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目的:观察蟾蜍灵对体外庆大霉素诱导的大鼠肾小管上皮细胞自噬的影响。方法:体外培养大鼠肾小管上皮细胞,分为正常对照组、庆大霉素组(2 mg/m L)、蟾蜍灵组(1×10~(-8)mol/L)、庆大霉素+蟾蜍灵组(庆大霉素2 mg/mL+蟾蜍灵1×10~(-8) mol/L)。应用透射电镜观察各组大鼠肾小管上皮细胞的自噬水平;Western blot检测庆大霉素及蟾蜍灵对肾小管上皮细胞自噬标志蛋白LC3Ⅱ、P62及肾损伤分子1(kindey injury molecule-1,KIM1)表达的影响。结果:透射电镜下观察:与对照组相比,庆大霉素组自噬相关形态指标自噬体、自噬泡等数量明显增加;蟾蜍灵干预后,细胞自噬体数量较庆大霉素组明显减少。Western blot结果显示,与对照组相比,庆大霉素组自噬标志蛋白LC3Ⅱ、P62、KIM1的蛋白相对表达量均升高(P<0.05),蟾蜍灵干预后,LC3Ⅱ、P62及KIM1蛋白相对表达量较庆大霉素组下降(P<0.05)。结论:庆大霉素可促进大鼠肾小管上皮细胞发生自噬、提高KIM1蛋白的表达;蟾蜍灵可部分缓解庆大霉素诱导的肾小管上皮细胞自噬的增加及KIM1蛋白表达量的提高,提示这可能是其发挥肾脏保护作用的机制之一。
Objective: To observe the effect of bufalin on gentamicin-induced autophagy in rat renal tubular epithelial cells in vitro. Methods: Rat renal tubular epithelial cells were cultured in vitro and divided into normal control group, gentamicin group (2 mg / m L), bufalin group (1 × 10 -8 mol / L) (+ Gentamicin 2 mg / mL + buprenorphine 1 × 10 ~ (-8) mol / L). The autophagy of renal tubular epithelial cells in each group was observed by transmission electron microscopy. The effects of gentamicin and bufalin on the expression of autophagy marker proteins LC3 Ⅱ, P62 and kindey injury molecule-1 , KIM1) expression. Results: Compared with the control group, the number of autophagy-related morphological indexes such as autophagosomes and autophagic vacuoles in gentamicin group was significantly increased as compared with the control group. The amount of autophagosomes in gentamicin group was significantly higher than that of gentamicin Group significantly reduced. Western blot results showed that compared with the control group, the protein relative expression of LC3Ⅱ, P62 and KIM1 in gentamicin group was increased (P <0.05), LC3 Ⅱ, P62 and KIM1 protein The relative expression decreased compared with gentamicin group (P <0.05). CONCLUSION: Gentamicin can promote autophagy in rat renal tubular epithelial cells and increase the expression of KIM1 protein. Bufalin can partially relieve the increase of gentamicin-induced autophagy in renal tubular epithelial cells and the increase of KIM1 protein expression , Suggesting that this may be one of its mechanisms to play a protective role in the kidney.