| BackgroundLeukemia is a common oncology disease,in our country,the incidence of leukemia can be ranked sixth in the incidence of various types of oncological diseases.At present,the medical treatment of leukemia mainly through chemotherapy(chemotherapy),with the continuous improvement of the medical level,continue to emerge new chemotherapy drugs and chemotherapy.However,no matter which chemotherapeutic agent is used,the chemotherapy regimen,MDR(multidrug resistance to chemotherapy)generated during the chemotherapy process may diminish the efficacy of the chemotherapy or even fail.The reasons for the emergence of MDR more complicated,a variety of genes involved.The main reason causing MDR is that the function of pumping tumor cells becomes stronger,and the content of P-glycoprotein(P-gp)in the cells is increased,which makes the drug concentration in the cells lower and the efficacy can not be exerted normally.Because Nrf2 can effectively regulate the synthesis of P-gp,and then affect the MDR of tumor cells,the study of Nrf2 inhibitors as an effective method to treat leukemia and inhibit the MDR of leukemia tumors was Domestic and foreign scholars conduct extensive research.Due to the current common drug-resistant reversal agent synthesized by chemicals,its single effect,and side effects are very detailed,to a large extent limit the application of space,and a wide range of Chinese medicine,efficacy extensive,and side effects,therefore,people will look Turn to traditional Chinese medicine research,try to find efficient drug resistance reversal agent from traditional Chinese medicine.The study found that bufalin(bufalin)can be extracted from the Chinese medicine toad,has a good inhibitory effect on the proliferation and resistance of leukemia cells.Endoplasmic reticulum stress(ERS)can induce cell proliferation to some extent,and when ERS intensity is too high,it can promote apoptosis.The use of this feature to study the mechanism of ERS promote apoptosis,can provide new ideas for the treatment of cancer.Activated IRE1? recruits tumor necrosis factor receptor associated factor 2(TRAF2),which forms IRE1-TRAF2-ASK1 protein complex and activates JNK or SAPK,eventually activating apoptosis signal.ObjectiveWe want to use bufalin as a research object,take its "poison attack" to treat multi-drug resistance of leukemia,to study its application in reducing leukemia multidrug resistance gene expression,and promote apoptosis of leukemia drug-resistant cells effect.The purpose of this experiment is to study the reversal effect of bufalin on K562 /A02 and the inhibitory effect of bufalin on the growth of K562 / A02,and to explore its possible mechanism.Methods and results 1?Resistant effect of bufalin on K562/A02The MTT assay showed that the drug resistance fold was 59.77 and the drug resistance reversal fold was 2.97 times.After ADM was detected by flow cytometry,the fluorescence intensity of ADM in K562/A02 cells was significantly increased after adding bufalin,however,the fluorescence intensity of Nrf2 siRNA group was significantly higher than that of K562/A02 + ADM + bufalin group(P <0.01);The expression of Nrf2-related genes and drug-resistant genes in bufalin-treated K562/A02 cells was detected by RT-PCR and western blot.The mRNA expression of Nrf2,and MDR1 in K562/A02 cells was significantly higher than that in K562 cells(P <0.01;P <0.01).The mRNA expression of Nrf2 and MDR1 was significantly lower than that of K562/A02 group(P <0.01);Western blot analysis showed that bufalin could inhibit the expression of Nrf2 and its target genes HO-1 and P-gp protein(P <0.01;P <0.01;P <0.01),while the detection of related proteins in Nrf2 siRNA group The expression of HO-1 and P-gp protein was significantly weaker than that of the non-interfering group(P>0.05),which was consistent with the results of RT-PCR,while the fluorescence intensity of K562/A02 cells was significantly increased After interference with Nrf2,the sensitivity of K562 / A02 to ADM increased.2?Effect of bufalin on apoptosis of K562/A02 cellsThe OD value of bufalin K562/A02 cells at the final concentrations of 0,12.5,25,50 and 100 nM were determined by MTT assay.The results showed that the inhibitory effect of bufalin on K562/A02 cells was time-dependent.Flow cytometry The apoptosis rate of bufalin group was significantly higher than that of K562/A02 cells(P <0.01);The mRNA expression of endoplasmic reticulum markers GRP78,94 in bufalin group was significantly increased by RT-PCR(P <0.01).However,the mRNA expression of Bax and Bcl-2 in the gene of bufalin was higher The ratio of Bax / Bcl-2 in Lingling group was higher than that in K562 / A02 group(P <0.01);The expression of Bax,cycto c and PAPR protein in bufalin group was significantly increased,while the expression of Bcl-2 was decreased(P <0.01;P <0.01;P <0.01;P <0.01).The expression of IREI?,TRAF2,JNK?p-JNK and Caspase-12 in the bufalin group were significantly increased(P <0.01;P <0.01;P <0.01;P <0.01);n order to illustrate the role of bufalin in inducing apoptosis of K562 /A02 cells,we performed IRE1? siRNA and JNK siRNA on the cells.Flow cytometry was used to detect the apoptosis of the cells in K562 /A02 cells.The apoptosis rate of the cells in the interference group was higher than that of normal The expression of JNK ?p-JNK and Caspase-12 mRNA in JNK siRNA group was significantly lower than that in IREI? siRNA group(P <0.01).The results showed that JNK and Caspase-12 mRNA were significantly reduce;The ratio of Bax / Bcl-2 mRNA in IRE1?siRNA and JNKsiRNA group was significantly lower than that in non-interfering group(P <0.01).The relevant protein detection of each interference group found that the results were consistent with the PCR results.Conclusion 1.Bufalin can reverse the drug resistance of K562 / A02 cells through Nrf2;2.Bufalin induces apoptosis through IRE1? / TRAF2 / JNK / Caspase-12 pathway... |
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