阅读说明：本技术 C3g在制备拮抗3-mcpd染毒造成的抗氧化系统破坏的拮抗剂中的应用 (Application of C3G in preparing antagonist for resisting damage of antioxidant system caused by 3-MCPD infection ) 是由 白卫滨 朱翠娟 孙建霞 蒋鑫炜 李旭升 李夏 焦睿 于 2017-07-07 设计创作，主要内容包括：本发明提供了C3G在制备拮抗3-MCPD染毒造成的抗氧化系统破坏的拮抗剂中的应用。本发明证实,C3G改善3-MCPD染毒造成的大鼠摄食减少,生长迟缓状况；并且可以有效拮抗3-MCPD染毒造成的大鼠抗氧化系统破坏,通过抑制MAPK信号通路激活调节支持细胞BTB结构功能,来减轻3-MCPD造成的生精上皮损伤,维持生精微环境稳定并调节生精细胞分裂与分化,改善生精障碍。同时C3G可以通过调节精子能量代谢相关酶,提高精子活性。调节3-MCPD染毒造成的大鼠性激素分泌紊乱,维持血清性激素与睾丸内环境中雄激素正常水平。本发明为以后花色苷对3-MCPD生殖毒性营养干预提供理论基础。(The invention provides application of C3G in preparing an antagonist for resisting the damage of an antioxidant system caused by 3-MCPD contamination. The invention proves that C3G improves the conditions of reduced food intake and growth retardation of rats caused by 3-MCPD infection; and can effectively antagonize the rat antioxidant system damage caused by 3-MCPD contamination, reduce the spermatogenic epithelial injury caused by 3-MCPD by inhibiting MAPK signal channel to activate and regulate the BTB structural function of the supporting cells, maintain the stability of spermatogenic microenvironment, regulate the division and differentiation of spermatogenic cells and improve the spermatogenic disorder. Meanwhile, C3G can improve sperm activity by regulating sperm energy metabolism related enzyme. Regulating the secretion disorder of rat sex hormone caused by 3-MCPD infection, and maintaining the normal level of androgen in serum sex hormone and testis environment. The invention provides theoretical basis for the later nutritional intervention of anthocyanin on reproduction toxicity of 3-MCPD.)

1. Application of cyanidin-3-O-glucoside in preparing antagonist for resisting damage of antioxidant system caused by 3-chloro-1, 2-propylene glycol contamination; the addition amount of the cyanidin-3-O-glucoside is 250-500 mg/kg of feed or medicine.

2. The use of claim 1, wherein said antagonizing comprises modulating the BTB structural function of a supporting cell by inhibiting MAPK signaling pathway activation, alleviating the damage to the spermatogenic epithelium caused by 3-chloro-1, 2-propanediol, maintaining the spermatogenic microenvironment stable and regulating the division and differentiation of spermatogenic cells, ameliorating spermatogenic disorders.

3. The use of claim 1, wherein said antagonizing comprises increasing sperm motility by modulating sperm energy metabolism-related enzymes.

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of C3G in preparation of an antagonist for resisting the damage of an antioxidant system caused by 3-MCPD contamination.

Background

3-chloro-1, 2-propanediol (3-MCPD) is widely available in food and has high toxicity, 3-chloro-1, 2-propanediol ester which is a metabolite of 3-chloro-1, 2-propanediol ester widely available in grease in vivo is 3-chloro-1, 2-propanediol, 3-MCPD is easily absorbed in digestive tract, is widely distributed in body fluid, and can pass through blood-testis barrier and blood-brain barrier to reach testis and brain. Pharmacokinetics indicates that: the 3-MCPD can be widely distributed in all parts of the body after being absorbed by the gastrointestinal tract, most chloropropanol in blood can be metabolized after 24 hours, but the accumulation in the brain can reach 20%, and the accumulation in the testis can reach 60%, so that the testis injury is caused.

cyanidin-3-O-glucoside (C3G) is one of the most abundant anthocyanins in common water-soluble anthocyanins, is the highest anthocyanin (68.3%) in black bean peel, and has strong antioxidant activity.

No protection effect of the anthocyanin on 3-MCPD reproductive injury is reported in the current research, and the research hopes to provide a theoretical basis for the later nutritional intervention of the anthocyanin on 3-MCPD reproductive toxicity.

Disclosure of Invention

The invention provides application of C3G in preparing an antagonist for resisting the damage of an antioxidant system caused by 3-MCPD contamination.

The invention adopts animal experiments, researches the protection effect of different doses of cyanidin-3-O-glucoside on 3-MCPD exposure induced rat testicle injury, and provides a theoretical basis for the nutrition intervention of cyanidin-3-O-glucoside on 3-MCPD reproductive toxicity.

In order to achieve the purpose, the invention adopts the following technical scheme:

application of cyanidin-3-O-glucoside in preparing antagonist for resisting damage of antioxidant system caused by 3-chloro-1, 2-propylene glycol contamination; the addition amount of the cyanidin-3-O-glucoside is 250-500 mg/kg of feed or medicine.

Preferably, the antagonism comprises activating and regulating the BTB structural function of a supporting cell by inhibiting a MAPK signal channel, relieving the damage of spermatogenic epithelium caused by 3-chloro-1, 2-propanediol, maintaining the stability of spermatogenic microenvironment, regulating the division and differentiation of spermatogenic cells and improving spermatogenic disorder.

Preferably, the antagonism comprises increasing sperm motility by modulating an enzyme associated with sperm energy metabolism.

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

the invention discovers that cyanidin-3-O-glucoside (C3G) can improve the conditions of reduced food intake and retarded growth of rats caused by 3-MCPD infection; and can effectively antagonize the rat antioxidant system damage caused by 3-MCPD contamination, reduce the spermatogenic epithelial injury caused by 3-MCPD by inhibiting MAPK signal channel to activate and regulate the BTB structural function of the supporting cells, maintain the stability of spermatogenic microenvironment, regulate the division and differentiation of spermatogenic cells and improve the spermatogenic disorder. Meanwhile, C3G can improve sperm activity by regulating sperm energy metabolism related enzyme. Regulating the secretion disorder of rat sex hormone caused by 3-MCPD infection, and maintaining the normal level of androgen in serum sex hormone and testis environment. The addition of low doses of 250mg/kg to medium doses of 500mg/kg in the feed or medicament showed a better protective effect, whereas the higher doses (1000mg/kg) were not significant.

