Application of IκBα phosphorylation inhibitor in the preparation of drugs for promoting sperm motility

Abstract

The present invention provides the use of an IκBα phosphorylation inhibitor in the preparation of a drug for promoting sperm motility. The present invention uses the IκBα phosphorylation inhibitor Bay117082 to promote human sperm motility, and achieves a relatively ideal therapeutic effect regardless of whether it is normal sperm or asthenozoospermia, not only providing new information and inspiration for the elucidation of the human sperm motility regulation mechanism, but also providing inspiration and basis for the clinical application of Bay117082 in improving the motility and quality of asthenozoospermia and frozen sperm, and has the potential to improve the prospects of assisted reproductive technology for asthenozoospermia patients, and has extremely high application value and social benefits.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to application of an IκBα phosphorylation inhibitor in the preparation of a drug for promoting sperm motility. Background Art

[0002] More and more studies have reported that the total amount, concentration, motility and motility of sperm are constantly decreasing. In clinical diagnosis, semen quality analysis is still an important indicator for evaluating male fertility. The types of male infertility semen mainly include azoospermia, oligospermia, asthenospermia, teratozoospermia, etc. Among them, asthenospermia, that is, sperm motility defects, are more common. Studies have shown that sperm motility is related to the success rate of fertilization. However, there are still a large number of patients with asthenospermia of unknown causes who find it difficult to conceive naturally by improving their lifestyle, taking oral medications and other treatments to improve sperm motility, and choose to conceive with assisted reproductive technology. Among assisted reproductive technologies, artificial insemination technology, which is closer to natural conception, is preferred, which increases the probability of fertilization by allowing sperm to directly reach a position closer to the egg cell. In addition, there is in vitro fertilization-embryo transfer technology, which uses sperm and eggs to combine in vitro to achieve fertilization and then transfer the embryo into the woman's uterus. Intracytoplasmic sperm injection uses artificial technology to replace the process of sperm penetrating the egg to achieve the purpose of fertilization, which can provide a further option for patients who have failed in vitro fertilization. The above-mentioned assisted reproductive technologies all require the collection, artificial processing and screening of the male's sperm. If the application of drugs can improve the motility of sperm and enhance the ability of sperm to penetrate eggs, it can improve the pregnancy rate and the success rate of assisted reproduction. And the improvement of sperm motility can enable patients to choose simpler techniques. For example, after the sperm motility of patients with asthenospermia is enhanced, artificial insemination can be successfully performed, and the invasive operation of in vitro fertilization-embryo transfer can be avoided. Activating sperm motility can also help screen sperm with stronger motility and healthier sperm for subsequent assisted reproductive applications. It is urgent to explore the mechanism of sperm motility and develop drugs to improve sperm motility, which is very important for improving my country's reproductive health.

[0003] There are two main types of normal sperm motility, namely, activated state and superactivated state. Activated sperm exhibit symmetrical, low-amplitude fluctuation, and linear motion, while superactivated motion exhibits asymmetrical, high-amplitude, and circular motion. Activated motion helps sperm to move in the female reproductive tract, while superactivated motion facilitates the separation of sperm from the fallopian tube epithelium after capacitation and facilitates the egg penetration reaction. Computer-assisted sperm analysis (CASA) can measure sperm motility parameters and reflect changes in sperm motility speed and pattern. Commonly used indicator parameters for human sperm superactivated motion include curvilinear velocity (VCL) and amplitude of lateral head displacement (ALH), and there is currently no unified standard.

[0004] Sperm movement requires not only complete basic structural units, but also energy supply. At present, reports on basic and clinical research show that the energy for human sperm movement mainly comes from glycolysis under anaerobic conditions and oxidative phosphorylation under aerobic conditions. The former produces 2 energy substances ATP in a complete reaction process, while the latter produces 32 ATP in a reaction process. In addition, there are sporadic reports suggesting that lipid substances can provide energy for sperm through fatty acid β-oxidation (a complete reaction can provide 128 ATP). Sperm contains rich lipid substances, which on the one hand provide sperm with good cell membrane fluidity and support the continuous movement of sperm. At the same time, sperm contains a large number of mitochondria and has the basis for using lipids such as fatty acids and phospholipids for energy metabolism reactions. Fatty acid oxidation energy supply can provide a large amount of ATP in a short time and has the potential to be an efficient energy substance for sperm to maintain movement, but currently research on the fatty acid oxidation function of human sperm is still rare.

