors weight were measured,and tumor inhibition rates were calculated;(8)The H1299 cell line was used to inoculate the right axilla of NSG nude mice to establish the H1299 human non-small cell lung cancer animal model.After 14 days of intervention with normal saline,low-dose platycodin D,and high-dose platycodin D The material was collected and immunohistochemical method was used to detect the expression changes of Ki-67 and Caspase-3 in tumor tissuesResults(1)Feiliuping ointment(phlegm-resolving group)can affect the CCK8 OD value of cells to a certain extent compared with blank serum,and Feiliuping ointment(phlegm-resolving group)can inhibit the proliferation of H1299 cells to a certain extent;After 48 hours of intervention with H1299 cells in the serum group containing Liuping ointment(phlegm-resolving group),the expression of P-JNK in the Feiliuping ointment(phlegm-reducing)group was 1.17±0.11,and that of the control group was 0.07±0.02.Compared with the control group,the expression of PUMA in the Feiliuping ointment(phlegm-resolving group)was significantly higher than that in the control group,and the result was statistically significant(P<0.05);the expression of PUMA in the Feiliuping ointment(phlegm-resolving group)was 1.46±0.05,and that of the control group was 0.19±0.04,and the result was statistically significant(P<0.05).The expression of Cleaved caspase-3 in the Feiliuping ointment(phlegm-resolving group)was 0.95±0.07,and that of the control group was 0.95±0.10.There is no significant difference between them;(2)The IC50 value of Platycodin D on H1299 cells is 7.9 ?mol/L;the IC50 value of Platycodin D on H2030 cells is 9.7 ?mol/L;the IC50 value of Platycodin D on A549 is 10.3?mol/L.(3)The results of flow cytometry showed that the proportion of apoptosis in Platycodin D group was greater than that in the control group,and the difference was statistically significant(P<0.01);Western Blot detection of H1299 cell apoptosis-related proteins showed that the PARP expression levels of cells in the 5 ?mol/L,10 ?mol/L,and 15 ?mol/L groups were 2.38±0.60,22.29±6.40,and 43.14±15.3 times than that of the 0 ?mol/L group,respectively.The 10 ?mol/L and 15 ?mol/L groups compared with the 0 ?mol/L group,the difference was significantly statistically significant(P<0.05);the expression of Cleaved caspase-3 in the 5 ?mol/L,10 ?mol/L,and 15 ?mol/L groups was 1.87 ± 0.40,19.86 ± 7.22,74.61 ± 6.88 times than that of the 0 ?mol/L group,respectively.The 10 ?mol/L and 15 ?mol/L groups are significantly different from the 0 ?mol/L group(P<0.0001);Western Blot detects H2030 cell apoptosis-related proteins.The results showed that the PARP expression levels of cells in the 5 ?mol/L,10 ?mol/L,and 15 ?mol/L groups were 2.82 ± 0.59,15.1±6.14,52.52±15.95 times than that of the 0 ?mol/L group,respectively.The difference between the 10?mol/L,15 ?mol/L group and the 0 ?mol/L group was statistically significant(P<0.01);the expression of Cleaved caspase-3 in the 5 ?mol/L,10 ?mol/L,and 15 ?mol/L groups were 2.24±0.75,29.08 ± 13.62,115.6±13.8 times than the of 0 ?mol/L group respectively.The 10?imol/L and 15 ?mol/L groups had significant differences compared with the 0 ?mol/L group(P<0.0001);(4)Western Blot detected H1299 cells.The results showed that after 48 hours of platycodin D intervention,the protein expression levels of P-JNK in H1299 cells in the 5?mol/L,10 ?mol/L,and 15 ?mol/L groups were 2.80 ± 0.45,17.96±0.27,29.5±1.82 times than that of the Omol/L group respectively.The 10 ?mol/L,15 ?mol/L group was significantly higher than the 0 ?mol/L group,the difference was statistically significant(P<0.0001);The protein expression levels of PUMA in H1299 cells in 5 ?mol/L,10?mol/L,15 ?mol/L group were 1.24 ± 0.1,7.97±3.07,12.08 ± 3.86 times that of the 0 ?mol/L group respectively.The 15 ?mol/L group was significantly higher than the 0 ?mol/L group,and the difference was statistically significant(P<0.05).The expressions of P-c-Jun and c-Jun in the 10 ?mol/L and 15 ?mol/L groups also increased compared with than in the 0 ?mol/L.The expression of JNK did not change significantly after the intervention of different concentrations of