CN115350196B - Biological preparation for reducing skin aging and improving skin injury repair - Google Patents
Biological preparation for reducing skin aging and improving skin injury repair Download PDFInfo
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- CN115350196B CN115350196B CN202211263779.7A CN202211263779A CN115350196B CN 115350196 B CN115350196 B CN 115350196B CN 202211263779 A CN202211263779 A CN 202211263779A CN 115350196 B CN115350196 B CN 115350196B
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Abstract
The invention provides a biological agent for reducing skin aging and improving skin injury repair, and belongs to the technical field of stem cells. The biological preparation provided by the invention contains the imperatorin and a pharmaceutically acceptable carrier. Experiments show that the imperatorin can effectively promote the proliferation of epidermal stem cells, reduce the senescence of the epidermal stem cells, reduce the protein expression of senescence-promoting proteins P21 and P16, promote the protein expression of senescence-inhibiting protein P53 and restore the osteogenic differentiation capacity of the senescent cells. Therefore, it is possible to prepare imperatorin as a biological agent for reducing skin aging and promoting wound repair, thereby using it for slowing skin aging and accelerating skin wound repair.
Description
Technical Field
The invention belongs to the technical field of skin stem cells, and particularly relates to a biological agent for reducing skin aging and improving skin injury repair.
Background
Aging of human skin is a complex process, mainly caused by the interaction of intrinsic mechanisms and extrinsic influences, and can be divided into intrinsic aging and extrinsic aging. Among them, intrinsic aging is the inevitable physiological and functional aging of the skin due to genetic factors, and the skin becomes rough, dry, and has increased fine wrinkles and skin atrophy as it ages. Extrinsic aging, also known as photoaging, is a skin extrinsic aging phenomenon caused by external factors such as air pollution, sun exposure, etc.
The skin stem cells are divided into epidermal stem cells and hair follicle stem cells, the epidermal stem cells are adult stem cells with self-renewal and multidirectional differentiation potential in the skin, and can be differentiated into fibroblasts, adipocytes, myoblasts or neuronal cells under different induction factors, so that the epidermal stem cells can be used for repairing skin, muscle and organ injuries. Skin aging is closely related to the reduction and aging of epidermal stem cells, which can differentiate into skin fibroblasts under specific conditions, thereby participating in the repair of skin tissue damage and reducing skin aging. Therefore, effectively increasing the proliferation of epidermal stem cells and slowing down the aging of epidermal stem cells will help to reduce the rate of aging of skin and help to repair skin damage.
Disclosure of Invention
The invention aims to provide a biological agent for reducing skin aging and improving skin injury repair, and provides a new function of the imperatorin.
In order to achieve the purpose, the invention provides the following technical scheme:
first, the present invention provides a biological agent for decreasing skin aging and improving skin damage repair, which contains imperatorin and a pharmaceutically acceptable carrier, and which promotes the proliferation of skin stem cells and decreases the aging of skin stem cells.
Preferably, the skin stem cells are epidermal stem cells.
Preferably, the imperatorin reduces the protein expression of senescence-promoting proteins P21 and P16, promotes the protein expression of senescence-inhibiting protein P53, and restores the differentiation ability of senescent cells.
Secondly, the invention provides the application of the imperatorin in preparing the biological preparation for reducing the skin stem cell aging.
Preferably, the skin stem cells are epidermal stem cells.
Preferably, the biological agent reduces senescence of epidermal stem cells, reduces protein expression of senescence-promoting proteins P21 and P16, promotes protein expression of senescence-inhibiting protein P53, restores differentiation ability of senescent cells.
Secondly, the present invention provides an application of the imperatorin in preparing a biological agent for skin injury repair, which repairs skin injury by promoting proliferation of epidermal stem cells.
Secondly, the invention provides the application of the imperatorin in preparing the biological preparation for promoting the proliferation of the epidermal stem cells.
Finally, the invention provides the application of the imperatorin in preparing the preparation for promoting the recovery of the osteogenic differentiation capacity of the aged epidermal stem cells.
The beneficial effects of the invention are:
the invention discovers that the imperatorin can promote the proliferation of epidermal stem cells, reduce the aging of the epidermal stem cells, reduce the protein expression of aging-promoting proteins P21 and P16, promote the protein expression of aging-inhibiting protein P53, and restore the differentiation capacity of aging cells, so that the imperatorin can be used for slowing down the aging of skin and accelerating the repair of skin injury.
