CN116590376A - Application of eNAMPT on the testis - Google Patents

Abstract

The invention discloses an application of eNAMPT on testis, which causes up-regulation of NF- κB and NLRP3 in testis macrophages by extracellular vesicles through the over-expressed eNAMPT, so that the macrophages are converted into a pro-inflammatory phenotype, thereby regulating and controlling the expression of senescence-associated genes, further affecting senescence and realizing the regulation and control of testis senescence. The invention lays a firm foundation for researching male reproductive aging mechanism, is favorable for deeply researching aging mechanism through a cell model and an animal model, diagnosing testis related diseases and developing a treatment strategy for resisting male reproductive aging or premature reproductive aging.

Description

Application of eNAMPT on the testis

technical field

The invention relates to the technical field of biomedicine, in particular to the application of eNAMPT on the testis.

Background technique

Aging is a comprehensive process of pathology, physiology and psychology of the whole body and various tissues and organs of the human body, which has a major impact on the physical and mental health of the individual, leading to a decline in the physical function and mental health of the individual, and an increase in the risk of disease and death. Aging leads to the aging of various tissues and organs, leading to related diseases. Common aging diseases include cancer, hypertension, cardiovascular disease, diabetes and various degenerative diseases. Various tissues and organs do not start the aging process when people enter old age. On the contrary, factors such as environment and lifestyle can lead to premature aging, and these tissues and organs will also enter the aging process in advance. The aging of the reproductive system starts earlier in the human body. For males, they have the ability to produce gametes throughout their life cycle, so they face the problem of reproductive aging in a longer period of time. The aging of the male reproductive system not only affects the individual's own reproductive ability, but also directly affects the functions of other organs, causing the function of other systems to decline, leading to health problems; at the same time, it will also cause relatively large psychological problems for the individual, affecting normal life; equally important is , the offspring produced will also have an increased risk of congenital diseases. Recent studies have found that in the testis, the aging process is closely related to the activation of inflammation; moreover, extracellular vesicles participate in intercellular communication and mediate immune responses and regulate the immune microenvironment. Therefore, an in-depth study of the changes in the immune microenvironment caused by the paracrine pathway will provide new ideas for the research and treatment of testicular aging.

Extracellular NAMPT (eNAMPT) is an important extracellular medium, and the abnormal level of eNAMPT is related to a variety of metabolic disorders. eNAMPT can regulate the pathogenesis of obesity and related diseases such as non-alcoholic fatty liver disease and oxidative stress by affecting type 2 diabetes response, apoptosis, lipid and glucose metabolism, inflammation and insulin resistance. In addition, eNAMPT circulates in functionally and structurally distinct monomers and dimers, and dimerized eNAMPT can promote NAD biosynthesis, while the role of monomers is not fully understood, but studies have demonstrated the elevation of monomeric eNAMPT Can play an important role in the pathogenesis of diet-induced diabetes through pro-inflammatory mechanisms. According to previous reports, in addition to enzymatic activity, eNAMPT acts as pro-inflammatory or anti-inflammatory cytokines in multiple signaling pathways, such as ERK1/2, IL-6-STAT3, PI3K-AKT, p38 MAPK, and NF-κB, which affect Expression of various cytokines such as TNF-α, IL-1β and TGF-β. Studies have pointed out that eNAMPT can activate the unique toll-like receptor 4 (TLR4) to induce NF-κB signaling pathway and lead to the activation of NLRP3, and TLR4 receptors also exist on the surface of testicular macrophages. Therefore, eNAMPT may play a regulatory role in the process of testicular aging. It is expressed in large quantities with age and accelerates the aging of mouse testis through the extracellular vesicle pathway, but its specific function and mechanism are not clear.

