“调理脾胃针刺法”对2型糖...n、desmin表达的影响_刘亮.pdf

第 39 卷第 2 期2023 年 2 月长春中医药大学学报Journal of Changchun University of Chinese MedicineVol.39 No.2Feb.2023152 DOI:10.13463/ki.cczyy.2023.02.009“调理脾胃针刺法”对 2 型糖尿病肾病大鼠足细胞中PCX、CD2AP、nephrin、desmin 表达的影响刘 亮1，董重阳2*，郝 华2*，胡锦华3，沈少华4，谭亚芹1，刘白雪1，王雄耀1（1.内蒙古医科大学中医学院针灸教研室，呼和浩特 010010；2.内蒙古医科大学中医学院中医基础理论教研室，呼和浩特 010010；3.天津市和平区新兴街社区卫生服务中心，天津 300070；4.内蒙古医科大学附属医院中医科，呼和浩特 010010）摘要：目的 研究“调理脾胃针刺法”对2型糖尿病肾病大鼠足细胞中足细胞标记蛋白（PCX）、CD2相关蛋白（CD2AP）、肾病蛋白（nephrin）、结蛋白（desmin）基因表达的影响，并阐明其可能的作用机制。方法 将 70 只健康雄性 SD 大鼠，随机分为模型组 52 只，空白组 18 只，将模型组大鼠喂养 8 周高脂高糖饲料之后，腹腔低剂量（30 mgkg-1）注射链脲佐菌素，建立 2 型糖尿病肾病大鼠模型。将造模成功的 36 只大鼠按照血糖值，以及尿蛋白定性结果构成比，采用分层随机的方法分为针刺组 4 周（n=6），模型组 4 周（n=6）；针刺组 8 周（n=6），模型组 8 周（n=6）；针刺组 12 周（n=6），模型组 12 周（n=6）。空白组随机分为空白组 4 周（n=6）、空白组 8 周（n=6）、空白组 12 周（n=6）。将针刺组穿着针刺罩衣固定，采用“调理脾胃针刺法”干预，分别针刺4周、8周及12周。模型组和空白组予相同抓取固定。观测各组干预不同周期后，测定 24h 尿蛋白；采用 HE 染色光镜下观察大鼠肾脏病理变化情况；采用荧光定量 PCR 法检测 PCX、CD2AP、nephrin 和 desmin mRNA 表达水平。结果 4 周、8 周及 12 周后各组组间比较，针刺组 24h 尿蛋白含量低于模型组（P 0.05）；肾脏 HE 染色，模型组肾小球增生，散在炎性细胞浸润，部分肾小管管腔变窄，系膜基质增生，基底膜轻度增厚，而针刺组上述病理变化均小于模型组（P 0.05）；4 周、8 周及 12 周后各组组间比较，针刺组 PCX、CD2AP 和 nephrin mRNA 表达显著高于模型组（P 0.01）；8 周、12 周后各组组间比较，针刺组 desmin 基因表达显著低于模型组（P 0.01）。结论“调理脾胃针刺法”可降低 2 型糖尿病肾病大鼠 24 h 尿蛋白含量，通过其肾脏足细胞 PCX、CD2AP、nephrin 蛋白及基因表达的水平，降低 desmin 蛋白及基因表达的水平，以改善 2 型糖尿病肾病大鼠足细胞损伤，减轻肾小球基底膜增厚，从而延缓 DN 的进展，其降低尿蛋白含量的作用机制可能与此相关。关键词：针刺；调理脾胃针刺法；足细胞；PCX；CD2AP；nephrin；desmin；2 型糖尿病肾病大鼠中图分类号：R259 文献标志码：A 文章编号：2095-6258(2023)02-0152-04Eff ects of acupuncture for regulating spleen and stomach on the expression of PCX,CD2AP,nephron and desmin in podocytes of rats with type 2 diabetic nephropathyLIU Liang1,DONG Chongyang2*,HAO Hua2*,HU Jinhua3,SHEN Shaohua4,TAN Yaqin1,LIU Baixue1,WANG Xiongyao1(1.Department of Acupuncture and Moxibustion,College of Traditional Chinese Medicine,Inner Mongolia Medical University,Hohhot 010010,China;2.Department of Basic Theory of Traditional Chinese Medicine,College of Traditional Chinese Medicine,Inner Mongolia Medical University,Hohhot 010010,China;3.Xinxing Street Community Health Service Center,Heping District,Tianjin 300070,China;4.Department of Traditional Chinese Medicine,Affi liated Hospital of Inner Mongolia Medical University,基金项目：内蒙古自治区自然科学基金面上项目（2019MS08134）；内蒙古医科大学科技百万工程项目（YKD2020KJBW017）作者简介：刘 亮（1983），女，博士，讲师，主要从事针灸治疗糖尿病及其并发症方面研究*通信作者：董重阳，副教授，副主任医师，硕士研究生导师，电子信箱- 郝 华，副教授，副主任医师，硕士研究生导师，电子信箱-实验研究第 39 卷第 2 期2023 年 2 月长春中医药大学学报Journal of Changchun University of Chinese MedicineVol.39 No.2Feb.2023153Hohhot 010010,China)Abstract:Objective To study the effects of acupuncture for regulating the spleen and the stomach on the gene expression of podocalyxin(PCX),CD2 associated protein(CD2AP),nephron,desmin in podocytes of rats with type 2 diabetic nephropathy,and to clarify its possible mechanisms.Methods Seventy healthy male SD rats were randomly divided into a model group(52 rats)and a blank group(18 rats).The model group was fed with high-fat and high-sugar diets for 8 weeks and then streptozotocin was injected intraperitoneally at a low dose(30 mgkg-1)to establish a rat model of type 2 diabetic nephropathy.According to their blood sugar levels and the composition ratios of the urine protein qualitative results,employing a stratified random method,the thirty-six successfully modeled rats were divided into an acupuncture group(4 weeks,n=6)and a model group(4 weeks,n=6);an acupuncture group(8 weeks,n=6)and a model group(8 weeks,n=6);an acupuncture group(12 weeks,n=6)and a model group(12 weeks,n=6).While the rats in the blank group were randomly divided into a blank group(4 weeks,n=6),a blank group(8 weeks,n=6),and a blank group(12 weeks,n=6).The rats in the acupuncture group were fixed in acupuncture gowns,and were treated with acupuncture for 4 weeks,8 weeks and 12 weeks,respectively.The model group and the blank group were fixed in the same way.After different periods of intervention,the 24-hour urine protein contents of rats were measured in each group,the pathological changes of the rats kidneys were observed by HE staining,and the mRNA expression levels of PCX,CD2AP,nephrin and desmin were observed by fluorescence quantitative PCR.Results After 4,8 and 12 weeks,the 24h urinary protein content in the acupuncture group was lower than that in the model group(P0.05).HE staining observed the pathological changes of glomerular hyperplasia,scattered inflammatory cells infiltration,narrowing of some tubules,mesangial matrix hyperplasia and mild thickening of basement membrane in the model group,while the above pathological changes were less noticeable in the acupuncture group(P0.05).After 4,8 and 12 weeks,the mRNA expression levels of PCX,CD2AP and nephrin in the acupuncture group were signifi cantly higher than that in the model group(P0.01).After 8 and 12 weeks,the gene expression level of desmin in the acupuncture group was signifi cantly lower than that in the model group(P0.01).Conclusion Acupuncture for regulating the spleen and the stomach can reduce the 24-hour urine protein content in rats with type 2 diabetic nephropathy.Through t