CN118286201A

CN118286201A - Application of dihydromyricetin in preparation of medicament for treating spermatogenic dysfunction

Info

Publication number: CN118286201A
Application number: CN202410234578.7A
Authority: CN
Inventors: 余建强; 海冬梅; 杨佳美; 马琳; 刘宁; 兰小兵; 杜娟
Original assignee: Ningxia Medical University
Current assignee: Ningxia Medical University
Filing date: 2024-03-01
Publication date: 2024-07-05

Abstract

The application provides an application of dihydromyricetin in preparing a medicament for treating spermatogenic dysfunction. Experimental results show that dihydromyricetin can remarkably improve the number and activity of sperms; can obviously inhibit the TGF-beta 3/p38 MAPK signal pathway in testis, and has a protective effect on mice with spermatogenic dysfunction.

Description

Application of dihydromyricetin in preparation of medicament for treating spermatogenic dysfunction

Technical Field

The application belongs to the field of reproductive system diseases, and particularly provides application of dihydromyricetin in preparation of a medicament for treating spermatogenic dysfunction.

Background

The incidence of infertility increases year by year with environmental pollution, pressure of life, delayed birth age, etc. At present, the infertility rate of China is up to about 12.5% -15%, namely, 1 pair of couples has infertility problem in 8 pairs of couples. Of the factors responsible for infertility, male and female factors account for 50% respectively. The etiology of male infertility is: semen abnormalities (57%), sexual dysfunction (23%), prostatitis (15%), varicoceles (5%). Dysspermia is the root cause of semen abnormalities. The dysspermia of the testes is complex in etiology and often not caused by a single factor, which is mainly related to medicine, environment, genetics, endocrine, infection, disease and lifestyle. At present, drugs are mainly administered for treating male spermatogenic dysfunction clinically, and the drugs are various in variety, but the curative effect is poor and the curative effect cannot be ensured. Therefore, the research and development of safe and effective medicaments for treating testicle dysspermia has great social value and research significance.

Modern researches have shown that the medicinal plants of wolfberry fruit, dodder seed, cistanche salsa, epimedium herb, cynomorium songaricum and the like have the protection effect on male spermatogenic dysfunction, and further researches prove that the medicinal active ingredients are mainly flavonoid compounds. Dihydromyricetin (dihydromyricetin, DHM) belongs to main flavonoid compounds in vine tea, and has various pharmacological effects of anti-inflammatory, antioxidant, blood sugar reducing, blood lipid regulating, etc. Research shows that DHM has protective effect on kidney injury; the DHM can obviously improve the kidney oxidative damage of rats with abnormal glucose tolerance, and has better protection effect on early kidney damage; DHM can ameliorate cisplatin-induced impairment of renal function. And researches show that the DHM has close relation with TGF-beta, and the DHM can regulate the TGF-beta to play a role in protecting other diseases. Therefore, the compound has extremely high potential value and social significance in developing the compound into a medicament for treating spermatogenic dysfunction.

Disclosure of Invention

The application adopts cyclophosphamide (cyclophosphamide, CTX) to induce a mouse model with spermatogenic dysfunction, systematically discusses the effect and the primary mechanism of improving male spermatogenic dysfunction by using the aspects of spermatogenic function, tissue morphology and the like, adopts acrolein (Acrolein, ACR) to induce a TM4 supporting cell damage model, deeply discusses the relationship between the protection effect of the DHM on the mouse with spermatogenic dysfunction and a blood-testosterone barrier TGF-beta 3/p38MAPK signal path by using a TGF-beta 3 specific inhibitor (S-7701), and provides a new approach for developing novel medicaments and medicament targets for treating male spermatogenic dysfunction.

In one aspect, the application provides the use of dihydromyricetin in the preparation of a medicament for treating spermatogenic dysfunction.

Further, dihydromyricetin in the medicine is the only active ingredient.

Further, the spermatogenic dysfunction is a loss of spermatogenic function.

Further, the spermatogenic dysfunction is a reduced sperm count and motility and a sperm malformation.

Further, the medicament is in an injection or oral dosage form.

Further, the medicament is in an oral dosage form.

In another aspect, the invention provides a non-therapeutic method of enhancing spermatogenic function and sperm motility comprising administering dihydromyricetin.

Further, the single application dose of the dihydromyricetin is 37.5-150mg/kg.

Further, the single application dose of the dihydromyricetin is 75-150mg/kg.

Further, the single application dose of dihydromyricetin is 150mg/kg.

On the other hand, the application provides application of dihydromyricetin in preparing medicines for inhibiting expression of TGF-beta 3, p38 MAPK and p-p38 MAPK proteins in a TGF-beta 3/p38 MAPK pathway in testis.