[0005] NF-κB (Nuclear factor kappa-light-chain-enhancer of activated B cells, NF-κB) is an important nuclear transcription factor in mammals. It can enter the nucleus to regulate the functions of multiple target genes and participate in the regulation of cell proliferation, apoptosis, cell stress and inflammatory response. It can bind to discrete DNA sequences through κB elements to promote or inhibit the expression of target genes. In mammals, NF-κB contains five subunits, namely RelA (p65), RelB, c-Rel, p50 (NF-κB1, the precursor is p105) and p52 (NF-κB2, the precursor is p100). The homo- or hetero-dimers of each subunit form NF-κB complexes to exert their functions. In most cell types, NF-κB is bound to IκBs (including IκBα, IκBβ, IκBε, IκBγ, and IκBδ), or stored in the cytoplasm in the form of inactive precursor proteins such as p100 or p105. When there is external stimulation or the action of related cytokines, IκBs proteins are degraded, and the NF-κB complex bound to them is released and enters the cell nucleus, binding to specific κB sites on DNA, thereby leading to transcriptional activation or inhibition of specific promoters and regulating the expression of specific genes.

[0006] IκBs play an important role in the NF-κB classical pathway. The degradation of IκBα leads to the transfer of NF-κB to the nucleus, causing the transcription of IκBα itself and a series of genes. Studies have reported that IκBα has the function of inhibiting apoptosis independently of the NF-κB pathway. IκBα is localized in the outer membrane of mitochondria and has the function of inhibiting apoptosis. On mitochondria, IκBα stabilizes the VDAC1 and hexokinase 2 complex, thereby inhibiting the recruitment of Bax and the release of cytochrome C-induced apoptosis. This inhibitory effect is independent of the NF-KB pathway.

[0007] Bacterial infection of the reproductive tract is one of the most serious causes of male infertility. There are also many studies on the effects of immune stimulants on sperm. The bacterial endotoxin LPS can be detected in human semen, and LPS can act on human and mouse sperm, inhibiting sperm motility, increasing sperm apoptosis, and reducing the in vivo and in vitro fertilization rates of mouse sperm. Some studies believe that LPS affects sperm motility by reducing intracellular cAMP. LPS has also been detected in samples from clinical infertility patients to increase the reactive oxygen species (ROS) in sperm. The molecular mechanism of LPS action on sperm has not yet been fully elucidated. The effect of inflammatory cytokines on sperm production may be the reason for the low fertilization potential of human sperm under inflammatory conditions.

[0008] Bay117082 is an irreversible inhibitor of IκBα phosphorylation. It can inhibit the phosphorylation of IκBα in a variety of cells and tissues, especially TNFa-induced phosphorylation, and block the activation of the NF-κB pathway. At the same time, Bay117082 also has other effects, such as broad-spectrum inhibitory activity on inflammatory signaling pathways, inhibition of phosphatase activity, inhibition of inflammasomes, inhibition of the ubiquitin system, etc. In anucleated mature erythrocytes, treatment with the IκBα phosphorylation inhibitor Bay117082 increased the intracellular calcium ion concentration and reduced reduced glutathione. Bay117082 inhibits IκBα phosphorylation in platelets, restoring fully diffused platelets to a spherical morphology. The pharmacological effects of Bay117082 in anti-inflammatory, anti-tumor, and neuroprotective effects have been studied extensively, but its effects on mature sperm have not been reported.

[0009] At present, the stimulants used in the research to regulate human sperm motility are mainly exogenous compounds such as pentoxifylline and caffeine; endogenous substances such as follicular fluid, progesterone, and lysophosphatidic acid, but these compounds take a long time to take effect and have a short duration of action; they have obvious non-specific effects; and have large side effects. The reason may be that the compounds are not efficient enough, the mechanism of target action is not clear enough, or they fail to act on the main motor regulation pathway of sperm motility. Summary of the invention

[0010] In order to overcome the defects in the prior art, the present invention provides the use of an IκBα phosphorylation inhibitor in the preparation of a drug for promoting sperm motility.

[0011] To achieve the above object, the present invention adopts the following technical solution:

[0012] The first aspect of the present invention is to provide the use of an IκBα phosphorylation inhibitor in the preparation of a medicament for promoting sperm motility. The medicament comprises an IκBα phosphorylation inhibitor and a pharmaceutically acceptable carrier or excipient thereof.

[0013] Furthermore, the above-mentioned IκBα phosphorylation inhibitor includes Bay117082.

[0014] Furthermore, the above sperm is normal sperm or weak sperm.

[0015] Furthermore, the above sperm is frozen sperm.

[0016] The second aspect of the present invention is to provide the use of an IκBα phosphorylation inhibitor in the preparation of a drug for promoting the fatty acid β-oxidation energy supply of sperm. The drug comprises an IκBα phosphorylation inhibitor and a pharmaceutically acceptable carrier or excipient thereof.

