Application of MLKL inhibitor in the preparation of preparations for promoting hair growth or improving androgenic alopecia

Abstract

The present invention discloses a new therapeutic target and small molecule inhibitor for promoting hair growth and improving androgenic alopecia. We found that the key molecule of necroptosis, the mixed lineage kinase domain-like protein (MLKL), plays a key role in the regulation of the hair cycle. The small molecule drug NSA is used to target and inhibit the activation of MLKL, which can accelerate the transition from the resting phase to the growth phase of the hair follicles on the back of mice, thereby promoting hair growth. Furthermore, we used dihydrotestosterone DHT to induce an AGA mouse model. In the AGA mouse model, it was found that inhibiting MLKL can antagonize the inhibitory effect of DHT on mouse hair growth, indicating that inhibiting MLKL may have a certain improvement effect on AGA. Therefore, MLKL can be used as a therapeutic target for promoting hair growth and improving AGA, and its inhibitor NSA can target and inhibit MLKL activity to promote hair growth and treat AGA.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and to a new therapeutic target for promoting hair growth and improving androgenetic alopecia (AGA). Specifically, it relates to the use of an inhibitor of the key molecule of necroptosis, the mixed lineage kinase domain-like protein (MLKL), in the preparation of a drug for promoting hair growth and improving AGA. Background Art

[0002] In recent years, the incidence of hair loss has increased year by year. A resident health survey shows that residents' concerns about hair loss have ranked seventh among health problems, and "hair volume anxiety" and baldness have become the hidden pain of many people. With the development of social economy, people's demand for treatment of hair loss has gradually increased. AGA is the most common type of hair loss, mainly manifested as the receding hairline of the forehead and (or) the progressive reduction and thinning of the hair on the top of the head, also known as male pattern baldness. Hair loss seriously affects the patient's appearance and mental health. Mainstream research believes that AGA is a genetic background in which testosterone is converted into dihydrotestosterone (Dihydrotestosterone, DHT) by 5α-reductase. DHT binds to androgen receptors on hair follicles, resulting in miniaturization of hair follicles, which eventually leads to a gradual decrease in hair follicle density. At present, the pathogenesis of AGA has not been fully elucidated, which limits its targeted treatment. Therefore, it is particularly important to explore its pathogenesis and guide the search for targeted treatment methods.

[0003] Although AGA is a very common disease, the approved treatment options are limited and the effects are poor. Currently, the drugs approved for the treatment of male AGA mainly include finasteride and minoxidil, but both require long-term use to be effective. The use of finasteride may cause adverse reactions such as decreased libido and impotence. The propylene glycol in the minoxidil solution may cause skin allergies; high sodium may cause water and sodium storage; minoxidil has the effect of dilating blood vessels, and some patients may experience symptoms such as palpitations and dizziness when using minoxidil for treatment. Spironolactone used for female AGA has the risk of causing irregular menstruation and electrolyte imbalance. Hair transplantation technology can redistribute hair and improve appearance, but it is an invasive operation and is expensive. In addition, the surgery uses autologous single hair follicles and hair follicle units for transplantation, which are limited in number and have unstable survival rates.

[0004] Through the above analysis, the problems and defects of the prior art are as follows:

[0005] (1) Among the existing methods for improving hair loss, oral and topical medications have many problems, such as serious side effects, slow onset of effect, unstable efficacy, and recurrence of the disease after discontinuation of medication.

[0006] (2) Among the existing methods for improving hair loss, surgical treatment is expensive and, because the surgery uses autologous single hair follicles and hair follicle units for transplantation, the number is limited and the survival rate is unstable.

[0007] Necrosulfonamide (NSA), N-[4-[[(3-methoxypyrazinyl)amino]sulfonyl]phenyl]-3-(5-nitro-2-thienyl)-2-acrylamide (Necrosulfonamide), CAS: 1360614-48-7, chemical formula: C 18 H 15 N 5 O 6 S 2 .

[0008] Necrosulfonamide inhibits MLKL-mediated necrosis by blocking the function of the N-terminal CC region of MLKL. It can inhibit downstream necrosis activated by RIP3. Necrosulfonamide has no effect on TNF-α and Smac mimetic-induced apoptosis in Panc-1 cells that do not express RIP3, even at a concentration of 5 μM. Necrosulfonamide effectively inhibits necrosis in human cells.

