小球藻提取物促进糖尿病小鼠皮肤创面愈合的作用研究

doi:10.11958/20230825

天津医药 ›› 2024, Vol. 52 ›› Issue (4): 337-345.doi: 10.11958/20230825

• 细胞与分子生物学 • 下一篇

小球藻提取物促进糖尿病小鼠皮肤创面愈合的作用研究

黄玉¹^,²(), 贺蕊莹³, 刘森¹, 陈开元¹, 李美运¹, 程键晔¹, 武艳¹^,^△()

1 牡丹江医学院生命科学学院（邮编157011）
2 武汉大学人民医院妇产科
3 湖北大学化学化工学院

收稿日期:2023-06-14 修回日期:2023-07-28 出版日期:2024-04-15 发布日期:2024-04-19
通讯作者: ^△E-mail：wuyan@mdjmu.edu.cn
作者简介:黄玉（1995），女，硕士在读，主要从事糖尿病创面方面研究。E-mail：18208946170@126.com
基金资助:
牡丹江市指导性科技计划项目(HT2022JG125)

Study on the effect of Chlorella extract on promoting skin wound healing in diabetic mice

HUANG Yu¹^,²(), HE Ruiying³, LIU Sen¹, CHEN Kaiyuan¹, LI Meiyun¹, CHENG Jianye¹, WU Yan¹^,^△()

1 School of Life Sciences, Mudanjiang Medical College, Mudanjiang 157011, China
2 Department of Obstetrics and Gynecology, Renmin Hospital of Wuhan University
3 School of Chemistry and Chemical Engineering, Hubei University

Received:2023-06-14 Revised:2023-07-28 Published:2024-04-15 Online:2024-04-19
Contact: ^△E-mail：wuyan@mdjmu.edu.cn

黄玉, 贺蕊莹, 刘森, 陈开元, 李美运, 程键晔, 武艳. 小球藻提取物促进糖尿病小鼠皮肤创面愈合的作用研究[J]. 天津医药, 2024, 52(4): 337-345.

HUANG Yu, HE Ruiying, LIU Sen, CHEN Kaiyuan, LI Meiyun, CHENG Jianye, WU Yan. Study on the effect of Chlorella extract on promoting skin wound healing in diabetic mice[J]. Tianjin Medical Journal, 2024, 52(4): 337-345.

摘要/Abstract

摘要：

目的探讨小球藻提取物（CE）对糖尿病小鼠皮肤创面的愈合作用。方法细胞毒性实验设置空白对照组（CON组），CE低（1 mg/L）、中（10 mg/L）和高（100 mg/L）剂量组；抗氧化实验分组增加了H₂O₂（100 μmol/L）组，各实验组增加了100 μmol/L H₂O₂；抗炎实验分组增加了脂多糖（1 mg/L），各实验组增加了1 mg/L脂多糖干预。采用MTT法检测CE的细胞毒性；荧光探针法检测CE的抗氧化作用；荧光定量PCR检测CE的抗炎作用。设置空白CON组、CE低（0.5 g/L）、中（5 g/L）和高（50 g/L）剂量组，采用平板菌落计数法检测CE的抗菌能力。腹腔注射链脲佐菌素法诱导糖尿病小鼠模型，并采用随机数字表法分为CON组、CE低（0.5 g/L）、中（5 g/L）和高（50 g/L）剂量组，每组12只。在小鼠脊柱两侧对称性各做一个直径6 mm的圆形创面进行给药处理，每日1次，连续14 d。分别进行拍照并取材，记录创面愈合情况并对皮肤组织进行组织学（7 d和14 d）及免疫荧光染色（7 d）检测。结果体外实验：CE各剂量组对NIH-3T3细胞都有良好的细胞相容性；与H₂O₂组相比，CE低、中和高剂量组的活性氧（ROS）荧光信号依次减弱；与脂多糖组相比，CE低、中和高剂量组CD86的表达降低，CD206的表达升高（P<0.05）；与CON组相比，CE低、中和高剂量组对细菌生长的抑制作用依次增强（P<0.05）。体内实验：术后第7天和第14天，CE组糖尿病小鼠创面的闭合速率加快，创面愈合效果明显；对创面组织分别进行HE染色和Masson染色发现，与CON组相比，CE低、中和高剂量组糖尿病小鼠创面有更多的胶原沉积。免疫荧光检测结果显示，与CON组相比，CE治疗后的创面的α平滑肌肌动蛋白（α-SMA）、血小板-内皮细胞黏附分子（CD31）及巨噬细胞甘露糖受体（CD206）水平升高，而白细胞分化抗原86（CD86）表达水平降低。结论 CE可有效促进糖尿病小鼠创面愈合，其机制可能与其抗氧化、抗炎和抗菌有关。

