CN115192709A

CN115192709A - Use of an inhibitor of O-GlcNAc glycosyltransferase for the manufacture of a medicament for the inhibition of spermatogenesis

Info

Publication number: CN115192709A
Application number: CN202210519206.XA
Authority: CN
Inventors: 李恩中; 李思强; 李云; 谌馥佳; 杨玉荣; 朱志坚; 王天阳; 吴科谕; 林梦珂
Original assignee: Huanghuai University
Current assignee: Huanghuai University
Priority date: 2022-05-12
Filing date: 2022-05-12
Publication date: 2022-10-18
Anticipated expiration: 2042-05-12
Also published as: CN115192709B

Abstract

The invention discloses application of an O-GlcNAc glycosyltransferase inhibitor in preparing a medicament for inhibiting spermatogenesis, belonging to the technical field of medicaments. The O-GlcNAc glycosylation modification of the testis protein of the mouse with abnormal spermatogenesis is reduced due to the cryptorchidism operation, after resveratrol is injected, the O-GlcNAc glycosylation level of the testis protein can be recovered to a certain level, and meanwhile, the spermatogenesis can also be recovered to a certain amount. However, when resveratrol and alloxan were injected simultaneously, alloxan inhibited the level of O-GlcNAc glycosylation of the testicular protein in resveratrol-treated cryptorchidism mice, thereby further inhibiting spermatogenesis in resveratrol-treated cryptorchidism mice. Therefore, the O-GlcNAc glycosyltransferase inhibitor can effectively inhibit the generation of sperms, has clear target and obvious effect of the inhibition effect, and can be applied to medicines for male contraception and contraception.

Description

Use of an inhibitor of O-GlcNAc glycosyltransferase for the manufacture of a medicament for the inhibition of spermatogenesis

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to application of an O-GlcNAc glycosyltransferase inhibitor in preparation of a medicine for inhibiting spermatogenesis.

Background

The O-GlcNAc modification is accomplished under a pair of reversible cycle enzyme modifications, in which O-GlcNAc glycosyltransferase (OGT, 110 kDa) is responsible for the transfer of GlcNAc to the Thr or Ser residue of the protein, while N-acetylglucosamine hydrolase (OGA, 130 kDa) participates in the reverse reaction, which complement each other to maintain a dynamic cycle. The dynamic equilibrium of the modification of O-GlcNAc glycosylation is crucial to maintain the normal physiological function of the body, and the disruption of the dynamic equilibrium will lead to physiological abnormality or even pathological disease of the body.

Although various methods of male birth control have been developed, their comfort and safety have hindered the development of male birth control. For example, vas deferens sterilization still faces problems of reproducibility, attached tea stasis, anti-sperm antibody formation, etc. Hormonal contraceptive drugs affect normal hormone metabolism in the body. The chemically synthesized oral contraceptive has still considerable side effects on human bodies. Spermatogenesis is a complex and precisely regulated process, once abnormality occurs, symptoms such as azoospermia, asthenospermia and oligospermia can be caused, and male sterility can be caused. At present, developing a safe and efficient medicine for inhibiting spermatogenesis aiming at the spermatogenesis process becomes a research hotspot of domestic and foreign researchers.

Disclosure of Invention

The invention aims to provide application of an O-GlcNAc glycosyltransferase inhibitor in preparing a medicine for inhibiting spermatogenesis and a medicine for inhibiting spermatogenesis.

The invention is realized by the following technical scheme:

in a first aspect, the present invention provides the use of an inhibitor of an O-GlcNAc glycosyltransferase in the manufacture of a medicament for the inhibition of spermatogenesis.

Further, in a preferred embodiment of the present invention, the above-mentioned inhibitor of O-GlcNAc glycosyltransferase comprises Alloxan (Alloxan). The English name O- (2-acetamido-2-deoxy-D-glucopyranosylidene) amino-N-phenylcarbanate, and the molecular formula is C ₄ H ₂ N ₂ O ₄ ·H ₂ O, molecular weight 160.08, structure shown below:

2,4,5,6(1H,3H)-Pyrimidinetetrone

in a second aspect, the present invention provides the use of an inhibitor of an O-GlcNAc glycosyltransferase in the manufacture of a medicament for male contraception.

