A composition for alleviating, repairing testicular damage caused by heat stress and use thereof

Abstract

The application belongs to the technical field of medicine, and particularly relates to a composition for alleviating and repairing testicular damage caused by heat stress and application thereof. The composition for alleviating and repairing testicular damage caused by heat stress comprises the following raw materials: lycopene inclusion compound, ginsenoside Re, modified chitosan nanoparticles and a solvent. The amount of the modified chitosan nanoparticles is 10-15 wt% of the ginsenoside Re. The application innovatively designs a combination formula with lycopene and ginsenoside Re as the core. Both of the two main effective components in the formula are natural plant or traditional Chinese medicine extract, which effectively avoids the conflict between the medicinal properties of traditional Chinese medicines and has good biological safety. The composition can significantly repair the testicular damage caused by heat stress and accelerate the recovery after heat stress damage.

Description

Technical Field

[0001] This invention belongs to the field of medical technology, specifically relating to a composition for alleviating and repairing testicular damage caused by heat stress and its application. Background Technology

[0002] The testes, an important component of the male reproductive system, are the organs that produce sperm and secrete androgens. Their parenchyma is mainly composed of seminiferous tubules, which play a crucial role in spermatogenesis and transformation, the maintenance of male secondary sexual characteristics and sexual function, and individual development. Spermatogenesis is a complex physiological process and a fundamental basis for male reproduction. The haploid sperm produced during meiosis in spermatogenesis are highly specialized in structure and have poor resilience, making them extremely susceptible to external environmental influences. Therefore, this process places strict requirements on maintaining the spermatogenic microenvironment within the seminiferous tubules, especially exhibiting high sensitivity to temperature.

[0003] However, various factors such as environment, lifestyle, and pathology can lead to elevated testicular temperature, inducing testicular heat stress (HS) damage and subsequently causing male reproductive diseases. Elevated scrotal temperature can cause testicular atrophy, massive apoptosis of spermatogenic cells leading to spermatogenesis arrest, reduced sperm count, impaired mature sperm motility, and acrosome function. Data shows that for every 1°C increase in testicular temperature, spermatogenesis efficiency decreases by approximately 14%. Extensive research has shown that maintaining a testicular temperature of 42°C or higher in mice for more than 20 minutes significantly causes testicular and spermatogenesis abnormalities, primarily due to damage to the blood-testis barrier caused by heat stress, which disrupts the homeostasis of the seminiferous tubule environment.

[0004] Sertoli cells, the main cell type within the seminiferous tubules, play a crucial role in supporting and nourishing germ cells. The blood-testis barrier, formed by tight junctions between Sertoli cells, is a vital barrier maintaining the spermatogenic microenvironment. During heat stress, this barrier is the first to break, leading to an imbalance in the spermatogenic microenvironment and subsequent severe damage to spermatogenic cells and testicular tissue. Although some studies show that this damage repairs itself for a period after the heat stress subsides, most cells within the seminiferous tubules inevitably undergo severe apoptosis during this period. Furthermore, it remains uncertain whether this reversible damage will have long-term effects on the male reproductive system. Some studies have found that mice subjected to a single heat stress injury experience rapid heat stress testicular damage that repairs itself within a short period; however, long-term observation has revealed secondary testicular atrophy in these mice. The specific mechanism of this reversible damage remains unclear.

[0005] In real life, there are many potential heat stress scenarios, such as environmental problems like high outdoor temperatures, high-temperature workplaces, and poor air circulation in enclosed spaces, as well as lifestyle habits like prolonged sitting, cycling, saunas, and hot baths. Pathological problems such as obesity, cryptorchidism, varicocele, and systemic fever can all easily induce heat stress damage to the male reproductive system. However, these issues have not received much attention from the public, and many people are unaware of the potential harm this problem can cause to men. Furthermore, there are few related products available or used clinically.