[0009] After searching, the following reports were found: CN202210601780.X discloses the application of ultrasound in inducing hair follicle cells and hair growth, specifically: using 30kHz ultrasound to inhibit the secretion level of dihydrotestosterone-induced hair loss inducing factors DKK1 and TGF-β1, inducing the proliferation and anti-apoptosis of human hair papilla cells hDPC and outer root sheath keratinocytes for hair loss treatment. CN202111668170.3 discloses the use of recombinant calreticulin in hair growth, hair cultivation, hair protection or hair loss prevention and related products. CN201410675650.6 discloses the application of microRNA-22 in hair development and androgen-induced hair loss. CN201810683534.7 discloses a new stem cell hair growth agent that promotes hair regeneration, specifically including PDGF, VEGF, and KGF, wherein the concentration of KGF is 856.73 μg / ml, the concentration of PDGF is 89.31 μg / ml, and the concentration of VEGF is 118.65 μg / ml. No relationship between NSA and AGA has been reported.

[0010] Therefore, in this field, it is of great significance to find new drugs for the treatment of hair loss by exploring the factors that affect changes in the hair cycle, finding the signal pathways that affect hair growth, and targeting key molecules to promote the transition from the hair resting phase to the growth phase. Summary of the invention

[0011] In view of the problems existing in the prior art, the present invention aims to provide a new therapeutic target - MLKL, a key molecule of necroptosis, and to study the relationship between Necrosulfonamide (NSA) and targeted inhibition of MLKL activity to promote hair regeneration in mice and improve AGA.

[0012] The present invention is implemented as follows: first, by constructing a small interfering RNA (siRNA) targeting MLKL, detecting the knockdown of siMLKL by RT-qPCR, injecting siMLKL into the mouse epidermis by intradermal injection, knocking down the expression of MLKL in the epidermis, observing the hair growth, detecting the expression activation of MLKL and the expression of β-Catenin, a key molecule in the hair growth-related signal Wnt signal, by RT-qPCR and Western Blot, to explore the role of MLKL in mouse hair growth. Further, by intradermal injection of MLKL inhibitor NSA, the activity of MLKL in the epidermis is inhibited, the hair growth is observed, and the expression activation of MLKL and the expression of β-Catenin are detected by RT-qPCR and Western Blot. It is found that targeted inhibition of MLKL can promote hair regeneration in mice, further verifying the key role of MLKL in hair regeneration.

[0013] The small molecule drug for promoting mouse hair regeneration provided by the present invention can be purchased directly, the working solution preparation method is simple, the operation is convenient, and it has a significant effect, which helps to alleviate the trend of hair loss in mice and / or improve the hair condition.

[0014] The technical solution of the present invention is: the present invention provides the use of MLKL inhibitors in the preparation of preparations for promoting hair growth or improving androgenic alopecia.

[0015] Preferably, the MLKL inhibitor is siMLKL or NSA.

[0016] Preferably, the siMLKL is shown in the sequence SEQ ID NO.1-SEQ ID NO.6.

[0017] Preferably, the concentration of NSA is 20uM-50uM.

[0018] Preferably, targeted inhibition of MLKL improves the inhibitory effect of DHT on hair growth in mice.

[0019] Compared with the prior art, the advantages of the present invention are:

[0020] The present invention provides a new idea of ​​using NSA, an inhibitor of MLKL, for hair research. NSA can accelerate the transition from the resting phase to the growth phase of hair follicles on the back of mice, thereby promoting hair growth and upregulating the expression of β-Catenin, a key signaling molecule for hair growth.

[0021] In addition, the drug was used in an AGA mouse model, and it was found that targeted inhibition of MLKL could improve the inhibitory effect of DHT on mouse hair growth, indicating that targeted inhibition of MLKL has a certain improvement effect on AGA, providing a new direction for the preparation of drugs to promote hair growth and improve AGA. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments of the present invention. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0023] Figure 1 RT-qPCR detected the knockdown of different siMLKL in HaCaT cells.

[0024] Figure 2 The experimental results of knocking down the key molecule of necroptosis MLKL on the promotion of hair growth in mice are shown in Figure 1. (A) The mice in each group that were intradermally injected with siMLKL after hair removal were photographed on the 0th, 8th, 11th, 13th, and 15th days of intervention, with 5 mice in each group, for a total of 20 mice. Compared with the negative control, topical NSA promoted the transition from the resting phase to the growth phase of hair growth in C57 mice. (B) Hematoxylin and eosin (H&E) stained tissue sections in transverse and longitudinal sections showed that the number and density of hair follicles in the skin of mice treated with NSA increased (scale: 1mm). (C) Cross-sectional hair follicle counts in different treatment groups. (D, E) Compared with the control, the mRNA expression of MLKL and β-Catenin in the hair follicles of mice treated with siMLKL and siNC was changed. (F, G) Compared with the control, the protein expression of MLKL, p-MLKL, β-Catenin, and GAPDH was analyzed in the hair follicles of mice treated with siMLKL and siNC.