In a third aspect, the present invention provides a medicament for inhibiting spermatogenesis, comprising an inhibitor of O-GlcNAc glycosyltransferase and a pharmaceutically acceptable excipient.

Further, in a preferred embodiment of the present invention, the inhibitor comprises alloxan.

Further, in a preferred embodiment of the present invention, the concentration of alloxan to be administered is 25 to 75. Mu.g/g.

Further, in a preferred embodiment of the present invention, the dosage form of the above-mentioned medicament includes injection, tablet, pill, capsule, granule, emulsion, solution or suspension.

Further, in a preferred embodiment of the present invention, the above-mentioned drugs are administered orally, intravenously or intramuscularly.

Compared with the prior art, the invention at least has the following technical effects:

compared with normal mice, the O-GlcNAc glycosylation modification of the testicular protein of the mouse with abnormal spermatogenesis caused by the cryptorchidism operation is reduced, and the O-GlcNAc glycosylation level of the testicular protein is obviously recovered along with the recovery of sperms after the resveratrol treatment. Meanwhile, an O-GlcNAc glycosyltransferase inhibitor (such as alloxan) can effectively inhibit the O-GlcNAc glycosylation level of the testis protein of the resveratrol-treated cryptorchidism mouse, thereby further inhibiting the spermatogenesis of the resveratrol-treated cryptorchidism mouse.

Because of the large number of sperm in normal experimental mice (usually 10) ⁸ one/mL), which poses great difficulty in counting experiments. In this regard, the inventors constructed a cryptorchidism model, treated the cryptorchidism model with resveratrol to enable spermatogenesis, but controlled the number of spermatozoa to a smaller extent (typically 10) by exploiting the effect of resveratrol in promoting the O-GlcNAc glycosylation level of the testicular protein ⁷ one/mL) to facilitate statistical observation of the effect of the test drug on spermatogenesis in mice.

Experiments show that the O-GlcNAc glycosyltransferase inhibitor can effectively inhibit the generation of sperms, has clear target and obvious effect of the inhibition effect, and is expected to be applied to male birth control and contraception medicaments.

Drawings

FIG. 1 is a graph showing the effect of subcutaneous injection of Alloxan (Alloxan) on sperm count in mice in example 1 of the present invention.

FIG. 2 is a graph showing the effect of subcutaneous injection of Alloxan on mouse testicular tissue in example 1 of the present invention.

FIG. 3 is a graph showing the effect of subcutaneous injection of Alloxan on mouse hormones in example 1 of the present invention: panel a shows the measurement of MDA levels in serum by ELISA; FIG. B shows the measurement of T level in serum by ELISA; FIG. C shows measurement of serum FSH level by ELISA; FIG. D shows the level of E in serum measured by ELISA; FIG. E shows the measurement of GnRH levels in serum by ELISA; FIG. F shows the measurement of LH levels in serum by ELISA; FIG. G shows the measurement of T-SOD level in serum by ELISA method.

FIG. 4 shows the expression level of O-GlcNAc glycosylation in example 2 of the present invention, in lane 1, marker, in

lanes

2, 5 and 8, CR, in

lanes

3, 6 and 9, CO, and in

lanes

4, 7 and 10, CRA. The primary antibody is O-linked N-acetylglucosamine, and is diluted according to the ratio of 1; the secondary antibody was coat Anti-Mouse IgG (H + L) GRP diluted as 1.

FIG. 5 shows the level of OGT expression in example 2 of the present invention, marker in lane 1, CR in

lanes

2, 5 and 8, CO in

lanes

3, 6 and 9, and CRA in

lanes

4, 7 and 10; the primary antibody is Anti-OGT rabbit polyclonal antibody, diluted according to 1; the secondary antibody was HRP-conjugated coat Anti-Rabbit IgG diluted in 1.