[0006] In recent years, research on treatment strategies for heat stress-induced damage to the male reproductive system has increased significantly, with related studies mainly focusing on efficacy evaluations of components such as natural product extracts, traditional Chinese medicines, and chemically synthesized substances. Among natural plant extracts, lycopene, as one of the most frequently reported components, has significant theoretical advantages compared to other components. First, it can actively accumulate in testicular tissue, mitigating oxidative stress damage caused by heat stress through its potent antioxidant and anti-inflammatory effects. Second, it can regulate cell apoptosis and proliferation by modulating the insulin-like growth factor 1 (IGF1) signaling pathway. Furthermore, it can effectively protect the level of tight junction protein (ZO-1), a key component of the blood-testis barrier, thereby protecting the intercellular connection structures and the integrity of the blood-testis barrier. However, most related research remains at the theoretical stage. Traditional Chinese medicinal herbs and their active ingredients also have certain connections with the repair of heat stress injury. Common components include icariin, wolfberry polysaccharides, ginsenoside Re (GRe), and astragalin. Numerous studies have shown their potential for rescuing heat stress-induced damage to animals or cells. However, most studies have only observed their antioxidant properties and protection of some key cellular functions, without exploring specific mechanisms or post-injury recovery. Furthermore, there may be conflicts between the properties of different Chinese medicinal herbs. In recent years, there has been considerable research on the use of chemically synthesized substances for the treatment of heat stress injury. Melatonin, nicotinamide mononucleotide, vitamin E, and N-acetylcysteine ​​have all been analyzed. Data shows that these compounds exert their effects through antioxidant, anti-inflammatory, methylation-regulating, and targeted regulation of apoptosis-related signaling pathways, effectively improving heat stress-induced damage to the male reproductive system and accelerating repair. However, chemically synthesized substances involve many complex chemical synthesis processes, resulting in high production costs.

[0007] Therefore, developing a composition to alleviate and repair testicular damage caused by heat stress in men can provide 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 men's daily reproductive health protection. Summary of the Invention

[0008] This invention discloses a composition for alleviating and repairing testicular damage caused by heat stress and its application. It innovatively designs a combination formula with lycopene and ginsenoside Re as the core. The two main active ingredients in this formula are extracts of natural plants or Chinese medicinal materials, which effectively avoids the conflict between the properties of Chinese medicines and has good biocompatibility. This composition can significantly repair testicular damage caused by heat stress and accelerate the recovery of testicular heat stress injury.

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

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

[0011] The amount of the modified chitosan nanoparticles used 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 complex are as follows:

[0016] After mixing vitamin E, lycopene, and hydroxypropyl-γ-cyclodextrin evenly, add the first portion of deionized water and grind for 1-2 hours in a water bath at 20-30℃. Then add the second portion of deionized water, centrifuge, and dry the supernatant to constant weight to obtain the 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 as follows:

[0021] (1) Dissolve chitosan in glacial acetic acid aqueous solution, adjust the pH value to 5-6, add chitosanase for enzymatic hydrolysis for 3-4 hours, filter, then ultrafilter the filtrate, freeze dry to obtain low molecular weight chitosan.

[0022] (2) Dissolve the low molecular weight chitosan from step (1) in poloxam aqueous solution, adjust the pH to 4.5-5.0, filter, add sodium tripolyphosphate aqueous solution under stirring, stir for 1.5-2h, purify, concentrate, freeze dry to obtain chitosan nanoparticles.

[0023] (3) Dissolve the chitosan nanoparticles from step (2) in an aqueous solution of glacial acetic acid, mix with an aqueous solution of glucosamine, adjust the pH to 5-5.5, react at 60-70℃ for 60-80h, let stand in an ice bath for 10-30min, dialyze to purify, and soak in distilled water for 1-3 days to obtain modified chitosan nanoparticles.

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

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

[0026] Preferably, the amount of chitosanase used is 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 chitosan nanoparticles to glucosamine is 1:3.25.

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

[0030] This invention selects lycopene and ginsenoside Re as effective components in a composition for treating heat stress injury in men. It is the first time that these two components have been applied and studied to reduce testicular damage caused by heat stress and accelerate the recovery rate of heat stress injury in men. From a theoretical perspective, lycopene and ginsenoside Re have excellent complementarity. Lycopene mainly reduces testicular damage caused by oxidative stress due to heat stress through its antioxidant effects and also reduces testicular damage caused by heat stress through its protective effect on key constituent proteins of the blood-testis barrier. Ginsenoside Re mainly affects testicular damage caused by heat stress and the post-damage repair process by protecting the thermal stability of key protein structures and regulating the stress particle dynamics process in heat stress injury repair. From a safety perspective, both lycopene and ginsenoside Re are extracts from natural plants or traditional Chinese medicines, avoiding unnecessary chemical synthesis processes and conflicts between the properties of traditional Chinese medicines. Furthermore, both components have a large dose safety window, and the selected doses are far below the toxic dose.

