参附注射液对肾小管上皮细胞缺氧再复氧损伤的改善作用及机制探讨

Alleviation and Action Mechanism of Shen-Fu Injection for Hypoxia/Reoxygenation Injury in Renal Tubular Epithelial Cell Injury

目的:探讨参附注射液(SF)对肾小管上皮细胞缺氧再复氧损伤的改善作用及机制。方法:将人近端肾小管上皮细胞系HK-2细胞分为5组:正常对照组、单纯缺氧再复氧模型组、低浓度参附组(1%)、中浓度参附组(2%)、高浓度参附组(5%)。观察各组细胞在缺氧4h再复氧8h刺激下的反应。用流式细胞仪碘化丙啶(PI)染色法和TUNEL检测细胞凋亡比例,激光共聚焦显微镜检测经Fluo-3AM标记的细胞内钙浓度,ELISA检测细胞培养液中MCP-1的浓度。结果:经流式细胞仪检测,缺氧再复氧刺激后细胞凋亡率达(32.97±1.19)%,明显高于正常对照组(P<0.01),SF可显著降低细胞凋亡率,并呈剂量依赖性(P<0.01),5%SF组凋亡率最低,与单纯模型组相比有统计学差异(P<0.01);TUNEL检测的细胞凋亡率变化趋势与流式细胞术一致;缺氧再复氧刺激后细胞内钙荧光值升高,达140.18±3.62,SF可降低胞内荧光值,并呈剂量依赖性(P<0.01),5%SF组荧光值最低61.77±4.82,与单纯模型组相比有统计学差异(P<0.01);HK-2细胞正常情况下分泌少量的MCP-1(12.04±0.44)pg/ml,在缺氧再复氧刺激下分泌量增加至(27.12±3.83)pg/ml。SF有抑制肾小管上皮细胞分泌MCP-1的作用(P<0.05),但未观察到剂量依赖性(P>0.05)。结论:参附注射液通过降低缺氧再复氧诱导的肾小管上皮细胞的凋亡率、钙超载及单核细胞趋化蛋白-1(MCP-1)的分泌发挥改善肾缺血再灌注损伤的作用。

Objective：To investigate the alleviation and action mechanism of Shen- Fu injection for hypoxia/reoxygenation injury in renal tubular epithelial cell injury. Methods：Renal tubular epithelia cells HK- 2 were dividec into 5 groups： the normal control group,the model group of hypoxia/reoxygenation, SF low dose group（ 1% ） ,SF middle dose group （2%） and SF high dose group （5%）.Alteration of each group was observed after hypoxia for 4h and then rcoxygenation for 8h. Apoptosis rate was measured with flow cytornetry,TUNEL and propidium iodide（PI） staining. Intracellular calcium concentration was measured with Fluo- 3AM marking uand confocal laser scanning microscopy. Concentraiton of MCP - 1 in culture matrix was mesured by ELISA. Resnlts： Apoptosis rate measured by flow cytometry of model group ceils reached （32.97 ± 1.19） %, which was obviously higher than that in the normal control group[ （3.60 ± 0.80） %, P 〈 0.01 ]. SF could decrease apoptosis rate in a dose - dependent manner. The apoptosis rate in the high dose group was the lowest, which was significant with that in the model group [ （ 11.93 ± 0.51 ） % vs, （32.97 ± 1.19） %, P 〈 0.01 ]. The alteration trend of atx^ptosis rate measured by TUNEI was consistent with that measured by flow cytometry. After hypoxia/reoxygenation stimulation, intracellular fluorescence value elevated, reaching 140.18 ± 3.62. SF could decrease the intracellular fluorescence value in a dose - dependent manner（P 〈 0.01 ）. The intracellular fluorescence value in the hight dose group was the lowest, reaching 61.77 ± 4.82, which was significant with that in the model group （P 〈 0.01 ）. Under nomal condition, HK - 2 cell secreted slight MCP - 1 at （ 12.04 ＋ 0.44） pg/ml, which increased to （27.12 ± 3.83） pg/ml after hypoxia/reoxygenation stimulation. SF was effective to inhibit MCP- lsecretion in the tubular epithelial cells（ P 〈 0.05）. Dosedependant effect was not observed（P〉0.05）. Conclusion： Shen- Fu injection allevitated renal ischemic reperfusion injury by decrease apoptosis induced by hypoxia/reoxygenation, calcium overload and excretion of MCP- 1.