Use of three compounds in the preparation of a product for reducing cadmium damage to the male reproductive system

Abstract

The present application relates to the field of assisted reproductive technology, and provides application of three kinds of compounds in preparation of products for reducing cadmium damage to male reproductive system, wherein the compounds are chlorogenic acid, L-glutamic acid or S-methyl-L-cysteine. The three kinds of compounds can effectively improve activity of mouse testicular interstitial cells in vitro, reduce oxidative stress level of the cells, reduce toxic damage of cadmium to the testicular interstitial cells, and the effect of different compounds is different. When chlorogenic acid and S-methyl-L-cysteine are used in combination, the effect of improving damage is better than that of using any one of the compounds alone, the activity of damaged testicular interstitial cells can be further improved, and oxidative damage can be relieved, which provides a new direction for developing products for reducing male reproductive damage caused by cadmium exposure, and has good development and application prospect.

Description

Technical Field

[0001] This invention relates to the field of assisted reproductive technology, and in particular to the application of three compounds in the preparation of products that reduce cadmium damage to the male reproductive system. Background Technology

[0002] Since the 1950s, the global incidence of infertility has been steadily increasing, evolving into a serious medical and social problem. Male factors account for approximately 40-50% of infertility cases in couples. Among these, cadmium pollution is one of the key factors leading to reduced male fertility. Related research has confirmed this in mouse, rat, pig, and goat models. Studies have found that even low doses of cadmium can cause damage to testicular interstitial cells, affect testosterone synthesis and sperm production, disrupt the balance of the reproductive system, and reduce sperm quality and quantity.

[0003] Selenium is an essential trace element for the human body. Studies have found that selenium plays a crucial role in the detoxification of heavy metals (mercury, cadmium, lead, manganese, etc.). Numerous studies have investigated selenium's role in reducing cadmium-induced damage to the male reproductive system, using models including mice, rats, and chickens. Research indicates that selenium's effect in reducing cadmium damage to the male reproductive system is closely related to its chemical form. For example, Wang Jiajun et al. treated rats with Nano-Se and sodium selenite, respectively, and found that Nano-Se showed a better effect on reducing cadmium-induced testicular tissue damage compared to sodium selenite. In recent years, research on the application of selenium-enriched foods in reducing cadmium damage has made some progress. Currently, there are many selenium-enriched foods on the market, including selenium-enriched rice, selenium-enriched probiotics, and selenium-enriched rapeseed.

[0004] Detection revealed a rich variety of compounds in selenium-enriched rapeseed. Among them, selenium-enriched rapeseed was particularly rich in methylselenocysteine, selenocysteine, sodium selenite, chlorogenic acid, L-glutamic acid, and S-methyl-L-cysteine. Determining the efficacy of these six substances in improving cadmium damage to the reproductive system and their synergistic effects is of great significance for preventing cadmium damage to male reproductive capacity. Summary of the Invention

[0005] The purpose of this invention is to provide the application of three compounds in the preparation of products that reduce cadmium damage to the male reproductive system.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the application of three compounds in the preparation of products that reduce cadmium damage to the male reproductive system, wherein the compounds are chlorogenic acid, L-glutamic acid or S-methyl-L-cysteine.

[0007] Preferably, the effective concentration of chlorogenic acid is 0.25~10 μg / L.

[0008] Preferably, the effective concentration of L-glutamic acid is 0.25~5 μg / L.

[0009] Preferably, the effective concentration of the S-methyl-L-cysteine ​​is 500~5000 μg / L.

[0010] This invention provides the application of three compounds in the preparation of products that reduce cadmium-induced damage to the male reproductive system. The compounds are chlorogenic acid, L-glutamic acid, or S-methyl-L-cysteine. All three compounds can effectively improve the activity of mouse testicular interstitial cells in vitro, reduce cellular oxidative stress levels, and decrease the toxic damage of cadmium to testicular interstitial cells. The effects of different compounds vary, with chlorogenic acid and S-methyl-L-cysteine ​​showing a better effect in improving damage than either compound alone. This combination can further enhance the vitality of damaged testicular interstitial cells and alleviate oxidative damage, providing a new direction for developing products that reduce male reproductive damage caused by cadmium exposure and demonstrating promising application prospects. Attached Figure Description