[0031] However, lycopene is a fat-soluble substance while ginsenoside Re is a water-soluble substance. The two components have significantly different solubilities, and simple mixing not only affects efficacy but also results in an extremely unstable composition that is difficult to preserve. This invention uses a controlled grinding method to encapsulate vitamin E and lycopene in hydroxypropyl-γ-cyclodextrin, forming a stable inclusion complex. This significantly improves the solubility and stability of both, and enhances the therapeutic effect in repairing heat stress damage in men. The applicant discovered that forming an inclusion complex with hydroxypropyl-γ-cyclodextrin significantly improves the solubility of fat-soluble vitamin E and lycopene in water. Simultaneously, the formation of the inclusion complex protects both from the effects of light, heat, and oxygen, improving their chemical stability, antioxidant activity, and efficacy. This may be because: firstly, vitamin E and lycopene are both potent antioxidants; when used together, they can produce a synergistic effect, scavenging free radicals and enhancing antioxidant effects; secondly, they can complement each other, enhancing cell protection and improving 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 gastrointestinal irritation, enhance their therapeutic effects, and reduce side effects.

[0032] Furthermore, although ginsenoside Re has good water solubility, its bioavailability needs improvement. This invention utilizes modified chitosan nanoparticles in conjunction with ginsenoside Re, which can improve the stability and bioavailability of ginsenoside Re. This invention first uses chitosanase to enzymatically hydrolyze high molecular weight chitosan to form low molecular weight chitosan, then processes it into nanoparticles with specific structural characteristics, and finally modifies them with glucosamine. Through ionic interactions, a polyelectrolyte complex is formed, resulting in modified chitosan nanoparticles with excellent biocompatibility and biodegradability. These nanoparticles can serve as an effective carrier for the encapsulation and controlled release of ginsenoside Re, improving its water solubility and bioavailability. In addition, these modified chitosan nanoparticles can also improve the stability of ginsenoside Re, preventing its degradation during storage and use, and enhancing its absorption and utilization in vivo.

[0033] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0034] 1. This invention provides a composition for alleviating and repairing testicular damage caused by heat stress. It innovatively designs a combination formula with lycopene and ginsenoside Re as the core. The two main active ingredients in this formula are extracts of natural plants or Chinese medicinal herbs, which effectively avoids the conflict between the properties of Chinese medicines and has good biocompatibility. This composition can significantly repair testicular damage caused by heat stress and accelerate the recovery of testicular heat stress injury.

[0035] 2. This invention refines vitamin E, lycopene, and hydroxypropyl-γ-cyclodextrin into an inclusion complex, which can significantly improve the solubility of vitamin E and lycopene in water. At the same time, the formation of the inclusion complex can protect the two from the effects of light, heat and oxygen, and improve their chemical stability, antioxidant activity and efficacy.

[0036] 3. This invention prepares a modified chitosan nanoparticle with excellent biocompatibility and biodegradability. It can serve as an effective carrier to encapsulate and control the release of ginsenoside Re, thereby improving the water solubility and bioavailability of ginsenoside Re. Attached Figure Description

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

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

[0039] Figure 3 The images show fluorescent staining images of testicular sections from mice in each experimental group at different time points during experiment 3.2.

[0040] Figure 4The expression levels of testicular-related proteins in mice of each experimental group at different time points during experiment 3.2. Detailed Implementation

[0041] Various exemplary embodiments of the present invention are now described in detail. This detailed description should not be considered as a limitation of the invention, but rather as a more detailed description of certain aspects, features, and embodiments of the invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, regarding numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in the invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0042] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of the invention. Various modifications and variations to the specific embodiments described in this specification are apparent to those skilled in the art without departing from the scope or spirit of the invention. Other embodiments derived from this specification will be apparent to those skilled in the art. This application specification and embodiments are merely exemplary.

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

[0044] It should be noted that the operations such as "grinding", "centrifugation", "filtration", "ultrafiltration", "stirring" and "freeze-drying" described in this invention are conventional operations for those skilled in the art, and should be selected according to actual operation.

[0045] The mass fractions mentioned in the following examples may be in grams, kilograms, tons, or other units of mass.

