Method for determining affinity and thermal stability of small molecule drugs and fibroblast growth factor 2, inhibitors and uses thereof

Abstract

The embodiments of the present disclosure disclose methods, inhibitors and uses for determining the affinity and thermal stability of small molecule drugs and fibroblast growth factor 2. A specific implementation of the method includes: chip activation of the CM5 chip; ligand fixation of the CM5 chip and fibroblast growth factor 2 after chip activation; flushing the chip surface of the CM5 chip after chip activation; controlling the dilution of the small molecule drug to bind to fibroblast growth factor 2; controlling the dilution of the small molecule drug to dissociate from fibroblast growth factor 2, and determining the cell affinity of the small molecule drug to fibroblast growth factor 2; performing cell heat treatment on the control group and the treatment group respectively; determining the thermal stability of the small molecule drug to fibroblast growth factor 2. This implementation effectively verifies that N-trans-feruloyl octopamine has excellent affinity and thermal stability as an inhibitory drug targeting fibroblast growth factor 2.

Description

Technical Field

[0001] The embodiments of the present disclosure relate to the field of biotechnology, and in particular to methods for determining the affinity and thermal stability of small molecule drugs and fibroblast growth factor 2, inhibitors, and uses thereof. Background Art

[0002] Pigmentation after sunburn is a common skin problem. The main cause is that the skin is overexposed to ultraviolet radiation, which leads to overactive melanocytes, resulting in the production and deposition of a large amount of melanin on the surface of the skin. At present, the main methods commonly used to treat pigmentation after sunburn are: method (1) using external whitening agents such as retinoic acid, kojic acid, arbutin (α-arbutin, β-arbutin, deoxyarbutin) to inhibit the activity of tyrosinase to achieve the purpose of inhibiting melanin production; method (2) through laser treatment, using laser of a specific wavelength to destroy melanin particles; method (3) using chemical substances such as fruit acid and salicylic acid to peel off the skin epidermis.

[0003] However, when the above method is adopted, the following technical problems often occur:

[0004] Method (1) has a long application cycle and is difficult to achieve the purpose of effectively inhibiting melanin in a short period of time. At the same time, due to its irritation, it is likely to induce new skin problems for people with sensitive skin.

[0005] Method (2) has a high cost of treatment and a high risk of recurrence;

[0006] Method (3) uses high concentrations of chemical substances such as fruit acid and salicylic acid. Although they have strong epidermal exfoliation ability, they are often accompanied by strong skin irritation. For people with sensitive skin, they are likely to induce new skin problems. At the same time, the thinning of the epidermis will also weaken the skin's ability to resist external invasion.

[0007] The above information disclosed in this Background section is only for enhancement of understanding of the background of the inventive concept and therefore it may contain information that does not form the prior art that is already known to a person of ordinary skill in the art. Summary of the invention

[0008] The content of this disclosure is used to introduce concepts in a brief form, which will be described in detail in the detailed implementation section below. The content of this disclosure is not intended to identify the key features or essential features of the technical solution claimed for protection, nor is it intended to limit the scope of the technical solution claimed for protection.

[0009] Some embodiments of the present disclosure provide methods for determining the affinity and thermal stability of small molecule drugs and fibroblast growth factor 2, inhibitors, and uses to solve one or more of the technical problems mentioned in the above background technology section.

