Composition for relieving and repairing testis injury caused by heat stress and application thereof

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a composition for relieving and repairing testis injury caused by heat stress and application of the composition. The composition for alleviating and repairing testis injury caused by heat stress comprises the following raw materials: a lycopene inclusion compound, ginsenoside Re, modified chitosan nanoparticles and a solvent, the use amount of the modified chitosan nanoparticles is 10 to 15 weight percent of the ginsenoside Re. A combined formula with lycopene and ginsenoside Re as cores is innovatively designed, the two main effective components in the formula are natural plant or traditional Chinese medicine extracts, conflicts between traditional Chinese medicine properties are effectively avoided, and the lycopene and ginsenoside Re combined formula has good biological safety and is suitable for being used as a traditional Chinese medicine preparation. The composition can be used for remarkably repairing testis injury caused by heat stress and accelerating recovery after heat stress injury.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to a composition for alleviating and repairing testicular damage caused by heat stress and an application thereof. Background Art

[0002] As an important part of the male reproductive system, the testicle is an organ that produces sperm and secretes androgens. Its substantial part is mainly composed of seminiferous tubules, which play an important role in the occurrence and deformation of sperm, the maintenance of male secondary sexual characteristics and sexual function, and the process of individual development. Spermatogenesis is a complex physiological process and an important basis for male reproduction. The haploid sperm produced by meiosis during spermatogenesis has a highly specialized structure and poor stress resistance, and is easily affected by the external environment. Therefore, this process has strict requirements for the maintenance of the spermatogenic microenvironment in the seminiferous tubules of the testicles, and is particularly sensitive to temperature.

[0003] However, due to various reasons such as environment, living habits, and pathology, testicular temperature can rise and induce testicular heat stress (HS) damage, which in turn causes male reproductive diseases. When the scrotal temperature rises, it can cause testicular atrophy, a large number of spermatogenic cells to undergo apoptosis, leading to spermatogenesis stagnation, a decrease in sperm count, and impaired motility and acrosome function of mature sperm. Data show that for every 1°C increase in testicular temperature, spermatogenesis efficiency decreases by about 14%. A large number of in-depth studies have shown that when the testicular temperature of mice is at 42°C for more than 20 minutes, it will significantly cause testicular and spermatogenesis abnormalities. The main reason is that the blood-testis barrier is damaged due to heat stress, which in turn destroys the homeostasis of the environment in the seminiferous tubules.

[0004] As the main cell type in the seminiferous tubules, the testicular supporting cells play an important role in supporting and nourishing germ cells. The blood-testis barrier formed by tight junctions between supporting cells is an important barrier to maintain the spermatogenic microenvironment. When heat stress occurs, it will first break, which will cause an imbalance in the homeostasis of the spermatogenic microenvironment and subsequent serious damage to spermatogenic cells and testicular tissues. Although some studies have shown that this damage will repair itself within a period of time after the heat stress disappears, the vast majority of cells in the seminiferous tubules will inevitably suffer from severe apoptosis during this period. At the same time, it is not certain whether this reversible damage will have long-term effects on the male reproductive system. Some studies have found that after a single heat stress injury to the mouse testicles, heat stress testicular damage will occur rapidly and will repair itself within a period of time. However, long-term observations have found that mice have secondary testicular atrophy, but the specific mechanism is still unclear.

[0005] There are many potential heat stress scenarios in real life, such as high temperature outdoor weather, high temperature workplace, poor air circulation in enclosed spaces and other environmental problems, sitting for long periods of time, cycling, saunas, hot baths and other lifestyle problems, obesity, cryptorchidism, varicocele, systemic fever and other pathological problems can easily induce heat stress damage to the male reproductive system, but the related issues have not received much attention from the public. Many people have not yet realized the potential harm this problem brings to men, and there are few related products and clinical applications.

[0006] In recent years, more and more research has been conducted on the treatment strategies for male reproductive system damage caused by heat stress. The relevant research mainly focuses on the efficacy evaluation of natural product extracts, traditional Chinese medicinal materials, chemical synthetic substances and other types of ingredients. Lycopene, as one of the most reported ingredients in natural plant extracts, has significant theoretical advantages over other ingredients. First, it can actively enrich testicular tissue to alleviate oxidative stress damage caused by heat stress with its strong antioxidant and anti-inflammatory effects. Secondly, it can regulate cell apoptosis and proliferation by regulating the insulin-like growth factor 1 (IGF1) signaling pathway. In addition, it can also effectively protect the level of tight junction protein (ZO-1), a key component protein of the blood-testis barrier, thereby protecting the problem of supporting cell-cell connection structure and the integrity of the blood-testis barrier. However, most of the relevant research is still in the theoretical stage. Traditional Chinese medicines and their active ingredients are also related to the repair of heat stress damage. Common ingredients include icariin, Lycium barbarum polysaccharides, ginsenoside Re (GRe), astragaloside, etc. A large number of studies have shown that they can be used to rescue animals or cell damage caused by heat stress, but most studies have only observed their antioxidant properties and the protection of some key cell functions, and have not studied and discussed the specific mechanisms and recovery after damage, and there may also be conflicts between the medicinal properties of Chinese medicines. In recent years, there have been many related studies on the use of chemical synthetic substances in the treatment of heat stress damage. Among them, melatonin, nicotinamide mononucleotide, vitamin E, N-acetylcysteine ​​and other ingredients have been studied and analyzed. The data show that this type of compound can play a role through antioxidant, anti-inflammatory, methylation regulation and targeted regulation of cell apoptosis-related signaling pathways, which can effectively improve the damage caused by heat stress to the male reproductive system and accelerate the repair of damage. However, chemical synthetic substances include many chemical synthesis processes, which are complicated to operate and have high production costs.