At present, there is no report on whether eNAMPT can regulate testicular aging. The present invention finds that eNAMPT can cause differential expression of NF-κB and NLRP3 in testicular macrophages through the extracellular vesicle pathway, thereby regulating the testicular immune microenvironment, and then regulating aging-related genes expression, thereby affecting aging. The invention lays a solid foundation for the research on the mechanism of male reproductive aging, and is beneficial to in-depth research on aging mechanism through cell models and animal models, diagnosis of testicular related diseases and development of anti-male reproductive aging or premature reproductive aging treatment strategies.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies in the prior art, provide a kind of application of eNAMPT on testis, obtain the new effect and new mechanism of eNAMPT regulating testicular aging by extracting interstitial extracellular vesicles of testis, for the research of male reproductive aging Mechanism research has laid a solid foundation, which is conducive to in-depth research on aging mechanisms through cell models and animal models, diagnosis of testicular-related diseases, and development of anti-male reproductive aging or reproductive premature aging treatment strategies.

In order to achieve the above object, the technical scheme provided by the present invention is as follows: the application of eNAMPT on the testis, the up-regulation of NF-κB and NLRP3 in the testicular macrophages caused by the overexpressed eNAMPT from the extracellular vesicles, so that the macrophages can move towards The pro-inflammatory phenotype is transformed, thereby regulating the expression of aging-related genes, thereby affecting aging, and realizing the regulation of testicular aging.

Further, firstly, the interstitial tissue fluid of the testis is collected, and then the extracellular vesicles in the interstitial tissue fluid of the testis are extracted using differential speed and ultracentrifugation, and then the extracellular vesicles of the interstitial tissue of the testis are subjected to protein extraction, liquid chromatography tandem mass spectrometry, Western blotting and proteomic GO or KEGG analysis revealed the differential expression of eNAMPT in the testicular aging process, and further realized the application of eNAMPT in testis through cell experiments and animal experiments.

Further, eNAMPT was overexpressed in stem cells by means of lentiviral transfection, and eNAMPT-overexpressed stem cells produced extracellular vesicles overexpressing eNAMPT during the culture process, and then the stem cells in the stem cell culture medium were extracted by differential speed and ultracentrifugation. Overexpress eNAMPT extracellular vesicles, and then use the overexpressed eNAMPT extracellular vesicles to realize the application of eNAMPT in testis through cell experiments and animal experiments.

Compared with the prior art, the present invention has the following advantages and beneficial effects:

1. The present invention can cause the differential expression of NF-κB and NLRP3 in testicular macrophages by overexpressing eNAMPT extracellular vesicles, thereby regulating the immune microenvironment of the testis, and then regulating the expression of aging-related genes, thereby affecting aging; the present invention obtains The new role and new mechanism of eNAMPT regulating testicular aging, the present invention has laid a solid foundation for the study of the mechanism of male reproductive aging, and is conducive to the in-depth study of aging mechanisms through cell models and animal models, the diagnosis of testicular related diseases and the development of anti-male reproductive aging or Treatment strategies for premature reproductive aging.

2. The present invention extracts the extracellular vesicles in the interstitial tissue fluid of the testis through differential speed and ultracentrifugation, and further discovers the difference of protein in the extracellular vesicles of the interstitium of the testis with age, thereby developing the extracellular vesicles of the interstitial cells of the testis. Application on the testicles.

3. In the present invention, by overexpressing eNAMPT in stem cells, and then extracting the overexpressed eNAMPT extracellular vesicles in the stem cell culture medium by differential speed and ultracentrifugation, the overexpressed eNAMPT extracellular vesicles derived from individual homologous stem cells can be used to develop Application on the testicles.

Description of drawings

FIG. 1 is a graph showing the results of extracellular vesicle extraction and proteomic analysis in the interstitial space of the testis in young and aged mice in Example 1.

Fig. 2 is a graph showing the results of overexpression of eNAMPT in testicular extracellular vesicles of aging mice regulating the expression of NF-κB and NLRP3 genes in testicular macrophages in Example 4.

Fig. 3 is a diagram of verification results of overexpression of eNAMPT extracellular vesicles regulating testicular aging-related gene levels in Example 7.