Platycodin D;Western Blot detected H2030 cells,the results showed after the intervention of Platycodin D for 48 hours.The protein expression levels of P-JNK in H2030 cells in the 5 ?mol/L,10 ?mol/L,15 ?mol/L group were 3.32 ± 0.73,31.99±11.63,and 20.5 ± 4.10 times than that of the 0?mol/L group respectively.Among them,the 10 ?mol/L and 15 ?mol/L groups were significantly higher than those in the 0 ?mol/L group.Statistical significance(P<0.05);5?mol/L,10 ?mol/L,15 ?mol/L group H2030 cell PUMA protein expression levels were 1.32 ±0.25,9.26 ± 2.99,10.97 ± 4.04 times than that of 0 ?mol/L group,of which 10 ?mol/L group/L,15 ?mol/L group was significantly higher than 0 ?mol/L group,the difference was statistically significant(P<0.05).The expressions of P-c-Jun and c-Jun in the 10 ?mol/L and 15 ?mol/L groups also increased compared with that in the 0 ?mol/L.The expression of JNK did not change significantly after intervention with different concentrations of Platycodin D;H1299 cells were detected by qPCR.After 48 hours of intervention,compared with the control group,the expression of PUMA mRNA in H1299 cells of Platycodin D group increased significantly,and the difference was statistically significant.(P<0.05).(5)Western Blot test results showed that the expression of JNK and JNK1 in the blank JNK1 silenced group was significantly lower than that of the blank negative control group,and the result was statistically significant(P<0.05);the expression of JNK and JNK2 in the blank JNK2 silenced group was more significant than that of the blank negative control group,and the result was statistically significant(P<0.05);The expression of JNK1,P-JNK,Pc-Jnn,c-Jnn,Cleaved caspase-3,PUMA in Platycodin D JNK1 silent group was significantly lower than that of Platycodin D negative control group.The results were statistically significant(P<0.05);the expression of JNK,JNK2,P-JNK,Pc-Jun,c-Jun,Cleaved caspase-3 in the platycodin D JNK2 silenced group was lower than that of the platycodin D negative control group.Only JNK and JNK2 were statistically significant(P<0.05).(6)The results of the dual luciferase reporter gene experiment showed that after 24 interventions with Platycodin D,the relative luciferase expression of AP-1 in the control group was 0.40±0.06,and the relative luciferase expression of Platycodin D group was 1.38 ± 0.26;After 48 hours of platycodin D intervention,the relative luciferase expression of AP-1 in the control group was 0.14 ± 0.02,and the relative luciferase expression of platycodin D group was 3.43±0.59;and the difference between the two groups after 48 hours of intervention was greater than that of the 24 hours of intervention.The above results are statistically significant(P<0.01).(7)After 14 days of intervention with different concentrations of Platycodin D,the average tumor weight of the control group was 0.76±0.02g,the average tumor weight of Platycodin D low-dose group was 0.48±0.05g,and the average tumor weight of Platycodin D high-dose group was 0.36±0.06g,the average tumor weight of the low-dose and high-dose Platycodin D group was significantly smaller than that of the control group,and the results were statistically significant.The tumor inhibition rate of Platycodin D low-dose group was 36.87%,and that of Platycodin D high-dose group was 52.49%.(8)After 14 days of intervention,the expression of Ki-67 in tumor tissues of the platycodin D low-dose group and the platycodin D high-dose group were significantly lower than those in the control group,the result is statistically significant;after 14 days of intervention,the expression of Cleaved caspase-3 in the tumor tissues of the platycodin D low-dose group and the platycodin D high-dose group was significantly higher than that of the control group,the results were statistically significant.Conclusions1.In Feiliuping ointment,phlegm-resolving drugs can activate the JNK/PUMA pathway of lung cancer cells.2.Platycodin D induces lung cancer apoptosis by regulating the JNK/PUMA pathway.3.The anti-tumor effect of the phlegm-resolving method can be achieved by regulating cell apoptosis. |
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