[0011] Figure 1 The effect of S-methyl-L-cysteine ​​on cell viability; Figure 2 The effect of chlorogenic acid on cell activity; Figure 3 The effect of co-treatment with chlorogenic acid and CdCl2 on cell viability; Figure 4 The effect of co-treatment with L-glutamate and CdCl2 on cell viability; Figure 5 The effect of co-treatment with S-methyl-L-cysteine ​​and CdCl2 on cell viability; Figure 6 The effect of co-treatment with chlorogenic acid, S-methyl-L-cysteine ​​and CdCl2 on cell viability; Figure 7 The effect of co-treatment with chlorogenic acid, S-methyl-L-cysteine ​​and CdCl2 on cell viability; Figure 8 The effect of co-treatment with chlorogenic acid, S-methyl-L-cysteine ​​and CdCl2 on cell viability; Figure 9 The effects of co-treatment with chlorogenic acid, S-methyl-L-cysteine ​​and CdCl2 on cell viability. Detailed Implementation

[0012] This invention provides the application of three compounds in the preparation of products that reduce cadmium damage to the male reproductive system, wherein the compounds are chlorogenic acid, L-glutamic acid, or S-methyl-L-cysteine.

[0013] In this invention, the effective concentration of chlorogenic acid is preferably 0.25~10 μg / L, and more preferably 1~5 μg / L.

[0014] In this invention, the effective concentration of L-glutamic acid is preferably 0.25~5 μg / L, and more preferably 1~2 μg / L.

[0015] In this invention, the effective concentration of S-methyl-L-cysteine ​​is preferably 500~5000 μg / L, and more preferably 1000~2000 μg / L.

[0016] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0017] The methylselenocysteine, selenocysteine, and sodium selenite provided in this invention were purchased from Shanghai Macklin Biochemical Technology Co., Ltd. (Shanghai, China); chlorogenic acid, L-glutamic acid, and S-methyl-L-cysteine ​​were purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China).

[0018] Example 1

[0019] 1. Effects of S-methyl-L-cysteine ​​on improving mouse testicular interstitial cells in vitro

[0020] When the cell density in the culture dish reaches approximately 80% and the cells are in normal condition, remove the culture medium with a pipette and wash the cells once with PBS. Then, add 1 mL of 0.25% trypsin for digestion. When the cells begin to detach, add 2 mL of TM4 cell culture medium to stop the digestion reaction. Next, use a pipette to detach the cells from the culture dish and transfer them to centrifuge tubes. Centrifuge (1200 rpm × 3 min), discard the supernatant, and add 3 mL of TM4 cell culture medium to resuspend the cells. Count the cells at 1 × 10⁶ cells per well. 4 Cells were seeded into 96-well plates. After 24 h of culture, the cells were treated with S-methyl-L-cysteine ​​at concentrations of 100, 500, 1000, 2000, and 5000 μg / mL and cultured for another 24 h. The culture medium was then removed, and 100 μL of freshly prepared medium containing 10% CCK-8 was added. The cells were incubated at 37°C for 40 min, and the absorbance was measured using a microplate reader.

[0021] Experimental results are as follows Figure 1 As shown, S-methyl-L-cysteine ​​significantly inhibited TM4 cells at concentrations of 2000 and 5000 μg / mL, with cell viability of 77.75% and 66.25%, respectively.

[0022] 2. Effects of chlorogenic acid on improving interstitial cells in mouse testes in vitro

[0023] When the cell density in the culture dish reaches approximately 80% and the cells are in normal condition, remove the culture medium with a pipette and wash the cells once with PBS. Then, add 1 mL of 0.25% trypsin for digestion. When the cells begin to detach, add 2 mL of TM4 cell culture medium to stop the digestion reaction. Next, use a pipette to detach the cells from the culture dish and transfer them to centrifuge tubes. Centrifuge (1200 rpm × 3 min), discard the supernatant, and add 3 mL of TM4 cell culture medium to resuspend the cells. Count the cells at 1 × 10⁶ cells per well. 4 Cells were seeded into 96-well plates. After 24 h of culture, the cells were treated with chlorogenic acid at concentrations of 0.25, 0.5, 1, 10, and 25 μg / mL and cultured for another 24 h. The culture medium was then removed, and 100 μL of freshly prepared medium containing 10% CCK-8 was added. The cells were incubated at 37°C for 40 min, and the absorbance was measured using a microplate reader.

[0024] Experimental results are as follows Figure 2 As shown, chlorogenic acid has no significant effect on TM4.