Fig. 4 is a diagram showing the results of verification of the anti-aging strategy proposed in Example 10 for overexpressing eNAMPT extracellular vesicle regulation in stem cells to affect aging.

Fig. 5 is a diagram of the validation results of Example 13 for evaluating Leydig's extracellular vesicles in young and aging individuals.

Detailed ways

In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with the examples and accompanying drawings. limit.

Example 1 (extraction and characterization of a Leydig cell extracellular vesicle)

Leydig extracellular vesicles were extracted from young mice and aging mice, and the successful extraction of extracellular vesicles was verified by transmission electron microscopy, western blot, and nanometer flow cytometry; Rat (young) Leydig's extracellular vesicles were quantitatively analyzed by 4DLabel-free non-labeled proteomics. The main steps include protein extraction, protein quantification, protein enzymatic hydrolysis, LC-MS/MS analysis, database retrieval and data analysis. The protein differences between the young mouse group and the aging mouse group were analyzed, and the significantly up-regulated proteins and the functions involved in the aging mice were analyzed by GO and KEGG. Specific steps are as follows:

1) C57BL/6 mice aged 3 months and 24 months were killed by neck dislocation, the scrotum of the mice was carefully cut open with scissors, and all the fat on the testes was removed from both testes.

2) Weigh each testis, then carefully tear a small opening on the albuginea of the testis, add 5 mL of PBS per gram of testis, and incubate at 4° C. for 45 minutes.

3) Centrifuge at 10,000 g for 15 min at 4°C to obtain the interstitial fluid of the testis. The interstitial fluid of the testis was subjected to gradient centrifugation to remove cell debris, centrifuged at 2000g for 10min at 4°C, 10000g for 30min, and sterilized by 0.22μm filtration before use.

4) Transfer to an ultracentrifuge tube and centrifuge at 200,000g for 2h, resuspend the pellet with PBS, and centrifuge at 200,000g for 2h, remove the supernatant, and obtain Leydig's extracellular vesicles.

5) Take 10 μL of the sample and drop it on the copper grid for precipitation for 1 min, and filter paper to absorb the floating liquid. Then draw 10 μL of ammonium molybdate solution and drop it on the copper grid for precipitation for 1 min, and filter paper absorbs the floating liquid. After several minutes of drying at room temperature, TEM imaging was performed at 100 kV.

6) Use RIPA lysate (containing protein phosphatase inhibitor and PMSF) instead of PBS to directly resuspend the extracellular vesicle pellet. The protein in the extracellular vesicles was extracted according to the aforementioned method, and after the protein concentration was determined using the BCA protein quantification kit, it was denatured, electrophoresed, and transferred to a membrane. Then the primary antibody was incubated overnight, and the secondary antibody was incubated the next day and exposed for imaging.

7) Dilute the sample to 90 μL, add 20 μL of fluorescently labeled antibodies (CD63, CD81, IgG) to 30 μL each, mix well, and incubate at 37° C. for 30 min in the dark. Add 1mL of pre-cooled PBS, 4°C, 110000g, and ultracentrifuge for 70min. Carefully remove the supernatant, add 1 mL of pre-cooled PBS, centrifuge again at 110,000 g at 4°C for 70 min. Carefully remove the supernatant and resuspend with 50 μL of pre-cooled PBS. Use the NanoFCM instrument to detect protein index results.

8) Add appropriate amount of SDT lysate to each sample, transfer to EP tube, then boil in water bath for 3 minutes, sonicate for 2 minutes, centrifuge at 16000g for 20 minutes at 4°C, take supernatant, and use BCA method for protein quantification. Take an appropriate amount of protein from each sample for FASP enzymatic hydrolysis, and then take an appropriate amount of peptides from each sample for chromatographic separation using the nanoliter flow rate Easy nLC 1200 chromatographic system.