[0010] In a first aspect, some embodiments of the present disclosure provide a method for determining the affinity and thermal stability of a small molecule drug and fibroblast growth factor 2, wherein the small molecule drug is an inhibitory drug targeting the fibroblast growth factor 2, and the small molecule drug is: N-trans-feruloyloctopamine, comprising: activating a CM5 chip; in response to completion of the activation, ligand fixation is performed on the CM5 chip and fibroblast growth factor 2 after the chip activation; in response to completion of the ligand fixation, the chip surface of the CM5 chip after the chip activation is rinsed with a pre-configured buffer to perform baseline signal correction; in response to completion of the baseline signal correction, at a preset flow rate, controlling the diluted small molecule drug to bind to the fibroblast growth factor 2 for a first preset time period; in response to completion of the binding, controlling The diluted small molecule drug is dissociated from fibroblast growth factor 2 for a second preset time, and the cell affinity of the small molecule drug and fibroblast growth factor 2 is determined at at least K concentration detection points, wherein K ≥ 5, and the corresponding concentration values ​​of the concentration detection points in the above K concentration detection points are continuous; within the preset temperature range, the control group and the treatment group are subjected to cell heat treatment for a third preset time, wherein the cells in the above control group have not been treated with small molecule drugs, and the cells in the above treatment group have been treated with small molecule drugs; in response to the end of the cell heat treatment, the thermal stability of the above small molecule drug and fibroblast growth factor 2 is determined according to the remaining amount of fibroblast growth factor 2 in the cells in the above control group and the remaining amount of fibroblast growth factor 2 in the cells in the above treatment group.

[0011] Optionally, the above-mentioned chip activation of the CM5 chip includes: on the first experimental channel, activating the above-mentioned CM5 chip for a preset time by a mixture of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide; on the second experimental channel, activating the above-mentioned CM5 chip for the preset time by a mixture of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide.

[0012] Optionally, the fibroblast growth factor 2 includes: a first fibroblast growth factor 2 and a second fibroblast growth factor 2; and in response to the activation being completed, the CM5 chip and the fibroblast growth factor 2 are fixed with a ligand according to the chip after activation, including: performing the following first processing step on the first experimental channel: diluting the first fibroblast growth factor 2 to a preset concentration by sodium acetate to obtain the diluted first fibroblast growth factor 2; in response to the response value corresponding to the CM5 chip after the chip activation being a first response value, the diluted first fibroblast growth factor 2 flows through the chip After the chip surface of the activated CM5 chip is blocked, the unreacted spots on the chip surface of the activated CM5 chip are blocked; the following second processing step is performed for the second experimental channel: the second fibroblast growth factor 2 is diluted to the preset concentration by sodium acetate to obtain the diluted second fibroblast growth factor 2; in response to the response value corresponding to the activated CM5 chip being the second response value, after the diluted second fibroblast growth factor 2 flows through the chip surface of the activated CM5 chip, the unreacted spots on the chip surface of the activated CM5 chip are blocked.

[0013] Optionally, after the CM5 chip is activated, the method further comprises: sealing the first reference channel, wherein the first reference channel corresponds to the first experimental channel; and sealing the second reference channel, wherein the second reference channel corresponds to the second experimental channel.

[0014] Optionally, the pre-configured buffer is configured by the following steps: respectively configuring a dimethyl sulfoxide solution of a first concentration and a dimethyl sulfoxide solution of a second concentration; configuring the pre-configured buffer according to the dimethyl sulfoxide solution of the first concentration and the dimethyl sulfoxide solution of the second concentration, wherein the concentration of the pre-configured buffer is a third concentration, and the first concentration < the third concentration < the second concentration.

[0015] In a second aspect, some embodiments of the present disclosure provide an inhibitor targeting fibroblast growth factor 2, wherein the inhibitor comprises: N-trans-feruloyloctopamine.

[0016] In a third aspect, some embodiments of the present disclosure provide a use of the inhibitor as described above in the second aspect in inhibiting melanocyte proliferation and production and preventing melanin deposition.

[0017] Optionally, the above-mentioned use of inhibiting melanocyte proliferation and production is the use of inhibiting fibroblast growth factor 2, down-regulating the expression of tyrosinase and melanin-induced transcription factor.

