Application of cyanidin-3-O-glucoside in preparation of medicine for treating/or preventing sperm quality reduction caused by cadmium
阅读说明：本技术 矢车菊素-3-o-葡萄糖苷在制备治疗/或预防镉引发的***质量下降的药物中的应用 (Application of cyanidin-3-O-glucoside in preparation of medicine for treating/or preventing sperm quality reduction caused by cadmium ) 是由 白卫滨 李旭升 蒋鑫炜 孙建霞 田灵敏 于 2019-10-11 设计创作，主要内容包括：本发明公开了矢车菊素-3-O-葡萄糖苷在制备治疗/或预防镉引发的精子质量下降的药物中的应用,本发明发现,矢车菊素-3-O-葡萄糖苷(Cyanidin-3-O-glucoside,C3G)可改善青春期Cd暴露造成的精子质量下降。C3G干预可通过组蛋白修饰缓解精子变形期的组蛋白-鱼精蛋白替换过程、改善睾丸抗氧化系统使得精子免受Cd氧化应激诱导的细胞凋亡,使得精子发生过程维持正常,保证精子的正常生成。(The invention discloses application of Cyanidin-3-O-glucoside in preparing a medicament for treating and/or preventing sperm quality reduction caused by cadmium, and discovers that Cyanidin-3-O-glucoside (C3G) can improve sperm quality reduction caused by Cd exposure in adolescence. C3G intervenes in the replacement process of histone-protamine which can relieve the sperm deformation period through histone modification, and improves a testicle antioxidant system to prevent the sperm from apoptosis induced by Cd oxidative stress, so that the spermatogenesis process is maintained to be normal, and the normal generation of the sperm is ensured.)
1. Application of cyanidin-3-O-glucose in preparation of medicine for treating/or preventing sperm quality reduction caused by Cd.
2. The use according to claim 1, wherein the decreased sperm quality is manifested as decreased sperm count, motility and motility.
3. The use according to claim 1, wherein the sperm cell quality reduction is due to a blocking of the spermatogenic process.
4. The use according to claim 3, wherein the spermatogenic process arrest is in the spermatogenic stage.
5. The use of claim 4, wherein the spermatogenic process block is due to failure of sperm nuclear histone-protamine replacement during sperm deformation induced by Cd exposure.
6. The use as claimed in claim 3, wherein the sperm quality degradation is due to apoptosis of sperm due to Cd oxidative stress following sperm deformation failure.
7. The use of claim 6, wherein the sperm apoptosis is transduced via DNA damage, MAPK signaling pathway and mitochondrial apoptosis signaling pathway.
8. Application of cyanidin-3-O-glucose in preparation of medicine for treating and/or preventing decrease of sperm number and/or motility and motor ability caused by Cd.
9. The use according to any one of claims 1 to 8, wherein the human is administered in a dose of 0.15mg/kg.bw to 10 mg/kg.bw.
Technical Field
The invention relates to the technical field of biological medicines, and in particular relates to application of cyanidin-3-O-glucoside in preparation of a medicine for treating and/or preventing sperm quality reduction caused by cadmium.
Background
Epidemiological studies show that the number and concentration of sperms in males are reduced by 50% in nearly 40 years, and the sterility rate is as high as 15%. Among these, exposure from environmental pollutants is one of the important reasons for the continuous increase in male infertility rates. Cadmium (Cadmium, Cd) is a heavy metal environmental pollutant that can enter the body through various ways such as food, water and air and accumulate, and can cause sperm quality reduction through various ways such as oxidative damage. The adolescent male reproductive system is in a developmental stage and is more susceptible to damage by Cd than in a sexual maturation stage. Cyanidin-3-O-glucoside (C3G) is used as a water-soluble anthocyanin widely distributed in plant fruits, has strong antioxidant activity, has a chelating effect on cadmium ions, and is a potential functional factor for antagonizing cadmium toxicity. No improvement effect of C3G on Cd-induced adolescent sperm quality reduction is reported in the current research, and the protection mechanism of the Cd-induced adolescent sperm quality reduction is not eliminated.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides the application of cyanidin-3-O-glucoside in preparing the medicine for treating and/or preventing the quality reduction of sperms caused by cadmium.
