Application of vincamine in preparation of medicine for treating testis ischemia-reperfusion injury

Abstract

The invention discloses application of vincamine in preparation of a medicine for treating testis ischemia-reperfusion injury. Research results show that vincamine can significantly improve the pathological structure of the testis, alleviate inflammatory response and oxidative stress injury, and inhibit pyroptosis of testis cells, so that the reproductive function of the testis is protected after testis torsion reduction; the action mechanism is that an NF-kappa B signal channel is inhibited by up-regulating GPR40. The invention provides a novel potential drug for testis ischemia reperfusion injury.

Description

TECHNICAL FIELD

[0001] The application relates to the field of biological medicine, and particularly relates to application of vincamine in preparation of a medicine for treating testicular ischemia-reperfusion injury. BACKGROUND

[0002] Testicular torsion (TT) is a common emergency disease in pediatric urology, with an annual incidence of about 5 / 10 million in men under the age of 25, and is one of the main causes of testicular loss in adolescents. The disease is caused by the sudden rotation of the testis along the longitudinal axis and the twisting of the spermatic cord, resulting in a significant decrease in local blood flow and tissue perfusion. If testicular detorsion (TD) and fixation surgery can be performed in time, it is helpful to preserve organ function. However, clinical studies have shown that even if the testis that can survive is salvaged by surgery, persistent testicular atrophy, massive loss of germ cells and infertility may still occur after the surgery, and the main reason is considered to be the inevitable ischemia / reperfusion (I / R) injury after the surgery.

[0003] Ischemia-reperfusion (I / R) injury is the main pathophysiological process after testicular torsion reduction, which can lead to persistent testicular atrophy and abnormal sperm morphology, and its specific mechanism is not clear, and the existing treatment means is limited. In the I / R process, the cascade reaction of inflammatory signals and the outbreak of reactive oxygen species (ROS) will induce cell death. This injury process involves multiple signaling pathways, including p21 / p53-dependent apoptosis, JNK pathway and NF-κB signaling pathway. However, the specific mechanism of causing the continuous amplification of the inflammatory response in I / R injury is not clear. Pyroptosis is a new type of programmed cell death, characterized by activation of NLRP3 inflammasome, cell membrane rupture and release of inflammatory factors. Pyroptosis is an important mechanism for aggravating inflammation and has been confirmed to exist in various I / R injury models. Clinically, inhibition of IL-1β release mediated by pyroptosis has been used to treat inflammatory diseases such as arthritis, so it is speculated that pyroptosis may play an important role in the inflammatory response of testicular I / R injury and become a potential therapeutic target.

[0004] At present, there have been many studies trying to apply antioxidants, anti-inflammatory drugs, calcium channel blockers and vasodilators to alleviate I / R injury, and these drugs can reduce tissue damage to a certain extent. However, so far no drug has been proven to effectively prevent the continuous loss of germ cells caused by I / R injury after surgery, and there is still a gap in postoperative treatment in the clinic, so it is of great significance to develop new treatment strategies. SUMMARY

[0005] The application aims at the above problems, and provides an application of vinblastine in preparation of a medicine for treating testicular ischemia-reperfusion injury.

[0006] In order to achieve the purpose, the application adopts the technical scheme of:

[0007] The application provides an application of vinblastine in preparation of a medicine for treating testicular ischemia-reperfusion injury.

[0008] Among them, vinblastine reduces ischemia-reperfusion-induced testicular inflammatory response and tissue damage, and improves testicular pathological structure.

[0009] Further, vinblastine reduces the levels of LDH, IL-1beta and IL-18, and restores the activity of SOD.

[0010] In the application technical scheme, vinblastine reduces testicular pyroptosis.

[0011] Among them, vinblastine reduces testicular pyroptosis by targeting GPR40.

[0012] Further, vinblastine inhibits GC1 and GC2 cell OGD / R-induced pyroptosis by targeting GPR40, and reduces GC1 and GC2 cell OGD / R-induced structural damage and inflammation.

[0013] In the application technical scheme, vinblastine plays a role by targeting GPR40 to inhibit the NF-kappa B signaling pathway.

[0014] Further, vinblastine inhibits GC1 and GC2 cell OGD / R-induced up-regulation of the NF-kappa B pathway by targeting GPR40.

[0015] In the application technical scheme, the testicular ischemia-reperfusion injury is testicular ischemia-reperfusion injury caused by testicular detorsion.

[0016] The application has the following beneficial effects:

[0017] The application establishes a rat TT and TD I / R injury in vivo model, and an oxygen-glucose deprivation / reoxygenation (OGD / R) in vitro model of reproductive cells. The potential therapeutic drug vincamine (Vin) is predicted by combining transcriptome sequencing data with CMap drug database, which is a G protein-coupled receptor 40 (GPR40) agonist. The results show that Vin can significantly improve the pathological structure of the testis, reduce the inflammatory response and oxidative stress damage, and inhibit testicular pyroptosis, thereby protecting the testicular reproductive function after testicular torsion reduction; the mechanism is to inhibit the NF-κB signaling pathway by up-regulating GPR40. The application provides a new potential drug for testicular ischemia-reperfusion injury. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Drug prediction and molecular docking are shown: (A) identification of hub genes; (B) prediction of potential drugs using key DEGs in the CMap database; (C) molecular docking of vincamine with GPR40 protein and visualization of hydrogen bond interactions.

