CN113648419A

CN113648419A - Application of c-Src/PI3K signal path in protection of reproductive injury

Info

Publication number: CN113648419A
Application number: CN202110793622.4A
Authority: CN
Inventors: 李环; 张晶; 徐小磊; 王洪艳
Original assignee: Beihua University
Current assignee: Beihua University
Priority date: 2021-07-12
Filing date: 2021-07-12
Publication date: 2021-11-16

Abstract

The invention relates to application of a c-Src/PI3K signal channel in reproductive injury protection, and relates to the technical fields of biotechnology and medicine. The invention provides application of a c-Src/PI3K signal channel as a target point in preparation of a medicine for treating reproductive injury, and provides a new action target point for developing a natural spermatogenic medicine.

Description

Application of c-Src/PI3K signal path in protection of reproductive injury

Technical Field

The invention relates to the technical field of biotechnology and medicine, in particular to application of a c-Src/PI3K signal path in reproductive injury protection.

Background

In recent years, the fertility rate of age-appropriate breeding couples has gradually decreased. There are data showing that about 15% of couples worldwide are affected by infertility, of which 20% to 30% are due to male infertility and resulting in infertility. The traditional Chinese medicine has unique advantages and better curative effect in the aspect of treating male infertility and spermatogenic disorder. In the medical treatment of infertility, ginseng known as the king of Chinese herbal medicine is a frequently selected medicine for treating infertility. It is reported in the literature that ginseng extract (GINST) treated by pectinase has an antioxidant effect and can effectively treat testicular damage of rats caused by heat stress, and Korean Red Ginseng (KRG) can prevent testicular damage caused by adriamycin and aging. Salvati G et al reported that 66 patients who took the ginseng crude extract had significantly increased sperm count and motility, increased testosterone levels, and exhibited different effects on different levels of the hypothalamus-pituitary-testicular axis. The effective components of ginseng include saponins, small molecular peptides, polypeptides, polysaccharides and the like, wherein the ginsenoside is the most important effective component of ginseng. The factors responsible for male infertility are manifold, with one major factor being environmental factors. There is evidence that environmental factors have a significant impact on the quantity and quality of semen. However, the intervention of ginsenoside on environmental toxicant-induced reproductive injury is not clear.

Disclosure of Invention

Aiming at the problems, the invention provides the application of a c-Src/PI3K signal channel as a target point in preparing a medicament for treating reproductive injury, and provides a new action target point for developing natural spermatogenic medicaments.

In order to achieve the aim, the invention provides application of a c-Src/PI3K signal pathway as a target point in preparing a medicament for treating reproductive injury.

Src kinase family (SFKs) are a family of non-receptor protein tyrosine kinases that play important roles in signal transduction and regulation of various biological processes such as cell proliferation, differentiation, and apoptosis. At present, the Src protein is researched to participate in intracellular signal transduction, a Src/PI3K/Akt pathway is a more critical one, and Src regulates a PI3K/Akt signal pathway, so that apoptosis can be inhibited, and cell survival can be regulated. The inventor discovers that the treatment of ginsenoside Rg3 can up-regulate the protein expression levels of BBT tight junction protein Cx43 and c-Src/PI3K/Akt signal pathway-related phosphorylated proteins p-Src, p-PI3K and p-Akt of a DBP infected mouse through Western blot analysis. To further confirm whether c-Src and PI3K are involved in the interference of ginsenoside Rg3 on DBP-induced damage of supporting cells, MBP and c-Src inhibitor PP2 and PI3K specific inhibitor LY294002 were used in vitro experiments to interfere with the phosphorylation process of T4 cells c-Src, PI3K and Akt. Through cell oxidation index detection, Western blot, immunofluorescence, laser confocal and other methods, the antioxidant enzyme activity is remarkably improved after the T4 cells treated by MBP (DBP in vivo active metabolite) are given ginsenoside Rg3, the MDA content of the oxidation product is reduced, the expression levels of p-Src, p-PI3K and p-Akt proteins in the cells and the expression level of BBT tight junction protein Cx43 protein are remarkably improved. Therefore, experiments prove that the c-Src/PI3K signal channel can be activated to play a role in reversing reproductive injury, namely the c-Src/PI3K signal channel can be used as a target point, and a novel medicine or a treatment mode aiming at the target point is designed.

In one embodiment, the c-Src/PI3K signaling pathway comprises a c-Src/PI3K/Akt signaling pathway.

In one embodiment, the treatment of reproductive injury results from abnormal expression of tight junction protein between testicular and sertoli cells.

The invention discloses application of a c-Src/PI3K signal pathway activator in preparing a medicament for treating reproductive injury. By adopting the activator, the c-Src/PI3K signal channel can be used as a target point to activate the c-Src/PI3K signal channel which is exposed and inhibited by DBP and MBP, the antioxidase activity of T4 cells is obviously improved, the continuity of cell connection is improved, the expression of tight junction protein between supporting cells of testis is recovered, the protection effect on the tight junction protein between the supporting cells is realized, and the effect of treating reproductive injury is finally achieved.