In another aspect, the application provides an application of dihydromyricetin in preparing a medicament for increasing expression levels of blood-testosterone barrier related proteins N-cadherin, occludin, claudin-11 and Cx43 proteins in testis tissue.

The structural formula of the dihydromyricetin is shown as a formula (1):

The medicament of the application can also contain various food/pharmaceutically acceptable auxiliary materials according to the requirement of the form/dosage form, including but not limited to coating materials, solvents, solubilizers, binders, stabilizers, antioxidants, pH regulators and flavoring agents, wherein the auxiliary materials can be selected by one skilled in the art according to the common general knowledge of food/pharmacy.

Non-therapeutic methods in the present application include, but are not limited to, scientific experimental methods, non-therapeutic purpose healthcare methods, and the like.

Advantageous effects

The invention proves for the first time that the dihydromyricetin has the function of treating mice with spermatogenic dysfunction, can be used for preparing the therapeutic drugs with spermatogenic dysfunction, and provides a new choice for the drugs. Specifically, dihydromyricetin can remarkably improve the number and activity of sperms; the dihydromyricetin can inhibit TGF-beta 3, p38 MAPK and p-p38 MAPK protein expression in TGF-beta 3/p38 MAPK pathway in testis, raise blood-testosterone barrier related protein N-cadherin, occludin, claudin-11 and Cx43 protein expression level in testis tissue, and improve spermatogenic dysfunction.

Drawings

FIG. 1 influence of DHM on sperm parameters of seminiferous dysfunctional miceN=10); part A: sperm count (billions); part B: sperm viability (%); c: sperm motility (%); ^*** P < 0.001 compared to the Control group and ^#P＜0.05,^##P＜0.01,^### P < 0.001 compared to the CTX group.

Fig. 2. Influence of dhm on testis tissue morphology in seminiferous dysfunctional mice.

Fig. 3 effect of dhm on blood-testosterone barrier in seminiferous dysfunctional mice.

FIG. 4 influence of DHM on blood-testosterone barrier-related protein expression in seminiferous dysfunctional miceN=6); part A: representative Western blot images of N-cadherein, 0ccludin, claudin-11, and Cx43 in different groups of testis tissues; part B: protein expression statistics of N-cadherin, occludin, claudin-11 and Cx 43; ^**P＜0.01,^*** P < 0.001 means comparing with the Control group, ^#P＜0.05,^### P < 0.001 means comparing with the CTX group.

FIG. 5 influence of DHM on expression of TGF-beta 3, p38 MAPK and p-p38 MAPK proteins in seminiferous dysfunctional miceN=6); part A: representative western blot images of TGF- β3, p38 MAPK, and p-p38 MAPK in different groups of testicular tissue; part B: protein expression statistics of TGF-beta 3, p38 MAPK and p-p38 MAPK; ^** P < 0.01 indicates comparison with the Control group, ^# P < 0.05 indicates comparison with the CTX group.

FIG. 6 effects of DHM on expression of Occludin and Claudin-11 proteins in seminiferous dysfunctional mice testisN=6); part A: representative fluorescence plots of Occludin and Claudin-11 protein expression in testis tissue; part B: a positive cell number statistical chart; ^*** P < 0.001 means comparing with the Control group, ^##P＜0.01,^### P < 0.001 means comparing with the CTX group.

FIG. 7 effect of different concentrations of ACR on the viability of TM 4-bearing cellsN=3); ^**P＜0.01,^*** P < 0.001 represents a comparison with the Control group.

FIG. 8 effects of different concentrations of DHM on the viability of TM 4-bearing cellsN=3); ^*P＜0.05,^** P < 0.01 indicates comparison with the Control group.

FIG. 9 effect of different concentrations of DHM on survival of ACR-induced TM 4-supporting cell injury modeln＝3)。

FIG. 10. Influence of DHM on ACR-induced TM4 supporting cell damage model cytoskeleton.

FIG. 11 effect of different concentrations of S-7701 on survival of ACR-induced TM4 support cell injury modelN=3); ^*** P < 0.00 indicates comparison with the Control group, ^# P < 0.05 indicates comparison with the ACR group.

FIG. 12 effect of DHM and S-7701 on survival of ACR-induced TM 4-supported cell injury modelN=3); ^*** P < 0.001 indicates comparison with the Control group, ^# P < 0.05 indicates comparison with the ACR group.