Drawings

FIG. 1 is a graph of the trend of C3G intervention on rat body weight change;

FIG. 2 is a trend of C3G intervention on rat feeding changes;

FIG. 3 shows the coefficient changes of the mouse organs before and after C3G intervention;

FIG. 4 is a pathological analysis of testis before and after C3G intervention;

FIG. 5 is a pathological analysis of the epididymal head before and after C3G intervention;

FIG. 6 is a pathological analysis of the epididymal tail before and after C3G intervention;

FIG. 7 shows the results of sperm motility/viability assays before and after C3G intervention;

FIG. 8 shows the results of the sperm kinetic parameters before and after C3G intervention;

FIG. 9 shows the results of total sperm count and teratogenicity measurements before and after C3G intervention;

FIG. 10 shows the effect on sperm morphology before and after C3G intervention;

FIG. 11 shows the change in testicular LDH and LDH-X activity before and after C3G intervention;

FIG. 12 is a graph of intratesticular changes in GSH and GSSG content before and after C3G intervention;

FIG. 13 shows the change in intratesticular SOD activity before and after C3G intervention;

FIG. 14 is a graph of the change in testicular MDA levels before and after C3G intervention;

FIG. 15 shows the change in serum inflammatory factor levels in rats before and after C3G intervention;

FIG. 16 is a graph of the immunological impact on sperm cells 8-0HdG before and after C3G intervention;

FIG. 17 shows the changes in serum hormone levels in rats before and after C3G intervention;

FIG. 18 is a graph of the effect on apoptosis of sperm cells TUNEL before and after C3G intervention;

FIG. 19 is a graph of the effect of changes in the apoptotic index of sperm cells before and after C3G intervention;

FIG. 20 shows the change in testis GAPDH protein expression before and after C3G intervention;

FIG. 21 shows the expression changes of testis ERK protein before and after C3G intervention;

FIG. 22 shows the change in testis p38 protein expression before and after C3G intervention;

FIG. 23 shows the change in testicular JNK protein expression before and after intervention with C3G;

FIG. 24 shows the expression changes of testis apoptosis-related protein before and after C3G intervention.

Detailed Description

The present invention is further described below in conjunction with the following detailed description and the appended drawings, wherein examples are illustrated in the accompanying drawings and described below, and some detailed implementations and specific operations are given. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Example 1

1.1 Experimental animals

54 male Wistar rats at 10 weeks of age were purchased from experimental animal centers in Guangdong province. Rats were housed in the SPF animal room of medical college of river-south university, alternating day and night for 12 hours. The temperature is maintained at 22-26 ℃, the relative humidity is 60-80%, and the food can be freely taken.

1.2 animal feed and reagents

The general AIN93-G feed is purchased from Jiangsumeidisen, the AIN93-G feed added with C3G is purchased from Jiangsumeidisen, and the formula of the AIN93-G feed added with C3G is shown in the table 1:

TABLE 1 AIN93-G feed formulation supplemented with C3G

Kind of feed	C3G content (mg/kg feed)
		Common AIN93-G feed	0
Low dose AIN93-G feed	250
		Medium-dose AIN93-G feed	500
High-dose AIN93-G feed	1000

The feed entrusts are processed by the biological medicine of Meidisen of Jiangsu

1.33-MCPD solution preparation

Accurately weighing 5g of 3-MCPD, dissolving in 500mL of normal saline, uniformly stirring, and storing at 4 ℃. Reformulation was performed once a week.

2. The experimental method comprises the following steps: adopts cyanidin-3-O-glucoside pure product with purity of 92%.

2.1 animal grouping and handling

After the rats were fed with the general AIN93-G ad libitum for three weeks, the rats were randomly divided into 6 groups of 9 animals by weight stratification, and the specific groups were as follows:

TABLE 2 animal groups

Group of rats	Kind of feed	3-MCPD
			Normal control	AIN93-G feed	-
3-chloro-1, 2-propanediol	AIN93-G feed	+
			High dose anthocyanin intervention	High-dose AIN93-G feed	+
Medium dose anthocyanin intervention	Medium-dose AIN93-G feed	+
			Low dose anthocyanin intervention	Low dose AIN93-G feed	+
Anthocyanin control	Medium-dose C3GAIN93-G feed	-

Rats were dosed daily with 3-MCPD in a gavage manner at a dose of 20mg/kg. The 3-MCPD preparation concentration is 5mg/mL, and the product is stored at 4 ℃. During the administration period, rats had free access to food and water. The body weight was weighed once every three days and the dose was adjusted. The feed was changed every two days and the food intake was recorded. The feed is stored at-20 deg.C and returned to room temperature when administered to animals.

2.2 animal specimen Collection and measurement

2.2.1 preparation of Experimental reagents

Testicular fixation (mDF): 30 percent of formaldehyde (37 to 40 percent), 15 percent of absolute ethyl alcohol, 5 percent of glacial acetic acid and 50 percent of distilled water.

SDS-PAGE gels (ingredients as shown in Table 3)

TABLE 3 SDS-PAGE gel formulation

Unit: mL

The electrophoresis gel is prepared by sequentially adding the components according to the sequence of a component table, wherein the volume of the four gel blocks is shown in the table. 30% acrylamide, Tris-HCl, 10% SDS, 10% ammonium persulfate, TEMED from kit.

Protein electrophoresis buffer (ingredients shown in Table 4)

TABLE 4 protein electrophoretic fluid formulation

Composition (I)	Dosage of
		Glycine (Gly)	14.4g
Tris-Base	3.03g
		SDS	1g
ddH2O	1000mL

Protein transfer buffer (composition shown in Table 5)

TABLE 5 protein transfer solution buffer formulation

Composition (I)	Dosage of
		Glycine (Gly)	14.4g
Tris-Base	3.03g
		SDS	1g
Methanol	200mL
		ddH2O	800mL

After magnetic stirring and mixing, the mixture is stored at 4 ℃.

Membrane washing buffer (TBST) (ingredients shown in Table 6)

TABLE 6 Membrane Wash buffer formulation

After magnetic stirring and mixing, the mixture is stored at 4 ℃.

5% milk sealing liquid (ingredients as shown in Table 7)

TABLE 75% MILK SEALING LIQUID FORMULATION

Composition (I)	Dosage of
		Defatted milk powder	5g
TBST	100mL

Shaking evenly, standing at 4 ℃ and taking supernatant.

2.2.2 immunohistochemical reagents

(1) Citrate buffer (pH 6.0)

Sodium citrate 1.47g was weighed and dissolved in 500mL ddH₂In O, 4M HCl is used for adjusting the pH value to 6.0, then 0.25mL of Tween-20 is added, and after uniform mixing, the mixture is refrigerated at 4 ℃ for standby.

(2) 5% BSA blocking solution

Weighing 2.5g BSA, dissolving in 50mL PBS, mixing well, and cold preserving for use.

2.3 concrete Experimental methods

2.3.1 obtaining samples of animal specimens

(1) Anaesthesia and heart blood sampling

Rats are continuously dosed for four weeks, and within 24 hours after the last dose, the rats are fasted and are not forbidden to be dosed for 12 hours. Rats were anesthetized with 2% sodium pentobarbital. Collecting blood by heart blood sampling, standing for 30min, centrifuging at 4 deg.C at 3000rpm/min for 15min, and collecting serum, and storing at-80 deg.C.

(2) Perfusion with physiological saline

1.5% EDTA-2Na physiological saline solution was prepared and pre-warmed in a 37 ℃ water bath, and 40mL was perfused into each rat heart.