[0025] Figure 3 This is the molecular formula diagram of NSA.

[0026] Figure 4The experimental results of the effect of NSA on the promotion of hair growth in mice are shown in Figure 1. (A) The mice in each group after hair removal were photographed on the 0th, 8th, 11th, 13th and 15th days of intervention, with 5 mice in each group, for a total of 20 mice. Compared with the negative control, topical NSA promoted the transition from the resting phase to the growing phase of hair growth in C57 mice. (B) Hematoxylin and eosin (H&E) stained tissue sections in transverse and longitudinal sections showed an increase in the number and density of hair follicles in the skin of mice treated with NSA (scale: 1 mm). (C) Cross-sectional hair follicle counts in different treatment groups. (D, E) mRNA expression of MLKL and β-Catenin in the hair follicles of mice treated with 20uM-NSA and 50uM-NSA compared with the control. (F, G) Western blot analysis of MLKL, p-MLKL, β-Catenin and GAPDH was analyzed in the hair follicles of mice treated with 20uM-NSA and 50uM-NSA compared with the control.

[0027] Figure 5 The experimental results of NSA improving the inhibitory effect of DHT on mouse hair growth, where (A) photos of the back skin of mice on days 0, 8, 11, 13, and 15 of different treatment groups. Compared with the negative control, topical NSA promoted hair growth in C57 mice, while DHT inhibited hair growth. Compared with mice treated with DHT, hair growth in mice treated with the NSA+DHT group showed an increasing trend. (B) Transverse and longitudinal sections of hematoxylin and eosin (H&E) stained tissue sections show the number and density of mouse hair follicles. (C) Cross-sectional hair follicle counts of different treatment groups in Figure B. (D) mRNA expression levels of MLKL in mouse hair follicles in different treatment groups. (E, F, G, H) WB analysis results of MLKL, p-MLKL, β-Catenin, and GAPDH were analyzed in mouse hair follicles treated with 50uM NSA, DHT, 50uM NSA+DHT, and minoxidil compared with the control.

[0028] Figure 6 It is a detailed experimental plan diagram. Implementation

[0029] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. Example

[0030] 1. Experimental plan:

[0031] The application embodiment of the present invention provides an application of a small molecule inhibitor for promoting hair regeneration in mice, and the application method of the small molecule drug for promoting hair regeneration in mice includes:

[0032] 1. Inhibitor configuration: NSA is an inhibitor of MLKL, a key molecule of necroptosis, purchased from Selleck, with a chemical formula of C18H15N5O6S2. Figure 3 As shown,

[0033] 2. In order to explore whether targeted inhibition of MLKL promotes hair growth in mice, we constructed a dorsal hair removal model of 7-week-old male C57 mice. After depilating the dorsal skin of the mice, siMLKL and negative control siNC were injected intradermally, with an injection dose of 2nmol / 20g body weight of mice, and the injection frequency was twice a week. Topical application of 3% minoxidil was used as a positive control once a day. The hair growth of the mice was observed every other day. The skin tissue of the mice was obtained on the 15th to 17th day, and the mRNA expression levels of MLKL and β-Catenin, a key signaling molecule for hair growth, were detected by qPCR. The protein levels of MLKL, P-MLKL, and β-Catenin were detected by WB. The number of hair follicles was observed by HE staining of the cross-section and longitudinal section.

[0034] 3. We used this drug to construct a hair loss model in 7-week-old C57 mice, and gave subcutaneous injections of 20uM, 50uMNSA, 300uL each time with saline containing 0.1% DMSO (negative control). The positive control group applied 3% minoxidil tincture once a day. The hair growth of the mice was observed every other day. The skin tissue of the mice was taken on the 15th to 17th day, and the mRNA expression levels of MLKL and β-Catenin were detected by qPCR. The protein levels of MLKL, P-MLKL, and β-Catenin were detected by WB. The cross-section and longitudinal section were HE stained to observe the number of hair follicles.

[0035] 4. Similarly, 7-week-old C57 mice were selected, the backs were depilated, and 0.1% DMSO-containing saline, 50uMNSA, 0.3ug / ml DHT, and 50uMNSA+0.3ug / ml DHT were subcutaneously injected, and the above indicators were tested again.