[0019] Figure 2 Vincamine reduces testicular damage, reduces inflammation and oxidative stress factors, reduces rat testicular pyroptosis and inhibits the activation of the NF-κb signaling pathway: (A) schematic diagram of the vincamine administration time point; (B) ELISA detection of the levels of LDH, SOD, IL-1β and IL-18 in the serum and testis of rats; (C-E) the thickness of the spermatogenic epithelium, the testicular organ coefficient and the Johnsen score of each group; (F) the gross morphological changes of the testis of each group; (G) H&E staining of the testis tissue morphology of the three groups ((a, b, c) scale = 250 μm, (d, e, f) scale = 100 μm); (H) transmission electron microscopy showing the changes in cell morphology and organelle structure of the sham operation group and the I / R group ((a-c) scale = 1 μm, (d-f) scale = 500 nm); (I) Western blot detection of the P-P65 / P65 and IKBα protein expression ratio of the three groups; (J) immunohistochemical staining to detect the expression of P-P65 in testicular tissue (scale = 100 μm); (K) Western blot detection of the NLRP3, ASC and N-GSDMD protein expression levels in vivo; (L) GSDMD and NLRP3 tissue immunofluorescence staining (scale = 50 μm); *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns, not significant.

[0020] Figure 3Vincristine activates GPR40 in vitro and in vivo: (A) Western blot analysis of GPR40 in testicular tissue; (B) Immunofluorescence staining of GPR40 in three groups of testicular tissue (scale bar = 50 μm); (C) Western blot analysis of GPR40 expression levels after treatment of GC1 cells with vincristine or vincristine + GW1100; (D) Immunofluorescence staining of GPR40 in GC1 cells (scale bar = 50 μm); (E) Western blot analysis of GPR40 expression levels after treatment of GC2 cell line with vincristine or vincristine + GW1100; (F) Immunofluorescence staining of GPR40 in GC2 cells (scale bar = 50 μm).

[0021] Figure 4 Vincristine attenuates pyroptosis and down-regulates NF-κb signaling pathway in GC1 and GC2 cells after OGD / R injury by targeting GPR40: (A, H) In vitro scanning electron micrographs of four groups of cells; ((a-d) scale bar = 2 μm, (e-h) scale bar = 1 μm, (m-p) scale bar = 500 nm); (B, I) In vitro transmission electron micrographs of four groups of cells; ((a-d) scale bar = 2 μm, (e-h) scale bar = 500 nm) (C, J) Comparison of MDA, SOD, LDH, IL-1β and IL-18 after OGD 4h / R 24h in vitro cultured using ELISA; (D, K) ROS production in GC1; (E, L) Cellular immunofluorescence staining of GSDMD in GC1 cell line (scale bar = 50 μm); (F, M) NLRP3, ASC, N-GSDMD protein expression levels (Western blot) in GC1 and GC2 cell lines; (G, N) P-P65 / P65 and IKBα protein expression (Western blot) in GC1 and GC2 cell lines. DETAILED DESCRIPTION

[0022] The application is further described in conjunction with the following examples without thereby limiting the application.

[0023] The experimental methods in the following examples are routine methods unless otherwise specified.

[0024] The present application studies the establishment of a testicular I / R injury model in vivo by surgically twisting and resetting the testes of experimental animals. RNA sequencing results show that inflammation and pyroptosis are significantly upregulated in I / R-injured testes, and the NF-κB signaling pathway is enriched. Combined with differential expression gene and CMap database analysis, it is predicted that Vincamine (Vin), a G protein-coupled receptor 40 (GPR40) agonist, has potential therapeutic effects. We gave Vin intervention in I / R-injured rats to evaluate its protective effect. To explore the mechanism of action, we further established an oxygen-glucose deprivation / reoxygenation (OGD / R) in vitro model in GC1 and GC2 cell lines, and applied Vin and GPR40 inhibitor GW1100, respectively. Western blot, enzyme-linked immunosorbent assay, immunohistochemistry and immunofluorescence were used to detect the expression levels of pyroptosis and NF-κB-related proteins. The results show that Vin can significantly improve the pathological structure of the testis, reduce inflammation and oxidative stress injury, and inhibit pyroptosis; its mechanism of action is to inhibit the NF-κB signaling pathway by upregulating GPR40. The application of GW1100 can eliminate this protective effect. The present application reveals that inflammation and pyroptosis are involved in the process of testicular I / R injury, and confirms that Vin can target GPR40 to reduce testicular pyroptosis. The specific research experiments are as follows:

[0025] Example 1

[0026] 1. Materials and methods

[0027] 1.1 Animals

[0028] The present animal study follows the relevant guidelines of the Declaration of Helsinki and has been approved by the Ethics Committee of the Children's Hospital Affiliated to Chongqing Medical University (Approval No. CHCMU-IACUC20250217003). Twenty-one male clean grade (SPF) Sprague-Dawley (SD) rats (4 weeks old, weighing 100-150 g) were selected and raised under SPF conditions with free access to food and water (polycarbonate cages, temperature 25±2℃, humidity 50±5%, light cycle half light / half dark).