关键词: 小球藻属, 糖尿病, 抗氧化剂, 抗菌药, 抗炎, 创面愈合

Abstract:

Objective To investigate the effect of Chlorella vulgaris extract (CE) on skin wound healing in diabetic mice. Methods The blank control group (CON group), CE low (1 mg/L), medium (10 mg/L) and high (100 mg/L) dose groups were set up in vitro cytotoxicity assay. The additional H₂O₂(100 μmol/L) group was set up for antioxidant experiments, and the concentration of H₂O₂ (100 μmol/L) in each experimental group was also added. The additional lipopolysaccharide (1 mg/L) group was set up for the anti-inflammatory experiments, and the concentration of lipopolysaccharide (1 mg/L) in each experimental group was also added. The cytotoxicity of CE was detected by MTT method. The antioxidant activity of CE was investigated by fluorescent probe assay. The anti-inflammatory effect of CE was detected by fluorescent quantitative PCR. The blank control group (CON group), CE low (0.5 g/L), medium (5 g/L) and high (50 g/L) dose groups were set up, and the antibacterial properties of CE were examined by plate colony counting method. The diabetic mouse model was induced by streptozotocin, and model mice were divided into the control group, the CE low (0.5 g/L), medium (5 g/L) and high (50 g/L) dose groups using the random number table method, with 12 mice in each group. After the model was successfully established, two 6 mm wounds were symmetrically created on each side of spine of mouse. The wounds were administered once daily for a total of 14 consecutive days. The wound healing was observed and photographed on days 0, 7 and 14 respectively. The wound samples were taken and processed for histology (7 d and 14 d) and immunofluorescence (7 d). Results In vitro experiments: CE low, medium and high dose groups showed good cytocompatibility with NIH-3T3 cells compared to the control group, and CE low, medium and high dose groups showed weaker reactive oxygen species (ROS) fluorescence signal compared to the H₂O₂ group. CE low, medium and high dose groups showed lower expression of CD86 and higher expression of CD206 compared to the control group (P<0.05). The inhibition of bacterial growth was enhanced in the CE low, medium and high dose groups compared to the control group (P<0.05). In vivo experiment results demonstrated that wound healing rates on the 7th and 14th day after operation were accelerated in the CE group, and the wound healing effect was obvious. Results of HE and Masson staining showed that there were more collagen deposition in wound in the CE low, medium and high dose groups compared to the control group. Results of the immunofluorescence assay showed that the higher expression levels of α-smooth muscle actin (α-SMA), platelet endothelial cell adhesion molecule-1 (CD31) and macrophage mannose receptor (CD206) after treatment, and the lower levels of leukocyte differentiation antigen 86 (CD86) in wound compared to the control group. Conclusion CE can effectively promote wound healing in diabetic mice, and the mechanism may be related to its antioxidant, anti-inflammatory and antibacterial effects.