(3) Taking materials

Dissect rapidly after perfusion, and take the epididymal tail on the right side for sperm determination. And taking testis, epididymis, kidney, liver and spleen, weighing and recording. Weighing right testis and kidney, quickly freezing in dry ice, and storing in-80 deg.C refrigerator for subsequent experiment. The left testis and epididymis are fixed in mDF fixing solution.

2.3.2 sperm motility assay

(1) The water bath was turned on to 37 ℃ in advance, and the DMEM medium was preheated.

(2) And turning on the sperm analyzer, and placing the sperm counting plate on an objective table for preheating.

(3) The right epididymis of the rat is picked up, fat is removed, the tail of the epididymis is cut off and washed in preheated physiological saline to remove blood stains. 5-6 gaps on the epididymis tail are quickly placed into a preheated 50mL DMEM medium to be released for 0.5 h. The medium was gently shaken during sperm release.

(4) After the release is finished, 10 μ L of sperm diluent is uniformly injected into a sperm counting plate for sperm activity and kinetic parameter measurement, and 1000 sperm are counted for each rat. The kinetic parameters were as follows:

VCL curve rate (μm/s), the time-averaged rate of the sperm head along its actual curve, i.e. the two-dimensional motion trajectory seen under the microscope, reflects sperm motility.

VSL linear velocity (μm/s), time-averaged velocity of linear motion of the sperm head between the position at which detection was started and the position at which it was last located.

VAP mean path velocity (μm/s), the time-averaged velocity as the sperm head moves along its mean path.

ALH sperm head yaw amplitude (μm) the amplitude of lateral displacement of the sperm head about its mean path is expressed as the mean value of the yaw.

LIN linearity, linearity of curve trace, VSL/VCL.

WOB wobble, the wobble value of the actual curve path with respect to the average path, VAP/VCL.

STR forward, straightness of the average path, VSL/VAP.

BCF whip frequency (Hz), the average frequency at which the sperm curve path crosses its average path.

MDA average angular displacement (degree), and time average absolute value of the instant turning angle of the sperm head along the curve track.

(5) The sperm morphology was photographed and the total number of sperm and the teratogenicity rate were counted, 1000 sperm per rat.

2.3.3 tissue fixation and HE staining

2.3.3.1 tissue fixation

The testis is put into mDF fixing solution at a ratio of 1:9, and fixed for more than 24 h. Cutting the head and tail of epididymis, and fixing in mDF fixing solution at a ratio of 1:9 for over 24 hr. After the fixation, paraffin embedding is performed, and paraffin sections are prepared.

2.3.3.2 tissue HE staining and photographing

(1) Baking sheet

The slices were baked in a 75 ℃ oven for 2 h.

(2) Dewaxing to water

Dewaxing the slices in xylene for 10min, replacing fresh xylene, dewaxing for 10min, sequentially soaking in anhydrous ethanol for 5min, 95% ethanol for 2min, 90% ethanol for 2min, 80% ethanol for 2min, 70% ethanol for 2min, and distilled water for 2 min.

(3) And (5) dyeing with hematoxylin for 5-10 min, and washing with running water.

(4) The color of the slices fades to light blue-red color after the color separation of 1 percent hydrochloric acid alcohol for several seconds, and the slices are washed clean by running water.

(5) And (4) carrying out eosin dyeing for 3-5 min, and washing with running water.

(6) And (3) dehydrating and transparency: soaking the slices in 70% ethanol for 2min, 80% ethanol for 2min, 90% ethanol for 2min, 95% ethanol for 2min, and anhydrous ethanol for 5min, and allowing xylene to be transparent twice for 10min each time.

(7) And (5) sealing the neutral gum.

(8) Take pictures under a 40-fold objective lens.

(9) Testis tissue was evaluated according to the Makler scoring criteria as follows:

TABLE 8 testis tissue score criteria

Total score of D + M + P + S

2.3.4 testis tissue-associated enzyme assay

2.3.4.1LDH/LDH-X Activity assay

The activity of the LDH can be calculated by utilizing the principle of colorimetry, namely, the pyruvic acid generated by the metabolism of the lactic acid and the 2, 4-dinitrophenylhydrazine dissolved in the acid act to generate the pyruvic acid-dinitrophenylhydrazone which is in grass yellow in an acid environment and brownish red in an alkaline solution, the color depth is in direct proportion to the concentration of the pyruvic acid, and the pyruvic acid-dinitrophenylhydrazone is colorimetric with the phenylhydrazine generated by the pyruvic acid with the standard concentration.

The method specifies the enzyme activity of generating 1 mu mol of pyruvic acid at 37 ℃ for 15min as a unit.

(1) Determination of LDH Activity

1) 10% tissue supernatant preparation

Testis tissues are accurately weighed and added with normal saline according to the mass/volume ratio of 1: 9. Homogenizing until no tissue block is visible, centrifuging at 3500rpm/min and 4 deg.C for 10min, and collecting supernatant. All sample manipulations were performed in an ice bath.

2) Reagents were prepared (see kit instructions).

Preparing a coenzyme 1 application solution: each powder is dissolved in 1.3mL of distilled water, and the mixture is shaken and mixed for standby.

0.2. mu. mol/mL pyruvic acid standard solution: the 4mmol/mL pyruvic acid standard solution is diluted by 10 times and is prepared for use.

3) Preparation of Standard Curve

The pyruvic acid standard solution is diluted by ultrapure water by 200 times, 100 times, 50 times, 20 times, 10 times, 5 times and 2 times in sequence. A 96-well plate was used, and blank wells and standard wells were set. Adding 25 mu L of ultrapure water into the blank hole at one time, uniformly mixing 25 mu L of substrate buffer solution, incubating at 37 ℃ for 15min, adding 25 mu L of 2, 4-dinitrophenylhydrazine, uniformly mixing, carrying out warm bath at 37 ℃ for 15min, adding 250 mu L of 0.4mol/L sodium hydroxide, uniformly mixing, and measuring the light absorption value at 450 nm; 5 mu L of ultrapure water is respectively added into the standard holes, 20 mu L of pyruvic acid standard liquid with different concentrations and 25 mu L of substrate buffer solution are respectively added into the pyruvic acid standard holes, the pyruvic acid standard liquids and the substrate buffer solution are evenly mixed and then bathed for 15min at 37 ℃,2, 4-dinitrophenylhydrazine is added into the pyruvic acid standard holes, 25 mu L of 2, 4-dinitrophenylhydrazine is added into the pyruvic acid standard holes, the pyruvic acid standard liquids and the substrate buffer solution are bathed for 15min at 37 ℃ after being evenly mixed and then added with 250 mu L of 0.4mol/L of sodium hydroxide, and the absorbance value is measured at 450nm after being evenly mixed. And drawing a standard curve by taking the light absorption value as a vertical coordinate and the corresponding standard concentration as a horizontal coordinate.

