Serum molecular marker for predicting damage of microwave electromagnetic radiation to male reproductive function and application

Abstract

The invention belongs to the technical field of medical biological detection, and particularly relates to a serum molecular marker for predicting damage of microwave electromagnetic radiation to male reproductive function and application. The invention discloses serum molecular markers (H1-1, H1-4, H1-5, Hp1bp3, Itgb2, Ncf2 and Rhoc) capable of detecting the male radiation related reproductive function injury for the first time, and the male radiation related reproductive function injury can be diagnosed by detecting the expression quantity of related molecules in serum. The condition that currently found markers for reproductive injury caused by electromagnetic radiation are few is made up, and the clinical application defect that most existing markers are proteins or metabolites found in testis and are not beneficial to convenient detection and large-scale application of the markers is overcome.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological detection, and relates to application of a serum molecular marker for predicting microwave electromagnetic radiation damage to the reproductive function of male mice in preparation of a radiation-related reproductive damage detection kit. BACKGROUND

[0002] Since the 1950s, with the rapid development of wireless communication technology, the level of non-ionizing electromagnetic radiation in our daily life environment has risen sharply. The highest power magnetic flux density is more than 1018 times the natural level, especially the radio frequency EMR (RF-EMR) with a frequency of 300 kHz to 300 GHz. Electromagnetic radiation has been listed as the fourth pollutant by the World Health Organization (WHO), and the International Agency for Research on Cancer (IARC) has also listed electromagnetic waves as “possibly carcinogenic to humans” (2B).

[0003] Electromagnetic radiation can be absorbed by organisms, triggering a series of biological and functional changes, such as cell proliferation, gene expression, DNA damage, and oxidative stress. The male reproductive system is very susceptible to electromagnetic radiation, and studies have shown that electromagnetic radiation is related to the decrease in progressive active sperm count, motility and survival rate, and the increase in reactive oxygen species (ROS) and abnormal sperm morphology. Non-ionizing radiation also causes a decrease in fertilization rate, a decrease in the number of spermatogenic cells and the induction of apoptosis, a decrease in sperm quality, and changes in testicular hormones, and can lead to fetal loss and developmental disorders during the embryonic period. There is evidence that Wi-Fi radiation from a laptop computer has a negative impact on sperm quality; men who use mobile phones have a decrease in sperm concentration, motility, normal morphology, and viability. However, sometimes the conclusions of different studies contradict each other, such as a study that suggests that low-frequency continuous EMR can activate adaptive mechanisms in GC-2 mouse spermatocytes, protecting their genomes from harmful effects, while pulsed EMR exposure leads to more severe damage and the formation of DNA breaks; the testis is very sensitive to radiation, and radiation received by the testis during the development stage can cause serious damage in the form of genotoxic effects. During the development stage of the testis, penetration depth is not the only influencing factor, other influencing factors also include exposure time, the number of non-developing cells exposed to EMR (the more non-developing cells, the greater the impact of radiation), and the water content of the organ (the greater the water content of the organ, the greater the impact of radiation). Literature has reported that morphological changes caused by EMR also depend on its type, dose, mode, and duration of exposure. Domestic studies have shown that the consequences of oxidative damage to testicular tissue caused by non-ionizing radiation have a dose-dependent effect, and the higher the field strength and the more the number of pulses, the higher the level of oxidative stress in testicular tissue, the lower the level of antioxidant, the higher the rate of apoptosis of spermatogenic cells, and the more difficult the damage repair.

[0004] The mechanism of the influence of electromagnetic radiation on the male reproductive system may include local non-thermal effects or oxidative stress caused by whole body irradiation, which is very complex and has not been clearly studied, so it is difficult to effectively protect the male reproductive system from damage caused by electromagnetic radiation, and there is a lack of effective intervention means. For complex diseases or effects, finding relevant biomarkers for their occurrence is an effective way to treat or intervene. At present, there are few biomarkers of reproductive damage caused by electromagnetic radiation, and most of them are proteins or metabolites found in the testis, which is not conducive to the convenient detection and large-scale application of biomarkers. We detected and jointly analyzed the testicular and serum proteomics of a mouse model after electromagnetic radiation, identified potential protein biomarkers in the serum that can predict testicular and sperm damage caused by electromagnetic radiation, making detection more convenient and efficient, which will provide important help for the early warning and protection of reproductive damage caused by electromagnetic radiation. SUMMARY

[0005] The present application is based on the above research, and aims to provide a serum biomarker for diagnosing male reproductive function damage, and to provide a new use of the serum biomarker, i.e. application in preparing a radiation-related reproductive damage detection kit or a weak sperm diagnosis kit.