[0025] 3. Effects of L-glutamate on improving interstitial cells in mouse testes in vitro

[0026] When the cell density in the culture dish reaches approximately 80% and the cells are in normal condition, remove the culture medium with a pipette and wash the cells once with PBS. Then, add 1 mL of 0.25% trypsin for digestion. When the cells begin to detach, add 2 mL of TM4 cell culture medium to stop the digestion reaction. Next, use a pipette to detach the cells from the culture dish and transfer them to centrifuge tubes. Centrifuge (1200 rpm × 3 min), discard the supernatant, and add 3 mL of TM4 cell culture medium to resuspend the cells. Count the cells at 1 × 10⁶ cells per well. 4 Cells were seeded into 96-well plates. After 24 h of culture, the cells were treated with L-glutamate at concentrations of 0.25, 0.5, 1, 5, and 10 μg / mL and cultured for another 24 h. The culture medium was then removed, and 100 μL of freshly prepared medium containing 10% CCK-8 was added. The cells were incubated at 37°C for 40 min, and the absorbance was measured using a microplate reader.

[0027] Example 2: Effects of three compounds on cell viability when co-treated with cadmium chloride on TM4 cells.

[0028] 1. Effects of chlorogenic acid on improving interstitial cells in mouse testes in vitro

[0029] When the cell density in the culture dish reaches approximately 80% and the cells are in normal condition, remove the culture medium with a pipette and wash the cells once with PBS. Then, add 1 mL of 0.25% trypsin for digestion. When the cells begin to detach, add 2 mL of TM4 cell culture medium to stop the digestion reaction. Next, use a pipette to detach the cells from the culture dish and transfer them to centrifuge tubes. Centrifuge (1200 rpm × 3 min), discard the supernatant, and add 3 mL of TM4 cell culture medium to resuspend the cells. Count the cells at 1 × 10⁶ cells per well. 4 Cells were seeded into 96-well plates. After 24 h of culture, the cells were treated with chlorogenic acid and cadmium chloride at concentrations of 0.25, 0.5, 1, 10, and 25 μg / mL and cultured for another 24 h. The culture medium was then removed, and 100 μL of freshly prepared medium containing 10% CCK-8 was added. The cells were incubated at 37°C for 40 min, and the absorbance was measured using a microplate reader.

[0030] The results are as follows Figure 3 As shown, compared with the cadmium chloride group, 0.25, 0.5, 1, and 10 μg / mL chlorogenic acid had a significant effect on reducing cadmium chloride-induced cell damage in TM4 cells, with cell viability rates of 55.08%, 56.95%, 58.02%, and 52.73%, respectively. 2. Effects of L-glutamate on improving interstitial cells in mouse testes in vitro When the cell density in the culture dish reaches approximately 80% and the cells are in normal condition, remove the culture medium with a pipette and wash the cells once with PBS. Then, add 1 mL of 0.25% trypsin for digestion. When the cells begin to detach, add 2 mL of TM4 cell culture medium to stop the digestion reaction. Next, use a pipette to detach the cells from the culture dish and transfer them to centrifuge tubes. Centrifuge (1200 rpm × 3 min), discard the supernatant, and add 3 mL of TM4 cell culture medium to resuspend the cells. Count the cells at 1 × 10⁶ cells per well. 4 Cells were seeded into 96-well plates. After 24 h of culture, the cells were treated with L-glutamate at concentrations of 0.25, 0.5, 1, 5, and 10 μg / mL and co-treated with cadmium chloride for another 24 h. The culture medium was then removed, and 100 μL of freshly prepared medium containing 10% CCK-8 was added. The cells were incubated at 37°C for 40 min, and the absorbance was measured using a microplate reader.