Drawings
FIG. 1 shows the effect of imperatorin, isoliquiritigenin, sinigrin and paeoniflorin on epidermal stem cell proliferation;
FIG. 2 is a graph of the effect of various concentrations of imperatorin on epidermal stem cell proliferation;
FIG. 3 is a cell diagram of the effect of 50. Mu. Mol/L of imperatorin on epidermal stem cell senescence;
wherein (a) is a control group, and (b) is a 50 mu mol/L imperatorin group;
FIG. 4 is a statistical plot of the effect of 50. Mu. Mol/L of imperatorin on epidermal stem cell senescence;
FIG. 5 shows the effect of 50. Mu. Mol/L of imperatorin on the senescence-associated proteins P21, P16 and P53 of epidermal stem cells;
FIG. 6 is a graph showing the effect of 50. Mu. Mol/L of imperatorin on ALP enzyme activity during the osteogenic differentiation of epidermal stem cells.
Detailed Description
The examples are provided for better illustration of the invention, but the invention is not limited to the examples. Therefore, those skilled in the art should make insubstantial modifications and adaptations to the embodiments of the present invention in light of the above teachings and remain within the scope of the invention.
Example 1
Different plant extracts were tested for epidermal stem cells.
(1) The epidermal stem cells of the P3 generation are inoculated in a 96-well cell culture plate according to the amount of 1000 cells/100 mu l;
(2) After overnight culture, the medium was removed and replaced with a medium containing 25 μmol/L of imperatorin, isoliquiritigenin, sinigrin, paeoniflorin, and a control group supplemented with medium only, each set of 3 replicates;
(3) After 72h of incubation, cck-8 was added and the OD 450nm was measured.
The results of the experiment are shown in FIG. 1, the OD value of the control group is 0.550 + -0.013, the OD value of the imperatorin group is 0.734 + -0.029, the OD value of the isoliquiritigenin group is 0.534 + -0.019, the OD value of the sinigrin is 0.588 + -0.031, and the OD value of the paeoniflorin group is 0.633 + -0.024.
It can be seen that the OD values of the paeoniflorin group and the imperatorin group are increased compared with the control group, wherein the proliferation promoting effect of the imperatorin group is more obvious, and therefore, the invention selects the imperatorin for subsequent experiments.
Example 2
The optimal concentration of the imperatorin for the proliferation promotion of the epidermal stem cells is detected.
(1) Inoculating the epidermal stem cells of the P3 generation into a 96-well cell culture plate according to the amount of 1000 cells/100 mu l;
(2) After overnight incubation, the medium was removed and replaced with a control containing 6.25,12.5, 25,50,100 μmol/L of imperatorin, and medium only, with 3 replicates per group set up;
(3) After 72h of incubation, cck-8 was added and the OD 450nm was measured.
The results of the experiment are shown in fig. 2, the control group is 0.555 ± 0.017; the content of 6.25 μmol/L imperatorin is 0.595 + -0.016; the content of the 12.5 mu mol/L imperatorin group is 0.633 +/-0.010; the 25 mu mol/L of the imperatorin group is 0.723 +/-0.014; the content of 50 μmol/L imperatorin is 0.782 + -0.024; the content of the 100 mu mol/L imperatorin group is 0.752 +/-0.020.
It can be seen that 50. Mu. Mol/L is the optimum concentration of imperatorin for promoting epidermal stem cell proliferation.
Example 3
The effect of imperatorin on epidermal stem cell senescence was examined.
(1) Inoculating epidermal stem cells of P12 generation into a 6-well plate, culturing overnight, replacing a control group with a common culture medium, and replacing an experimental group with 50 mu mol/L of imperatorin;
(2) Culturing for 48h, removing the culture medium, and adding a stationary liquid for fixing the cells at room temperature for 15min;
(3) After fixation, PBS is added to wash the cells for 2 times, beta-galactosidase staining solution is added, then a sealing film is used to seal the cell culture plate, and CO-free cells are placed 2 The incubator of (1) is incubated overnight;
(4) After the staining is finished, the cells are placed under an inverted microscope for observation and the cell positive rate is calculated.
The results of the experiment are shown in FIGS. 3 and 4, the positive rate of the control group is 75%, and the positive rate after 50. Mu. Mol/L of the imperatorin is 47%.
It can be seen that the aging of cells can be reduced by the treatment with imperatorin.
Example 4
And (3) detecting the influence of the imperatorin on the epidermal stem cell senescence-associated protein.