[0018] The above-mentioned embodiments of the present disclosure have the following beneficial effects: through the affinity and thermal stability determination methods of the small molecule drugs and fibroblast growth factor 2 of some embodiments of the present disclosure, it is effectively verified that N-trans-feruloyl octopamine, as an inhibitory drug targeting fibroblast growth factor 2, has excellent affinity and thermal stability, that is, N-trans-feruloyl octopamine can effectively inhibit fibroblast growth factor 2, and down-regulate the effects of tyrosinase and melanin-inducing transcription factor (MITF, Microphthalmia transcription factor). Compared with the use of topical whitening agents, laser treatment and chemical substances to peel the skin epidermis, the present disclosure uses N-trans-feruloyl octopamine as an inhibitory drug for fibroblast growth factor 2, which can act on fibroblast growth factor 2 in a targeted manner to achieve the purpose of directly inhibiting the proliferation and generation of melanocytes and preventing melanin deposition, and therefore has the effects of short application cycle, low treatment cost, and reduced irritation to the skin. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The above and other features, advantages and aspects of the embodiments of the present disclosure will become more apparent with reference to the following detailed description in conjunction with the accompanying drawings. Throughout the accompanying drawings, the same or similar reference numerals represent the same or similar elements. It should be understood that the drawings are schematic and the elements are not necessarily drawn to scale.

[0020] Figure 1 is the binding sensorgram of fibroblast growth factor 2 and N- trans -feruloyloctopamine;

[0021] Figure 2 is the steady-state fitting diagram of fibroblast growth factor 2 and N-trans-feruloyloctopamine;

[0022] Figure 3 It is a molecular docking visualization diagram of fibroblast growth factor 2 and N-trans-feruloyloctopamine;

[0023] Figure 4 This is a comparison chart of the thermal stability results of fibroblast growth factor 2 and N-trans-feruloyloctopamine;

[0024] Figure 5 This is a comparison of the color changes of the back skin of mice in the irradiation group and the non-irradiation group;

[0025] Figure 6 It is a schematic diagram of the Fontana-Masson staining results of mouse skin;

[0026] Figure 7 This is a comparison chart of the color changes of the mouse back skin in the control group, model group and treatment group. DETAILED DESCRIPTION

[0027] Embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. Although certain embodiments of the present disclosure are shown in the accompanying drawings, it should be understood that the present disclosure can be implemented in various forms and should not be construed as being limited to the embodiments set forth herein. On the contrary, these embodiments are provided to provide a more thorough and complete understanding of the present disclosure. It should be understood that the drawings and embodiments of the present disclosure are only for exemplary purposes and are not intended to limit the scope of protection of the present disclosure.

[0028] It should also be noted that, for ease of description, only the parts related to the invention are shown in the drawings. In the absence of conflict, the embodiments and features in the embodiments of the present disclosure can be combined with each other.

[0029] It should be noted that the modifications of "one" and "plurality" mentioned in the present disclosure are illustrative rather than restrictive, and those skilled in the art should understand that unless otherwise clearly indicated in the context, it should be understood as "one or more".

[0030] It should be noted that the term “about” mentioned in the present disclosure, when used to define a numerical value, means ±5% of the specified numerical value.

[0031] It should be noted that the "normal temperature" mentioned in the present disclosure refers to the ambient temperature, that is, no special heating or cooling treatment has been performed.

[0032] Explanation of terms

[0033] The term "N-trans-feruloyloctopamine", also known as N-trans-Feruloyloctopamine.

[0034] The term "Fibroblast Growth Factor 2", also known as FGF2 (Fibroblast Growth Factor), is one of the important members of the fibroblast growth factor (FGFs) family. It is an effective regulator of cell proliferation and differentiation functions and plays an important role in the normal development of body stem cells, tissue maintenance, wound repair and angiogenesis.

[0035] The term "1-ethyl-3-(3-dimethylaminopropyl)carbodiimide", also known as "EDC", is a commonly used organic synthesis reagent.

[0036] The term "N-hydroxysuccinimide", also known as "NHS", is a commonly used chemical reagent.

[0037] The term "dimethyl sulfoxide", also known as "DMSO", is a polar aprotic solvent.

[0038] The term "PBST" is a phosphate buffered saline Tween.

[0039] The present disclosure will be described in detail below with reference to the accompanying drawings.