4) Determination of sample concentration in preliminary experiments

5) According to the standard curve, when the tissue sample concentration in the preliminary experiment is selected to be 0.1%, the formal experiment measurement is carried out. Blank holes, standard holes, measurement holes and control holes are arranged, and the sample adding sequence is as follows:

TABLE 9 LDH Activity assay sample addition sequence

Mixing, standing at room temperature for 5min, and measuring absorbance at 450 nm. The 0.2 mu mol/mL pyruvic acid standard solution, the substrate buffer solution, the coenzyme 1,2, 4-dinitrophenylhydrazine and the sodium hydroxide solution in the reagent come from a kit.

6) BCA assay determination of protein concentration in tissue homogenates: diluting tissue homogenate with 10% of physiological saline to the concentration of 2% for protein quantification; ② 2mg/mL protein standard is respectively diluted into 1mg/mL, 0.5mg/mL, 0.25mg/mL, 0.125mg/mL, 0.0625mg/mL, 0.03125mg/mL and 0mg/mL by ultrapure water; preparing a BCA working solution, and preparing A, B working solution according to a ratio of 50:1 for use; fourthly, adding 10 mu L of standard substance and sample to be detected into a 96-well plate, then adding 200 mu L of working solution into each well, setting three multiple wells for each sample, uniformly mixing, and incubating for 30min at 37 ℃; measuring absorbance value at 562nm, drawing standard curve, and calculating tissue sample protein concentration according to the standard curve.

7) Tissue LDH activity was calculated according to the following formula:

(2) LDH-X Activity assay

LDH-X has thermal stability, and the tissue homogenate is subjected to water bath at 65 ℃ for 30 min. The subsequent assay method was the same as the LDH activity assay.

2.3.4.2GSH/GSSG content determination

The circulating reaction of DNTB is utilized to measure the content of total glutathione and oxidized glutathione in testis tissues.

(1) Determination of T-GSH content

1) Preparing a reagent: reagents required for the determination of T-GSH were formulated with reference to kit instructions.

2) Preparation of tissue supernatant: accurately weighing the tissue weight, adding homogenate at a mass/volume ratio of 1:4, homogenizing, centrifuging at 3500rpm/min4 deg.C for 10min, and collecting supernatant and storing at low temperature for use.

3) Preparation of Standard Curve

Dissolving GSH standard substance in ultrapure water, preparing 1mmol/L stock solution, and diluting to the following concentration: 0. mu. mol/L, 25. mu. mol/L, 50. mu. mol/L, 100. mu. mol/L. Adding reagents in sequence according to the kit specification, uniformly mixing and accurately timing, reading an absorbance value A1 at 405nm for 30s, standing for 10min at room temperature, and reading an absorbance value A2 again for 10min for 30 s. A standard curve was prepared with Δ A (A1-A2) as the ordinate and the concentration as the abscissa.

4) Determination of sample dilution concentration in preliminary experiments

5) According to the standard curve, when the tissue sample concentration in the preliminary experiment is 1.25%, the actual experiment measurement is carried out, 50 mu mol/L GSH standard substance is taken as a standard control, and absorbance values A1 and A2 are measured at 405nm, and delta A is calculated.

6) The T-GSH content was calculated according to the following formula:

T-GSH content (μmol/L) ═ sample measurement Δ a value/GSH standard Δ a value × standard concentration (50 μmol/L) × dilution factor before sample measurement

(2) GSSG content determination

1) The required reagents were formulated with reference to kit instructions.

2) Preparation of Standard Curve

GSSG was dissolved in ultrapure water to prepare 1mmol/L mother liquor, which was diluted to the following concentrations: 0. mu. mol/L, 25. mu. mol/L, 50. mu. mol/L, 100. mu. mol/L. Using a 1.5mL EP tube, 100. mu.L of the standard was added, and then the other reagents were added in order according to the instructions, followed by vortexing for 1min and incubation at 37 ℃ for 30 min. 10 μ L of the sample was measured, and absorbance values A1 and A2 were measured by the T-GSH method, and a standard curve was prepared with Δ A as the ordinate and the concentration as the abscissa.

3) Determination of sample dilution concentration in preliminary experiments

4) According to the standard curve, when the tissue sample concentration in the preliminary experiment is 1.25%, the actual experiment measurement is carried out, the light absorption values A1 and A2 are measured by using 50 mu mol/L GSSG standard substance as a standard control, and the delta A is calculated.

5) The GSSG content was calculated according to the following formula:

GSSG content (μmol/L) — sample assay Δ a value/GSSG standard Δ a value × standard concentration (50 μmol/L) × dilution factor before sample assay

(3) Calculation of GSH content

The GSH content was calculated according to the following formula:

GSH(μmol/L)＝T-GSH－2×GSSG

2.3.4.3SOD Activity measurement

The WST-1(2- (4-Iodophenyl) -5- (2, 4-disulphophenyl) -2H-tetrazolium) method is adopted, namely WST-1 can react with superoxide anions generated by xanthine oxidase to generate water-soluble formazan dye, and the reaction step can be inhibited by SOD. The enzyme activity of SOD can be calculated by colorimetric analysis of WST-1 product.

SOD activity definition: the amount of the enzyme consumed in the reaction system at an inhibition rate of S0D of 50% was one activity unit (U).

(1) Tissue supernatant preparation.

(2) The BCA method determines the protein concentration in the tissue homogenate.

(3) Reagent preparation

Substrate application solution: the buffer solution and the substrate stock solution are prepared in a ratio of 200:1, are used as they are and are stored at low temperature.

Enzyme working solution: the enzyme diluent and the enzyme stock solution are prepared according to the proportion of 10:1, are used as the raw materials when being prepared, and are stored at low temperature.

(4) Determination of sample dilution concentration in preliminary experiments

(5) The tissue homogenate concentration at 50% inhibition in the preliminary experiment was selected for the official experiment. The inhibition of each group was calculated to be about 50% at a tissue homogenate concentration of 0.2%.

(6) Each group of samples was diluted to 0.2% with physiological saline. Loading was performed using 96-well plates and with reference to kit instructions: adding 20 mu L of ultrapure water and 20 mu L of enzyme working solution into the control hole; 20 mu L of sample to be detected and 20 mu L of enzyme working solution are respectively added into the measuring hole; adding 20 mu L of ultrapure water and 20 mu L of enzyme diluent into the control blank hole; and (3) adding 20 mu L of a sample to be detected (only one hole is made) and 20 mu L of enzyme diluent into a blank hole, then adding 200 mu L of substrate application solution into each hole, uniformly mixing, incubating at 37 ℃ for 20min, and measuring the absorbance value at 450 nm. The SOD inhibition was calculated according to the following formula:

SOD inhibition% ((a control-a control blank) - (a assay-a assay blank))/(a control-a control blank) × 100%

The SOD activity was calculated according to the following formula:

SOD activity (U/mgprot) ═ SOD inhibition rate ÷ 50% × 12 (reaction system dilution factor) ÷ sample protein concentration (mgprot/mL)

2.3.5 testis MDA level assay

(1) Tissue supernatant preparation.

(2) The BCA method determines the protein concentration in the tissue homogenate.

