丹蛭降糖胶囊通过下调Notch1/NICD/MAML1信号通路改善糖尿病肾病大鼠肾脏血管内皮功能

doi:10.12122/j.issn.1673-4254.2025.10.21

摘要/Abstract

摘要：

目的探讨丹蛭降糖胶囊通过Notch信号通路对糖尿病肾病（DN）模型大鼠肾脏血管内皮损伤进行修复的反应机制。方法随机将大鼠分为正常组、模型组、丹蛭降糖胶囊低、中、高剂量组、抑制剂组（n=10）。除正常组外，其余各组大鼠采取左肾切除术后高糖高脂饲料联合链脲佐菌素（STZ）注射建立DN模型。丹蛭降糖胶囊低、中、高剂量组分别按0.315、0.63、1.26 g/（kg·d）给药，抑制剂组以γ-分泌酶抑制剂DAPT20 mg/（kg·d）溶解于50%的CMC-Na溶液中隔天给药，正常组和模型组使用同等浓度生理盐水每天灌胃，连续给药4周。给药结束后用ELISA试剂盒检测肌酐（CRE）、尿素氮（BUN）、尿微量白蛋白（m ALB）水平，用透射电镜观察肾组织病理学变化，免疫组化法测量血管内皮生长因子（VEGF）、内皮素-1（ET-1）表达水平，Western blotting法测量血管内皮标志物CD31、Notch信号通路中Notch1、NICD、MAML1的蛋白表达，并比较各组数据。结果与正常组大鼠比较，模型组大鼠CRE、BUN、m ALB水平升高（P<0.01），电镜下肾脏出现病理损伤，肾脏组织中VEGF、ET-1、CD31表达升高（P<0.01），Notch1、NICD、MAML1水平上调（P<0.01）；与模型组大鼠比较，丹蛭降糖胶囊组和抑制剂组的CRE、BUN、m ALB水平降低（P<0.05），电镜下肾脏病理变化也有明显好转，肾脏组织中VEGF、ET-1、CD31表达减少（P<0.05），Notch1、NICD、MAML1水平下调（P<0.05），其中丹蛭降糖胶囊高剂量组效果最好。结论丹蛭降糖胶囊治疗DN的机制可能与抑制肾组织中NICD、MAML1介导的Notch信号通路有关，进而调控内皮细胞VEGF、ET-1水平来改善大鼠血管内皮过度增殖、功能障碍的损伤状态。

关键词: 丹蛭降糖胶囊, 糖尿病肾病, Notch信号通路, 血管内皮功能, 肾功能

Abstract:

Objective To investigate the therapeutic mechanism of Danzhi Jiangtang Capsule (DZJTC) for repairing renal vascular endothelial injury in rats with diabetic nephropathy (DN). Methods Fifty male SD rat models of DN, established by left nephrectomy, high-sugar and high-fat diet and streptozotocin injection, were randomized into DN model group, low-, medium-, and high-dose DZJTC treatment groups, and DAPT (a γ-secretase inhibitor) treatment group, with 10 rats with normal feeding as the control group. DZJTC was administered by daily gavage at 0.315, 0.63, or 1.26 g/kg, and DAPT (20 mg/kg, dissolved in 50% CMC-Na solution) was given by gavage every other day for 4 weeks; normal saline was given in the control and model groups. After treatment, the levels of creatinine (CRE), blood urea nitrogen (BUN), and microalbuminuria (mALB) were detected with ELISA, and renal pathologies were observed by transmission electron microscopy. Renal expressions of vascular endothelial growth factor (VEGF) and endothelin-1 (ET-1) were measured by immunohistochemistry, and the protein expressions of CD31 and Notch signaling pathway components were detected using Western blotting. Results The rat models of DN showed significantly increased CRE, BUN, and mALB levels, obvious renal pathologies under electron microscopy, increased renal VEGF, ET-1 and CD31 expressions, and upregulated Notch1, NICD, and MAML1 protein levels. Treatment with DZJTC at the 3 doses and DAPT significantly reduced CRE, BUN, and mALB levels, improved renal pathology, decreased VEGF, ET-1 and CD31 expressions, and lowered Notch1, NICD and MAML1 levels, and the effects were the most pronounced with high-dose DZJTC. Conclusion DZJTC ameliorates hyperproliferation and dysfunction of renal vascular endothelium in DN rats possibly by regulating renal VEGF and ET-1 levels via inhibiting NICD- and MAML1-mediated Notch signaling pathway.

