Due to the deterioration of the environment, life pressure and other factors, the incidence of infertility in the population is gradually increasing [1-2], causing great trouble to the families of the patients. Male infertility is mainly manifested in the testicular tissue oxidative damage, in which the sperm count and motility are the main factors. Currently used drugs to improve reproductive performance with high side effects, from the ocean to improve reproductive performance and less toxic side effects of natural drugs has become the focus of attention. Cyanobacteria are rich in cyanobacterial protein (C-PC), which has antioxidant and antitumor effects[3-6] , but little research has been done on its role in the field of reproductive immunity. Reactive oxygen species (ROS) is a kind of substance produced by the metabolism of biological system that can cause oxidative reaction in the body[7-9] , which mainly includes superoxide radicals, hydroxyl radicals and so on.
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When intracellular ROS production increases, it can lead to protein, lipid and nucleic acid damage, which can have harmful effects on cellular structure[10] . Studies have shown that the accumulation of ROS in cells can lead to cytoskeletal disorganization[11] , dysfunction of the antioxidant system[12-13] and cellular apoptosis[14] . Reproductive cells produce ROS during normal metabolic processes and gradually accumulate them, leading to the development of oxidative stress, which in turn leads to germ cell damage, reduces germ cell quality and quantity, and affects embryonic development[15] . Oxidative stress has been recognized as one of the major mechanisms of potential cellular aging[16-17] , and its negative effects on the human reproductive process are no longer a subject of debate[18] . In the present study, the protective effect of C-PC on H2O2-induced oxidative damage of mouse spermatogonia GC-1 spg and its mechanism were investigated, aiming to provide a theoretical basis for the development and utilization of C-PC in the field of reproductive immunity.
1 Materials and methods
1 . 1 MATERIALS
C-PC was purchased from Taizhou Binmei Biotechnology Co., Ltd; mouse spermatogonia GC-1 spg was purchased from Wuhan Punosai Life Science and Technology Co., Ltd; DMEM medium was purchased from Hyclone, Inc; fetal bovine serum and trypsin digest were purchased from BI, Inc; penicillin-streptomycin mixture was purchased from Beijing Solepol Technology Co. Ltd; BCA protein quantification kit was purchased from China bio-sharp; H2O2 was purchased from Sigma; Annexin V-PI cell apoptosis detection kit and ROS detection kit were purchased from Shanghai Biyuntian Biotechnology Ltd; RIP-1 was purchased from Chengdu Zhengneng Bio-technology Ltd; RIP-3 was purchased from Santa.
1 .2 Cell culture and processing
The GC-1 spg was inoculated with a mixture of 0.10 volume fraction fetal bovine serum and 0.5 volume fraction fetal bovine serum. GC-1 spg was inoculated in high sugar DMEM medium containing 0.10 v/v fetal bovine serum and 0.01 v/v penicillin. GC-1 spg was inoculated in high sugar DMEM medium containing 0.10 v/v fetal bovine serum and 0.01 penicillin by volume, and incubated at 37 ℃ in a cell culture incubator with 0.05 CO2 by volume. The cells were incubated at 37 ℃ in a cell culture incubator containing 0.05 CO2 by volume. The cells were divided into control group (group A), model group (group B), C-PC low dose group (group C), C-PC medium dose group (group D) and C-PC high dose group (group E). The density of GC-1 spg was adjusted to 7 × 107 cells/L, and then the cells were inoculated into 96-well (100 μL per well) or 6-well culture plates (2 mL per well). After overnight walling of the cells, the cells were added into the culture solution for regular culture in Groups A and B, and the cells were added into the culture solution for regular culture in Groups C, D, and E with 0.25, 0.25, 0.25, 0.25, 0.25, 0.25, 0.25, 0.25, 0.25, 0.25 and 0.25 respectively. Groups C, D and E were incubated with 0.25, 0.50, 1.50 and 0.50 mL of culture medium, respectively. Groups C, D and E were pre-treated with 0.25, 0.50 and 1.00 g/L C-PC. Groups C, D, and E were pretreated with 0.25, 0.50, and 1.00 g/L C-PC for 24 h. Group A removed the culture medium and replaced it with fresh culture medium, and Groups B, C, D, and E removed the culture medium and added fresh culture medium containing 600 μmol/L H2O2 to continue the incubation for 24 h, and then proceeded to the subsequent experiments.
1 .3 Cell proliferation rate by CCK-8 assay
After the cells were processed, the medium was aspirated and 110 μL of medium containing 0.10 CCK-8 v/v was added. Add 110 μL of medium containing 0.10 CCK-8 v/v. After incubation for 2 h, the absorbance at 450 nm was measured by enzyme assay (A).