The implementation results are shown in Figure 1. See A in Fig. 1 , this example extracts extracellular vesicles from Leydigm of young mice and aged mice. See B in Figure 1, the successful extraction of extracellular vesicles was confirmed by transmission electron microscopy, western blot and nanometer flow cytometry. See Figure 1, C, clustering analysis of Leydigm extracellular vesicles in young mice and aged mice shows that the samples are consistent and the results are stable. See D in Fig. 1, through proteomics quantitative analysis, the proteome differences in testicular extracellular vesicles of aged mice were determined. See E in Figure 1, the metabolism of nicotinate and nicotinamide showed very strong enrichment factors after further analysis by KEGG functional enrichment analysis. See F in Figure 1. The metabolism of nicotinate and nicotinamide in extracellular vesicle proteins and related proteins in NAD+ biosynthesis were further analyzed. significantly doubled, indicating that eNAMPT is associated with aging.

Example 2

The difference from Example 1 is that the ultracentrifugation speed is replaced by 100,000 g from 200,000 g, and other preparation conditions remain consistent with Example 1. Leydig extracellular vesicles were extracted from young mice and aging mice, and the successful extraction of extracellular vesicles was verified by transmission electron microscopy, western blot, and nanometer flow cytometry.

Example 3

The difference from Example 1 is that the ultracentrifugation time is replaced from 2h to 1h, and other preparation conditions remain consistent with Example 1. Leydig extracellular vesicles were extracted from young mice and aging mice, and the successful extraction of extracellular vesicles was verified by transmission electron microscopy, western blot, and nanometer flow cytometry.

Embodiment 4 (the research method of the effect and mechanism of eNAMPT regulating testicular aging)

1) C57BL/6 mice aged 3 months and 24 months were killed by neck dislocation, the scrotum of the mice was carefully cut open with scissors, and all the fat on the testes was removed from both testes.

2) Weigh each testis, then carefully tear a small opening on the albuginea of the testis, add 5 mL of PBS per gram of testis, and incubate at 4° C. for 45 minutes.

3) Centrifuge at 10,000 g for 15 min at 4°C to obtain the interstitial fluid of the testis. The interstitial fluid of the testis was subjected to gradient centrifugation to remove cell debris, centrifuged at 2000g for 10min at 4°C, 10000g for 30min, and sterilized by 0.22μm filtration before use.

4) Transfer to an ultracentrifuge tube and centrifuge at 200,000g for 2h, resuspend the pellet with PBS, and centrifuge at 200,000g for 2h, remove the supernatant, and obtain Leydig's extracellular vesicles.

5) The above samples and normal saline were co-incubated with the testicular macrophages respectively, and after 24 hours, the cells were taken for qPCR detection.

6) The above samples and normal saline were injected into the testes of young mice respectively, and the mice were sacrificed three days later and the tissues were taken for qPCR detection.

7) The above samples and normal saline were injected into the testes of young mice respectively. Three days later, the mice were sacrificed to take the tissues, digested with type IV collagenase at 37° C. for 15 minutes, and filtered with a cell sieve to obtain testicular single cell suspension. Cell suspension was labeled with Anti-MouseF4/80 FITC antibody and PE-Cyanine7 Anti-Mo CD45 antibody. After washing away the antibody, put it on ice, and then use flow cytometry to detect.

8) The blood of the mouse was collected through the orbit, and the red blood cells were removed by using the red blood cell lysate, and the blood cells were removed by centrifugation, and then resuspended with PBS to obtain a single cell suspension. Cell suspension was labeled with Anti-Human/Mouse CD11b APC antibody and Anti-Mouse Ly-6G(Gr-1) Alexa Fluor488 antibody. After washing away the antibody, put it on ice, and then use flow cytometry to detect.

9) The macrophages were treated with eNAMPT recombinant protein 5ng/ml, and the cells were taken 24 hours later for qPCR detection.