[0036] SiRNA sequences:

[0037]

[0038] Primer sequences:

[0039]

[0040] The method for preparing the MLKL inhibitor for promoting hair regeneration in mice comprises the following steps:

[0041] NSA is an inhibitor of MLKL, a key molecule of necroptosis, purchased from Selleck Company, with a chemical formula of C18H15N5O6S2. Figure 1 As shown, the molecular weight is 461.47.

[0042] First, accurately weigh 4.6147 mg of NSA powder and place it in an EP tube. Add 1 ml of sterile DMSO to the EP tube and shake well to obtain the mother solution. The concentration of the mother solution is 10 mM. When using, take a certain amount of the mother solution and add physiological saline to dilute it according to the volume ratio so that the final working solution concentration is 20 uM or 50 uM.

[0043] The small molecule drug was dissolved in pure DMSO to prepare a stock solution with a concentration of 10 mM. When used, a certain amount of the stock solution was taken and diluted with physiological saline according to the volume ratio to make the final working solution concentration 20uM, 50uM, and injected intradermally into the back skin of the mouse depilatory model once every other day.

[0044] 2. Experimental results:

[0045] Evidence of relevant effects of the embodiments. The embodiments of the present invention have achieved some positive effects during the development or use process, and indeed have great advantages over the prior art. The following content is described in conjunction with the data, charts, etc. of the test process.

[0046] 1. Screening of siRNA targeting MLKL in HaCaT cells

[0047] In order to explore whether MLKL, a key molecule of necroptosis, is involved in the regulation of the hair cycle and its role in hair growth. We constructed three siRNAs (Small-interfering RNA, siRNA) targeting MLKL to knock down the MLKL gene in HaCaT cells, and detected the mRNA and protein expression levels of MLKL in HaCaT cells by RT-qPCR to evaluate its transfection efficiency. The results showed that compared with the control group, the key molecule of necroptosis MLKL was successfully knocked down in HaCaT cells, among which siRNA-RIPK3-03 was the most efficient in silencing the expression of MLKL (P<0.05) (see Figure 1 ). Therefore, we selected siRNA-MLKL-03 for subsequent experiments.

[0048] 2. Knockdown of MLKL in the back skin of mice promotes hair growth

[0049] In order to explore whether intradermal transfection of siMLK in C57 mice promotes hair growth, we established a dorsal hair removal model of 7-week-old male C57 mice. After depilating the dorsal skin of the mice, siMLKL and negative control siNC were injected intradermally, respectively, with an injection dose of 2nmol / 20g body weight of mice, and the injection frequency was twice a week. Topical application of 3% minoxidil was used as a positive control, once a day. The hair growth on the back of the mice was observed every other day. Compared with the control group, the back skin of the mice in the siMLK group and the minoxidil group turned black earlier, and the hair growth of the siMLK group was faster than that of the siNC group. On the 15th day after local intradermal injection of siMLKL, it can be observed that most of the hair of the mice in the NSA group entered the growth phase earlier than the control group. Therefore, intradermal injection of siMLKL promotes hair growth in C57 mice. Further counting of hair follicles in the cross-sections of hematoxylin and eosin (H&E)-stained tissue sections in different treatment groups revealed that the hair follicle density in the siMLKL group was significantly higher than that in the control group ( P < 0.05 ) (see Figure 2 A, B, C).

[0050] We further extracted RNA from the epidermis of each group of mice and used RT-qPCR to detect the mRNA expression of MLKL and β-Catenin, a key signaling molecule for hair growth, in the epidermis of mice after siMLKL transfection. The results showed that compared with the control group, the mRNA expression level of MLKL in the epidermis of the mice in the siMLKL group was downregulated (see Figure 2 D), β-Catenin mRNA expression level is upregulated ( Figure 2 E), the difference was statistically significant (P<0.05). Using WB technology to detect the expression levels of MLKL, p-MLKL, and β-catenin proteins, it was found that the protein expression levels of MLKL and pMLKL in the back skin of mice were downregulated, and the expression level of β-catenin, a key protein in the Wnt / β-catenin signaling pathway, was upregulated (P<0.05) ( Figure 2 F, G, H).