(3) Sample application

After reagents are prepared according to the kit specification, a 10mL EP tube is adopted for sample adding, a blank tube, a standard tube, a sample determination tube and a sample control tube are arranged, wherein the blank tube, the standard tube and the sample control tube are respectively provided with one tube. Adding 100 mu L of absolute ethyl alcohol into a blank tube, adding 100 mu L of 10nmol/mL tetraethoxypropane into a standard tube, respectively adding 100 mu L of a sample to be detected into a sample measuring tube and a reference tube, adding prepared reagents according to the specification of the kit, uniformly mixing, carrying out water bath at 95 ℃ for 40min (a test tube opening needs to be sealed by a sealing film, a small hole is punched on a tube cover), taking out, cooling by running water, and centrifuging at 3500rpm/min for 10 min. The supernatant was collected and measured for absorbance at 532nm in a 96-well plate.

(4) Calculated according to the following formula:

2.3.6 serum hormone level determination

Follicle Stimulating Hormone (FSH) and inhibin B (iNH-B), testosterone (T), Dihydrotestosterone (DHT) and Luteinizing Hormone (LH) in rat serum were measured by ELISA (kit available from Wuhan Huamei Biometrics, Inc.).

2.3.7 serum inflammatory factor assay

The serum inflammatory factors TNF-alpha, IL-6 and IFN-gamma of the rat are measured by an ELISA method (an ELISA kit is purchased from Xinbo-Sheng biological Co., Ltd.).

2.3.8TUNEL method for detecting tissue apoptosis

(1) And (6) baking the slices.

(2) Dewaxing and returning water.

(3) Perforating by proteinase K treatment

mu.L of proteinase K (containing no DNase) at 20. mu.g/ml was added dropwise to each section, and the sections were exposed to 27 ℃ for 20min and washed 3 min. times.3 times with PBS.

(4) Incubating at low temperature in 3% hydrogen peroxide for 20min, inactivating endogenous catalase in the tissue, and washing with PBS for 3min × 3 times.

(5) Preparing a biotin labeling solution: in a ratio TdT: prepared according to a ratio of 1:24, and is used as it is. 30 μ L of biotin labeling solution was added dropwise to each section, and incubated at 37 ℃ for 1h in a wet box. PBS was washed once after incubation.

(6) The labeled reaction stop solution was added dropwise, incubated at room temperature for 10min, and washed with PBS 3min X3 times.

(7) In the ratio Streptavidin-HRP: preparing a secondary antibody working solution from a diluent of 1:49, uniformly mixing, dropwise adding 30 mu L of each section, incubating in a room-temperature wet box for 30min, and washing with PBS for 3min and 3 times.

(8) And (5) DAB color development. After mixing the developing solution A, B at an equal ratio, 200. mu.L of the developing solution was added dropwise to each section, and the staining time was determined by observing under a microscope. After staining, PBS was washed clean.

(9) And (5) dyeing with hematoxylin. And (5) observing under a microscope, and determining the dyeing time. After staining, PBS was washed clean.

(10) And (5) dehydrating and transparent.

(11) And sealing the neutral gum, and taking a picture by a microscope for observation.

(12) 400 spermatogenic cells were counted per rat.

2.3.9Western Blot analysis of related proteins

2.3.9.1 protein sample preparation

(1) Tissue protein extraction: accurately weighing testis tissue, adding RIPA lysate (RIPA: Cocktail: PMSF ═ 100:1:1) at a ratio of 1:9, homogenizing until no visible solid is present, centrifuging at 3500rpm/min at 4 deg.C for 20min, and collecting supernatant, and storing at low temperature for later use.

(2) The tissue homogenate was diluted 10-fold with ultrapure water, and the protein concentration was measured by the BCA method.

(3) Protein sample preparation: referring to the results of the preliminary experiment, the total amount of protein loading was determined to be 45. mu.g, and the volume of the protein sample was determined to be 15. mu.L. Protein concentration was determined by BCA and the volume of tissue homogenate required was calculated for a total protein amount of 40 μ g. Each sample was added with Loading Buffer (5X) in a volume of 1/5% of the total volume of the protein sample, and when the volume of the tissue homogenate and the total volume of the Loading Buffer were less than 15. mu.L, ultrapure water was added to make up for 15. mu.L.

(4) The protein sample is placed in boiling water for more than 7min to denature the protein and stored at-20 ℃.

2.3.9.2 protein electrophoresis

(1) Preparation of SDS-PAGE gel

After the glue preparation device is installed, pure water is filled in the glass plate to test the leakage of the device, and the leakage test is successful when the liquid level does not fall within 5 min. Preparing 10% separation gel according to the formula, uniformly adding into the glass plate until the liquid surface is 2cm away from the short glass plate, rapidly adding anhydrous ethanol, coagulating the separation gel at room temperature for 30min, discarding the above anhydrous ethanol, drying with filter paper, adding 5% concentrated gel, rapidly inserting into a comb, and coagulating at room temperature for 40 min.

(2) Disassembling the glass rubber plate, installing and placing the glass rubber plate into an electrophoresis tank, filling electrophoresis buffer solution into the electrophoresis tank, vertically pulling out a comb, sequentially adding the Protein sample and the Protein Ladder, starting constant-pressure electrophoresis, concentrating the gel for 45V for about 45min, and separating the gel for 110V for about 70 min.

2.3.9.3 transfer film

(1) The membrane buffer was prepared in advance and pre-cooled in a refrigerator at 4 ℃.

(2) And (3) immersing the PVDF membrane with the proper size in methanol for activation for 10 min.

(3) Opening the black cathode side of the rotary membrane clamp, and sequentially placing sponge, filter paper, glue, PVDF membrane, filter paper and sponge to form a sandwich structure. Air bubbles between the rubber block and the PVDF membrane are driven out, and the membrane rotating clamp is clamped. The whole process is carried out in a transmembrane buffer.

(4) And (3) loading the film transferring clamp into a film transferring groove, fully filling the film transferring liquid (an ice bag can be placed in the film transferring groove to ensure low temperature), turning on a power supply, regulating the current to 260mA, and starting constant film transferring for 85 min. The entire film transfer was carried out in an ice bath. 2.4.9.4 milk closure

(1) Carefully remove the PVDF membrane into the incubation box, TBST buffer in 3 washing times

(2) 5% skimmed milk blocking solution was prepared according to the formulation in 3.2.5, added to the incubation box, and blocked on a vertical shaker for 1.5h at room temperature.

2.3.9.5 antibody incubation

(1) Primary antibody incubation

1) The blocking solution was discarded and TBST washed 3 times

2) Primary antibody was diluted according to the antibody specification, added to the incubation chamber and incubated overnight on a vertical shaker at 4 ℃.

(2) Incubation with secondary antibody

1) The primary antibody was recovered and the membrane washed with TBST 5 times 7min each time on a horizontal shaker.

2) The secondary antibody was diluted with 5% skim milk, added to the incubation box, and incubated on a vertical shaker at room temperature for 1 h.

2.3.9.6ECL development

(1) The secondary antibody was discarded and the membrane washed with TBST 5 times for 7min each on a horizontal shaker.