[0017] Furthermore, the above-mentioned IκBα phosphorylation inhibitor includes Bay117082.

[0018] Furthermore, the above sperm is normal sperm or weak sperm.

[0019] Furthermore, the above sperm is frozen sperm.

[0020] The third aspect of the present invention is to provide a medicine or a pharmaceutical composition for promoting sperm motility, which comprises an IκBα phosphorylation inhibitor and a pharmaceutically acceptable carrier or excipient thereof.

[0021] Furthermore, the above-mentioned IκBα phosphorylation inhibitor includes Bay117082.

[0022] Furthermore, the above sperm is normal sperm or weak sperm.

[0023] Furthermore, the above sperm is frozen sperm.

[0024] The present invention adopts the above technical solution, and has the following technical effects compared with the prior art:

[0025] The present invention uses an IκBα phosphorylation inhibitor to promote human sperm motility, and achieves a relatively ideal therapeutic effect regardless of normal sperm or asthenozoospermia. It not only provides new information and inspiration for the elucidation of the regulatory mechanism of human sperm motility, but also provides inspiration and basis for the clinical application of Bay117082 in improving the motility and quality of asthenospermia and frozen sperm. It has the potential to improve the prospects of assisted reproductive technology for asthenospermia patients and has extremely high application value and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The expression distribution of protein components in the NF-κB pathway in human sperm and their activation in sperm capacitation fluid are shown; A: Western blot detection of proteins in the NF-κB pathway, 1, 2, and 3 represent sperm samples from three different people, and α-Tubulin is used as a spotting internal reference; B: Western blot detection of the phosphorylation levels of IKKA / B, RELA, and IκBα proteins in the NF-κB pathway before and after capacitation fluid stimulation, and α-Tubulin is used as a spotting internal reference; C~F: Indirect immunofluorescence detection of the localization of IKKA, IKKB, IκBα, and RELA proteins in the NF-κB pathway on sperm, DAPI: staining indicates the location of the cell nucleus;

[0027] Figure 2The figure shows that IκB A phosphorylation regulates the movement of human sperm; A: Changes in IκBα phosphorylation after human sperm was treated with Bay117082, α-Tubulin was used as the internal standard for spotting; B, C: Changes in the movement parameters VCL (B) and ALH (C) of human sperm after Bay117082 treatment; D: Changes in IκBα phosphorylation after human sperm was treated with LPS, α-Tubulin was used as the internal standard for spotting; E, F: Changes in the movement parameters VCL (B) and ALH (C) of human sperm after LPS treatment; *p<0.05, **p<0.01, ***p<0.001, ns, no significant difference;

[0028] Figure 3 The IκBα inhibitor Bay117082 changes the direction of sperm movement; A-B: The movement trajectory of sperm before (A) and after (B) treatment with Bay117082; C, G: The changes in movement parameters of normal sperm with VCL < 100μm / s (C) and VCL > 100μm / s (G) before and after treatment with Bay117082; E, I: Weakly motile sperm with VCL < 100μm / s (E) and VCL > 100μm / s (I) D, F, H, J: Changes in motility parameters before and after treatment with Bay117082 in normal sperm and weakly motile sperm; VCL: curvilinear velocity, VAP: average path velocity; VSL: linear velocity, LIN: linear index, STR: linear index, WOB: wobble index, ALH: lateral swing amplitude; *p<0.05, **p<0.01, ***p<0.001;

[0029] Figure 4 The figure shows the changes in the metabolic levels of human sperm before and after Bay117082 treatment; A: Changes in ATP levels before and after Bay117082 treatment; B: Changes in principal components, square points are the control group without Bay117082 treatment, and triangle points are the treatment group; C: Heat map display of metabolite levels before and after Bay117082 treatment; D, E, F, G: Changes in oleic acid, adrenic acid, stearic acid, and palmitic acid levels before and after Bay117082 treatment; H: Pathway enrichment analysis, the reference database is SMPDB (The Small Molecule Pathway Database): https: / / smpdb.ca / ;

[0030] Figure 5The results show that Bay117082 promotes human sperm motility independently of sugar metabolism-related pathways; A, B: Changes in VCL (A) and ALH (B) after human sperm is treated with Bay117082 in the absence of sugars such as glucose, pyruvate, and lactate in the culture medium; C: Effects of glycolysis inhibitor α-ChI on the percentage of sperm motility; D: Changes in VCL after human sperm is treated with Bay117082 in the presence of α-ChI (ACH); *p<0.05, **p<0.01, ***p<0.001;