In one embodiment, the activator of the c-Src/PI3K signal pathway can repair reproductive injury by up-regulating the expression level of c-Src/PI3K/Akt pathway-related phosphorylated proteins.

In one embodiment, the phosphorylated protein comprises at least one of the following proteins: p-Src protein, p-PI3K protein, p-Akt protein.

In one embodiment, the c-Src/PI3K signaling pathway activator effects restoration of reproductive injury by reversing testicular tissue apoptosis. The activator has obvious protective effect on cells affected by MBP-induced cytotoxicity, and reverses MBP-induced proliferation inhibition and apoptosis.

In one embodiment, the activator comprises ginsenoside Rg 3. Ginsenoside Rg3 can activate DBP and MBP exposure-inhibited c-Src/PI3K signal pathway, improve continuity of cell connection, and protect tight junction protein between testis supporting cells.

The invention also discloses application of the activator in preparing a medicament for preventing and/or treating reproductive injury.

Compared with the prior art, the invention has the following beneficial effects:

according to the invention, a mouse testis model treated by 35d DBP contamination is constructed, and the protein expression quantity of c-Src/PI3K/Akt pathway-related phosphorylated proteins p-Src, p-AKT and p-PI3K of a mouse testis tissue is found to be obviously reduced; the protein expression level of the BTB tight junction related protein Cx43 is also obviously reduced, which shows that DBP destroys the blood testis barrier and influences the spermatogenic function; after the intervention of Rg3, the expression level of the protein is remarkably increased (P <0.05), which indicates that the intervention of Rg3 can regulate and control the expression of testis supporting cell signal-related protein through a c-Src/PI3K signal channel, improve Sertoli cell connection damage caused by DBP exposure, and further improve the spermatogenic microenvironment.

Meanwhile, in a cell experiment, MBP and c-Src inhibitors PP2 and a PI3K specific inhibitor LY294002 are respectively used for interfering the phosphorylation processes of c-Src, PI3K and Akt, the influence of the MBP and c-Src inhibitors PP2 and the PI3K specific inhibitor LY294002 on the expression of p-Src, p-PI3K, p-Akt and BBT tight junction protein Cx43 in T4 cells is observed through Western blot detection, and biochemical indexes of oxidative stress of the T4 cells are detected. The results show that MBP reduces the activity of SOD of T4 cells, increases the content of MDA, obviously reduces the expression level of p-Src, p-PI3K and p-Akt proteins in the cells, simultaneously obviously reduces the expression of Cx43 protein of BBT tight junction protein in T4 cells, and the inhibition effect is consistent with the effect of c-Src inhibitor PP2 and PI3K specific inhibitor LY294002 on T4. After Rg3 is given, the antioxidant enzyme activity of T4 cells is obviously improved, the MDA content of an oxidation product is reduced, the expression levels of p-Src, p-PI3K and p-Akt proteins in the cells and the expression of BBT tight junction protein Cx43 protein are obviously improved, and the immunofluorescence result is basically consistent with the protein expression of a Western Blot experiment result. Further, when the support cells are stimulated by MBP, the c-Src/PI3K signal channel can be inhibited, the phosphorylation of c-Src, PI3K and Akt is weakened, multiple downstream channels of the cells are disturbed, and then the TM4 cells are damaged, and Rg3 can improve the c-Src phosphorylation expression, activate the c-Src/PI3K/Akt signal channel, increase the expression of the tight junction protein Cx43, improve the tight junction among cells, and further improve the damage of the TM4 cells caused by MBP.

The research shows that the application of the c-Src/PI3K signal channel as a target point in preparing the medicine for treating reproductive injury provides a new action target point for developing natural spermatogenic medicines.

Drawings

FIG. 1 is a graph showing the results of staining testis tissue HE in example 1;

FIG. 2 is a Westernblot result chart showing the effect of Rg3 on expression of c-Src/PI3K/Akt signal pathway-related protein in testis tissue of a DBP-infected mouse in example 1;

FIG. 3 is a graph showing the effect of Rg3 on the expression of c-Src/PI3K/Akt signaling pathway-related protein p-Src (Tyr416) in the testis tissue of mice infected with DBP in example 1;

FIG. 4 is a graph showing the effect of Rg3 on the expression of c-Src/PI3K/Akt signal pathway-related protein p-PI3K in the testis tissue of mice infected with DBP in example 1;

FIG. 5 is a graph showing the effect of Rg3 on the expression of c-Src/PI3K/Akt signal pathway-related protein p-Akt (Ser473) in testis tissue of mice infected with DBP in example 1;

