Huang Hai, Chen Li, Wu Huan, Jin Jing-Song
Clinical College of Chinese Medicine, Hubei University of Chinese Medicine, Wuhan 430061, China; Department of Rehabilitation, Hubei Provincial Hospital of Chinese Medicine, Wuhan 430074.
College of Acupuncture-moxibustion and Orthopedics-traumatology, Hubei University of Chinese Medicine, Wuhan 430061.
Zhen Ci Yan Jiu. 2022 Feb 25;47(2):108-14. doi: 10.13702/j.1000-0607.20210329.
To observe the effect of electroacupuncture(EA)on renal function and autophagy-related protein expression in diabetic nephropathy (DN) rats，so as to explore its mechanisms underlying improvement of DN.
Male Wistar rats were randomly divided into normal control，DN model and EA groups (=8 rats in each group). The DN model was established by high-fat diet (for 4 weeks) + streptozotocin (STZ，35 mg/kg，i.p.). EA (2 Hz/15 Hz，1 mA) was applied to bilateral "Shenshu"(BL23)and "Zusanli"(ST36)for 20 min，5 times a week for 6 consecutive weeks. The urine volume in 24 h (24 h-UV) was recorded，the levels of urine protein in 24 h (24 h-UP)，blood urea nitrogen (BUN) and serum creatinine (Scr) were detected by using an automatic biochemical analyzer，and the fasting blood glucose (FBG) was detected using rapid blood glucose paper test. The histopathological changes of glomerulus of the left kidney were observed after periodic acid-Schiff stain (PAS) and the ultra-microstructural changes，the numbers of autophagosomes and autophagic vacuoles of the right kidney were observed by transmission electronic microscope (TEM). The expression and localization of podocyte marker protein Nephrin was detected by using immunofluorescence assay，and the expression levels of autophagy-related proteins microtubule associated protein 1 (LC3)，Beclin-1，p62 and Nephrin in the kidney tissue measured by Western blot.
Before treatment,compared with the normal control group，the levels of 24 h-UV，24 h-UP and FBG were significantly increased (<0.01) in the model and EA groups. After treatment, compared with the normal group，the levels of 24 h-UV，24 h-UP，FBG，BUN，Scr and p62 protein expression were signi-ficantly increased (<0.01)，and the expression levels of LC3Ⅱ，Beclin-1 and Nephrin proteins and ratio of LC3Ⅱ/Ⅰ were significantly decreased (<0.01) in the model group. Compared with the model group，the levels of 24 h-UV，24 h-UP，FBG，BUN and p62 were apparently decreased (<0.01，<0.05)，while the expression levels of LC3Ⅱ，Beclin-1 and Nephrin proteins and ratio of LC3Ⅱ/Ⅰ were obviously up-regulated (<0.01，<0.05) in the EA group. No significant changes were found in the level of Scr after EA (>0.05) and in the expression levels of LC3 Ⅰ after modeling and EA (>0.05). Outcomes of PAS showed blurred structure of the glomerulus with obvious proliferation of glomerular mesangial cells，increase of cell matrix，and thickening of basement membrane of the glomerulus and renal tubule，atrophy of epithelia cells of renal tubules and infiltration of inflammatory cells in the renal interstitum; and TEM revealed fusion and exfoliation of some foot processes，bareness or thickening of some basement membrane，and reduction of number of autophagosomes or autophagobubbles in podocytes in the model group，which was relatively milder in the EA group.
EA can effectively alleviate kidney damage in DN rats，which may be related to its function in facilitating autophagy in the kidney.
观察电针（EA）对糖尿病肾病（DN）大鼠肾功能及自噬相关蛋白表达的影响，以探讨其改善DN的作用机制。
雄性Wistar大鼠随机分为正常对照组、DN模型组和EA组（每组8只）。采用高脂饮食（4周）+链脲佐菌素（STZ，35mg/kg，腹腔注射）建立DN模型。将EA（2Hz/15Hz，1mA）施加于双侧“肾俞”（BL23）和“足三里”（ST36），每次20分钟，每周5次，连续6周。记录24小时尿量（24h-UV），用自动生化分析仪检测24小时尿蛋白（24h-UP）、血尿素氮（BUN）和血清肌酐（Scr）水平，用快速血糖试纸检测空腹血糖（FBG）。经高碘酸-希夫染色（PAS）后观察左肾肾小球的组织病理学变化，用透射电子显微镜（TEM）观察右肾的超微结构变化、自噬体和自噬泡数量。采用免疫荧光法检测足细胞标志物蛋白Nephrin的表达及定位，用蛋白质免疫印迹法检测肾组织中自噬相关蛋白微管相关蛋白1轻链3（LC3）、Beclin-1、p62和Nephrin的表达水平。
治疗前，与正常对照组相比，模型组和EA组的24h-UV、24h-UP和FBG水平显著升高（P<0.01）。治疗后，与正常组相比，模型组的24h-UV、24h-UP、FBG,BUN,Scr和p62蛋白表达水平显著升高（P<0.01），LC3Ⅱ、Beclin-1和Nephrin蛋白表达水平及LC3Ⅱ/Ⅰ比值显著降低（P<0.01）。与模型组相比，EA组的24h-UV、24h-UP、FBG、BUN和p62水平明显降低（P<0.01，P<0.05），而LC3Ⅱ、Beclin-1和Nephrin蛋白表达水平及LC3Ⅱ/Ⅰ比值明显上调（P<0.01，P<0.05）。EA治疗后Scr水平无明显变化（P>0.05），建模及EA后LC3Ⅰ表达水平无明显变化（P>0.05）。PAS结果显示肾小球结构模糊，肾小球系膜细胞明显增生，细胞基质增多，肾小球和肾小管基底膜增厚，肾小管上皮细胞萎缩，肾间质有炎性细胞浸润；TEM显示模型组部分足突融合、脱落，部分基底膜裸露或增厚，足细胞中自噬体或自噬泡数量减少，EA组相对较轻。
EA可有效减轻DN大鼠肾损伤，其机制可能与促进肾脏自噬有关。