[0031] Figure 6 The results showed that Bay117082 inhibited IκBα phosphorylation and significantly downregulated the content of acetyl-CoA carboxylase ACC; A: Expression of fatty acid β-oxidation-related proteins CPT1A, ACSL1, ACADL, ACADVL and HADHA in sperm; BF: Distribution and localization of proteins CPT1A, ACSL1, ACADL, ACADVL and HADHA in human sperm; G: Effect of protein CPT1 inhibitor Etomoxir (Eto) on the percentage of sperm motility; H: VCL expression in human sperm after Bay117082 treatment in the presence of Eto I: Protein hybridization shows the changes of AMPK, ACC and their phosphorylation after Bay117082 treatment of human sperm, α-Tubulin was used as the internal reference; J: Quantitative analysis of the changes of ACC and its phosphorylation after Bay117082 treatment of human sperm; K: Analysis of the changes of ACC phosphorylation and ACC protein ratio after Bay117082 treatment of human sperm; L: Quantitative analysis of the changes of ACC and its phosphorylation after Bay117082 treatment of human sperm; M: Distribution and localization of protein ACC in human sperm; N: NADH / NAD after Bay117082 treatment of human sperm + Proportional change analysis;

[0032] Figure 7 Shows the effect of Bay117082 on the motility of IκBα knockout sperm; A: Protein hybridization shows the knockout of IκBα in sperm; B: Indirect immunofluorescence shows the knockout of IκBα in sperm; C, D: The effect of Bay117082 on the motility parameters VCL (C) and ALH (D) of IκBα knockout sperm; *p<0.05, ***p<0.001, ns no significant difference;

[0033] Figure 8 The hypothesis of the mechanism by which Bay117082 inhibits IκBα phosphorylation and significantly promotes human sperm motility is shown. DETAILED DESCRIPTION

[0034] In the early experiments, the inventor used a small molecule compound library to study the effects of various small molecules on mouse sperm, and screened out Bay117082, an IκBα phosphorylation inhibitor that can significantly promote sperm motility. Subsequently, through drug experiments on human sperm, mouse models and other studies, it was found that the compound can significantly improve the motility of human sperm in a short period of time, and can promote motility for a long time. Its mechanism is to promote the energy supply of sperm fatty acid β-oxidation. The present invention is the first to use the IκBα phosphorylation inhibitor Bay117082 to promote human sperm motility, and has achieved a relatively ideal therapeutic effect regardless of normal sperm or weak sperm. For the first time, the knowledge of non-transcriptional regulation of the NF-κB signaling pathway in sperm was explored, deepening the understanding of the new functions and mechanisms of NF-κB.

[0035] The present invention is described in detail and specifically below through specific embodiments and drawings to provide a better understanding of the present invention, but the following embodiments do not limit the scope of the present invention.

[0036] The methods in the examples are conventional methods unless otherwise specified, and the reagents used are conventional commercial reagents or reagents prepared according to conventional methods unless otherwise specified.

[0037] Example 1

[0038] 1. Experimental materials and methods:

[0039] (1) Reagents:

[0040] Sperm capacitation fluid (Biggers, Whitten, and Whittingham medium, BWW), the composition is: 95mM sodium chloride, 4.8mM potassium chloride, 1.7mM calcium chloride, 1.2mM magnesium sulfate, 1.2mM potassium dihydrogen phosphate, 5.6mM glucose, 20mM sodium lactate, 0.25mM sodium pyruvate, 25mM sodium bicarbonate. Add 3.5mg / ml bovine serum albumin after preparation the day before use, adjust the pH to 7.2-7.4, adjust the osmotic pressure to 270-300mOsm / L, put it in a 5% CO2 incubator overnight and use it the next day. Alternatively, sperm capacitation fluid such as human fallopian tube fluid (HTF) can be used for sperm washing, incubation and culture. LPS, Bay117082, Etomoxir, α-chlorohydrin were purchased from Sigma.

[0041] (2) Clinical sperm sample processing:

[0042] After abstaining from sex for 2-7 days, the patient ejaculates by masturbation in the hospital's semen collection room and collects the sperm in a semen collection cup to collect the semen completely. After sampling, the sample container is kept in an environment of 20-37°C and then sent to the inspection department or laboratory. The specimen container is placed on the laboratory table or incubator and maintained at a constant temperature of 37°C to liquefy the semen for 30-60 minutes. Semen evaluation, motility measurement and other routine experiments are then performed. The remaining clinical sperm sample is centrifuged to remove seminal plasma, and the sperm is washed with BWW and the sperm concentration is adjusted to 2×10 6 / ml~50×10 6 / ml for subsequent motility testing. After mixing the sample, divide it into several equal parts, add different concentrations of drugs according to the experimental design, gently invert the sample to mix, incubate it in a 37℃ incubator for a specific time, and detect sperm motility to evaluate the effect of the drug on sperm motility.

