阅读说明：本技术 一种促进头发生长和防治脱发的木脂素化合物及其应用 (Lignan compound for promoting hair growth and preventing and treating alopecia and application thereof ) 是由 李吉 邓智利 周应军 于 2020-12-17 设计创作，主要内容包括：本发明公开了一种促进头发生长和防治脱发的木脂素化合物及其应用,涉及木脂素化合物领域,具体为一种促进头发生长和防治脱发的木脂素化合物及其应用,包括以下步骤：S1、材料准备；S2、人毛囊的分离培养；S3、分离培养hDPC；S4、MTS分析；S5、总RNA分离、cDNA合成和实时PCR；S6、免疫印迹试验；S7、统计分析。在促进头发生长和防治脱发的木脂素化合物及其应用中,发现了VB-1增强了人真皮乳头细胞中Wnt/β-catenin信号,并显著促进了hDPCs的增殖,VB-1显示出促进头发生长的作用,表明它可作为脱发的一种新的治疗方法。(The invention discloses a lignan compound for promoting hair growth and preventing and treating alopecia and application thereof, relates to the field of lignan compounds, and particularly relates to a lignan compound for promoting hair growth and preventing and treating alopecia and application thereof, wherein the lignan compound comprises the following steps: s1, preparing materials; s2, separating and culturing human hair follicles; s3, separating and culturing hDPC; s4, MTS analysis; s5, total RNA separation, cDNA synthesis and real-time PCR; s6, immunoblotting test; and S7, statistical analysis. In the lignan compound for promoting hair growth and preventing and treating alopecia and the application thereof, VB-1 is found to enhance Wnt/beta-catenin signals in human dermal papilla cells and remarkably promote the proliferation of hDPCs, and the VB-1 shows the effect of promoting hair growth, thereby indicating that the lignan compound can be used as a novel treatment method for alopecia.)

1. A lignan compound for promoting hair growth and preventing hair loss, comprising the steps of:

s1, preparing materials;

s2, separating and culturing human hair follicles;

s3, separating and culturing hDPC;

s4, MTS analysis;

s5, total RNA separation, cDNA synthesis and real-time PCR;

s6, immunoblotting test;

and S7, statistical analysis.

2. The lignan compound for promoting hair growth and preventing hair loss according to claim 1, wherein in the step S1, the material preparation, the natural compound lignan-1.

3. The lignan compound for promoting hair growth and preventing hair loss according to claim 1, wherein the isolation culture of human hair follicles in step S2 comprises the steps of:

s201, separating hair follicles at non-alopecia parts from hair transplantation operation of androgenetic alopecia patients;

s202, removing adipose tissues around hair follicles under an optical microscope with scissors and forceps, and culturing in William' S E medium for 14 days supplemented with 10mg/mL insulin, 2mM L-glutamine, 10ng/mL hydrocortisone, and 100U/mL streptomycin at 37 ℃ in a 5% CO2 cell incubator;

s203, incubating the hair follicle in a William' S E culture medium by using VB1, and soaking the hair follicle in PBS at 37 ℃ for photographing every 48 hours;

s204, in all experiments, VB-1 culture medium was changed to fresh culture medium every other day, 150 hair follicles in the anagen phase were isolated from three different volunteers, and cultured with VB-1 at different concentrations, and the experiments were repeated 5 times, and 6 times for each concentration group.

4. The lignan compound for promoting hair growth and preventing hair loss according to claim 1, wherein the step S3 of isolating and culturing the hdcp comprises the steps of:

s301, adding 10% (v/v) fetal calf serum, 100U/mL penicillin and 100mg/mL streptomycin into Dulbecco' S modified Eagle medium, and culturing the separated hDPC;

s302, replacing the culture medium every 2 days;

s303, when the degree of cell fusion is 80-90%, hDPCs are harvested with 0.25% (w/v) trypsin-EDTA at a ratio of 1: 3, 3-5 passages were used in this study.

5. The lignan compound for promoting hair growth and preventing hair loss according to claim 1, wherein the step S4 of the MTS assay comprises the steps of:

s401, spreading the cells in a 96-well culture dish, adding 20ul of MTS working solution into the culture dish after 4 hours, and shaking up and mixing the cells;

s402, the 96-well plate was incubated at 37 ℃ in a 5% CO2 incubator for 1h, and OD490nm values were recorded at subsequent 12h, 24h, 48h and 72h time points.

