川陈皮素对急性肾损伤大鼠的改善作用及机制研究

doi:10.11958/20221287

天津医药 ›› 2023, Vol. 51 ›› Issue (5): 498-503.doi: 10.11958/20221287

川陈皮素对急性肾损伤大鼠的改善作用及机制研究

耿永芝¹(), 杨利², 李国伟¹, 张金涛¹, 程晓磊¹, 檀立端¹^,^△()

1.承德市中心医院急诊科（邮编067000）
2.宽城满族自治县医院普外科

收稿日期:2022-08-18 修回日期:2022-09-28 出版日期:2023-05-15 发布日期:2023-05-05
通讯作者: △E-mail:ceinf60@163.com
作者简介:耿永芝（1983），女，主治医师，主要从事急诊医学方面研究。E-mail：gengyongzhi1983@163.com
基金资助:
河北省承德市科学技术研究与发展计划项目(202006A155)

Study on the improvement effect and mechanism of nobiletin on rats with acute kidney injury

GENG Yongzhi¹(), YANG Li², LI Guowei¹, ZHANG Jintao¹, CHENG Xiaolei¹, TAN Liduan¹^,^△()

1. Department of Emergency, Chengde Central Hospital, Chengde 067000, China
2. Department of General Surgery, Kuancheng Manchu Autonomous County Hospital

Received:2022-08-18 Revised:2022-09-28 Published:2023-05-15 Online:2023-05-05
Contact: △E-mail:ceinf60@163.com

耿永芝, 杨利, 李国伟, 张金涛, 程晓磊, 檀立端. 川陈皮素对急性肾损伤大鼠的改善作用及机制研究[J]. 天津医药, 2023, 51(5): 498-503.

GENG Yongzhi, YANG Li, LI Guowei, ZHANG Jintao, CHENG Xiaolei, TAN Liduan. Study on the improvement effect and mechanism of nobiletin on rats with acute kidney injury[J]. Tianjin Medical Journal, 2023, 51(5): 498-503.

摘要/Abstract

摘要：

目的探究川陈皮素（NOB）通过调节沉默信息调节因子1（SIRT-1）/叉头框转录因子O3a（FOXO3a）通路介导的自噬对急性肾损伤（AKI）大鼠的改善作用及其机制。方法采用一次性腹腔注射顺铂（20 mg/kg）的方法构建AKI大鼠模型。将60只SPF级SD雄性大鼠采用随机数字表法分为对照组（Control组）、AKI模型组（Model组）、低剂量NOB组（NOB-L组，200 mg/kg）、高剂量NOB组（NOB-H组，400 mg/kg）和高剂量NOB+SIRT-1抑制剂EX527组（NOB-H+EX527组，400 mg/kg NOB+5 mg/kg EX527），每组12只。末次给药后收集大鼠24 h尿液，检测24 h尿微量白蛋白含量、尿N-乙酰-β-D-氨基葡萄糖甘酶（NAG）含量和尿β2微球蛋白（β2-MG）含量；酶联免疫吸附试验（ELISA）检测大鼠血清中尿素氮（BUN）、血肌酐（Scr）和肾脏中超氧化物歧化酶（SOD）、丙二醛（MDA）水平；HE染色观察大鼠肾小管组织病理变化；TUNEL染色法检测肾小管细胞凋亡；实时荧光定量PCR（qPCR）法检测大鼠肾脏组织中SIRT1和FOXO3a mRNA表达；Western blot检测大鼠肾脏组织中SIRT1/FOXO3a通路和自噬相关蛋白的表达。结果与Control组相比，Model组大鼠肾脏组织病理损伤严重，24 h尿微量白蛋白、尿NAG、尿β2-MG、血BUN、血Scr、肾小管损伤评分、肾脏MDA水平、细胞凋亡率升高，肾脏SOD水平、SIRT1/FOXO3a通路和自噬相关蛋白表达降低（P<0.05）；与Model组相比，NOB-L组和NOB-H组大鼠肾脏组织病理损伤减轻，24 h尿微量白蛋白、尿NAG、尿β2-MG、血BUN、血Scr、肾小管损伤评分、肾脏MDA水平、细胞凋亡率降低，肾脏SOD水平、SIRT1/FOXO3a通路和自噬相关蛋白表达升高（P<0.05）。结论 NOB可能通过激活SIRT-1/FOXO3a通路，促进自噬，进而改善大鼠的AKI。