[0040] Experimental method description

[0041] The method for determining the affinity and thermal stability of a small molecule drug and fibroblast growth factor 2 involved in the present disclosure comprises the following steps:

[0042] The first step is to activate the CM5 chip.

[0043] Among them, the CM5 chip carries a hydroxymethyl glucose matrix covalently attached to the gold surface, which can covalently couple molecules to the chip surface of the CM5 chip by utilizing the available amino, thiol, aldehyde or carboxyl groups on the ligand.

[0044] Optionally, activating the CM5 chip may include the following steps:

[0045] In the first sub-step, on the first experimental channel, the CM5 chip is activated for a preset time using a mixture of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide.

[0046] Among them, the first experimental channel is the FC2 channel. The mass ratio of the mixture composed of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide is 1:1. The above preset time length is 420 seconds. In practice, in surface ion resonance technology, multiple channels are usually set. For example, common channels include: experimental channel, reference channel and regeneration channel. Among them, the experimental channel is used to fix the ligand and detect the interaction with the analyte. Specifically, the ligand can be fixed in the experimental channel, and analyte solutions of different concentrations can be injected, and the binding and dissociation processes can be monitored. The reference channel is used to eliminate nonspecific binding and background signals. Specifically, no ligand is fixed in the reference channel, and only the same processing steps as the experimental channel are performed, that is, the signal corresponding to the reference channel can be subtracted in the subsequent analysis to obtain a signal change for the experimental channel without noise interference. The regeneration channel is used to remove the bound analytes on the surface of the CM5 chip, so that the chip surface returns to its initial state. Specifically, in multiple cycle experiments, cleaning the chip surface with an appropriate regeneration agent can ensure that each experiment is carried out under the same experimental conditions to ensure the validity of the experimental conclusions.

[0047] In the second sub-step, on the second experimental channel, the CM5 chip is activated for the preset time using a mixture of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide.

[0048] The second experimental channel is the FC4 channel. The mass ratio of the mixture of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide is 1:1. The above preset time length is 420 seconds.

[0049] In the second step, in response to the completion of activation, the CM5 chip and fibroblast growth factor 2 are immobilized as ligands after chip activation.

[0050] Optionally, in the experiment disclosed herein, the fibroblast growth factor 2 includes: a first fibroblast growth factor 2 and a second fibroblast growth factor 2, wherein the first fibroblast growth factor 2 is FGF2, and the second fibroblast growth factor 2 is Dow FGF2.

[0051] Optionally, in response to the activation being completed, the CM5 chip and fibroblast growth factor 2 are immobilized with ligand after the chip activation, and the following steps may be included:

[0052] The first sub-step is to perform the following first processing step on the first experimental channel:

[0053] Step 1: dilute the first fibroblast growth factor 2 to a preset concentration using sodium acetate to obtain diluted first fibroblast growth factor 2.

[0054] The pH value of sodium acetate is 4.0. The preset concentration is 40 μg / mL. In practice, the first fibroblast growth factor 2 is diluted to 40 μg / mL by 10 mM sodium acetate to obtain the diluted first fibroblast growth factor 2.

[0055] Step 2: In response to the response value corresponding to the CM5 chip after the chip activation being the first response value, after the diluted first fibroblast growth factor 2 flows through the chip surface of the CM5 chip after the chip activation, the unreacted points on the chip surface of the CM5 chip after the chip activation are blocked.

[0056] The first response value is 9030 U. The response value is a dimensionless unit used to quantify the refractive index change on the chip surface.

[0057] The second sub-step is to perform the following second processing steps for the second experimental channel:

[0058] Step 1: dilute the second fibroblast growth factor 2 to the preset concentration using sodium acetate to obtain the diluted second fibroblast growth factor 2.

[0059] The pH value of sodium acetate is 4.0. The preset concentration is 40 μg / mL. In practice, the second fibroblast growth factor 2 is diluted to 40 μg / mL by 10 mM sodium acetate to obtain the diluted second fibroblast growth factor 2.