The first purpose of the invention is to provide the application of cyanidin-3-O-glucose in preparing the medicine for treating and/or preventing sperm quality reduction caused by Cd.
The second purpose of the invention is to provide the application of cyanidin-3-O-glucose in preparing the medicine for treating and/or preventing the reduction of sperm quantity and/or activity and movement ability caused by Cd.
In order to achieve the purpose, the invention is realized by the following technical scheme:
the invention adopts animal experiments, researches on the regulation and control effect of a certain dose of cyanidin-3-O-glucoside on the reduction of the sperm quality of adolescent mice caused by Cd exposure, and discovers that compared with a Cd injury group, the ingestion of C3G obviously improves the sperm quantity, improves the sperm activity and survival rate, and effectively improves the sperm quality. Adolescent oral Cd exposure can be modified by epigenetic abnormalities in histones, which in turn leads to blocking of histone-protamine replacement, resulting in the arrest of spermatogenesis in the sperm metaplasia phase. And the intervention of C3G can maintain normal histone modification, effectively relieve histone-protamine block, and make sperm deformation process smoothly proceed. In addition, sperm deformation failure caused by adolescent Cd exposure makes the sperm nucleus susceptible to DNA damage mediated by Cd-induced oxidative stress, leading to sperm cell apoptosis.
The invention therefore claims the following:
application of cyanidin-3-O-glucose in preparation of medicine for treating/or preventing sperm quality reduction caused by Cd.
Preferably, the sperm quality reduction is manifested as a reduction in sperm count, motility, and motility.
The number of sperm is expressed by sperm density.
The motion capability includes a sperm linear motion rate (VSL), an Average path rate (VAP), an Amplitude of sperm lateral displacement (VLH), a curve rate (VCL), a whip frequency (bcat-cross frequency, BCF), and an Average angular displacement.
Preferably, the sperm quality reduction is due to a blocking of the spermatogenic process.
More preferably, the spermatogenic process block occurs during the spermatogenic stage.
More preferably, the spermatogenic process arrest is due to failure of sperm nuclear histone-protamine replacement during sperm deformation induced by Cd exposure.
More preferably, the Cd exposure occurs during puberty.
More preferably, the sperm quality degradation is due to apoptosis of sperm due to Cd oxidative stress following failure of sperm deformation.
More preferably, the sperm apoptosis is transduced via DNA damage, MAPK signaling pathways, and mitochondrial apoptosis signaling pathways.
The invention also claims the application of cyanidin-3-O-glucose in the preparation of a medicament for treating and/or preventing the reduction of sperm number and/or motility and movement capacity caused by Cd. Preferably, the dosage of the human body is 0.15mg/kg.bw to 10 mg/kg.bw.
The dosage is converted according to the dosage of 150-1000 mg/kg of C3G in 1kg of mouse feed ingested by mice and the dosage effect of mice-human bodies
Compared with the prior art, the invention has the following beneficial effects:
the Cyanidin-3-O-glucoside (Cyanidin-3-O-glucoside, C3G) can improve the quality reduction of sperms caused by Cd exposure in adolescence. C3G intervenes in the replacement process of histone-protamine which can relieve the sperm deformation period through histone modification, and improves a testicle antioxidant system to prevent the sperm from apoptosis induced by Cd oxidative stress, so that the spermatogenesis process is maintained to be normal, and the normal generation of the sperm is ensured.
Drawings
FIG. 1 shows the weight and coefficient changes of testis and epididymis; a is the change in testicular weight; b is the change of the testis weight coefficient; c is the weight change of the epididymis; d is the weight coefficient change of the epididymis.
FIG. 2 shows the change in the anogenital distance of mice.
FIG. 3 is H & E staining of testis and epididymis tissue sections (200X); a is testis H & E staining picture; b is an H & E staining picture of the epididymis head; c is an H & E staining picture of the epididymis tail.
FIG. 4 is mouse testis lumen Johnsen's score.
FIG. 5 shows the morphological teratogenicity and density of sperm; a is a sperm photo; b is the change in sperm count; c is sperm aberration rate change.
FIG. 6 shows sperm motility and survival rate; a is sperm motility change; and B is the change of sperm motility.