[0007] Therefore, the development of a composition for alleviating and repairing testicular damage caused by heat stress in men can provide certain data support for the diagnosis and treatment of clinical heat stress-related diseases and basic translational research, and can also provide a feasible option for the protection of men's daily reproductive health. Summary of the invention

[0008] The present invention discloses a composition for alleviating and repairing testicular damage caused by heat stress and its application. An innovative combination formula with lycopene and ginsenoside Re as the core is designed. The two main active ingredients in the formula are both natural plant or Chinese herbal medicine extracts, which effectively avoids the conflict between the medicinal properties of Chinese herbal medicines and has good biosafety. The composition can significantly repair testicular damage caused by heat stress and accelerate the recovery of testicular heat stress damage.

[0009] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0010] The first aspect of the present invention provides a composition for alleviating and repairing testicular damage caused by heat stress, comprising the following raw materials: lycopene inclusion compound, ginsenoside Re, modified chitosan nanoparticles, and solvent;

[0011] The amount of the modified chitosan nanoparticles is 10-15 wt % of ginsenoside Re.

[0012] In some embodiments, the solvent includes but is not limited to pure water or physiological saline.

[0013] In some embodiments, the molar ratio of lycopene to ginsenoside Re in the lycopene inclusion complex is (1-4):(1-4).

[0014] Preferably, the molar ratio of lycopene to ginsenoside Re in the lycopene inclusion complex is 1:1.

[0015] In some embodiments, the preparation steps of the lycopene inclusion compound are:

[0016] After vitamin E, lycopene and hydroxypropyl-γ-cyclodextrin are mixed evenly, the first portion of deionized water is added, and the mixture is ground in a water bath at 20-30° C. for 1-2 hours. The second portion of deionized water is added, and the mixture is centrifuged. The supernatant is dried to a constant weight to obtain a lycopene inclusion complex.

[0017] In some embodiments, the mass ratio of vitamin E, lycopene, and hydroxypropyl-γ-cyclodextrin is 1:(1-1.5):(80-90).

[0018] Preferably, the mass ratio of vitamin E, lycopene and hydroxypropyl-γ-cyclodextrin is 1:1.25:85.

[0019] In some embodiments, the amount of the first portion of deionized water added is 2-3 times the total mass of vitamin E, lycopene, and hydroxypropyl-γ-cyclodextrin.

[0020] In some embodiments, the preparation steps of the modified chitosan nanoparticles are:

[0021] (1) dissolving chitosan in an aqueous solution of glacial acetic acid, adjusting the pH value to 5-6, adding chitosanase for enzymatic hydrolysis for 3-4 hours, filtering, and then ultrafiltering the filtrate, and freeze-drying to obtain low molecular weight chitosan;

[0022] (2) dissolving the low molecular weight chitosan obtained in step (1) in a poloxamer aqueous solution, adjusting the pH to 4.5-5.0, filtering, adding a sodium tripolyphosphate aqueous solution under stirring, stirring for 1.5-2 hours, purifying, concentrating, and freeze-drying to obtain chitosan nanoparticles;

[0023] (3) The chitosan nanoparticles prepared in step (2) are dissolved in an aqueous solution of glacial acetic acid, mixed with an aqueous solution of glucosamine, the pH value is adjusted to 5-5.5, reacted at 60-70° C. for 60-80 h, allowed to stand in an ice bath for 10-30 min, dialyzed for purification, and soaked in distilled water for 1-3 days to obtain modified chitosan nanoparticles.

[0024] In some embodiments, the chitosan has a deacetylation degree of ≥90% and a dynamic viscosity of 300-500 mPa·s at 20°C.

[0025] In some embodiments, the chitosanase has an activity of ≥100,000 U / g, and its usage is 1-1.5 wt % of the chitosan.

[0026] Preferably, the chitosanase is used in an amount of 1.25 wt % of chitosan.

[0027] In some embodiments, the mass ratio of the chitosan nanoparticles to glucosamine is 1:(3.2-3.5).

[0028] Preferably, the mass ratio of the chitosan nanoparticles to glucosamine is 1:3.25.

[0029] The second aspect of the present invention provides an application of the composition described in the above scheme in the field of biomedicine.

[0030] The present invention selects lycopene and ginsenoside Re as the effective ingredients for treating heat stress injury in men in the composition, and for the first time applies and studies these two ingredients in reducing testicular damage caused by heat stress in men and accelerating the recovery rate of heat stress injury. From a theoretical perspective, lycopene and ginsenoside Re have excellent complementarity, wherein lycopene mainly reduces the damage of oxidative stress to the testicles caused by heat stress through antioxidant effects, and also reduces testicular damage caused by heat stress through its protective effect on key components of the blood-testis barrier protein; while ginsenoside Re mainly affects testicular damage caused by heat stress and the post-injury repair process by exerting a protective effect on the thermal stability of key protein structures and regulating the dynamic process of stress particles in the repair of heat stress injury. From a safety perspective, lycopene and ginsenoside Re are both natural plant or Chinese herbal medicine extracts, which not only avoid unnecessary chemical synthesis processes, but also avoid conflicts between the medicinal properties of Chinese medicines. At the same time, both ingredients have a large dosage safety window, and the selected dosages are far less than toxic doses.