[0029] 1.2 Animal experimental design

[0030] Rats were randomly divided into three groups (7 rats per group): (1) sham operation group; (2) I / R group; (3) I / R+Vin group. The I / R group and the I / R+Vin group were established by surgery to establish the testicular I / R injury model. After intraperitoneal injection of 5 ml / kg 20% urethane anesthesia, a vertical incision was made on the left side of the scrotum, the testis and spermatic cord were separated, and they were rotated counterclockwise by 720° and sutured with 5 / 0 silk thread to the scrotal cavity. After suturing the incision for 4 hours, anesthesia was performed again and the incision suture was removed, and the twisted testis and spermatic cord were reduced. The sham operation group only exposed the testis without twisting and reduction. Penicillin was given for 3 consecutive days after the operation to prevent infection. The I / R+Vin group was orally gavaged with Vin (30 mg / kg) 30 minutes before reduction and once a day for 7 consecutive days after the operation, and the other two groups were gavaged with the same volume of normal saline in the same way. On the 8th day after the operation, the rats were sacrificed, the testes were fixed with 4% paraformaldehyde (PFA) for subsequent hematoxylin-eosin (H&E) staining and immunofluorescence (IF) detection; blood was collected and centrifuged at 4°C to separate serum for further biochemical detection.

[0031] 1.3 Histological examination

[0032] The testicular tissue was dehydrated, paraffin-embedded, and sectioned at a thickness of 4 μm. After deparaffinization and hydration, the sections were stained according to the standard operating procedure. Optical microscopy (Nikon, Japan) was used to obtain images, and the Johnsen scoring method was used to evaluate the degree of testicular tissue damage and spermatogenesis.

[0033] 1.4 RNA sequencing analysis

[0034] To explore the potential mechanisms and screen for therapeutic drugs, the left testicular tissue samples from the sham operation group and the I / R group (3 rats per group, a total of 6 rats) were collected for transcriptome analysis. High-throughput sequencing was completed by Hangzhou Leikang Biological Technology Co., Ltd. using the Illumina NovaSeq 5000 platform. The R language edgeR software package (R 4.2.0) was used to analyze differentially expressed genes (DEGs) with a screening condition of p<0.05 and |logFC|>2, and low-abundance genes were excluded. The ggplot2 software package was used to draw a volcano plot, and the clusterProfiler software package was used for gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Gene set enrichment analysis (GSEA) was used to identify possible involved signaling pathways. Inflammation-related genes (IRGs) were obtained from the GeneCards website (access time: February 4, 2024, https: / / www.genecards.org / ) and intersected with differentially expressed genes, and the intersection results were displayed in the form of a heat map.

[0035] 1.5 Identification of key differentially expressed genes (DEGs)

[0036] To identify the key differentially expressed genes (DEGs), the DEGs were first uploaded to STRING database to construct protein-protein interaction (PPI) network, and the MCC algorithm was used to screen hub genes, followed by visual processing using Cytoscape. Finally, the top 170 hub genes were selected as key DEGs.

[0037] 1.6 Drug prediction

[0038] Connectivity Map (CMap) is a widely used drug database in pharmacological research. By sequencing the transcriptome of a large number of different cells after drug treatment, researchers can find potential therapeutic drugs by comparing the key differentially expressed genes (DEGs) screened by themselves with the CMap database data. To alleviate testicular ischemia / reperfusion (I / R) injury, DEGs were analyzed in association with the CMap database to predict potential drugs.

[0039] 1.7 Molecular docking

[0040] The three-dimensional structure of the drug was derived from the PubChem database (https: / / pubchem.ncbi.nlm.nih.gov / , accessed on February 4, 2024). The GPR40 protein structure (PDB ID: 5tzr) was downloaded from the RCSB PDB database (http: / / www.rcsb.org / ). Molecular docking was performed using AutoDockTools-1.5.7 software. First, the GPR40 protein crystal structure was preprocessed, including removing water molecules, adding hydrogen, and removing ligands. Then, the Vina program was used to complete the molecular docking of GPR40 and vinblastine (Vin). The binding affinity was used to evaluate the strength of the interaction, and the lower the binding affinity value, the more stable the interaction. The final results were visualized using PyMOL 3.0. 1.8 Immunohistochemical staining (IHC)

[0041] The immunohistochemical staining procedure of testis tissue sections was as follows: first, deparaffinization with xylene, followed by hydration with gradient decreasing ethanol concentrations. After antigen retrieval by microwave heating, endogenous peroxidase activity was eliminated using 3% hydrogen peroxide, followed by blocking the sections with 0.5% BSA (Solarbio, Beijing, China) for 1 hour. Next, the sections were incubated with primary antibodies overnight at 4°C, including NLRP3 (Invitrogen, MA5-32255), GSDMD (Proteintech, 20770-1-AP), ASC (Zenbio, 340097), and phosphorylated P65 (P-P65, Zenbio, 310013). After washing with phosphate-buffered saline (PBS), the secondary antibody (Zenbio, 511203) was incubated for 1 hour. Then, color development was performed using 3,3'-diaminobenzidine (DAB, Abeam, Cambridge, UK), and finally, hematoxylin counterstaining was performed.

[0042] 1.9 Tissue immunofluorescence staining (IF)

[0043] The deparaffinization and hydration steps of testis tissue sections were as described in the “IHC staining” section. Then, the sections were immersed in a sodium citrate solution for 15 minutes for antigen retrieval. Next, the cells were permeabilized with 0.1% Triton (Keshi, China) for 15 minutes, followed by blocking the sections with BSA for 1 hour. Then, the sections were incubated with primary antibodies overnight at 4°C, including ASC, GSDMD, NLRP3, and GPR40 (Abclonal, A16379). Next, the sections were incubated with a fluorescein-labeled goat anti-rabbit secondary antibody for 1 hour at room temperature in the dark. Finally, the nuclei were counterstained with Hoechst 33342 (Beyotime, P0113) for 30 minutes. Images were captured and analyzed using a fluorescence microscope imaging system (KEYENCE, BZ-X800).