FIG. 7 is a parameter of sperm motility; a is the change of the linear motion velocity VSL; b is the change in the average path rate, VAP; c is the variation of the sperm head side swing amplitude VLH; d is the change in curve rate VCL; e is the change in the whiplash frequency BCF; f is the change of the average angular displacement MAD; g is the change in the linearity LIN of the sperm; h is the forward STR variation.
FIG. 8 shows PAS staining of testicular seminiferous epithelium at different stages.
FIG. 9 shows the ratio of sperm to supporting cells at the late stage of sperm cell deformation.
FIG. 10 is the protein expression levels of histones H2A and H2B in testis; a is immunohistochemical staining for H2A and H2B; b is an electrophoretogram of H2A and H2B; c is the change of the expression level of H2A and H2B.
FIG. 11 shows mRNA expression levels of transition protein and protamine in testis; a is the mRNA expression level of
FIG. 12 is the protein expression levels of ubiquitinated histones ub-H2A and ub-H2B in testis.
FIG. 13 is the mRNA expression levels in testis for related ubiquitin ligases and bindases of histones H2A and H2B; a is mRNA level of histone H2A related ubiquitin enzyme; b is mRNA level of histone H2B related ubiquitin enzyme.
FIG. 14 is testis tissue HDAC, HAT content; a is the HDAC content; b is HAT content.
FIG. 15 shows the expression of testis histones H4 and acetylated histones H4 at different sites.
FIG. 16 shows protein level expression of Histone tissue histones H3K9me1 and
FIG. 17 shows protein level expression of testis tissue G9 a.
FIG. 18 is testis tissue MDA levels.
FIG. 19 is testis tissue GSH and GSSG levels; a is the content change of GSH; b is the content change of GSSG; c is the ratio change of GSH and GSSG.
FIG. 20 shows the activity of SOD in testis tissue.
FIG. 21 shows the expression of 8-OHdG in round sperm.
Figure 22 is apoptotic TUNEL staining in testis.
FIG. 23 is the expression of ERK protein in testis; a is an electrophoretogram of ERK and p-ERK; b is the change in ERK content; c is the content change of p-ERK; d is the ratio of p-ERK to ERK.
FIG. 24 is expression of JNK protein in testis; a is an electrophoretogram of JNK and p-JNK; b is the change of JNK content; c is content change of p-JNK; d is p-JNK and the proportion change of the JNK.
FIG. 25 shows the expression of p38 protein in testis; a is an electrophoretogram of p38 and p-p 38; b is the change of p38 content; c is content change of p-p 38; d is the ratio change of p-p38 and p 38.
FIG. 26 is the expression of p53 protein in testis; a is an electrophoretogram of p53 and p-p 53; b is the change of p53 content; c is content change of p-p 53; d is the ratio change of p-p53 and p 53.
FIG. 27 is expression of Bcl-2 family protein in testis; a is an electrophoretogram of Bax, Bcl-2 and Bad; b is the content change of Bax; c is the content change of Bcl-2; d is the content change of Bad.
FIG. 28 is the expression of cleared-
Detailed Description
The invention is described in further detail below with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
First, experimental material
1. Laboratory animal
Kunming male mice, 96 mice at 3 weeks of age, were purchased from the center of laboratory animals in Guangdong province. The experimental animals are raised in animal rooms of Chinese university and are alternately lighted day and night for 12 hours. The temperature is kept between 22 and 26 ℃, the relative humidity is 60 to 80 percent, and the food can be freely taken.
2. Animal feed
The basic formula of the animal feed is a common AIN93-G feed formula, and 500mg/kg of DietC3G is additionally added into a Cd + C3G group. The feed is manufactured by Jiangsu Meditson biological medicine limited company and is preserved at the temperature of-20 ℃.
3. Primer usage information
Table 1 PCR primer information:
all primers were synthesized by Shanghai Bioengineering, Inc.
Second, Experimental methods
1. Epididymal tail sperm parameter determination
The left epididymis tail is cut into pieces and is rapidly placed in 1.0mL of preheated DMEM medium, the materials are released for 30min at 37 ℃, 10.0 mu L of sperm suspension is added into a sperm counting plate after being fully mixed, and continuous frame dynamic photographing and single frame static photographing of sperms are carried out by utilizing a computer-assisted sperm analysis system, so that the method is used for measuring sperm motility, motion related parameters, sperm quantity, aberration rate and morphological analysis.