[0031] However, lycopene is a fat-soluble substance while ginsenoside Re is a water-soluble substance. The solubility of the two components is quite different. Simple blending not only affects the efficacy, but also the system of the composition is extremely unstable, which is not conducive to preservation. The present invention includes vitamin E and lycopene in hydroxypropyl-γ-cyclodextrin by controlling the grinding method to form a stable inclusion compound, which can significantly improve the solubility and stability of the two, and improve the efficacy of repairing male heat stress damage. The applicant found that by forming an inclusion compound with hydroxypropyl-γ-cyclodextrin, the solubility of fat-soluble vitamin E and lycopene in water can be significantly improved; at the same time, the formation of the inclusion compound can also protect the two from the influence of light, heat and oxygen, and improve their chemical stability, antioxidant activity and efficacy. This may be due to: first, vitamin E and lycopene are both powerful antioxidants. When used together, on the one hand, they can produce a synergistic effect, scavenge free radicals, and improve the antioxidant effect; on the other hand, they can complement each other, enhance the protective effect on cells, and improve reproductive function. Secondly, the inclusion complex formed by using hydroxypropyl-γ-cyclodextrin can improve the bioavailability, pharmacokinetic properties and targeted release of vitamin E and lycopene, reduce their irritation to the gastrointestinal tract, improve their therapeutic effects and reduce side effects.

[0032] In addition, although ginsenoside Re has good water solubility, its bioavailability needs to be improved. The present invention uses modified chitosan nanoparticles together with ginsenoside Re to improve the stability and bioavailability of ginsenoside Re. The present invention first uses chitosanase to enzymolyze high molecular weight chitosan to form low molecular weight chitosan, and then makes it into nanoparticles with specific structural characteristics, and then uses glucosamine to modify it, and forms a polyelectrolyte complex through ionic interaction. The obtained modified chitosan nanoparticles have excellent biocompatibility and biodegradability, and can be used as an effective carrier to achieve the encapsulation and controlled release of ginsenoside Re, and improve the water solubility and bioavailability of ginsenoside Re. In addition, the modified chitosan nanoparticles can also improve the stability of ginsenoside Re, prevent its degradation during storage and use, and improve its absorption and utilization in vivo.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] 1. The present invention provides a composition for alleviating and repairing testicular damage caused by heat stress. An innovative combination formula with lycopene and ginsenoside Re as the core is designed. The two main active ingredients in the formula are both natural plant or Chinese herbal medicine extracts, which effectively avoids the conflict between the medicinal properties of Chinese herbal medicines and has good biosafety. The composition can significantly repair testicular damage caused by heat stress and accelerate the recovery of testicular heat stress damage.

[0035] 2. The present invention can significantly improve the solubility of vitamin E and lycopene in water by preparing an inclusion compound with vitamin E, lycopene and hydroxypropyl-γ-cyclodextrin; at the same time, the formation of the inclusion compound can also protect the two from the influence of light, heat and oxygen, thereby improving their chemical stability, antioxidant activity and efficacy.

[0036] 3. The present invention prepares a modified chitosan nanoparticle with excellent biocompatibility and biodegradability, which can be used as an effective carrier to achieve the encapsulation and controlled release of ginsenoside Re, thereby improving the water solubility and bioavailability of ginsenoside Re. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 HE staining images of testicular sections of mice in each experimental group at different times in experiment 3.2;

[0038] Figure 2 The testicular sections of mice in each experimental group at different time points in the experiment 3.2 are TUNEL staining images;

[0039] Figure 3 The fluorescence staining images of the testicular sections of mice in each experimental group at different time points in the experiment 3.2;

[0040] Figure 4This is the expression level of testicular tissue-related proteins in each experimental group of mice at different time points in experiment 3.2. DETAILED DESCRIPTION

[0041] Now describe in detail various exemplary embodiments of the present invention, this detailed description should not be considered as limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present invention. It should be understood that the terms described in the present invention are only for describing a particular embodiment, and are not used to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the range.

[0042] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. Various modifications and variations may be made to the specific embodiments of the present invention specification without departing from the scope or spirit of the present invention, which will be apparent to those skilled in the art. Other embodiments obtained from the present invention specification will be apparent to the technician. The present application specification and examples are exemplary only.

[0043] The words “include,” “including,” “have,” or “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0044] It should be noted that the operations of "grinding", "centrifugation", "filtration", "ultrafiltration", "stirring" and "freeze drying" described in the present invention are routine operations for those skilled in the art and can be selected according to actual operations.

[0045] The mass fractions described in the following embodiments may be grams, kilograms, tons or other mass units.

[0046] The poloxamer used in the present invention is poloxamer 188, and the dialysis bag used for dialysis has a MWCO of 3000Da; the neutral gum (10004160) used was purchased from Shanghai Hushi Co., Ltd.; the Tunel kit used was purchased from Novozyme Biotechnology Co., Ltd. (A113); the DAPI, lysis buffer (P0013), and BCA kit (P0012) used were all purchased from Bio-Tech Biotechnology Co., Ltd. (P0131); the ZO-1 (21773-1-AP), G3B P1 (13057-2-AP) was purchased from Wuhan Sanying Biotechnology Co., Ltd.; the electrophoresis buffer (G2142) and transfer buffer (G2028) used were purchased from Sevier Biotechnology Co., Ltd.; the ECL luminescent liquid used was purchased from Shanghai Tianneng Life Science Co., Ltd. (180-501); the deacetylation degree of the chitosan used = 90%, and the dynamic viscosity at 20°C is 400mPa·s; the chitosanase activity used = 100,000 U / g; the other related reagents used can be purchased from the market.

[0047] Preparation Example 1

[0048] The preparation steps of lycopene inclusion compound are as follows:

[0049] Take 0.1g vitamin E, 0.125g lycopene, and 8.5g hydroxypropyl-γ-cyclodextrin and mix them evenly, then add 21.8g deionized water, grind them for 1.5h in a 25°C water bath at a grinding speed of 150r / min, then add 10g deionized water, centrifuge, and dry the obtained supernatant at 45°C to constant weight to obtain a lycopene inclusion complex.

