蝉花菌丝联合海昆肾喜胶囊对糖尿病肾小管上皮细胞自噬-溶酶体通路的影响

doi:10.3877/cma.j.issn.2095-3216.2022.04.006

中华肾病研究电子杂志 ›› 2022, Vol. 11 ›› Issue (04) : 212 -218. doi: 10.3877/cma.j.issn.2095-3216.2022.04.006

论著

蝉花菌丝联合海昆肾喜胶囊对糖尿病肾小管上皮细胞自噬-溶酶体通路的影响

贾英民¹, 张术姣², 耿运玲², 曹梓静², 王耀献², 吕仁和², 刘玉宁², 刘伟敬²^,^†(

)

^1. 101300　北京，北京中医医院顺义医院
^2. 100007　北京，北京中医药大学东直门医院、肾病研究所

收稿日期:2022-05-11 出版日期:2022-08-28
通信作者: 刘伟敬
基金资助:
国家自然科学基金项目(82074361); 第六批顺义区人才工作创新项目暨北京中医医院顺义医院中医药重点学科人才团队建设项目

Effect of Cordyceps sobolifera mycelium combined with Haikun Shenxi capsule on autophagy-lysosome pathway of diabetic renal tubular epithelial cells

Yingmin Jia¹, Shujiao Zhang², Yunling Geng², Zijing Cao², Yaoxian Wang², Renhe Lv², Yuning Liu², Weijing Liu²^,^†()

^1. Shunyi Hospital, Beijing Hospital of Traditional Chinese Medicine, Beijing 101300
^2. Dongzhimen Hospital, Institute of Nephrology, Beijing University of Chinese Medicine, Beijing 100007; China

Received:2022-05-11 Published:2022-08-28
Corresponding author: Weijing Liu

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贾英民, 张术姣, 耿运玲, 曹梓静, 王耀献, 吕仁和, 刘玉宁, 刘伟敬. 蝉花菌丝联合海昆肾喜胶囊对糖尿病肾小管上皮细胞自噬-溶酶体通路的影响[J]. 中华肾病研究电子杂志, 2022, 11(04): 212-218.

Yingmin Jia, Shujiao Zhang, Yunling Geng, Zijing Cao, Yaoxian Wang, Renhe Lv, Yuning Liu, Weijing Liu. Effect of Cordyceps sobolifera mycelium combined with Haikun Shenxi capsule on autophagy-lysosome pathway of diabetic renal tubular epithelial cells[J]. Chinese Journal of Kidney Disease Investigation(Electronic Edition), 2022, 11(04): 212-218.

目的

探讨蝉花菌丝联合海昆肾喜胶囊对糖尿病肾病(DN)肾小管上皮细胞自噬-溶酶体的影响与机制。

方法

将24只大鼠腹腔单次注射链脲佐菌素(STZ)诱导SD大鼠高血糖模型后，随机分为模型组(DN)和治疗组(DN+蝉花菌丝联合海昆肾喜胶囊)，用药24周；另外10只未诱导高血糖大鼠设为对照组。检测各组大鼠肾功能指标，取肾皮质行组织病理学检查，采用Western印迹检测大鼠分离肾小管的胶原蛋白-Ⅲ、α-平滑肌肌动蛋白(α-SMA)、转录因子EB(TFEB)、自噬泡标记蛋白LC3和自噬底物p62表达。将HK-2细胞分为牛血清白蛋白(Co-BSA)组、Co-BSA治疗组、糖基化终末产物-BSA(AGEs-BSA)组及AGEs-BSA-治疗组。Western印迹检测各组细胞p62、TFEB表达；以TFEB质粒转染HK-2细胞，通过RT-PCR检测过表达效率，观察各组HK-2细胞溶酶体红色荧光探针荧光强度，并检测各组细胞组织蛋白酶活性。

结果

与对照组大鼠相比，模型组大鼠的血肌酐(Scr)、24 h尿蛋白水平增高、LC3、p62、胶原蛋白-Ⅲ和α-SMA表达明显增多，而肾小球滤过率(GFR)、TFEB表达明显减少。与模型组大鼠相比，治疗组大鼠的Scr、24 h尿蛋白均明显降低，GFR明显升高，LC3、p62、胶原蛋白-Ⅲ和α-SMA表达明显减少，TFEB表达明显增多。在HK-2细胞中，TFEB和p62的表达情况与在大鼠肾组织中相似。与Co-BSA组相比，AGEs-BSA组的探针荧光强度和组织蛋白酶活性明显减弱；过表达TFEB后，AGEs-BSA组的荧光强度和组织蛋白酶活性均明显增强。

结论

蝉花菌丝联合海昆肾喜胶囊对糖尿病肾小管上皮细胞可能具有上调TFEB表达、调节自噬-溶酶体通路的作用。

关键词: 蝉花菌丝, 海昆肾喜胶囊, 糖尿病肾病, 转录因子EB, 自噬

Objective

To investigate the effect of Cordyceps sobolifera mycelium combined with Haikun Shenxi capsule on autophagy-lysosome of diabetic renal tubular epithelial cells.