FIG. 6 is a graph showing the effect of Rg3 on the expression of c-Src/PI3K/Akt signal pathway-related protein p-Akt (Thr308) in mouse testis tissue infected with DBP in example 1;

FIG. 7 is a graph showing the results of immunohistochemical detection in example 1;

FIG. 8 is a Westernblot result chart showing the effect of Rg3 on the expression of BTB tight junction-related protein in testis tissue of DBP-infected mouse in example 1;

FIG. 9 is a graph showing the effect of Rg3 on the expression of BTB tight junction-related protein Cx43 in testis tissue of mice infected with DBP in example 1;

FIG. 10 is a graph showing the effect of Rg3 on the expression of BTB tight junction-related protein E-cadherin in testis tissue of mice infected with DBP in example 1;

FIG. 11 is a graph showing the effect of Rg3 on the expression of ZO-1, a BTB tight junction-related protein in testis tissue of mice infected with DBP in example 1;

FIG. 12 is a graph showing the effect of Rg3 on the expression of testicular tissue skeletal protein Vimentin in DBP-infected mice in example 1;

FIG. 13 is a graph showing the results of measuring the viability of T4 cells in each group after different concentrations of Rg3 were applied by the CCK-8 method in example 2;

FIG. 14 is a graph showing the results of testing the cell viability of groups of MBP-treated T4 cells after applying different concentrations of Rg3 according to the CCK-8 method in example 2;

FIG. 15 is a graph showing the results of measuring the viability of T4 cells of each group treated with different concentrations of LY294002 by the CCK-8 method in example 2;

FIG. 16 is a graph showing the results of measuring the viability of T4 cells in each group treated with different concentrations of PP2 by the CCK-8 method in example 2;

FIG. 17 is a graph showing the results of detecting apoptosis of each group of T4 cells by flow cytometry in example 2;

FIG. 18 is a Westernblot result chart showing the effect of Rg3 on the c-Src/PI3K/Akt signaling pathway-related protein expression in MBP-induced TM4 cell injury in example 2;

FIG. 19 is a graph showing the effect of Rg3 on MBP-induced TM4 cell damage in example 2, showing the effect of c-Src/PI3K/Akt signaling pathway-associated protein Cx43 expression;

FIG. 20 is a graph showing the effect of Rg3 on MBP-induced TM4 cell injury c-Src/PI3K/Akt signaling pathway-related protein p-Src (Tyr416) expression in example 2;

FIG. 21 is a graph showing the effect of Rg3 on the expression of c-Src/PI3K/Akt signaling pathway-related protein p-PI3K in MBP-induced TM4 cell injury in example 2;

FIG. 22 is a graph showing the effect of Rg3 on the expression of c-Src/PI3K/Akt signaling pathway-related protein p-Akt (Thr308) in MBP-induced TM4 cell injury in example 2;

FIG. 23 is a graph showing the effect of Rg3 on the expression of c-Src/PI3K/Akt signaling pathway-associated protein p-Akt (Ser473) in MBP-induced TM4 cell injury in example 2;

FIG. 24 is a graph showing the results of immunofluorescence in example 2 showing expression of relevant factors in c-Src/PI3K/Akt signaling pathway in TM4 cells.

Detailed Description

To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

Defining:

the PI3K of the invention: refer to phosphatidylinositol-3-kinase.

Akt: refers to protein kinase B.

DBP: refers to dibutyl phthalate.

c-Src: refers to the SFKs signal protein uniquely expressed in GJ

GJ: the gap junction in three types of cell junctions in the testis of mammals is formed by the butt joint of GJ protein hemichannels on adjacent cell membranes, is a chemical signal channel between supporting cells and between supporting cell-germ cells, and allows molecules with the molecular weight less than 1.2kDa between the adjacent cells to pass through.

Cx 43: the protein is found as a tumor inhibiting factor, is a connexin with abundant and widely distributed quantity, participates in forming a gap connection channel, provides a connection path for tumor cells and vascular endothelial cells, and the function of the gap connection channel is regulated and controlled by Cx43 expression.

Rg 3: ginsenoside Rg3 is a specific saponin in Ginseng radix Rubri.

Reagents, materials, equipment sources:

rg3 (alatin), dbp (santa cruz), monobutyl phthalate (alatin), Giemsa (Sigma usa), antibodies P-src, P-AKT (ser473) (Genen tex), P-AKT (thr308) (CST), P-PI3K, Cx43, E-cadherin, Zol and vimentin (bioorld), ECL luminescence kit (seikagaku), DAPI (petit clouds), mouse serum Luteinizing Hormone (LH), Follicle Stimulating Hormone (FSH), testosterone (T), radioimmunity kit (shanghai lajiang life); CCK8 cell proliferation and cytotoxicity detection kit, PP2, LY294002(MCE), MDA, SOD, LDH, SDH detection kit (Nanjing institute of bioengineering, China), WLJY-9000 color sperm quality detection system (Beijing Webber), digital camera (DP20, Olympus, Tokyo, Japan), FACS420 flow cytometer (Becton-Dickinson, USA), enzyme labeler (Molecular Devices), biomicroscope (BX53, Olympus), 6-week-old male C57BL/6J mice (Jilin laboratory center), mouse testis supporting cell strain TM4 (ATCC company), microscope (DP20, Olympus university).