[0006] The present application first constructs a male mouse model of electromagnetic radiation, then detects the sex hormone level, sperm parameters, testicular morphology and function of the male mouse to determine the negative effects of electromagnetic radiation on the male mouse; then finds the differential proteins of the testis after electromagnetic radiation through testicular proteomics and performs functional clustering analysis, and simultaneously performs joint analysis of serum proteomics and testicular proteomics to explore common differential proteins; finally, the correlation analysis of the common differential proteins and sperm parameters is performed, and the proteins (H1-1, H1-4, H1-5, Hp1bp3, Itgb2, Ncf2 and Rhoc) with statistical differences in the correlation analysis are determined as potential biomarkers for predicting male reproductive function damage caused by electromagnetic radiation. Thus, potential serum molecular diagnostic biomarkers for weak sperm and radiation-related reproductive damage are provided, and effective and simple clinical diagnosis is achieved by detecting the serum of patients.

[0007] Specifically, the present application provides the following technical solutions:

[0008] In a first aspect, the present application provides a serum biomarker combination for radiation-related reproductive damage, which is composed of H1-1, H1-4, H1-5, Hp1bp3, Itgb2, Ncf2 and Rhoc. The seven serum molecules are differentially expressed in serum and testis, and are significantly correlated with the concentration, motility and proportion of forward motility sperm.

[0009] In the second aspect of the present application, the serum molecular marker combination is used in the preparation of a kit for detecting radiation-related reproductive damage. Preferably, the radiation-related reproductive damage is reproductive damage caused by non-ionizing electromagnetic radiation.

[0010] Since the radiation-related reproductive damage is also a component of asthenozoospermia, the serum molecular marker combination is also used in the preparation of a kit for diagnosing asthenozoospermia. When diagnosing asthenozoospermia, whether the radiation-related damage is a triggering factor of asthenozoospermia can be determined by detecting the serum molecular markers.

[0011] Preferably, the kit contains reagents for detecting the expression levels of H1-1, H1-4, H1-5, Hp1bp3, Itgb2, Ncf2 and Rhoc in the serum sample.

[0012] Further, the method for detecting the expression levels of H1-1, H1-4, H1-5, Hp1bp3, Itgb2, Ncf2 and Rhoc in the serum sample can be selected from the prior art, and any method capable of detecting gene or protein expression levels is suitable for the present application.

[0013] Therefore, the reagents for detecting the expression levels of H1-1, H1-4, H1-5, Hp1bp3, Itgb2, Ncf2 and Rhoc in the serum sample are selected from one or more detection techniques or methods in the following group: ELISA detection method, chemiluminescent immunoassay method, protein chip technology, in situ hybridization method, Northern blotting method, RT-PCR, real-time quantitative PCR method.

[0014] In the third aspect of the present application, a kit for detecting radiation-related reproductive damage contains reagents for detecting the expression levels of H1-1, H1-4, H1-5, Hp1bp3, Itgb2, Ncf2 and Rhoc in the serum sample.

[0015] Preferably, the kit contains one or more detection techniques or methods in the following group: ELISA detection method, chemiluminescent immunoassay method, protein chip technology, in situ hybridization method, Northern blotting method, RT-PCR, real-time quantitative PCR method.

