The Malignant Transformation Effect Of HEEC By AFB₁ Exposure And Its Relationship With P38 MAPK Signaling Pathway

Objective:Long-term continuous low concentration of aflatoxin B₁（AFB₁）was used to treat human normal esophageal epithelial cells（HEEC）to 30 generations.p38 MAPK protein specific inhibitor（SB203580）was used to inhibit the p38 MAPK signaling pathway in 30 generations of infected cells.The changes in cell growth vitality,cycle,apoptosis and other biological functions was observed.The expression of epithelial-mesenchymal transition（EMT）-related proteins,cycle-related proteins,and p38 MAPK signaling pathway related proteins was detected.This study aimed to explore the carcinogenic effect of AFB₁ and its relationship with p38 MAPK signaling pathway in the process of HEEC malignant transformation,and further explore the relevant molecular mechanism of AFB₁ in the occurrence of esophageal cancer,lay a foundation for the etiology of esophageal cancer,and provide a certain theoretical basis for preventing the toxicity of AFB₁ in food to the esophagus.Methods:（1）Study on the malignant transformation of HEEC induced by long-term exposure to low-concentration AFB₁:After HEEC was exposed to culture medium containing 0,0.01,0.1,1,10,100,and 1000μmol/L AFB₁ for 24 h,the effect on the activity of HEEC was tested.Subsequently,1μmol/L AFB₁ was selected to continuously infect HEEC cells for 30 generations.Uninfected HEEC was set as the control group,the growth morphology of the cells was observed at intervals of 5generations.The CCK8 test was used to detect the effect of AFB₁ on cell viability of different generations.Flow cytometry was used to detect cell apoptosis,cell cycle,and the Transwell was used to detect the changes in the number of invasions and migrations of 30 generations of infected cells.（2）Study on the toxicity of p38 MAPK signaling pathway in HEEC malignant transformation cell line:The 30 generations of AFB₁ infected cells were exposed to 0,1,10,100,and 1000μmol/L SB203580 for 24 h,and the effect of SB203580 on cell viability was detected.Subsequent exposure with 0,25μmol/L SB203580 for 24 h,CCK8 test was used to detect the effect of SB203580 on cell viability,and flow cytometry was used to detect the changes of cell apoptosis and cell cycle.（3）Function-related protein expression of HEEC malignantly transformed cell lines:After culturing normal HEEC cells,a 30-generation cell line containing 1μmol/L AFB₁,and a 30-generation cell line containing 1μmol/L AFB₁+25μmol/L p38 protein inhibitor for 24 hours,the protein was detected by Western blot,including EMT proteins:E-cadherin,N-cadherin,cell cycle related proteins:cyclin A2,cyclin D1 and CDK2,p38MAPK signaling pathway related protein p38,phosphorylated p38expression.Results:1.Study on the malignant transformation of HEEC induced by long-term exposure to low-concentration AFB₁:（1）When HEEC was exposed to different concentrations of AFB₁ for 24 h,the cell viability gradually decreased in a dose-dependent manner,and the half inhibitory concentration（IC 50）was 169.3μmol/L.The AFB₁ concentration with less damage to cells（1μmol/L）was chosen to treat HEEC for a long time.（2）Observation of cell morphology found that with the increase of AFB₁ exposure generation,the cell volume became larger,the shape was loose,slender,accompanied by pseudopodia formation,and similar EMT changes appeared,which was significantly different from the control group.（3）Compared with the control group,the CCK8 test results showed that the cell proliferation rate was increased in the early stage（5th and 10th generations）after exposure（P<0.01）,and there was no statistically significant difference between the 15th generation in the mid-term exposure（P>0.05）.The cell proliferation rate was significantly increased at the 20th generation in the mid-stage and late-stage（25th and 30th generations）exposure（P<0.01）.Under the long-term low-concentration of AFB₁ treatment,the proliferation ability of cells has experienced a process of first increasing,then inhibition and then increasing,and the 30th generation has the strongest proliferation ability.（4）The cell cycle test results showed that compared with the control group,the ratio of cells in the G0/G1 phase of the cells at the initial stage of exposure（5th and 10th generations）increased,while the ratio of cells in the S phase decreased.The proliferation index PI=（S+G2/M）/（G0/G1+S+G2/M）decreased（P<0.05）,and there was no significant difference in the ratio of G0/G1 and PI of cells in the mid-stage（15th and 20th generations）exposure（P>0.05）.In the late stage of exposure（25th and 30th generations）,the ratio of cells in G0/G1 phase decreased,the ratio of cells in S phase increased,and PI increased（P<0.05）.Under the long-term low concentration of AFB₁ treatment,the cell cycle experienced blockade G0/G1 phase,which then promotes the process of cell cycle differentiation from G0/G1 phase to S phase.（5）The results of cell apoptosis test showed that compared with the control group,with the increase of the number of infected passages,the total cell apoptosis rate showed a gradual change.The pre-exposure gradually increased,and the total apoptosis rate of the 15th generation cells was the highest,and subsequently,the apoptosis rate showed a downward trend.Compared with the control group,there was no statistically significant difference in the 30th generation cell apoptosis rate（P>0.05）.