Reagents, materials and equipment used in the embodiment are all commercially available sources unless otherwise specified; unless otherwise specified, all the experimental methods are routine in the art.

Example 1

Mouse validation experiment.

1. And preparing and grouping animal models.

30 SPF-grade male C57BL/6J mice, 6 weeks old, weigh 20. + -.2 g. The experimental animals were divided into a control group, a DBP group (400mg/kg), and an Rg3+ DBP group (400mg/kgDBP +20mg/kgRG 3). Administration methods animals in the DBP group and DBP + Rg3 group were gavaged with 400mg/kg DBP (dissolved in corn oil) at 8 am each day. Rg3 group and DBP + Rg3 group were administered with Rg320mg/kg (dissolved in normal saline) intraperitoneally at 2 am of the day, control group was administered with an equal volume of corn oil for intragastric administration at 8 am of the day, and all experimental animals were administered with the drug continuously for 35 days after 2 am of the day. After the experimental period is finished, the patient is anesthetized by ether, 10mL of blood is taken from the abdominal aorta,standing at room temperature, and then 2500r.min^-1Centrifuging for 25min, collecting serum, and storing in refrigerator at 4 deg.C. Sacrificed and the testis and epididymis are left for sample analysis.

2. Mouse sperm quality analysis.

(1) Detecting the sperm density, sperm motility and total deformity rate of the mouse sperm.

Taking bilateral epididymis of a mouse, and preparing sperm filtrate: placing epididymis into a plate, adding 3mL of physiological saline at 37 ℃, shearing epididymis tissues along a tube cavity, placing for 1-2min to prepare a sperm suspension, dripping the sperm suspension onto a pre-warmed sperm counting plate, and performing sperm quality analysis by using a WLJY-9000 type sperm quality detection system. Taking 4 visual fields, and completing detection within 2 min. Detection indexes are as follows: sperm density (1X 10)⁶counts/mL), sperm motility (%), total distortion (%).

(2) Rg3 has effects on sperm density, sperm motility and sperm teratospermia of DBP infected mice.

The results are shown in the table below, and the sperm density and sperm motility of each group are reduced compared with the control group by the sperm density detection of each group, and the DBP group has statistical significance (P is less than 0.01); compared with the DBP group, the DBP + Rg3 group has obviously different sperm density and sperm motility (P is less than 0.01); there was no significant difference (P > 0.05) between the groups in the total teratospermia.

TABLE 1 Rg3 Effect on sperm density, sperm motility and sperm teratospermia in DBP-infected mice

Grouping	Sperm density (10)⁶/mL)	Sperm motility (%)	Sperm abnormality rate (%)
				Control group	42.34±2.70	32.83±2.45	13.14±2.71
DBP	28.71±2.31^**	19.64±2.34^**	13.95±2.92
				DBP+Rg3	39.13±3.64^##	26.25±2.72^##	12.93±2.59

P <0.01vs. control; # P <0.01vs. DBP group.

3. Mouse serum sex hormone level determination.

(1) And detecting the contents of testosterone (T), Follicle Stimulating Hormone (FSH) and Luteinizing Hormone (LH) in the serum of the mouse.

The ELISA kit is adopted to detect the serum testosterone (T), Follicle Stimulating Hormone (FSH) and Luteinizing Hormone (LH) contents of each group of mice.

(2) The effect of Rg3 on serum reproductive hormones in DBP-infected mice.

The results are shown in the following table, compared with the control group, the serum Luteinizing Hormone (LH) and testosterone (T) of the mice in the DBP group are reduced, the Follicle Stimulating Hormone (FSH) is increased, and the difference has statistical significance (P is less than 0.01); compared with the DBP group, the DBP + Rg3 group has the advantages that the testosterone (T) and Luteinizing Hormone (LH) levels are increased, the Follicle Stimulating Hormone (FSH) falls back, and the difference is obvious (P is less than 0.01).

TABLE 2 Effect of Rg3 on serum reproductive hormones in DBP-infected mice

Grouping	LH(IU/L)	T(nmol/L)	FSH(IU/L)
				Control group	169.80±17.60	37.67±3.87	38.01±3.56
DBP	118.36±10.50^**	17.74±3.11^**	63.48±7.15^**
				DBP+Rg3	147.73±15.79^##	23.18±2.44^##	52.04±4.07^##

P <0.01vs. control; # P <0.01vs. DBP group.

4. Histopathological examination of testis.

(1) Histopathological examination of testis.