[0046] The poloxam used in this invention is poloxam 188, and the dialysis bag used for dialysis has an MWCO of 3000 Da; the neutral resin (10004160) used was purchased from Shanghai Hushi Co., Ltd.; the Tunel kit used was purchased from Novizan Biotechnology Co., Ltd. (A113); the DAPI, lysis buffer (P0013), and BCA kit (P0012) used were all purchased from Beyotime Biotechnology Co., Ltd. (P0131). ZO-1 (21773-1-AP) and G3B were also purchased from Beyotime Biotechnology Co., Ltd. (P0131). P1 (13057-2-AP) was purchased from Wuhan Sanying Biotechnology Co., Ltd.; the electrophoresis buffer (G2142) and transfer buffer (G2028) used were purchased from Savill Biotechnology Co., Ltd.; the ECL luminescence solution used was purchased from Shanghai Tianneng Life Science Co., Ltd. (180-501); the degree of deacetylation of the chitosan used was 90%, and the dynamic viscosity at 20℃ was 400 mPa·s; the activity of the chitosanase used was 100,000 U / g; all other related reagents used were available from the market.

[0047] Preparation Example 1

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

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

[0050] Preparation Example 2

[0051] The preparation steps for the lycopene inclusion complex were the same as in Preparation Example 1, except that 0.5g of vitamin E was used.

[0052] Preparation Example 3

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

[0054] Preparation Example 4

[0055] The preparation steps for the lycopene inclusion complex were the same as in Preparation Example 1, except that vitamin E was not added.

[0056] Preparation Example 5

[0057] The preparation steps for the lycopene inclusion complex were the same as those in Preparation Example 1, except that an equal mass of hydroxypropyl-β-cyclodextrin was used instead of hydroxypropyl-γ-cyclodextrin.

[0058] Preparation Example 6

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

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

[0061] (2) Take 1g of low molecular weight chitosan from step (1) and dissolve it in 1L of 0.005wt% poloxam aqueous solution. Adjust the pH to 5.0, filter, and add 400mL of 0.1wt% sodium tripolyphosphate aqueous solution dropwise at a rate of 20mL / h under stirring conditions. Stir at 1500rpm and 30℃ for 1.5h, purify by ultrafiltration, concentrate, freeze dry, and obtain chitosan nanoparticles.

[0062] (3) Take 1g of chitosan nanoparticles from step (2) and dissolve them in 150mL of 1wt% glacial acetic acid aqueous solution. Mix them with 50mL of aqueous solution containing 3.25g of glucosamine. Adjust the pH to 5 and react at 65℃ for 70h. Then, let them stand in an ice bath at 0℃ for 20min, dialyze to purify them, and soak them in distilled water for 2 days to obtain modified chitosan nanoparticles.

[0063] Preparation Example 7

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

[0065] Preparation Example 8

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

[0067] Preparation Example 9

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

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

[0070] (2) Take 1g of low molecular weight chitosan from step (1) and dissolve it in 1L of 0.005wt% poloxam aqueous solution. Adjust the pH to 5.0, filter, and add 400mL of 0.1wt% sodium tripolyphosphate aqueous solution dropwise at a rate of 20mL / h under stirring conditions. Stir at 1500rpm and 30℃ for 1.5h, purify by ultrafiltration, concentrate, freeze dry, and obtain chitosan nanoparticles.

[0071] Example 1

[0072] A composition for mitigating and repairing testicular damage caused by heat stress, comprising the following ingredients: lycopene inclusion complex, ginsenoside Re, modified chitosan nanoparticles, and purified water;

[0073] The lycopene inclusion complex contains lycopene and ginsenoside Re in a molar ratio of 1:1, both with a concentration of 5 nM.

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

[0075] The lycopene inclusion complex used was obtained from Preparation Example 1; the modified chitosan nanoparticles used were obtained from Preparation Example 6.

[0076] Example 2

[0077] A composition for mitigating and repairing testicular damage caused by heat stress, comprising the following ingredients: lycopene inclusion complex, ginsenoside Re, modified chitosan nanoparticles, and purified water;

[0078] The lycopene inclusion complex contains lycopene and ginsenoside Re in a molar ratio of 1:2 and concentrations of 5 nM and 10 nM, respectively.