The implementation results are shown in Figure 2. As shown in Figure 2, A, the use of eNAMPT-overexpressed Leydig cells to stimulate macrophages can significantly up-regulate the expression of NLRP3 and inflammasome-related genes. See B in Figure 2, injecting young mice with Leydig cells from aging mice with high expression of eNAMPT can significantly up-regulate the expression of NLRP3 and inflammasome-related genes in the testis, and at the same time up-regulate the expression of aging-related genes. See Figure 2, C. In order to exclude the influence of other substances in extracellular vesicles, macrophages were treated with eNAMPT recombinant protein, which can significantly up-regulate NF-κB, NLRP3 and aging-related genes, which is consistent with the results of extracellular vesicles . See D in Figure 2, extracellular vesicles overexpressing eNAMPT can significantly up-regulate inflammatory cells in testis and blood.

Example 5

The difference from Example 4 is that the eNAMPT recombinant protein was replaced from 5 ng/ml to 500 ng/ml, and other conditions remained consistent with Example 4.

Example 6

The difference from Example 4 is that the eNAMPT recombinant protein was replaced from 5 ng/ml to 250 ng/ml, and other conditions remained consistent with Example 4.

Example 7 (Method for constructing stem cells overexpressing eNAMPT extracellular vesicles)

1) Extract testis-derived stem cells and culture them in a 37°C cell incubator until they are stable.

2) Stem cells were transfected with SV40T for immortalization, and fresh medium was replaced 24-72 hours after transfection.

3) Puromycin was used to screen the immortalized stem cells, and the fluorescence was detected by flow cytometry.

4) Transfect the stem cells with eNAMPT lentivirus, and continue culturing the cells with ordinary medium after the cells are stabilized.

5) Collect the cell supernatant medium. Centrifuge at 2000g for 10min at 4°C, centrifuge at 10000g for 30min, filter and sterilize, transfer to an ultracentrifuge tube and centrifuge at 200000g for 2h, resuspend the pellet in PBS and centrifuge at 200000g for 2h, remove the supernatant, and extract the extracellular vesicles overexpressing eNAMPT Bubble.

6) and use western blot to test.

7) The extracellular vesicles above 1 μg/ml were used for co-culture with testicular macrophages, and the cells were used for qPCR detection after 24 hours.

The results of the implementation are shown in A in Figure 3, where testis-derived stem cells are extracted; in B in Figure 3, stem cells are immortalized to make them easy to be used for culturing and producing extracellular vesicles; in C in Figure 3, immortalized cells are screened out The transformed stem cells indicate that the immortalized stem cells used for the production of extracellular vesicles were successfully constructed; see D in the accompanying drawing 3, and the extracellular vesicles overexpressing eNAMPT were successfully constructed by western blot; see E in the accompanying drawing 3, through qPCR proved that overexpression of eNAMPT extracellular vesicles can up-regulate NF-κB and NLRP3 genes, thereby up-regulating aging-related genes, and then affecting aging. It was shown that overexpression of eNAMPT extracellular vesicles could induce testicular senescence.

Example 8

The difference from Example 7 is that the extracellular vesicles overexpressing eNAMPT were replaced from 1 μg/ml to 10 μg/ml, and other conditions remained the same as in Example 7.

Example 9

The difference from Example 7 is that the extracellular vesicles overexpressing eNAMPT were replaced from 1 μg/ml to 100 μg/ml, and other conditions remained the same as in Example 7.

Embodiment 10 (Treat the testis after induction of aging by anti-inflammatory drugs)

1) Collect the cell supernatant medium. Centrifuge at 2000g for 10min at 4°C, centrifuge at 10000g for 30min, filter and sterilize, transfer to an ultracentrifuge tube and centrifuge at 200000g for 2h, resuspend the pellet in PBS and centrifuge at 200000g for 2h, remove the supernatant, and extract the extracellular vesicles overexpressing eNAMPT Bubble.

2) The eNAMPT recombinant protein was used to inject the testes of young mice, and the anti-inflammatory drug curcumin was injected at the same time.

3) After 3 days, the tissues were collected for ELISA to detect the levels of inflammatory factors and qPCR to detect the levels of aging-related genes.