FIG. 13 influence of DHM and S-7701 on apoptosis in ACR-induced TM 4-supported cell injury modelN=3); upper part: apoptosis images; the lower part: a statistical graph of apoptosis rate; ^*** P < 0.001 indicates comparison with the Control group, ^### P < 0.001 indicates comparison with the ACR group.

FIG. 14 influence of DHM and S-7701 on ACR-induced expression level of TGF-beta 3/p38 MAPK signaling pathway-related protein in TM4 supporting cellsN=6); part A: representative Western blot images of TGF-. Beta.3, p38MAPK, and p-p38MAPK in cells were supported by different groups of TM 4; part B: protein expression statistics of TGF-beta 3, p38MAPK and p-p38 MAPK; ^*P＜0.05,^*** P < 0.001 indicates comparison with the Control group, ^# P < 0.05 indicates comparison with the CTX group.

FIG. 15 effects of DHM and S-7701 on ACR-induced expression levels of protein associated with the TM4 support cell Claudin-11 signaling pathwayN=6); part A: representative Western blot images of Claudin-11 in different groups of TM4 supporting cells; part B: protein expression statistics of Claudin-11; ^*** P < 0.001 indicates comparison with the Control group, ^#P＜0.05,^## P < 0.01 indicates comparison with the CTX group.

Detailed Description

The present invention will be described in further detail with reference to examples, in which dihydromyricetin is used as a compound represented by the aforementioned formula (1), which are commercially available. The single application dose of the dihydromyricetin is 37.5mg/kg, 75mg/kg and 150mg/kg of mice, and the dosage form of the medicament is an oral dosage form.

Example 1 Experimental materials

Animal treatment

120 Male ICR mice, 18-22g weight, purchased from the university of Ningxia medical laboratory animal center, laboratory animal license number: SCXK (Ning) 2020-0001, the raising environment is at 22-24 ℃, the humidity is 45% -65%, the light and shade period is 12h, and the mice can drink and eat freely.

Experimental medicine and instrument

The main drugs and reagents that will be involved in this experiment include: dihydromyricetin (purchased from Shanghai leaf biology company, purity not less than 98%); levocarnitine (available from ALFASIGMA company in the united states); cyclophosphamide (available from Jiangsu Shengdi medicine Co., ltd.); saline (available from Tianjin metallocene chemical reagent plant); EZ-Link Sulfo-NHS-LC biotin (available from Thermo FISHER SCIENTIFIC Co.); alexa Fluor ^TM 568 conjugate (available from beijing eunikang); GAPDH goat anti-rabbit, anti-rabbit IgG antibodies, FITC goat anti-rabbit, p38 MAPK, N-cadherin, and Connexin 43 (available from proteintech company, usa); rabbit anti-TGF-. Beta.3, claudin-11 and Occludin (available from abcam, UK); rabbit anti-p 38 MAPK (available from CELL SIGNALING company, usa); DAPI (purchased from beijing china fir bridge biotechnology company); whole protein extraction kit (purchased from south kyoki biosome); whole protein quantification kit (purchased from south tokyo biology company); skim milk powder (purchased from beijing soybao technologies); PVDF membranes (available from Millipore corporation, usa); ECL luminescent solution (available from Advansta company); marker (10-180 kDa) (from Thermo filter); animal testis tissue fixative (available from Servicebio limited); FITC-phalloidin (available from MESGEM company); apoptosis assay kit (purchased from bi yun tian).

The main instruments that will be involved in this experiment include: sperm analyzer (purchased from soning songjinlun corporation); microplate reader (commercially available from Thermo scientific); an electric thermostatic water bath (purchased from Shanghai precision laboratory equipment Co., ltd.); full-automatic sample cryomill (purchased from Shanghai Net communication technologies Co., ltd.); SDS-PAGE electrophoresis apparatus (from Bio-RAD Co., U.S.A.); visible fluorescence imager (available from Auzre Biosystems, usa); electrothermal constant temperature forced air drying oven (purchased from Shanghai Lang laboratory equipment Co., ltd.); fluorescence microscopy (OLYMPUS); optical microscopy (OLYMPUS); electrophoresis apparatus, electrotransport apparatus (Powerpac basic, bio-Rad Co., U.S.A.); high-speed low-temperature centrifuges (Eppendorf, germany); cell incubator (available from Thermo FISHER SCIENTIFIC company); ultra clean bench (available from air technologies, inc. Of safe, su zhou); flow assay apoptosis detector (available from Sysmex-partec Co.).