[0060] Step 2: In response to the response value of the CM5 chip after the chip activation being the second response value, after the diluted second fibroblast growth factor 2 flows through the chip surface of the CM5 chip after the chip activation, the unreacted points on the chip surface of the CM5 chip after the chip activation are sealed.

[0061] Among them, the second response value is 8930U.

[0062] Optionally, after activating the CM5 chip, the method further comprises:

[0063] The first sub-step is to close the first reference channel.

[0064] The first reference channel corresponds to the first experimental channel. In practice, the first reference channel is the FC1 channel. The reference channel is used to eliminate nonspecific binding and background signals. Specifically, no ligand is fixed in the reference channel, and only the same processing steps as the experimental channel are performed, that is, the signal corresponding to the reference channel can be subtracted in the subsequent analysis to obtain the signal change for the experimental channel without noise interference.

[0065] The second sub-step is to close the second reference channel.

[0066] The second reference channel corresponds to the second experimental channel. In practice, the second reference channel is the FC3 channel.

[0067] In the third step, in response to the completion of the ligand immobilization, the chip surface of the CM5 chip after the chip activation is rinsed with a pre-configured buffer to perform baseline signal correction.

[0068] Optionally, a pre-prepared buffer is prepared by the following steps:

[0069] In the first sub-step, a dimethyl sulfoxide solution with a first concentration and a dimethyl sulfoxide solution with a second concentration are prepared respectively.

[0070] In practice, the first concentration is 4.5% and the second concentration is 5.8%.

[0071] As an example, a first concentration of a dimethyl sulfoxide solution is prepared by adding 45 mL of a dimethyl sulfoxide solution (DMSO) to 1 L of 1×PBST. A second concentration of a dimethyl sulfoxide solution is prepared by adding 58 mL of a dimethyl sulfoxide solution (DMSO) to 1 L of 1×PBST.

[0072] The second sub-step is to prepare the pre-prepared buffer solution according to the dimethyl sulfoxide solution of the first concentration and the dimethyl sulfoxide solution of the second concentration.

[0073] The concentration of the pre-configured buffer is the third concentration, and the first concentration is less than the third concentration and less than the second concentration. In practice, the third concentration is 5%.

[0074] As an example, assuming that a dimethyl sulfoxide solution of a first concentration in volume V1 and a dimethyl sulfoxide solution of a second concentration in volume V2 are required to prepare a pre-configured buffer, the following equation is obtained:

[0075]

[0076] The solution is:

[0077]

[0078] That is, to prepare 100 mL of the pre-configured buffer solution of the third concentration, approximately 61.54 mL of the dimethyl sulfoxide solution of the first concentration and 38.46 mL of the dimethyl sulfoxide solution of the second concentration are required to be uniformly mixed.

[0079] In the fourth step, in response to the completion of the baseline signal correction, the diluted small molecule drug is controlled to bind to the fibroblast growth factor 2 for a first preset time period at a preset flow rate.

[0080] The preset flow rate is 50 μL / min. The first preset time length is 60 seconds.

[0081] In response to the completion of the binding, the diluted small molecule drug and fibroblast growth factor 2 are controlled to dissociate for a second preset time period, and the cell affinity of the small molecule drug and fibroblast growth factor 2 is determined at at least K concentration detection points.

[0082] Wherein, K≥5, the concentration values ​​corresponding to the concentration detection points in the above K concentration detection points are continuous. The second preset time length is 60 seconds.

[0083] In practice, first, a small molecule drug: N-trans-feruloyl octopamine is diluted 20 times using a 1.05×PBST solution. Then, N-trans-feruloyl octopamine is further diluted to 25 μM using a running buffer. In addition, a 2-fold gradient dilution is performed in sequence. In addition, after the dilution is completed, N-trans-feruloyl octopamine flows through the first experimental channel and the first reference channel to detect with the first fibroblast growth factor 2 (FGF2).