The results of the implementation are shown in Figure 4. ELISA and qPCR prove that anti-inflammatory treatment can block the release of inflammatory factors, thereby improving the upregulation of aging-related genes and having anti-aging effects. Because eNAMPT activates NLRP3 by up-regulating NF-κB, thereby releasing inflammatory factors, leading to changes in the immune microenvironment, and then up-regulating aging-related genes to affect aging. The use of anti-inflammatory drugs can improve the expression of aging-related genes by reducing the expression of inflammatory genes.

Example 11

The difference from Example 10 is that curcumin is replaced by resveratrol, and other conditions remain consistent with Example 8.

Example 12

The difference from Example 11 is that curcumin is replaced by rapamycin, and other conditions remain consistent with Example 8.

Example 13 (diagnosis using Leydig's extracellular vesicles)

1) C57BL/6 mice aged 3 months and 24 months were killed by neck dislocation, the scrotum of the mice was carefully cut open with scissors, and all the fat on the testes was removed from both testes.

2) Weigh each testis, then carefully tear a small opening on the albuginea of the testis, add 5 mL of PBS per gram of testis, and incubate at 4° C. for 45 minutes.

3) Centrifuge at 10,000 g for 15 min at 4°C to obtain the interstitial fluid of the testis. The interstitial fluid of the testis was subjected to gradient centrifugation to remove cell debris, centrifuged at 2000g for 10min at 4°C, 10000g for 30min, and sterilized by 0.22μm filtration before use.

4) Transfer to an ultracentrifuge tube and centrifuge at 200,000g for 2h, resuspend the pellet with PBS, and centrifuge at 200,000g for 2h, remove the supernatant, and obtain Leydig's extracellular vesicles.

5) Use RIPA lysate (containing protein phosphatase inhibitor and PMSF) instead of PBS to directly resuspend the extracellular vesicle pellet. The protein in the extracellular vesicles was extracted according to the aforementioned method, and after the protein concentration was determined using the BCA protein quantification kit, it was denatured, electrophoresed, and transferred to a membrane. Then the primary antibody was incubated overnight, and the secondary antibody was incubated the next day and exposed for imaging.

The results of the implementation are shown in Figure 5. The eNAMPT monomer content expressed in the extracellular vesicles of the Leydigm of the testes in aging mice was significantly increased, which can be used as a criterion for judging testicular aging. According to this method, other related diseases of the testis can be judged according to specific indicators.

The implementation examples described above are only preferred embodiments of the present invention, and are not intended to limit the scope of the present invention. Therefore, all changes made according to the shape and principle of the present invention should be covered within the scope of protection of the present invention.

Claims (3)

1. The application of eNAMPT on the testis is characterized in that the up-regulation of NF-κB and NLRP3 in the testicular macrophages is caused by the extracellular vesicles through the overexpressed eNAMPT, so that the macrophages are transformed into a pro-inflammatory phenotype, thereby Regulate the expression of aging-related genes, and then affect aging, and realize the regulation of testicular aging. 2. the application of eNAMPT according to claim 1 on testis, is characterized in that, at first, collect interstitial fluid of testis, then use differential speed, ultracentrifugation to extract the extracellular vesicles in interstitial fluid of testis, subsequently The differential expression of eNAMPT in testicular aging process was found by protein extraction, liquid chromatography tandem mass spectrometry, western blotting and proteomic GO or KEGG analysis of Leydig Leydig's extracellular vesicles. Further cell experiments and animal experiments The method realizes the application of eNAMPT on the testis. 3. The application of eNAMPT on testis according to claim 1, characterized in that, by using lentiviral transfection means to overexpress eNAMPT in stem cells, the stem cells overexpressing eNAMPT produce cells overexpressing eNAMPT in the culture process Extracellular vesicles, and then extract the overexpressed eNAMPT extracellular vesicles in the stem cell culture medium by differential speed and ultracentrifugation, and then use the overexpressed eNAMPT extracellular vesicles to realize the application of eNAMPT in the testis through cell experiments and animal experiments .