(2) ECL luminous liquid is prepared according to the proportion of 1:1(ECL A liquid: ECL B liquid), and is developed in an automatic developing exposure instrument. The grey levels of the protein bands of interest were analyzed using ImageJ image analysis software.

(3) And removing blots on the PVDF membrane by using a primary antibody removing solution and a secondary antibody removing solution, and continuously incubating other target proteins.

2.4.10 immunohistochemical detection of related proteins and DNA Damage

Immunohistochemistry was used to detect Claudin-11, Ocplus, N-cadherin, Androgen Receptor (AR) and 8-hydroxydeoxyguanine (8-hydroxy-2' -deoxyguanosine, 8-OhdG) in testis.

(1) And (6) baking the slices. (2) Dewaxed to water. (3) Antigen retrieval

The sodium citrate buffer was heated to near boiling in a microwave oven and the slides were placed in the sodium citrate buffer and heated for 20 min. After being taken out, the mixture was cooled with running water, washed twice with pure water and then washed 3min × 2 times with PBS.

(4) Incubating at low temperature in 3% hydrogen peroxide for 20min, inactivating endogenous catalase in the tissue, and washing with PBS for 3min × 3 times.

(5) Blocking with 5% BSA for 30 min.

(6) Primary antibody was incubated overnight at 4 ℃ and washed 5min X3 times with PBS.

(7) The secondary antibody was incubated at room temperature for 2h and washed 5min × 3 times with PBS.

(8) DAB staining for 1 min.

(9) Soaking in pure water for 1 min.

(10) And (5) dehydrating and transparent. See 2.4.3.2(6)

(11) And sealing the neutral gum, and taking a picture by a microscope for observation.

2.4 statistical analysis

The results of the experimental data were statistically analyzed using Graph Pad prism6.0 and the results are expressed as Mean ± SEM. The One-way ANOVE is analyzed by adopting One-way variance for comparison among groups; the weight of the rat and the damage degree of the testicular seminiferous epithelium are scored by using Mann-Whitney t-test; the sperm motility index analysis was performed by Kruskal-Wallis Test. Significant differences were noted for # p < 0.05, # p < 0.01, # p < 0.001, # p < 0.05, # p < 0.01 and # p < 0.001.

Example 2: results and analysis:

1. behavioral manifestations during animal feeding

During the administration period of rats, the control group is good in growth state and energetic, the administration group is low in mental state, and each dose of C3G intervention group is good in mental state and energetic. Food intake in each group decreased slightly within two weeks of dosing, food intake stabilized after the second week, and water intake in each group decreased during dosing. No mortality occurred in the rats of each group during the administration period. In addition, due to the fact that individual data are abnormal due to the fact that other external factors such as serum hemolysis, sperm measurement errors and the like occur in the rat processing process, the rats in each group are removed, and the number of the rats is more than or equal to 6.

2. Changes in body weight and food intake of rats

2.1 rat body weight changes

The Control group and the M-C3G Control group were well-grown for four weeks after continuous administration, and there was no significant difference between the two groups. The slow weight increase of the 3-MCPD group is obviously different from that of a control group, the dry prognosis of the C3G is realized by different doses, the slow weight increase of rats caused by the 3-MCPD is obviously improved by the medium dose and the low dose C3G intervention group, and the differential analysis is significant. The improvement effect of the high dose C3G intervention group on the slow weight gain in rats was not evident, and the rate of weight gain in rats was normal within the first week of administration, after which there was a delay in weight gain (see figure 1).

The weight change of the rats before and after intervention is shown in table 1, compared with the Control group, the weight change of the 3-MCPD group is obviously reduced, the low-dose group C3G intervention group has an obvious effect of increasing the weight change of the rats, and the rats are basically recovered to the level of the Control group.

TABLE 10 weight change before and after intervention in rats

Unit: g

Group of	Before intervention	After intervention	Amount of change
				Normal control	414.8±9.607	466.9±9.89	55.33±3.77
3-chloro-1, 2-propanediol	4.5.4±5.409	448.9±7.24	42.83±3.07#
				High dose anthocyanins	407.5±7.028	453.2±8.537	45.73±3.986
Medium-dose anthocyanin	427.7±3.428	475.1±3.798	47.37±1.15
				Low dose anthocyanins	419.4±5.232	476±6.307	56.60±2.41*
Anthocyanin control	414.3±5.046	475.2±7.607	60.96±3.88*

2.2 rat food intake changes

The food intake of rats in each group decreased during the administration period, and then remained stable and increased. The food intake of the 3-MCPD group, the high-dose C3G intervention group and the low-dose C3G intervention group is obviously reduced within two weeks after administration, the food intake of the 3-MCPD group and the low-dose C3G intervention group is increased to the level of the Control group after two weeks, and the food intake of the high-dose C3G intervention group is also increased but is slightly increased. The Control group and the M-C3G group showed consistent changes in food intake, and C3G had no effect on the food intake of rats within a certain dosage range (see FIG. 2). Statistical analysis of the food intake of rats in each group showed no phase significant difference.

2.3 rat organ coefficient changes

Rats were sacrificed four weeks after dosing, organs were taken and the wet-to-weight ratio of each organ, i.e. organ coefficient, was calculated. Compared with the Control group, the 3-MCPD group has obviously reduced testis organ coefficient, the medium-dose and low-dose C3G intervention group has obviously increased testis organ coefficient, and the statistical analysis has significance. Therefore, the testis weight reduction caused by the medium dose and the low dose of C3G is caused by 3-MCPD, and the testis weight reduction has a better improvement effect. The high dose of C3G slightly improved the reduction of testicular organ coefficients by 3-MCPD, but was not statistically significant compared to the 3-MCPD group.

The weight of the epididymis of a rat is obviously reduced after 3-MCPD is infected with virus, the organ coefficient of the epididymis is obviously reduced compared with that of a Control group, the intervention effect of low-dose C3G is obvious after different doses of C3G are adopted, and the organ coefficient of the epididymis is close to the level of the Control group. The medium dose C3G group also showed significant improvement in epididymal weight loss, but there was no significant difference in statistical analysis. Compared with the medium-dose and low-dose C3G groups, the high-dose group C3G had no improvement effect on the decrease in organ coefficient caused by 3-MCPD. 3-MCPD and various doses of C3G had no effect on liver, kidney, and spleen organ coefficients (FIG. 3).

