淫羊藿苷保护脑死亡大鼠肾损伤的作用及机制研究

doi:10.3877/cma.j.issn.1674-3903.2017.03.004

中华移植杂志(电子版) ›› 2017, Vol. 11 ›› Issue (03) : 143 -147. doi: 10.3877/cma.j.issn.1674-3903.2017.03.004

所属专题：文献；

论著

淫羊藿苷保护脑死亡大鼠肾损伤的作用及机制研究

刘岩峰¹, 熊韫祎², 王东³, 陈文芳⁴, 苏乔⁴, 李雯雯⁴, 黄慧婷⁴, 刘龙山⁴^,^†(

), 王长希⁵

^1. 518001　深圳，暨南大学第二附属医院泌尿外科；510080　广州，中山大学附属第一医院
^2. 510260　广州医科大学附属第二医院移植科
^3. 510080　广州，中山大学附属第一医院
^5. 510080　广州，中山大学附属第一医院；510080　广州，广东省器官捐献与移植免疫重点实验室

收稿日期:2017-07-11 出版日期:2017-08-25
通信作者: 刘龙山
基金资助:
国家自然科学基金(81670680); 广东省科技计划项目(2014B020212006，2015B020226002); 广东省自然科学基金(2015A030313135); 广州市科技计划项目(2014Y2－00114); 广东省器官捐献与移植免疫重点实验室基金(2013A 061401007)

Protective effect of icariin on brain-death induced kidney injury and its mechanisms in rats

Yanfeng Liu¹, Yunyi Xiong², Dong Wang³, Wenfang Chen⁴, Qiao Su⁴, Wenwen Li⁴, Huiting Huang⁴, Longshan Liu⁴^,^†(), Changxi Wang⁵

^1. Department of Urology, the Second Affiliated Hospital, JiNan University, Shenzhen 518001, China; The First Affiliated Hospital of Sun Yat-Sen University, Guangzhou 510080, China
^2. Department of Organ Transplantation, the Second Affiliated Hospital, Guangzhou Medical University, Guangzhou 510260, China
^4. The First Affiliated Hospital of Sun Yat-Sen University, Guangzhou 510080, China
^5. The First Affiliated Hospital of Sun Yat-Sen University, Guangzhou 510080, China;Guangdong Provincial Key Laboratory on Organ Donation and Transplant Immunology, Guangzhou 510080, China

Received:2017-07-11 Published:2017-08-25
Corresponding author: Longshan Liu
About author:
Corresponding author: Liu Longshan, Email: liulongshan2003@163.com

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引用本文：

刘岩峰, 熊韫祎, 王东, 陈文芳, 苏乔, 李雯雯, 黄慧婷, 刘龙山, 王长希. 淫羊藿苷保护脑死亡大鼠肾损伤的作用及机制研究[J]. 中华移植杂志(电子版), 2017, 11(03): 143-147.

Yanfeng Liu, Yunyi Xiong, Dong Wang, Wenfang Chen, Qiao Su, Wenwen Li, Huiting Huang, Longshan Liu, Changxi Wang. Protective effect of icariin on brain-death induced kidney injury and its mechanisms in rats[J]. Chinese Journal of Transplantation(Electronic Edition), 2017, 11(03): 143-147.

目的

探讨淫羊藿苷对脑死亡大鼠肾损伤的保护作用及其作用机制。

方法

30只SD大鼠被随机分为3组，每组各10只，分别为脑死亡组、治疗组和假手术组。脑死亡组大鼠在诱导脑死亡后不接受任何治疗；治疗组大鼠在诱导脑死亡后腹腔注射淫羊藿苷，剂量为100 mg/kg；假手术组保持颅内压正常并在麻醉状态下接受机械通气6 h。脑死亡组和治疗组分别在诱导脑死亡6 h后处死大鼠，假手术组在机械通气6 h后处死大鼠，分别取血清和肾组织进行检测。计量资料以均数±标准差(±s)表示，采用单因素方差分析比较3组大鼠血清肌酐、尿素氮、Paller评分、CD68^＋巨噬细胞计数、促炎因子(IL-1β、IL-6、TNF-α)及趋化因子(MCP-1、IP-10)的转录水平，组间两两比较采用LSD法。P<0.05为差异有统计学意义。

结果

淫羊藿苷治疗组脑死亡大鼠血清尿素氮和血清肌酐水平明显降低，分别为(10.0±1.5)、(92±12)mmol/L，与其他两组比较差异均有统计学意义(P均<0.05)；组织病理学示肾小管损伤评分明显降低，透射电镜示线粒体和内质网损伤减轻。淫羊藿苷可明显降低脑死亡大鼠肾组织内促炎因子(IL-1β、IL-6、TNF-α)和趋化因子(MCP-1、IP-10)的转录水平及CD68^＋巨噬细胞浸润，并明显降低c-Jun氨基末端激酶、p38丝裂原活化蛋白激酶和信号传导与转录激活因子3蛋白质磷酸化水平。

结论

淫羊藿苷能减轻脑死亡大鼠肾损伤，保护肾功能，可能通过抑制炎性反应和SAPK、JAK-STAT信号通路发挥作用。

关键词: 脑死亡, 炎性损伤, 淫羊藿苷, 信号通路

Objective

To investigate the protective effect of icariin on brain-death induced kidney injury in rats and its mechanisms.