6. The lignan compound for promoting hair growth and preventing hair loss according to claim 1, wherein the step S7, statistical analysis, comprises the steps of: statistical analysis used graphpadprism7.0 software and used the t-test.

7. The use of the lignan compound for promoting hair growth and preventing hair loss according to any one of claims 1 to 6 for hair regeneration.

Technical Field

The invention relates to the technical field of lignan compounds, in particular to a lignan compound for promoting hair growth and preventing and treating alopecia and application thereof.

Background

The hair follicle is a complex micro-organ composed of epidermal and mesenchymal (dermal) components that undergoes cycles of catagen, telogen and anagen phases throughout the maturation phase. This hair cycle is based on the ability of Hair Follicle Stem Cells (HFSCs), which are slowly circulating, yet retain the label. The hair follicle stem cells are located in a position called a bulge, and briefly exit the resting state to initiate the growth phase. Activation and differentiation of HFSCs is mainly controlled by a special group of mesenchymal cells, which are located at the bottom of the hair follicle, called the Dermal Papilla (DP). With the initiation of the anagen phase of the follicle, the stem cells in the bulge are activated to promote the growth of new follicles in response to DP signaling.

In the postnatal hair cycle, Dermal Papilla Cells (DPCs) act as signaling centers, controlling the proliferation, migration and differentiation of peripheral epithelial stem/progenitor cells to complete the hair regeneration process. In addition, DPCs have hair follicle-inducing ability through interaction with adjacent epithelial stem cells. Several signaling pathways, particularly the Wnt/β -catenin signaling pathway, have been shown to play a key role in the development of new hair follicles and the initiation of hair growth. A number of Wnt ligands and inhibitors expressed in DPCs are critical for regulating hair growth. The deletion of β -catenin in DP may induce the anagen phase of the follicle prematurely and prevent regeneration of the follicle.

Several alopecia conditions are characterized by failure of the hair follicle to re-enter the anagen phase of the cycle, especially in the case of androgenetic alopecia, by ectopic activation of androgen receptor signaling in the hair follicle, primarily in DP, altering the expression of hair growth-related paracrine factors (e.g., DKK1, Wnts, and TGF- β). Dysregulation of these paracrine factors impairs proliferation and differentiation of hair follicle stem/stromal cells, leading to a shortened anagen phase and to progressive hair follicle miniaturization, a major feature of androgenetic alopecia. Thus, DP is considered to be a major therapeutic target for androgenetic alopecia. Current pharmacological treatment of androgenetic alopecia has focused primarily on preventing further alopecia. However, the development of drugs to activate HFSCs proliferation and to restart the hair cycle has not been well established.

The natural compound lignan (VB-1) is a group of complex polyphenol antioxidants widely present in plants. Lignans are widely used in Chinese herbal medicines and have been shown to have various pharmacological effects, such as anti-inflammatory, antioxidant and anticancer activities. Clinical studies have shown that lignans have a potential role in cancer prevention. Some clinical trials have demonstrated that lignans also inhibit the development of certain cancers. For example, some studies have shown that low risk of ovarian and prostate cancer is associated with a high lignan intake diet. This may be why the mediterranean diet (olives have a higher lignin content) is associated with a lower incidence of cancer. Lignans have been studied for their anti-cancer properties in cancer cells cultured in vitro. In these studies, purified VB-l prevented cancer cell proliferation during the G2/M phase of the cell cycle and induced apoptosis effectively over a range of concentrations. Although VB-1 has been shown to have anti-cancer activity, its pharmacological effects are not clear. The pharmacological value of the compound is worthy of further research.

Here, we evaluated the effect of VB-1 on hair growth. We demonstrated that VB-1 promotes the proliferation of hDPC in a concentration-dependent manner over a range of concentrations. Among the hair growth related genes studied, DKKL was significantly reduced in VB-1 treated hDPC (human dermal papilla cells). Through immunoblot analysis, we found that the activity of beta-catenin is increased, and the expression of AXIN2 is reduced, which indicates that VB-1 promotes Wnt/beta-catenin signal transduction in hDPCs. In addition, VB-1 enhanced the expression of the DP tag gene in hDPC. Furthermore, we found that VB-1 promotes the growth of human hair follicles in organ culture experiments. Taken together, these findings suggest that VB-1 promotes hair growth and may be a novel approach to treating alopecia.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a lignan compound for promoting hair growth and preventing and treating alopecia and application thereof, and solves the problems in the background technology.

