益智仁激活PPARα介导的自噬作用改善糖尿病肾病  

Alpiniae oxyphyllae fructus ameliorates diabetic kidney disease through activation of PPARα⁃mediated autophagy

作　　者：颜子杰 康渝 刘书蔓 陈柏岑 马天鹏[1] 肖曼[3] 谢毅强[1] YAN Zijie;KANG Yu;LIU Shuman;CHEN Bocen;MA Tianpeng;XIAO Man;XIE Yiqiang(College of Traditional Chinese Medicine,Hainan Medical University,Haikou 571199,China;Heilongjiang Academy of Traditional Chinese Medicine,Harbin 150001,China;School of Basic Medical Sciences,Hainan Medical University,Haikou 571199,China)

机构地区：[1]海南医学院中医学院,海南海口571199 [2]黑龙江省中医药科学院,黑龙江哈尔滨150001 [3]海南医学院基础医学院,海南海口571199

出　　处：《海南医学院学报》2024年第17期1281-1289,共9页Journal of Hainan Medical University

基　　金：国家自然科学基金-面上项目(82174334);代谢性疾病中医药调控四川省重点实验2022年开放基金(SZKF202203)。

摘　　要：目的:利用体内和体外模型研究益智仁对糖尿病肾病(diabetic kidney disease,DKD)的潜在保护作用,并探讨其作用机制。方法:体内实验将6只db/m小鼠作为正常组,30只db/db小鼠分为模型组、益智仁低剂量组、益智仁中剂量组、益智仁高剂量组、卡格列净组,每组6只。正常组和模型组给予生理盐水,治疗组小鼠分别给予相应药物。治疗8周后,收集血清、尿液和肾脏组织样本。测定随机血糖、24 h尿微量白蛋白、肌酐、尿素氮、甘油三酯和总胆固醇含量。通过HE染色观察肾组织的病理变化。采用60 mmol/L葡萄糖干预HK-2细胞72 h构建DKD体外模型,同时给予不同浓度益智仁含药血清(2%、5%、10%)干预24 h后,通过CCK8法测定细胞活力以及透射电镜观察HK-2细胞中自噬溶酶体的数量。采用RT-qPCR和Western blot检测小鼠及细胞中PPARα、LC3BⅡ、Beclin1、P62 mRNA及蛋白的表达水平。结果:体内实验表明,与正常组相比,模型组的血糖、24 h尿微量白蛋白、肌酐、尿素氮、甘油三酯和总胆固醇含量升高(P<0.0001);与模型组相比,经益智仁治疗后上述生化指标含量降低(P<0.05,P<0.01,P<0.001,P<0.0001)。HE染色显示,模型组肾小球肿胀加重,基底膜增厚,肾小管肿胀且管腔变窄。药物干预后,肾脏病理变化均有不同程度的减轻。且中、高剂量益智仁的疗效均优于低剂量。体外实验表明,益智仁含药血清可提高高糖胁迫下HK-2细胞活力,且10%的剂量效果最佳(P<0.01)。透射电镜发现,与正常组相比,高糖组HK-2细胞中自噬溶酶体数量降低,脂滴增加;经益智仁干预后,自噬溶酶体数量增加,脂滴减少。体内和体外研究中RT-qPCR和Western blot结果一致,表明益智仁能够提高PPARαmRNA和蛋白(P<0.05,P<0.01,P<0.001)水平,促进Beclin1、LC3BⅡ和降低P62蛋白表达水平(P<0.05,P<0.01,P<0.001)。结论:益智仁可能通过激活PPARα介导的自噬作用改善DKD。Objective:To investigate the potential protective effects of Alpiniae oxyphyllae fructus against diabetic kidney dis⁃ease using in vivo and in vitro models and to explore its mechanism of action.Methods:In the in vivo experiments,6 db/m mice were taken as the normal group;30 db/db mice were divided into 5 groups of 6 mice each:model,low dose Alpiniae oxyphyllae fructus,medium dose Alpiniae oxyphyllae fructus,high dose Alpiniae oxyphyllae fructus,and canagliflozin⁃treated.Saline was given to the normal and model groups,and the mice in the treatment groups were given the corresponding drugs respectively.After 8 weeks of treatment,serum,urine and kidney tissue samples were collected.Random blood glucose,24 h urine microalbumin,creatinine,urea nitrogen,triglyceride and total cholesterol levels were determined.Pathological changes in renal tissues were ob⁃served by HE staining.An in vitro model of diabetic kidney disease was constructed by intervening HK⁃2 cells with 60 mmol/L glucose for 72 h.After 24 h of intervention with different concentrations of Alpiniae oxyphyllae fructus⁃containing serum(2%,5%and 10%),the cell viability was determined by CCK8 assay and the number of autophagic lysosomes in HK⁃2 cells was ob⁃served by transmission electron microscopy.The expression levels of PPARα,LC3BⅡ,Beclin1,P62 mRNA and protein in mice and cells were detected by RT⁃qPCR and western blot.Results:In vivo experiments showed that the levels of random blood glucose,24 h urine microalbumin,creatinine,urea nitrogen,triglyceride and total cholesterol were increased in the model group compared with the normal group(P<0.0001);the levels of the above biochemical indexes were decreased in the drug group com⁃pared with the model group after treatment with Alpiniae oxyphyllae fructus(P<0.05,P<0.01,P<0.001,P<0.0001).HE staining showed that in the model group glomerular swelling increased,basement membrane thickened,renal tubules swollen and lumen narrowed.After the drug intervention,the pathological changes of the ki

关 键 词：益智仁 糖尿病肾病 PPARΑ 自噬 中医药 

分 类 号：R285.5[医药卫生—中药学]