摘要
目的:探讨参芪地黄汤对糖尿病肾病(DKD)的治疗作用及其机制,重点考察其对花生四烯酸相关性铁死亡的影响。方法:将60只C57BL/6小鼠分为正常组10只,造模组50只,其中43只造模成功。将造模成功小鼠分为模型组,参芪地黄汤低、中、高剂量组,达格列净组,达格列净组予以达格列净药液(0.13 mg·kg^(-1))灌胃,参芪地黄汤组分别予以参芪地黄汤水溶液(4.68、9.36、18.72 g·kg^(-1))灌胃,正常组与模型组分别予以等容生理盐水灌胃。电子天平和血糖仪分别监测小鼠体质量和小鼠尾尖空腹血糖变化;酶联免疫吸附测定法(ELISA)检测小鼠血清肌酐(Scr)和尿素氮(BUN)水平;苏木素-伊红(HE)染色、马松染色(Masson)、过碘酸雪夫(PAS)染色检测肾组织病理变化;倒置荧光显微镜观察冰冻切片中活性氧(ROS)的荧光强度,以评估肾脏组织中的亚铁离子(Fe^(2+))及脂质过氧化水平;对小鼠肾组织谷胱甘肽过氧化物酶4(GPX4)、酰基辅酶A合成酶长链家族成员4(ACSL4)免疫荧光染色,以检测其定位定量表达;蛋白免疫印迹法(Western blot)检测铁死亡关键蛋白如GPX4、胱氨酸谷氨酸反向转运蛋白(xCT)和花生四烯酸代谢通路相关蛋白如ACSL4、溶血磷脂酰胆碱酰基转移酶3(LPCAT3)、花生四烯酸-15-脂氧合酶(ALOX15)的表达水平;实时荧光定量聚合酶链式反应(Real-time PCR)检测小鼠肾组织中铁死亡关键蛋白如溶质载体家族7成员11(SLC7A11)、GPX4及花生四烯酸代谢通路相关mRNA的表达。结果:与正常组比较,DKD模型组小鼠体质量显著下降(P<0.01),血糖水平显著升高(P<0.01),24 h尿蛋白、血清Scr和BUN水平均显著升高(P<0.01),肾小球系膜细胞增生、基膜增厚、肾小管萎缩和肾小管间质炎症细胞浸润等严重的病理损伤,肾组织中Fe^(2+)、丙二醛(MDA)、脂质过氧化物(LPO)及ROS水平显著升高(P<0.01),GPX4、xCT显著降低(P<0.01),ACSL4、LPCAT3、ALOX15显著升高(P<0.01),GPX4、SLC7A11 mRNA均显著降低(P<0.01),ACSL4、LPCAT3、ALOX15 mRNA显著升高(P<0.01)。与模型组比较,参芪地黄汤低、中、高剂量组和达格列净组小鼠体质量显著升高(P<0.01),血糖水平显著下降(P<0.01),24 h尿蛋白、血清Scr水平均显著降低(P<0.01),肾小球和肾小管病变均有不同程度减少,肾小球和肾小管纤维化程度均有不同程度降低,参芪地黄汤高剂量组和达格列净组小鼠Fe^(2+)、MDA、LPO、ROS水平均显著降低(P<0.01),参芪地黄汤中、高剂量组和达格列净组小鼠肾组织中GPX4、xCT显著增加(P<0.01),ACSL4、LPCAT3、ALOX15明显下降(P<0.05,P<0.01),ACSL4、LPCAT3、ALOX15 mRNA显著下降(P<0.01)。结论:参芪地黄汤能够通过抑制铁死亡来改善DKD,其机制可能与降低铁离子水平、抑制脂质过氧化、上调GPX4表达及下调ACSL4表达有关。该研究为中医药治疗DKD提供了新的思路和理论依据,并为开发新型DKD治疗药物提供了潜在靶点。
Objective:To investigate the therapeutic effects and mechanism of Shenqi Dihuang decoction(SQDHD)on diabetic kidney disease(DKD),with a focus on its impact on arachidonic acid-related ferroptosis.Methods:Sixty C57BL/6 mice were allocated into a normal group(n=10)and a modeling group(n=50),with 43 mice successfully modeled.The successfully modeled mice were further allocated into model,low-,medium-,and high-dose(4.68,9.36,and 18.72 g·kg^(-1),respectively)SQDHD,and dapagliflozin(0.13 mg·kg^(-1))groups.The drug treatment groups were administrated with corresponding agents by gavage,and the normal and model groups were administrated with equal volumes of normal saline by gavage.An electronic balance and a glucometer were used to monitor the body weight and fasting blood glucose level from the tail tip,respectively.Serum creatinine(Scr)and blood urea nitrogen(BUN)levels were measured by enzyme-linked immunosorbent assay(ELISA).Histopathological changes in the renal tissue were assessed by hematoxylin-eosin staining,Masson staining,and periodic acid-Schiff(PAS)staining.The fluorescence intensity of reactive oxygen species(ROS)in frozen sections was observed by an inverted fluorescence microscope to evaluate the levels of ferrous ions(Fe^(2+))and lipid peroxidation in the renal tissue.Immunofluorescence staining of glutathione peroxidase 4(GPX4)and acyl-CoA synthetase long-chain family member 4(ACSL4)in the renal