复方丹参注射液减轻缺氧/复氧性HK-2细胞损伤的作用及机制

Role and mechanism of Salvia miltiorrhiza Bunge in alleviating injury induced by hypoxia/reoxygenation in HK-2 cells

摘要:

目的：探讨丹参注射液减轻缺氧/复氧性人肾近曲小管上皮细胞（HK-2）细胞损伤的作用机制。方法：以人近曲肾小管上皮细胞为研究对象，采用液体石蜡覆盖法建立缺氧/复氧模型。实验分为3组：对照组、损伤组和丹参组，每个组均设立7个时间点：缺氧4，12，24 h和缺氧24 h后复氧4，12，24，48 h，其中丹参组设立4个浓度亚组（生药浓度）：0.05%，0.10%，0.15%，0.20%。在倒置相差显微镜下观察细胞的形态学改变，MTT法进行活细胞计数，生化法检测培养上清液中乳酸脱氢酶（LDH）含量，放射免疫法检测肿瘤坏死因子α（TNF-α）的含量。结果：缺氧处理后，损伤组在各时间点细胞数量均明显降低，在缺氧24 h达最低值；与损伤组比较，在丹参各组中，HK-2细胞数量较多，其中以0.10%丹参组细胞数量在各时间点均为最高。与对照组比较，损伤组LDH，TNF-α水平升高，在缺氧24 h/复氧4 h达最高值；与损伤组相比，丹参组在缺氧/复氧过程各时间点中LDH，TNF-α水平降低。结论：复方丹参注射液对HK-2细胞缺氧／复氧性损伤具有保护作用，作用机制可能与丹参注射液抑制炎症细胞因子的产生有关。［中国当代儿科杂志，2007，9（6）：559-562］

Abstract:

OBJECTIVE: Salvia miltorrhiza Bunge (SMB) is a traditional Chinese herb, which is considered to promote blood flow and remove blood stasis. This study examined whether SMB can alleviate injury induced by hypoxia/reoxygenation (H/R) in human kidney proxiamal tubular cells-2 (HK-2 cells).MethodsThere were 3 experimental groups: control, H/R injury and SMB-treated H/R injury. H/R injury of HK-2 cells was induced by first covering the cells with and then removing liquid paraffin wax. Different concentrations of compound SMB solution (0.05%, 0.10%, 0.15% or 0.20%) were administered to the SMB-treated H/R injury group before the hypoxic injury. After 4, 12 and 24 hrs of hypoxia and 4, 12, 24 and 48 hrs of reoxygenation, morphologic changes of HK-2 cells were observed under an inverted microscope. Cell viability was measured by the MTT method. Lactate dehydrogenase (LDH) activity in the culture supernatants was assayed using biochemical methods; TNF-α levels were determined by radioimmunoassay (RIA). RESULTS: The number of HK-2 cells was significantly reduced in the H/R injury group after hypoxia, and reached a nadir 24 hrs after hypoxia treatment. Various concentrations of SMB-treated groups showed significantly greater number of HK-2 cells than the H/R injury group. SMB solution (0.10%) produced the best effect. The levels of LDH and TNF-α in the H/R injury group were significantly increased, and reached a peak between 24 hrs of hypoxia and 4 hrs of reoxygenation when compared to the control group. Pre-treating with 0.10% SMB resulted in significantly lower levels of LDH and TNF-α than in the untreated H/R injury group at various time points of H/R.CONCLUSIONS: SMB has protective effects against H/R injury of HK-2 cells, possibly through inhibition of inflammatory cytokines.［Chin J Contemp Pediatr, 2007, 9 (6):559-562］