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PURPOSE: Radiation cystitis (RC) is a complex and common complication after radiotherapy for pelvic cancer. Icariside II (ICAII) is a flavonoid compound extracted from Epimedium, a traditional Chinese medicine, with various pharmacological activities. The aim of the present study was to investigate the cysto-protective effects of ICAII in RC rats and its possible mechanisms. MATERIALS AND METHODS: A rat model of induced radiation cystitis using pelvic X-ray irradiation was used, and bladder function was assessed by bladder volume and bladder leakage point pressure (LPP) after ICAII treatment. HE and Masson stains were used to assess the histopathological changes in the bladder. IL-6, TNF-α, IL-10, IL-4 and IL-1ß were measured by ELISA to assess the level of inflammation. The gene-level changes in ICAII-treated RC were observed by transcriptome sequencing, and then the potential targets of action and biological mechanisms were explored by PPI, GO and KEGG enrichment analysis of the differentially expressed genes. Finally, the predicted targets of action were experimentally validated using immunohistochemistry, RT-qPCR, molecular docking and CETSA. RESULTS: ICAII significantly increased bladder volume and the LPP, ameliorated pathological damage to bladder tissues, decreased the levels of IL-6, TNF-α, and IL-1ß, and increased the levels of IL-10 and IL-4 in radiation-injured rats. A total of 90 differentially expressed genes were obtained by transcriptome sequencing, and PPI analysis identified H3F3C, ISG15, SPP1, and LCN2 as possible potential targets of action. GO and KEGG analyses revealed that these differentially expressed genes were mainly enriched in the pathways metabolism of xenobiotics by cytochrome P450, arachidonic acid metabolism, Staphylococcus aureus infection and chemical carcinogenesis - reactive oxygen species. Experimental validation showed that ICAII could significantly increase the expression of H3F3C and ISG15 and inhibit the expression of SPP1 and LCN2. ICAII binds well to H3F3C, ISG15, SPP1 and LCN2, with the best binding ability to H3F3C. Furthermore, ICAII inhibited the protein degradation of H3F3C in bladder epithelial cells. CONCLUSIONS: ICAII may alleviate the bladder inflammatory response and inhibit the fibrosis process of bladder tissues through the regulation of H3F3C, ISG15, SPP1, and LCN2 targets and has a protective effect on the bladder of radioinjured rats. In particular, H3F3C may be one of the most promising therapeutic targets.
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Cancer and diabetes are significant diseases that pose a threat to human health. Their interconnection is complex, particularly when they coexist, often necessitating multiple therapeutic approaches to attain remission. Sodium-glucose cotransporter protein two inhibitors (SGLT-2i) emerged as a treatment for hyperglycemia, but subsequently exhibited noteworthy extra-glycemic properties, such as being registered for the treatment of heart failure and chronic kidney disease, especially with co-existing albuminuria, prompting its assessment as a potential treatment for various non-metabolic diseases. Considering its overall tolerability and established use in diabetes management, SGLT-2i may be a promising candidate for cancer therapy and as a supplementary component to conventional treatments. This narrative review aimed to examine the potential roles and mechanisms of SGLT-2i in the management of diverse types of cancer. Future investigations should focus on elucidating the antitumor efficacy of individual SGLT-2i in different cancer types and exploring the underlying mechanisms. Additionally, clinical trials to evaluate the safety and feasibility of incorporating SGLT-2i into the treatment regimen of specific cancer patients and determining appropriate dosage combinations with established antitumor agents would be of significant interest.
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Nitrogen (N) fertilization is crucial for maintaining plant productivity. Clonal plants can share resources between connected ramets through clonal integration influencing microbial communities and regulating soil biogeochemical cycling, especially in the rhizosphere. However, the effect of various N fertilization practices on microbial communities in the rhizosphere of clonal ramets remain unknown. In this study, clonal fragments of Moso bamboo (Phyllostachys edulis), consisting of a parent ramet, an offspring ramet, and an interconnecting rhizome, were established in the field. NH4NO3 solution was applied to the parent, offspring ramets or rhizomes to investigate the effect of fertilization practices on the structure and function of rhizosphere microbial communities. The differences in N availability, microbial biomass and community composition, and abundance of nitrifying genes among rhizosphere soils of ramets gradually decreased during the rapid growth of Moso bamboo, irrespective of fertilization practice. The soil N availability variation, particularly in NO3-, caused by fertilization practices altered the rhizosphere microbial community. Soil N availability and stable microbial biomass N in parent fertilization were the highest, being 9.0 % and 18.7 %, as well as 60.8 % and 90.4 % higher than rhizome and offspring fertilizations, respectively. The microbial network nodes and links in rhizome fertilization were 1.8 and 7.5 times higher than in parent and offspring fertilization, respectively. However, the diversity of bacterial community and abundance of nitrifying and denitrifying genes were the highest in offspring fertilization among three practices, which may be associated with increased N loss. Collectively, the rhizosphere microbial community characteristics depended on fertilization practices by altering the clonal integration of N in Moso bamboo. Parent and rhizome fertilization were favorable for N retention in plant-soil system and resulted in more stable microbial functions than offspring fertilization. Our findings provide new insights into precision fertilization for the sustainable Moso bamboo forest management.
