小檗碱预先给药对缺氧／复氧诱导人肾小管上皮细胞凋亡的影响

Effect of berberine pretreatment on hypoxia/reoxygenation-induced apoptosis in human renal tubular epi- thelial cells

目的探讨小檗碱预先给药对缺氧／复氧诱导人肾小管上皮细胞凋亡的影响。方法培养人肾小管上皮细胞（HK-2），以1×10^6个／ml密度接种于培养皿（2ml／皿）和96孔培养板（200μl/孔），采用随机数字表法，将其分为4组（n=30）：正常对照组（c组）、小檗碱组（B组）、缺氧／复氧组（H／R组）和缺氧，复氧＋小檗碱组（H／R＋B组）。B组和H／R＋B组加入10μmol／L小檗碱孵育2h，随后H／R组和H／R＋B组进行缺氧24h复氧3h。测定细胞活力、细胞凋亡率、丙二醛（MDA）、超氧化物歧化酶（SOD）、caspase-3、活化caspase-3、细胞色素c、葡萄糖调节蛋白78（GRP78）和C／EBP同源蛋白（CHOP）表达，并计算Bax表达与Bcl-2表达的比值（Bax／Bcl-2）。结果与c组比较，H／R组和H／R＋B组细胞活力、SOD活性和caspase-3表达降低，细胞凋亡率、MDA浓度和Bax／Bcl-2升高，活化caspase-3、细胞色素c、GRP78和CHOP表达上调（P〈0．05），B组上述指标差异无统计学意义（P〉0．05）；与H／R组比较，H／R＋B组细胞活力、SOD活性和caspase-3表达升高，细胞凋亡率、MDA浓度和Bax／Bcl-2降低，活化caspase-3、细胞色素c、GRP78和CHOP表达下调（P〈0．05）。结论小檗碱预先给药可抑制缺氧／复氧诱导人肾小管上皮细胞凋亡，其机制与抑制线粒体应激通路和内质网应激通路有关。

Objective To investigate the effect of berberine pretreatment on hypoxia/reoxygenation （H/ R）-induced apoptosis in human renal tubular epithelial cells. Methods The human renal tubular epithelial cells were cultured and seeded in culture dishes （2 ml/dish） or 96-well plates （200 μl/well） with the density of 1 ×10^6/ml. The cells were then randomly divided into 4 groups （ n = 30 each） ： normal control group （group C）, ber- berine group （group B）, H/R group and H/R ＋ berberine group （H/R ＋ B group）. In groups B and H/R ＋ B, berberine 10 μmol/L was added to the culture medium and the cells were incubated for 2 h. Groups H/R and H/R ＋ B were then exposed to 94% N2-5% CO2-1%O2 for24 h followed by 3 h reoxygenation. The cell viability, ap- optotic rate and level of malondialdehyde （MDA） and superoxide dismutase （SOD） were detected. The expression of caspase-3, activated caspase-3, cytochrome c, glucose-regulated protein 78 （GRP78） and C/EBP homologous protein （CHOP） was determined. The ratio of Bax/Bcl-2 was ealeulated. Results Compared with group C, the cell viability, SOD activity and easpase-3 expression were signifieantly decreased, the apoptotic rate, Bax/Bel-2 ratio and concentration of MDA were increased, and the expression of activated easpase-3, cytoehromc c, GRP78 and CHOP was up-regulated in groups H/R and H/R ＋ B （ P 〈 0.05）. Compared with group H/R, the cell viabili- ty, SOD activity and caspase-3 expression were significantly increased, the apoptotie rate, Bax/Bcl-2 ratio and concentration of MDA were decreased, and the expression of activated caspase-3, cytochrome c, GRP78 andCHOP was down-regulated in group H/R ＋ B （ P 〈 0.05）. Conclusion Berberine pretreatment can inhibit HIR- induced apoptosis in human renal tubular epithelial cells, and inhibition of mitochondrial stress pathway and endo- plasmic reticttlum stress pathway is involved in the mechanism.