[0050] Preparation Example 2

[0051] The preparation steps of the lycopene inclusion compound are the same as those in Preparation Example 1, except that 0.5 g of vitamin E is used.

[0052] Preparation Example 3

[0053] The preparation steps of the lycopene inclusion compound are the same as those in Preparation Example 1, except that the first portion of deionized water used is 15 g.

[0054] Preparation Example 4

[0055] The preparation steps of the lycopene inclusion compound are the same as those of Preparation Example 1, except that vitamin E is not added.

[0056] Preparation Example 5

[0057] The preparation steps of the lycopene inclusion compound are the same as those of Preparation Example 1, except that an equal amount of hydroxypropyl-β-cyclodextrin is used instead of hydroxypropyl-γ-cyclodextrin.

[0058] Preparation Example 6

[0059] The preparation steps of modified chitosan nanoparticles are:

[0060] (1) Dissolve 4 g of chitosan in 100 mL of 1 wt% glacial acetic acid aqueous solution, adjust the pH value to 5.5, add 0.05 g of chitosanase for enzymatic hydrolysis for 3.5 h, filter, and then ultrafilter the filtrate and freeze-dry to obtain low molecular weight chitosan;

[0061] (2) 1 g of the low molecular weight chitosan prepared in step (1) was dissolved in 1 L of a 0.005 wt% poloxamer aqueous solution, the pH was adjusted to 5.0, and the mixture was filtered. Under stirring conditions, 400 mL of a 0.1 wt% sodium tripolyphosphate aqueous solution was added dropwise at a rate of 20 mL / h, and the mixture was stirred at 1500 rpm and 30° C. for 1.5 h. The mixture was purified by ultrafiltration, concentrated, and freeze-dried to obtain chitosan nanoparticles.

[0062] (3) Take 1 g of the chitosan nanoparticles prepared in step (2) and dissolve it in 150 mL of a 1 wt% glacial acetic acid aqueous solution, mix it with 50 mL of an aqueous solution containing 3.25 g of glucosamine, adjust the pH to 5, react at 65° C. for 70 h, let it stand in an ice bath at 0° C. for 20 min, dialysis purification, and soak it in distilled water for 2 days to obtain modified chitosan nanoparticles.

[0063] Preparation Example 7

[0064] The preparation steps of modified chitosan nanoparticles are the same as those in Preparation Example 6, except that 0.025 g of chitosanase is used.

[0065] Preparation Example 8

[0066] The preparation steps of modified chitosan nanoparticles are the same as those in Preparation Example 6, except that 3 g of glucosamine is used.

[0067] Preparation Example 9

[0068] The preparation steps of chitosan nanoparticles are as follows:

[0069] (1) Dissolve 4 g of chitosan in 100 mL of 1 wt% glacial acetic acid aqueous solution, adjust the pH value to 5.5, add 0.05 g of chitosanase for enzymatic hydrolysis for 3.5 h, filter, and then ultrafilter the filtrate and freeze-dry to obtain low molecular weight chitosan;

[0070] (2) Take 1 g of the low molecular weight chitosan of step (1) and dissolve it in 1 L of 0.005 wt% poloxamer aqueous solution, adjust the pH to 5.0, filter, and under stirring, drop 400 mL of 0.1 wt% sodium tripolyphosphate aqueous solution at a rate of 20 mL / h, stir at 1500 rpm and 30° C. for 1.5 h, purify by ultrafiltration, concentrate, and freeze-dry to obtain chitosan nanoparticles.

[0071] Example 1

[0072] A composition for alleviating and repairing testicular damage caused by heat stress, comprising the following raw materials: lycopene inclusion compound, ginsenoside Re, modified chitosan nanoparticles, and pure water;

[0073] The molar ratio of lycopene and ginsenoside Re in the lycopene inclusion compound is 1:1, and the concentration is 5 nM;

[0074] The amount of the modified chitosan nanoparticles is 12 wt % of ginsenoside Re.

[0075] The lycopene inclusion compound used is obtained from Preparation Example 1; the modified chitosan nanoparticles used are obtained from Preparation Example 6.

[0076] Example 2

[0077] A composition for alleviating and repairing testicular damage caused by heat stress, comprising the following raw materials: lycopene inclusion compound, ginsenoside Re, modified chitosan nanoparticles, and pure water;

[0078] The molar ratio of lycopene and ginsenoside Re in the lycopene inclusion compound is 1:2, and the concentrations are 5 nM and 10 nM respectively;

[0079] The amount of the modified chitosan nanoparticles is 10 wt % of ginsenoside Re.

[0080] The lycopene inclusion compound used is obtained from Preparation Example 1; the modified chitosan nanoparticles used are obtained from Preparation Example 6.

[0081] Example 3

[0082] A composition for alleviating and repairing testicular damage caused by heat stress, comprising the following raw materials: lycopene inclusion compound, ginsenoside Re, modified chitosan nanoparticles, and pure water;

[0083] The molar ratio of lycopene and ginsenoside Re in the lycopene inclusion compound is 2:1, and the concentrations are 10 nM and 5 nM respectively;

[0084] The amount of the modified chitosan nanoparticles is 15 wt % of ginsenoside Re.

[0085] The lycopene inclusion compound used is obtained from Preparation Example 1; the modified chitosan nanoparticles used are obtained from Preparation Example 6.