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

[0080] The lycopene inclusion complex used was obtained from Preparation Example 1; the modified chitosan nanoparticles used were obtained from Preparation Example 6.

[0081] Example 3

[0082] A composition for mitigating and repairing testicular damage caused by heat stress, comprising the following ingredients: lycopene inclusion complex, ginsenoside Re, modified chitosan nanoparticles, and purified water;

[0083] The lycopene inclusion complex contains lycopene and ginsenoside Re in a molar ratio of 2:1 and concentrations of 10 nM and 5 nM, respectively.

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

[0085] The lycopene inclusion complex used was obtained from Preparation Example 1; the modified chitosan nanoparticles used were obtained from Preparation Example 6.

[0086] Example 4

[0087] A composition for mitigating and repairing testicular damage caused by heat stress, comprising the following ingredients: lycopene inclusion complex, ginsenoside Re, modified chitosan nanoparticles, and purified water;

[0088] The lycopene inclusion complex contains lycopene and ginsenoside Re in a molar ratio of 1:4 and concentrations of 2.5 nM and 10 nM, respectively.

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

[0090] The lycopene inclusion complex used was obtained from Preparation Example 1; the modified chitosan nanoparticles used were obtained from Preparation Example 6.

[0091] Example 5

[0092] A composition for mitigating and repairing testicular damage caused by heat stress, comprising the following ingredients: lycopene inclusion complex, ginsenoside Re, modified chitosan nanoparticles, and purified water;

[0093] The lycopene inclusion complex contains lycopene and ginsenoside Re in a molar ratio of 4:1, with concentrations of 10 nM and 2.5 nM, respectively.

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

[0095] The lycopene inclusion complex used was obtained from Preparation Example 1; the modified chitosan nanoparticles used were obtained from Preparation Example 6.

[0096] Example 6

[0097] A composition for mitigating and repairing testicular damage caused by heat stress, comprising the following ingredients: lycopene inclusion complex, ginsenoside Re, modified chitosan nanoparticles, and purified water;

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

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

[0100] The lycopene inclusion complex used was obtained from Preparation Example 1; the modified chitosan nanoparticles used were obtained from Preparation Example 6.

[0101] Performance testing

[0102] 1. The following tests were performed on the lycopene inclusion complexes prepared in Examples 1-5:

[0103] (1) Lycopene inclusion rate test: Take 1g of lycopene inclusion complex m0, dissolve it in 10mL of toluene and record it as V0. Dilute it by a certain factor according to the actual concentration and 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] Plotting the standard curve: Take lycopene and dilute it to 50 mL with toluene to prepare test solutions of 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 μg / mL respectively. Use toluene as the reference solution and measure its absorbance value at 484 nm. Repeat three times and take the average value to plot the standard curve.

[0105] The lycopene content is calculated using the following formula: Content (μg / mg) = (n × C × V0) / m0;

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

[0107] Where m is the total mass of the lycopene inclusion complex in g; m 初 The initial mass (mg) of lycopene added during the preparation of the inclusion complex.

[0108] (2) Stability: Take 1g of lycopene complex inclusion complex, and prepare a sample solution with a lycopene mass concentration of 15μg / mL using toluene according to the lycopene content obtained in (1). Use this as the initial mass concentration. After heating this sample solution in a water bath at 40℃ for 48h, measure the absorbance of the solution at a wavelength of 484nm. Calculate the lycopene content according to the formula for calculating the content above, and then calculate the lycopene retention rate according to the following formula: Retention rate (%) = 100 × (mass concentration of remaining lycopene in the solution / initial mass concentration of lycopene). Prepare a control sample solution with 15μg / mL using lycopene and toluene, and determine its retention rate after water bath heating to be 22.5%.

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

[0110] Table 1

[0111]

[0112]

[0113] Table 1 shows that 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 changed, the amount of deionized water added during grinding in Preparation Example 3 changed, and vitamin E was not added in Preparation Example 4. All of these changes affected the inclusion of lycopene by hydroxypropyl-γ-cyclodextrin and the stability of the inclusion complex. In Preparation Example 5, hydroxypropyl-β-cyclodextrin was used instead of hydroxypropyl-γ-cyclodextrin. The inclusion effect of hydroxypropyl-β-cyclodextrin on lycopene was worse than that of hydroxypropyl-γ-cyclodextrin, which also affected the stability of the lycopene inclusion complex.