[0044] 1.10 Cell culture and in vitro treatment

[0045] The GC-1 and GC-2 cell lines were purchased from Procell Life Science & Technology (Wuhan, China). Cells from 5 to 15 passages were used in the experiments. Cells were cultured in a humidified incubator (37 °C, 5% CO2) in Dulbecco’s Modified Eagle Medium (DMEM, Gibco, USA) containing 10% fetal bovine serum (VivaCell, Shanghai, China), 1% penicillin-streptomycin. The sugar-free DMEM was purchased from Gibco (USA). Vincamine (CAS No: 1617-90-9, MCE, HY-B1021, USA) and GPR40 antagonist GW1100 (CAS No: 306974-70-9, MCE, HY-50691, USA) were dissolved in dimethyl sulfoxide (DMSO, MCE, HY-Y0320, USA) with a final DMSO concentration less than 0.1%.

[0046] The in vitro experiment was divided into four groups: (1) control group; (2) OGD / R group; (3) OGD / R+Vin group; (4) OGD / R+Vin+GW1100 group. The OGD / R model was established as follows: on the second day after cell seeding, the medium was replaced with sugar-free DMEM and the cells were placed in a hypoxic incubator (5% CO2, 1% O2, 94% N2) for 4 hours, then returned to the regular DMEM and normoxic environment (5% CO2, 21% O2, 74% N2) for recovery. The OGD / R+Vin group used the Vin-containing medium after cell attachment to establish the OGD / R model. The OGD / R+Vin+GW1100 group was treated with GW1100 (10 μM) for 5 hours to inhibit GPR40 expression before using the Vin-containing medium. The same concentration of DMSO was added to the medium of the control and OGD / R groups.

[0047] 1.11 Scanning electron microscope (SEM)

[0048] Testicular tissues and cells were pre-fixed in 3% glutaraldehyde, followed by post-fixation with 1% osmium tetroxide. The samples were dehydrated with decreasing concentrations of ethanol, followed by drying in a critical point drying instrument (Quorum, K850). The dried samples were attached to conductive tape and placed in an ion sputtering instrument (JEOL, Smart Coater) for gold spraying. Image acquisition was performed using a scanning electron microscope (JEOL, JSM-IT700HR, Japan).

[0049] 1.12 Transmission electron microscope (TEM)

[0050] After fixation as described in the SEM section, the samples were dehydrated with acetone solution, then embedded in EPON 812 resin and sectioned with a diamond scalpel. The sections were then stained with uranyl salt and lead citrate. Images were acquired using a transmission electron microscope (JEOL, JEM-1400FLASH, Japan).

[0051] 1.13 Cell viability assay

[0052] Cell viability and the inhibitory effect of different drug concentrations on cells under different hypoxia durations were assessed using the Cell Counting Kit-8 (CCK8, MCE, HY-K0301). GC-1 and GC-2 cells were seeded at a density of 5000 cells per well in 96-well plates. In the hypoxia assay, glucose-containing DMEM was replaced with glucose-free DMEM, and cells were cultured in a hypoxic incubator for 0, 1, 2, 3, 4, 5, and 6 hours. In the drug inhibition assay, different concentrations of drug (10, 20, 50, 100, 200, and 500 μM) were added after cell adhesion and cultured for 24 hours. After washing twice with PBS, 10% CCK8-prepared DMEM was added, and cells were cultured in the dark for 2 hours. The optical density (OD) at 450 nm was measured using a spectrophotometer.

[0053] 1.14 Enzyme-linked immunosorbent assay (ELISA)

[0054] The target molecules, including lactate dehydrogenase (LDH), superoxide dismutase (SOD), malondialdehyde (MDA), interleukin-1β (IL-1β), and interleukin-18 (IL-18), were quantitatively detected in rat serum, testicular tissue, and cell culture supernatant using an ELISA kit (Jiancheng, Nanjing).

[0055] 1.15 Detection of Intracellular Reactive Oxygen Species (ROS)

[0056] Intracellular ROS were detected using the H2DCFDA probe (MCE, HY-D0940). GC-1 and GC-2 cells were seeded at a density of 5 × 10^5 cells / well in 6-well plates. After treatment, cells were incubated with 10 μM H2DCFDA probe for 30 min. Images were acquired using a fluorescence microscope (Nikon, Japan), and fluorescence intensity was quantitatively analyzed using ImageJ software.

[0057] 1.16 Cell Immunofluorescence Staining

[0058] After cells were seeded in 24-well plates, they were fixed with 4% paraformaldehyde for 30 min and blocked with 0.5% BSA for 1 h. Then, cells were incubated with primary antibodies (GPR40, NLRP3, GSDMD, ASC) at 4°C overnight, followed by incubation with secondary antibodies diluted at 1:200 for 1 h. The staining and intensity analysis steps were described in the section of “IHC staining”.