In order to achieve the purpose, the invention is realized by the following technical scheme: a lignan compound for promoting hair growth and preventing and treating alopecia and its application, comprising the steps of:

s1, preparing materials;

s2, separating and culturing human hair follicles;

s3, separating and culturing hDPC;

s4, MTS analysis;

s5, total RNA separation, cDNA synthesis and real-time PCR;

s6, immunoblotting test;

and S7, statistical analysis.

Optionally, in the step S1, in the material preparation, the natural compound lignan-1.

Optionally, in the step S2, the isolated culture of the human hair follicle includes the following steps:

s201, separating hair follicles at non-alopecia parts from hair transplantation operation of androgenetic alopecia patients;

s202, removing adipose tissues around hair follicles under an optical microscope with scissors and forceps, and culturing in William' S E medium for 14 days supplemented with 10mg/mL insulin, 2mM L-glutamine, 10ng/mL hydrocortisone, and 100U/mL streptomycin at 37 ℃ in a 5% CO2 cell incubator;

s203, incubating the hair follicle in a William' S E culture medium by using VB1, and soaking the hair follicle in PBS at 37 ℃ for photographing every 48 hours;

s204, in all experiments, VB-1 culture medium was changed to fresh culture medium every other day, 150 hair follicles in the anagen phase were isolated from three different volunteers, and cultured with VB-1 at different concentrations, and the experiments were repeated 5 times, and 6 times for each concentration group.

Optionally, in the step S3, the isolated culture of hdcp, includes the following steps:

s301, adding 10% (v/v) fetal calf serum, 100U/mL penicillin and 100mg/mL streptomycin into Dulbecco' S modified Eagle medium, and culturing the separated hDPC;

s302, replacing the culture medium every 2 days;

s303, when the degree of cell fusion is 80-90%, hDPCs are harvested with 0.25% (w/v) trypsin-EDTA at a ratio of 1: 3, 3-5 passages were used in this study.

Optionally, the step S4 and the MTS analysis include the following steps:

s401, spreading the cells in a 96-well culture dish, adding 20ul of MTS working solution into the culture dish after 4 hours, and shaking up and mixing the cells;

s402, the 96-well plate was incubated at 37 ℃ in a 5% CO2 incubator for 1h, and OD490nm values were recorded at subsequent 12h, 24h, 48h and 72h time points.

Optionally, the step S7 and the statistical analysis include the following steps: the step 7 of statistical analysis comprises the following steps: statistical analysis used graphpadprism7.0 software and used the t-test.

Optionally, the use of the lignan compound for promoting hair growth and preventing hair loss in hair regeneration.

The invention provides a lignan compound for promoting hair growth and preventing and treating alopecia and application thereof, and the lignan compound has the following beneficial effects:

1. the lignan compound for promoting hair growth and preventing and treating alopecia and the application thereof detect the proliferation condition of hDPCs through cell counting and continuous cell culture; detecting expression of lefl, wnt5a, bmp2, bmp4, alpl and vcan by using an RT-PCR method; detecting the expression of dkkl, tgf-01, active beta-catenin and AXIN2 by RT-PCR or immunoblotting; measuring hair growth in the absence or presence of VB-1; as a result, we demonstrated that VB-1 significantly promotes the proliferation of hdcp in a concentration-dependent manner over a range of concentrations; among the hair growth related genes studied, dkkl was significantly down-regulated in VB-1 treated hdcp; increased activity of β -catenin and decreased levels of AXIN2 protein indicate that VB-1 promotes Wnt/β -catenin signaling in hDPCs in vitro, and that DP tag gene expression is also up-regulated following VB-1 treatment; VB-1 is further proved to promote the growth of human hair follicles through in vitro hair follicle organ culture experiments.

2. In the lignan compound for promoting hair growth and preventing and treating alopecia and the application thereof, VB-1 is found to obviously enhance Wnt/beta-catenin signals in human dermal papilla cells and obviously promote the proliferation of hDPCs, and VB-1 shows the effect of promoting hair growth, which indicates that the lignan compound can be used as a new treatment method for alopecia.