Experimental grouping and administration

Animal level: after 3 days of adaptive feeding in clean-grade feeding room of male adult ICR mice weighing 24-28g, they were equally divided into Control group, CTX group, levocarnitine (Levocarnitine, LC) group and DHM 37.5mg/kg, 75mg/kg, 150mg/kg group according to the body weight gradient sampling method. Except normal feeding, the animals in each group are subjected to intraperitoneal injection of CTX 120mg/kg once a week, and a male mouse spermatogenic dysfunction model is constructed. LC (3.03 mL/kg) was infused in the stomach for the LC group, and DHM 37.5mg/kg, 75mg/kg, 150mg/kg was infused in the stomach for the dihydromyricetin (37.5 mg/kg, 75mg/kg, 150 mg/kg) groups, 20 each. The LC group and the DHM group are continuously administered for 2 weeks at a fixed time point of morning, and the control group and the model group mice are respectively administered with physiological saline with the same volume; intragastric administration at an administration volume of 0.1ml/10 g; the rest of the experimental conditions were the same.

Cell level: experiments can be performed with a cell density of about 80%. The cell is digested with pancreatin and then given in groups, and the specific group administration is decided according to the experimental contents (see experimental procedures (seven) to (nine) for details). The cells were incubated in a cell incubator at 37℃with 5% CO ₂ after manipulation.

Example 2 organ coefficient measurement

The experimental method comprises the following steps:

The mice of each group were weighed and recorded 24h after the last dose. After the experiment is finished, the mice are sacrificed, the abdomen of the mice is rapidly cut off by surgical scissors, the testes on the two sides of the mice are fully exposed, surrounding adipose tissues and connective tissues are removed, the testes, epididymis and seminal vesicle glands are taken out, and the weight of the mice is accurately weighed by an electronic balance after the surface blood is sucked and recorded.

Experimental results:

As a result, as shown in Table 1, the organ coefficients of the testis, epididymis and seminal vesicle glands were significantly decreased (P < 0.001) in the CTX group of mice compared with the Control group. The organ factor ratio of the reproductive organs of mice treated with DHM (75 mg/kg, 150 mg/kg) was significantly increased (P < 0.05, P < 0.001), and the LC treatment group also significantly increased the organ factor ratio of the reproductive organs (P < 0.001). The DHM is suggested to have a certain improvement effect on reproductive organs of mice with spermatogenic dysfunction.

TABLE 1 DHM influence on organ coefficients of reproductive organs of seminiferous dysfunctional micen＝10)

^*** P < 0.001 to Control group, ^#P＜0.05,^### P < 0.001 to CTX group

Example 3 sperm parameter determination

The experimental method comprises the following steps:

After the epididymis is taken out, the epididymis tail is cut off. Cutting epididymis into EP tube (preheated to 37 ℃ in advance) containing 2mL of physiological saline by using dissecting scissors, incubating at 37 ℃ for 5min, filtering with a 200-mesh nylon net to obtain a sperm stock solution, lightly blowing and mixing, diluting and mixing with physiological saline (sperm stock solution: physiological saline=1:4), uniformly mixing the obtained suspension, taking 10 mu L of the suspension on a sperm counting plate, and analyzing parameters such as sperm quantity, sperm activity, sperm motility and the like by using a sperm analyzer.

Experimental results:

Compared with the Control group, the number of sperms, the sperm viability and the sperm motility of the CTX group mice are obviously reduced (P is less than 0.001); compared with the CTX group, the sperm parameters of the CTX+LC group mice are significantly increased (P < 0.01, P < 0.001); the CTX+DHM 75mg/kg and CTX+DHM 150mg/kg groups showed significantly increased sperm count, viability and vigor (P < 0.05, P < 0.01, P < 0.001) compared to the CTX group, as shown in FIG. 1.

Changes in tissue morphology of testes

The experimental method comprises the following steps:

After the experiment, the mice were sacrificed, the abdomen of the mice was rapidly cut with surgical scissors, the bilateral testes were fully exposed, the surrounding adipose tissues and connective tissues were removed, the testes were taken out, immersed in animal testis tissue fixative solution, sent to Servicebio company for HE staining and white cutting.

Experimental results

Under an optical microscope, the testis tissue form of the Control group mouse is complete, interstitial cells are not shed, 5-7 layers of spermatogenic cells are visible in the seminiferous tubule, the cells are closely and orderly arranged, and a large number of mature sperms can be observed in the cavity (see figure 2). The CTX group interstitial cells fall off, the seminiferous tubules shrink, the arrangement of each grade of seminiferous cells is irregular, and the seminiferous cell layers are obviously reduced; LC group and DHM 150mg/kg testis tissue morphology are superior to CTX group, the seminiferous tubules are orderly arranged, the endophytic sperm cell layer of the seminiferous tubules is obviously increased, the arrangement is orderly and compact, and the number of the mature sperms in the cavity is more.