Methods

After a single intraperitoneal injection of streptozotocin (STZ) to induce the hyperglycemia model in 24 SD rats, the rats were randomly divided into the model group (DN) and the treatment group with a treatment period of 24 weeks, while another 10 rats without hyperglycemia induction were as the control group. The renal function indexes of the rats were detected, and the renal cortex was taken for histopathological examination, while the expressions of collagen-Ⅲ, α-smooth muscle actin (α-SMA), transcription factor EB (TFEB), autophagic vacuole marker protein LC3, and autophagy substrate p62 in the separated tubules were detected by Western blot. The renal tubular HK-2 cells were divided into Co-BSA group, Co-BSA drug group, advanced glycation end products-BSA (AGEs-BSA) group, and AGEs-BSA-drug group. Western blot was used to detect the expressions of p62 and TFEB in each group of cells. The TFEB plasmid was transfected into the HK-2 cells, and the overexpression efficiency was detected by RT-PCR. The fluorescence intensity of lysosomal red fluorescent probe was observed, and the cathepsin activity was measured.

Results

Compared with the control group, the model group showed higher levels of serum creatinine (Scr) and 24h urine protein, more expressions of LC3, p62, collagen-Ⅲ, and α-SMA, but lower levels of glomerular filtration rate (GFR) and TFEB expression. Compared with the model group, the treatment group showed lower levels of Scr and 24h urinary protein, less expressions of LC3, p62, collagen-Ⅲ, and α-SMA, but higher levels of GFR and TFEB expression. In the HK-2 cells, the expressions of TFEB and p62 were similar to those in kidneys of the rats. Compared with the BSA group, the AGEs-BSA group displayed lower levels of the probe fluorescence intensity and cathepsin activity. After overexpression of TFEB, the AGEs-BSA group′s fluorescence intensity and cathepsin activity were significantly enhanced.

Conclusion

Cordyceps sobolifera mycelium combined with Haikun Shenxi capsule might have the effect of up-regulating TFEB expression and regulating the autophagy-lysosome pathway of diabetic tubular epithelial cells.

Key words: Cordyceps sobolifera mycelium, Haikun Shenxi capsule, Diabetic nephropathy, Transcription factor EB, Autophagy

表1 蝉花菌丝联合海昆肾喜对DN大鼠一般指标的影响(±s)

检测指标	对照组	模型组	治疗组
Scr (mg/dl)	0.53±0.05	1.31±0.16^a	0.89±0.09^b
BUN (mg/dl)	35.4±3.6	58.9±6.1^a	47.8±4.9
24 h尿蛋白(mg)	12.0±1.4	24.1±2.5^a	15.8±1.7^b
GFR (ml/min)	1.41±0.12	0.71±0.08^a	1.19±0.10^b
肾重/体重×1 000	4.92±0.51	8.14±0.85^a	6.99±0.72

表1 蝉花菌丝联合海昆肾喜对DN大鼠一般指标的影响(±s)

检测指标	对照组	模型组	治疗组
Scr (mg/dl)	0.53±0.05	1.31±0.16^a	0.89±0.09^b
BUN (mg/dl)	35.4±3.6	58.9±6.1^a	47.8±4.9
24 h尿蛋白(mg)	12.0±1.4	24.1±2.5^a	15.8±1.7^b
GFR (ml/min)	1.41±0.12	0.71±0.08^a	1.19±0.10^b
肾重/体重×1 000	4.92±0.51	8.14±0.85^a	6.99±0.72

图1 三组大鼠自噬应激对抗肾间质纤维化观测注：A：各组大鼠肾组织LC3免疫荧光染色(×400)及分析；B：各组大鼠肾组织p62免疫组织化学染色(×400)及分析；C：Western印迹检测各组大鼠肾组织纤维化标志物；与对照组相比，^aP<0.05；与模型组相比，^bP<0.05

图2 Western印迹检测肾组织及HK-2细胞核转录因子EB和p62蛋白表达量注：A：Western印迹检测各组大鼠肾组织TFEB和p62蛋白；B：Western印迹检测各组HK-2细胞TFEB和p62蛋白；与对照组或Co-BSA组相比，^aP<0.05；与模型组或AGEs-BSA组相比，^bP<0.05

图3 RT-PCR检测HK-2细胞中核转录因子EB mRNA水平及各组HK-2细胞溶酶体红色荧光探针染色和平均荧光强度注：A：RT-PCR检测HK-2细胞TFEB mRNA表达水平；B：溶酶体红色荧光探针染色的平均荧光强度：与对照组相比，^aP<0.05

图4 各组HK-2细胞组织蛋白酶活性检测注：A：各组HK-2细胞组织蛋白酶B活性检测；B：各组HK-2细胞组织蛋白酶L活性检测；C：各组HK-2细胞组织蛋白酶D活性检测；与对照组相比，^aP<0.05

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