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

doi:10.11958/20230140

Abstract

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

CLC Number:

R285.5，R349.6

Figures/Tables 8

References 21

[1]	BOYKO E J, ZELNICK L R, BRAFFETT B H, et al. Risk of foot ulcer and lower-extremity amputation among participants in the diabetes control and complications trial/epidemiology of diabetes interventions and complications study[J]. Diabetes Care, 2022, 45(2):357-364. doi:10.2337/dc21-1816.
[2]	AWASTHI A, SINGH S K, KUMAR B, et al. Treatment strategies against diabetic foot ulcer: success so far and the road ahead[J]. Curr Diabetes Rev, 2021, 17(4):421-436. doi:10.2174/1573399816999201102125537.
[3]	WAN R, WEISSMAN J P, GRUNDMAN K, et al. Diabetic wound healing:The impact of diabetes on myofibroblast activity and its potential therapeutic treatments[J]. Wound Repair Regen, 2021, 29(4):573-581. doi:10.1111/wrr.12954.
[4]	曹明明, 车琳琳, 朱路文. 金丝桃苷药理作用及机制研究进展[J]. 辽宁中医药大学学报, 2022, 24(6):150-155.
	CAO M M, CHE L L, ZHU L W. Research progress on the pharmacological action and mechanism of hyperoside[J]. Journal of Liaoning University of Traditional Chinese Medicine, 2022, 24(6):150-155. doi:10.13194/j.issn.1673-842x.2022.06.033.
[5]	夏骏, 姚晓丽, 刘潭, 等. 金丝桃苷改善脓毒症模型大鼠心肌损伤作用机制研究[J]. 中国药业, 2021, 30(5):21-25.
	XIA J, YAO X L, LIU T, et al. Mechanism of hyperoside improving myocardial injury in model rats with sepsis[J]. China Pharmaceuticals, 2021, 30(5):21-25. doi:10.3969/j.issn.1006-4931.2021.05.006.
[6]	张元丽, 王素利, 张宇, 等. 金丝桃苷调控胰岛素受体底物1/磷脂酰肌醇3-激酶/蛋白激酶B信号通路对糖尿病肾病大鼠肾组织损伤的保护作用[J]. 中国临床药理学杂志, 2023, 39(2):241-245.
	ZHANG Y L, WANG S L, ZHANG Y, et al. Protective effect of hyperoside on renal tissue damage in rats with diabetic nephropathy by regulating the insulin receptor substrate 1 /phosphatidylinositol 3 -kinase /protein kinase B signaling pathway[J]. Chin J Clin Pharmacol, 2023, 39(2):241-245. doi:10.13699/j.cnki.1001-6821.2023.02.019.
[7]	邓九红, 郑超, 王声遥, 等. 醋酸泼尼松对糖尿病肾病模型大鼠肾功能、肾脏炎性反应及AMPK/SIRT1信号通路的影响[J]. 基础医学与临床, 2022, 42(2):243-248.
	DENG J H, ZHENG C, WANG S Y, et al. Effects of prednisone acetate on renal function,renal inflammation and AMPK/SIRT1 signaling pathway in diabetic nephropathy rats[J]. Basic and Clinical Medicine, 2022, 42(2):243-248. doi:10.3969/j.issn.1001-6325.2022.02.007.
[8]	JIA W, BAI T, ZENG J, et al. Combined administration of metformin and atorvastatin attenuates diabetic cardiomyopathy by inhibiting inflammation, apoptosis, and oxidative stress in type 2 diabetic mice[J]. Front Cell Dev Biol, 2021, 9:634900-634913. doi:10.3389/fcell.2021.634900.
[9]	景亮, 祁永章. 红景天苷对糖尿病足溃疡大鼠Nrf2/Keap1信号通路及伤口愈合的影响[J]. 中国比较医学杂志, 2021, 31(8):48-54.
	JING L, QI Y Z. Effects of salidroside on the Nrf2/Keap1 signaling pathway and wound healing in rats with diabetic foot ulcer[J]. Chin J Comp Med, 2021, 31(8):48-54. doi:10.3969/j.issn.1671-7856.2021.08.007.
[10]	郭晓, 曲凤霞, 辛越, 等. 金丝桃苷对心力衰竭大鼠肝纤维化的影响及其分子机制[J]. 山东医药, 2021, 61(2):40-45.
	GUO X, QU F X, XIN Y, et al. Effect and mechanism of hyperin on liver fibrosis in rats with heart failure[J]. Shandong Medical Journal, 2021, 61(2):40-45. doi:10.3969/j.issn.1002-266X.2021.02.009.
[11]	于泽洋, 李天博, 王江宁. 复方芪参提取物对糖尿病足溃疡模型大鼠创面愈合及HIF-1α/VEGF/VEGFR2通路的影响[J]. 河北医学, 2021, 27(3):374-379.
