金丝桃苷对糖尿病足溃疡大鼠炎症反应、创面愈合及AMPK/SIRT1信号通路的影响

doi:10.11958/20230140

天津医药 ›› 2023, Vol. 51 ›› Issue (11): 1205-1210.doi: 10.11958/20230140

金丝桃苷对糖尿病足溃疡大鼠炎症反应、创面愈合及AMPK/SIRT1信号通路的影响

雷慧¹(), 鲍亚玲¹^,^△(), 鲍喜静², 马君²

1.张家口学院护理学院（邮编075000）
2.张家口市第一医院内分泌科

收稿日期:2023-02-20 修回日期:2023-05-03 出版日期:2023-11-15 发布日期:2023-11-07
通讯作者: ^△E-mail：527199577@qq.com
作者简介:雷慧（1979），女，副教授，主要从事中药药理学方面研究。E-mail：leihui6372@163.com
基金资助:
河北省医学科学研究课题计划项目(20211151)

Effects of hyperin on inflammatory reaction, wound healing and AMPK/SIRT1 signaling pathway in diabetic foot ulcer model rats

LEI Hui¹(), BAO Yaling¹^,^△(), BAO Xijing², MA Jun²

1. Nursing College of Zhangjiakou University, Zhangjiakou 075000, China
2. Department of Endocrinology, Zhangjiakou First Hospital

Received:2023-02-20 Revised:2023-05-03 Published:2023-11-15 Online:2023-11-07
Contact: ^△E-mail：527199577@qq.com

雷慧, 鲍亚玲, 鲍喜静, 马君. 金丝桃苷对糖尿病足溃疡大鼠炎症反应、创面愈合及AMPK/SIRT1信号通路的影响[J]. 天津医药, 2023, 51(11): 1205-1210.

LEI Hui, BAO Yaling, BAO Xijing, MA Jun. Effects of hyperin on inflammatory reaction, wound healing and AMPK/SIRT1 signaling pathway in diabetic foot ulcer model rats[J]. Tianjin Medical Journal, 2023, 51(11): 1205-1210.

摘要/Abstract

摘要：

目的探究金丝桃苷对糖尿病足溃疡（DFU）大鼠炎症反应、创面愈合以及对腺苷酸活化蛋白激酶（AMPK）/沉默信息调节因子1（SIRT1）信号通路的影响。方法构建DFU大鼠模型，并将建模成功的48只大鼠随机分为模型组，金丝桃苷低（100 mg/kg）、高（200 mg/kg）剂量组，二甲双胍（200 mg/kg）组，每组12只；另取12只大鼠作为对照组。各组大鼠给予相应药物干预，每日1次，连续4周。采用血糖仪分别检测大鼠给药前后空腹血糖（FBG）水平；酶联免疫吸附试验检测大鼠血清中白细胞介素6（IL-6）、肿瘤坏死因子α（TNF-α）水平；计算大鼠创面愈合率；苏木素-伊红（HE）染色观察大鼠创面肉芽组织病理学变化；荧光定量PCR检测创面肉芽组织中AMPK、SIRT1 mRNA表达水平；蛋白免疫印迹法检测AMPK、SIRT1蛋白表达水平。结果对照组大鼠创面肉芽组织细胞结构完整，排列整齐，少量炎性细胞浸润；与对照组相比，模型组大鼠创面肉芽组织成纤维细胞及毛细血管坏死严重，伴随大量炎性细胞浸润，FBG、血清TNF-α、IL-6水平升高（P<0.05），创面愈合率、AMPK、SIRT1 mRNA和蛋白表达水平降低（P<0.05）；与模型组相比，金丝桃苷低、高剂量组创面肉芽组织病变程度依次减轻，FBG、血清中TNF-α、IL-6水平依次降低（P<0.05），创面愈合率、AMPK、SIRT1 mRNA和蛋白表达水平依次升高（P<0.05）；二甲双胍组和金丝桃苷高剂量组肉芽组织病理学变化及各项指标比较差异无统计学意义（P>0.05）。结论金丝桃苷可减轻DFU大鼠炎症反应，加快创面愈合，其机制可能与激活AMPK/SIRT1信号通路有关。

关键词: 金丝桃苷, 糖尿病足, 足溃疡, 炎症, 腺苷酸活化蛋白激酶, 沉默信息调节因子1

Abstract:

Objective To explore the effect of hyperin on inflammatory reaction, wound healing and AMP-activated protein kinase (AMPK)/silent information regulator 1 (SIRT1) signaling pathway in diabetic foot ulcer (DFU) rats. Methods The DFU rat model was established, and 48 rats with successful DFU model were randomly divided into the model group, the hyperin low dose (100 mg/kg) group, the hyperin high dose (200 mg/kg) group and the metformin (200 mg/kg) group, with 12 rats in each group. Another 12 rats were taken as the control group. Rats of each group were given corresponding drug intervention, once a day for 4 weeks. Fasting blood glucose (FBG) levels of rats were detected by glucose meter after modeling (before administration) and after administration. Serum levels of interleukin (IL-6) and tumor necrosis factor-α (TNF-α) were detected by enzyme linked immunosorbent assay. The wound healing rate of rats was calculated by taking pictures. Hematoxylin-eosin staining was used to observe pathological changes of wound granulation tissue. Fluorescence quantitative PCR was used to detect mRNA expression levels of AMPK and SIRT1 in wound granulation tissue of rats. Western blot assay was used to detect protein expression levels of AMPK and SIRT1 in wound granulation tissue of rats. Results In the control group, the granulation tissue cells on the wound surface of rats were intact and orderly arranged, with less inflammatory cell infiltration. Compared with the control group, the necrosis of fibroblasts and capillaries in wound granulation tissue was severe, accompanied by a large number of inflammatory cell infiltration, and FBG,TNF-α and IL-6 levels in serum were significantly increased (P<0.05). The wound healing rate, the mRNA and protein expression levels of AMPK and SIRT1 in wound granulation tissue were significantly decreased (P<0.05). Compared with the model group, the lesion degree of wound granulation tissue was reduced in turn in the hyperin low dose group and the hyperin high dose group, and the FBG, serum levels of TNF-α and IL-6 were decreased in turn as well (P<0.05). The wound healing rate, the mRNA and protein expression levels of AMPK and SIRT1 in wound granulation tissue were increased in turn (P<0.05). There were no significant differences in pathological changes of the wound granulation tissue and other indicators between the metformin group and the hyperin high dose group (P>0.05). Conclusion Hyperin can reduce inflammatory reaction and accelerate wound healing in DFU rats, and its mechanism may be related to the activation of AMPK/SIRT1 signal pathway.

Key words: hyperin, diabetic foot, foot ulcer, inflammation, AMP-activated protein kinase, silent information regulator 1

中图分类号:

R285.5，R349.6

图/表 8

表1 qPCR引物序列

Tab.1 Primer sequence for qPCR

基因名称	引物序列（5′→3′）	产物大小/bp
AMPK	上游：TCGATCGATCGATCGATGGC	191
	下游：AGCTAGCTAGCTAGCATTCT	191
SIRT1	上游：TAGCTAGCTAGTATTCCGA	164
	下游：GCTAGCTAGCTATCAAGTT	164
GAPDH	上游：AGCTAGCTAGCTGTCGGA	176
	下游：GCGAGCTAGCTGACTAAC	176

表2 各组大鼠给药前后FBG水平比较（n=12，mmol/L，$\bar{x}±s$）

Tab.2 Comparison of FBG levels of rats between five groups

组别	给药前	给药后
对照组	5.03±0.71	5.11±0.63
模型组	22.35±4.19^a	21.86±4.05^a
金丝桃苷低剂量组	21.97±4.02^a	15.97±3.11^b
金丝桃苷高剂量组	22.58±3.98^a	9.69±1.93^bc
二甲双胍组	21.86±3.67^a	9.85±2.06^bc
F	55.700^＊＊	73.685^＊＊

表3 各组大鼠血清中TNF-α、IL-6水平比较（n=12，ng/L，$\bar{x}±s$）

Tab.3 Comparison of serum levels of TNF-α and IL-6 between five groups

组别	TNF-α	IL-6
对照组	12.24±1.55	15.59±1.24
模型组	25.46±2.68^a	35.64±3.36^a
金丝桃苷低剂量组	20.49±2.12^b	27.24±2.93^b
金丝桃苷高剂量组	15.27±2.04^bc	19.88±2.05^bc
二甲双胍组	15.42±1.97^bc	20.03±2.31^bc
F	73.833^＊＊	120.640^＊＊

图1 各组大鼠给药后伤口愈合情况

Fig.1 Wound healing of rats in each group after administration

图2 各组大鼠创面肉芽组织病理学变化情况（HE染色，×400）

Fig.2 Histopathological changes of wound granulation tissue of rats in each group (HE staining, ×400)

表4 各组大鼠创面肉芽组织中AMPK、SIRT1 mRNA表达水平比较（n=12，$\bar{x}±s$）

Tab.4 Comparison of mRNA expression levels of AMPK and SIRT1 in wound granulation tissue of rats between five groups

组别	AMPK mRNA	SIRT1 mRNA
对照组	1.00±0.00	1.00±0.00
模型组	0.31±0.02^a	0.34±0.03^a
金丝桃苷低剂量组	0.52±0.04^b	0.55±0.06^b
金丝桃苷高剂量组	0.71±0.06^bc	0.85±0.08^bc
二甲双胍组	0.72±0.08^bc	0.87±0.10^bc
F	329.350^＊＊	208.909^＊＊

图3 各组大鼠创面肉芽组织中AMPK、SIRT1蛋白免疫印迹图 A：对照组；B：模型组；C：金丝桃苷低剂量组；D：金丝桃苷高剂量组；E：二甲双胍组。

Fig.3 Western blot assay of AMPK and SIRT1 in wound granulation tissue of rats in each group

表5 各组大鼠创面肉芽组织中AMPK、SIRT1蛋白表达水平比较（n=12，$\bar{x}±s$）

Tab.5 Comparison of protein expression levels of AMPK and SIRT1 in wound granulation tissue of rats between five groups

组别	AMPK	SIRT1
对照组	0.95±0.15	1.34±0.17
模型组	0.18±0.03^a	0.31±0.04^a
金丝桃苷低剂量组	0.39±0.05^b	0.57±0.07^b
金丝桃苷高剂量组	0.72±0.09^bc	0.98±0.11^bc
二甲双胍组	0.75±0.09^bc	1.03±0.10^bc
F	135.349^＊＊	171.997^＊＊

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金丝桃苷对糖尿病足溃疡大鼠炎症反应、创面愈合及AMPK/SIRT1信号通路的影响

Effects of hyperin on inflammatory reaction, wound healing and AMPK/SIRT1 signaling pathway in diabetic foot ulcer model rats

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