[0084] Optionally, first, small molecule drugs: OM (Octyl Mannoside), NF (N-Feruloyloctopamine, an alkaloid formed by ester bonding of synephrine and ferulic acid), SEC (Sulfate Ester of Chondroitin) are diluted 20 times using 1.05×PBST solution. Then, OM and NF are further diluted to 25 μM using Running Buffer. Next, SEC is diluted to 100 μM using Running Buffer. In addition, 2-fold gradient dilutions are performed in sequence. In addition, after the dilution is completed, OM and NF flow through the first experimental channel and the first reference channel, respectively, to detect with the first fibroblast growth factor 2 (FGF2); and SEC flows through the second experimental channel and the second reference channel to detect with the second fibroblast growth factor 2 (Dow FGF2).

[0085] As an example, five concentration detection points can be selected for kinetic 1:1 binding analysis and steady state analysis. Figure 1 The binding sensorgrams of fibroblast growth factor 2 and N-trans-feruloyloctopamine are shown, and Figure 2 The steady-state fitting diagram of fibroblast growth factor 2 and N-trans-feruloyl octopamine shown in the figure shows that the response value increases in the binding stage and decreases in the dissociation stage. Specifically, when the affinity constant is 6.148E-06, the square sum error of the response value is 0.00187, that is, the experimental data and the simulation process have a good fit. It can be seen that fibroblast growth factor 2 and N-trans-feruloyl octopamine have a strong binding affinity.

[0086] As yet another example, see Figure 3 The molecular docking visualization diagram of fibroblast growth factor 2 and N-trans-feruloyl octopamine is shown, wherein the binding energy of fibroblast growth factor 2 and N-trans-feruloyl octopamine is -5.6 kcal / mol, indicating that the two have a strong binding affinity, wherein the main binding sites include Arg82 site and Tyr125 site, that is, hydrogen bonds and π-π stacking effects are formed.

[0087] Optionally, after dissociation, the analyte (small molecule drug) on ​​the chip surface of the CM5 chip can be removed by a regeneration buffer to restore the chip surface of the CM5 chip to its initial state. In practice, common regeneration buffers may include: acidic regeneration buffer, alkaline regeneration buffer, denaturant, salt solution, detergent. Specifically, the acidic regeneration buffer is a buffer with a lower pH value. The alkaline regeneration buffer is a buffer with a higher pH value. For example, a 1×PBST solution containing a 5% concentration of dimethyl sulfoxide (DMSO) solution can be selected as a regeneration buffer, and the chip surface of the CM5 chip is regenerated at a flow rate of 30 μL / min with a regeneration time of 30 seconds.

[0088] The fifth step is to perform cell heat treatment for a third preset time period on the control group and the treatment group respectively within the preset temperature range.

[0089] Among them, the cells in the above control group have not been treated with small molecule drugs, and the cells in the above treatment group have been treated with small molecule drugs. In practice, HaCat cells (human immortalized epidermal cells) are used for cells in the treatment group and the control group. Among them, the cells in the treatment group are treated with 10μM N-trans-feruloyloctopamine for 2 hours. The preset temperature range is 44℃~65℃. The third preset time length is 3 minutes.

[0090] The sixth step is to determine the thermal stability of the small molecule drug and fibroblast growth factor 2 in response to the completion of the cell heat treatment, based on the remaining amount of fibroblast growth factor 2 in the cells of the control group and the remaining amount of fibroblast growth factor 2 in the cells of the treatment group, respectively.

[0091] In practice, the remaining amount of fibroblast growth factor 2 in the cells of the control group and the remaining amount of fibroblast growth factor 2 in the cells of the above-mentioned treatment group at each temperature point are detected by Western blot.

[0092] As an example, see Figure 4 The thermal stability results of fibroblast growth factor 2 and N-trans-feruloyl octopamine are compared in the figure, wherein the deformation temperature of fibroblast growth factor 2 in the treatment group is approximately 3 degrees Celsius higher than that in the control group, indicating that the combination of fibroblast growth factor 2 and N-trans-feruloyl octopamine improves the thermal stability of fibroblast growth factor 2.

[0093] Animal experiment verification

[0094] Among them, the animal experiment verification used a mouse post-sunburn pigmentation model to verify the in vivo efficacy of N-trans-feruloyl octopamine.