[0086] Example 4

[0087] A composition for alleviating and repairing testicular damage caused by heat stress, comprising the following raw materials: lycopene inclusion compound, ginsenoside Re, modified chitosan nanoparticles, and pure water;

[0088] The molar ratio of lycopene and ginsenoside Re in the lycopene inclusion compound is 1:4, and the concentrations are 2.5 nM and 10 nM respectively;

[0089] The amount of the modified chitosan nanoparticles is 11 wt % of ginsenoside Re.

[0090] The lycopene inclusion compound used is obtained from Preparation Example 1; the modified chitosan nanoparticles used are obtained from Preparation Example 6.

[0091] Example 5

[0092] A composition for alleviating and repairing testicular damage caused by heat stress, comprising the following raw materials: lycopene inclusion compound, ginsenoside Re, modified chitosan nanoparticles, and pure water;

[0093] The molar ratio of lycopene and ginsenoside Re in the lycopene inclusion compound is 4:1, and the concentrations are 10 nM and 2.5 nM respectively;

[0094] The amount of the modified chitosan nanoparticles is 14 wt % of ginsenoside Re.

[0095] The lycopene inclusion compound used is obtained from Preparation Example 1; the modified chitosan nanoparticles used are obtained from Preparation Example 6.

[0096] Example 6

[0097] A composition for alleviating and repairing testicular damage caused by heat stress, comprising the following raw materials: lycopene inclusion compound, ginsenoside Re, modified chitosan nanoparticles, and pure water;

[0098] The molar ratio of lycopene to ginsenoside Re in the lycopene inclusion compound is 1:1;

[0099] The amount of the modified chitosan nanoparticles is 12 wt % of ginsenoside Re.

[0100] The lycopene inclusion compound used is obtained from Preparation Example 1; the modified chitosan nanoparticles used are obtained from Preparation Example 6.

[0101] Performance Testing

[0102] 1. The lycopene inclusion complex of Preparation Example 1-5 was subjected to the following tests:

[0103] (1) Lycopene inclusion rate test: Take 1g of lycopene inclusion complex m0, dissolve it in 10mL of toluene, record it as V0, dilute it by a certain multiple according to the actual concentration, record it as n, measure the absorbance at 484nm, repeat three times and take the average value, substitute the absorbance value into the standard curve to obtain the lycopene concentration C;

[0104] Drawing of standard curve: Take lycopene and dilute it to 50mL with toluene, prepare test solutions of 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0μg / mL respectively, use toluene as reference solution, measure the absorbance at 484nm, repeat three times and take the average value to draw a standard curve.

[0105] The lycopene content was calculated according to the following formula: content (μg / mg) = (n×C×V0) / m0;

[0106] The lycopene inclusion rate was calculated according to the following formula: Inclusion rate (%) = 100 × (content × m) / m 初 ;

[0107] Wherein, m is the total mass of lycopene inclusion complex / g; m 初 is the initial mass of lycopene added during the preparation of the inclusion complex / mg.

[0108] (2) Stability: Take 1g of lycopene complex inclusion compound, and prepare a sample solution with a lycopene mass concentration of 15 μg / mL using toluene according to the lycopene content obtained in (1), and take this as the initial mass concentration. After heating the sample solution in a water bath at 40°C for 48 hours, measure the absorbance of the solution at a wavelength of 484nm, calculate the lycopene content according to the above calculation formula, and then calculate the lycopene preservation rate according to the following formula: preservation rate (%) = 100 × (mass concentration of remaining lycopene in the solution / initial mass concentration of lycopene). Lycopene and toluene are used to prepare a 15 μg / mL control sample solution, and its preservation rate after heating in a water bath is measured to be 22.5%.

[0109] The specific test results are shown in Table 1.

[0110] Table 1

[0111]

[0112]

[0113] As shown in Table 1, the lycopene inclusion complex obtained in Preparation Example 1 has a high inclusion rate of lycopene and good stability. Compared with Preparation Example 1, the amount of vitamin E used in Preparation Example 2 is changed, the deionized water added during grinding in Preparation Example 3 is changed, and vitamin E is not added in Preparation Example 4, all of which will affect the inclusion of lycopene by hydroxypropyl-γ-cyclodextrin and the stability of the inclusion complex; hydroxypropyl-β-cyclodextrin is used instead of hydroxypropyl-γ-cyclodextrin in Preparation Example 5, and the inclusion effect of hydroxypropyl-β-cyclodextrin on lycopene is worse than that of hydroxypropyl-γ-cyclodextrin, which will also affect the stability of the lycopene inclusion complex.

[0114] 2. Carry out Caco-2 cell transport experiment on the modified chitosan nanoparticles / chitosan nanoparticles of Preparation Examples 6-9:

[0115] The successfully constructed Caco-2 model was selected for transport experiments. Before the transport experiment, the DMEM culture medium was removed and the monolayer cells were washed twice with preheated PBS solution; 0.5 mL of HBSS solution with pH = 6.5 was added to the AP side, and 1.5 mL of HBSS solution with pH = 7.4 was added to the BL side, and the cells were placed in an incubator for 30 minutes of equilibration. After the equilibration, the blank equilibration solution on the AP side was replaced with the test solution. The test solutions were grouped as follows: blank HBSS solution, Group 1 was 50 μg / mL ginsenoside Re solution, Group 2 was 50 μg / mL ginsenoside Re solution + 7.5 μg / mL modified chitosan nanoparticles (Preparation Example 6) solution, Group 3 was 50 μg / mL ginsenoside Re solution + 7.5 μg / mL modified chitosan nanoparticles (Preparation Example 7) solution, Group 4 was 50 μg / mL ginsenoside Re solution + 7.5 μg / mL modified chitosan nanoparticles (Preparation Example 8) solution, and Group 5 was 50 μg / mL ginsenoside Re solution + 7.5 μg / mL modified chitosan nanoparticles (Preparation Example 9) solution. 1.5 mL of blank HBSS solution was added to the BL side as a receiving pool.