Key words: chlorella, diabetes mellitus, antioxidants, anti-bacterial agents, anti-inflammatory, wound

中图分类号:

R587.2，R285.5

图/表 11

表1 CD86、IL-1β、TNF-α、CD206、IL-10、ARG-1和β-actin引物序列

Tab.1 Primer sequences for CD86, IL-1β, TNF-α, CD206, IL-10, ARG-1 and β-actin

基因名称	引物序列（5′→3′）	产物大小/bp
CD86	上游：ACGGAGTCAATGAAGATTTCCT	151
	下游：GATTCGGCTTCTTGTGACATAC	151
IL-1β	上游：CTCCATGAGCTTTGTACAAGG	245
	下游：TGCTGATGTACCAGTTGGGG	245
TNF-α	上游：CCCTCACACTCAGATCATCTTCT	61
	下游：GCTACGACGTGGGCTACAG	61
CD206	上游：CCTATGAAAATTGGGCTTACGG	131
	下游：CTGACAAATCCAGTTGTTGAGG	131
IL-10	上游：GTAGAAGTGATGCCCCAGGC	187
	下游：CACCTTGGTCTTGGAGCTTATT	187
ARG-1	上游：TTGGGTGGATGCTCACACTG	67
	下游：TTGCCCATGCAGATTCCC	67
β-actin	上游：CTTTGCAGCTCCTTCGTTGC	150
	下游：ACGATGGAGGGGAATACAGC	150

图1 采用DCFH-DA荧光探针对各组NIH-3T3细胞中的ROS检测（×200）

Fig.1 ROS results detected by DCFH-DA fluorescent probe assay in NIH-3T3 cells in each group (×200)

表2 各组巨噬细胞中CD86、IL-1β、TNF-α、CD206、IL-10和Arg-1的表达水平比较（n=6，$\bar{x}±s$）

Tab.2 Comparison of expression levels of CD86, IL-1β, TNF-α, CD206, IL-10 and Arg-1 in macrophages between the five groups

组别	CD86	IL-1β	TNF-α
CON组	0.98±0.02	0.97±0.02	0.98±0.01
LPS组	2.60±0.08^a	1.79±0.02^a	1.76±0.03^a
LPS+CE低剂量组	2.23±0.06^b	1.68±0.03^b	1.55±0.02^b
LPS+CE中剂量组	1.87±0.09^bc	1.50±0.03^bc	1.49±0.07^bc
LPS+CE高剂量组	1.36±0.03^bcd	1.40±0.02^bcd	1.17±0.02^bcd
F	322.100^＊＊	548.400^＊＊	220.700^＊＊
组别	CD206	IL-10	Arg-1
CON组	0.96±0.02	0.98±0.01	0.97±0.01
LPS组	0.56±0.03^a	0.73±0.04^a	0.69±0.03^a
LPS+CE低剂量组	0.63±0.02^ab	0.76±0.01^b	0.77±0.01^b
LPS+CE中剂量组	0.72±0.02^bc	0.81±0.01^bc	0.81±0.03^bc
LPS+CE高剂量组	0.89±0.03^bcd	0.87±0.01^bcd	0.92±0.03^bcd
F	151.200^＊＊	94.250^＊＊	72.950^＊＊

表3 各组金黄色葡萄球菌和大肠杆菌存活率比较（n=3，%，$\bar{x}±s$）

Tab.3 Comparison of survival rate of Staphylococcus aureus and Escherichia coli between the four group

组别	金黄色葡萄球菌存活率	大肠杆菌存活率
CON组	100.00±0.00	100.00±0.00
CE低剂量组	34.83±1.14^a	24.57±0.70^a
CE中剂量组	23.07±1.88^ab	15.70±0.40^ab
CE高剂量组	0.00±0.00^abc	0.00±0.00^abc
F	4 578.000^＊＊	36 314.000^＊＊

图2 小球藻提取物对金黄色葡萄球菌和大肠杆菌的抑菌实验

Fig.2 Bacteriostatic experiment of Chlorella extract against Staphylococcus aureus and Escherichia coli

表4 各组小鼠皮肤损伤后不同时间点创面愈合率比较（n=6，%，$\bar{x}±s$）

Tab.4 Comparison of wound healing rate at different time points after skin injury between the four groups