[0059] 1.17 Western blot

[0060] The testis tissue and cell proteins were extracted using RIPA lysis buffer (Beyotime, P0013B) containing 10% protease inhibitor (MCE, HY-K0010) and phosphatase inhibitor (MCE, HY-K0021). The protein concentration was determined by bicinchoninic acid (BCA, Epizyme Biotech, zj102) method. The proteins were separated by SDS-PAGE loading buffer (Beyotime, P0015L) and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, ISEQ00010, USA) after electrophoresis. After blocking the membranes in blocking buffer (NCM, P30500, China), the primary antibodies were incubated at 4°C overnight, including NLRP3 (CST, D4D8T), ASC, caspase-1 (Proteintech, 22915-1-AP), GSDMD, IL-1β (Abeam, ab254360), IKBa (Zenbio, R380682), P65 (Zenbio, 250060), phosphorylated NF-κb p65 (P-P65, CST, 3033T), and β-actin (Huabio, EM21002). Then, the secondary antibodies, goat anti-rabbit IgG H&L (HRP, Zenbio, 511203) or goat anti-mouse IgG H&L (HRP, Zenbio, 511103), were incubated according to the species of the primary antibodies. Finally, the enhanced chemiluminescence substrate (ECL, Thermo, 37070) was used for development, and the ImageJ software was used for quantification. The protein expression was normalized to β-actin.

[0061] 1.18 Statistical analysis

[0062] All experiments were repeated at least three times. The data were expressed as mean ± standard deviation (SD), and the statistical analysis was performed using GraphPad Prism software (8.0.2, CA, USA). Student’s t test was used for comparison between two groups, and one-way ANOVA was used for comparison among multiple groups. The difference was considered statistically significant when the p value was less than 0.05.

[0063] 2. Results

[0064] 2.1 Testicular torsion and detorsion cause testicular injury in rats

[0065] To determine whether germ cell injury occurs after testicular torsion and detorsion, we established a rat model of testicular ischemia / reperfusion (I / R) by surgery. After surgery, the testis volume and organ coefficient of the I / R group decreased significantly. By H&E staining to assess the morphological injury of seminiferous tubules, the testis of the I / R group showed twisted seminiferous tubules, disordered tissue structure, and abnormal germ cell shedding compared with the sham group, with a significantly thinned epithelium, increased inflammatory cell infiltration, and reduced Johnsen score. Using scanning electron microscopy (SEM), we observed abnormal morphology of germ cells in the I / R group, including increased vesicles and damaged cell membranes. Transmission electron microscopy (TEM) showed that the endoplasmic reticulum and mitochondria were significantly swollen, with broken mitochondrial cristae and a large number of cell vacuoles. In summary, the results showed that testicular torsion and detorsion significantly damaged the testis.

[0066] 2.2 Testicular I / R induces pyroptosis and enrichment of NF-κB pathway in tissues

[0067] To further explore the potential mechanisms of testicular I / R injury, we performed differential expression analysis and enrichment analysis on the testicular tissue transcriptome. Differential analysis identified 705 differentially expressed genes, of which 389 were downregulated and 416 were upregulated. In particular, the upregulated differentially expressed genes included inflammatory factors closely related to the pyroptosis process, such as NLRP3 and IL-1β. Based on the differentially expressed genes, GO and KEGG enrichment analysis was performed, and the results showed that it was mainly related to the inflammatory process, especially the positive regulation of IL-1β. In addition, metabolic processes related to testicular I / R injury (such as peroxisome, purine, arachidonic acid, glutathione, linoleic acid, and alpha-linolenic acid metabolism) and inflammation-related signaling pathways (TNF, NOD-like receptor, Toll-like receptor, and IL-6 signaling pathways) were also enriched. In addition, GSEA analysis results showed that the differential genes were significantly enriched in the NF-κB signaling pathway and inflammatory response. These results suggest that testicular I / R injury may be related to inflammation activation, pyroptosis, and the NF-κB signaling pathway. Subsequent Western blot and immunohistochemical results also confirmed this.

[0068] 2.3 CMap predicts long-acting mebendazole as a potential therapeutic drug based on transcriptome sequencing

[0069] To explore the role of inflammatory response in testicular I / R, the differentially expressed genes (DEGs) were intersected with the inflammation-related genes (IRGs) in GSEA gene sets. The intersection results were displayed in a heatmap, showing that a large number of inflammation-related genes were significantly up-regulated. Subsequently, the DEGs were uploaded to the STRING database to construct a protein interaction network, which was visualized by Cytoscape. The top 10 key genes (IL-1β, IL-6, Nos2, Ptgs2, Stat3, Icam-1, Ccl2, Ccl3, IL-1α, Serpine1) were screened out by using the MCC algorithm Figure 1 A). Among them, IL-1β is a potent pro-inflammatory factor involved in the process of inflammation and immune response. The precursor protein pro-IL-1β can be induced by TLR or NF-κB pathway, and activated by caspase-1 mediated by NLRP3 inflammasome. After IL-1β interacts with IL-1R1, it can up-regulate inflammation-related factors and signaling pathways. Under the stimulation of inflammatory factors such as IL-1β and TNF-α, the expression of NOS2 and Ptgs2 increases, both of which are regulated by NF-κB, leading to inflammation amplification. In addition, NOS2 produces a large amount of nitric oxide, which can form peroxynitrite with superoxide anion, causing lipid peroxidation and DNA damage. At the same time, the expression of chemokines and adhesion molecules can further recruit immune cells. The above results suggest that testicular I / R injury may be related to the IL-1β-related inflammatory cascade.