[0031] The results are as follows Figure 4 As shown, compared with the cadmium chloride group, 0.25, 0.5, 1 and 5 μg / mL L-glutamate had a significant effect on reducing cadmium chloride-damaged cells in TM4 cells, with cell viability rates of 66.19%, 67.71%, 65.54% and 65.27%, respectively. 3. Effects of S-methyl-L-cysteine ​​on improving mouse testicular interstitial cells in vitro When the cell density in the culture dish reaches approximately 80% and the cells are in normal condition, remove the culture medium with a pipette and wash the cells once with PBS. Then, add 1 mL of 0.25% trypsin for digestion. When the cells begin to detach, add 2 mL of TM4 cell culture medium to stop the digestion reaction. Next, use a pipette to detach the cells from the culture dish and transfer them to centrifuge tubes. Centrifuge (1200 rpm × 3 min), discard the supernatant, and add 3 mL of TM4 cell culture medium to resuspend the cells. Count the cells at 1 × 10⁶ cells per well. 4 Cells were seeded into 96-well plates. After 24 h of culture, the cells were treated with S-methyl-L-cysteine ​​at concentrations of 100, 500, 1000, 2000, and 5000 μg / mL and co-treated with cadmium chloride for another 24 h. The culture medium was then removed, and 100 μL of freshly prepared medium containing 10% CCK-8 was added. The cells were incubated at 37°C for 40 min, and the absorbance was measured using a microplate reader.

[0032] The results are as follows Figure 5 As shown, compared with the cadmium chloride group, 500, 1000, 2000 and 5000 μg / mL S-methyl-L-cysteine ​​significantly reduced cadmium chloride damage in TM4 cells, with cell viability rates of 60.00%, 82.33%, 94.67% and 76.67%, respectively.

[0033] In summary, chlorogenic acid, L-glutamic acid, and S-methyl-L-cysteine ​​can all significantly reduce cadmium chloride damage in TM4 cells.

[0034] Example 3: Effects of chlorogenic acid and S-methyl-L-cysteine ​​on improving mouse testicular interstitial cells in vitro

[0035] When the cell density in the culture dish reaches approximately 80% and the cells are in normal condition, remove the culture medium with a pipette and wash the cells once with PBS. Then, add 1 mL of 0.25% trypsin for digestion. When the cells begin to detach, add 2 mL of TM4 cell culture medium to stop the digestion reaction. Next, use a pipette to detach the cells from the culture dish and transfer them to centrifuge tubes. Centrifuge (1200 rpm × 3 min), discard the supernatant, and add 3 mL of TM4 cell culture medium to resuspend the cells. Count the cells at 1 × 10⁶ cells per well. 4Cells were seeded into 96-well plates. After 24 h of culture, the cells were treated with chlorogenic acid at concentrations of 0.5, 1, and 10 μg / mL, and S-methyl-L-cysteine ​​at concentrations of 1000 and 2000 μg / mL, along with cadmium chloride, and cultured for another 24 h. The culture medium was then removed, and 100 μL of freshly prepared medium containing 10% CCK-8 was added. The cells were incubated at 37°C for 40 min, and the absorbance was measured using a microplate reader.

[0036] The results are as follows Figures 6-9 As shown, the combination of the two drugs can significantly reduce cadmium chloride damage in TM4 cells; Chlorogenic acid at concentrations of 0.5, 1, and 10 μg / mL, when co-treated with 1000 μg / mL S-methyl-L-cysteine, showed a significant synergistic effect compared to its single action, with cell viability rates of 127.99%, 123.55%, and 119.72%, respectively. The results are as follows: Figures 7-9 As shown, chlorogenic acid has no synergistic effect with L-glutamic acid, and L-glutamic acid has no synergistic effect with S-methyl-L-cysteine.

[0037] As shown in the above embodiments, this invention provides the application of three compounds in the preparation of products that reduce cadmium damage to the male reproductive system. These compounds are chlorogenic acid, L-glutamic acid, or S-methyl-L-cysteine. All three compounds of this invention can effectively improve the activity of mouse testicular interstitial cells in vitro, reduce cellular oxidative stress levels, and decrease the toxic damage of cadmium to testicular interstitial cells. The effects of different compounds vary, with chlorogenic acid and S-methyl-L-cysteine ​​showing a better effect in improving damage than either compound alone. This combination can further enhance the vitality of damaged testicular interstitial cells and alleviate oxidative damage, providing a new direction for developing products that reduce male reproductive damage caused by cadmium exposure and demonstrating promising development and application prospects.

[0038] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of three compounds in the preparation of products that reduce cadmium damage to the male reproductive system, characterized in that, The compounds are chlorogenic acid, L-glutamic acid, or S-methyl-L-cysteine.

2. The application according to claim 1, characterized in that, The effective concentration of chlorogenic acid is 0.25~10 μg / L.

3. The application according to claim 1, characterized in that, The effective concentration of L-glutamic acid is 0.25~5 μg / L.

4. The application according to claim 1, characterized in that, The effective concentration of the S-methyl-L-cysteine ​​is 500~5000 μg / L.

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