[0051] 3. MLKL inhibitor NSA promotes hair growth in mice

[0052] The previous study found that intradermal injection of siMLKL promotes hair growth in C57 mice. Furthermore, in order to find a simple substance to prepare to replace siMLKL, we first applied NSA, a key MLKL inhibitor of necroptosis, to hair research to explore whether NSA has a promoting effect on hair growth in vivo. We constructed a back hair removal model for 7-week-old male C57 mice. After depilation, the mice were given subcutaneous injections of 0.1% DMSO-containing saline, 20uM NSA, and 50uM NSA every other day. Topical application of 3% minoxidil was used as a positive control, and photos were taken every other day to observe hair growth. Compared with the control group, the back skin of mice in the NSA group and the minoxidil group turned black earlier, and the hair growth in the 50uMNSA group was faster than that in the 20uM group. On the 15th day after local intradermal injection of NSA, it can be observed that most of the hair of the mice in the NSA group entered the growth phase earlier than that in the control group ( Figure 4 A). Thus, NSA significantly promoted hair growth in C57 mice.

[0053] Further staining of the tissue with hematoxylin and eosin (H&E) showed that the density and number of skin hair follicles in the mice treated with NSA increased, and the promoting effect was more obvious in the mice treated with 50uMNSA ( Figure 4 A, B, and C). In the animal model, RT-qPCR results showed that there was no significant change in the mRNA level of MLKL in the control group and the NSA group ( Figure 4 D) NSA upregulated the mRNA expression level of β-Catenin ( Figure 4 E). Using WB technology to detect the expression levels of MLKL, p-MLKL, and β-catenin proteins, it was found that the protein level of MLKL in the back skin of mice did not change significantly, the protein expression level of pMLKL was downregulated, and the expression level of β-catenin, a key protein in the Wnt / β-catenin signaling pathway, was upregulated (P<0.05) ( Figure 4 F, G, H).

[0054] 4. MLKL inhibitor NSA improves the inhibitory effect of DHT on hair growth in mice

[0055] The 7-week-old male C57 mice with hair loss model were randomly divided into four groups, with five mice in each group. Physiological saline containing 0.1% DMSO, 0.3ug / ml DHT, 50uMNSA and 50uM NSA+DHT were subcutaneously administered, injected once every other day, and 3% minoxidil was applied topically as a positive control. The hair growth on the back of the mice was photographed on days 0, 8, 11, 13, and 15. The results showed that on the 15th day, the back skin of the NSA group darkened most significantly, and the hair grew faster, followed by the minoxidil group, both of which were higher than the negative control group, while most of the back skin of the DHT group was still pink, and the hair in the NSA+DHT group grew significantly faster than the DHT group ( Figure 5 A). Therefore, NSA can significantly accelerate the transition of hair follicles from the resting phase to the growth phase and promote hair growth in C57 mice. Hematoxylin and eosin (H&E) stained tissues showed that the density and number of hair follicles increased in mice treated with NSA and minoxidil compared with the control group, and the density and number of hair follicles increased in mice treated with NSA+DHT compared with DHT-treated mice ( Figure 5 B, C). MLKL mRNA levels did not change ( Figure 5 D). The MLKL protein expression level did not change significantly in any treatment group, the p-MLKL protein expression level was downregulated in the NSA and NSA+DHT groups, and the β-catenin protein expression level was upregulated ( Figure 5 E, 5F, 5G, 5H).

[0056] 3. Conclusion:

[0057] 1. The present invention first discovered that targeted inhibition of MLKL, a key molecule of necroptosis, can regulate the hair cycle, promote the transition from the resting phase to the growth phase in mice, and promote hair growth, which is a possible new target for the treatment of hair loss diseases. DHT can cause hair cycle abnormalities by causing apoptosis of hair papilla cells, and is therefore often used to construct a mouse AGA model. In order to explore whether MLKL inhibitors have an effect on AGA, we used DHT to induce an AGA mouse model and found that the use of MLKL inhibitor NSA can antagonize the inhibitory effect of DHT on mouse hair growth.

[0058] 2. The present invention provides a new idea of ​​using MLKL inhibitors to promote hair growth for the first time. NSA, an inhibitor of MLKL, a key molecule of necroptosis, is used in hair research and it is found that it can promote hair growth and improve AGA.

[0059] The above is a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, can make equivalent replacements or changes according to the technical solutions and concepts of the present invention, which should be covered by the protection scope of the claims of the present invention.

Claims

1. Use of a MLKL inhibitor in the preparation of a preparation for promoting hair growth or improving androgenic alopecia, wherein the MLKL inhibitor is siMLKL, and the siMLKL is shown in the sequence SEQ ID NO.5-SEQ ID NO.6.

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