[0116] After the above treatment, the Transwell was placed in a cell culture incubator, and 100 μL of samples were taken from the BL side at 30, 60, 90, and 120 min, respectively, and an equal volume of blank HBSS solution was added. The content of ginsenoside Re in the samples was measured by UPLC-MS / MS, and the permeability of ginsenoside Re was expressed as the apparent permeability coefficient (Papp), which was calculated according to the following formula: Papp = ΔQ / (ΔtAC0); where ΔQ / Δt is the permeation rate, A is the effective membrane area of ​​the Transwell chamber, and C0 is the initial mass concentration of ginsenoside Re.

[0117] The specific test results are shown in Table 2.

[0118] Table 2

[0119]

[0120]

[0121] It can be seen from Table 2 that the modified chitosan nanoparticles obtained in Preparation Example 6 can significantly promote the permeability of ginsenoside Re; compared with Preparation Example 6, the preparation process of the modified chitosan nanoparticles in Preparation Examples 7-8 has changed, which changes the particle size and performance of the modified chitosan nanoparticles, affecting their permeability to ginsenoside Re; in Preparation Example 9, glucosamine was not used to modify the chitosan nanoparticles, and the permeability of the chitosan nanoparticles to ginsenoside Re was not significantly improved.

[0122] 3. The following performance tests were performed on the compositions obtained in the examples:

[0123] 3.1 Cell test: Cell experiments were performed on the compositions obtained in Examples 1-5. Cell suspensions (100 μL / well) were inoculated in a 96-well plate, and the culture plates were placed in an incubator for pre-culture at 37°C for 24 hours; the compositions to be tested in Examples 1-5 were added to the culture plates, and the culture plates were placed in an incubator for incubation at 37°C for 24 hours; 10 μL of CCK8 solution was added to each well, and the culture plates were placed in an incubator for incubation at 37°C for 4 hours; the absorbance at 450 nm was determined using an ELISA instrument, and each group was measured three times to take the average value. An equal volume of 0.9 wt% normal saline was used instead of the composition (NC).

[0124] The specific test results are shown in Table 3.

[0125] Table 3

[0126]

[0127] As shown in Table 3, when the dosage of lycopene Lyc / ginsenoside GRe is 5nM / 5nM, the cell viability reaches the maximum value, so the composition (LR03) for alleviating and repairing testicular damage caused by heat stress obtained in Example 6 was used to carry out the following animal experiments.

[0128] 3.2 Animal testing

[0129] Sixty C57B6J mice weighing 22±2.5g were selected and randomly divided into 12 groups, with 5 mice in each group, including HS groups on days 1, 7, 14, and 21 (model group, mice were placed in a water bath at 42℃ for 20min to induce heat stress); HS+GRe groups on days 1, 7, 14, and 21 (mice were placed in a water bath at 42℃ for 20min and then fed with ginsenoside Re at a feeding amount of 42.24μmol / kg); The HS+LR03 group on days 14 and 21 (the mice were placed in a water bath at 42°C for 20 min, and then fed with the composition of Example 6, with the feeding amount controlled to be 42.24 μmol / kg for both ginsenoside Re and lycopene Lyc), was given the drug by oral gavage once a day; after modeling, samples were collected from 5 mice in each group on days 2, 8, 15, and 22, and the testicular weights (bilateral) were recorded and photographed, and one side was fixed with MDF and the other side was frozen at -20°C.

[0130] During the modeling process, double antibodies were added to the water bath system to reduce infection of the wounds of mice after anesthesia. At the same time, the mice were placed in a 50mL centrifuge tube and their limbs were fixed to ensure that only the testicles and the limbs below were fully immersed in water. Heat stress mice were successfully constructed, and no infection occurred subsequently.

[0131] (1) HE staining

[0132] The fixed testicles were embedded in paraffin and then sliced, and the paraffin sections were stained with HE to observe the histological changes of the seminiferous tubules. The specific operation is as follows:

[0133] The sections were dewaxed in a 65°C oven for 2 hours; the sections were immersed in xylene and incubated at 37°C twice for 15 minutes each time; incubated in anhydrous ethanol twice for 2 minutes each time; incubated in a 90wt% ethanol aqueous solution for 2 minutes, incubated in an 80wt% ethanol aqueous solution for 2 minutes; incubated in a 70wt% ethanol aqueous solution for 2 minutes; rinsed with dH2O for 10 minutes; stained with hematoxylin for 5 minutes; rinsed with running water for 10 minutes, differentiated with a 1wt% hydrochloric acid aqueous solution for 30 seconds, and rinsed with running water for 10 minutes; stained with eosin for 2.5 minutes; the sections were immersed in a 70wt% ethanol aqueous solution and incubated for 2 minutes; incubated in an 80wt% ethanol aqueous solution for 2 minutes; incubated in a 90wt% ethanol aqueous solution for 2 minutes; incubated in anhydrous ethanol twice for 2 minutes each time; incubated in xylene twice for 15 minutes each time; and sealed with a neutral resin.

[0134] For specific test results, see Figure 1 .

[0135] from Figure 1It can be seen that heat stress stimulation will cause damage to the seminiferous tubules of mice, and a large number of spermatogenic cells will undergo apoptosis and fall off in the testis, forming a tubular cavity with only supporting cells. The degree of damage reached a peak on the 7th day, and then there was significant spontaneous recovery of damage on the 21st day. The test group fed with ginsenoside Re and the test group fed with the composition of Example 1 can significantly reduce the maximum degree of damage on the 7th day, and achieve better recovery effects on the 21st day, and the HS+LR03 group (the group administered with Example 1) has the best recovery effect.

