缺氧诱导因子-1α抑 制剂YC-1改善糖尿病肾病小鼠肾脏损伤的机制研究

doi:10.3969/j.issn.1674-8115.2023.09.003

上海交通大学学报（医学版） ›› 2023, Vol. 43 ›› Issue (9): 1089-1098.doi: 10.3969/j.issn.1674-8115.2023.09.003

• 论著 · 基础研究 • 上一篇

缺氧诱导因子-1α抑制剂YC-1改善糖尿病肾病小鼠肾脏损伤的机制研究

贾君杰(), 邢海帆, 张群子, 刘奇烨, 汪年松, 范瑛()

上海交通大学医学院附属第六人民医院肾内科，上海 200233

收稿日期:2023-04-14 接受日期:2023-08-10 出版日期:2023-09-28 发布日期:2023-09-28
通讯作者: 范瑛 E-mail:jekun0610@gmail.com;fanyingsh@126.com
作者简介:贾君杰（1997—），男，博士生；电子信箱：jekun0610@gmail.com。
基金资助:
国家自然科学基金(81870468);上海交通大学医学院“双百人”项目(20191833);上海交通大学“交大之星”医工交叉重点项目(YG2023ZD21)

Renal protective effect and mechanism research of hypoxia inducible factor-1α inhibitor YC-1 in diabetic nephropathy mice

JIA Junjie(), XING Haifan, ZHANG Qunzi, LIU Qiye, WANG Niansong, FAN Ying()

Department of Nephrology, Shanghai Sixth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200233, China

Received:2023-04-14 Accepted:2023-08-10 Online:2023-09-28 Published:2023-09-28
Contact: FAN Ying E-mail:jekun0610@gmail.com;fanyingsh@126.com
Supported by:
National Natural Science Foundation of China(81870468);“Two-hundred Talents” Program of Shanghai Jiao Tong University School of Medicine(20191833);Medical Engineering Cross Research Foundation of the Star Program of Shanghai Jiao Tong University(YG2023ZD21)

摘要/Abstract

摘要：

目的·研究缺氧诱导因子-1α（hypoxia inducible factor-1α，HIF-1α）抑制剂YC-1对小鼠糖尿病肾病（diabetic nephropathy，DN）进展的影响及潜在机制。方法·将10周龄的雄性db/db小鼠（DN模型）和同窝野生型（WT）小鼠按是否给予YC-1分为4组，即WT组、WT+YC-1组、DB组、DB+YC-1组，每组6只。YC-1干预组予以YC-1（20 mg/kg，1次/d）腹腔注射8周，非干预组同时予以等体积二甲基亚砜腹腔注射。干预8周后，检测小鼠血糖、体质量和肾脏质量，并收集血清、尿液、肾组织标本。检测小鼠血肌酐、尿白蛋白/肌酐比（urinary albumin-to-creatinine ratio，UACR）、尿中性粒细胞明胶酶相关脂质运载蛋白（neutropil gelatinase-associated lipocalin，NGAL）水平。肾脏行苏木精-伊红（H-E）染色、过碘酸-雪夫（PAS）染色观察组织病理损伤；马松（Masson）染色检测纤维化情况，免疫组织化学（免疫组化）法检测Ⅰ型胶原蛋白，Western blotting检测α-平滑肌肌动蛋白（α-smooth muscle actin，α-SMA）水平；免疫组化法和Western blotting检测HIF-1α表达；TUNEL染色和Western blotting检测细胞凋亡水平；试剂盒检测肾脏超氧化物歧化酶（superoxide dismutase，SOD）活性和丙二醛（malondialdehyde，MDA）含量；Western blotting检测内质网应激（endoplasmic reticulum stress，ERS）标志物免疫球蛋白重链结合蛋白（immunoglobulin heavy chain binding protein，BiP；又称GRP78）、磷酸化蛋白激酶样内质网激酶（phospho-protein kinase R-like endoplasmic reticulum kinase，p-PERK）、总PERK、磷酸化真核起始因子2α（phospho-eukaryotic initiation factor 2α，p-eIF2α）、总eIF2α、激活转录因子4（activating transcription factor 4，ATF4）和C/EBP同源蛋白（C/EBP homologous protein，CHOP）的表达。结果·与WT组小鼠相比，DB组小鼠血糖升高，肾功能下降，肾脏病理损伤和纤维化加重，肾脏HIF-1α表达、氧化应激和ERS激活程度增加。与DB组小鼠相比，DB+YC-1组小鼠血糖无明显变化，但肾/体质量比、血肌酐、UACR、尿NGAL水平显著下降，肾脏病理损伤和纤维化程度显著减轻，Ⅰ型胶原蛋白和α-SMA表达显著降低，肾脏HIF-1α表达显著降低，肾脏TUNEL阳性细胞数减少，促凋亡蛋白BAX和活化的胱天蛋白酶（cleaved caspase-3）表达显著下降，抑制凋亡蛋白BCL-2表达显著升高，肾脏SOD活性显著升高，MDA含量显著降低，肾脏ERS标志物GRP78、p-PERK、p-eIF2α、ATF4和CHOP表达显著下降（均P<0.05）。结论·HIF-1α抑制剂YC-1能够改善DN小鼠肾脏氧化应激和ERS的异常激活，抑制细胞凋亡和肾脏纤维化，减轻肾脏病理损伤，保护肾功能。

