PbK173青蒿素敏感性不同虫株的染虫红细胞柔韧性差异分析

Differential Analysis of Erythrocyte Flexibility of PbK173 Artemisinin-sensitive Strains

目的:检测对青蒿素类药物敏感程度不同的伯氏疟原虫K173(PbK173)染虫红细胞柔韧性差异,并探索其差异的深层机制。方法:采用102只SPF级雄性C57小鼠随机分为3组,空白组和青蒿素抗性株(PbK173-R)组每组30只,青蒿素敏感株(PbK173-S)组42只。除空白组30只外,其余接种1×10^(7)个PbK173-S/PbK173-R的染虫红细胞建立鼠疟模型。其中给药期和恢复期(空白组、PbK173-R/PbK173-S)给予双氢青蒿素(DHA,40 mg·kg^(-1)),咯萘啶(MD,6 mg·kg^(-1))连续给药4 d。PbK173-S/PbK173-R组虫率≥20%,取外周血;给药结束第1天(给药期)和给药结束第5天(恢复期)取外周血和各脏器,检测各组血液参数和脏器指数;红细胞渗透脆性法检测各组外周血红细胞渗透脆性;蛋白免疫印迹法检测红细胞膜上的Piezo1、Band3蛋白的水平变化。结果:给药期和恢复期,PbK173-S MD组与DHA组差异均无统计学意义;给药期MD组PbK173-S与PbK173-R各血液学参数差异均无统计学意义,但恢复期MD组,PbK173-R染虫鼠的红细胞计数、血红蛋白浓度、红细胞压积均明显高于PbK173-S染虫鼠(P<0.05);与空白组比较,PbK173-S/PbK173-R组渗透脆性显著增强(P<0.01),且PbK173-S组的渗透脆性显著强于PbK173-R组(P<0.01);给药期PbK173-S组红细胞渗透脆性显著强于给药期空白组和给药期PbK173-R组(P<0.01);恢复期PbK173-R组红细胞渗透脆性明显高于给药期空白组和恢复期PbK173-S组(P<0.05);与空白组比较,PbK173-S组红细胞膜上的Piezo1蛋白和Band3蛋白显著降低(P<0.01);与PbK173-R组比较,PbK173-S组红细胞膜上的Piezo1蛋白和Band3蛋白显著降低(P<0.01)。结论:对青蒿素类药物敏感程度不同的PbK173染虫红细胞的柔韧性存在差异,疟原虫感染红细胞能够明显降低红细胞膜上的Piezo1蛋白、Band3蛋白水平;且正常小鼠、PbK173-R染虫鼠、PbK173-S染虫鼠红细胞柔韧性依次下降。

Objective:To detect the flexibility differences of Plasmodium berghei K173(PbK173)-infected red blood cells with varying degrees of sensitivity to artemisinin-based drugs and to preliminarily explore the underlying mechanisms of the differences.Method:A total of 102 specific-pathogenfree(SPF) male C57BL/6 mice were randomly divided into three groups,with 30 mice each in the control group and PbK173-resistant(PbK173-R) group,and 42 mice in the PbK173-sensitive(PbK173-S) group.Except for the control group,the rest groups were vaccinated with 1×10^(7) PbK173-S/PbK173-R infected red blood cells to establish a mouse malaria model.During the administration and recovery periods(control group,PbK173-R/PbK173-S),dihydroartemisinin(DHA,40 mg·kg^(-1)) and malaridine(MD,6 mg·kg^(-1)) were administered continuously for four days.Peripheral blood was taken from the PbK173-S/PbK173-R groups with an infection rate equal to or greater than 20%.Peripheral blood and each organ were taken on the first day at the end of administration(dosing period) and on the fifth day at the end of administration(recovery period),and blood parameters and organ indices of each group were examined.The osmotic fragility of peripheral blood red blood cells in each group was detected using the red blood cell osmotic fragility test.Western blot was applied to determine the levels of Piezo1 and Band3 proteins in the red blood cell membrane.Result:During the administration and recovery periods,there were no significant differences between the PbK173-S MD group and the DHA group.During the administration period,there were no significant differences in hematological parameters between PbK173-S and PbK173-R in the MD group.However,during the recovery period,the red blood cell count,hemoglobin concentration and hematocrit of the PbK173-R group were significantly higher than those of the PbK173-S group(P<0.05) in the MD group.Compared with that of the control group,the osmotic fragility of the PbK173-S/PbK173-R groups was significantly enhanced(P<0.01),and the osmotic fragility of the PbK173-S group was significantly stronger than that of the PbK173-R group(P<0.01).The osmotic fragility of red blood cells in the PbK173-S group during the administration period was significantly stronger than that in the control group and PbK173-R group during the administration period(P<0.01).The osmotic fragility of red blood cells in the PbK173-R group during the recovery period was significantly higher than that in the control group during the administration period and the PbK173-S group during the recovery period(P<0.05).Compared with those in the control group,the Piezo1 protein and Band3 protein in the red blood cell membrane of the PbK173-S group were significantly reduced(P<0.01).Compared with those in the PbK173-R group,the Piezo1 protein and Band 3 protein in the red blood cell membrane of the PbK173-S group were significantly reduced.Conclusion:The flexibility of PbK173-infected red blood cells with different sensitivities to artemisinins differed.Plasmodium-infected red blood cells significantly reduced the levels of Piezo1 and Band3 proteins in the red blood cell membrane,and the erythrocyte flexibility exhibited a decreasing trend in the following order:normal group,PbK173-R group,and PbK173-S group.