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

doi:10.11958/20230825

Abstract

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

CLC Number:

R587.2，R285.5

Figures/Tables 11

References 19

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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

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

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^＊＊

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^＊＊

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^＊＊