2. Hematoxylin-eosin staining
Taking testis and epididymis paraffin embedded wax blocks, slicing at the thickness of 5 μm, and baking in an oven at 70 deg.C for 2 h. Gradient deparaffinization to water, hematoxylin staining for 35s, eosin staining for 3s, and then dehydration to clear. Mixing neutral gum and fresh xylene at a ratio of 4:1, sealing, and naturally drying. And (6) microscopic examination and photographing.
3. Testis spermatogenic epithelium Johnsen's score
Johnsen's score was performed on testicular luminal seminiferous epithelium with reference to standards, and no less than 50 seminiferous tubules were analyzed per mouse.
4. Periodic acid-Schiff base dyeing
The testis seminiferous tubules were stained with periodic acid schiff base staining kit (Nanjing Bega corporation) according to the kit instructions.
5. Immunohistochemistry
After baking and dewaxing the testis slices, adopting microwave antigen to repair. Sections were placed in 3% hydrogen peroxide, incubated for 25min at room temperature in the dark to block endogenous peroxidase, and then washed 3 times in a PBS shaker. The tissue position is circled by an immunohistochemical pen, 3% BSA is dripped to uniformly cover the tissue, and the tissue is sealed for 30min at room temperature. After the confining liquid is discarded, primary antibody diluted according to a certain proportion is dripped, and the section is placed in a wet box for incubation overnight at 4 ℃. The primary antibody was discarded and the slide was washed 3 times with PBS shaker. Adding corresponding species of secondary antibody dropwise, and incubating at room temperature for 50 min. Discarding the secondary antibody, washing the slide for 3 times by using a PBS shaker, dripping DAB color developing solution, immediately observing by using a microscope to control the color developing time, counterstaining with hematoxylin for 35s, and washing for 5min by using running water. Dehydrating and sealing, performing microscopic examination, and taking a picture.
6. Immunofluorescence
After baking and dewaxing the testis slices, adopting microwave antigen to repair. The tissue position is circled by an immunohistochemical pen, an autofluorescence quencher is added for 5min, and the tissue is washed by running water for 10 min. Dropwise adding 3% BSA, sealing at room temperature for 5min, removing the sealing solution, dropwise adding diluted primary antibody according to a certain proportion, and placing the slices in a wet box for incubation at 4 ℃ overnight. Discarding the primary antibody, washing the slide with PBS for 3 times by using a shaking table, dropwise adding the corresponding species of fluorescent secondary antibody, and incubating for 50min at room temperature in a dark place. The secondary antibody was discarded, the slide was washed 3 times with PBS shaker, and DAPI staining solution was added dropwise, followed by incubation in the dark at room temperature for 10 min. The slides were washed 3 times with PBS shaker and mounted with anti-fluorescence quenching mounting medium. The slices were placed under a fluorescence microscope, excited with the corresponding excitation wavelength, observed and images collected.
7. Protein extraction and immunoblotting
(1) Tissue protein extraction
Animal tissues are accurately weighed, protein lysate is added in a ratio of 1:9, and homogenization is carried out until no visible solid exists. (the homogenate of samples at 4 degrees C, 14000g under the conditions of centrifugation for 10min, supernatant for use, BCA method to the supernatant protein concentration determination, according to the supernatant protein concentration, 2.0 u g/. mu.L protein concentration dilution, dilution using 1/5 final volume protein buffer (5 x), the remaining volume with PBS filling, vortex and shake the sample, 100 degrees C boiling 7min, -20 degrees C storage for use.
(1) Western blot
Preparing SDS-PAGE gel: the reagents used for preparing the gel are all from the kit. According to the molecular weight of the target protein to be separated, deionized water, 30% Arc-Bis, Tris-HCl (pH 8.8 or pH 6.8), 10% SDS and 10% ammonium persulfate are mixed in sequence, TEMED is added before use, and separation gel and concentrated gel with different concentrations are prepared.
Protein electrophoresis: preparing separation gel and concentrated gel, adding electrophoresis buffer solution, sequentially adding protein sample and protein ladder to start constant-pressure electrophoresis, and stopping when bromophenol blue reaches the bottom of gel.
