| Objective:Environmental factors play an important role in the development of Primary Biliary Cholangitis(PBC).This study intends to explore the effects and possible mechanisms of 3,3?,4,4?,5-pentachlorobiphenyl(PCB126)on natural killer(NK)cells from peripheral blood in patients with PBC.Methods:Twenty PBC patients admitted to the department of rheumatology and twenty healthy subjects at the same time in our hospital were selected in the study.NK cells were isolated by magnetic beads sorting.The separation efficiency of NK cells and the expression levels of NK cells surface molecules and lysis-associated molecules with PCB126 were detected by flow cytometry.The aryl hydrocarbon receptor(AhR)expression and cytokines in NK cells were determined by RT-PCR,Western Blot and ELISA.The cytotoxicity of NK cells against human intrahepatic biliary epithelial cells(HiBEC)was assessed by CCK8 assay and flow cytometry.Results:NK cells were isolated from peripheral blood by magnetic beads sorting,and the purity of CD3~-CD56~+NK cells was beyond 95%.The experimental results showed that the IL6 m RNA(0.993±0.223 vs 0.660±0.150,p>0.05),AhR mRNA(2.230±0.371 vs 1.595±0.372,p>0.05)and CYP1A1 m RNA(1.270±0.189 vs 1.062±0.268,p>0.05)in NK cells from PBC patients were both increased compared with healthy subjects absent PCB126 stimulation,without statistical differences.After PCB126 stimulation,the IL6 mRNA(1.811±0.202 vs 1.129±0.120,p<0.01),AhR mRNA(4.884±0.794 vs 3.067±0.379,p<0.05)and CYP1A1 mRNA(2.207±0.271 vs1.430±0.263,p<0.05)in NK cells from PBC patients were significantly higher than healthy subjects,with statistically significant differences.On the protein level,it was found that without PCB126 stimulation,the AhR protein(0.839±0.109 vs 0.645±0.134,p>0.05)and CYP1A1 protein(0.773±0.169 vs 0.612±0.237,p>0.05)in NK cells from PBC patients were slightly higher than healthy subjects,and the difference was not statistically significant.After PCB126 stimulation,the AhR protein(1.786±0.146 vs 1.247±0.098,p<0.05)and CYP1A1 protein(1.911±0.100 vs 1.347±0.108,p<0.05)in NK cells from PBC patients were significantly higher than healthy subjects,and the difference was statistically significant.Without PCB126 stimulation,there were no significant difference in the percentage of CD69~+NK cells(21.867±1.534%vs 16.167±1.357%,p>0.05)and NKp44~+NK cells(39.097±2.000%vs37.550±1.156%,p>0.05)between PBC patients and healthy subjects.After PCB126 stimulation,the percentage of CD69~+NK cells(60.867±2.458%vs 42.733±2.401%,p<0.01)and NKp44~+NK cells(69.757±2.165%vs 45.833±2.441%,p<0.01)from PBC patients were significantly higher than healthy subjects.Without PCB126 stimulation,the percentage of Perforin~+NK cells(35.030±1.573%vs 32.780±1.571%,p>0.05)and Granzyme B~+NK cells(41.370±1.554%vs 36.893±1.861%,p>0.05)from PBC patients were higher than healthy subjects,but the difference was not statistically significant.After PCB126 stimulation,the percentage of Perforin~+NK cells(47.497±2.361%vs 39.297±1.237%,p<0.05)and Granzyme B~+NK cells(50.700±1.440%vs 44.383±1.697%,p<0.05)from PBC patients were significantly higher than healthy subjects,and the difference was statistically significant.The expression levels of IFN-?(156.517±7.362 pg/ml vs 145.542±27.444 pg/ml,p>0.05)and TNF-?(71.238±4.691 pg/ml vs 59.917±3.197 pg/ml,p>0.05)in NK cells of PBC patients were slightly higher than healthy subjects without PCB126 stimulation.However,after PCB126 stimulation,there was a more obvious increase trend of expression levels of IFN-?(214.23±9.644 pg/ml vs 177.16±12.217 pg/ml,p<0.05),TNF-?(95.983±4.993 pg/ml vs 69.255±4.331 pg/ml,p<0.001)in NK cells from PBC patients.And compared with healthy subjects after PCB126 stimulation,the difference was statistically significant.In healthy subjects,the cytotoxicity of NK cells and the apoptosis of HiBEC were enhanced after PCB126 stimulation.In PBC patients,the cytotoxicity of NK cells and the apoptosis of HiBEC with PCB126 stimulation were both enhanced compared with PCB126 unstimulation.Moreover,it was found that in the co-culture system of NK cells of PBC patients and HiBEC both stimulated with PCB126,the cytotoxicity of NK cells and the apoptosis of HiBEC were the strongest.Conclusion:1.PCB126 induced up-regulation of AhR,CYP1A1 and IL6 in NK cells from PBC patients,and suggested that PCB126 might activate AhR pathway of NK cells.2.PCB126 induced the activation of NK cells,and promoted the expression of lysis-associated molecules and inflammatory factors in PBC patients through AhR.3.The cytotoxicity of NK cells and the ability of inducing HiBEC apoptosis were enhanced in PBC patients after PCB126 stimulation,PCB126 might accelerate the progression of PBC disease through direct toxicity and indirect toxicity that enhance the cytotoxicity of NK cells against HiBEC. |
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