EXAMPLE 4 integrity of the blood-testosterone barrier

The experimental method comprises the following steps:

3 mice from the Control group, CTX group and CTX+DHM (150 mg/kg) group were anesthetized to expose testis tissue. 50 μl EZ-Link Sulfo-NHS-LC biotin was injected under the testicular leucorrhea at different sites, the testicular tissue was repositioned, and the wound was covered with cotton alcohol cloth. After 50min, mice were sacrificed and their testis tissues were embedded in Tissue Tek OCT for frozen sections. Sections (5 μm) were fixed in 4% paraformaldehyde for 40min, washed 3 times with PBST buffer, goat serum blocking solution blocked for 30min, and incubated with Alexa Fluor ^TM 568 conjugate at room temperature for 2.5h, blocked with DAPI-containing blocking agent, and observed under fluorescent microscope.

Experimental results:

as shown in fig. 3, control group mice BTB is complete; CTX group BTB integrity is compromised and biotin (red fluorescence) penetrates BTB into the seminiferous tubules; the DHM 150mg/kg group had a recovery of BTB integrity and a reduced amount of biotin into the seminiferous tubules compared to the CTX group.

Example 5 Western blot detection of expression levels of blood-testosterone Barrier and TGF-beta 3/p38 MAPK Signal pathway-related proteins in mouse testis

The experimental method comprises the following steps:

And (3) extracting total protein by using a Kaiyi total protein extraction kit, and determining the total protein concentration of a sample and calibrating the unified protein concentration by using a BCA protein content detection kit. SDS-polyacrylamide gel electrophoresis, and wet transfer was performed with PVDF membrane. After the film transfer is finished, the film is put into 5% skim milk powder sealing liquid for sealing for 2 hours. Blocking end incubation 5% nonfat milk powder diluted primary antibody p38MAPK(1∶1000)、N-cadherin(1∶1000)、Connexin 43(1∶1000)、TGF-β3(1∶2000)、Claudin-11(1∶1000)、Occludin(1∶1000) and p-p38 MAPK (1:1000), overnight at 4℃and membrane wash incubation of secondary antibody for 2h. The membrane was washed 3 times with 10min each with PBST. The exposure was performed by dropping a protein chemiluminescent agent (ECL). And calculating the gray value of the target protein by adopting Image J Image analysis software, and correcting errors by utilizing the corresponding internal reference gray value.

Experimental results:

expression level of blood-testosterone barrier-related protein

Compared with the Control group, the expression level of the blood-testosterone barrier related proteins N-cadherin, occludin, claudin-11 and Cx43 of the CTX group mice is obviously reduced (P is less than 0.01 and P is less than 0.001); after 150mg/kg of DHM treatment, the expression levels of N-cadherin, occludin, claudin-11 and Cx43 proteins were significantly increased (P < 0.05, P < 0.001) compared to the CTX group, see FIG. 4.

Expression level of TGF-beta 3/p38 MAPK signaling pathway-related proteins

Compared with the Control group, the expression level of TGF-beta 3, P38 MAPK and P-P38 MAPK proteins in testes of the CTX group mice is obviously increased (P is less than 0.01); after 150mg/kg of DHM treatment, the expression levels of TGF-beta 3, P38 MAPK and P-P38 MAPK proteins were significantly reduced (P < 0.05) compared to the CTX group, as shown in FIG. 5.

EXAMPLE 6 immunofluorescence detection of expression levels of Occludin and Claudin-11 proteins in mouse testis

The experimental method comprises the following steps:

immunofluorescence was used to detect expression of testis tissue Occludin and Claudin-11 protein. Taking a mouse testis paraffin slice, dewaxing, and soaking the slice with xylene for 3 times/5 min; soaking and washing the slices with absolute ethyl alcohol for 2 times/10 min; the sections were then rinsed with 95% ethanol 2 times/10 min and moistened in distilled water 2 times/5 min. Care should be taken to avoid drying out of the tissue sections during this process.

Antigen exposure. At room temperature, the sections were immersed in 1mM EDTA (0.372 g of EDTA (C ₁₀H₁₄N₂O₈Na₂·2H₂ O) was weighed and dissolved in 1L of distilled water when 1L was prepared), and the pH was adjusted to 6.0.). The slide immersed in EDTA was heated to near boiling by a microwave oven and held at 95-99℃for about 10 minutes. The slide was removed and allowed to cool naturally on a laboratory bench for 30 minutes. Rinse 3 times/5 min in distilled water and 3 times/5 min in PBS.