[0016] Compared with the prior art, the present application has the following advantages:

[0017] This invention discloses for the first time a serum molecular marker capable of detecting radiation-related reproductive dysfunction in men. Diagnosis of male radiation-related reproductive dysfunction can be made by detecting the expression levels of relevant molecules in the serum. This not only addresses the current scarcity of markers for reproductive damage caused by electromagnetic radiation, but also overcomes the shortcomings of current markers, which are mostly proteins or metabolites found in the testes, hindering convenient detection and large-scale clinical application. Attached Figure Description

[0018] Figure 1 The following figures show the results of 30 days of 3.2 GHz pulsed electromagnetic radiation (8 hours / day) damaging the reproductive function of male mice: A. Continuous monitoring of food intake; B. Continuous monitoring of body weight; C. Sex hormone levels; D. Computer-aided semen analysis (CASA) analysis of sperm motility, concentration, and morphology; E. HE staining and weight of the testes; F. Fluorescent staining of testicular spermatogenesis-related proteins and testicular TUNEL staining.

[0019] Figure 2 The following are the results of proteomic analysis of the testes after electromagnetic radiation: A. Principal component analysis (PCA) plot; B. Volcano plot of differentially expressed proteins; C. GO cluster analysis of downregulated proteins; D. GO cluster analysis of upregulated proteins; E. KEGG pathway analysis of downregulated proteins; F. KEGG pathway analysis of upregulated proteins; G. Expression heatmap of spermatogenesis-related proteins; H. Bar chart of expression levels of spermatogenesis-related proteins.

[0020] Figure 3 The analysis of serum molecular markers of electromagnetic radiation-induced reproductive dysfunction in male mice is presented: A. Volcano plot of differentially expressed proteins in serum proteomics after electromagnetic radiation; B. Heatmap of protein expression in serum proteomics; C. Venn diagram of differentially expressed proteins in serum and testicular proteomics; D. Commonly differentially expressed proteins in serum and testicular proteomics; E. Bar chart of expression levels of commonly differentially expressed proteins; F. Correlation analysis of expression levels of commonly differentially expressed proteins with sperm parameters. Detailed Implementation

[0021] The present invention will now be described in detail with reference to the embodiments and accompanying drawings, but the implementation of the present invention is not limited thereto.

[0022] All reagents and raw materials used in this invention are commercially available or can be prepared according to literature methods. Experimental methods in the following examples, unless otherwise specified, are generally performed under conventional conditions as described in Sambrook et al., *Molecular Cloning: A Laboratory Guide* (New York: Cold Spring Harbor Laboratory Press, 1989), or under conventional conditions, or as recommended by the manufacturer.

[0023] The purpose of this invention is to provide serum molecular markers for predicting the damage of microwave electromagnetic radiation to the reproductive function of male mice, which can be used for early warning of microwave electromagnetic radiation-induced male fertility damage.

[0024] The method for determining the molecular markers of this invention is as follows:

[0025] 1) Construct an electromagnetic radiation-induced male mouse model;

[0026] 2) Detect sex hormone levels, sperm parameters, testicular morphology and function in male mice;

[0027] 3) Testicular proteomics identified differentially expressed proteins in the testes after electromagnetic radiation and performed functional cluster analysis.

[0028] 4) Combined analysis of serum proteomics and testicular proteomics to identify common differentially expressed proteins;

[0029] 5) Correlation analysis was performed between common differential proteins and sperm parameters, and proteins with statistically significant differences in the correlation analysis were identified as potential markers for predicting electromagnetic radiation-induced damage to male reproductive function.

[0030] To make the above description of the present invention more apparent and understandable, the present invention will be described in detail with reference to the embodiments and accompanying drawings, but this is not intended to limit the present invention.

[0031] I. Assessment of the impact of 3.2 GHz electromagnetic pulse radiation on the reproductive function of male mice

[0032] (I) Research Methods

[0033] 1. Laboratory animal husbandry:

[0034] Twenty 8-week-old male balb / c mice (approximately 25 g in weight) were purchased from GemPharmatech. The mice were housed in a controlled environment with a 12-hour light / 12-hour dark cycle at a room temperature of 24°C ± 2°C. The animals were in a stable condition and had free access to standard commercial pelleted feed and water. Five animals were placed in each cage in standard ventilated polypropylene cages with dry rice husks as bedding. Twenty randomly selected mice were divided into a sham control group and an electromagnetic radiation (EMR) group, with ten mice in each group. Mice in both experimental groups were continuously exposed to 3.2 GHz (pulsed wave) microwave radiation for 8 hours daily from 9:00 AM to 5:00 PM for four weeks. Blood glucose, food intake, and body weight were measured weekly.