Methods

Thirty SD rats were randomly divided into three groups, including sham group, control group (brain death) and experiment group (brain death with icariin treatment). Icariin was intraperitoneally given at the dose of 100 mg/kg right after brain death. Sham group were anaesthetized and treated with mechanical ventilation for 6 hours at the condition of normal intracranial pressure. The rats in control group and experiment group were sacrificed 6 hours after brain death and rats in sham group were sacrificed 6 hours after mechanical ventilation, and peripheral blood and kidneys were collected and detected. Measurement data was represented as (±s). One-way analysis of variance was used to compare serum creatinine, urea nitrogen, Paller score, CD68⁺ macrophage count and transcriptional levels of proinflammatory factors (IL-1β, IL-6, TNF-α) and chemokines (MCP-1, IP-10) among the 3 groups, least significant difference method was used to compare the above indexes between any two groups, difference was statistically significant when P<0.05.

Results

The levels of serum urine nitrogen and creatinine in experiment group were (10.0±1.5) and (92±12) mmol/L, respectively, which were significantly lower than control group and higher than sham group (P all<0.05). Icariin decreased the paller scores of tubular injury and alleviated mitochondria and endoplasmic reticulum injury. Icariin significantly decreased the mRNA levels of proinflammatory factors (IL-1β, IL-6, TNF-α) and chemokines (MCP-1, IP-10) , infiltration degree of macrophages, and phosphorylation levels of JNK, p38-MAPK and STAT3 in kidney tissues of experiment group.

Conclusions

Icariin protects renal function by alleviation of brain-death induced kidney injury, which might by inhibition of inflammation and SAPK and JAK-STAT signal pathways.

Key words: Brain death, Inflammatory injury, Icariin, Signal pathway

表1 目的基因实时荧光定量PCR引物

目的基因	上游引物(5’-3’)	下游引物(5’-3’)
IL-1β	ATGACCTGTTCTTTGAGGCTGAC	CTTGTCGAGATGCTGCTGTGA
IL-6	AACTCCATCTGCCCTTCAGGAACA	AAGGCAGTGGCTGTCAACAACATC
TNF-α	GGTGTCTGTGCCTCAGCCTCT	GGGAACTTCTCCTCCTTGTTGG
MCP-1	GCCTGTTGTTCACAGTTGCTG	GACTCATTGGGATCATCTTGC
IP-10	TGAAAGCGGTGAGCCAAAGAA	CTGGGTAAAGGGAGGTGGAGA
β-actin	AGATTACTGCCCTGGCTCCTA	ATCGTACTCCTGCTTGCTGAT

表1 目的基因实时荧光定量PCR引物

目的基因	上游引物(5’-3’)	下游引物(5’-3’)
IL-1β	ATGACCTGTTCTTTGAGGCTGAC	CTTGTCGAGATGCTGCTGTGA
IL-6	AACTCCATCTGCCCTTCAGGAACA	AAGGCAGTGGCTGTCAACAACATC
TNF-α	GGTGTCTGTGCCTCAGCCTCT	GGGAACTTCTCCTCCTTGTTGG
MCP-1	GCCTGTTGTTCACAGTTGCTG	GACTCATTGGGATCATCTTGC
IP-10	TGAAAGCGGTGAGCCAAAGAA	CTGGGTAAAGGGAGGTGGAGA
β-actin	AGATTACTGCCCTGGCTCCTA	ATCGTACTCCTGCTTGCTGAT

图1 3组大鼠肾组织病理学表现(HE×200)

表2 3组大鼠肾功能(±s)

组别	只数	血清肌酐(mmol/L)	血清尿素氮(mmol/L)	Paller评分(分)
脑死亡组	10	121±15^a	12.4±1.9^a	242±42^a
治疗组	10	92±12^ab	10.0±1.5^ab	1 680±240^ab
假手术组	10	43±12	4.2±1.1	50±100

表2 3组大鼠肾功能(±s)

组别	只数	血清肌酐(mmol/L)	血清尿素氮(mmol/L)	Paller评分(分)
脑死亡组	10	121±15^a	12.4±1.9^a	242±42^a
治疗组	10	92±12^ab	10.0±1.5^ab	1 680±240^ab
假手术组	10	43±12	4.2±1.1	50±100

图2 3组大鼠肾组织超微结构(×5 000)

表3 3组大鼠肾组织促炎因子、趋化因子表达和CD68^＋巨噬细胞浸润情况(±s)

组别	只数	IL-1β	IL-6	TNF-α	MCP-1	IP-10	CD68^＋(个)
脑死亡组	10	9.4±1.67^a	12.8±3.24^a	3.8±1.39^a	4.2±1.55^a	22.1±3.41^a	46.7±10.2^a
治疗组	10	5.1±0.73^ab	5.5±1.65^ab	2.5±0.81^ab	2.4±0.64^ab	8.3±2.58^ab	18.7±3.3^ab
假手术组	10	1.0±0.14	1.0±0.15	1.0±0.27	1.0±0.24	1.0±0.20	3.8±2.9

表3 3组大鼠肾组织促炎因子、趋化因子表达和CD68^＋巨噬细胞浸润情况(±s)

组别	只数	IL-1β	IL-6	TNF-α	MCP-1	IP-10	CD68^＋(个)
脑死亡组	10	9.4±1.67^a	12.8±3.24^a	3.8±1.39^a	4.2±1.55^a	22.1±3.41^a	46.7±10.2^a
治疗组	10	5.1±0.73^ab	5.5±1.65^ab	2.5±0.81^ab	2.4±0.64^ab	8.3±2.58^ab	18.7±3.3^ab
假手术组	10	1.0±0.14	1.0±0.15	1.0±0.27	1.0±0.24	1.0±0.20	3.8±2.9

图3 3组大鼠肾组织内JNK、p38-MAPK、STAT3蛋白质磷酸化水平

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