“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

Effects of acupuncture for regulating spleen and stomach on the expression of PCX, CD2AP, nephron and desmin in podocytes of rats with type 2 diabetic nephropathy

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 mg·kg-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。

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 mg·kg-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(8weeks, 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(P <0.05). 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(P <0.05). 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(P<0.01). 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(P<0.01). 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 the protein and gene expression levels of PCX, CD2AP and nephrin in their renal podocytes, the protein and gene expression level of desmin can be reduced, so as to improve their podocyte injury, reduce the thickening of glomerular basement membrane, and thus delay the progress of DN, to which its mechanism on lowering the urine protein content may be related.