Key words: Danzhi Jiangtang Capsule, diabetic nephropathy, Notch signaling pathway, vascular endothelium function, renal function

朱思佳, 马竞成, 郑玉娇, 吴传云, 赵建根, 李玲秀, 汪莉, 周雪梅. 丹蛭降糖胶囊通过下调Notch1/NICD/MAML1信号通路改善糖尿病肾病大鼠肾脏血管内皮功能[J]. 南方医科大学学报, 2025, 45(10): 2250-2257.

Sijia ZHU, Jingcheng MA, Yujiao ZHENG, Chuanyun WU, Jiangen ZHAO, Lingxiu LI, Li WANG, Xuemei ZHOU. Danzhi Jiangtang Capsule improves renal vascular endothelial function in rats with diabetic nephropathy by downregulating the Notch1/NICD/MAML1 signaling pathway[J]. Journal of Southern Medical University, 2025, 45(10): 2250-2257.

图/表 6

图1 各组大鼠肾组织病理学变化

Fig.1 Histopathological changes in renal tissue of rats in each group (Original magnification: ×25 000). A: Con group and Mod group; B: DZJT-L group and DZJT-M group; C: DZJT-H group and DAPT group.

图2 各组大鼠CRE、BUN、mALB水平比较

Fig.2 Comparison of CRE (A), BUN (B), and mALB (C) levels in rats among the groups. *P<0.01 vs Con group. #P<0.05, ##P<0.01 vs Mod group.

图3 各组大鼠肾组织ET-1、VEGF免疫组化结果

Fig.3 Immunohistochemical results of ET-1 (A) and VEGF (B) in the renal tissues of rats in each group (red arrows indicate positive staining).

图4 各组大鼠肾组织ET-1、VEGF平均光密度值比较

Fig.4 Comparison of mean optical density values of ET-1 (A) and VEGF (B) in renal tissue of rats among the groups. *P<0.01 vs Con group. ##P<0.01 vs Mod group.

图5 各组大鼠CD31、Notch1、NICD、MAML1的Western blotting条带

Fig.5 Protein bands of CD31, Notch1, NICD and MAML1 by Western blotting in rat renal tissues in each group.

图6 各组大鼠肾组织CD31、Notch1、NICD、MAML1蛋白水平比较

Fig.6 Comparison of CD31 (A), Notch1 (B), NICD (C), and MAML1 (D) protein expression levels in renal tissues among the groups. *P<0.01 vs Con group. #P<0.05, ##P<0.01 vs Mod group.