FIG. 6 shows the level of OGA expression in example 2 of the present invention, marker in lane 1, CR in

lanes

2, 5 and 8, CO in

lanes

3, 6 and 9, and CRA in

lanes

4, 7 and 10; primary antibody is MGEA5 Ab, diluted as 1; the secondary antibody was HRP-conjugated Goat Anti-Rabbit IgG diluted as 1.

Detailed Description

Embodiments of the present invention will be described in detail with reference to the following examples, but those skilled in the art will understand that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the present invention, and that the specific conditions not specified in the examples are carried out according to conventional conditions or conditions suggested by the manufacturer, and that the reagents or equipment used are not specified by the manufacturer, and are all conventional products available through commercial purchase.

The following describes the embodiments of the present invention in detail. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are given by way of illustration and explanation only, not limitation.

Example 1

Construction of alloxan as O-GlcNAc glycosyltransferase inhibitor in preparation of cryptorchidism mouse model for inhibiting Resveratrol (RSV) treatment

In order to examine the inhibitory effect of Alloxan as an O-GlcNAc glycosyltransferase inhibitor, the inventors constructed a cryptorchidism mouse model, performed resveratrol administration to intervene in mouse spermatogenesis, and simultaneously intraperitoneally injected Alloxan (Alloxan) to observe the effect of Alloxan (Alloxan) as an O-GlcNAc glycosyltransferase inhibitor on resveratrol O-GlcNAc glycosylation.

(1) Molding: cryptorchidism operation is performed on normal mice

When the cryptorchidism model is constructed, an incision of about 1 cm needs to be made in the outer skin of the abdominal cavity of a mouse, then the muscle layer is opened, the fat heads on two sides are found and then pulled out of the body of the mouse, the testicle leading belts are broken, then the fat heads are bound together, and finally the muscle layer and the outer skin are sequentially sutured. The mouse testis exists in the abdominal cavity, and the method can be used for constructing the cryptorchidism azoospermia model.

(2) Animal grouping and administration

Healthy Balb/C mice, male, 2-3 weeks old, purchased from Zhengzhou university institute for pharmaceutical research. Mice were randomly divided into 3 groups, respectively: cryptorchidism control group (CO group), resveratrol control group (CR group) and alloxan experimental group (CRA group). The CO group treatment method is intragastric administration of soybean oil every day, the CR group treatment method is intragastric administration of 200mg/kg resveratrol every day, and the CRA group treatment method is intraabdominal injection of 50mg/kg alloxan (using normal saline as a solvent) every other day and intragastric administration of 200mg/kg resveratrol every day. The above experimental period was 35d, and mice were weighed and recorded once a week from the first day of dosing.

(3) Collecting and processing a sample:

after 35d, mice were bled from the eyeball followed by cervical drainage to sacrifice the mice, bilateral testes were removed and recorded by weighing. And (3) freezing one testis in liquid nitrogen at a medium speed, transferring to an ultralow temperature refrigerator for storage, and placing the other testis in a stationary liquid for subsequent H & E staining analysis.

When taking the testis, collecting epididymis beside the testis, and completely cutting in an EP tube containing warm PBS. Counting the number of sperms by a Malan sperm counting instrument, and analyzing to find no sperms in the CO group, recover the sperm production capability of the CR group mice, and ensure that the number of the sperms is 6.67 multiplied by 10 ⁷ Mice in CRA group had spermatozoa but in significantly lower numbers than in CR group at individual/mL, the number of spermatozoa was 3.12X 10 ⁴ One per mL. As shown in fig. 1, it is thus demonstrated that the number of mouse sperm was significantly reduced after alloxan treatment.