关键词: 川陈皮素, 急性肾损伤, 顺铂, 自噬相关蛋白质类, SIRT-1/FOXO3a

Abstract:

Objective To investigate the ameliorating effect and mechanism of nobiletin (NOB) by regulating the autophagy mediated by silent information regulator 1 (SIRT-1)/forkhead box transcription factor O3a (FOXO3a) pathway in rats with acute kidney injury (AKI). Methods A rat model of AKI was established by a one-time intraperitoneal injection of cisplatin (20 mg/kg). Sixty SPF SD male rats were randomly grouped by random number table into the control group, the AKI model group (model group), the low-dose NOB group (NOB-L group, 200 mg/kg), the high-dose NOB group (NOB-H group, 400 mg/kg) and the high-dose NOB+SIRT-1 inhibitor EX527 group (NOB-H+EX527 group, 400 mg/kg NOB+5 mg/kg EX527), with 12 rats in each group. The 24-hour urine of rats was collected after the last administration. The 24-hour urine microalbumin content, urine N-acetyl-β-D-glucosaminidase (NAG) content and urinary β2-microglobulin (β2-MG) content were detected. The serum levels of urea nitrogen (BUN) and creatinine (Scr), levels of superoxide dismutase (SOD) and malondialdehyde (MDA) in kidney were detected by enzyme-linked immunosorbent assay (ELISA). HE staining was used to observe the pathological changes of renal tubules. TUNEL staining was applied to observe the apoptosis of rat kidney tissue. SIRT1 and FOXO3a mRNA expression levels in rat kidney tissue were measured by qPCR. Western blot assay was applied to detect the expression of SIRT1/FOXO3a pathway and autophagy-related proteins in rat kidney tissue. Results Compared with the control group, there was severe renal tissue pathological injury in the model group, and the 24 h urine microalbumin content, urine NAG, urine β2-MG, BUN, Scr, renal tubular injury score, renal MDA level and apoptosis rate were obviously increased. The renal SOD level, SIRT1/FOXO3a pathway and autophagy-related protein expression were obviously decreased (P<0.05). Compared with the model group, the pathological injury of kidney tissue was reduced in the NOB-L group and the NOB-H group, and the 24 h urine microalbumin content, urine NAG, urine β2-MG, BUN, Scr, renal tubular injury score, renal MDA level and apoptosis rate were obviously decreased. The renal SOD level, SIRT1/FOXO3a pathway and autophagy-related protein expression were obviously increased (P<0.05). Conclusion NOB may promote autophagy by activating the SIRT-1/FOXO3a pathway, thereby improving acute kidney injury in rats.

Key words: nobiletin, acute kidney injury, cisplatin, autophagy-related proteins, SIRT-1/FOXO3a

中图分类号:

R285.5

图/表 8

参考文献 25

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基因名称	引物序列（5'→3'）	产物大小（bp）
SIRT1	上游：CAGTTCCAGCCATCTCTGTG	158
SIRT1	下游：GCAACCTGCTCCAAGGTATC	158
FOXO3a	上游：GCAAACCCTCTCGGACTCTC	116
FOXO3a	下游：TTCCCCACATTCAAACCAAC	116
GAPDH	上游：ACAGCAACAGGGTGGTGGAC	197
GAPDH	下游：TTTGAGGGTGCAGCG AACTT	197