[0070] Based on the above genes, CMap was used to screen potential drugs to alleviate testicular injury. The results showed that vinblastine (Vin) was considered to have potential therapeutic effect with a small score Figure 1 B). Vinblastine has been reported to inhibit NLRP3 inflammasome activation and down-regulate NF-κB pathway, which is consistent with our sequencing results, so vinblastine is selected and its therapeutic effect is verified. Vinblastine has been reported as an allosteric modulator of GPR40, but its binding ability and specific binding site have not been reported, so vinblastine is docked with GPR40 molecule. The binding energy is -6.2 kcal / mol, indicating strong binding affinity. The three-dimensional structure model shows that vinblastine forms hydrogen bonds with three residues (Val84, Leu135 and Phe142) of GPR40 protein, and is located outside the binding site Figure 1 C).

[0071] 2.4 Vinblastine improves the pathological structure of ischemia-reperfusion (I / R) testis and inhibits inflammatory response

[0072] To evaluate the effect of vinblastine (Vin) on I / R testis, animal experiments were performed Figure 2 A). Compared with the I / R group, the testis volume and organ coefficient of the I / R+Vin group increased Figure 2F, C). H&E staining of I / R+Vin group showed that the thickness of spermatogonia increased, the arrangement of germ cells was regular, and the Johnsen score was improved Figure 2 G, D, E). Transmission electron microscopy observation found that after vinblastine treatment, the swelling of mitochondria and endoplasmic reticulum was reduced, the cell vacuoles were significantly reduced, and the membrane damage was alleviated Figure 2 H). Testicular I / R injury is closely related to the continuous expansion of inflammation and the aggravation of oxidative stress. To further determine the anti-inflammatory and antioxidant activity of vinblastine, the levels of IL-1β, IL-18, LDH and SOD were detected by ELISA. The results showed that I / R promoted the release of LDH and the production of inflammatory factors IL-1β and IL-18, and reduced the activity of antioxidant enzyme SOD; while vinblastine reduced the levels of LDH, IL-1β and IL-18, and restored the activity of SOD Figure 2 B). These findings indicate that vinblastine can reduce the inflammatory response and tissue damage induced by I / R in testis.

[0073] 2.5 Vinblastine reduces pyroptosis in testicular I / R and inhibits the NF-κB signaling pathway

[0074] NLRP3 inflammasome is composed of sensor protein NLRP3, adapter protein ASC and effector protein caspase-1. The activation of inflammasome can increase the expression of its components and pro-IL-1β. Western blot and immunofluorescence staining results showed that the protein expression of NLRP3, ASC and caspase-1 was increased in the I / R group, suggesting that the assembly and activation of NLRP3 inflammasome were enhanced, and vinblastine could reverse this phenomenon in vivo. Subsequently, caspase-1 was cleaved and activated in NLRP3 inflammasome. On the one hand, inflammatory caspase cleaves the junction region of GSDMD, releasing the active N-terminal fragment, which forms a cell membrane pore through oligomerization; on the other hand, caspase-1 cleaves pro-inflammatory factors pro-IL-1β and pro-IL-18 into mature forms, which are released through GSDMD pores, further exacerbating the inflammatory response. In this study, the protein expression of GSDMD, N-GSDMD, pro-IL-1β and IL-1β was increased in I / R testis, and vinblastine significantly inhibited these expressions Figure 4 K, L).

[0075] The transcription of NLRP3 can be induced by the NF-κB signaling pathway. To verify the mechanism of vinblastine in inhibiting pyroptosis, the phosphorylation level of P65 and the degradation of IKBα were detected. Western blot and immunohistochemical results showed that the overexpression of phosphorylated P65 and the decrease of IKBα expression in I / R testis suggested that the NF-κB pathway was upregulated, and vinblastine could inhibit the activation of the pathway Figure 4 I, J).

[0076] 2.6OGD / R induces pyroptosis and activates NF-κB signaling in GC1 and GC2 cells

[0077] To further explore the mechanism of vinblastine alleviating testicular I / R injury, hypoxia-ischemia-reoxygenation (OGD / R) model was established in GC1 and GC2 cell lines. CCK8 detection showed that cell proliferation decreased significantly after 4 hours of hypoxia. After reoxygenation, inflammatory factors and oxidative stress markers continued to accumulate, especially IL-1β and IL-18 reached a peak at OGD / R 24h, so OGD4h / R24h was selected for cell experiments. Scanning electron microscopy of OGD / R groups of GC1 and GC2 showed a large number of pyroptosis bodies, transmission electron microscopy showed obvious cell vacuolization, organelle swelling and membrane damage. Subsequent Western blotting showed that NLRP3, ASC, caspase-1, GSDMD and IL-1β were up-regulated. P-P65 was overexpressed and IKBα was down-regulated after OGD / R. Therefore, it is speculated that inflammation, pyroptosis and NF-κB signaling pathway are involved in OGD / R-induced germ cell damage.

[0078] 2.7Vinblastine activates GPR40 in vitro and in vivo

[0079] It has been reported that vinblastine is a GPR40 agonist, and GPR40 may inhibit the activation of NLRP3 inflammasome

[44] . To further clarify the role of GPR40 in vinblastine alleviating testicular pyroptosis, vinblastine and GPR40 inhibitor GW1100 were used, and GPR40 expression in vitro and in vivo was detected. Western blotting and immunofluorescence staining showed that GPR40 was lowly expressed in I / R group, and vinblastine could increase its expression ( Figure 3 A). Tissue immunofluorescence staining showed that GPR40 was mainly increased in I / R+Vin group in germ cells ( Figure 3 B). In vitro experiments, GPR40 was lowly expressed in OGD / R group, vinblastine restored its expression, and GW1100 inhibited the expression of GPR40 induced by vinblastine ( Figure 3 C-F).