(4) Experimental method and result for mouse testis histopathological morphology research

Paraffin embedding, slicing and baking:

soaking mouse testis in 50% ethanol for 30min, and dehydrating with gradient ethanol, wherein the dehydration step comprises soaking mouse testis in 70% ethanol for 10min → 80% ethanol for 10min → 95% ethanol for 10min → 100% ethanol for I for 10min → 100% ethanol for II for 10min;

xylene is transparent: alcohol + xylene (volume ratio of alcohol to xylene 3: 1) (10 min) → alcohol + xylene 2:1 (10 min), paraffin was melted simultaneously (two paraffin waxes were placed in two containers, one melted and immediately placed in the oven, the other melted for approximately ten minutes, after which the two containers were mixed, which is old wax new wax 1) → alcohol + xylene 1:1 (10 min) → xylene I (10 min) → xylene II (10 min).

Paraffin embedding: xylene + paraffin 3:1 (20 min) → xylene + paraffin 2:1 (20 min) → xylene + paraffin 1:1 (20 min) → paraffin (30 min) → embedding (20 min).

Tissue section: preparing a water bath of the glyceroprotein and 37 ℃, cutting the tissue into 5 μm sections with a microtome, placing the sections in the water bath for about three minutes, smearing the glyceroprotein on the slide, placing the sections on the slide with the glyceroprotein side of the slide, and finally placing the slide in an oven at 60 ℃ and baking for 1 hour.

HE staining: xylene I dewaxing (5 min) → xylene II dewaxing (5 min) → 100% alcohol (5 min) → 95% alcohol (5 min) → 80% alcohol (5 min) → 70% alcohol (5 min) → water washing (5 min) → eosin (2-7 s) → water rinsing, observation slice color → hematoxylin (2-7 s) → water rinsing, observation slice color, and photographing sampling → hydrochloric acid alcohol decoloring (2-7 s) → water rinsing, observation slice color → 100 alcohol (2 min) → xylene I (2 min) → xylene II (2 min) → neutral gum sealing, observation slice, and photographing sampling.

Experimental results and analysis:

as shown in fig. 2, testis was sectioned-stained and observed under a nikon microscope. In the cryptorchidism model, the testis of a mouse is sutured in the abdominal cavity, the temperature in the abdominal cavity is higher than that of the scrotum, and the testis, the spermatocyte and the spermatogonial cells at all levels are obviously not suitable for the development of the testis, the spermatocyte and the spermatocyte, so that the endogenous spermatocyte of a CO group can be seen in a slice and removed, a cavity is formed in the seminiferous tubule, the spermatocyte is not seen, and cell fragments are obvious; however, the CR group showed that after the spermatogonial stem cells were cleared, spermatocytes and spermatogonial cells at all levels began to appear and line up neatly after administering 200mg/kg resveratrol, with clearly visible sperm present, and no cavity in the seminiferous tubules, thus resveratrol was shown to promote spermatogenesis; CRA group showed that after intraperitoneal injection of alloxan, spermatocyte in testis was significantly reduced, and seminal tubule had partial cavity and small amount of cell debris, compared with CR group. This suggests that alloxan significantly destroys germ cells in the seminiferous tubules, thereby inhibiting the spermatogenesis process.

(5) Analysis of serum hormone levels in mice:

the obtained blood is taken off line at a low speed after being kept overnight at 4 ℃, and then the levels of various hormones such as Malondialdehyde (MDA), testosterone (T), follicle Stimulating Hormone (FSH), estrogen (E), gonadotropin releasing hormone (GnRH), luteinizing Hormone (LH), total superoxide dismutase (T-SOD) and the like in the blood serum are detected strictly according to the specification of a gene American ELISA kit.

Experimental results and analysis:

as shown in fig. 3, the inventors measured the levels of hormones such as Malondialdehyde (MDA), testosterone (T), follicle Stimulating Hormone (FSH), estrogen (E), gonadotropin-releasing hormone (GnRH), luteinizing Hormone (LH), total superoxide dismutase (T-SOD), etc. in serum by ELISA kit.