2.4 histopathological analysis of testis and epididymis

2.4.1 analysis of testis histopathology HE

After rat testis is fixed, paraffin embedding is carried out and sections are made, and after HE staining, the tissue morphology of each group of rat testis is observed under a microscope and is photographed and analyzed (figure 4). The rat testis tissues of the Control group and the M-C3G Control group are regularly and tightly arranged, the support cell structure of the spermatogenic epithelium is complete, all levels of spermatogenic cells are clearly visible, and the lumen of the seminiferous tubule is filled with sperms. Compared with the Control group, the 3-MCPD group has the advantages that seminiferous tubules shrink, the diameter of the tubules is obviously reduced, the interstitium is widened, the seminiferous epithelium almost disappears, only a small amount of seminiferous cells are retained in the basal layer, vacuoles with different sizes (shown by blue arrows) appear among the seminiferous epithelium supporting cells, multinucleated giant cells are formed (not shown in the figure), mature sperms do not exist in the lumen, and the seminiferous tubules almost become a cavity. The medium-dose and low-dose C3G intervention groups have the advantages that damage to seminiferous epithelium is obviously improved, the atrophy degree of seminiferous tubules is weakened, the inner diameter is basically normal, the supporting cell structure of the seminiferous epithelium is more compact than that of the 3-MCPD group, vacuoles disappear, the seminiferous cells are kept about 3 layers, mature sperms appear in most of the canalicular lumens of the low-dose C3G intervention group, and desquamated spermatocytes (shown by black arrows) exist in individual canals. Compared with other C3G, the high dose of C3G did not improve the damaged seminal tubules.

Makler scoring of testis tissue showed atrophy of seminal tubules in 3-MCPD group, but within the normal range, the total lesion score was significantly different from that in Control group. After intervention of different doses of C3G, the medium dose and the low dose of C3G have obvious improvement on spermatogenic epithelial injury caused by 3-MCPD, and statistical analysis shows significant difference.

TABLE 11 rat testis tissue Makler score

Grouping	D	M	P	S	D+M+P+S
						Normal control	5	5	5	5	19.79±0.1020
3-chloro-1, 2-propanediol	5	4	1	2	11.49±0.4883###
						High dose anthocyanins	5	4	1	2	12.61±0.6463###
Medium-dose anthocyanin	5	5	3	3	15.81±1.050#**
						Low dose anthocyanins	5	5	3	4	17.58±0.7466#***
Anthocyanin control	5	4	5	5	18.71±0.2625***

2.4.2 Observation of epididymis histopathology of HE

As shown in figure 5, the epididymal head tissue structure was observed and analyzed under an optical microscope, the epididymal structures of the Control group and the M-C3G group were neat, the epithelial basal cells were closely arranged, the cilia of the highly columnar epithelial cells were clear, the free edges were neat, a large number of mature sperm were visible in the tube, and individually exfoliated spermatogenic cells appeared. In the 3-MCPD group, epididymis atrophy, epididymal duct interstitial enlargement and duct wall thickening are obviously seen, the duct almost becomes a cavity, and epithelial cell cilia are messy and irregular. In the medium-dose and low-dose C3G intervention groups, epididymal epithelium was significantly improved, and mature sperm appeared in part of epididymal lumen.

As shown in figure 6, the epididymal tail histopathology was observed and analyzed, and the epididymis morphology of the Control group and the M-C3G Control group was consistent with the epididymal head tissue structure, the main cell structure was orderly, the height of the high columnar epithelial cells was reduced, the resting cilia were shortened, the lumen was filled with sperm, and the exfoliated spermatogenic cells appeared individually. The epididymis tube shape change of the 3-MCPD group and the high-dose C3G intervention group is consistent with the head of the epididymis, mature sperms do not exist in the epididymis tube cavity, epididymis tube epithelium is degenerated, the height of high columnar cells is increased, large pieces of fusion appears in the tube cavity and is a homogeneous substance, and vacuole-like change (black arrows) appears in the cells. The epididymal epithelial cell morphology is improved after the intervention of the medium-dose C3G and the low-dose C3G, basal cells and high columnar cells are basically orderly and closely arranged, and mature sperms are arranged in partial lumens.

The activity of epididymal tail sperm is measured by CASA technology, the sperm activity rate and activity of each group are shown in figure 7, and the sperm activity rate and activity of rats in the Control group and the M-C3G group are higher. The 3-MCPD dry prognosis sperm motility and the survival rate are almost zero. Different doses of C3G are adopted for intervention, the medium-dose group and the low-dose group C3G have better recovery effect on sperm motility rate and activity, 4 rats in each group have better improved activity, and the medium-dose group C3G has significant difference compared with the 3-MCPD group.

The effect of 3-MCPD on sperm kinetic parameters as shown in fig. 8, the decrease in each index after 3-MCPD contamination is almost zero for sperm linear Velocity (VSL), sperm curvilinear Velocity (VCL), sperm pathway Velocity (VAP), sperm whipping frequency (BCF), and sperm Mean Angular Displacement (MAD). Sperm motility was slightly improved after the intervention in the medium and low dose groups, C3G, but there was no difference in the statistical analysis. Sperm lateral swing Amplitude (ALH), significantly reduced after 3-MCPD exposure, and was not improved after each dose of C3G intervention. 3-MCPD has no influence on sperm motility (WOB), Linearity (LIN) and tropism (STR), which indicates that 3-MCPD has no influence on sperm movement mode, and specific numerical values are not listed.

C3G maintaining sperm morphology and density

And (4) taking epididymal tail sperms to measure the sperm density, calculating the total number of the sperms, and counting the sperm teratogenesis rate. The total number of sperm in rats sharply decreases after 3-MCPD infection as shown in FIG. 9 (left), the sperm aberration rate is as high as 100%, and normal sperm are not seen in the 3-MCPD group as shown in FIG. 10, and the sperm density is very low. The total number of sperm was significantly increased after the intervention in the medium dose group C3G, with a difference in statistical analysis compared to the 3-MCPD group. The low dose group C3G also significantly increased the total number of sperm, but there was no difference in the statistical analysis. The medium-dose and low-dose C3G intervention groups both have a reducing effect on the teratocarcinology rate of rats, but the statistical analysis shows no difference. The high-dose C3G intervention group has no improvement effect on the total sperm count and sperm teratospermia of rats.

The teratospermia caused by the 3-MCPD infection is mostly head malformation, and the sperms are headless or unhooked (see red arrows marked in figure 10), so the sperms have no activity. After medium and low dose C3G dry pretreatment, sperm teratogenesis decreased and normal morphology sperm increased (shown by blue arrows in fig. 10).

C3G regulates testicular LDH/LDH-X changes

As shown in FIG. 11, the determination of LDH and LDH-X changes in rat testis tissue showed that LDH enzyme activity was significantly increased after 3-MCPD dry prognosis, and decreased in the low dose group compared with the 3-MCPD group after different doses of C3G dry prognosis, but was not significant by statistical analysis. LDH-X enzyme activity of testis tissues is not obviously changed after 3-MCPD is infected, and LDH-X activity can be obviously improved by a medium-dose C3G intervention group, a low-dose C3G intervention group and a C3G control group. Comparing the LDH-X ratio with the LDH ratio of each group, the LDH-X ratio with the LDH ratio is obviously reduced after the 3-MCPD is infected with the virus, the LDH-X ratio with the LDH ratio is obviously improved after the intervention of the medium dose and the low dose of C3G, and the LDH-X ratio with the LDH ratio is obviously improved compared with the Control group in the C3G Control group. The decreased LDH-X ratio results in sperm maturation and suppression of sperm motility in rats. C3G can significantly improve LDH-X activity to maintain spermatogenesis and maturation.