组别	第7天	第14天
CON组	30.57±1.19	54.08±1.64
CE低剂量组	34.34±1.13^a	61.55±1.04^a
CE中剂量组	45.70±2.06^ab	74.08±1.43^ab
CE高剂量组	55.89±1.71^abc	97.23±1.35^abc
F	160.900^＊＊	563.100^＊＊

图3 小鼠创面皮肤形态学观察 A：不同治疗后不同时间点小鼠皮肤创面的代表性图像；B：小鼠创面愈合过程的模拟图。

Fig.3 Morphological observation of wound skin in mice

图4 各组小鼠术后创面皮肤的组织形态学观察（×200）

Fig.4 Histomorphology observation of postoperative wound skin in each group of mice (×200)

表5 各组小鼠术后第7天创面皮肤组织中α-SMA、CD31、CD86和CD206的表达水平比较（n= 6，$\bar{x}±s$）

Tab.5 Comparison of expression levels of α-SMA, CD31, CD86 and CD206 in the postoperative traumatic skin tissue between the four groups

组别	α-SMA	CD31	CD86	CD206
CON组	6.63±1.53	38.12±2.06	43.17±1.50	18.11±1.86
CE低剂量组 CE中剂量组	15.33±1.53^a 26.67±2.08^ab	45.36±1.81^a 58.67±2.04^ab	21.37±1.040^a 15.26±1.59^ab	24.74±1.77^a 35.61±1.28^ab
CE高剂量组	38.67±2.52^abc	68.80±3.80^abc	8.06±1.48^abc	41.40±1.11^abc
F	153.700^＊＊	86.090^＊＊	342.000^＊＊	140.000^＊＊

图5 各组术后第7天小鼠创面皮肤组织中α-SMΑ、CD31的表达情况（免疫荧光染色，×200）

Fig.5 Expression of α-SMA and CD31 in wound skin on day 7 after surgery in each group (immunofluorescence staining, ×200)

图6 各组术后第7天小鼠创面皮肤组织中CD86、CD206的表达情况（免疫荧光染色，×200）

Fig.6 Expression of CD86 and CD206 in wound skin on day 7 after surgery in each group (immunofluorescence staining, ×200)