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Microbiota , Nitrogênio , Rizoma , Poaceae , Microbiologia do Solo , Solo , FertilizaçãoRESUMO
BACKGROUND: Hematologic diseases have seriously threatened human health. Although hematopoietic stem cell transplantation (HSCT) is an effective curative option, the complications, especially graft-versus-host disease (GVHD), are a big problem. METHODS: TNF-α pretreatment of hematopoietic stem cells. Apoptosis was detected by flow cytometry, Transwell, and wound healing assays were used to assess cell migration and invasion, E-selectin expression was observed by fluorescence imaging, the levels of NO were measured by a kit, the expression of Ecadherin, MMP2, and MMP9 was detected in cells by qRT-PCR, and western blot was used to analyze the expression of E-cadherin, CXCL12, MCP-1, MCP-3, MMP2, and MMP9. RESULTS: TNF-α induces a high apoptosis rate of CD3, CD19, and CD133 and a low apoptosis rate of CD34. The level of Fas and TNF-R1 was significantly high than that of TNF-R2. HSCs treated with TNF- α declined the invasion and migration of HUVECs. E-selectin, MMP2 and MMP9 mRNA levels of HUVECs and MMP2, CXCL12, MCP-1, and MCP-3 were decreased after HSCs-TNF-α treatment, while the E-cadherin mRNA and protein level of HUVECs was enhanced with HSCs-TNF-α treatment. CONCLUSION: TNF-α pretreated HSCs can lead to reduced levels of migration, adhesion, and chemokines of HUVECs, thereby declining the inflammatory response and GVHD.
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Doença Enxerto-Hospedeiro , Transplante de Células-Tronco Hematopoéticas , Humanos , Fator de Necrose Tumoral alfa/farmacologia , Selectina E/metabolismo , Metaloproteinase 2 da Matriz , Metaloproteinase 9 da Matriz , Doença Enxerto-Hospedeiro/prevenção & controle , Caderinas , Células-Tronco Hematopoéticas/metabolismo , RNA Mensageiro , Transplante de Células-Tronco Hematopoéticas/métodosRESUMO
BACKGROUND: Erectile dysfunction is common among the complications of diabetes mellitus. Shaofu Zhuyu decoction (SFZYD) is commonly used to treat diabetic mellitus erectile dysfunction (DMED). However, its main active components and specific mechanism are still unknown. PURPOSE: To confirm the activity of SFZYD in improving DMED, explore the main active components of SFZYD, and clarify the underlying mechanism. METHODS: A diabetic rat model was induced with streptozotocin (STZ). After intragastric administration, erectile function was assessed by the maximum intracavernous pressure (ICPmax)/mean arterial pressure (MAP). Corpus cavernosum fibrosis was evaluated by Masson staining, and ELISA methods were used to determine the serum levels of IL-6, TNF-α, IL-10, IL-4 and IL-1ß to evaluate inflammation. Then, the main active components of SFZYD were identified by UPLCâMS/MS. Finally, the target and biological mechanism of SFZYD in improving DMED were predicted by combined network pharmacology and transcriptomics, which was also validated by molecular docking and cellular thermal shift assay (CETSA) experiments. RESULTS: SFZYD significantly improved erectile dysfunction and inhibited inflammatory responses and local tissue fibrosis in diabetic rats. A total of 1846 active components were identified by UPLCâMS/MS, and isorhamnetin was the main active component. The transcriptomic results were used to identify differentially expressed genes among the control, DM and SFZYD groups, and 1264 differentially expressed genes were obtained from the intersection. The network pharmacology results showed that SFZYD acts on core targets such as AKT1, ALB, HSP90AA1 and ESR1 through core components such as isorhamnetin, quercetin and chrysophanic acid. Further combined analysis revealed that multiple targets, such as CYP1B1, DPP4, NOS2 and LCN2, as well as the regulation of the PI3K-AKT signaling pathway, may be important mechanisms by which SFZYD improves DMED. Molecular docking verification showed that isorhamnetin, the key component of SFZYD, has good binding ability with several core targets, and its binding ability with CYP1B1 was the strongest. The CETSA results showed that isorhamnetin binds to CYP1B1 in CCECs. CONCLUSION: SFZYD improves DMED, inhibits the inflammatory response and alleviates local tissue fibrosis. The combined application of transcriptomic, network pharmacology, molecular docking and CETSA approaches was helpful for revealing the mechanism by which SFZYD improves DMED, which may be related to the regulation of CYP1B1 and the PI3K-Akt signaling pathway.