关键词: 糖尿病肾病, 缺氧诱导因子-1α, 氧化应激, 内质网应激

Abstract:

Objective ·To investigate the effect of hypoxia inducible factor-1α (HIF-1α) inhibitor YC-1 on the progression of diabetic nephropathy (DN) in mice and the potential mechanism. Methods ·Ten-week-old male db/db mice (DN model) and their nondiabetic wild-type (WT) littermates were divided into 4 groups (n=6) according to whether treated with YC-1 or not: WT group, WT+YC-1 group, DB group, and DB+YC-1 group. The treatment groups were intraperitoneally injected with YC-1 (20 mg·kg^-1) once a day, while the non-treatment groups received the same volumes of DMSO injection. After a total of 8 weeks of intervention, blood glucose, body weight, and kidney weight of all mice were measured. Serum, urine and kidney tissue samples were harvested. Serum creatinine, urinary albumin-to-creatinine ratio (UACR), and urine neutropil gelatinase-associated lipocalin (NGAL) levels were detected. The kidneys were stained with ?hemat?oxyli?n-eosin (H-E) and periodic acid-Schiff (PAS) to observe the pathological changes. Masson staining was used to detect fibrosis, collagen-Ⅰ was detected by immunohistochemistry, and α-smooth muscle actin (α-SMA) was detected by Western blotting. The expression of HIF-1α was detected by both Western blotting and immunohistochemistry. TUNEL staining and Western blotting for apoptosis-related proteins were used to observe the cell apoptosis level. Superoxide dismutase (SOD) activity and malondialdehyde (MDA) level were detected by the kits. Endoplasmic reticulum stress (ERS) markers, including immunoglobulin heavy chain binding protein (BiP, also known as GRP78), phospho-protein kinase R-like endoplasmic reticulum kinase (p-PERK), total PERK, phospho-eukaryotic initiation factor 2α (p-eIF2α), total eIF2α, activating transcription factor 4 (ATF4), and C/EBP homologous protein (CHOP), were determined by Western blotting. Results ·Compared with the WT group, the DB group showed significant rise of blood glucose, loss of renal function, severe kidney histopathology injuries and kidney fibrosis, increase of renal HIF-1α expression, and aggravated oxidative stress and ERS. Whilst there were no significant changes in blood glucose, YC-1 treatment notably reduced kidney weight/body weight ratio, serum creatinine, UACR, and urine NGAL levels in db/db mice. YC-1 treatment ameliorated kidney histopathology injuries and kidney fibrosis, and decreased the expressions of collagen-Ⅰ and α-SMA. YC-1 treatment also reduced the number of TUNEL positive cells, the expression of HIF-1α and pro-apoptotic proteins including BAX and cleaved caspase-3, and MDA level in the kidneys of db/db mice, while promoting anti-apoptotic protein BCL-2 expression and SOD activity. The expressions of ERS markers GRP78, p-PERK, p-eIF2α, ATF4, and CHOP were likewise significantly decreased in DB+YC-1 group. Conclusion ·HIF-1α inhibitor YC-1 inhibits oxidative stress and abnormal activation of ERS, improving cell apoptosis and fibrosis in the kidneys of DN mice, which would attenuate the aggravation of pathological damage and loss of kidney function.

Key words: diabetic nephropathy (DN), hypoxia inducible factor-1α (HIF-1α), oxidative stress, endoplasmic reticulum stress (ERS)

中图分类号:

R587.2

贾君杰, 邢海帆, 张群子, 刘奇烨, 汪年松, 范瑛. 缺氧诱导因子-1α抑制剂YC-1改善糖尿病肾病小鼠肾脏损伤的机制研究[J]. 上海交通大学学报（医学版）, 2023, 43(9): 1089-1098.