参考文献 19

[1]	CAO W, PENG S, YAO Y, et al. A nanofibrous membrane loaded with doxycycline and printed with conductive hydrogel strips promotes diabetic wound healing in vivo[J]. Acta Biomater, 2022, 152:60-73. doi:10.1016/j.actbio.2022.08.048.
[2]	WANG X, YUAN C X, XU B, et al. Diabetic foot ulcers:Classification,risk factors and management[J]. World J Diabetes, 2022, 13(12):1049-1065. doi:10.4239/wjd.v13.i12.1049.
[3]	KIM K, MAHAJAN A, PATEL K, et al. Materials and cytokines in the healing of diabetic foot ulcers[J]. Adv Ther(Weinh), 2021, 4(9):2100075. doi:10.1002/adtp.202100075.
[4]	王一鸣, 朱朝军, 孙旭, 等. 化腐再生法治疗糖尿病足疮面标准操作流程的制定及实践[J]. 中国中西医结合外科杂志, 2024, 30(1):5-8.
	WANG Y M, ZHU C J, SUN X, et al. Formulation and practice of the standard operating procedures for the treatment of diabetes foot sore with the method of decay and regeneration[J]. Chinese Journal of Integrated Traditional Chinese and Western Medicine Surgery, 2024, 30(1):5-8. doi:10.3969/j.issn.1007-6948.2024.01.001.
[5]	ZHENG S Y, WAN X X, KAMBEY P A, et al. Therapeutic role of growth factors in treating diabetic wound[J]. World J Diabetes, 2023, 14(4):364-395. doi:10.4239/wjd.v14.i4.364.
[6]	USLU K, TANSUKER H D, TABARU A, et al. Investigation of the effects of thrombocyte-rich plasma,systemic ozone and hyperbaric oxygen treatment on intraoral wound healing in rats:experimental study[J]. Eur Arch Otorhinolaryngol, 2020, 277(6):1771-1777. doi:10.1007/s00405-020-05872-5.
[7]	ZHANG Z, ZHANG W, XU Y, et al. Efficacy of hyperbaric oxygen therapy for diabetic foot ulcers:An updated systematic review and meta-analysis[J]. Asian J Surg, 2022, 45(1):68-78. doi:10.1016/j.asjsur.2021.07.047.
[8]	BITO T, OKUMURA E, FUJISHIMA M, et al. Potential of Chlorella as a dietary supplement to promote human health[J]. Nutrients, 2020, 12(9):2524. doi:10.3390/nu12092524.
[9]	CHEN S, WANG L, FENG W, et al. Sulfonamides-induced oxidative stress in freshwater microalga Chlorella vulgaris:Evaluation of growth,photosynthesis,antioxidants,ultrastructure,and nucleic acids[J]. Sci Rep, 2020, 10(1):8243. doi:10.1038/s41598-020-65219-2.
[10]	ZAHRAN E, ELBAHNASWY S, RISHA E, et al. Antioxidative and immunoprotective potential of Chlorella vulgaris dietary supplementation against chlorpyrifos-induced toxicity in Nile tilapia[J]. Fish Physiol Biochem, 2020, 46(4):1549-1560. doi:10.1007/s10695-020-00814-8.
[11]	ZHOU J, WANG M, BÄUERL C, et al. The impact of liquid-pressurized extracts of Spirulina,Chlorella and Phaedactylum tricornutum on in vitro antioxidant,antiinflammatory and bacterial growth effects and gut microbiota modulation[J]. Food Chem, 2023, 401:134083. doi:10.1016/j.foodchem.2022.134083.
[12]	ZHOU X, GUO Y, YANG K, et al. The signaling pathways of traditional Chinese medicine in promoting diabetic wound healing[J]. JEthnopharmacol, 2022, 282:114662.
[13]	YANG J, CHU Z, JIANG Y, et al. Multifunctional hyaluronic acid microneedle patch embedded by Cerium/Zinc-based composites for accelerating diabetes wound healing[J]. Adv Healthc Mater, 2023, 22:e2300725. doi:10.1002/adhm.202300725.
[14]	WU H, YANG P, LI A, et al. Chlorella sp.-ameliorated undesirable microenvironment promotes diabetic wound healing[J]. Acta Pharm Sin B, 2023, 13(1):410-424. doi:10.1016/j.apsb.2022.06.012.
[15]	QIAN B, LI J, GUO K, et al. Antioxidant biocompatible composite collagen dressing for diabetic wound healing in rat model[J]. Regen Biomater, 2021, 8(2):rbab003. doi: 10.1093/rb/rbab003.
[16]	LI H, CHENG K, WONG C, et al. Evaluation of antioxidant capacity and total phenolic content of different fractions of selected microalgae[J]. Food Chemistry, 2007, 102(3):771-776.
[17]	MONTERO-LOBATO Z, VÁZQUEZ M, NAVARRO F, et al. Chemically-induced production of anti-inflammatory molecules in microalgae[J]. Mar Drugs, 2018, 16(12):478.
[18]	ZHANG R, CHEN J, MAO X, et al. Anti-inflammatory and anti-aging evaluation of pigment-protein complex extracted from Chlorella pyrenoidosa[J]. Mar Drugs, 2019, 17(10):586.
[19]	HWANG H R, LEE E S, KANG S M, et al. Effect of antimicrobial photodynamic therapy with Chlorella and Curcuma extract on Streptococcus mutans biofilms[J]. Photodiagnosis Photodyn Ther, 2021, 35:102411. doi:10.1016/j.pdpdt.2021.102411.

小球藻提取物促进糖尿病小鼠皮肤创面愈合的作用研究

Study on the effect of Chlorella extract on promoting skin wound healing in diabetic mice

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[15]	涂静, 夏晨曦, 李婷. 2型糖尿病亚临床周围神经病变与TIR的相关性及危险因素探讨[J]. 天津医药, 2024, 52(11): 1188-1192.