[0043] (3) Measurement of sperm motility using computer-assisted sperm motility analyzer (CASA)

[0044] CASA can measure various parameters of sperm movement, including movement percentage, forward movement percentage, average path velocity, linear velocity, curve velocity, lateral swing amplitude, whipping frequency, forwardness, linearity, etc., which can more comprehensively reflect the changes in sperm movement patterns.

[0045] (4) Western blot (WB) and indirect immunofluorescence (IF) were used to determine the expression and distribution of proteins. Anti-IκB-α (C-21): SC-371, anti-IκB-α (H-4): sc-1643, anti-IκB-β (C-20): SC-945, anti-IKKγ (B-3): SC-8032 were purchased from Santa Cruz Biotechnology. Anti-phosphotyrosine monoclonal antibody 4G10, anti-α-tubulin monoclonal antibody: T6199 were purchased from Merck Millipore Corporation. NF-κB p65(D14E12)XP Rabbit mAb,Phospho-NF-κB p65(Ser536)(93H1)Rabbit mAb,RelB(D7D7W)Rabbit mAb,c-Rel(D4Y6M)Rabbit mAb,NF-κB1 p105 / p50(D4P4D)Rabbit mAb,NF-κB2p100 / p52(D7A9K)Rabbit mAb,IKKα(3G12)Mouse mAb,IKKβ(D30C6)Rabbit mAb,Phospho-IKKα / β(Ser176 / 180)(16A6)Rabbit mAb,Phospho-IκBα(Ser32)(14D4)Rabbit mAb,Acetyl-CoA Carboxylase(C83B10)Rabbit mAb 3676,Phospho-Acetyl-CoA Carboxylase(Ser79)(D7D11)Rabbit mAb 11818,Phospho-AMPKα(Thr172)(40H9)Rabbit mAb 2535,AMPKα(D5A2)Rabbit mAb 5831,anti-rabbit IgG horseradish peroxidase linked antibody, anti-mouse IgG horseradish peroxidase linked antibody, purchased from CellSignaling Technology Corporation.ACSL1 Polyclonal antibody (13989-1-AP), ACADL-Specific Polyclonal antibody: 17526-1-AP, ACADVL Polyclonal Antibody: 14527-1-AP, HADHA Polyclonal antibody: 10758-1-AP were purchased from Proteintech. Anti-CPT1A antibody (ab128568) was purchased from Abcam. Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488: A32790, Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488: A32766 were purchased from Thermo Scientific.

[0046] (5) Non-targeted metabolomics of sperm. Liquid chromatography-mass spectrometry (LC-MS) was used. The non-targeted metabolomics analysis process is as follows: ① Prepare and collect sperm samples treated with Bay117082; ② Obtain analytical standards, optimize product ion collision energy to obtain the best signal, and then determine the MRM transition values ​​of the metabolites studied in the triple quadrupole MS system. ③ Use quantitative analysis software to process the data, obtain a report, and perform statistical analysis and interpretation.

[0047] (6) Sperm ATP, NADH / NAD + Detection: ATP detection was performed using the Bio-Tech Enhanced ATP Detection Kit, as described in the instructions. NADH / NAD+ detection was performed using the Nanjing Jiancheng Bioengineering Institute kit for coenzyme I (NAD + / NADH) content test kit (colorimetric method) detection, refer to the instructions.

[0048] (7) Construction of IκBα conditional knockout mouse model. The brief process is as follows: ① Use the LoxP vector system to construct the LoxP-IκBα recombinant vector, and use EcoRV to identify the vector to verify whether the construction is successful; ② The LoxP-IκBα plasmid is linearized by NotI digestion, and the digestion product is purified after a series of treatments for use; ③ The purified linearized digestion product is introduced into ES cells by electroporation. ④ Screening positive clones. ⑤ PCR identification of the positive ES cell genome, only clones with recombination in both arms enter the next step of the experiment. ⑥ Inject the above-mentioned ES positive clones that meet the conditions into blastocysts. The source of blastocysts is C57 mice that are naturally conceived after superovulation and develop in vivo to the blastocyst stage; ⑦ The blastocysts injected with positive ES cells are transplanted into the uterus of synchronously mated pseudo-pregnant female mice; ⑧ Select mature male mice with a chimerism rate greater than 50% from the F0 generation mice born and mate with normal C57 female mice, and select the positive offspring mice with both arms for subsequent experiments. After obtaining LoxP-IκBα mice, they were mated with Stra8-Cre mice to finally obtain IκBα conditional knockout mice.