[0035] 2. Electromagnetic radiation exposure parameters:

[0036] Laboratory mice exposed to 3.2 GHz pulsed microwave radiation had a total average power density of 0.0248 mW / cm². 2 The total average specific absorptivity (SAR) was 0.0146 W / kg. The SAR value along the body length parallel to the electric field was estimated based on the mouse's actual position. (0.0248 mW / cm) 2 Exposure at total average power density did not lead to an increase in ambient temperature in the animal cage or rectal temperature in mice.

[0037] 3. Computer-Assisted Sperm Analysis (CASA)

[0038] One side of the epididymal tail was taken and placed in 1 ml of preheated Hank's solution at 37°C. The sperm was released by cutting 4-5 times with scissors. The sperm suspension to be tested was incubated in a 37°C water bath for 10-15 minutes. 10 μL of sperm was collected and placed on preheated CASA slides and coverslips. Real-time observation was performed under a microscope. Sperm motility parameters were collected from 7 different fields of view for each sample.

[0039] 4. Sex hormone testing

[0040] Serum levels of FSH, LH, estradiol, testosterone, and inhibin B were quantified using a mouse FSH ELISA kit (Aviva Systems Biology, OKDD03124), a mouse LH ELISA kit, and a mouse estradiol ELISA kit. All ELISAs were run on 96-well plates and analyzed by colorimetric readings using a Synergy Neo microplate reader (BioTek Instruments).

[0041] 5. Immunofluorescence (paraffin sections)

[0042] The slides were placed in a 60°C oven for approximately 15 minutes, then immersed in xylene, 100% ethanol, 95% ethanol, 85% ethanol, and 70% ethanol for dewaxing and rehydration. Antigen retrieval was performed by boiling in Tris-EDTA buffer (pH=9.0) for 20 minutes. Blocking was performed at room temperature with 5% donkey serum (Jackson, USA, 017-000-121) for 1 hour; incubation was then performed overnight at 4°C with the primary antibody. The secondary antibody was diluted 1:400 and incubated at room temperature for 1 hour. Scanning and analysis were performed using a Nano Zoomer S60 digital slide scanner.

[0043] (II) Research Results

[0044] Studies have found that after four weeks of exposure to 3.2 GHz electromagnetic pulses, male balb / c mice showed no significant changes in body weight or food intake. Figure 1 A, B), elevated levels of reproductive hormones FSH, LH, estradiol, testosterone, inhibin B, progesterone, and prolactin ( Figure 1 C), CASA results showed that sperm concentration, motility, and the proportion of progressively motile sperm significantly decreased after electromagnetic radiation. Parameters reflecting sperm motility, including linear velocity (VSL), curvilinear velocity (VCL), and average path velocity (VAP), decreased, while the sperm DNA fragmentation index (DFI) significantly increased. Figure 1 D); In the irradiated group, the testicular weight and testicular body weight ratio decreased, and certain changes occurred in the pathological tissue structure: the diameter of the seminiferous tubules decreased, the intercellular spaces of the spermatogenic cells increased, the arrangement of spermatogenic cells became disordered, and vacuoles were more common in the seminiferous tubules. Figure 1 E). Immunofluorescence staining of the testes showed a significant decrease in the number of spermatogonia (DDX4), Sertoli cells (SOX9), and sperm (ACROSIN) after electromagnetic radiation, while the number of inactivated spermatogonial stem cells (STRA8) relatively increased. Tunel imaging showed an increased level of apoptosis in the testes. Figure 1 F). The above results indicate that 4 weeks of 3.2 GHz electromagnetic pulse radiation caused significant damage to sperm quality and testicular function in male mice.

[0045] II. Combined analysis of testicular and serum proteomics revealed serum protein markers associated with reproductive function impairment in male mice caused by 3.2 G Hz electromagnetic pulse radiation.