(1) Inoculating epidermal stem cells of P9 generation into a 6-well plate, culturing overnight, replacing a control group with a common culture medium, and replacing an experimental group with 50 mu mol/L of imperatorin;
(2) After culturing for 48h, removing the culture medium, adding 80ul of protein lysate, scraping off the cells by using a cell scraper, transferring the lysate containing the cells into an EP (EP) tube, and crushing the cells by using an ultrasonic cell crusher;
(3) Centrifuging at 4 deg.C and 12000rpm for 20min, and collecting supernatant to new EP tube;
(4) Detecting the protein concentration by using a BCA method according to the kit specification, and adding a 5 xSDS loading buffer solution to keep the concentration of each group of protein samples consistent;
(5) Assembling a glue preparation bracket, preparing 12% of lower layer glue and 5% of upper layer glue, assembling an electrophoresis device after the preparation is finished, and starting electrophoresis;
(6) Performing electrophoresis for 30min by using constant voltage 80V for upper layer concentrated gel, performing electrophoresis for about 60min by using constant voltage 120V for lower layer separation gel, and stopping electrophoresis when a bromophenol blue strip moves to a position about 1cm away from the bottom of the separation gel;
(7) Placing the glue in an electric rotating clamp, manufacturing a membrane rotating clamp according to a sandwich model, removing all bubbles in the process, installing an electric rotating device, placing the electric rotating device in an ice box, pouring membrane rotating liquid, adopting a constant current of 250mA, and completing membrane rotating after electric rotating for 90 min;
(8) After the electro-conversion is finished, taking out the PVDF membrane, putting the PVDF membrane into 5% of skimmed milk powder, and sealing the PVDF membrane on a shaking table for 1 hour;
(9) According to the corresponding protein size, incubating P21, P16, P53, beta-actin, and performing shake culture overnight at 4 ℃;
(10) Absorbing primary antibody, washing the membrane with PBS for three times, each time for 5min, incubating secondary antibody, and keeping the temperature at room temperature for 1.5h;
(11) The secondary antibody was discarded and the membrane washed three times with PBS for 5min each time for development exposure.
The results of the experiment are shown in fig. 5, and after the epidermal stem cells of P9 generation are treated with the imperatorin, the protein expression of the senescence promoting proteins P21 and P16 in the cells is obviously reduced, and the protein expression of the senescence inhibitory protein P53 is obviously increased.
The above results indicate that the imperatorin can effectively inhibit the senescence-promoting proteins P21 and P16 and promote the expression of the senescence-inhibiting protein P53.
Example 5
And (3) detecting the influence of the imperatorin on the activity of ALP enzyme in the osteogenic differentiation process of the epidermal stem cells.
(1) Inoculating the P3 generation epidermal stem cells and the P9 generation epidermal stem cells in a 6-well plate, wherein 3 multiple wells are arranged in the P3 generation and 6 multiple wells are arranged in the P9 generation;
(2) After overnight culture, the culture medium was changed for P3 generation as a control group, half of P9 generation as experiment group 1 and half as experiment group 2, respectively, the culture medium containing 50. Mu. Mol/L imperatorin;
(3) After culturing for 48 hours, removing the culture medium, and replacing the culture medium with an osteogenic differentiation medium for culturing;
(4) After culturing for 7d, removing the culture medium, washing cells with PBS, adding 200ul Triton X-100 per well, and treating on ice for 30min;
(5) Collecting lysed cells, centrifuging to collect supernatant, adding the supernatant into a 96-well plate, and adding 10ul of the supernatant, 50ul of chromogenic substrate and 40ul of ALP enzyme detection buffer into each well;
(6) After mixing, the mixture was placed in an microplate reader to detect the OD value at 450 nm.
The results of the experiment are shown in FIG. 6, in which the OD value of the control group is 0.451. + -. 0.023, the OD value of the experimental group is 0.224. + -. 0.015, and the OD value of the experimental group 2 is 0.325. + -. 0.016.
The above results indicate that the ALP enzyme activity of senescent cells upon osteogenic differentiation can be increased after the treatment with imperatorin, thereby improving the osteogenic differentiation ability of senescent cells.
Finally, although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that various changes and modifications may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (3)
1. Application of imperatorin in preparing biological preparation for reducing skin stem cell aging is provided.
2. Use according to claim 1, characterized in that the skin stem cells are epidermal stem cells.
3. The use of claim 1, wherein the biological agent reduces senescence in epidermal stem cells, reduces protein expression of senescence-promoting proteins P21 and P16, promotes protein expression of senescence-inhibiting protein P53, restores the differentiation ability of senescent cells.
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| CN114939123A (en) * | 2022-06-29 | 2022-08-26 | 山东第一医科大学附属省立医院(山东省立医院) | Therapeutic preparation for promoting wound repair |
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