基因名称	引物序列（5'→3'）	产物大小（bp）
SIRT1	上游：CAGTTCCAGCCATCTCTGTG	158
SIRT1	下游：GCAACCTGCTCCAAGGTATC	158
FOXO3a	上游：GCAAACCCTCTCGGACTCTC	116
FOXO3a	下游：TTCCCCACATTCAAACCAAC	116
GAPDH	上游：ACAGCAACAGGGTGGTGGAC	197
GAPDH	下游：TTTGAGGGTGCAGCG AACTT	197

组别	24 h尿微量白蛋白（mg）	尿NAG （U/L）	尿β2-MG （mg/L）
Control组	7.86±2.55	1.44±0.18	6.54±0.96
Model组	92.64±12.58^a	4.67±1.25^a	52.42±6.85^a
NOB-L组	68.25±9.68^b	3.37±0.59^b	34.66±4.20^b
NOB-H组	25.13±3.74^bc	1.95±0.34^bc	21.37±2.46^bc
NOB-H+EX527组	56.12±8.26^d	2.89±0.45^d	32.94±2.61^d
F	265.892^＊＊	60.064^＊＊	295.741^＊＊

组别	24 h尿微量白蛋白（mg）	尿NAG （U/L）	尿β2-MG （mg/L）
Control组	7.86±2.55	1.44±0.18	6.54±0.96
Model组	92.64±12.58^a	4.67±1.25^a	52.42±6.85^a
NOB-L组	68.25±9.68^b	3.37±0.59^b	34.66±4.20^b
NOB-H组	25.13±3.74^bc	1.95±0.34^bc	21.37±2.46^bc
NOB-H+EX527组	56.12±8.26^d	2.89±0.45^d	32.94±2.61^d
F	265.892^＊＊	60.064^＊＊	295.741^＊＊

组别	BUN（mmol/L）	Scr（μmol/L）
Control组	2.58±0.54	18.63±2.57
Model组	8.84±1.46^a	82.51±10.23^a
NOB-L组	6.72±1.34^b	60.16±7.44^b
NOB-H组	3.89±0.70^bc	42.56±6.30^bc
NOB-H+EX527组	5.24±1.25^d	56.27±8.82^d
F	109.769^＊＊	135.065^＊＊

川陈皮素对急性肾损伤大鼠的改善作用及机制研究

Study on the improvement effect and mechanism of nobiletin on rats with acute kidney injury

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参考文献 25

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组别	SOD （U/mL）	MDA （nmol/L）	SIRT1	FOXO3a
Control组	285.27±35.42	2.54±0.68	1.00±0.00	1.00±0.00
Model组	140.54±25.49^a	9.10±1.14^a	0.37±0.07^a	0.42±0.03^a
NOB-L组	190.65±25.61^b	7.57±0.85^b	0.55±0.06^b	0.60±0.08^b
NOB-H组	248.40±33.04^bc	4.38±0.49^bc	0.78±0.04^bc	0.82±0.06^bc
NOB-H+EX527组	180.35±27.27^d	6.80±0.77^d	0.62±0.09^d	0.64±0.04^d
F	36.728^＊＊	113.609^＊＊	159.341^＊＊	289.526^＊＊

组别	SIRT1	FOXO3a	LC3-Ⅱ/ LC3-Ⅰ	Beclin-1
Control组	0.79±0.04	0.86±0.06	2.12±0.18	0.56±0.04
Model组	0.25±0.02^a	0.32±0.04^a	0.56±0.06^a	0.10±0.01^a
NOB-L组	0.46±0.05^b	0.50±0.02^b	1.08±0.04^b	0.23±0.02^b
NOB-H组	0.62±0.03^bc	0.71±0.05^bc	1.83±0.07^bc	0.42±0.02^bc
NOB-H+EX527组	0.49±0.03^d	0.53±0.03^d	1.14±0.05^d	0.30±0.02^d
F	390.386^＊＊	306.840^＊＊	226.787^＊＊	793.818^＊＊

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