The left testis of each mouse is fixed in 4% paraformaldehyde solution, and testis tissue sections are prepared by conventional dehydration, paraffin embedding, sectioning and hematoxylin-eosin (HE) staining. The layer, morphological structure and lumen damage degree of the spermatogenic epithelial cells are observed under 200 times of an optical microscope.

(2) And (6) analyzing results.

HE staining results are shown in figure 1: the control group had normal testicular tissue structure, intact seminiferous epithelium structure, intact basement membrane, abundant intracavity sperm, and uniformly distributed interstitial cells between seminiferous tubules. The testis tissue structure of the DBP group is obviously changed, cells in a lumen fall off, the number of supporting cells is reduced, and the sperm in the lumen is rare; compared with the DBP group, the testis structure of the rats in the DBP + Rg3 group tends to be normal, the arrangement of the seminiferous tubules is more compact and regular, no cast-off cells exist in the lumen, the supporting cell structure is normal, and the number of the sperms in the lumen is increased.

5. And (3) detecting the expression of the testis tissue related protein.

(1) And detecting the expression of the testis tissue related protein.

Collecting testis tissue of about 100mg from each mouse, placing in 4 deg.C pre-cooled lysis buffer, ultrasonically mixing at 4 deg.C (6s × 6 times), incubating at 4 deg.C for 30min, and culturing at 2500r.min^-1Centrifuging for 10min × 3 times, and subpackaging the supernatant; the protein content was determined by the Bradford method and the proteins were separated by 10% SDS-PAGE using standard bovine serum albumin. Each well was filled with 60. mu.g of protein and electrophoresed at a constant voltage of 160V until the bottom of the gel was implanted. After blocking with 5% bovine serum albumin for 2h, add 1: 1000 Rabbit anti-mouse Cx43, anti-E-cadherin, anti-Zol, anti-Vimentin, anti-p-PI 3K, anti-p-Akt (Thr308), anti-p-Akt (Ser473), anti-p-Src, shaken at 37 ℃ for 1h, washed 3 times with phosphate buffer-Tween 20 for 10 min. A secondary antibody (1: 5000) labeled with horseradish peroxidase was added, shaken at 37 ℃ for 1h, washed 3 times in PBST, and then irradiated with a chemiluminescent agent. The band size was quantified using AlphaInnotech software.

(2) Influence of Rg3 on expression of c-Src/PI3K/Akt pathway-related protein in testis tissue of DBP-infected mouse.

The Westernblot results show: as shown in FIGS. 2-6, compared with the control group, the model group has significantly reduced protein expression levels of c-Src/PI3K/Akt pathway-related phosphorylated proteins P-Src (Tyr416), P-Akt (Thr308), P-Akt (Ser473) and P-PI3K in the DBP-exposed rat testis tissue (P < 0.05); compared with the model group, the protein expression of P-Src (Tyr416), P-Akt (Thr308), P-Akt (Ser473) and P-PI3K is obviously up-regulated after Rg3 intervention (P < 0.05).

(3) The effect of Rg3 on the expression of BTB tight junction related protein in testis tissue of mice infected with DBP.

The Westernblot results show: as shown in FIGS. 8-12, the protein expression levels of BTB tight junction related proteins Cx43, ZO-1, E-cadherin and skeleton protein Vimentin in the DBP-exposed rat testis tissues were also significantly reduced (P <0.05) compared with the control group; compared with a model group, the expression level of the protein is remarkably increased after Rg3 intervention (P < 0.05).

6. And (4) carrying out immunohistochemical detection.

(1) And (4) carrying out immunohistochemical detection.

Dewaxing testis paraffin section with xylene, serial ethanol rehydration, 3% hydrogen peroxide treatment, 0.25% trypsin incubation at 37 deg.C for 10min, extracting antigen. The sliced goat serum was incubated for 10min at room temperature with blocking. Sections were then incubated overnight at 4 ℃ and primary antibodies (1:100 dilution) included Cx43, E-cadherin, Zol and anti-Vimentin. After 3 washes with PBS, HRP-labeled goat anti-rabbit IgG (1:100 dilution) was incubated, and then HRP was revealed with streptomyces antibiotics peroxidase and DAB. Finally, the sections were stained with hematoxylin. Images were collected by microscope.

(2) And (6) analyzing results.

Immunohistochemistry showed mouse testis tissue BTB tight junction associated protein expression, as shown in figure 7.

Immunohistochemistry shows that the result is consistent with a Westernblot result of Rg3 on the expression of BTB tight junction related protein of testis tissues of mice infected with DBP: the expression level of proteins of BTB tight junction related proteins Cx43, ZO-1, E-cadherin and framework protein Vimentin of a mouse testis tissue exposed by DBP is obviously reduced, and the expression level of the proteins is obviously increased after the intervention of Rg 3.

Example 2

T4 cell validation experiment.

