| Objective Glioma is the most common primary malignant tumor of the central nervous system(CNS),accounting for 50%-60% of intracranial tumors.The traditional treatment is surgery combined with radiotherapy and chemotherapy.However,the recurrence rate is high and the prognosis is poor.The median survival time of low-grade glioma patients is 3-5 years,while that of high-grade glioma patients is only 1 year,which poses a great threat to people’s health.China is a region with high incidence of glioma.The number of patients and deaths is increasing year by year.The occurrence and development of glioma involve cell signal transduction.Like other tumors,the disorder of proto-oncogene and anti-oncogene results in excessive proliferation of tumour cells.Recent studies have shown that one of the reasons for the poor prognosis of high-grade glioma is its resistance to chemotherapeutic drugs.Nrf2 is a research hotspot in recent years,and its role in anti-oxidative stress and anti-cancer has attracted wide attention.This study will explore the relationship between Keap1-Nrf2 signaling pathway and multidrug resistance protein in human glioma(U87 cell line)and the effect of low frequency and low intensity ultrasound combined with low dose curcumin on multidrug resistance protein in human glioma cells through Keap1-Nrf2 pathway.Method in this study,U87 cells were divided into low-frequency ultrasound group at the early stage.U87 cells were divided into six groups: 0,3.0,50.4,83.4,142.0 and 290.0 m W/cm2 according to different intensity of action.The irradiation time was 60 seconds.Four curcumin groups: 0,5,10,20,40 umol/l.The absorbance of U87 cells was measured by CCK8 method,and the proliferation rate was calculated.U87 cells apoptotic rate was higher and necrosis rate was lower when the intensity of low frequency ultrasound was 83.4 m W/cm2,the duration of action was 60 s and curcumin concentration was 5 umol/l.This condition was the best parameter for U87 cells apoptotic induced by low frequency ultrasound combined with low dose curcumin.U87 cells were divided into four groups: control group,low dose curcumin group(C5),low frequency low intensity ultrasound group(U83.4)and low frequency low intensity ultrasound combined with low dose curcumin group(C5U83.4).The absorbance of U87 cells in each group was measured by CCK8 method,and the proliferation rate was calculated.The expression of Nrf2,Keap1,ABCG2 and P-gp protein in U87 cells was detected by immunofluorescence staining and Western Blot respectively.The expression of Nrf2,Keap1,ABCG2 and P-gp in U87 cells was detected by fluorescence quantitative reverse transcription-polymerase chain reaction q RT-PCR.U87 cells were divided into control group,Scrambled group and Nrf2-down group.Lentivirus-mediated Nrf2 silencing reduced the expression of Nrf2 in U87 cells.Quantitative RT-PCR was used to detect the transfection efficiency of si-Nrf2 and the difference of expression of Nrf2 in each group.The expressions of Keap1,ABCG2 and P-gp in U87 cells after transfection of si-Nrf2 were detected respectively.CCK8 method was used to detect the absorbance value of cells in each group and calculate the proliferation rate.Immunofluorescence staining and Western Blot were used to detect the expression of Keap1,Nrf2,ABCG2 and P-gp protein in U87 cells of each group after transfection of si-Nrf2,and the gray values of each group were obtained.SPSS 19.0 was used for statistical analysis.Result 1.Proliferation inhibition rate: In the low-frequency ultrasound group,compared with the control group,low-frequency ultrasound with intensity < 50.4 m W/cm2 had no significant inhibitory effect on U87 cell proliferation,while low-intensity ultrasound with intensity < 83.4 m W/cm2 inhibited cell proliferation significantly,and with the increase of ultrasound intensity,the proliferation rate decreased significantly,and the proliferation inhibition increased significantly in a intensity-dependent manner.In the curcumin group,compared with the control group,curcumin at 5 umol/L had no significant inhibitory effect on the proliferation of U87 cells,but curcumin significantly inhibited the proliferation of U87 cells when the concentration was more than 10 umol/L.With the increase of curcumin concentration,the proliferation rate decreased significantly,in a concentration-dependent manner.In the subsequent combined effect experiment,C5U83.4 group had significant inhibition on U87 cell proliferation compared with the control group and the individual action group(C5 and U83.4 groups).The results showed that the combined group had significant inhibition on U87 cell proliferation compared with the control group(P < 0.05).2.Pre-transfection protein content: Immunofluorescence staining,Western Blot and q RT-PCR were used to determine the intracellular protein content of control group,low dose curcumin group(C5),low frequency and low intensity ultrasound group(U83.4)and low frequency and low intensity ultrasound combined with curcumin group(C5U83.4).The expression of Nrf2,Keap1,ABCG2,P-gp and GAPDH protein was observed.Result The relative expression of Nrf2,Keap1,ABCG2,P-gp and GAPDH were analyzed according to the measured gray value.Nrf2,ABCG2 and P-gp were highly expressed in the control group,while Keap1 was low.The expression of Nrf2,ABCG2 and P-gp in C5U83.4 group was significantly decreased,while the expression of Keap1 was significantly increased in C5U83.4 group compared with the control group and C5 and U83.4 group alone.Compared with the control group,the changes of protein expression in the combined group were significant(P < 0.05).3.Transfection efficiency of si-Nrf2: The expression of Nrf2 was detected by q RT-PCR in three groups: control group,Scrambled group and Nrf2-down group.The expression of Nrf2 in Nrf2-down group was significantly lower than that in control group and Scrambled group.There was no difference in the expression of Nrf2 between control group and Scrambled group.Compared with the control group,the expression of Nrf2 in Nrf2-down group was significantly changed by one-way ANOVA(P < 0.05).4.Protein content after transfection: Immunofluorescence staining,Western Blot and q RT-PCR were used to determine the intracellular protein content of control,Scrambled and Nrf2-down groups.The expressions of Nrf2,Keap1,ABCG2,P-gp and GAPDH were observed.The results were based on the gray values.The relative expression levels of Nrf2,Keap1,ABCG2,P-gp and GAPDH were analyzed.Nrf2,ABCG2,P-gp were highly expressed in Control group and Scrambled group,while Keap1 was low.Compared with control group and Scrambled group,the expression of Nrf2,ABCG2 and P-gp in Nrf2-down group decreased significantly,while the expression of Keap1 increased significantly.Compared with the control group,the expression of Nrf2-down histone protein in each group was significantly changed by one-way ANOVA(P < 0.05).Conclusion 1.Compared with Control group,C5U83.4 group had obvious inhibitory effect on U87 cell proliferation,and was stronger than C5 and U83.4 group on U87 cell proliferation.2.Nrf2 and Keap1 may also be negatively correlated in U87 cells,and the changes of Nrf2 protein level can affect the changes of ABCG2 and P-gp protein content to a certain extent,while low-frequency low-intensity ultrasound combined with low-dose curcumin may significantly reverse the drug resistance of glioma.3.After the down-regulation of Nrf2 protein level by si-Nrf2,it was confirmed that there was a correlation between Nrf2 and Keap1 expression in U87 cells,constituting the Keap1-Nrf2 pathway,which played a certain role in the complex biological behavior of glioma and might participate in the process of glioma resistance.When the down-regulation of Nrf2 protein level,the expression of ABCG2 and P-gp protein was down-regulated.4.Low-frequency ultrasound combined with low-dose curcumin may reduce the expression of multidrug resistance protein in human glioma cells through the Keap1-Nrf2 signaling pathway. |
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