[0049] 2. Experimental Design

[0050] 1. Expression and function of NFκB pathway in human sperm

[0051] (1) Normal sperm samples were collected for NFκB pathway detection. The presence and content of each component were detected by immunoblotting, and the expression and localization of each protein were detected by immunofluorescence.

[0052] (2) The expression and activation of the sperm NFκB pathway in PBS and capacitation fluid were detected by immunoblotting to explore whether this pathway might be related to sperm motility.

[0053] 2. Effects of Bay117082 on human sperm motility and sperm quality

[0054] (1) Application of normal sperm samples: Use normal human sperm to conduct measurements to explore the dose and time effects of LPS and Bay117082, and to determine the optimal action time and concentration on human sperm.

[0055] (2) Application of weak sperm samples:

[0056] We found that Bay117082 can promote sperm motility, so we collected asthenospermia samples to test whether Bay can improve sperm motility in patients with asthenospermia in order to evaluate its efficacy and potential value in clinical application.

[0057] (3) Detect the effect of Bay117082 on other sperm functions, including sperm ATP, sperm NADH / NAD +level, which helps to explore its molecular mechanism of action.

[0058] 3. Non-targeted metabolomics analysis of sperm after Bay117082 treatment: Through the observation of sperm motility and the detection of ATP, we speculated that Bay117082 may change sperm motility by affecting sperm metabolism, so paired sperm samples were collected for non-targeted metabolomics testing to find possible mechanisms of action.

[0059] 4. Effects of Bay117082 treatment on sperm metabolism

[0060] (1) Use BWW medium without glucose, lactose and fructose to culture sperm, or use the glycolysis inhibitor α-chlorohydrin to test whether the Bay effect depends on the glycolysis pathway.

[0061] (2) Detection of fatty acid oxidation pathway: Western blot, IF, etc. were used to detect the expression of fatty acid oxidation pathway in sperm, and the inhibitor Etomoxir was used to observe whether the effect of Bay117082 depends on the fatty acid oxidation pathway.

[0062] 5. Construction of IKBα conditional knockout mouse model

[0063] The aim was to investigate whether Bay117082 could promote motility in knockout mice and to clarify whether the target of Bay117082 on sperm was to inhibit IκBα phosphorylation.

[0064] 3. Results

[0065] (1) Expression distribution of NF-κB signaling pathway protein components in sperm.

[0066] Normal human sperm samples were purified and collected, and Western blotting revealed that most NF-κB pathway components were expressed in human sperm cells, including IKKA, IKKB, IKKγ, p105 / 50, p100 / 52, RELA / B, c-REL, IκBα and other proteins, such as Figure 1 As shown in A. Indirect immunofluorescence assay showed that IKKA, IKKB, IκBα, RELA, etc. were located in the middle of the sperm tail, such as Figure 1 C to F. Among them, the phosphorylation of proteins such as IKKA / B, RELA, and IκBα is constitutive and can be further enhanced by the energy-harvesting fluid BWW, such as Figure 1 As shown in B.

[0067] (2) Phosphorylation of IκBα protein in the NF-κB pathway regulates human sperm motility.

[0068] Sperm was treated with Bay117082, a key protein in the NF-κB pathway, and it was found that Bay117082 could inhibit IκBα phosphorylation in a dose-dependent manner. Figure 2 A. At the same time, Bay117082 can promote sperm motility and increase the hyperactivated motility-related parameters VCL (such as Figure 2 B) and ALH (such as Figure 2 C), suggesting that IκBα phosphorylation inhibition can enhance sperm motility. In contrast, the use of IκBα phosphorylation promoter LPS can significantly increase the level of IκBα phosphorylation, such as Figure 2 D, and reduced sperm hyperactivation movement parameters, such as Figure 2 E to F. This shows that phosphorylation of IκBα, a key protein in the NF-κB pathway, can regulate the motility of human sperm.

[0069] (3) Bay117082 inhibits IκBα phosphorylation and can change human sperm motility from linear to curvilinear.