[0095] First, the model of pigmentation after sunburn in mice is described as follows:

[0096] Selection and preparation of experimental animals: 6- to 8-week-old male SPF-level C57BL / 6J mice (mice source: provided by Beijing Weitonglihua Experimental Animal Technology Co., Ltd., license number: SCXK (Beijing) 2021-0006) were selected for a 7-day adaptation period. The mice were kept in a 12-hour light-dark cycle environment and were free to eat. The ambient temperature was: 22±2℃. The humidity was: 50±10%.

[0097] Preparation before modeling the mouse sunburn pigmentation model: 24 hours before modeling, remove a 4×2 cm area on the back of the mouse 2 Then 16 mice were randomly divided into 2 groups (irradiation group and non-irradiation group), wherein each of the irradiation group and non-irradiation group contained 8 mice.

[0098] UV irradiation requirements for the irradiation group:

[0099] UV lamp: Use peak wavelength of 311nm and dosage of 0.45mw / cm 2 UV lamp (UV lamp source: ROYAL PHILIPS, Netherlands).

[0100] Irradiation distance: The ultraviolet lamp was 20 cm away from the mouse's back skin.

[0101] Irradiation time: 30 minutes for the first irradiation; subsequent irradiation is carried out every other day, 10 minutes each time. The irradiation cycle is 8 days, with a total of 4 irradiations.

[0102] Observation results: After the irradiation, the mice were observed until the 14th day, and the color changes of the back skin of the mice were recorded. The skin tissue sections were stained to quantitatively analyze the melanin content.

[0103] The above-mentioned mouse model of pigmentation after sunburn is another inventive point of the present disclosure. In practice, pigmentation after sunburn is a common skin problem. Research on its occurrence mechanism and treatment methods often requires a reliable animal model to carry out corresponding animal experiments. However, the common guinea pig model, although the skin structure of guinea pigs is similar to that of humans, is difficult to raise and has high costs. The rat model, although simple to operate, the skin structure of rats is quite different from that of humans, and it is impossible to effectively characterize whether the experimental results are applicable to humans. The nude mouse model, although easy to observe skin changes, cannot effectively simulate normal immune responses due to certain immune deficiencies. Therefore, the present disclosure selected mice with skin structures similar to those of humans as the animal source, and set a 14-day observation period and corresponding mouse processing methods in combination with the experiment. Therefore, the mouse model of pigmentation after sunburn disclosed in the present invention has the advantages of short modeling cycle and stable experimental results.

[0104] As an example, Figure 5 The comparison chart of the back skin color changes of mice in the irradiated group and the non-irradiated group shows the back skin color changes of mice in the irradiated group and the non-irradiated group observed and recorded at 1 day (1day), 7 days (7day) and 14 days (14day).

[0105] As another example, a control group, a model group, and a treatment group were set up to record the color changes of the back skin of mice, and the skin tissue sections were stained to quantitatively analyze the melanin content. Among them, the mice in the treatment group were treated with a 10% concentration of N-trans-feruloyloctopamine gel, which was topically applied to the back area of ​​the mice with shaved hair twice a day for 2 weeks. For details, see Figure 6 Schematic representation of the results of Fontana-Masson staining of mouse skin, showing an approximately 80% reduction in melanin content. Figure 7 The comparison chart shows the color changes of the back skin of mice in the control group, model group and treatment group. Analysis found that N-trans-feruloyl octopamine can effectively inhibit the formation of pigmentation after sunburn.

[0106] The above descriptions are only some preferred embodiments of the present disclosure and an explanation of the technical principles used. Those skilled in the art should understand that the scope of the invention involved in the embodiments of the present disclosure is not limited to the technical solutions formed by a specific combination of the above-mentioned technical features, but should also cover other technical solutions formed by any combination of the above-mentioned technical features or their equivalent features without departing from the above-mentioned inventive concept. For example, the above-mentioned features are replaced with the technical features with similar functions disclosed in the embodiments of the present disclosure (but not limited to) and the technical solutions formed.