. The cell proliferation rate was calculated. Cell proliferation rate (%) = (Experiment 0- White group A)/(Control group A - White group A) ×
1 .4 DCFH-DA Staining for Determination of Intracellular ROS Levels
Dichlorofluorescent yellow diacetate (DCFH-DA) fluorescent probe was diluted with serum-free culture solution at 1:1,500 and the final concentration was 7.5 μmol/L . The final concentration was 7.5 μmol/L. After each group of cells was treated, the culture medium was discarded, and 1 mL of diluted DCFH-DA was added to each well of a 6-well plate and incubated in a cell culture incubator at 37 ℃ for 20 min. Wash the cells with serum-free cell culture medium for 3 times, add serum-free cell culture medium and observe the green fluorescence intensity under a fluorescence microscope.
1 .5 Detection of cell necrosis by flow cytometry
After the cells were processed, the culture medium was discarded, and the cells were digested with trypsin without ED-TA and collected by centrifugation, washed twice with pre-cooled PBS, and collected by centrifugation. Resuspend the cells by adding 100 μL of binding buffer, add 5 μL of Annexin V-FITC and 10 μL of PI Stai- ning Solution, mix well, incubate for 15 min at room temperature away from light, and then add 400 μL of binding buffer. Mix well and place on ice, then analyze immediately by flow cytometry.
1.6 Western blot assay for RIP-1 and RIP-3 expression in cells
After the cells of each group were processed, the medium was discarded, the cells were washed three times with pre-cooled PBS, and 200 μL of protein lysate (prepared at the ratio of RIPA:PMSF:IP of 100:1:1) was added to each group, and the total cellular proteins were extracted, and the concentration of the proteins was measured by the BCA method. The proteins were separated on SDS-PAGE and transferred to a PVDF membrane, which was closed with 50 g/L skimmed milk powder prepared with TBST for 2 h. The membrane was incubated at 4 ℃ for 2 h. The membrane was incubated with primary antibody (1:1,000) for 12 h at 4 ℃, and GAPDH (1:2,000) was used as internal reference. The membrane was washed three times with TBST for 10 min each time. The membrane was incubated with secondary antibody (1:1,000) for 2 h at room temperature and washed three times with TBST for 10 min each time. ECL development. Protein bands were analyzed by Image J software, and the relative expression of target proteins was calculated by the gray value of target protein/gray value of internal reference protein.
2 Results
2 . 1 Determination of GC-1 spg proliferative activity in each group
The results of the CCK-8 assay showed that the cells in groups A, B, C, D, and E were not affected by the presence of the CCK-8 method.
The proliferative activity was 100.00 ± 5.0%. 00 ± 5 . (100.00 ± 5.09) %, (64.91 ± 4.0) % and (64.91 ± 4.0) %, respectively. 91 ± 4 . (64.91 ± 4.75) %, (76.25 ± 4.09) 25 ± 4 . 94) %, (78 . 69 ± 8 . 69 ± 8 . 54) % and . 6 groups,q,. There,P.C(q2 .2 The effect of C-PC on ROS levels in GC-1 spg. Under the fluorescence microscope, the intensity of green fluorescence in the cells of group B was significantly increased compared with that of group A. The intensity of green fluorescence in the cells of groups C, D, and E was significantly decreased compared with that of group B (Fig. 1).
2 .3 Effect of C-PC on H 2 O 2-induced necrosis of GC-1 spg
The results of flow cytometry showed that the cell necrosis rates of group A ~ E were 0.004 ± 0.004 ± 0.004 ± 0.004 ± 0.004 ± 0.004 respectively. 004 ± 0 . 0.001, 0.195 ± 0.001, 0.001 and 0.001 respectively. 195 ± 0 . 0.004, 0.195 ± 0.004, and 0.166 ± 0.001 respectively. 166 ±0 . 002 , 0 . 153 ± 0 . 003 , 0 . 092 ±0 . 0.092±0.003 . Compared with group A, there was a significant increase in necrotic cells in group B (F = 206.90, q = 60.0%). 90 , q = 60 . 07 , P <0 . 05); Groups C, D and E all showed a significant decrease in necrotic cells compared with Group B. The difference was statistically significant (q = 9.09 ~ 32.09, P < 0.05). 09 ~ 32 . 41 ,P <0.05) . The differences were statistically significant (q = 9.09 ~ 32.41, P < 0.05). See Fig.
2 .4 Effect of C-PC on H 2 O 2-induced expression of GC-1 spg necrosis-associated proteins
Western blot results showed that the relative expression of RIP-1 protein in the cells of group A~E was 1.013 ± 0.5 mm, respectively. 013 ± 0 . 014, 1 . 1.947 ± 0.014, 1.947 ± 0.014, 1.947 ± 0.014, 1.947 ± 0.014 and 1.947 ± 0.014 respectively. 038, 1.140 ± 0.025 140 ± 0 . 021 , 0 . 877 ± 0 . 017 , 0 . 517 ± 0 . 012 . The relative expression of RIP-3 protein was 0 . 930 ± 0 . 012 , 1 . 623 ± 0 . 052 , 0 . 883 ± 0 . 015 , 0 . 603 ±0 . 015 , 0 . 430 ±0 . 0 . 430 ±0 . 015 . Compared with group A, the relative expression of RIP-1 and RIP-3 proteins in the cells of group B was significantly higher, and the differences were all significant (F = 541.2, 297.2, 297.2, 297.2, 297.2, 297.2, 297.2, 297.2). F = 541.2, 297.5, both significant. The differences were significant (F = 541.2, 297.5, q = 27.97 ~ 62.5). 97 ~ 62 . 98 ,P＜0.05). 05) . See Fig.