参考文献 33

[1]	汪四海, 方朝晖, 倪英群, 等.丹蛭降糖胶囊对糖尿病肾病大鼠肾脏TGF-β1/Smad3信号通路和AGEs/RAGE水平的影响[J].中华中医药杂志, 2021, 36(4): 2019-24.
[2]	周凯旋. 补阳还五汤治疗糖尿病肾病的作用机制及药效物质基础研究[D]. 长春: 长春中医药大学, 2024.
[3]	李建平, 朱德礼, 马贞, 等.丹芪益肾方对糖尿病肾病患者临床疗效及肾保护作用机制研究[J].广州中医药大学学报,2025, 42(2):350-7.
[4]	Mukherjee M, Fogarty E, Janga M, et al. Notch signaling in kidney development, maintenance, and disease[J]. Biomolecules, 2019, 9(11): 692. doi：10.3390/biom9110692
[5]	Fernández-Chacón M, García-González I, Mühleder S, et al. Role of Notch in endothelial biology[J]. Angiogenesis, 2021, 24(2): 237-50. doi：10.1007/s10456-021-09793-7
[6]	孙立平, 周霞,刘炬, 等. 从调控血管内皮生长因子及其受体激活Notch信号通路角度探讨生地梓醇促血管新生及神经功能重塑作用[J]. 实用心脑肺血管病杂志, 2021, 29(11): 63-8.
[7]	方朝晖, 陈志. 丹蛭降糖胶囊治疗2型糖尿病实验研究进展[J]. 安徽中医药大学学报, 2019, 38(2): 91-4.
[8]	周雪梅, 徐从书, 王凯, 等. 益气养阴活血中药对糖尿病肾病大鼠Notch/Hes1通路与血管内皮CD34、CD144的影响[J]. 南方医科大学学报, 2019, 39(7): 855-60.
[9]	Cheng H, Chen L, Hu XX, et al. Knockdown of MAML1 inhibits proliferation and induces apoptosis of T-cell acute lymphoblastic leukemia cells through SP1-dependent inactivation of TRIM59[J]. J Cell Physiol, 2019, 234(4): 5186-95. doi：10.1002/jcp.27323
[10]	尚立芝, 季书, 李耀洋, 等. 二陈汤加味通过Jagged1/Notch1/Hes1信号通路对慢性阻塞性肺疾病的抗炎机制[J]. 中国实验方剂学杂志, 2023, 29(9): 109-118.
[11]	路建饶, 陈晛, 陈杰. 传统医学对糖尿病肾病发病机制的研究进展[J]. 时珍国医国药, 2018, 29(2): 415-7.
[12]	王筠禹, 王帆竞, 张皖豫, 等.方朝晖基于《不居集》膈消论治思想治疗气阴两虚夹瘀型糖尿病周围神经病变经验[J].山西中医药大学学报, 2025, 26(5): 529-32+538.
[13]	鲍陶陶, 孟利亚, 黄展鹏, 等. 基于TLRs/MyD88信号通路探讨丹蛭降糖胶囊对糖尿病肾病大鼠的作用机制研究[J]. 时珍国医国药,2024, 35(9): 2076-80.
[14]	王奕霏, 卢慧蕴, 丰心月, 等. 太子参化学成分及生物活性研究进展[J]. 食品安全质量检测学报, 2024, 15(7): 122-32.
[15]	伍琳琳, 李志鹏, 曹世杰, 等. 牡丹皮活性成分改善糖尿病及其并发症的研究进展[J]. 中草药, 2022, 53(13): 4162-9.
[16]	陆春红, 许惠琴, 刘凯, 等. 生地环烯醚萜苷类成分对AGEs损伤HUVEC的保护作用[J]. 南京中医药大学学报, 2015, 31(1): 55-9.
[17]	马旺霞, 王斌.通络药水蛭及配伍防治糖尿病肾病的机制探析[J].中医临床研究, 2024, 16(18): 61-6.
[18]	黄长盛, 谭婷婷, 邢聘婷. 菟丝子对妊娠期糖尿病患者糖脂代谢影响的临床研究[J]. 现代中西医结合杂志, 2016, 25(20): 2199-201.
[19]	董莹莹, 张秀媛, 王洁. 泽泻多糖激活PPAR-γ/LXR-α/ABCG1信号通路发挥对糖尿病肾病大鼠的保护作用[J]. 药物评价研究, 2023, 46(7): 1480-7.
[20]	Fan W, Zeng SH, Wang XT, et al. A feedback loop driven by H3K9 lactylation and HDAC2 in endothelial cells regulates VEGF-induced angiogenesis[J]. Genome Biol, 2024, 25(1): 165. doi：10.1186/s13059-024-03308-5
[21]	Jiang YY, Li JH, Zhang J, et al. Serum VEGF as a predictive marker of glycemic control and diabetic nephropathy in Chinese older adults with type 2 diabetes mellitus[J]. Front Endocrinol (Lausanne), 2023, 14: 1274025. doi：10.3389/fendo.2023.1274025