（6）The results of cell migration and invasion experiments showed that compared with the control group,the number of cell migration and invasion at the 30th generation of the infection increased,and the difference was statistically significant（P<0.05）.2.Study on the toxicity of p38 MAPK signaling pathway in HEEC malignant transformation cell line:（1）After 30 generations of AFB₁ treated cells were exposed to different concentrations of SB203580for 24 hours.As the concentration increased,the cell viability first decreased,then increased and then decreased（P<0.01）,with an IC 50 value of 85.36μmol/L.1/3 concentration of IC 50 value was chosen:25μmol/L was used to intervene in AFB₁ infected 30 generations of cells.（2）The CCK8 test results showed that compared with the 30-generation cell group,the cell viability of the SB203580group was reduced,and the difference was statistically significant（P<0.01）.（3）The cell cycle test results showed that compared with the 30-generation cell group,the ratio of G0/G1 phase cells in the SB203580 group increased,the ratio of S phase cells decreased,the ratio of G2/M phase cells decreased,and PI decreased.The differences were statistically significant（P<0.05）.（4）The apoptosis test results showed that compared with the 30-generation cell group,the total apoptosis rate of the SB203580 group increased（P<0.05）.3.Function-related protein expression of HEEC malignantly transformed cell lines:（1）The results of detection of EMT-related proteins showed that the difference of the expression of E-cadherin protein in the control group,the 30 generation cell group and the SB203580 group was statistically significant（P<0.05）.There was no statistically significant difference in N-cadherin protein（P>0.05）.Compared with the control group,the expression of E-cadherin protein in the 30-generation cell group was reduced（P<0.05）.Compared with the 30-generation cell group,the expression of N-cadherin protein in the p38 protein inhibitor intervention cell group increased（P<0.05）.（2）The detection of cell cycle-related proteins showed that there were statistically significant differences in the expression of cyclin A2 and cyclin D1 in the control group,the 30-generation cell group and the SB203580 group（P<0.05）,and the difference in the expression of CDK2 protein was not statistically significant.（P>0.05）.Compared with the control group,the expression of cyclin A2 protein in the 30-generation cell group was slightly increased（P<0.05）.Compared with the 30-generation cell group,the expression of cyclin A2 protein in the SB203580 group decreased（P<0.05）,and the expression of cyclin D1 protein increased（P<0.05）.（3）The expression of p38 and p-p38 proteins in the control group,the 30generation cell group and the SB203580 group were significantly different（P<0.05）.Compared with the control group,the expression of p-p38 protein in the 30-generation cell group was increased（P<0.05）.Compared with the 30-generation cell group,the expression of p38 and p-p38 protein in the SB203580 group was significantly reduced（P<0.05）.Conclusion:（1）AFB₁ can inhibit the activity of HEEC in a dose-dependent manner.After 30 passages of cells infected with 1μmol/L AFB₁,the cell morphology changes similar to EMT;the cell proliferation rate increases;the cell cycle differentiates from G0/G1 phase to S phase;there is no significant change in the total cell apoptosis rate;cell migration and invasion capabilities are enhanced,and there is a tendency for malignant transformation.（2）Under the intervention of low-dose p38 protein inhibitor,the cell survival rate increased temporarily.As the concentration gradually increased,the cell survival rate decreased and the inhibitory effect increased.25μmol/L p38 protein inhibitor treated 1μmol/L AFB₁-infected cells for30 generations,the cell viability decreased,the G0/G1 phase was blocked,the apoptosis rate increased,and it inhibited cell proliferation.SB203580 can inhibit the proliferation of cells infected with AFB₁for 30 generations.Therefore,AFB₁can pass through the p38 MAPK signaling pathway,leading to an increase in the proliferation capacity of HEEC and a tendency for malignant transformation.（3）1μmol/L AFB₁ can down-regulate the expression of E-cadherin protein and up-regulate the expression of cyclin A2 protein during 30 generations of HEEC exposure,causing changes in indicators such as cycle,thereby promoting cell proliferation.At the same time,by up-regulating the expression of p-p38 protein,it shows that AFB₁ can act on the p38 MAPK signaling pathway to play its role.25μmol/L SB203580 can inhibit the p38 MAPK signaling pathway by down-regulating the expression of p38 protein and p-p38 protein.And 25μmol/L SB203580 can up-regulate the expression of E-cadherin protein and down-regulate the expression of cyclin A2 protein,thereby inhibiting cell proliferation.Therefore,AFB₁ can down-regulate the expression of E-cadherin protein and up-regulate the expression of cyclin A2 protein by acting on the p38 MAPK signaling pathway.This results in EMT-like changes in cell morphology,cell cycle transition from G0/G1 phase to S phase,increased invasion and migration capacity,increased cell proliferation capacity,and a trend of malignant transformation.