CN111450106A

CN111450106A - Application of ginsenoside in repairing wound surface

Info

Publication number: CN111450106A
Application number: CN202010425898.2A
Authority: CN
Inventors: 杨延龙; 陈婷如; 苏永华; 殷子斐
Original assignee: Shanghai Changhai Hospital
Current assignee: Shanghai Changhai Hospital
Priority date: 2020-05-19
Filing date: 2020-05-19
Publication date: 2020-07-28

Abstract

The invention relates to application of ginsenoside Ro in preparation of a skin wound repair drug. The skin wound is skin wound caused by keratinocyte injury or skin wound caused by keratinocyte destruction. The invention has the advantages that: according to HaCaT human keratinocyte experiments, the Ro has an obvious proliferation effect on HaCaT cells, and the proliferation rate of the Ro is reduced along with the increase of time; when the concentration is gradually increased, the proliferation rate is also increased. After the ginsenoside Ro treatment for 16h, no obvious difference in migration distance can be observed in the Ro treatment group compared with the cells of the control group under a light microscope. After Ro treatment, the proportion of HaCaT G0/G1 decreased, the ratio of S-phase cells did not change much, the ratio of G2/M cells slightly increased, but no obvious time or dose dependent trend appeared.

Description

Application of ginsenoside in repairing wound surface

Technical Field

The invention relates to the technical field of medicines, in particular to application of ginsenoside in wound repair.

Background

The skin is an important organ of the human body and consists of epidermis, dermis and subcutaneous tissue parts, and the basic functions of the skin have five aspects: protection function, body temperature regulation function, feeling function, perspiration function and absorption function. And has important physical, chemical and biological barrier functions. The wound surface is the damage of normal skin (tissue) caused by external injury factors such as surgical operation, external force, heat, current, chemical substances, low temperature and internal factors of the body such as local blood supply obstacle; it is often accompanied by a breakdown in the integrity of the skin and the loss of a certain amount of normal tissue, while the normal function of the skin is impaired, also known as a wound or trauma, wound repair, i.e. wound repair. Keratinocytes are epithelial cells that synthesize keratin. These cells are the main body of epidermis, and gradually proliferate and differentiate from the deep layer of epidermis, and in keratinized keratinocytes, the cell nucleus and organelles completely disappear, and the cells also lose physiological functions and fall off.

Keratinocytes ultimately produce keratin, which can be generally divided into four layers, namely the basal layer, the spinous layer, the granular layer, and the stratum corneum, during its evolution into keratinocytes. The first three or two layers have been referred to as the germinal layer or the marmata layer. In addition, a clear layer is visible below the stratum corneum in certain areas, particularly the palmoplantar area.

Ginsenoside (Ginsenoside) is a steroid compound, triterpene saponin. Mainly exists in ginseng medicinal materials. Chinese patent document CN111012790A discloses an application of ginsenoside Rh2 in treating acute myocardial infarction. Chinese patent document CN110559308A discloses the application of ginsenoside Re in preparing medicines for treating and preventing inflammation-related diseases. However, the application of ginsenoside in repairing wound surfaces is not reported at present.

Disclosure of Invention

The invention aims to provide the application of ginsenoside Ro in preparing a skin wound repair medicine aiming at the defects in the prior art.

In order to achieve the purpose, the invention adopts the technical scheme that:

application of ginsenoside Ro in preparing medicine for repairing skin wound is provided.

Further priority scheme: the skin wound is skin wound caused by keratinocyte injury or skin wound caused by keratinocyte destruction

The invention has the advantages that: according to HaCaT human keratinocyte experiments, the Ro has an obvious proliferation effect on HaCaT cells, and the proliferation rate of the Ro is reduced along with the increase of time; when the concentration is gradually increased, the proliferation rate is also increased. After the ginsenoside Ro treatment for 16h, no obvious difference in migration distance can be observed in the Ro treatment group compared with the cells of the control group under a light microscope. After Ro treatment, the proportion of HaCaT G0/G1 decreased, the ratio of S-phase cells did not change much, the ratio of G2/M cells slightly increased, but no obvious time or dose dependent trend appeared. Can be used for preparing medicines for repairing wound related diseases.

Drawings

FIG. 1 shows that Ro has a significant proliferative effect on HaCaT cells.

FIG. 2 is the effect of ginsenosides Ro on HaCaT migration.

Detailed Description

The following examples are provided to illustrate specific embodiments of the present invention.

Example 1

1. Materials and methods

1.1 materials ginsenoside Ro, Shanghai Huo biological science and technology limited; MTT, Sigma; RPMI-1640 medium, Hyclone; microplate reader, BioTek corp.