[0136] (2) TUNEL staining

[0137] The fixed testes were embedded in paraffin and then sliced, and TUNEL staining was performed on the paraffin sections to observe the changes in seminiferous tubule apoptosis. The specific operation is as follows:

[0138] The sections were placed in a 65°C oven for dewaxing for 2 h; the sections were immersed in xylene and incubated at 37°C twice for 15 min each time; incubated in anhydrous ethanol twice for 2 min each time; incubated in 90wt% ethanol aqueous solution for 2 min, incubated in 80wt% ethanol aqueous solution for 2 min; incubated in 70wt% ethanol aqueous solution for 2 min; rinsed with dH2O for 10 min; diluted 2 mg / mL Proteinase K solution with 1×PBS at a ratio of 1:100, 100 μL of the diluted Proteinase K solution was added to each sample, incubated at 25°C for 20 min, washed 3 times with PBS solution for 5 min each time; then 100 μL of 1×EquilibrationBuffer was added to cover the sample area to be tested, and equilibrated at 25°C for 20 min; after the equilibration, the Equilibration Buffer was aspirated, and then 50 μL of TdT was added to the sample, incubated at 25°C for 60 min, washed 3 times with PBS for 5 min each time; and sealed with DAPI.

[0139] For specific test results, see Figure 2 .

[0140] from Figure 2 It can be seen that heat stress stimulation will lead to apoptosis in the lumen of seminiferous tubules (TUNEL). The level of lumen apoptosis in the heat stress HS group reached a peak on the 7th day, and then recovered with time; while the HS+GRe group and HS+LR03 group had the highest level of lumen apoptosis on the 1st day, and then recovered with time, and the HS+LR03 group (the group administered with medicine in Example 1) had the best effect.

[0141] (3) Perform fluorescent staining on paraffin sections to detect the protein levels of ZO-1 and G3BP1. The specific operation is as follows:

[0142] The sections were dewaxed in a 65°C oven for 2 h, immersed in xylene, incubated twice at 37°C for 15 min each time; incubated twice in anhydrous ethanol for 2 min each time; incubated in 90wt% ethanol aqueous solution for 2 min, incubated in 80wt% ethanol aqueous solution for 2 min; incubated in 70wt% ethanol aqueous solution for 2 min; rinsed with dH2O for 10 min; incubated in 0.5% Triton at 37°C for 15 min, incubated at 25°C for 30 min, washed 3 times with PBS solution for 5 min each time; blocked with 2.5% BSA at 25°C for 2 h; incubated with primary antibody at 4°C for 12 h, washed 3 times with PBS for 5 min each time; incubated with secondary antibody at 25°C in the dark for 2 h, washed 3 times with PBS for 5 min each time; and sealed with DAPI.

[0143] For specific test results, see Figure 3 .

[0144] ZO-1 is a key structural unit of the blood-testis barrier. The blood-testis barrier can effectively maintain the stability of the physical and chemical environment for sperm formation in the testis, which plays an extremely important role in sperm formation and testicular development. Figure 3 It can be seen that after heat stress stimulation, ZO-1 levels decreased, and then ZO-1 recovered over time, with a significant recovery effect at 14 days; G3BP1 is a key protein in the assembly of stress granules. Stress granules are involved in the recruitment, degradation, and protein remodeling of extensive protein damage repair in cells after heat stress injury. It is crucial for cell damage repair after heat stress injury. The increase in G3BP1 levels represents more severe heat stress injury, and also indicates that the repair process after injury is not complete. Figure 3 As shown, after heat stress stimulation, G3BP1 aggregated and then degraded over time, with a significant degradation effect at 14 days.

[0145] The HS+GRe group and the HS+LR03 group were able to significantly inhibit the aggregation of G3BP1 and the decrease of ZO-1 after heat stress stimulation due to feeding ginsenoside Re and the composition of Example 1. And the degradation of G3BP1 and the recovery of ZO-1 in the HS+LR03 group were better than those in the HS+GRe group. G3BP1 is an indicator protein of stress granules. The aggregation of G3BP1 proves the formation of stress granules, and the degradation of G3BP1 proves the degradation of stress granules; ZO-1 is an important protein in the formation of the blood-testis barrier BTB. The reduction of ZO-1 proves the damage of the blood-testis barrier, and the recovery of ZO-1 proves the repair of the blood-testis barrier. Ginsenoside Re and the composition of Example 1 can accelerate the degradation of stress granules and the repair of the blood-testis barrier, thereby protecting the seminiferous tubules, and the composition of Example 1 has the best effect.

[0146] (4) Extract tissue proteins from frozen testes and use immunoblotting to analyze the changes in related proteins.

[0147] The specific operations are as follows:

[0148] a. Protein extraction: Take mouse testicles, remove the capsule, add lysis buffer, place on ice and sonicate every 30 seconds until the tissue becomes liquid, centrifuge at 4°C, and transfer the upper wash to a 1.5 ml EP tube;

[0149] b. Protein concentration determination: Use the BCA kit to prepare the working solution, make a standard curve, add samples, add 200 μL of the working solution to each well, incubate at 37°C for 30 min, detect the absorbance with an enzyme-labeled instrument, calculate the protein concentration, and denature the protein at 95°C for 5 min.