Fig. 3A shows the MDA level in serum, MDA represents the degree of cellular oxidative damage, and the MDA level in the CO group mice is significantly lower than that in the CR group mice (P < 0.01), which is statistically significant.

Fig. 3B shows the level of T in serum, which is positively correlated with spermatogenic potential, and the level of T in CR group mice was significantly higher than in CO and CRA groups (P < 0.01).

Fig. 3C and 3D show that the FSH and E levels in serum, which are too high to significantly inhibit T content, are negatively correlated with spermatogenic ability, and the FSH and E levels in CO group mice are significantly higher than those in CR group (P < 0.01), while the FSH and E levels in CR group mice are significantly lower than those in CRA group (P < 0.01), and the E levels in CR group mice are significantly lower than those in CRA group (P < 0.05).

Fig. 3E shows GnRH levels in serum, with increased GnRH levels favoring spermatogenesis, with GnRH levels significantly lower in CO group mice than in CR group (P < 0.01), and GnRH levels significantly higher in CR group mice than in CRA group (P < 0.05).

Fig. 3F shows the LH level in serum, which promoted testicular support cell development and further testosterone secretion, and the LH level was significantly lower in CO mice than in CR (P < 0.01).

FIG. 3G shows the level of T-SOD in serum, which is characteristic of the antioxidant capacity of cells, and the LH level in mice in CO group is significantly higher than that in CR group (P < 0.01).

The rest of the unspecified representations showed no significant differences and were not statistically significant (P > 0.05). The results show that the testis of the cryptorchidism mouse has oxidative damage, the sperm generation process is obstructed, and the sperm is recovered to a certain extent after intervention of resveratrol. The administration of alloxan caused oxidative damage to the testes of mice, which indicates that alloxan caused sperm disorders by oxidative damage to mice.

Example 2

The detection method, the result and the analysis of the change of the protein level of resveratrol O-GlcNAc glycosylation by alloxan comprise the following steps:

the inventors further performed Western-Blot experiments to examine the effect of Alloxan (Alloxan) as an inhibitor of O-GlcNAc glycosyltransferase on the protein levels of resveratrol for O-GlcNAc glycosylation.

(1) Tissue treatment:

taking one testis from each group of the ultralow temperature refrigerator, and weighing the testis: RIPA lysate volume = 1. Transferring the supernatant obtained by the above treatment to an environment with the temperature of-20 ℃ for subpackage and preservation.

(2) Drawing a standard curve:

using the BCA kit, the procedures were performed in accordance with the instructions of the kit of assist in san Biotech, inc., as shown in FIG. 4.

(3) Correcting internal parameters:

SDS-PAGE electrophoresis, membrane conversion and immunoreaction are sequentially carried out, and finally, an image is observed and stored under a ChemiDoc MP full-function imaging system, as shown in figure 5.

(4) And (3) analyzing an experimental result:

(4.1) O-GlcNAc glycosylation expression level analysis:

as shown in FIG. 4, O-GlcNAc glycosylation was most expressed in mice testis protein in CR, followed by CRA and then CO. The expression level of O-GlcNAc glycosylation of the mouse testicular protein processed by the cryptorchidism is reduced, and the expression level of O-GlcNAc glycosylation of the testicular protein is obviously increased after the intervention model of the resveratrol drug is passed. Meanwhile, the result also indicates that the alloxan inhibits the spermatogenesis of the mouse by down-regulating the expression of O-GlcNAc glycosylation of the testosterone protein.

(4.2) analysis of OGT expression Activity:

as shown in FIG. 5, O-GlcNAc glycosyltransferase (OGT), which has a molecular weight of approximately 110kDa, acts to transfer N-acetylglucosamine to proteins. CR had the highest OGT expression activity, followed by CO, then CRA. This suggests that alloxan acts as an inhibitor of OGT and inhibits spermatogenesis by inhibiting OGT expression to down-regulate O-GlcNAc glycosylation.