[0080] 2.8Vinblastine alleviates OGD / R-induced structural damage and inflammation in GC1 and GC2 cells by targeting GPR40

[0081] To determine whether the protective effect of vinblastine on germ cells is through the regulation of GPR40, the damage of 4 groups of cells was detected in vitro. Scanning electron microscopy showed that vinblastine reduced the formation of pyroptosis bodies, reduced membrane pores, and restored the structure of villi, while GPR40 inhibition led to the production of pyroptosis bodies ( Figure 4 A, H). Transmission electron microscopy showed that vinblastine alleviated cell vacuolization and membrane and mitochondrial damage, and GW1100 counteracted this protective effect ( Figure 4B, I). The inflammatory factors and oxidative stress indicators of the four groups of cells at different reoxygenation time points were detected. After OGD 4h / R 24h, vincristine significantly reduced MDA, LDH, IL-1β and IL-18, and increased SOD activity, and GW1100 reversed the effect Figure 4 C, J). ROS generation is an important factor for NLRP3 activation

[42] . Using the H2DCFDA probe, it was found that vincristine could reduce the ROS burst induced by OGD / R, and GPR40 inhibition promoted ROS production Figure 4 D, K). In summary, vincristine protects GC1 and GC2 cells from OGD / R injury by targeting GPR40, and inhibits inflammation and oxidative stress.

[0082] 2.9 Vincristine inhibits pyroptosis of GC1 and GC2 cells induced by OGD / R by targeting GPR40

[0083] Western blot and immunofluorescence results showed that vincristine reduced the expression of NLRP3 inflammasome components (NLRP3, pro-caspase-1 and ASC), and inhibited caspase-1 activation. In addition, vincristine inhibited the production and cleavage of GSDMD and pro-IL-1β, and reduced GSDMD pore formation and pro-inflammatory factor release. After GPR40 was inhibited, the protective effect of vincristine was eliminated Figure 4 E, F, L, M). Therefore, vincristine reduces pyroptosis of GC1 and GC2 cells by increasing GPR40 expression.

[0084] 2.10 Vincristine inhibits the upregulation of the NF-κB pathway in GC1 and GC2 cells induced by OGD / R by targeting GPR40

[0085] Western blot results showed that vincristine reduced IKBα degradation and P65 phosphorylation. After GPR40 inhibitor GW1100 treatment, IKBα degradation and P65 phosphorylation increased Figure 4 G, N). NF-κB is considered to be the core mediator of NLRP3 inflammasome activation signals, so our results show that vincristine downregulates the NF-κB pathway by targeting GPR40 to inhibit pyroptosis of GC1 and GC2 cells.

[0086] 3. Analysis and summary

[0087] Testicular torsion leads to acute tissue ischemia, and subsequent detorsion surgery inevitably causes reperfusion injury, which is considered to be the cause of persistent testicular atrophy (TA), loss of germ cells, and abnormal sperm morphology. It has been reported that the incidence of postoperative TA is more than 50%, and more than 60% of patients have abnormal sperm motility or morphology, and patients who undergo orchiectomy have better sperm motility compared to those who undergo testicular fixation. However, the specific molecular mechanisms of testicular ischemia-reperfusion (I / R) injury are not fully understood, and there is currently a lack of clinically effective treatments. Therefore, a deep understanding of the underlying mechanisms of testicular I / R injury and the discovery of new therapeutic drugs are crucial for the development of effective therapies. In this study, we predicted vinblastine (Vin) as a potential drug for treating testicular I / R injury through transcriptome sequencing and bioinformatics analysis. The results of in vivo and in vitro experiments showed that vinblastine inhibited pyroptosis of germ cells and activation of the NF-κB signaling pathway by targeting GPR40.

[0088] Adolescence is a critical period for testicular development and effective establishment of spermatogenesis, but it is also one of the periods with a high incidence of testicular torsion in children, and postoperative infertility remains one of the most important sequelae of testicular torsion.

[0089] Vinblastine is a monoterpenoid indole alkaloid extracted from Catharanthus roseus and can be safely used as a dietary supplement. Due to its vasodilatory and antioxidant effects, vinblastine has been used clinically to treat ischemic brain disease and ischemic optic neuropathy. Recent studies have found that vinblastine and its derivatives can alleviate various I / R-related diseases, including kidney, retina, intestinal, brain, and liver injury. Our study found that vinblastine improved the pathological structure of the testis and reduced inflammation and oxidative stress damage after I / R injury. Literature reports that vinblastine is an agonist of GPR40, and its protective effect may be achieved by targeting GPR40. The molecular docking results in our study showed that it stably binds to GPR40. By inhibiting GPR40 in an in vitro model, our study results showed that vinblastine exerts a good protective effect on the testis by targeting GPR40.

[0090] GPR40, also known as free fatty acid receptor 1 (FFAR1), belongs to the G protein-coupled receptor (GPCR) family and has high affinity for unsaturated free fatty acids. Because GPR40 is expressed in pancreatic beta cells and regulates insulin release, it is considered a potential target for the treatment of type 2 diabetes. Recent studies have shown that GPR40 has anti-inflammatory and antioxidant biological activities in various tissues, and can alleviate various inflammatory diseases, including nervous system inflammation, periodontitis, and rheumatoid arthritis. Previous studies have reported that GPR40 can inhibit the activation of NLRP3 inflammasome, but the specific mechanism is not clear, which may involve multiple processes. The present invention found that the expression of GPR40 in I / R testis and OGD / R germ cells was reduced, and the expression of GPR40 was restored after administration of meclizine, which reduced oxidative stress damage and inflammatory response; while using GW1100 to inhibit the expression of GPR40, the protective effect of meclizine disappeared. In summary, meclizine alleviates germ cell damage by targeting GPR40. To our knowledge, there is no related research on GPR40 in testicular tissue, and we first explored the role of GPR40 in alleviating testicular I / R injury.