[0114] 2. Caco-2 cell transport experiments were conducted on the modified chitosan nanoparticles / chitosan nanoparticles prepared in 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. The cells were incubated for equilibration for 30 min. After 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 nanoparticle solution (Preparation Example 6), Group 3 was 50 μg / mL ginsenoside Re solution + 7.5 μg / mL modified chitosan nanoparticle solution (Preparation Example 7), Group 4 was 50 μg / mL ginsenoside Re solution + 7.5 μg / mL modified chitosan nanoparticle solution (Preparation Example 8), and Group 5 was 50 μg / mL ginsenoside Re solution + 7.5 μg / mL modified chitosan nanoparticle solution (Preparation Example 9). 1.5 mL of blank HBSS solution was added to the BL side as a receiving cell.

[0116] After the above treatment, the Transwell was placed in a cell culture incubator, and 100 μL 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 ginsenoside Re content in the samples was determined by UPLC-MS / MS. The permeability of ginsenoside Re was expressed as the apparent permeability coefficient (Papp), 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] As shown in Table 2, 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 changed, which altered the particle size and properties of the modified chitosan nanoparticles and affected 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 Assays: Cell assays were performed on the compositions obtained in Examples 1-5. Cell suspension (100 μL / well) was seeded in 96-well plates, and the plates were pre-cultured at 37°C for 24 h. The test compositions from Examples 1-5 were added to the plates, and the plates were incubated at 37°C for 24 h. Then, 10 μL of CCK8 solution was added to each well, and the plates were incubated at 37°C for another 4 h. The absorbance at 450 nm was measured using a microplate reader, with three measurements taken for each group, and the average value was recorded. An equal volume of 0.9 wt% physiological 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, cell viability reached its maximum when the dosage of lycopene (Lyc) / ginsenoside GRe was 5 nM / 5 nM. Therefore, the following animal experiments were conducted using the composition (LR03) obtained in Example 6, which alleviates and repairs testicular damage caused by heat stress.

[0128] 3.2 Animal Experiments

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

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

[0131] (1) HE staining

[0132] Fixed testes were embedded in paraffin and sectioned. The paraffin sections were stained with hematoxylin and eosin (HE) to observe histological changes in the seminiferous tubules. The specific procedures are as follows:

[0133] Dewax the sections in a 65℃ oven for 2 hours; immerse the sections in xylene and incubate twice at 37℃ for 15 minutes each time; incubate twice in anhydrous ethanol for 2 minutes each time; incubate in 90wt% ethanol aqueous solution for 2 minutes, incubate in 80wt% ethanol aqueous solution for 2 minutes; incubate in 70wt% ethanol aqueous solution for 2 minutes; rinse with dH2O for 10 minutes; stain with hematoxylin for 5 minutes; rinse with running water for 10 minutes; differentiate with 1wt% hydrochloric acid aqueous solution for 30 seconds; rinse with running water for 10 minutes; stain with eosin for 2.5 minutes; immerse the sections in 70wt% ethanol aqueous solution for 2 minutes; incubate in 80wt% ethanol aqueous solution for 2 minutes; incubate in 90wt% ethanol aqueous solution for 2 minutes; incubate twice in anhydrous ethanol for 2 minutes each time; incubate twice in xylene for 15 minutes each time; mount with neutral resin.

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

[0135] from Figure 1It can be seen that heat stress stimulation damages the seminiferous tubules of mice, resulting in the apoptosis and shedding of a large number of spermatogenic cells in the testes, forming tubules resembling only supporting cells. The degree of damage peaks on day 7, followed by significant spontaneous recovery on day 21. Feeding the ginsenoside Re experimental group and the experimental group fed the composition of Example 1 significantly reduced the maximum degree of damage on day 7, achieving better recovery on day 21, with the HS+LR03 group (the group treated in Example 1) showing the best recovery effect.