Drawings

FIG. 1 is a schematic diagram of the present invention showing that VB-1 promotes the proliferation of hDPC;

FIG. 2 is a schematic diagram showing that the expression of the characteristic gene of hDPC is increased in FIG. 2VB-1 according to the present invention;

FIG. 3 is a schematic diagram of the present invention FIG. 3VB-1 for promoting Wnt/beta-catenin signaling in hDPC;

FIG. 4 is a schematic drawing showing the elongation of the hair shaft of the present invention in FIG. 4VB-1 to promote culture of hair follicles.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments.

Referring to fig. 1 to 4, the present invention provides a technical solution: a lignan compound for promoting hair growth and preventing and treating alopecia and its application, comprising the steps of:

s1, preparing materials;

s2, separating and culturing human hair follicles;

s3, separating and culturing hDPC;

s4, MTS analysis;

s5, total RNA separation, cDNA synthesis and real-time PCR;

s6, immunoblotting test;

and S7, statistical analysis.

In the present invention, in step S1 and material preparation, lignan-1 (VB-1), a natural compound, is used.

In the present invention, the isolation culture of human hair follicles in step S2 includes the steps of:

s201, separating hair follicles at non-alopecia parts from hair transplantation operation of androgenetic alopecia patients;

s202, removing adipose tissues around hair follicles under an optical microscope with scissors and forceps, and culturing in William' S E medium for 14 days supplemented with 10mg/mL insulin, 2mM L-glutamine, 10ng/mL hydrocortisone, and 100U/mL streptomycin at 37 ℃ in a 5% CO2 cell incubator;

s203, incubating the hair follicle in a William' S E culture medium by using VB1, and soaking the hair follicle in PBS at 37 ℃ for photographing every 48 hours;

s204, in all experiments, VB-1 culture medium was changed to fresh culture medium every other day, 150 hair follicles in the anagen phase were isolated from three different volunteers, and cultured with VB-1 at different concentrations, and the experiments were repeated 5 times, and 6 times for each concentration group.

In the present invention, the step S3 of isolating and culturing hdcp comprises the steps of:

s301, adding 10% (v/v) fetal calf serum, 100U/mL penicillin and 100mg/mL streptomycin into Dulbecco' S modified Eagle medium, and culturing the separated hDPC;

s302, replacing the culture medium every 2 days;

s303, when the degree of cell fusion is 80-90%, hDPCs are harvested with 0.25% (w/v) trypsin-EDTA at a ratio of 1: 3, 3-5 passages were used in this study.

In the present invention, the step S4 and the MTS analysis include the steps of:

s401, spreading the cells in a 96-well culture dish, adding 20ul of MTS working solution into the culture dish after 4 hours, and shaking up and mixing the cells;

s402, the 96-well plate was incubated at 37 ℃ in a 5% CO2 incubator for 1h, and OD490nm values were recorded at subsequent 12h, 24h, 48h and 72h time points.

In the present invention, the step S7 and the statistical analysis include the following steps: statistical analysis used graphpadprism7.0 software and used the t-test, where P <0.05 was considered statistically different and P <0.01 was significantly different.

In the present invention, a lignan compound for promoting hair growth and preventing hair loss is used for hair regeneration.

Results

VB-1 promotes proliferation of human dermal papilla cells:

to evaluate the effect of VB-1 on cultured hDPCs, we first examined the proliferation of cells treated with different doses of VB-1(0, 0.01, 0.1, 1 and 10. mu.M); within 8 days of culture, 0.1. mu.M of VB-1 group amplified hDPCs more than the control group (0. mu.M), with no significant difference between 0.01 and 1. mu.M; however, high concentrations of VB-1(10 μ M) significantly inhibited the proliferation of hDPCs and possibly increased apoptosis of cells (fig. 1, 1A and 1B); these results were further confirmed by the MTS method for different doses of VB-1 treated hDPCs (FIG. 1, 1D); interestingly, we also found that 0.1. mu.M VB-1 was involved in colony growth (FIG. 1, 1A), indicating an increased hair-inducing ability of hDPCs; also, the number of days per stage of culture of hDPC was lower than that of the other groups in the medium supplemented with 0.1. mu.M VB-1 (FIG. 1, 1C); overall, these dose-dependent effects of VB-1 on LEFL, wnt5a, bmp2, bmp4, alpl and vcan mRNA expression in 24h of hDPC in culture (0-10 μ M); data are shown as the ratio of the respective gene expression to GAPDH mRNA expression; the experiment was performed in three terms; data are reported as mean + SEM, checked with T-test; denotes p <0.05, denotes p < 0.01; the results show that VB-1 promotes the proliferation of hDPCs, and improves the hair induction capability of the cells.

