原儿茶醛对环磷酰胺诱导急性肾损伤的保护作用及机制研究

Preventive mechanism of protocatechuic aldehyde on cyclophosphamideinduced acute kidney injury via anti-apoptosis

目的研究原儿茶醛对环磷酰胺(CTX)所致急性肾损伤的预防作用及其机制。方法将24只SPF级雄性昆明种小鼠随机分为对照组、模型组及原儿茶醛低、高剂量(15、30mg·kg^(-1))组,每组各6只。对照组和模型组ig给予0.9%氯化钠溶液,原儿茶醛低、高剂量组每日1次ig对应药物。连续给药14d,末次给药1h后,除对照组外,其余各组单次ip给药CTX(200mg·kg^(-1))。次日,在称体质量麻醉后取血和肾脏,分离血清,检测小鼠血清肌酐(CRE)、超氧化物歧化酶(SOD)和过氧化氢酶(CAT)等生化指标;采用苏木素-伊红(HE)染色观察肾脏病理损伤情况;采用荧光染色检测小鼠肾脏TUNEL的表达情况;采用Westernblotting检测B淋巴细胞瘤2(Bcl-2)和Bcl-2相关X蛋白(BAX)等凋亡相关蛋白的表达情况。结果与对照组比较,模型组小鼠的肾脏指数显著降低(P<0.01);血清CRE水平显著升高(P<0.001);血清SOD、CAT、谷胱甘肽过氧化物酶(GSH-Px)水平显著降低(P<0.05、0.01、0.001),丙二醛(MDA)水平显著升高(P<0.01);模型组小鼠肾脏细胞排列错乱、可见明显的炎症细胞浸润,伴随肾小管空泡变性和肾小管扩张;模型组小鼠肾脏TUNEL阳性表达显著增多(P<0.001);且肾脏天冬氨酸特异性半胱氨酸蛋白酶3(Caspase-3)和Bcl-2的表达显著下降(P<0.01、0.001),而cleaved-Caspase3、BAX蛋白的表达显著上升(P<0.01、0.001)。与模型组比较,原儿茶醛低、高剂量组小鼠肾脏指数均显著增加(P<0.05);原儿茶醛高剂量组小鼠血清中的CRE水平明显降低(P<0.001);原儿茶醛低、高剂量组小鼠血清中SOD、CAT、GSH-Px水平显著上升(P<0.05、0.01、0.001),MDA水平显著下降(P<0.05;原儿茶醛低、高剂量组肾脏组织细胞排列比较规整,炎症细胞浸润及肾小管空泡变性和肾小管扩张等病理改变少见;原儿茶醛低、高剂量组肾脏TUNEL阳性表达有所下降(P<0.01、0.001),Caspase-3和Bcl-2蛋白表达显著上升(P<0.05、0.01、0.001)),cleaved-Caspase-3和BAX蛋白表达显著下降(P<0.01、0.001),呈剂量相关性。结论原儿茶醛可预防CTX所致急性肾损伤,其机制可能与抗细胞凋亡有关。

Objective To explore the preventive effect and mechanism of protocatechuic aldehyde(PAL)on acute kidney injury induced by cyclophosphamide(CTX).Methods Twenty-four SPF male KM mice were randomly divided into normal control group,model group,low,high dose PAL group(15,30 mg·kg^(-1)),with six mice in each group.Except for the normal control group and model group,which were given normal saline by gavage,the mice of other groups were given corresponding drugs by gavage once daily.After 14 d,all the mice were given a single intraperitoneal injection of CTX(200mg·kg^(-1))except for those of normal control group which were injected with normal saline.On the next day,after weighing and anesthesia,blood and kidneys were obtained.The serum was separated to detect biochemical indicators such as creatinine(CRE),superoxide dismutase(SOD),and catalase(CAT).Hematoxylin eosin(HE)staining was conducted to observe the changes in renal pathological damage.Fluorescence staining was made to detect the expression of TUNEL in mice kidneys.Western blotting was adopted to detect the expression of apoptosis-related proteins such as B-cell lymphoma-2(Bcl-2)and Bcl-2-related X protein(BAX).Results Compared with the control group,the kidney index of the mice in the model group was significantly decreased(P<0.01).The serum CRE was significantly increased(P<0.001).The levels of serum SOD,CAT,and GSH-Px were significantly reduced(p<0.05,0.01,0.001),while the level of MDA was significantly increased(P<0.01).The kidney cells of the mice in model group were arranged in a disordered manner,with obvious infiltration of inflammatory cells,accompanied by tubular vacuolar degeneration and tubular dilation.The positive expression of TUNEL in the mice kidneys of the model group were significantly increased(P<0.001).Moreover,the expressions of Caspase-3 and Bcl-2 in the kidneys were significantly decreased(P<0.01,0.001),while the expressions of cleaved-Caspase-3 and BAX were significantly increased(P<0.01,0.001).Compared with the model group,the kidney indexes of the mice in PAL-treated groups were significantly increased(P<0.05).The serum CRE level of the mice in H-PAL-treated group was significantly reduced(P<0.001).The levels of SOD,CAT,and GSH-Px in the serum of the mice in PAL-treated groups were significantly increased(P<0.05,0.01,0.001),while the levels of MDA were significantly decreased(P<0.05).The arrangement of kidney cells in PAL treatment groups was relatively regular,with rare pathological changes such as inflammatory cell infiltration,tubular vacuolar degeneration,and tubular dilation.The positive expression of TUNEL in the kidneys of the PAL treatment groups was decreased(P<0.01,0.001),the expressions of Caspase-3 and Bcl-2 were obviously increased(P<0.05,0.01,0.001),and the expressions of cleaved-Caspase-3 and BAX were remarkably decreased(P<0.01,0.001)in a dose-dependent manner.Conclusion PAL could prevent acute kidney injury caused by CTX,and its mechanism might be related to anti-cell apoptosis.