1. And (4) culturing and grouping the cells.

In order to confirm the damage effect of the c-Src/PI3K signal pathway on sperms by DBP and the interference protection of ginsenoside Rg3, the effects of a PI3K specific inhibitor LY294002 and a c-Src inhibitor PP2 on T4 cells are added in an in vitro experimental design. In addition, since DBP is mainly absorbed into the body in the form of its metabolite, and DBP is more toxic and more active than DBP after metabolizing the product MBP in the body, a series of experiments using MBP were conducted in cellular experiments. Cells were divided into 8 groups: DMSO group, MBP group, Rg3 group, MBP + Rg3 group, MBP + LY294002 group, MBP + Rg3+ LY294002 group, MBP + PP2 group, MBP + Rg3+ PP2 group. Each group of cells was routinely cultured in DMEM/F12 medium containing 10% (volume fraction) fetal bovine serum at 37 ℃ in 95% (volume fraction) air and 5% (volume fraction) carbon dioxide. And (5) changing the liquid or passaging according to the cell state and density every 3 days, and taking the cells in the logarithmic growth phase for experiment.

2. And (5) detecting the cell viability.

(1) The viability of each group of T4 cells was examined by the CCK-8 method.

Each group of cells was tested for viability using a cell counting kit. The cell density was adjusted to 5X 10 in each group of TM4 cells in logarithmic growth phase with good growth state⁴Putting into 96-well plate, 100ul cell suspension per well, setting blank group, culturing overnight at 37 deg.C (adding 100ul sterile PBS into the hole around the cell well); adding 10ul CCK-8 into each hole of each group of cells, and culturing at 37 ℃ for 4 h; and measuring the light absorption value OD450 of each hole by using a microplate reader.

(2) And (6) analyzing results.

As shown in FIGS. 13-16, to investigate whether c-Src and PI3K are involved in MBP toxicity to the supporting cells, MBP, PP2 and LY294002 were used at doses within the concentration range where no significant cytotoxicity was observed, and MBP dose (100. mu.M), PP2 (1. mu. mol/L) and LY294002 (5. mu. mol/L) and ginsenoside Rg3 dose of 20. mu.g/mL were selected as the doses used in the subsequent experiments based on the CCK-8 assay results.

3. And (4) detecting cell apoptosis.

(1) Apoptosis was detected in each group of T4 cells using flow cytometry.

And detecting the apoptosis of each group of cells by using an annexin V-FITC/PI double-staining apoptosis kit. Digesting the cells with 0.25% pancreatin without EDTA, collecting the groups after terminating the digestion, and treating the groupsCentrifuging the groups of the finished cells at 1500rpm for 5min, removing supernatant, adding PBS for resuspension, rinsing the cells with PBS for 2 times at 1500rpm for 5 min; collecting 5X 10⁵The cells were resuspended in 500. mu.L of staining buffer, 5. mu.L of LPI and 5. mu.L of annexin V-FITC were added and mixed well, and incubated for 15min at room temperature in the absence of light. A total of 20000 cells were collected for each group using FACScan flow cytometry.

(2) And (6) analyzing results.

The results are shown in fig. 17, Rg3 has a significant protective effect on MBP-induced cytotoxicity, and MBP-induced proliferation inhibition and apoptosis are reversed, and MBP-induced apoptosis is exacerbated after inhibition of LY294002 and PP2 pathways.

4. And (5) detecting cell biochemical indexes.

(1) And (5) detecting cell biochemical indexes.

MDA, SOD, SDH and LDH detection kits are adopted to carry out MDA, SOD, SDH and LDH level and activity determination of each group of cells according to the instructions.

(2) Oxidative toxicity effects of Rg3 on MBP-induced T4 cells.

The activity of SOD indirectly reflects the ability of organism to eliminate oxygen free radical, MDA is the final product of lipid peroxidation accumulated by intracellular oxidative damage, and the content of MDA can indirectly reflect the intensity of lipid peroxidation in organism and the damage degree of free radical to tissue cell. MBP obviously reduces the activity of T4 cell SOD and increases the content of MDA. After administration of Rg3, T4 cells showed a marked increase in antioxidant activity, while the oxidation product MDA content decreased, as shown in the table below.

TABLE 3 oxidative toxicity effects of Rg3 on MBP-induced T4 cells

P<0.01vs.the DMSO group；^#P<0.05，^##P<0.01vs.MBP group；^∧∧P<0.01vs.Rg3 group；^&P<0.05，^&&P<0.01vs.MBP+Rg3 group。

5. And (3) detecting the expression of c-Src/PI3K/Akt pathway related protein of the T4 cells.

(1) Westernblotting examined the expression of p-PI3K, anti-p-Akt (Thr308), anti-p-Akt (Ser473) and anti-p-Src in cells.