[0070] After normal human sperm was treated with Bay117082, it was observed that the sperm velocity increased and the motion trajectory changed from linear motion to curved motion. Figure 3 As shown in A and B. Normal sperm were divided into high and low groups based on VCL values. When VCL was low, that is, VCL < 100 μm / s, after Bay117082 treatment, the VCL value of its motion parameter increased significantly from 81.757 ± 7.744 μm / s to 101.4965 ± 18.47 μm / s (p < 0.001), and VSL decreased from 40.804 ± 10.0231 μm / s to 31.7145 μm / s (p = 0.005). STR decreased from 72.32±14.4440% to 55.166±14.7576% (p<0.001), LIN decreased from 54.531±13.9815% to 39.864±13.4947% (p<0.01), and WOB decreased from 60.847±5.2122% to 56.185±3.4463% (p<0.01). Figure 3C. When the VCL value was high, that is, VCL>100 μm / s, it could not be significantly improved after Bay117082 treatment (before treatment: 111.9934±13.3063 μm / s, after treatment: 113.9119±15.2855, p=0.594), but its linear motion still changed significantly, and VSL decreased from 46.1531±11.2285 μm / s to 35 .4716±10.0448μm / s (p<0.001), STR decreased from 66.4966±16.0793% to 53.6875±16.0094% (p<0.01), LIN decreased from 50.3209±13.8960% to 38.854±11.2721% (p<0.001), and VAP and WOB showed no significant changes. Figure 3 G. The results show that regardless of the basic VCL of sperm, Bay117082 affects the movement of sperm, that is, the forward movement is transformed into a curved hyper-activated movement mode. We obtained similar results in the asthenospermia samples, that is, Bay117082 also has a significant effect on activating and changing the movement direction of asthenospermia samples, such as Figure 3 E and I. In addition, in all cases, Bay117082 was able to significantly increase the level of sperm lateral swing amplitude ALH, as shown in Figure 3 As shown in D, F, H, and J.

[0071] (4) Bay117082 significantly promotes metabolome changes during human sperm motility.

[0072] After human sperm was treated with Bay117082, ATP levels increased significantly, such as Figure 4 As shown in A. We then collected 20 normal human sperm samples and treated them with Bay117082 in BWW for 10 minutes before collecting the samples for non-targeted metabolomics analysis. The results showed that after Bay117082 treatment, the samples were clearly divided into groups, such as Figure 4 B. And some metabolites showed obvious down-regulation and up-regulation, such as Figure 4 C, further analysis revealed that the fatty acid molecules were significantly reduced, such as Figure 4 As shown in D, E, F, and G. Through enrichment pathway analysis, it was found that Bay117082 treatment led to β-oxidation of long-chain fatty acids, such as Figure 4 H. The results suggest that Bay117082 significantly promotes the motility of human sperm, which may be mainly achieved by enhancing the oxidation of fatty acids and increasing energy ATP.

[0073] (5) Bay117082 significantly promotes human sperm motility independent of the glucose metabolism pathway.

[0074] In BWW medium without glucose, pyruvate, or lactate (BWW-GPL), Bay117082 was still able to promote the motility parameter VCL ( Figure 5 A) and ALH( Figure 5 B), suggesting that Bay117082 significantly promotes human sperm motility independent of exogenous carbohydrates. Studies have reported that the glycolysis inhibitor α-ChI can block glycolysis and reduce the motility percentage of sperm. We have also confirmed this effect, such as Figure 5 C. However, under the condition of blocking glycolysis, Bay117082 can still significantly promote human sperm motility, as shown in Figure 5 D. The results further confirmed that Bay117082 promoted human sperm motility independently of the sugar metabolism pathway.

[0075] (6) Bay117082 significantly promoted human sperm motility by reducing the expression of acetyl-CoA carboxylase and promoting β-oxidation.

[0076] To further explore the role of Bay117082 in accelerating the β-oxidation process and promoting sperm motility, we detected key enzymes in the β-oxidation process. We found that the proteins CPT1A, ACSL1, ACADL, ACADVL, and HADHA related to fatty acid β-oxidation were highly expressed in sperm, such as Figure 6 As shown in A, they are mainly located in the mitochondria, such as Figure 6 B to F. Studies have reported that the protein CPT1 inhibitor Etomoxir (Eto) can reduce the motility percentage of human sperm, and we have also confirmed this effect, such as Figure 6 G. And under the condition that Eto blocks CPT1, Bay117082 can no longer promote human sperm motility, as shown in Figure 6 H. The results further confirmed that Bay117082 promoted human sperm motility dependent on the CPT1 protein-mediated pathway.

[0077] In the upstream of CPT1 in the fatty acid β-oxidation process, acetyl-CoA carboxylase (ACC) can catalyze acetyl-CoA to form malonyl-CoA, which can inhibit the activity of CPT1 and thus weaken the fatty acid β-oxidation process. AMPK itself can phosphorylate ACC to inactivate it, thereby regulating the content of malonyl-CoA. In this study, it was found that ACC is located in the mitochondria of human sperm, such as Figure 6 M. After Bay117082 treatment of human sperm, AMPK and its phosphorylation did not change significantly ( Figure 6 I, L), while the content of ACC decreased significantly, and the ratio of ACC phosphorylation to ACC also showed an upward trend ( Figure 6I, J, K), these changes will lead to a decrease in malonyl-CoA. Therefore, the results suggest that Bay117082 leads to a decrease in ACC, which in turn reduces the inhibition of CPT1, thereby activating the β-oxidation process of fatty acids. In addition, it was further found that NADH / NAD + The significant increase in content also indicates that the β-oxidation process is enhanced, such as Figure 6 As shown in N.