[0136] (2) TUNEL staining

[0137] Fixed testes were embedded in paraffin and sectioned. TUNEL staining was performed on the paraffin sections to observe changes in seminiferous tubule apoptosis. The specific procedures are as follows:

[0138] Dewaxing was performed in a 65°C oven for 2 hours. The sections were then immersed in xylene and incubated twice at 37°C for 15 minutes each time. They were then incubated twice in anhydrous ethanol for 2 minutes each time. Finally, they were incubated in 90 wt% ethanol aqueous solution for 2 minutes, 80 wt% ethanol aqueous solution for 2 minutes, and 70 wt% ethanol aqueous solution for 2 minutes. The sections were rinsed with dH₂O for 10 minutes. Proteinase K solution (2 mg / mL) was diluted 1:100 with 1×PBS. 100 μL of the diluted Proteinase K solution was added to each sample and incubated at 25°C for 20 minutes. The samples were washed three times with PBS for 5 minutes each time. 100 μL of 1×Equilibration Buffer was added to cover the sample area, and the samples were equilibrated at 25°C for 20 minutes. After equilibration, the Equilibration Buffer was removed, and 50 μL of TdT was added to the sample. The samples were incubated at 25°C for 60 minutes and washed three times with PBS for 5 minutes each time. The sections were then mounted with DAPI.

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

[0140] from Figure 2 It can be seen that heat stress stimulation leads to seminiferous tubule lumen apoptosis (TUNEL). The heat stress HS group reached the peak level of lumen apoptosis on day 7, and then recovered over time; while the HS+GRe group and HS+LR03 group had the highest lumen apoptosis level on day 1, and then recovered over time. The HS+LR03 group (the drug administration group in Example 1) had the best effect.

[0141] (3) Fluorescent staining was performed on paraffin sections to detect changes in the protein levels of ZO-1 and G3BP1. The specific procedures are as follows:

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

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

[0144] ZO-1, as a key structural unit of the blood-testis barrier, plays a crucial role in maintaining the stability of the physicochemical environment for spermatogenesis within the testes, and is essential for spermatogenesis and testicular development. Figure 3 It can be seen that ZO-1 levels decreased after heat stress stimulation, and then recovered over time, showing a significant recovery effect at 14 days. G3BP1, as a key protein in stress granule assembly, plays a crucial role in the recruitment, degradation, and protein remodeling of stress granules during the extensive intracellular protein damage repair process following heat stress injury. Elevated G3BP1 levels indicate more severe heat stress injury and also suggest that the repair process has not been completed. Figure 3 As shown, G3BP1 aggregates after heat stress, and then degrades over time, showing a significant degradation effect after 14 days.

[0145] Both the HS+GRe group and the HS+LR03 group showed significant inhibition of G3BP1 aggregation and ZO-1 decrease after heat stress stimulation due to the administration of ginsenoside Re and the composition of Example 1. Furthermore, the HS+LR03 group exhibited better effects than the HS+GRe group in both G3BP1 degradation and ZO-1 recovery. G3BP1 is an indicator protein of stress granules; G3BP1 aggregation indicates stress granule formation, while G3BP1 degradation indicates stress granule degradation. ZO-1 is an important protein in the formation of the blood-testis barrier (BTB); a decrease in ZO-1 indicates damage to the BTB, while a recovery of ZO-1 indicates BTB repair. Both ginsenoside Re and the composition of Example 1 can accelerate the degradation of stress granules and the repair of the BTB, thereby protecting the seminiferous tubules, with the composition of Example 1 showing the best effect.

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

[0147] The specific steps are as follows:

[0148] a. Protein extraction: Mouse testes were taken, the capsule was removed, lysis buffer was added, and the mixture was sonicated on ice every 30 seconds until the tissue was liquid. The tissue was centrifuged at 4°C, and the supernatant was transferred to a 1.5 ml EP tube.

[0149] b. Protein concentration determination: Using the BCA kit, prepare the working solution, create a standard curve, add the sample, add 200 μL of working solution to each well, incubate at 37℃ for 30 min, detect the absorbance with a microplate reader, calculate the protein concentration, and denature the protein at 95℃ for 5 min.

[0150] c. Western Blot: Prepare electrophoresis buffer, transfer buffer, and gel. Calculate the loading volume based on the sample concentration (30 ng / well); Marker (3 μL), electrophoresis at 85V constant voltage until the Marker bands are completely separated, about 30 min; switch to 125V constant voltage electrophoresis until the bottom band of the Marker band is close to the bottom of the gel, about 1 h. PVDF membrane (6×9cm): Immerse in methanol for 30s (to awaken the membrane), wash 3 times with dd H2O, cut a corner as a marker, and immerse in transfer buffer. Place the membrane in the following order from bottom to top: white clamp - sponge - filter paper - PVDF membrane - gel - filter paper - sponge - black clamp, removing all air. Transfer at 300mA for 100min, block with 5% skim milk at room temperature for 2h, cut the desired band, wash the membrane 3 times with TBST for 5min each time, prepare the primary antibody according to the instructions, and incubate overnight at 4℃; wash the membrane 3 times with TBST for 5min each time; prepare the secondary antibody according to the instructions, and incubate at room temperature for 2h; wash the membrane 3 times with TBST for 5min each time.

