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[摘要]
目的 探究龙血竭胶囊对肾缺血再灌注损伤的大鼠的保护作用及其机制。方法 将50只SD大鼠随机分为假手术组、模型组和龙血竭胶囊0.081、0.162、0.324 g/kg组,每组10只。龙血竭胶囊组将各剂量药物分别溶解于生理盐水中,ig 10 mL/kg(人临床等效剂量),模型组和假手术组大鼠灌胃给予相应剂量的生理盐水。7 d后,除假手术组外,另外4组大鼠都建立肾缺血再灌注损伤模型。肾脏缺血再灌注后24 h,检测血清肌酐(Scr)、尿素氮(BUN)、白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)和干扰素-γ(IFN-γ)水平。TUNEL法检测肾小管上皮细胞凋亡率。采用免疫组化法检测肾组织FAS、FASL蛋白的表达,Western blotting法检测细胞凋亡相关蛋白的表达。结果 与模型组大鼠比较,龙血竭胶囊各剂量组大鼠血清中的Scr、BUN显著降低(P<0.01),且龙血竭胶囊0.324 g/kg组大鼠血清中的IL-6、TNF-α、IFN-γ显著下降(P<0.05)。与模型组大鼠比较,龙血竭胶囊组大鼠细胞凋亡率均有所下降,但龙血竭胶囊0.324 g/kg组下降更为显著(P<0.01)。与模型组比较,龙血竭胶囊组FAS和FASL蛋白的表达量均显著降低(P<0.05),且降低的程度具有剂量相关性,剂量越大降低的趋势就越明显。与模型组比较,龙血竭胶囊各剂量组的大鼠Bax、Bad、Caspase-3、Caspase-8、Caspase-9蛋白的表达量降低,而Bcl-2、VEGF蛋白的表达量升高;龙血竭胶囊0.081 g/kg时Caspase-3、Caspase-8、Caspase-9、Bcl-2、VEGF及龙血竭胶囊0.162 g/kg时Caspase-3的蛋白表达与模型组的差异无统计学意义,而其余剂量给药组大鼠蛋白的表达与其差异均具有统计学意义(P<0.05)。结论 龙血竭胶囊对肾缺血再灌注损伤大鼠具有保护作用,其机制可能是调节炎症因子从而抑制炎症反应,以及调节细胞凋亡和增殖的相关蛋白表达量来减轻组织损伤。
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[Abstract]
Objective To investigate protective effect and its mechanism of Longxuejie Capsules on renal ischemia reperfusion injury in rats.Methods SD rats were divided into Sham group, model group, and Longxuejie Capsules (0.081, 0.162, and 0.324 g/kg) groups, and each group had 10 rats. In Longxuejie Capsules group, each dose of drugs was dissolved in normal saline, respectively, and ig administered with Longxuejie Capsules 10 mL/kg (human clinical equivalent dose) for 7 d. And the rats in model group and Sham groups were ig administered with corresponding doses of saline. After treatment for 7 d, renal ischemia reperfusion injury models were established in all the other 4 groups of rats except the Sham group. Twenty-four hours after renal ischemia reperfusion, the levels of Scr, BUN, IL-6, TNF-α, and IFN-γ were measured. TUNEL assay was used to detect the apoptosis rate of renal tubular epithelial cells. Immunohistochemistry was used to detect the expression of FAS and FASL proteins in renal tissue, and Western blot ting method was used to detect the expression of apoptosis-related proteins.Results Compared with model group, serum Scr and BUN in each dose group of Longxuejie Capsules groups were significantly decreased (P<0.01), and the IL-6, TNF-α and IFN-γ in Longxuejie Capsules (0.324 g/kg) group were significantly decreased (P<0.05). Compared with model group, the apoptosis rates of renal tubular epithelial cells in Longxuejie Capsules groups were decreased, but the apoptotic rate in Longxuejie Capsules (0.324 g/kg) group was more significantly decreased (P<0.01). Compared with model group, the expression levels of FAS and FASL protein in Longxuejie Capsules groups were significantly reduced, and the degree of reduction was dose-dependent. And the larger dose, the more obvious trend of reduction is. Compared with model group, the expression levels of Bax, Bad, caspase-3, caspase-8, and caspase-9 in each dose group of Longxuejie Capsules were decreased, but the expression levels of bcl-2 and VEGF were increased. There was no significant difference in the protein expression of Caspase-3, Caspase-8, Caspase-9, Bcl-2, and VEGF at 0.081 g/kg, and the protein expression of Caspase-3 at 0.162 g/kg of Longxuejie Capsules groups. While the protein expression of the other dose groups were statistically significant (P<0.05).Conclusion Longxuejie Capsules has protective effect on renal ischemia reperfusion injury in rats, which may be caused by the regulation of inflammatory factors to inhibit the inflammatory response, and the regulation of apoptosis and proliferation of related protein expression to reduce tissue damage.
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[基金项目]
国家临床重点专科基金资助项目(财社[2013]239号)