C3G testis protection antioxidant reduction system

C3G improving GSH and GSSG expression in testis

As shown in figure 12, the results of the determination of the content of GSH and GSSG in the testis of the rat show that the expression of GSH and GSSG in the testis of the rat is obviously reduced after the 3-MCPD is infected with the virus, and the proportion of GSH and GSSG is not obviously influenced. The middle and low dose C3G intervention group and the C3G control group significantly increased intratesticular GSH levels. For intratesticular GSSG expression, the medium dose and the low dose of C3G obviously play an improvement role, and the GSSG expression of the C3G control group is not changed compared with the normal control group. GSH and GSSG can be converted in vivo, the peroxide removed by GSH is oxidized into GSSH, and the GSSG can be reduced into GSH by organism. However, the GSH/GSSG is not obviously influenced after the 3-MCPD is infected with the virus, and the GSH/GSSG ratio is obviously improved in the medium dosage C3G intervention group compared with the Control group. The dosage of C3G in the prescription can obviously improve the level of antioxidant enzyme in the body.

C3G improving SOD activity

SOD catalyzes the excess superoxide anion free radical of organism to be H2O2, and GSH further reduces H2O2 to be H2O. GSH and SOD maintain the oxidation-reduction balance of the organism together. The SOD enzyme activity in the testis is measured, and the result shows that the SOD enzyme activity in the testis is obviously reduced after the 3-MCPD is infected with the virus, and the SOD enzyme activity is obviously improved after the dry prognosis is carried out by C3G with different doses. Compared with the 3-MCPD group, the medium-dose and low-dose C3G intervention groups have significant difference, and the high-dose C3G intervention group also has enhancement effect on SOD enzyme activity, but has no difference in statistical analysis. C3G was effective in improving the decrease in SOD enzyme activity in rats caused by 3-MCPD, and the low dose C3G group was most effective (see FIG. 13).

Testis MDA level

As shown in FIG. 14, the MDA level in rat testis was measured, and it was shown that no increase in MDA level was caused after 3-MCPD infection, and that each dose of C3G had no effect on the change in MDA level. Indicating that 3-MCPD did not cause testicular lipid peroxidation damage.

C3G reducing serum inflammatory factor levels

As shown in FIG. 15, the ELISA method measured changes in the levels of the inflammatory factors TNF-. alpha.IFN-. gamma.and IL-6 in the serum of rats. The results show that the levels of TNF-alpha and IFN-gamma in serum are increased after 3-MCPD is infected with virus, and the low-dose C3G dry-control group has obvious improvement effect on the TNF-alpha. The prognosis of each dose of C3G dry test has an improvement effect on the serum IFN-gamma level of rats, wherein the effect of the medium dose group C3G intervention group is more obvious.

C3G attenuation of spermatogenic cell DNA damage

As shown in FIG. 16, 3-MCPD caused oxidative stress in testis to produce excessive ROS, damaging DNA, while 8-OHdG was a marker product of sensitive ROS damaging DNA. The immunohistochemical method detects the expression of 8-OHdG in testis tissue and analyzes the DNA damage condition of spermatogenic epithelial cells. The results show that the 8-OHdG in the control group is less expressed, and the positive expression in the testis tissues is increased after the 3-MCPD is infected, and the positive expression is mainly distributed in spermatogonium. The C3G intervention group rats have reduced 8-OHdG positive expression in testis tissues, particularly the 8-OHdG expression in spermatogonium is obviously reduced, but the 8-OHdG positive expression is also realized in spermatids, which shows that C3G has protective effect on spermatids obtained in the prophase of the spermatids and antagonizes the damage effect of oxidative stress caused by 3-MCPD on spermatid DNA. Three doses of C3G were effective in reducing DNA damage.

C3G regulation of sex hormone secretion As shown in FIG. 17, ELISA measurement of the content change of FSH, LH, T, DHT and iNH-B in rat serum shows that 3-MCPD can inhibit the secretion of FSH and iNH-B, and has no influence on the level change of T, DHT and LH. The low dose group C3G had a significant improvement in decreased secretion of FSH and iNH-B. The statistical analysis had differences.

C3G inhibiting spermatogenic apoptosis caused by 3-MCPD

The TUNEL method is adopted to detect the apoptosis of spermatogenic cells in testicular spermatogenic epithelium, and the result shows that the apoptosis of the spermatogenic cells is intensified after 3-MCPD is infected with virus. The apoptosis of the sperm cells was significantly improved after the intervention of the medium and low dose of C3G (fig. 18). By calculating the apoptosis indexes of the spermatogenic cells of each group, the result shows that the effect of inhibiting the apoptosis of the spermatogenic cells by the low-dose C3G is most obvious, and the statistical analysis has significant difference (figure 19).

3G regulates testis-associated protein changes

C3G Down-regulated GAPDH expression

After 3-MCPD is infected, spermatogenic cell apoptosis of rats is increased, and the change of GAPDH protein in testis tissues of rats is detected by Western Blot, so that the GAPDH expression is obviously up-regulated after 3-MCPD is infected, which is probably caused by oxidative stress caused by 3-MCPD. C3G could significantly down-regulate GAPDH protein expression, with medium and low doses of C3G providing better regulation of GAPDH expression (fig. 20).

C3G inhibition of MAPK pathway activation

MAPK signal channels can regulate cell growth, proliferation, differentiation and apoptosis, can also regulate testis spermatogenic epithelial structure, and play an important role in maintaining spermatogenic process. ERK1(44kDa), P-ERK1(44kDa) are obviously up-regulated, ERK2(42kDa) is not obviously changed, and P-ERK2(42kDa) is up-regulated in rat testis after 3-MCPD is infected, which indicates that 3-MCPD mainly activates the ERK1 pathway. The C3G intervention group rats restored normal intratesticular ERK and p-ERK levels. p38 and p-p38 were both up-regulated after 3-MCPD infection, and p38 and p-p38 were both down-regulated to normal levels in rats in the C3G intervention group. However, JNK1(46kDa) and p-JNK1(46kDa) were upregulated after 3-MCPD contamination in the JNK signaling pathway, while inhibiting JNK2(54kDa) expression. C3G intervention group rats JNK expression and activation restored normal levels. Of the three different doses of C3G, the medium and low doses of C3G were better.

As the activation and the up-regulation of ERK and JNK1 proteins, the expression of Bax, Bad and p53 proteins in the downstream pathway of the protein is obviously down-regulated after 3-MCPD is infected, and the expression of anti-apoptotic protein Bcl-2 is obviously up-regulated. Inhibition of Bax protein expression in the testis results in atrophy of the seminiferous tubules of the testis, apoptosis of spermatogenic cells and appearance of multinucleated giant cells. The p53 protein is involved in regulating meiosis of spermatocytes, and when the expression of the protein is inhibited, multinucleated giant cells are caused to appear, and spermatogenic cell apoptosis is initiated. C3G intervened in the recovery of the expression of Bax, Bad, Bcl-2 and p53 proteins in rat testis to normal level (shown in FIGS. 21-24).

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.