VB-1 improves the hair-inducing properties of hDPCs:

previous studies have shown that DPCs play an essential role in vitro hair follicle reconstruction assays; however, DPC quickly lose hair-inducing properties during culture, greatly limiting their use in hair restructuring; to determine whether VB-1 affected the hair-inducing ability of hDPC, we treated cultured hDPC with different concentrations of VB-1(0, 0.01, 0.1, 1 and 10 μ M); our results show that the Wnt signal-related genes for DP, LEF1 and Wnt5a, ALL and VCAN were significantly up-regulated in 0.1 μ M VB-1 treated hDPC, while in other groups and VB-1 the expression of bmp2 and bmp4 was also increased, and two additional markers were added; these results indicate that VB-1 can promote hair-inducing ability of hDPC.

VB-1 activates the Wnt/beta-catenin signal in HDPC:

to determine the role of VB-1 in hair growth, we investigated its effect on the expression of hair growth-related genes in hDPC. We found that dkk1 was significantly down-regulated under 0.1 μ M VB-1 treatment and up-regulated in the presence of 10 μ M VB-1 compared to the control group (FIG. 3, 3A); however, the expression of another hair growth related gene tgf- β was not statistically significant (FIG. 3, 3B); immunoblot analysis, according to FIG. 3, VB-1 promotes Wnt/β -catenin signaling in hDPC, where (A) the effect of VB-1 concentration-dependent effects (0-10 μ M) on the expression of dkl mRNA of human DPCs cultured for 24 h; (B) the effect of VB-1 concentration-dependent effects (0-10. mu.M) on tgf-p mRNA expression in hDPCs cultured for 24 h; (C) performing immunoblot analysis on the expression of active beta-catenin in hDPCs treated with VB-1(0-10 mu M) for 24 h; (D) quantitative analysis of the expression of the active beta-catenin protein; (E) real-time PCR analysis of AXIN2 gene expression in hDPC treated with VB-1(0-10 μ M); (F) immunoblot analysis of AXIN2 expression in hDPC treated with VB-1 (0-10. mu.M); (G) quantitative analysis of AXIN2 protein expression; data are mean ± SEM; t-test was used, where p <0.05, p <0.01 and "ns" indicated no significant difference.

The results showed that the level of active β -catenin protein was increased in 0.1 μ M VB-1 treated hDPC and decreased in high concentration VB-1 treated hDPC (FIG. 3, 3C); in addition, mRNA levels of negative regulator axin2 of Wnt/β -catenin signal were reduced in 0.1 μ M VB-1 treated hDPC; high concentrations of VB-1(1 and 10. mu.M) increased expression of axin2 (FIG. 3, 3E); immunoblot analysis confirmed that VB-1 is dose-dependent on inhibition of AXIN2 (FIGS. 3, 3F and 3G); these results indicate that VB-1 induces Wnt/β -catenin signaling activation in HDPCs in a concentration-dependent manner over a range of concentrations.

VB1 promotes hair shaft elongation of cultured human hair follicles:

since DP is critical for regulation of hair growth, we further explored the possible effect of VB-1 on hair elongation, isolated human scalp hair follicles were cultured in the presence of different doses of VB-1 for 14 days, fig. 4VB-1 promotes elongation of the hair axis in cultured hair follicles, (a) typical pictures of hair on days 0, 2, 4, 6, 8, 10, 12, 14; (B) data are shown as the elongated length of the follicles treated with VB-1; data are mean ± SEM; the test was performed using T-test, where p <0.05 and p < 0.01.

In the absence or presence of VB-1, we found that 0.1. mu.M VB-1 significantly promoted elongation of the hair shaft in cultured human hair follicles (FIG. 4, FIGS. 4A and 4B).

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be able to cover the technical scope of the present invention and the equivalent alternatives or modifications according to the technical solution and the inventive concept of the present invention within the technical scope of the present invention.