[22]	Tan HT, Chen JX, Li YC, et al. Glabridin, a bioactive component of licorice, ameliorates diabetic nephropathy by regulating ferroptosis and the VEGF/Akt/ERK pathways[J]. Mol Med, 2022, 28(1): 58. doi：10.1186/s10020-022-00481-w
[23]	Funahashi Y, Shawber CJ, Sharma A, et al. Notch modulates VEGF action in endothelial cells by inducing Matrix Metalloprotease activity[J]. Vasc Cell, 2011, 3(1): 2. doi：10.1186/2045-824x-3-2
[24]	Zhang SH, Li XD, Liu SY, et al. Research progress on the role of ET-1 in diabetic kidney disease[J]. J Cell Physiol, 2023, 238(6): 1183-92. doi：10.1002/jcp.31023
[25]	Morand J, Briançon-Marjollet A, Lemarie E, et al. Zinc deficiency promotes endothelin secretion and endothelial cell migration through nuclear hypoxia-inducible factor-1 translocation[J]. Am J Physiol Cell Physiol, 2019, 317(2): C270-6. doi：10.1152/ajpcell.00460.2018
[26]	Abdelsaid M, Williams R, Hardigan T, et al. Linagliptin attenuates diabetes-induced cerebral pathological neovascularization in a blood glucose-independent manner: Potential role of ET-1[J]. Life Sci, 2016, 159: 83-9. doi：10.1016/j.lfs.2015.11.026
[27]	Yuan W, Qian M, Li ZX, et al. Endothelin-1 activates the Notch signaling pathway and promotes tumorigenesis in giant cell tumor of the spine[J]. Spine: Phila Pa 1976, 2019, 44(17): E1000-9. doi：10.1097/brs.0000000000003044
[28]	Denize T, Farah S, Cimadamore A, et al. Biomarkers of angiogenesis and clinical outcomes to cabozantinib and everolimus in patients with metastatic renal cell carcinoma from the phase III METEOR trial[J]. Clin Cancer Res, 2022, 28(4): 748-55. doi：10.1158/1078-0432.ccr-21-3088
[29]	张菊云, 陈绵雄, 华炳红, 等. 毛兰素调节Slit2/Robo1信号通路对糖尿病肾病模型大鼠肾小球内皮细胞血管生成的影响[J]. 中国药理学与毒理学杂志, 2024, 38(3): 177-82.
[30]	Raza Q, Nadeem T, Youn SW, et al. Notch signaling regulates UNC5B to suppress endothelial proliferation, migration, junction activity, and retinal plexus branching[J]. Sci Rep, 2024, 14(1): 13603. doi：10.1038/s41598-024-64375-z
[31]	Li YT, Tan P, Liu QP, et al. MiRNA-133a-3p attenuates renal tubular epithelial cell injury via targeting MALM1 and suppressing the Notch signaling pathway in diabetic nephropathy[J]. Cell Biochem Biophys, 2024, 82(3): 2401-11. doi：10.1007/s12013-024-01351-4
[32]	Zhang AF, Fang HW, Chen J, et al. Role of VEGF-A and LRG1 in abnormal angiogenesis associated with diabetic nephropathy[J]. Front Physiol, 2020, 11: 1064. doi：10.3389/fphys.2020.01064
[33]	Gopalakrishna D, Pennington S, Karaa A, et al. ET-1 stimulates superoxide production by ENOS following exposure of vascular endothelial cells to endotoxin[J]. Shock, 2016, 46(1): 60-6. doi：10.1097/shk.0000000000000576