[0046] (I) Research Methods

[0047] 1. Testicular proteomics analysis

[0048] Proteomics analysis was performed on testicular samples from 3 sham control groups and 3 EMR groups by Jingjie Biotechnology Co., Ltd. The specific method was as follows: Testicular tissue proteins were extracted, and protein concentrations were determined using a BCA kit. Equal amounts of protein were digested from each sample using trypsin. Trypsin peptides were dissolved in solvent A and directly loaded onto a self-made reversed-phase analytical column. The mobile phase consisted of solvent A (0.1% formic acid, 2% acetonitrile / water solution) and solvent B (0.1% formic acid-acetonitrile solution). The peptide separation gradient was as follows: 0–14 min, 6%–24% B; 14–16 min, 24%–35% B; 16–18 min, 35%–80% B; 18–20 min, 80% B, at a constant flow rate of 500 nl / min on a NanoElute UHPLC system (Bruker Daltonics). The peptides were then processed using a capillary source and analyzed by timsTOF Pro mass spectrometry. Full MS scans were set to 300-1500 (MS / MS scan range) and 20 PASEF (MS / MS mode) – MS / MS scans were acquired per cycle. The MS / MS scan range was set to 400-850, and the isolation window was set to 7 m / z. DIA data were processed using the DIA-NN search engine (v.1.8). Tandem mass spectra were searched based on Mus_musculus-10090_SP_20241202.fasta (17236 entries), which is linked to the reverse decoy database. Trypsin / P was designated as the cleavage enzyme, allowing a maximum of one deletion cleavage. N-terminal Met excision and urea methylation on Cys were designated as fixation modifications. FDR was adjusted to <1%.

[0049] 2. Serum proteomics analysis

[0050] Serum samples from 8 sham control groups and 8 EMR groups were subjected to proteomics analysis, performed by Jingjie Biotechnology Co., Ltd. The specific method was as follows: 100 µL of centrifuged blood sample was transferred to pre-washed magnetic nanomaterials and incubated at 37°C for 1 hour at 1200 rpm on a constant-temperature mixer. After incubation, the magnetic beads were washed three times with washing buffer. 150 μL of enzyme digestion buffer was added to each magnetic bead, followed by trypsin at a final concentration of 10 ng / μL, and digestion was performed overnight at 37°C. Dithiothreitol (DTT) was added to a final concentration of 5 mM, and reduction was carried out at 56°C for 30 min. Iodoacetamide (IAM) was then added to a final concentration of 11 mM, and incubation was carried out at room temperature in the dark for 15 min. The samples were desalted according to the C18 ZipTips instructions, freeze-dried, and then used for LC-MS / MS analysis.

[0051] Peptides were dissolved in mobile phase A of liquid chromatography and then separated using a Vanquish Neo ultra-high performance liquid chromatography (UHPLC) system. Mobile phase A was an aqueous solution containing 0.1% formic acid; mobile phase B was an aqueous solution containing 0.1% formic acid and 80% acetonitrile. The liquid phase gradient settings were: 0–1.6 min, 4%–22.5% B; 1.6–2.0 min, 22.5%–35% B; 2.0–2.6 min, 35%–55% B; 2.6–2.7 min, 55%–99% B; 2.7–6.8 min, 99% B; 6.8–7.6 min, 99% B, with the flow rate maintained at 300 nL / min. After separation by the UHPLC system, the peptides were injected into an NSI ion source for ionization and then analyzed by Orbitrap Astral mass spectrometry. The ion source voltage was set to 1900 V. Peptide precursor ions were detected and analyzed using an Orbitrap detector, while secondary fragment ions were detected and analyzed using an Astral detector. The primary mass spectrometry scan range was set to 480-780 m / z, with a scan resolution of 240,000. The secondary mass spectrometry scan range had a fixed starting point of 150 m / z, with a secondary scan resolution of 80,000. Data acquisition was performed using a data-independent scan (DIA) procedure, where peptide ions from multiple consecutive m / z windows were followed by fragmentation in the HCD collision cell after the primary scan, using 25% of the fragmentation energy, and then analyzed sequentially by secondary mass spectrometry. To improve the efficiency of the mass spectrometer, automatic gain control (AGC) was set to 500%, and the maximum injection time was set to 3 ms.