Claims

1. A method for determining the affinity and thermal stability of a small molecule drug and fibroblast growth factor 2, wherein: The small molecule drug is an inhibitory drug targeting the fibroblast growth factor 2, and the small molecule drug is: N-trans-feruloyl octopamine, including: Activate the CM5 chip; In response to the completion of activation, the CM5 chip and fibroblast growth factor 2 were immobilized according to the chip activation; In response to the ligand being fixed, the chip surface of the activated CM5 chip is washed with a pre-configured buffer to perform baseline signal correction; In response to the completion of the baseline signal correction, the diluted small molecule drug is controlled to bind to the fibroblast growth factor 2 for a first preset time period at a preset flow rate; In response to the end of the binding, controlling the diluted small molecule drug and the fibroblast growth factor 2 to dissociate for a second preset time, and determining the cell affinity of the small molecule drug and the fibroblast growth factor 2 at at least K concentration detection points, wherein K ≥ 5, and the concentration detection points in the K concentration detection points have continuous corresponding concentration values; In a preset temperature range, performing a cell heat treatment for a third preset time on a control group and a treatment group, respectively, wherein the cells in the control group have not been treated with a small molecule drug, and the cells in the treatment group have been treated with a small molecule drug; In response to the end of the cell heat treatment, the thermal stability of the small molecule drug and fibroblast growth factor 2 is determined based on the remaining amount of fibroblast growth factor 2 in the cells in the control group and the remaining amount of fibroblast growth factor 2 in the cells in the treatment group.

2. The method according to claim 1, wherein: The CM5 chip activation comprises: On the first experimental channel, the CM5 chip is activated for a preset time by a mixture of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide; On the second experimental channel, the CM5 chip is activated for the preset time period by a mixture of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide.

3. The method according to claim 2, wherein: The fibroblast growth factor 2 includes: a first fibroblast growth factor 2 and a second fibroblast growth factor 2; and In response to the activation being completed, the CM5 chip and fibroblast growth factor 2 are fixed with ligands after the chip activation, including: The following first processing steps are performed for the first experimental channel: Diluting the first fibroblast growth factor 2 to a preset concentration using sodium acetate to obtain a diluted first fibroblast growth factor 2; In response to the response value corresponding to the activated CM5 chip being the first response value, after the diluted first fibroblast growth factor 2 flows through the chip surface of the activated CM5 chip, unreacted spots on the chip surface of the activated CM5 chip are blocked; The following second processing steps are performed for the second experimental channel: Diluting the second fibroblast growth factor 2 to the preset concentration using sodium acetate to obtain the diluted second fibroblast growth factor 2; In response to the response value corresponding to the activated CM5 chip being a second response value, after the diluted second fibroblast growth factor 2 flows through the chip surface of the activated CM5 chip, the unreacted spots on the chip surface of the activated CM5 chip are blocked.

4. The method according to claim 3, wherein: After activating the CM5 chip, the method further includes: Closing a first reference channel, wherein the first reference channel corresponds to the first experimental channel; A second reference channel is sealed, wherein the second reference channel corresponds to the second experimental channel.

5. The method according to claim 4, wherein: The pre-configured buffer is configured by the following steps: respectively preparing a dimethyl sulfoxide solution of a first concentration and a dimethyl sulfoxide solution of a second concentration; The pre-configured buffer solution is configured according to the dimethyl sulfoxide solution of the first concentration and the dimethyl sulfoxide solution of the second concentration, wherein the concentration of the pre-configured buffer solution is a third concentration, and the first concentration is less than the third concentration and less than the second concentration.

6. Use of an inhibitor in inhibiting melanocyte proliferation and production and preventing melanin deposition, wherein: The inhibitor comprises: N-trans-feruloyl octopamine, and the use of inhibiting the proliferation and generation of melanocytes is to inhibit fibroblast growth factor 2 and down-regulate the expression of tyrosinase and melanin-induced transcription factor.

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