基于内质网应激诱导的细胞凋亡探究黄蜀葵花总黄酮减轻糖尿病肾小管病的作用和机制

Effects and mechanisms of total flavones of Abelmoschus manihot in attenuating diabetic tubulopathy by targeting endoplasmic reticulum stress-induced cell apoptosis

糖尿病肾脏疾病(diabetic kidney disease, DKD)患者的肾小管损伤可能是与肾小球和微血管病变同时出现的,并且在其肾损伤进展中发挥着重要的作用,目前被称之为“糖尿病肾小管病(diabetic tubulopathy, DT)”。为了探究传统治肾中药黄蜀葵花的提取物——黄蜀葵花总黄酮(total flavones of Abelmoschus manihot,TFA)在体内减轻DT的多靶点治疗作用和药理机制,笔者将全部大鼠随机分为4组,即正常对照组(正常组)、DT模型组(模型组)、DT模型+TFA组(TFA组)以及DT模型+罗格列酮(rosiglitazone, ROS)组(ROS组)。通过综合措施建立基于DKD的大鼠DT模型。在DT模型鼠成模后,每天用双蒸水、TFA混悬液、ROS混悬液分别给4组大鼠连续灌胃。在给药6周后,处死全部大鼠,分别收集其尿液、血液以及肾组织等标本而进行各项指标的检测,并观察TFA和ROS对DT模型鼠血液、尿液生化指标,肾小管损伤指标,肾小管上皮细胞凋亡和内质网应激(endoplasmic reticulum stress, ERS)指标以及肾组织蛋白激酶样内质网激酶(protein kinase-like endoplasmic reticulum kinase, PERK)-真核翻译起始因子2α(eukaryotic translation initiation factor 2α,eIF2α)-激活转录因子4(activating transcription factor 4,ATF4)-同源蛋白(C/EBP homologous protein C/EBP,CHOP)信号通路活性的影响。结果表明,DT模型鼠出现了肾小管上皮细胞肥大,肾小管增生、闭塞以及肾间质细胞外基质和胶原沉积;同时,还出现了明显的肾小管损伤标志分子表达程度和蛋白表达水平的变化;此外,出现了肾小管性尿蛋白的异常升高。经TFA和ROS干预,模型鼠尿蛋白、肾小管损伤特征、肾小管上皮细胞凋亡和ERS特征以及肾组织PERK-eIF2α-ATF4-CHOP信号通路活性均得到不同程度的改善;其中,对于肾小管/间质病理性改变,TFA的作用优于ROS。简言之,借助DT模型鼠,该研究阐明,TFA在体内能通过抑制肾小管ERS诱导的细胞凋亡而多靶点地减轻DT,其作用和机制与抑制肾组织PERK-eIF2α-ATF4-CHOP信号通路活性有关。这些发现为TFA在DT临床治疗领域的应用提供了初步的药理学证据。

Renal tubular injury in patients with diabetic kidney disease(DKD)may be accompanied by glomerular and microvascular diseases.It plays a critical role in the progression of renal damage in DKD,and is now known as diabetic tubulopathy(DT).To explore the multi-targeted therapeutic effects and pharmacological mechanisms in vivo of total flavones of Abelmoschus manihot(TFA),an extract from traditional Chinese medicine for treating kidney disease,in attenuating DT,the authors randomly divided all rats into four groups:a normal control group(normal group),a DT model group(model group),a DT model+TFA-treated group(TFA group)and a DT model+rosiglitazone(ROS)-treated group(ROS group).The DT rat model was established based on the DKD rat model by means of integrated measures.After successful modeling,the rats in the four groups were continuously given double-distilled water,TFA suspension,and ROS suspension,respectively by gavage every day.After 6 weeks of treatment,all rats were sacrificed,and the samples of their urine,blood,and kidneys were collected.The effects of TFA and ROS on various indicators related to urine and blood biochemistry,renal tubular injury,renal tubular epithelial cell apoptosis and endoplasmic reticulum stress(ERS),as well as the activation of the protein kinase R-like endoplasmic reticulum kinase(PERK)-eukaryotic translation initiation factor 2α(eIF2α)-activating transcription factor 4(ATF4)-C/EBP homologous protein(CHOP)signaling pathway in the kidney of the DT model rats were investigated.The results indicated that hypertrophy of renal tubular epithelial cells,renal tubular hyperplasia and occlusion,as well as interstitial extracellular matrix and collagen deposition occurred in the DT model rats.Moreover,significant changes were found in the expression degree and the protein expression level of renal tubular injury markers.In addition,there was an abnormal increase in tubular urine proteins.After TFA or ROS treatment,urine protein,the characteristics of renal tubular injury,renal tubular epithelial cell apoptosis and ERS,as well as the activation of the PERK-eIF2α-ATF4-CHOP signaling pathway in the kidney of the DT model rats were improved to varying degrees.Therein,TFA was superior to ROS in affecting the pathological changes in renal tubule/interstitium.In short,with the DT model rats,this study demonstrated that TFA could attenuate DT by multiple targets through inhibiting renal tubular ERS-induced cell apoptosis in vivo,and its effect and mechanism were related to suppressing the activation of the PERK-eIF2α-ATF4-CHOP signaling pathway in the kidney.These findings provided preliminary pharmacological evidence for the application of TFA in the clinical treatment of DT.