JIA Junjie, XING Haifan, ZHANG Qunzi, LIU Qiye, WANG Niansong, FAN Ying. Renal protective effect and mechanism research of hypoxia inducible factor-1α inhibitor YC-1 in diabetic nephropathy mice[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2023, 43(9): 1089-1098.

图/表 7

表1 YC-1对WT和db/db小鼠一般情况和肾功能指标的影响（n=6）

Tab 1 Effect of YC-1 on general physical signs and kidney function indexes in the WT and db/db mice （n=6）

Index	WT group	WT+YC-1 group	DB group	DB+YC-1 group
RBG/(mmol·L^-1)	8.10±0.39	8.25±0.43	28.57±1.18^①	29.15±1.29^①
BW/g	27.47±0.58	25.98±0.65	56.72±1.05^①	54.53±1.02^①
KW/BW/(mg·g^-1)	9.97±0.23	10.19±0.23	12.25±0.22^①	11.31±0.14^①②
Scr/(mg·dL^-1)	0.239±0.010	0.263±0.012	0.473±0.017^①	0.414±0.016^①③
UACR/(μg·mg^-1)	61.68±12.18	67.46±10.71	1 445.61±63.10^①	663.94±60.19^①②
uNGAL/(μg·mL^-1)	23.70±1.09	23.04±0.95	118.66±2.98^①	86.02±3.44^①②

图1 YC-1对db/db小鼠肾脏组织病理损伤的影响Note： A. Renal histology evaluations of different groups were performed with H-E staining and PAS staining (×200, scale bar=50 μm). B. Mesangial matrix index expressed as the ratio of mesangial matrix area to glomerular tuft area. C. Analysis of tubular injury score. ①P=0.000, compared with WT group; ②P=0.000, compared with DB group.

Fig 1 Effect of YC-1 on kidney histopathology injuries in the db/db mice

图2 YC-1对db/db小鼠肾脏纤维化的影响Note： A. Extracellular matrix accumulation and collagen fiber deposition were determined with Masson trichrome staining and immunohistochemical staining of collagen-Ⅰ (×200, scale bar=50 μm). B. Western blotting analysis and quantification of the expression of the pro?brotic molecule α-SMA. ①P=0.000, compared with the WT group; ②P=0.009, compared with the DB group.

Fig 2 Effect of YC-1 on kidney fibrosis in the db/db mice

图3 YC-1对db/db小鼠肾脏HIF-1α表达的影响Note： A. Immunohistochemical staining of HIF-1α in kidney tissues (×200, scale bar=50 μm). B. Western blotting analysis and quantification of HIF-1α expression. ①P=0.000, compared with the WT group; ②P=0.000, compared with the DB group.

Fig 3 Effect of YC-1 on HIF-1α expression in the kidneys of db/db mice

图4 YC-1对db/db小鼠肾脏细胞凋亡的影响Note： A. Representative TUNEL staining photographs (×200, scale bar=50 μm) and quantification analysis of TUNEL positive cells under each high power field. The data are presented as boxplot. B. Western blotting analysis and quantification of the expression of BAX, BCL-2, and cleaved caspase-3. ①P=0.000, ④P=0.004, compared with the WT group; ②P=0.011, ③P=0.000, ⑤P=0.021, compared with the DB group.

Fig 4 Effect of YC-1 on cell apoptosis in the kidneys of the db/db mice

表2 YC-1对db/db小鼠肾脏氧化应激的影响（n=6）

Tab 2 Effect of YC-1 on oxidative stress in the kidneys of db/db mice （n=6）

Index	WT group	WT+YC-1 group	DB group	DB+YC-1 group
MDA level/(nmol·mg^-1)	6.69±0.53	5.78±0.82	14.05±1.56^①	9.65±0.71^②
SOD activity/(U·mg^-1)	117.28±11.90	115.29±6.72	41.71±3.19^①	79.40±2.52^③

图5 YC-1对db/db小鼠肾脏ERS的影响Note： A. Representative Western blotting bands. t-PERK—total-PERK; t-eIF2α—total-eIF2α. B. Quantification of Western blotting bands. ①P=0.000, ⑤P=0.035, compared with the WT group; ②P=0.011, ③P=0.000, ④P=0.002, ⑥P=0.048, compared with the DB group.

Fig 5 Effect of YC-1 on ERS in the kidneys of db/db mice

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缺氧诱导因子-1α抑制剂YC-1改善糖尿病肾病小鼠肾脏损伤的机制研究

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缺氧诱导因子-1α抑 制剂YC-1改善糖尿病肾病小鼠肾脏损伤的机制研究

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