1.2 cell culture cell line HaCaT human keratinocytes, preserved in the laboratory of the second department of military medical university's subsidiary Long-sea Hospital of traditional Chinese medicine, 1640 culture medium was added with 10% Fetal Bovine Serum (FBS), 100. mu.g/m L penicillin, 100. mu.g/m L streptomycin, 95% air and 5% CO at 37 ℃ in₂Culturing under the condition of (1), and taking cells in logarithmic growth phase for experiment.

1.2 Experimental methods

1.2.1MTT method for detecting cell activity HaCaT is digested into single cells by pancreatin-EDTA, 4000/well of the cells are added into a 96-well plate, each well is 100 mu L, after the cells are adhered to the wall, old culture solution is abandoned, culture solution (80 mu M, 40 mu M, 20 mu M, 10 mu M and 5 mu M) of ginsenoside Ro with different concentrations is added into an experimental group, culture solution is added into a control group, 6 multiple wells are formed at each concentration, and solution is simultaneously establishedBottom group, each well is loaded with 100 mu L sample, put at 37 ℃ and 5% CO₂The incubator was further incubated for 24h and 48h, the newly prepared MTT solution 5mg/m L was added to a 96-well plate at 10. mu. L per well, the plate was again placed in the incubator for 4h, the triple solution was added at 100. mu. L per well, the plate was again placed in the incubator overnight, the OD value was measured using a microplate reader (Biotek Elx-800, USA) at a measurement wavelength of 570nm, and the cell inhibition rate was calculated according to the formula [1- (Experimental group average OD value-control group average OD value)/(control group average OD value-background average OD value) ], wherein]× 100%, the experiment was repeated 3 times.

1.2.2 Prior to plating for scratch test, 3 transverse marker lines were made at the bottom of each well of a 24-well plate using a black marker pen, HaCaT was digested into single cells using pancreatin-EDTA, and cells 2 × 10 were plated⁵Adding each hole into a 12-hole plate, wherein each hole is 1M L, carrying out an experiment after cells adhere to the wall and grow and fuse to 80-90%, using a 100 mu L gun head to be vertical to an original transverse marking line at the bottom of the plate, marking 3 longitudinal scratches, washing HaCaT cells for 3 times by using PBS (phosphate buffer solution) to remove cells falling off in the marking process, adding a culture solution containing Ro into an experimental group to enable the final concentration to be 40 mu M, adding the culture solution into a control group, setting 3 parallel holes in each group, and recording the cell migration distance at the intersection of the scratches and the marking line under an inverted phase difference microscope after culturing for 16 h.

1.2.3 flow assay of cell cycle HaCaT was digested into single cells using pancreatin-EDTA, cells 6 × 10⁴adding/M L into 6-well plate, 2M L per well, adding Ro (60 μ M, 30 μ M, 10 μ M) with different concentrations into experimental group after cell adherence, not adding control group, culturing for 12h and 24h, digesting and collecting cells, and culturing with Ca-free medium²⁺、Mg²⁺Washing with PBS for 2 times, shaking to disperse cells, fixing with 4 deg.C pre-cooled 75% ethanol for more than 18h, centrifuging at 1000rpm for 5min before detecting cell cycle by flow-type machine, removing ethanol, washing with PBS for 1 time, adding 10 μ g/m L RNase at 37 deg.C for 20min, adding PI50 μ g/m L, dyeing at room temperature in dark for more than 10min, measuring with flow cytometer (BD, USA), and analyzing cell cycle distribution by software.

2. Experimental results 2.1 Effect of ginsenoside Ro on HaCaT proliferation is shown in Table 1

And displaying according to the result: ro has obvious proliferation effect on HaCaT cells, and the proliferation rate of the Ro tends to decrease along with the increase of time; when the concentration is gradually increased, the proliferation rate is also increased. See fig. 1.

2.2 Effect of ginsenoside Ro on HaCaT migration after being treated with ginsenoside Ro for 16h, no obvious difference in migration distance was observed in the Ro treated group compared with the cells in the control group under the light microscope. See fig. 2.

2.3 Effect of ginsenoside Ro on HaCaT cell cycle, see Table 2.

The experimental results show that: after Ro treatment, the proportion of HaCaT G0/G1 decreased, the ratio of S-phase cells did not change much, the ratio of G2/M cells slightly increased, but no obvious time or dose dependent trend appeared.

The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and additions can be made without departing from the method of the present invention, and these modifications and additions should also be regarded as the protection scope of the present invention.

Claims

1. Application of ginsenoside Ro in preparing medicine for repairing skin wound is provided.

2. Use according to claim 1, wherein the skin wound is a skin wound caused by keratinocyte injury or a skin wound caused by keratinocyte destruction.