Immunostaining was performed. The samples were blocked with goat serum blocking solution in a 37 ℃ incubator for 60 minutes. Prior to the end of blocking, the primary antibody was diluted with PBS buffer as indicated in the instructions (Occludin:1:300, claudin-11:1:200). Removing the sealing liquid, and adding diluted primary antibody. Incubate overnight at 4 ℃. The primary antibody was thrown off and rinsed 5 times/5 min with PBS. 7. Fluorescein-labeled secondary antibody (1:50) was diluted in PBS and the samples were incubated with the diluted antibody in an incubator at 37℃for 1 hour in the absence of light. The sections were rinsed 5 times/5 min with PBS. The slide was covered after coating with DAPI-containing caplets. The samples were immediately observed under a fluorescence microscope with excitation light of the corresponding wavelength.

Experimental results:

The expression of Occludin and Claudin-11 proteins on the support cells in the testes was observed by immunofluorescence. As shown in FIG. 6, the expression levels of Occludin and Claudin-11 proteins on the supporting cells in testis were significantly reduced (P < 0.001) in the CTX group mice compared with the Control group; following 150mg/kg of DHM treatment, the expression levels of Occludin and Claudin-11 proteins were significantly increased (P < 0.01, P < 0.001) compared to the CTX group.

EXAMPLE 7 CCK-8 detection of TM 4-supporting cell viability

The experimental method comprises the following steps:

Observing that the cell density is about 80%, and then carrying out digestion and passage; preheating a complete culture medium, PBS and pancreatin to 37 ℃ in advance; taking out the culture dish needing passage, sucking and discarding the old culture medium, adding 2mL of PBS to wash the cells for 1 time, and discarding the PBS; 1mL of pancreatin is added for digestion until the cells completely fall off, and 3mL of complete culture medium is immediately added for stopping digestion; transferring into a 5mL sterile centrifuge tube, centrifuging in a 1000g centrifuge for 5min, discarding supernatant, adding 4mL complete culture medium, and blow-mixing; adjusting the concentration of the cell suspension to 1×10 ⁵ with a complete culture medium, plating the cell suspension on a 96-well plate, adding 100 μl of the cell suspension and 100 μl of the complete culture medium containing DHM (1 time of uniformly mixing the cell suspension every 3 wells, setting blank wells), adding 100 μl of PBS around the 96-well plate, and culturing in a cell culture box at 37deg.C with 5% CO ₂ for 12 hr; the old medium was aspirated, a Control group (Control group) was set, and serum-free medium containing ACR was added to each of the remaining wells, and the culture was continued in a cell incubator at 37 ℃ with 5% co ₂ for 3 hours. Old medium was aspirated and 100. Mu.L of diluted CCK-8 (CCK-8: serum free medium=1:9) was added to each well, incubated in an incubator for 1.5h, and absorbance (OD) was measured at 450 nm; the calculation formula is as follows: cell viability% = (experimental well OD value-blank well OD value)/(control well OD value-blank well OD value).

Experimental results:

Effect of different concentrations of acrolein on TM 4-supporting cell viability

Compared with the Control group, the survival rate of the TM4 supporting cells (P < 0.01 and P < 0.001) can be remarkably reduced after the TM4 supporting cells are molded for 3 hours by giving ACR series concentration, as shown in figure 7. The experiment finally determines that the ACR-induced TM4 support cell damage model concentration is 100. Mu.M.

Effect of Dihydromyricetin at different concentrations on TM 4-supporting cell viability

After the DHM series concentration of the TM4 supporting cells is given, compared with the Control group, the DHM concentration is between 10 and 100 mu M, so that the survival rate of the TM4 supporting cells is not obviously influenced; when the DHM concentration reached 200. Mu.M, the TM 4-supported cell viability was significantly reduced (P < 0.05, P < 0.01), see FIG. 8.

Effect of Dihydromyricetin at different concentrations on survival of the acrolein-induced TM4 support cell injury model

Compared with the Control group, the TM4 support cells can obviously reduce the cell survival rate (P < 0.001) after 100 mu M ACR modeling; DHM concentrations between 30-100 μm significantly increased the survival of TM4 supporting cells (P < 0.05, P < 0.001) compared to ACR groups after administration of DHM series concentrations, see figure 9. The final effective DHM concentration for this experiment was 40. Mu.M with a subthreshold dose of 20. Mu.M.

