苏木酮A调控JNK通路减轻大鼠肾脏缺血再灌注损伤

Sappanone A attenuates renal ischemia-reperfusion injury in rats by regulating JNK signal pathway

本研究旨在探讨苏木酮A(sappanone A,SA)调控大鼠肾脏缺血再灌注损伤(ischemia-reperfusion injury,IRI)的作用和机制。动物实验已获得苏州市吴江区儿童医院伦理委员会批准(批准号:2022010)。首先利用苏木精−伊红染色法(hematoxylin-eosin staining,H&E)观察大鼠肾组织形态学变化并进行肾损伤评分;提取血清检测肌酐(serum creatinine,SCr)、尿素氮(blood urea nitrogen,BUN)和胱抑素C(cystatin C,Cys C)的含量;通过TUNEL染色进一步分析苏木酮A对IRI引起的肾小管上皮细胞凋亡情况的影响;免疫印迹法(Western blot)检测肾组织中p-JNK/JNK、p-ERK/ERK、Bcl2、Bax和cleaved-caspase 3的蛋白表达水平。最后,通过以上研究方法明确JNK激活剂茴香霉素(anisomycin,Ani)是否可以逆转苏木酮A对大鼠IRI的保护作用。结果显示,苏木酮A明显减轻IRI引起的肾小管损伤,减少血清中SCr、BUN和Cys C的含量。TUNEL染色显示,苏木酮A明显减少IRI引起的肾小管上皮细胞凋亡。Western blot检测肾组织表明,苏木酮A明显促进凋亡抑制蛋白Bcl2的表达,抑制凋亡促进蛋白Bax和cleaved-caspase 3的表达,进一步分析显示苏木酮A不影响ERK的磷酸化,而抑制JNK的磷酸化。最后,通过H&E染色、血清学检测、TUNEL染色和免疫印迹法证实JNK激活剂茴香霉素可以逆转苏木酮A对大鼠IRI的保护作用。上述研究结果表明,苏木酮A通过抑制JNK磷酸化减轻大鼠肾脏缺血再灌注损伤。

This study aimed to investigate the role and mechanism of sappanone A(SA)in regulating renal ischemia-reperfusion injury(IRI)in rats.The animal experiment has been approved by the Ethics Committee of Suzhou Wujiang District Children's Hospital(approval number:2022010).First,hematoxylin-eosin(H&E)staining was used to evaluate the effects of SA on IRI,and renal damage was scored.Serum creatinine(SCr),blood urea nitrogen(BUN)and cystatin C(Cystatin C)were analyzed.The effect of sappanone A on the apoptosis of renal tubular epithelial cells induced by IRI was analyzed by TUNEL staining.Protein expression levels of p-JNK/JNK,p-ERK/ERK,Bcl2,Bax and cleaved-caspase 3 in renal tissues were detected by Western blot.Finally,H&E staining,serological analysis,TUNEL staining and Western blot were used to determine whether JNK activator anisomycin could reverse the effect of SA on IRI in rats.The results showed SA significantly reduced the renal tubule injury caused by ischemia-reperfusion,and decreased the level of SCr,BUN and Cys C in serum.TUNEL staining showed that SA significantly reduced the apoptosis of renal tubular epithelial cells induced by IRI.Western blot analysis of kidney tissue showed that SA significantly promoted the expression of apoptosis inhibiting protein Bcl2 and inhibited the expression of apoptosis-promoting proteins Bax and cleaved-caspase 3.Further analysis elucidated that SA did not affect the phosphorylation of ERK but decreased the phosphorylation of JNK.Finally,H&E staining,serological analysis,TUNEL staining and Western blot confirmed that JNK activator anisomycin could reverse the alleviating effect of SA on IRI in rats.The above findings suggest that SA could alleviate IRI in rats by inhibiting JNK phosphorylation.