[0091] According to previous reports, the present invention established an immature rat testicular ischemia-reperfusion (I / R) model by surgical torsion and detorsion to better simulate clinical conditions. Testicular damage was verified by histology, including a significant reduction in germ cells in the seminiferous tubules observed by H&E staining, and germ cell death and organelle damage observed under an electron microscope. Inflammation cell infiltration in H&E staining and sequencing results showing up-regulation of inflammation-related genes (IRGs) suggest that testicular damage is closely related to inflammation. Pyroptosis and NF-κB signaling pathways play a key role in inflammation.

[0092] Pyroptosis is a special form of inflammatory cell death, characterized by cell membrane damage, elevated expression of related proteins, and release of IL-1β and IL-18. Ning et al. (2021) and He et al. (2023) reported pyroptosis in testicular I / R injury, which is consistent with our sequencing results, indicating that pyroptosis plays an important role in testicular I / R injury. NLRP3 is a key factor in mediating pyroptosis, which can perceive a variety of stimuli (such as pathogens, metabolites and environmental toxins), and mediate the activation of caspase-1, thereby cleaving and activating inflammatory cytokines. The activation of NLRP3 can be regulated by G protein-coupled receptor (GPCR) signaling cascade in the early stage, and many studies have shown that GPR40 has an inhibitory effect on the activation of NLRP3. In the present study, after administration of long-chang anis, the formation of NLRP3 inflammasome and GSDMD pore and the content of IL-1β were decreased, while the expression of GPR40 was up-regulated. After inhibiting GPR40 in vitro, the expression levels of the above proteins were increased. Therefore, long-chang anis inhibits pyroptosis in testicular I / R injury by targeting GPR40.

[0093] The NF-κB signaling pathway is involved in immune and inflammatory responses and is believed to play a key role in tissue I / R injury. During inflammation, this pathway is over-activated, NF-κB is transferred to the nucleus and binds to DNA, promoting high expression of inflammation-related genes (IRGs). In particular, as the core mediator of NLRP3 inflammasome activation signals, NF-κB induces the transcriptional expression of NLRP3 and precursor IL-1β, initiating the pyroptosis process. In addition, NF-κB also triggers the transcription of various pro-inflammatory factors (such as COX2, NOS2, IL-6, TNF-α and ICAM-1), further amplifying the inflammatory response. Previous studies have reported that long-chang anis alleviated bleomycin-induced pulmonary fibrosis and streptozotocin-induced diabetic neuropathy by inhibiting the NF-κB pathway. To further explore the mechanism of long-chang anis in reducing testicular pyroptosis, we detected the expression levels of P-P65 and IKBα. The results showed that after the application of long-chang anis, the P-P65 / P65 ratio increased and the IKBα level decreased; while after inhibiting GPR40, the expression of the NF-κB pathway was up-regulated. The above results suggest that regulating the NF-κB pathway by regulating GPR40 is one of the mechanisms by which long-chang anis alleviates testicular pyroptosis.

[0094] In summary, the present application studies reveal the role of pyroptosis and inflammation in testicular torsion and detorsion-induced ischemia / reperfusion (I / R) injury through transcriptome sequencing, and in vivo and in vitro experiments confirm the anti-pyroptosis effect of vinblastine (Vin), including inhibition of inflammatory response, activation of NLRP3 inflammasome, formation of GSDMD pore, and further inflammation amplification caused by IL-1β. Mechanistically, vinblastine plays a role by targeting GPR40 to inhibit the NF-κB signaling pathway. In summary, these findings suggest that vinblastine is a potential drug for relieving testicular I / R injury.

Claims

1. Application of vincristine in the preparation of drugs for treating testicular ischemia-reperfusion injury.

2. The application according to claim 1, characterized in that: Vincristine reduces ischemia-reperfusion-induced testicular inflammation and tissue damage, and improves testicular pathological structure.

3. The application according to claim 2, characterized in that: Vincristine reduced LDH, IL-1β, and IL-18 levels and restored SOD activity.

4. The application according to claim 1, characterized in that: Vincristine reduces pyroptosis of testicular cells.

5. The application according to claim 4, characterized in that: Vincristine reduces testicular cell pyroptosis by targeting GPR40.

6. The application according to claim 5, characterized in that: Vincristine inhibits OGD / R-induced pyroptosis in GC1 and GC2 cells by targeting GPR40, thereby alleviating OGD / R-induced structural damage and inflammation in GC1 and GC2 cells.

7. The application according to claim 1, characterized in that: Vincristine exerts its effect by targeting GPR40, thereby inhibiting the NF-κB signaling pathway.

8. The application according to claim 7, characterized in that: Vincristine inhibits the upregulation of the OGD / R-induced NF-κB pathway in GC1 and GC2 cells by targeting GPR40.

9. The application according to any one of claims 1 to 8, characterized in that: The testicular ischemia-reperfusion injury referred to is testicular ischemia-reperfusion injury caused by testicular torsion reduction surgery.

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