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Diabetes Mellitus Experimental , Disfunção Erétil , Masculino , Humanos , Ratos , Animais , Disfunção Erétil/tratamento farmacológico , Disfunção Erétil/etiologia , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/metabolismo , Ratos Sprague-Dawley , Transcriptoma , Cromatografia Líquida , Simulação de Acoplamento Molecular , Farmacologia em Rede , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espectrometria de Massas em Tandem , FibroseRESUMO
BACKGROUND: Erectile dysfunction (ED) and weakness of the penis are processes related to hemodynamic alteration. Low-intensity pulsed ultrasound (LIPUS), as a new mechanical modality for the treatment of ED, deserves to be explored in depth for the biomechanical mechanisms it exerts. OBJECTIVE: The aim of this study was to explore the role of YAP/TAZ-mediated mechanotransduction in mechanical therapy for the treatment of neurogenic erectile dysfunction (NED). MATERIALS AND METHODS: Forty-two male SD rats (12 w old) were randomly divided into sham-operated (n = 14), bilateral cavernous nerve injury (BCNI, n = 14), and LIPUS-treated (n = 14) groups. Intracavernosal pressure/mean arterial pressure (ICP/MAP) was measured 14 and 28 days after treatment. Penile tissue specimens were collected for pathological examination, and the changes in YAP, TAZ, connective tissue growth factor (CTGF), CYR61, LATS1, and p38 mitogen-activated protein kinase expression levels were assessed by Western blot, real-time quantitative polymerase chain reaction (RT-qPCR) and immunological staining. RESULTS: Compared with BCNI, LIPUS significantly improved ICP/MAP levels and enhanced histopathological changes. The penile expression levels of YAP, TAZ, CTGF, and CYR61 were significantly downregulated in the BCNI group (p < 0.01), and LIPUS upregulated the expression levels of these proteins (p < 0.05). The expression levels of p-LATS1 and LATS1 were not significantly different among the groups (p > 0.05). Interestingly, the expression level of p-p38/p38 significantly increased in BCNI rats (p < 0.05), which was reversed by LIPUS treatment (p < 0.05). However, the p38 inhibitor SB203580 did not change the expression of YAP/TAZ in rat primary smooth muscle cells or mouse MOVAS cells (p > 0.05). DISCUSSION AND CONCLUSION: LIPUS can effectively improve penile erectile function in NED rats. The underlying mechanism may be related to the regulation of YAP/TAZ-mediated mechanotransduction. However, the upstream regulatory signal may differ from the classical Hippo pathway.
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Disfunção Erétil , Mecanotransdução Celular , Traumatismos do Sistema Nervoso , Animais , Masculino , Camundongos , Ratos , Modelos Animais de Doenças , Ereção Peniana , Pênis/patologia , Proteínas Serina-Treonina Quinases , Ratos Sprague-Dawley , Traumatismos do Sistema Nervoso/patologia , Ondas UltrassônicasRESUMO
This work aims to investigate the therapeutic effect of ursolic acid (UA) plus insulin (In) on diabetic nephropathy (DN) in streptozotocin (STZ)-induced T1DM rats. The experimental groups and operational details are as follows: A total of thirty-two SD rats were divided into four groups: the DN model group (DN, n = 8), DN + In treatment group (DN + In, n = 8), DN + In + UA administration group (DN + In + UA, n = 8), and negative control group (control, n = 8). After 8 weeks, changes in renal function indices and pathological damage were assessed. Additionally, oxidative stress-, apoptosis-, and fibrosis-related proteins in kidney tissue were measured. Compared with the control group, the vehicle group showed higher levels of creatine, blood urea nitrogen, urinary protein, apoptosis, and lipid peroxidation; lower superoxide dismutase levels; more severe levels of pathological kidney damage and renal fibrosis; and a deepened degree of EMT and EndMT. Better outcomes were achieved with the combined treatment than with insulin-only treatment. The improvement of TGF-ß1, phosphorylated p38 MAPK, FGFR1, SIRT3 and DPP-4 expression levels in renal tissues after combination therapy was greater than that after insulin-only treatment. This study shows that the combination of insulin and UA significantly improved the pathological changes in the renal tissue of T1DM rats, and the underlying mechanism may be related to improving apoptosis and oxidative stress by regulating p38 MAPK, SIRT3, DPP-4 and FGFR1 levels, thereby blocking TGF-ß signaling pathway activation and inhibiting EMT and EndMT processes.