[0151] d. Exposure and development: Prepare and use immediately by dropping ECL luminescent liquid onto the membrane, exposing it with a gel imaging system and saving the image.

[0152] For specific test results, please 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 mice in all groups increased over time, showing a significant increase at 21 days. ZO-1, as a key structural unit of the blood-testis barrier, plays a crucial role in maintaining the stability of the physicochemical environment for spermatogenesis within the testes, thus ensuring spermatogenesis and testicular development. Conversely, the G3BP1 protein level decreased over time, showing a significant decrease at 21 days. G3BP1, a key protein in stress granule assembly, plays a vital role in protein recruitment, degradation, and remodeling during the extensive intracellular protein damage repair process following heat stress injury. Elevated G3BP1 levels indicate more severe heat stress injury and suggest that the repair process has not yet been completed. The results showed that the blood-testis barrier structure in each group was significantly low immediately after heat stress stimulation, and gradually recovered over time. The recovery speed and degree of the HS+LR03 group were significantly improved. Moreover, the HS+LR03 group (the group treated in Example 1) was able to significantly reduce the level of stress particles in the testes after heat stress stimulation, and at the same time accelerate the degradation speed and degree of stress particles.

[0154] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present application in any way. Although the present application discloses the preferred embodiment as described above, it is not intended to limit the present application. Any changes or modifications made by those skilled in the art without departing from the scope of the technical solution of the present application using the disclosed technical content are equivalent to equivalent implementation cases. 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 scope of the technical solution of the present invention are still within the scope of the technical solution.

Claims

1. A composition for mitigating and repairing testicular damage caused by heat stress, characterized in that, The ingredients include: lycopene inclusion complex, ginsenoside Re, modified chitosan nanoparticles, and solvent; The amount of the modified chitosan nanoparticles used is 10-15 wt% of ginsenoside Re; The molar ratio of lycopene to ginsenoside Re in the lycopene inclusion complex is 1:1; The preparation steps of the lycopene inclusion complex are as follows: Take vitamin E, lycopene, and hydroxypropyl-γ-cyclodextrin, mix them evenly, add the first part of deionized water, grind them in a water bath at 20-30℃ for 1-2 hours, then add the second part of deionized water, centrifuge, and dry the supernatant to constant weight to obtain lycopene inclusion complex. The mass ratio of vitamin E, lycopene, and hydroxypropyl-γ-cyclodextrin is 1:1.25:85; The preparation steps of the modified chitosan nanoparticles are as follows: (1) Chitosan was dissolved in glacial acetic acid aqueous solution, the pH was adjusted to 5-6, chitosanase was added for enzymatic hydrolysis for 3-4 hours, filtered, and the filtrate was ultrafiltered and freeze-dried to obtain low molecular weight chitosan; the amount of chitosanase used was 1.25 wt% of chitosan; (2) Dissolve the low molecular weight chitosan from step (1) in poloxam aqueous solution, adjust the pH to 4.5-5.0, filter, add sodium tripolyphosphate aqueous solution under stirring, stir for 1.5-2 h, purify, concentrate, freeze dry to obtain chitosan nanoparticles. (3) Dissolve the chitosan nanoparticles from step (2) in an aqueous solution of glacial acetic acid, mix with an aqueous solution of glucosamine, adjust the pH to 5-5.5, react at 60-70℃ for 60-80h, let stand in an ice bath for 10-30min, dialyze to purify, soak in distilled water for 1-3 days to obtain modified chitosan nanoparticles.

2. 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.

3. The composition according to claim 1, characterized in that, The chitosan has a degree of deacetylation ≥90% and a dynamic viscosity of 300-500 mPa·s at 20°C.

4. The composition according to claim 1, characterized in that, The chitosanase activity is ≥100,000 U / g.

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

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