3 Discussion
It is well known that oxidative stress is detrimental to human health, leading to the development of a number of diseases, such as cardiovascular diseases, neurological diseases, renal diseases, and delayed sexual maturation[19-21] . Excessive accumulation of ROS causes cellular oxidative stress, a mechanism that has been implicated as a potential contributor to a variety of other common diseases such as endometriosis, ovarian cancer, and polycystic ovaries in women, as well as a potential causative factor for sperm DNA damage and sperm apoptosis in men[22] . Since oxidative stress is recognized as one of the major mechanisms underlying aging[16-17] , reducing oxidative stress in germ cells can effectively enhance germ cell viability. Studies have shown that C-PC is an effective scavenger of peroxyl radicals[23] .
In the present study, after GC-1 spg was treated with H2O2, the cell viability was significantly decreased and the necrosis rate was increased, and the treatment with C-PC at all dose groups could increase the cell viability and decrease the necrosis rate, and reverse the damage to the normal state. The C-PC pretreatment could protect GC-1 spg from the oxidative damage of H2O2, which indicated that the C-PC pretreatment could protect GC-1 spg from the oxidative damage of H2O2.
ROS play an important role in regulating various cellular functions, and under normal conditions, intracellular homeostasis is balanced by endogenous free radicals or antioxidant defense systems[24-25] . However, an excess of ROS will eventually lead to an imbalance in the antioxidant system and induce cellular oxidative damage. After the cells were treated with H2O2, an excessive amount of ROS was produced in the cells, resulting in a decrease in the cellular antioxidant capacity[26] . In order to show that C-PC can reverse the oxidative damage of GC-1 spg, the present study examined the level of ROS in H2O2-treated GC-1 spg. In this study, the intracellular ROS levels in GC-1 spg induced by H2O2 were significantly higher than those in the control group; compared with the model group, the C-PC low, medium and high dose groups could effectively reduce the intracellular ROS levels. These results suggest that C-PC reduces oxidative stress by lowering intracellular ROS levels.
Programmed necrosis is a new type of cell death that is regulated by death signals and is not dependent on Caspase[27] , and the receptor-interacting protein RIP is a key component of it, which is an important indicator of programmed necrosis. The programmed necrosis inhibitor Nec-1 functions as the kinase component of RIP-1 and RIP-3. HA-NUS et al.[28] demonstrated that, after oxidative stress, the nuclear and plasma membranes are disrupted and intracellular ATP is depleted, which are the main characteristics of programmed necrosis. Knockdown of RIP-3 or use of Nec-1, an inhibitor of RIP, can rescue cell death induced by oxidative stress, indicating that RIP is essential for oxidative stress-induced programmed cell necrosis. Recent studies have shown that programmed necrosis is also a major mechanism of retinal pigment epithelial cell death under oxidative stress[28-29] .
In the present study, a GC-1 spg oxidative damage model was developed using H2O2, and the results showed that H2O2 induced programmed necrosis in GC-1 spg, and RIP-1 and RIP-3 were the key molecules in H2O2-induced programmed necrosis, indicating that RIP-1 and RIP-3-mediated programmed necrosis were involved in H2O2-induced oxidative damage in GC-1 spg.
At the cellular level, it was confirmed that H2O2 up-regulated the expression of RIP-1 and RIP-3 proteins in GC-1 spg while causing GC-1 spg injury. This indicates that C-PC pretreatment of GC-1 spg can inhibit the H2O2-induced up-regulation of RIP-1 and RIP-3 proteins, and at the same time, it can significantly inhibit the H2O2-induced cell injury, increase cell viability, and reduce the formation of intracellular ROS. Therefore, inhibition of programmed necrosis may be another important mechanism by which C-PC protects GC-1 spg.
In conclusion, it is suggested that H2O2 induced oxidative damage and RIP-1 and RIP-3-mediated programmed cell necrosis in GC-1 spg. This study showed that C-PC had a significant protective effect on H2O2-induced oxidative damage in GC-1 spg, and its effect may be related to the ability of C-PC to inhibit oxidative stress-induced programmed necrosis by significantly decreasing the level of ROS, down-regulating the expression of necrotic pathway proteins RIP-1 and RIP-3, and decreasing the rate of necrotic cell death in GC-1 spg. This study provides a theoretical basis for the development and utilization of C-PC in the field of reproduction, and has a better clinical application prospect.
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