紫草素抑制缺血再灌注肾损伤后肾小管细胞的增殖和迁移

Shikonin inhibited the proliferation and migration of the renal tubular cells after the renal ischemia-reperfusion injury

目的探讨丙酮酸激酶M2(PKM2)抑制剂紫草素对肾脏缺血再灌注损伤(IRI)后肾小管修复的影响。方法8周龄雄性C57BL/6J小鼠30只随机分为6组:假手术的Sham溶剂组和Sham紫草素组,双侧肾脏IRI模型的bIRI溶剂1 d组、bIRI紫草素1 d组、bIRI溶剂3 d组、bIRI紫草素3 d组。肾脏IRI模型建立,采用双侧肾蒂夹闭28 min处理,术后4 h腹腔注射3 mg/kg紫草素或溶剂,术后1 d或3 d处死小鼠留取样本。检测各组小鼠血清肌酐、尿素氮水平;通过PAS染色观察肾组织病理学改变;免疫荧光检测丙酮酸激酶M2、增殖细胞核抗原(PCNA)、翅荚百脉根凝集素(LTL)表达;Western印迹检测肾脏的PCNA表达。体外实验,人近端肾小管上皮HK2细胞分成8组:对照组(不做缺氧处理)、模型组(缺氧/复氧处理)、模型+紫草素剂量组(缺氧/复氧后给予0.5μM、1.0μM、1.5μM、2.0μM、3.0μM、7.0μM紫草素干预)。采用细胞计数试剂盒检测细胞存活率、细胞生长抑制率。细胞划痕实验检测细胞迁移力。结果肾小管损伤面积评分:bIRI紫草素1 d组高于bIRI溶剂1 d组(P<0.01),bIRI紫草素3 d组高于bIRI溶剂3 d组(P=0.0337),而假手术的Sham溶剂组和Sham紫草素组之间没有明显差别。免疫荧光(P=0.0331)和免疫印迹(P=0.0228)结果均显示,bIRI紫草素3 d组小鼠肾组织的PCNA表达较bIRI溶剂3 d组明显减少。在体外实验中,与模型组比较,模型+紫草素剂量组(1.5μM、2.0μM、3.0μM、7.0μM紫草素干预)的HK2细胞存活率显著降低(P<0.001),细胞生长抑制率升高(P<0.001)。细胞划痕实验显示,1.5μM紫草素能够显著抑制复氧后肾小管细胞的迁移(P<0.001)。结论肾脏IRI损伤后,紫草素可能通过减少肾小管上皮细胞的再生和迁移来抑制肾小管修复,从而加重肾损伤。

Objective To investigate the effect of pyruvate kinase M2(PKM2)inhibitor shikonin on renal tubular repair after renal ischemia-reperfusion injury(IRI).Methods Thirty 8-week-old male C57 BL/6 J mice were randomly divided into 6 groups:sham solvent group(sham-operation plus solvent),sham shikonin group(sham-operation plus shikonin);bIRI solvent 1 d group(bilateral IRI plus solvent 1 d)and bIRI shikonin 1 d group(bilateral IRI plus shikonin 1 d);bIRI solvent 3 d group(bilateral IRI plus solvent 3 d),bIRI shikonin 3 d group(bilateral IRI plus shikonin 3 d).To establish the renal IRI model,bilateral renal pedicles were clipped for 28 min,and 3 mg/kg shikonin or solvent was intraperitoneally injected 4 h after the operation.The mice were sacrificed 1 or 3 days after the operation for samples collection.The serum creatinine and blood urea nitrogen levels of mice in each group were measured.The renal histopathological changes were observed by PAS staining.The expressions of PKM2,proliferating cell nuclear antigen(PCNA),and Lotus tetragonolobus lectin(LTL)were detected by immunofluorescence.Western blotting was used to detect renal PCNA expression.For the in vitro experiments,the human proximal tubular epithelial HK2 cells were divided into 8 groups:control group(without hypoxia treatment),model group(with hypoxia/reoxygenation treatment),shikonin doses groups(hypoxia/reoxygenation plus shikonin of 0.5μM,1.0μM,1.5μM,2.0μM,3.0μM,and 7.0μM).The cell survival rate and cell growth inhibition rate were detected by the cell counting kit 8(CCK-8).The scratch wound healing assay was applied to detect cell migration ability.Results The renal tubular injury area scores in the b IRI shikonin 1 d group and the b IRI shikonin 3 d group,were higher than those in the b IRI solvent 1 d group and b IRI solvent 3 d group,respectively(P=0.0337),while the renal tubular injury area scores in the sham solvent group and the sham shikonin group did not differ from each other significantly.Both immunofluorescence(P=0.0331)and western blotting(P=0.0228)results showed that the expression of PCNA in the kidneys of mice was significantly lower in the b IRI shikonin 3 d group than in the b IRI solvent 3 d group.During the in vitro experiments,compared with the model group,the shikonin doses groups(1.5μM,2.0μM,3.0μM,and 7.0μM)showed significantly lower survival rates of HK2 cells(P<0.001),as well as higher rates of cell growth inhibition(P<0.001).The scratch wound healing assay displayed that shikonin of 1.5μM could obviously inhibit the migration of renal tubular HK2 cells after reoxygenation(P<0.001).Conclusion After renal IRI injury,shikonin may inhibit the renal tubular repair process through reducing the regeneration and migration of renal tubular epithelial cells,thereby aggravating the renal injury.