Wet film transfer: opening a film transferring clamp in the film transferring liquid to enable the black cathode surface of the film transferring clamp to face downwards, sequentially placing sponge, filter paper, glue, a PVDF film, filter paper and sponge, closing the film transferring clamp, placing the film transferring clamp in an electric transferring groove, and adding the film transferring liquid precooled at 4 ℃. And (5) setting membrane transferring conditions according to different molecular weight requirements, and performing constant-current membrane transferring.
And (3) sealing: and taking out the PVDF membrane, washing for 1 time by using TBST buffer solution, adding 5% skimmed milk powder, and sealing for 1.5h by using a shaking table at room temperature.
Primary antibody incubation: discarding the confining liquid, washing with TBST buffer solution for 3 times, each time for 5min, discarding the washing liquid, adding diluted primary antibody, and incubating overnight at 4 deg.C in a shaking table.
And (3) secondary antibody incubation: recovering primary antibody, washing with TBST buffer solution for 3 times, each time for 10min, adding diluted secondary antibody of corresponding species, and incubating for 1h at room temperature in a shaking table.
And (3) developing: the secondary antibody was discarded and washed 3 times for 10min in TBST buffer. Adding ECL luminous liquid for reaction, and developing in an automatic developing exposure instrument. Stripe grayscale Analysis was performed using Clinx Chemi Analysis software.
8. RNA extraction and qRT-PCR
(1) Tissue RNA extraction
The tissue was soaked in 1mL Trizol and homogenized in a tissue grinder until no visible solids were present. Adding
(2) Reverse transcription of RNA
The reagents used for RNA reverse transcription were all from Takara kit. The DNA removal reaction and the reverse transcription reaction were performed according to the kit instructions.
(3) Real-time fluorescent quantitative PCR
The reaction system was prepared as shown in Table 2, and amplification was carried out under the following conditions. Pre-denaturation (Repeat: 1): at 95 ℃ for 30 s; PCR reaction (Repeat: 40): 95 ℃ for 5 s; 30s at 60 ℃; discovery.
TABLE 2 Real Time PCR reaction System
(4) qPCR result processing
According to the Ct value obtained by the reaction, the Ct is obtained(purpose)-Ct(internal reference)When the difference is equal to Δ Ct, Δ Δ Ct and 2 are obtained by inter-group calculation-ΔΔCt。
9. TUNEL apoptosis assay
Baking and dewaxing testis slice, circling out tissue area with immunohistochemical pen, dripping proteinase K working solution into the circle, and incubating at 37 deg.C for 25 min. The liquid was discarded and the slides were washed 3 times with a PBS shaker. And (3) dropwise adding a membrane breaking working solution, incubating at normal temperature for 20min, discarding the solution, and washing the slide for 3 times by using a PBS shaking table. TdT and dUTP were mixed at 1:9, added dropwise to the tissue, the sections were placed in a wet box and incubated at 37 ℃ for 2 h. The liquid was discarded, the slide was washed 3 times with a shaker, the DAPI stain was added dropwise, and incubation was carried out for 10min at room temperature in the dark. The liquid was discarded, the slides were washed 3 times with PBS shaker, mounted with anti-fluorescence quenching mounting medium, observed under excitation microscopy and photographed.
10. Testis tissue MDA, SOD, GSH level determination
The activity of the total superoxide dismutase (SOD) and the level of the reduced Glutathione (Glutathione) are all carried out according to the instruction of a determination kit (Nanjing institute of bioengineering).
11. ELSIA assay for HDT and HDAC in testis tissue
Histone Acetylase (HAT) and Histone Deacetylase (HDAC) were measured according to the instructions of an ELISA kit (jiangsu enzyme immunoassay biotechnology limited).
12. Data processing and statistical analysis
Western Blot bands were analyzed using Clinx Chemi Analysis software, ELISA assay standard curves were fitted using Curve Expert, comparisons of inter-data set differences were analyzed using SPSS 22.0 and GraphPad Prism7.0, and statistical plots were drawn using GraphPad Prism 7.0. Testis score and sperm data results were expressed using Median and limit values (mediawith range), and group comparisons were performed using non-parametric rank sum test (Kruskal-WallisTest); all remaining data results are expressed as Mean ± standard deviation (Mean ± SD) and comparisons between groups were performed using One-way ANOVA. P <0.05 was statistically significant difference, and p <0.01 indicated that the difference was very significant.