Effect of different concentrations of S-7701 on survival of the acrolein-induced TM4 support cell damage model

Compared with the Control group, the cell viability of the TM4 supporting cells is significantly reduced (P < 0.001) after 100 mu M ACR modeling; following administration of the S-7701 series, the S-7702 concentration significantly increased the survival rate of the TM4 supporting cells (P < 0.05) between 40 and 80. Mu.M compared to the ACR group, as shown in FIG. 11. The experiment finally determines that the subthreshold dose of S-7701 is 20. Mu.M.

Effect of Dihydromyricetin and S-7701 on survival of the acrolein-induced TM4 support cell injury model

As shown in FIG. 12, the TM4 support cells showed significantly lower cell viability (P < 0.001) after 100. Mu.M ACR modeling compared to the Control group; compared with ACR group, after 20 mu M DHM and 20 mu M S-7701 are independently administered, the survival rate of the supporting cells is not affected significantly; after 20 mu M DHM combined with 20 mu M S-7701, compared with ACR group, the survival rate of TM4 supporting cells can be obviously improved (P is less than 0.05).

Example 8F-actin staining to observe the Effect of DHM on the cytoskeleton of ACR-induced TM 4-supporting cell injury model

The experimental method comprises the following steps:

Observing that the cell density is about 80%, and then carrying out digestion and passage; preheating a complete culture medium, PBS and pancreatin to 37 ℃ in advance; taking out the culture dish needing passage, sucking and discarding the old culture medium, adding 2mL of PBS to wash the cells for 1 time, and discarding the PBS; 1mL of pancreatin is added for digestion until the cells completely fall off, and 3mL of complete culture medium is immediately added for stopping digestion; transferring into a 5mL sterile centrifuge tube, centrifuging in a 1000g centrifuge for 5min, discarding supernatant, adding 4mL complete culture medium, and blow-mixing; taking 12-hole plates, putting the cell climbing slices into each hole, adjusting the concentration of the cell suspension to 1X 10 ² by using a complete culture medium, adding 1mL of the complete culture medium containing DHM into each hole, and putting the mixture into a cell incubator with 5% CO2 and 37 ℃ for culturing for 12 hours; the old culture medium is sucked and removed, the serum-free culture medium containing ACR is added into each of the other holes except the control hole, and the cells are placed into a cell incubator with 5 percent CO ₂ and 37 ℃ for continuous culture for 3 hours; the old medium was aspirated and cells were washed 2 times with PBS and fixed for 10min with 4% formaldehyde added to each well; cells were washed 3 times with PBS and permeabilized with PBS containing 0.1% Triton-x-100 for 4min; the cells were washed 3 times with PBS and incubated with goat serum blocking solution containing FITC-phalloidin (1:150) at room temperature in the dark for 1h; cells were washed 3 times with PBS, blocked with DAPI-containing blocking agent, and observed under a microscope.

Experimental results:

f-actin staining shows that actin filaments of the Control group TM4 supporting cells are distributed strongly at the cell cortex, and the cell skeleton is complete; compared with the Control group, the actin filaments of the ACR treated group cells are distributed weakly; the DHM 40 μm group TM4 support cells showed an enhanced actin filament distribution compared to ACR group, as shown in fig. 10.

EXAMPLE 9 flow cytometry detection of TM 4-supported apoptosis rate

The experimental method comprises the following steps:

observing that the cell density is about 80%, and then carrying out digestion and passage; preheating a complete culture medium, PBS and pancreatin to 37 ℃ in advance; taking out the culture dish needing passage, sucking and discarding the old culture medium, adding 2mL of PBS to wash the cells for 1 time, and discarding the PBS; 1mL of pancreatin is added for digestion until the cells completely fall off, and 3mL of complete culture medium is immediately added for stopping digestion; transferring into a 5mL sterile centrifuge tube, centrifuging in a 1000g centrifuge for 5min, discarding supernatant, adding 4mL complete culture medium, and blow-mixing; taking 4 culture dishes with the thickness of 60mm, and averaging the cell suspension in each dish, wherein 2 dishes are added with 6mL of complete culture medium, and the culture medium is gently mixed uniformly to be set as a Control group and an ACR group; adding 6mL of complete culture medium containing DHM effective dose and DHM+S-7701 double-threshold dose into the rest 2 dishes, gently mixing, and culturing in a cell culture incubator with 5% CO2 at 37deg.C for 12 hr; the old medium was aspirated, and serum-free medium containing ACR (ACR concentration was 3.5.4.1. Determined) was added to each dish except for the Control group, and the culture was continued in a cell incubator at 37 ℃ for 3 hours with 5% co 2. Taking out 4 dishes of cells, discarding old culture medium, washing the cells with PBS for 1 time, adding 2mL of pancreatin for digestion until the cells completely fall off, and immediately adding 4mL of complete culture medium for stopping digestion; transferring into a 10mL sterile centrifuge tube, centrifuging in a 1000g centrifuge for 5min, discarding supernatant, adding 1mL PBS to wash cells, transferring the cell suspension into a 1.5mL sterile centrifuge tube, centrifuging for 5min, and repeating for 3 times; 1X 106 cells were taken, 200. Mu. LAnnexin V binding cells were added, then 5. Mu.L of Annexin V-FITC staining solution was added, after gentle mixing, 10. Mu.L of PI staining solution was added, incubated at room temperature for 25min, and then placed on ice for immediate analysis by flow cytometry.