tissue was performed to detect their localization and expression.Western blot was employed to assess the expression levels of key ferroptosis proteins such as GPX4 and cystine/glutamate antiporter(xCT),as well as the arachidonic acid metabolic pathway-related proteins,including ACSL4,lysophosphatidylcholine acyltransferase 3(LPCAT3),and arachidonate 15-lipoxygenase(ALOX15).Real-time PCR was employed to measure the mRNA levels of key ferroptosis proteins,including solute carrier family 7 member 11(SLC7A11)and GPX4,as well as arachidonic acid metabolism-related factors(ACSL4,LPCAT3,and ALOX15)in the renal tissue.Results:Compared with the normal group,DKD model mice exhibited a decrease in body weight(P<0.01),increases in levels of blood glucose(P<0.01),24-hour urinary protein,Scr,and BUN(P<0.01),along with severe pathological changes,such as mesangial cell proliferation,basement membrane thickening,tubular atrophy,and interstitial inflammatory cell infiltration.In addition,the modeling elevated the levels of Fe^(2+),MDA,LPO,and ROS(P<0.01),lowered the GPX4 and xCT levels(P<0.01),raised the ACSL4,LPCAT3,and ALOX15 levels(P<0.01),down-regulated the mRNA levels of GPX4 and SLC7A11(P<0.01),and up-regulated the mRNA levels of ACSL4,LPCAT3,and ALOX15(P<0.01)in the renal tissue.Compared with the model group,low-,medium-,and high-dose SQDHD groups and the dapagliflozin group showed an increase in body weight(P<0.01),decreases in levels of blood glucose(P<0.01),24-hour urinary protein,and Scr(P<0.01),alleviated pathological changes in glomeruli and tubules,and reduced degree of glomerular and tubular fibrosis.The high-dose SQDHD group and the dapagliflozin group showed reductions in Fe^(2+),MDA,LPO,and ROS levels(P<0.01).The medium-and high-dose SQDHD groups and the dapagliflozin group exhibited increased levels of GPX4 and xCT(P<0.01),decreased levels of ACSL4,LPCAT3,and ALOX15(P<0.05,P<0.01),and down-regulated mRNA levels of ACSL4,LPCAT3,and ALOX15(P<0.01).Conclusion:SQDHD ameliorates DKD by inhibiting ferroptosis potentially by reducing iron ion levels,inhibiting lipid peroxidation,upregulating GPX4 expression,and down-regulating ACSL4 expression.This study provides new insights and a theoretical basis for the treatment of DKD with traditional Chinese medicine and identifies potential targets for developing novel therapeutics for DKD.
作者
吴源陶
王智槟
傅馨莹
邹晓玲
胡文孝
邹译娴
冯君
WU Yuantao;WANG Zhibin;FU Xinying;ZOU Xiaoling;HU Wenxiao;ZOU Yixian*;FENG Jun(The First Affiliated Hospital of Hunan University of Chinese Medicine,Changsha 410007,China;Graduate School,Hunan University of Chinese Medicine,Changsha 410208,China)
出处
《中国实验方剂学杂志》
北大核心
2025年第12期140-149,共10页
Chinese Journal of Experimental Traditional Medical Formulae
基金
湖南省自然科学基金面上项目(2024JJ5317)
湖南省中医药管理局项目(B2023019)
湖南中医药大学校院联合基金项目(2023XYLH028)。