[0150] c. Western Blot: Prepare electrophoresis buffer, transfer buffer, gel, calculate the sample loading amount according to the sample concentration (30 ng / well); MARKER (3 μL), 85V constant voltage electrophoresis until the MARKER is completely separated, about 30 min; change to 125V constant voltage electrophoresis until the bottom band of the MARKER is close to the bottom of the gel, about 1 h. PVDF membrane (6×9cm): soak in methanol for 30s (wake up the membrane), wash 3 times with dd H2O, cut the corner as a mark, soak in transfer buffer, and place in the order from bottom to top: white clips-sponge-filter paper-PVDF membrane-gel-filter paper-sponge-black clips, exhaust all air; transfer at 300mA for 100min, block with 5% skim milk at room temperature for 2h, cut the required bands, wash the membrane 3 times with TBST, 5min each time; prepare the primary antibody according to the instructions, incubate at 4℃ overnight; wash the membrane 3 times with TBST, 5min each time; prepare the secondary antibody according to the instructions, incubate at room temperature for 2h; wash the membrane 3 times with TBST, 5min each time;

[0151] d. Develop with an exposure instrument; drop ECL luminescent liquid on the membrane immediately after preparation and use, and expose and save the image using a gel imaging system.

[0152] For specific test results, see Figure 4 .

[0153] Depend on Figure 4It can be seen that after heat stress stimulation, the ZO-1 protein level in the testicular tissue of each group of mice increased over time and increased significantly on the 21st day. ZO-1 is a key structural unit of the blood-testis barrier. The blood-testis barrier can effectively maintain the stability of the physical and chemical environment for sperm formation in the testis, which plays an extremely important role in sperm formation and testicular development; the G3BP1 protein level decreased over time and decreased significantly on the 21st day. G3BP1 is a key protein for the assembly of stress granules. Stress granules are involved in the recruitment, degradation, and remodeling of proteins in the process of extensive protein damage repair in cells after heat stress injury. It is crucial for the repair of cell damage after heat stress injury. The increase in G3BP1 levels represents more severe heat stress injury, and also indicates that the repair process after injury is not complete. The implementation results showed that the blood-testis barrier structure of each group showed a significant low level immediately after heat stress stimulation, and then gradually recovered over time. The recovery speed and degree of the HS+LR03 group were significantly improved; and the HS+LR03 group (the group administered in Example 1) was able to significantly reduce the level of stress granules in the testis after heat stress stimulation, and at the same time accelerate the degradation speed and degree of stress granules.

[0154] The above is only a preferred embodiment of the present invention and does not constitute any form of limitation to the present application. Although the present application is disclosed as above in the preferred embodiment, it is not intended to limit the present application. Any technician familiar with the profession, without departing from the scope of the technical solution of the present application, using the technical content disclosed above to make slight changes or modifications are equivalent to equivalent implementation cases. However, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution.

Claims

1. A composition for alleviating and repairing testicular damage caused by heat stress, characterized in that: The invention comprises the following raw materials: lycopene inclusion compound, ginsenoside Re, modified chitosan nanoparticles and solvent; The amount of the modified chitosan nanoparticles is 10-15 wt % of ginsenoside Re.

2. The composition according to claim 1, characterized in that The molar ratio of lycopene to ginsenoside Re in the lycopene inclusion compound is (1-4):(1-4).

3. The composition according to claim 1, characterized in that The preparation steps of the lycopene inclusion compound are as follows: After vitamin E, lycopene and hydroxypropyl-γ-cyclodextrin are mixed evenly, the first portion of deionized water is added, and the mixture is ground in a water bath at 20-30° C. for 1-2 hours. The second portion of deionized water is added, and the mixture is centrifuged. The supernatant is dried to a constant weight to obtain a lycopene inclusion complex.

4. The composition according to claim 1, characterized in that The mass ratio of the vitamin E, lycopene and hydroxypropyl-γ-cyclodextrin is 1:(1-1.5):(80-90).

5. The composition according to claim 1, characterized in that The amount of the first portion of deionized water added is 2-3 times the total mass of vitamin E, lycopene, and hydroxypropyl-γ-cyclodextrin.

6. The composition according to claim 1, characterized in that The preparation steps of the modified chitosan nanoparticles are: (1) dissolving chitosan in an aqueous solution of glacial acetic acid, adjusting the pH value to 5-6, adding chitosanase for enzymatic hydrolysis for 3-4 hours, filtering, and then ultrafiltering the filtrate, and freeze-drying to obtain low molecular weight chitosan; (2) dissolving the low molecular weight chitosan obtained in step (1) in a poloxamer aqueous solution, adjusting the pH to 4.5-5.0, filtering, adding a sodium tripolyphosphate aqueous solution under stirring, stirring for 1.5-2 hours, purifying, concentrating, and freeze-drying to obtain chitosan nanoparticles; (3) The chitosan nanoparticles prepared in step (2) are dissolved in an aqueous solution of glacial acetic acid, mixed with an aqueous solution of glucosamine, the pH value is adjusted to 5-5.5, reacted at 60-70° C. for 60-80 h, allowed to stand in an ice bath for 10-30 min, dialyzed for purification, and soaked in distilled water for 1-3 days to obtain modified chitosan nanoparticles.

7. The composition according to claim 6, characterized in that The deacetylation degree of the chitosan is ≥90%, and the dynamic viscosity at 20° C. is 300-500 mPa·s.

8. The composition according to claim 6, characterized in that The chitosanase has an activity of ≥100,000 U / g and is used in an amount of 1-1.5 wt% of the chitosan.

9. The composition according to claim 6, characterized in that The mass ratio of the chitosan nanoparticles to glucosamine is 1:(3.2-3.5).

10. Use of the composition for alleviating and repairing testicular damage caused by heat stress as claimed in any one of claims 1 to 9 in the field of biomedicine.

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