[0052] 3. Statistical Analysis

[0053] SPSS 18.0 software was used for analysis. Quantitative data were expressed as mean ± standard deviation, and t-tests were used for intergroup comparisons. Pearson's test was used for correlation analysis, and P < 0.05 was considered statistically significant.

[0054] (II) Research Results

[0055] Proteomic analysis of the testes after 3.2 GHz electromagnetic radiation: Principal component analysis (PCA) showed significant changes in testicular proteins after electromagnetic radiation. Figure 2 A); Using Fold change = 1.5 or 1 / 1.5, P < 0.05 as the screening criteria, after electromagnetic radiation, the expression of 102 proteins in the testes was downregulated and the expression of 99 proteins was upregulated. Figure 2 B). GO cluster analysis showed that the downregulated proteins were significantly enriched in pathways related to spermatogenesis and sperm flagellar motility, such as Spermatogenesis, Flagellated sperm motility, and Sperm fibrous sheath. Figure 2The expression levels of spermatogenesis-related proteins enriched (C, 2E) are as follows: Figure 2 As shown in G and 2H, the upregulated proteins are enriched in pathways related to DNA damage repair. Figure 2 D, 2F).

[0056] After 3.2 GHz electromagnetic radiation, 281 proteins were significantly downregulated and 149 proteins were significantly upregulated in serum. Figure 3 A, B), after intersecting the differentially expressed proteins in serum and testes, it was found that the expression levels of H1-1, H1-4, H1-5, H3-5, Hp1bp3, Itgb2, Macroh2a2, Mdk, Ncf2, Plod2, and Rhoc proteins were significantly altered in both serum and testes of mice after EMR. Figure 3 We performed Pearson correlation regression analysis on these 11 proteins and sperm parameters, and found that H1-1, H1-4, H1-5, Hp1bp3, Itgb2, Ncf2, and Rhoc were significantly correlated with sperm concentration, motility, and the proportion of progressively motile sperm. They can be considered as potential protein markers in serum related to testicular damage after electromagnetic radiation.

[0057] The undescribed parts of this invention are the same as or implemented using existing technology. The applicant declares that this invention is illustrated through the above embodiments, but the invention is not limited to the above detailed methods, i.e., it does not mean that the invention must rely on the above detailed methods to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the product of this invention, additions of auxiliary components, and selection of specific methods all fall within the protection and disclosure scope of this invention.

Claims

1. The application of serum molecular marker combinations in the preparation of a kit for detecting radiation-related reproductive damage, characterized in that, The serum molecular marker combination consists of H1-1, H1-4, H1-5, Hp1bp3, Itgb2, Ncf2, and Rhoc.

2. The application of serum molecular marker combinations in the preparation of a diagnostic kit for asthenospermia, characterized in that, The serum molecular marker combination consists of H1-1, H1-4, H1-5, Hp1bp3, Itgb2, Ncf2, and Rhoc.

3. The application according to claim 1, characterized in that, The radiation-related reproductive damage refers to reproductive damage caused by non-ionizing electromagnetic radiation.

4. The application according to claim 1 or 2, characterized in that, The kit contains reagents for detecting the expression levels of H1-1, H1-4, H1-5, Hp1bp3, Itgb2, Ncf2, and Rhoc in serum samples.

5. The application according to claim 4, characterized in that, The reagents used to detect the expression levels of H1-1, H1-4, H1-5, Hp1bp3, Itgb2, Ncf2, and Rhoc in serum samples are selected from one or more of the following detection techniques or methods: proteomics analysis, ELISA detection methods, chemiluminescent immunoassay methods, protein chip technology, in situ hybridization, Northern blot, RT-PCR, and real-time quantitative PCR.

6. A radiation-related reproductive injury detection kit, characterized in that, This kit contains reagents for detecting the expression levels of H1-1, H1-4, H1-5, Hp1bp3, Itgb2, Ncf2, and Rhoc in serum samples.

7. The reagent kit according to claim 6, characterized in that, The kit contains reagents from one or more of the following detection technologies or methods: proteomics analysis, ELISA detection methods, chemiluminescent immunoassay methods, protein chip technology, in situ hybridization, Northern blot, RT-PCR, and real-time quantitative PCR.

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