The groups of TM4 cells were harvested, washed 3 times with ice PBS, added with lysis buffer (containing protease and phosphatase inhibitors) and lysed on ice for 30 min. The suspension was centrifuged at 12000 Xg for 10min and the supernatant was taken. Protein concentration was measured by BCA method, each histone was adjusted to the same concentration with the lysis buffer, and 5 Xloading buffer was added and boiled at 100 ℃ for 10min to denature the protein. The denatured proteins (20. mu.g per loading well) were separated by SDS-PAGE (6% -10% gel) and wet-transferred onto PVDF membrane for 2 h. 5% (volume fraction) bovine serum albumin in TBST configuration was blocked for 1h at room temperature. Primary antibodies were diluted with TBST (1: 1000Cx43 antibody, anti-p-PI 3K, anti-p-Akt (1: 1000) anti-p-Src) at a volume ratio of 1: 1000 and incubated overnight at 4 ℃. The TBST was rinsed 3 times for 10min each. The goat anti-rabbit and anti-mouse IgG secondary antibodies are incubated for 1h at room temperature, and rinsed 3 times in TBST, 10min each time. And preparing an ECL chemiluminescence reagent, uniformly mixing, and coating on the PVDF membrane. The NGENE exposure system takes a picture.

The results of the WesternBlot assay are shown in FIGS. 18-23, and compared with the control group, the protein expression levels of P-Src, P-Akt (Thr308), P-Akt (Ser473) and P-PI3K of T4 cells in the MBP group are all obviously reduced (P < 0.05); BTB tight junction associated protein Cx43 was also lower than the control group; after the Rg3 is dried, the expression of the protein is obviously increased (P <0.05), and the difference has statistical significance.

(2) The immunofluorescence double-label detects the expression of p-Src and p-Akt of T4 cells.

Fixing the cell-plated slide in a culture plate with 4% paraformaldehyde for 15min, allowing 0.5% TritonX-100 (prepared with PBS) to permeate at room temperature for 20min, and sealing with serum at room temperature for 30 min. Sufficient diluted primary antibody was added dropwise to each slide and placed in a wet box and incubated overnight at 4 ℃. Adding diluted fluorescent secondary antibody dropwise, incubating at 37 deg.C for 1h in a wet box, adding normal goat serum dropwise onto the slide, and sealing at room temperature for 30 min. And then dropwise adding the diluted primary antibody, after the addition of the primary antibody, incubating in a 4 ℃ wet box in a dark place overnight, dropwise adding the diluted fluorescent secondary antibody, incubating in the wet box at 37 ℃ for 1h, dropwise adding DAPI, incubating in the dark place for 5min, staining a specimen for nucleus, finally sealing the specimen by using a sealing liquid containing an anti-fluorescence quencher, and observing and acquiring an image under a confocal microscope.

The immunofluorescence double-label detection result is shown in FIG. 24, and the immunofluorescence result verifies the expression result of WesternBlot: compared with the blank group, the MBP model group has obviously reduced expression of p-Src, p-Akt (Thr308), p-Akt (Ser473) and p-PI3K proteins, and has obviously increased expression of c-Src protein after the dry prognosis.

The experiments show that the invention successfully prepares the spermatogenic disorder model by using DBP and simultaneously gives Rg3, and the results show that Rg3 can reverse abnormal expression of tight junction protein between testis supporting cells caused by DBP, so that the arrangement of seminiferous tubules of rat testis is regular, the structure is complete, the structure and the function of testis are recovered, and the quantity and the activity of sperms are improved. The results of the invention preliminarily show that Rg3 improves the spermatogenesis dysfunction, inhibits apoptosis, reverses the reduction of sperm number, enhances the vitality of the damaged sperm and has the spermatogenic effect. The cell experiment shows that the active metabolite MBP of DBP can reduce the proliferation of the supporting cell, increase the apoptosis rate, reduce the expression level of the gap junction (Cx43) protein in the supporting cell, damage the shape, and reduce the activity of testis specific biochemical enzymes, namely Succinate Dehydrogenase (SDH) and Lactate Dehydrogenase (LDH), which are used for the mature development of testis and the perfect function and shape of sperm. The MBP can inhibit the oxidative respiration of the supporting cells and inhibit the energy transfer process, so that the supporting cells can not provide nutrition and metabolic support for the sperm cells, and the blood testis barrier is damaged. After the Rg3 is given, the proliferation of T4 supporting cells is increased, the apoptosis rate is reduced, the expression level of Cx43 protein is increased, the cell structure is complete, the activities of SDH and LDH enzymes are improved, and the Rg3 is prompted to play an important role in restoring the normal activities of the supporting cells by improving the activity of the energy metabolism related enzymes of the supporting cells, promoting sperm maturation, reducing the damage of the supporting cells and restoring the integrity of cell membranes. According to the invention, the mouse testis is infected with 35d DBP, and the protein expression level of c-Src/PI3K/Akt pathway-related phosphorylated proteins p-Src, p-AKT and p-PI3K in mouse testis tissues is obviously reduced; the protein expression level of the BTB tight junction related protein Cx43 is also obviously reduced, which shows that DBP destroys the blood testis barrier and influences the spermatogenic function; after the intervention of Rg3, the expression level of the protein is obviously increased (P < 0.05). Similar results are obtained in vitro experiments, which show that Rg3 intervention can regulate and control the expression of testis support cell signal-related protein through a c-Src/PI3K signal channel, improve Sertoli cell junction injury caused by DBP exposure, and further improve the spermatogenic microenvironment.