Experimental results:

As shown in FIG. 13, the apoptosis rate of the TM4 support cells was significantly increased (P < 0.001) after 100. Mu.M ACR modeling, and significantly decreased (P < 0.001) after the combination of DHM 40. Mu.M and 20. Mu.M DHM in the treatment of 20. Mu. M S-7701, as compared to the Control group.

EXAMPLE 10 Western Blot detection of expression levels of Claudin-11 and TGF-. Beta.3/p 38 MAPK Signal pathway-associated proteins in TM 4-supporting cells

The experimental method comprises the following steps:

And (3) extracting cellular proteins by using a Kelly whole protein extraction kit, and determining the concentration of the cellular proteins in the sample by using a BCA protein content detection kit and calibrating the unified concentration of the proteins. SDS-polyacrylamide gel electrophoresis was carried out by wet transfer of nitrocellulose (PVDF) membrane. After the film transfer is finished, the film is put into 5% skim milk powder sealing liquid for sealing for 2 hours. Blocking end incubation Primary antibody p38MAPK (1:1000), TGF-. Beta.3 (1:2000), claudin-11 (1:1000) and p-p38MAPK (1:1000) diluted with 5% nonfat milk powder were incubated overnight at 4℃and secondary antibody was incubated for 2h by washing. The membrane was washed 3 times with 10min each with PBST. The exposure was performed by dropping a protein chemiluminescent agent (ECL). And calculating the gray value of the target protein by adopting Image J Image analysis software, and correcting errors by utilizing the corresponding internal reference gray value.

Experimental results:

TGF-beta 3/p38MAPK signaling pathway related protein expression levels

Compared with Control group, after 100 mu M ACR modeling, TM4 support the expression level of intracellular TGF-beta 3, P38MAPK and P-P38MAPK proteins to be obviously increased (P is less than 0.05 and P is less than 0.001); after administration of 40. Mu.M DHM and 20. Mu.M DHM in combination with 20. Mu. M S-7701, the expression levels of TGF-. Beta.3, P38MAPK and P-P38MAPK proteins were significantly reduced (P < 0.05) compared to the ACR group, as shown in FIG. 14.

Claudin-11 expression level

As shown in FIG. 15, the expression level of Claudin-11 protein in the TM4 supporting cells was significantly reduced (P < 0.001) after 100. Mu.M ACR modeling was performed as compared with the Control group; after treatment with 40. Mu.M DHM and 20. Mu.M DHM in combination with 20. Mu. M S-7701, the expression level of Claudin-11 protein was significantly increased (P < 0.01) compared to the ACR group.

It is to be understood that the above examples of the present invention are provided by way of illustration only and are not intended to limit the scope of the invention. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While remaining within the scope of the invention, obvious variations or modifications are incorporated by reference herein.

Claims

1. Use of dihydromyricetin in preparing medicine for treating spermatogenic dysfunction is provided.

2. The use according to claim 1, wherein dihydromyricetin in the medicament is the only active ingredient.

3. The use according to claim 1 or 2, wherein the spermatogenic dysfunction is a loss of spermatogenic function.

4. The use according to claim 1 or 2, wherein the spermatogenic dysfunction is a reduced sperm count and motility and a sperm malformation.

5. Application of dihydromyricetin in preparing medicine for inhibiting TGF-beta 3, p38 MAPK and p-p38 MAPK protein expression in testicle internal TGF-beta 3/p38 MAPK pathway.

6. Application of dihydromyricetin in preparing medicine for increasing blood-testosterone barrier related protein N-cadherin, occludin, claudin-11 and Cx43 protein expression level in testis tissue is provided.

7. The use according to any one of claims 1-6, wherein the medicament is in an injectable or oral dosage form.

8. A non-therapeutic method of enhancing spermatogenic function and sperm motility comprising administering dihydromyricetin.

9. The method of claim 9, wherein the single application dose of dihydromyricetin is 37.5-150mg/kg.

10. The method of claim 10, wherein the single application dose of dihydromyricetin is 75-150mg/kg.