(4.32) OGA expression level analysis:

n-acetylglucosaminidase, OGA, has a molecular weight of approximately 130kDa and transfers GlcNAc to proteins. As shown in fig. 6, there was no significant change in OGA expression in the three groups of mice testis, indicating that alloxan had no significant effect on OGA expression, further indicating that alloxan inhibits spermatogenesis by inhibiting OGT expression to down-regulate O-GlcNAc glycosylation.

Example 3

Construction of alloxan as O-GlcNAc glycosyltransferase inhibitor in preparation of normal mouse inhibition model

To examine the inhibitory effect of Alloxan as an inhibitor of O-GlcNAc glycosyltransferase, the inventors injected Alloxan (Alloxan) intraperitoneally into normal mice and observed the effect of Alloxan (Alloxan) as an inhibitor of O-GlcNAc glycosyltransferase on O-GlcNAc glycosylation.

(1) Animal grouping and administration

Healthy Balb/C mice, male, 2-3 weeks old, were purchased from Zhengzhou university institute for pharmaceuticals. Mice were randomly divided into 3 groups, respectively: control group (BO group), and alloxan experimental group (BRA group). BO group treatment method is daily intragastric administration of soybean oil, and BRA group treatment method is every other day intraperitoneal injection of 50mg/kg alloxan (using physiological saline as solvent). The above experimental period was 35d, and mice were weighed and recorded once a week from the first day of dosing.

(3) Collecting and processing a sample:

after 35d, mouse eyedrops were bled, followed by cervical draining to sacrifice mice, bilateral testicles were removed and the weight records were weighed. When taking the testis, collecting epididymis beside the testis, and completely cutting in an EP tube containing warm PBS. The sperm count is counted by a Malan sperm counting instrument, and the analysis shows that the number of the BO group sperm is 12.13 multiplied by 10 ⁷ one/mL, BRA group mice had sperm production, but the number was significantly lower than BO group, the sperm number was 8.12 × 10 ⁵ one/mL. This demonstrates that the number of sperm in mice is significantly reduced after treatment with alloxan.

In summary, compared to normal mice, the O-GlcNAc glycosylation modification of the testicular protein of the spermatogenic abnormal mice by the cryptorchidism surgery is reduced, and after resveratrol is injected, the O-GlcNAc glycosylation level of the testicular protein is restored to a certain level, and meanwhile, spermatogenesis is also restored to a certain amount. However, when resveratrol and alloxan were injected simultaneously, alloxan inhibited the O-GlcNAc glycosylation level of the testis protein in resveratrol-treated cryptorchidism mice, thereby further inhibiting spermatogenesis in resveratrol-treated cryptorchidism mice. Therefore, the O-GlcNAc glycosyltransferase inhibitor can effectively inhibit the generation of sperms, has clear target and obvious effect, and can be applied to male birth control and contraception medicaments.

Finally, it should be noted that: the above description is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

Use of an inhibitor of an o-GlcNAc glycosyltransferase in the manufacture of a medicament for inhibiting spermatogenesis.
2. Use according to claim 1, wherein the inhibitor of an O-GlcNAc glycosyltransferase comprises alloxan.
Use of an inhibitor of an o-GlcNAc glycosyltransferase in the manufacture of a medicament for male contraception.
4. A medicament for inhibiting spermatogenesis, said medicament comprising an inhibitor of O-GlcNAc glycosyltransferase and a pharmaceutically acceptable excipient.
5. The medicament of claim 4, wherein the inhibitor comprises alloxan.
6. The medicament according to claim 5, wherein the alloxan is administered in a concentration of 25 to 75 μ g/g.
7. The medicament of claim 4, wherein the medicament is in a dosage form selected from the group consisting of injection, tablet, pill, capsule, granule, emulsion, solution and suspension.
8. The medicament of claim 4, wherein the administration of the medicament comprises oral, intravenous or intramuscular injection.