	YU Z Y, LI T B, WANG J N. Effects of compound Qishen extract on wound healing and HIF-1α/VEGF/VEGFR2 pathway in diabetic foot ulcer model rats[J]. Hebei Medicine, 2021, 27(3):374-379. doi:10.3969/j.issn.1006-6233.2021.03.005.
[12]	LAUWERS P, DIRINCK E, VaN BOUWEL S, et al. Malnutrition and its relation with diabetic foot ulcer severity and outcome: a review[J]. Acta Clin Belg, 2022, 77(1):79-85. doi:10.1080/17843286.2020.1800315.
[13]	DÖRR S, FREIER F, SCHLECHT M, et al. Bacterial diversity and inflammatory response at first-time visit in younger and older individuals with diabetic foot infection(DFI)[J]. Acta Diabetol, 2021, 58(2):181-189. doi:10.1007/s00592-020-01587-5.
[14]	MARÔNEK M, MARAFINI I, GARDLÍK R, et al. Metalloproteinases in inflammatory bowel diseases[J]. J Inflamm Res, 2021, 14(1):1029-1041. doi:10.2147/JIR.S288280.
[15]	WANG Q, WEI H C, ZHOU S J, et al. Hyperoside:A review on its sources,biological activities, and molecular mechanisms[J]. Phytother Res, 2022, 36(7):2779-2802. doi:10.1002/ptr.7478.
[16]	朱妍妍, 王桐生, 戴宁, 等. 金丝桃苷通过激活Keap1/Nrf2/HO-1通路保护小鼠GC-2细胞的氧化损伤[J]. 南方医科大学学报, 2022, 42(5):673-680.
	ZHU Y Y, WANG T S, DAI N, et al. Hyperoside protects mouse spermatocytes GC-2 cells from oxidative damage by activating the Keap1/Nrf2/HO-1 pathway[J]. J South Med Univ, 2022, 42(5):673-680. doi:10.12122/j.issn.1673-4254.2022.05.07.
[17]	申玲君, 张海瑞, 邵伟, 等. 金丝桃苷下调微小RNA-199a对脂多糖诱导的人肺泡上皮细胞的影响[J]. 安徽医药, 2022, 26(2):217-221.
	SHEN L J, ZHANG H R, SHAO W, et al. Effect of hypericin on LPS-induced HPAEpiC cells by down-regulating miR-199a[J]. Anhui Medical and Pharmaceutical Journal, 2022, 26(2):217-221. doi:10.3969/j.issn.1009-6469.2022.02.002.
[18]	王旭东, 赵玉良, 史为涛, 等. 黄芪多糖对脓毒症急性肾损伤大鼠AMPK/SIRT1信号通路介导的肾上皮细胞能量代谢的影响[J]. 中国医院药学杂志, 2021, 41(21):2181-2185.
	WANG X D, ZHAO Y L, SHI W T, et al. Effects of astragalus polysaccharides on energy metabolism of renal epithelial cells mediated by AMPK/SIRT1 signaling pathway in rats with septic acute kidney injury[J]. Chin J Hosp Pharm, 2021, 41(21):2181-2185. doi:10.13286/j.1001-5213.2021.21.05.
[19]	张蕴, 韩新生, 张洪阳, 等. N-乙酰半胱氨酸通过AMPK/SIRT1途径抑制缺氧诱导的大鼠脑血管内皮细胞损伤[J]. 中国动脉硬化杂志, 2020, 28(2):107-112.
	ZHANG Y, HAN X S, ZHANG H Y, et al. N-acetylcysteine inhibits hypoxia-induced cerobro-vascular endothelial cell injury via AMPK/SIRT1 pathway[J]. Chin J Arterioscler, 2020, 28(2):107-112. doi:10.3969/j.issn.1007-3949.2020.02.004.
[20]	LI L, CHEN J, ZHOU Y, et al. Artesunate alleviates diabetic retinopathy by activating autophagy via the regulation of AMPK/SIRT1 pathway[J]. Arch Physiol Biochem, 2023, 129(4):943-950. doi:10.1080/13813455.2021.1887266.
[21]	XUE W, MAO J, CHEN Q, et al. Mogroside ⅢE alleviates high glucose-induced inflammation, oxidative stress and apoptosis of podocytes by the activation of AMPK/SIRT1 signaling pathway[J]. Diabetes Metab Syndr Obes, 2020, 13:3821-3830. doi:10.2147/DMSO.S276184.

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

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

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^＊＊

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^＊＊

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^＊＊