In order to further verify whether the c-Src/PI3K pathway is involved in the interference effect of Rg3 on DBP-induced damage of support cells, MBP and c-Src inhibitors PP2 and a PI3K specific inhibitor LY294002 are respectively used for interfering the phosphorylation processes of c-Src, PI3K and Akt in a cell experiment, the influence of the MBP and c-Src inhibitors PP2 and the P3K specific inhibitor LY294002 on P-Src, P-PI3K and P-Akt and BBT tight junction protein Cx43 protein expression in T4 cells is observed through Westernblot detection, and biochemical indicators of T4 cell oxidative stress are detected. The research result shows that MBP reduces the activity of SOD of T4 cells, increases the content of MDA, obviously reduces the expression level of p-Src, p-PI3K and p-Akt proteins in the cells, simultaneously obviously reduces the expression of Cx43 protein of BBT tight junction protein in T4 cells, and the inhibition effect is consistent with the effect of c-Src inhibitor PP2 and PI3K specific inhibitor LY294002 on T4. After Rg3 is given, the antioxidant enzyme activity of T4 cells is obviously improved, the MDA content of oxidation products is reduced, and the expression levels of p-Src, p-PI3K and p-Akt proteins and the expression of BBT tight junction protein Cx43 protein in the cells are obviously improved. The immunofluorescence result is basically consistent with the protein expression of the WesternBlot experiment result, the fluorescence expression of p-Src and p-Akt proteins in the MBP group is obviously reduced, and the fluorescence expression of the p-Src and p-Akt proteins is obviously increased after Rg3 is given. The immunofluorescence results further indicate that when a supporting cell is stimulated by MBP, the c-Src/PI3K signal pathway can be inhibited, phosphorylation of c-Src, PI3K and AKT is weakened, multiple downstream pathways of the cell are disturbed, and then TM4 cell damage is caused, and Rg3 can improve the TM4 cell damage caused by MBP by promoting c-Src phosphorylation expression, activating c-Src/PI3K/Akt signal pathway, increasing expression of tight junction protein Cx43 and improving tight junction among cells. The research result shows that the active metabolite MBP of DBP can influence the expression of connexin of testicular support cells through a c-Src/PI3K/Akt signal pathway, further influence BBT and influence reproductive function. And Rg3 recovers the expression of T4 cell blood testis barrier tight junction protein Cx43 caused by MBP, and is realized by enhancing the antioxidant capacity of cells and influencing the activity of c-Src/PI3K/Akt signal channels.

In conclusion, Rg3 intervention has a certain repairing effect on the damage of DBP and MBP exposure male mice reproductive capacity. The mechanism of action may be: when cells are stimulated by oxidative stress caused by DBP and MBP exposure, a c-Src/PI3K signal channel is inhibited, the action of factors downstream of the channel is influenced, the connection between supporting cells is broken, and the permeability of a blood testis barrier is increased, so that a spermatogenic microenvironment is changed. Rg3 intervention may activate DBP and MBP exposure inhibition c-Src/PI3K signal pathways, improve continuity of cell connection, and realize the protective effect of Rg3 on supporting cell spermatogenic injury.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

Use of the c-Src/PI3K signalling pathway as a target in the manufacture of a medicament for the treatment of reproductive injury.
2. The use of claim 1, wherein said c-Src/PI3K signaling pathway comprises a c-Src/PI3K/Akt signaling pathway.
3. The use according to claim 1, wherein the treatment of reproductive damage is caused by abnormal expression of tight junction protein between sertoli cells.
Use of a c-Src/PI3K signalling pathway activator in the manufacture of a medicament for the treatment of reproductive injury.
5. The use as claimed in claim 4, wherein the activator of the c-Src/PI3K signaling pathway is effective in repairing reproductive injury by up-regulating the expression level of c-Src/PI3K/Akt pathway-related phosphorylated proteins.
6. The use according to claim 5, wherein the phosphorylated protein comprises at least one of the following proteins: p-Src protein, p-PI3K protein, p-Akt protein.
7. The use of claim 4 wherein the c-Src/PI3K signaling pathway activator effects restoration of reproductive injury by reversing testicular tissue apoptosis.
8. The use as claimed in claim 7 wherein the c-Src/PI3K signalling pathway activator comprises ginsenoside Rg 3.