[0078] (7) Bay failed to promote sperm motility in IκBα conditional knockout mice.

[0079] Using flox heterozygous mice (Nfkbia flox / + ) and Stra8 Cre + Mice were crossed to obtain the experimental group mice that were homozygous for flox and Cre positive (the mice were abbreviated as: Nfkbia flox / flox :Cre + , the proportion of this mouse offspring is 1 / 8) and the flox homozygous and Cre-negative control group mice (Nfkbia flox / flox The proportion of this mouse in the offspring was 1 / 8). The mouse genotype was identified by PCR amplification and electrophoresis, and the knockout efficiency of IκBα protein in sperm was verified by WB and IF ( Figure 7 A, B). The VCL of spermatozoa in the control mice significantly increased after Bay117082 treatment, while the VCL of spermatozoa in the Nfkbia cKO mice did not increase significantly. Figure 7 As shown in C and D. This suggests that IκBα protein is the main target protein of Bay117082 that causes changes in sperm motility.

[0080] (8) The mechanism of action of Bay117082 in inhibiting IκBα phosphorylation and significantly promoting sperm motility

[0081] Through in vitro pharmacology and IκBα knockout mouse models, we proposed the hypothesis that Bay117082 inhibits IκBα phosphorylation to regulate human sperm motility. Figure 8 As shown in the figure, Bay117082 inhibited IκBα phosphorylation and significantly downregulated the content of ACC, which may have reduced the level of malonyl-CoA and reduced its inhibition on CPT1, thereby accelerating fatty acid β-oxidation, increasing ATP energy levels, and enhancing sperm motility.

[0082] Verification Example

[0083] 1. Normal sperm sample 1, after centrifugation to remove seminal plasma, was resuspended with BWW, and then aliquoted, one sample was treated with Bay117082, and the other sample was not treated, and then cultured at 37°C for 10 minutes before sperm motility test. The test data of the two sperm samples are as follows:

[0084]

[0085]

[0086] The sperm motility velocity VCL (μm / s) increased from 85.35 to 105.02, and ALH (μm) increased from 4.59 to 6.008, indicating that the sperm motility curvilinearity was significantly improved and the motility velocity was accelerated; the linear motion-related parameters VSL, VAP, LIN, and STR all decreased to a certain extent, indicating that the linearity was weakened and the sperm tended to be in a super-activated motility state.

[0087] 2. Sample 2 of asthenozoospermia was centrifuged to remove seminal plasma and then resuspended with BWW. After aliquoting, one sample was treated with Bay117082 and the other sample was not treated. After incubation at 37°C for 10 minutes, sperm motility test was performed. The test data of the two sperm samples are as follows:

[0088]

[0089] The sperm motility velocity VCL increased from 82.95 (μm / s) to 116.03 (μm / s), and ALH increased from 4.89 (μm) to 6.61 (μm), indicating that the sperm motility curvilinearity was significantly improved and the motility velocity was accelerated; the linear motion-related parameters VSL, VAP, LIN, and STR all decreased to a certain extent, indicating that the linearity was weakened and the sperm tended to be in a super-activated motility state.

[0090] 3. Normal sperm sample 3, after centrifugation to remove seminal plasma, was resuspended with BWW, and then aliquoted, one sample was treated with Bay117082, and the other sample was not treated, and then cultured at 37°C for 10 minutes before sperm motility test. The test data of the two sperm samples are as follows:

[0091]

[0092] The basal value of sperm motility VCL was relatively high. After Bay117082 treatment, it only increased from 120.6 (μm / s) to 125.49 (μm / s), but ALH increased from 6.61 (μm) to 7.76 (μm), which still indicated that the curvilinearity of sperm motility was significantly improved and the movement was activated. The linear motion-related parameters VSL, VAP, LIN, and STR all decreased to a certain extent, indicating that the linearity was weakened and the sperm tended to be in a super-activated movement state.

[0093] The specific embodiments of the present invention are described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modification and substitution of the present invention are also within the scope of the present invention. Therefore, the equalization and modification made without departing from the spirit and scope of the present invention should be included in the scope of the present invention.

Claims

1. Application of IκBα phosphorylation inhibitor Bay117082 in the preparation of drugs for the treatment of asthenospermia.

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