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Background: We explored the preventive effect and mechanism of YS-10, a novel synthesized flavonoid derivative based on the structure of icariside II (ICA II), on a rat model of radiation-induced erectile-dysfunction (Ri-ED). Methods: Eighteen 10-week-old male Sprague-Dawley (SD) rats were randomly divided into 3 groups. Six rats were used as the control group (Control), and the remaining 12 were given a single X-ray irradiation of 20 Gy in the prostate and then randomly divided into the radiation injury group (Ri-ED group) and YS-10 treatment group (Ri-ED+YS-10, 2.5 mg/kg/day). After 4 weeks of drug administration and a 2-week drug washout period in the YS-10 treatment group, the erectile function of the animals was evaluated, and the tissues were collected for histopathological analysis and detection of oxidative stress indicators. Results: After radiation injury, the ratio of maximum intracavernosal pressure (ICP) to mean arterial pressure (MAP), the number of neuronal nitric oxide synthase (n-NOS) positive nerve fibers in the penis cavernosa, endothelial cell content, and n-NOS and endothelial nitric oxide synthase (e-NOS) proteins in the Ri-ED group were significantly lower than those in control group. Compared with the control group, the Ri-ED group had lower superoxide dismutase (SOD) levels and higher malondialdehyde (MDA) levels. Compared with the Ri-ED group, the YS-10 group had a significant increase in the ratio of ICP/MAP in the corpus cavernosum (0.59±0.06 vs. 0.43±0.06, P<0.01), the number of n-NOS positive nerve fibers, and the content of endothelial cells. The protein content of n-NOS and e-NOS in the corpus cavernosum increased and could significantly reduce the level of MDA (2.67±0.27 vs. 3.25±0.21, P<0.05). Conclusions: As a novel ICA II derivative, YS-10 could significantly improve the erectile dysfunction and pathological damage in rats caused by radiation injury, and its mechanism may be related to the improvement of radiation-induced oxidative stress.
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The China Spallation Neutron Source project Phase-II aims to deliver 500 kW beam power to the spallation target. To meet the beam power requirement, an RF-driven negative hydrogen ion source with an external-antenna has been developed. In order to optimize the beam transmission through the radio frequency quadrupole and the downstream linac, the low energy beam transport line needs to be carefully studied and the transverse emittance is focused in this paper. With computational simulation and experimental verification, the emittance growth caused by nonlinear magnetic fields of the solenoid and the residual magnetic fields at the measuring position has been carefully analyzed. The measurement uncertainty of the double-slit scanner has also been quantitatively estimated. Using the same plasma-beam boundary setting, the beam extraction system is also optimized with particle tracking simulation in CST PARTICLE STUDIO.
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OBJECTIVE: To explore the preventive and therapeutic effects of Icariside â ¡ (ICAâ ¡) on radiation injury-induced ED (Ri-ED) in rats. METHODS: Twenty-four 10-week-old male SD rats received exposure of the prostate to single X-ray irradiation of 20 Gy, and were randomly equally divided into an Ri-ED group (6 survived and 6 died) and a treatment group treated with ICAâ ¡ at 4.5 mg/kg/d (9 survived and 3 died). Another 6 SD rats were taken as negative controls. After 4 weeks of continuous intragastric administration and 2 weeks of drug elution, the erectile function of the rats was evaluated by measurement of the maximum intracavernous pressure / mean arterial pressure (ICPmax/MAP), and the penile tissues were subjected to immunofluorescence staining, immunohistochemistry, Masson's trichrome staining, Western blot and detection of oxidative stress indicators. RESULTS: Compared with the negative controls, the rats in the Ri-ED group showed significant decreases in ICPmax/MAP (0.76 ± 0.09 vs 0.42 ± 0.06, P < 0.01), the number of nNOS-positive nerve fibers in the corpus cavernosum (10.17 ± 2.64 vs 3.17 ± 1.72, P < 0.01), the content of endothelial cells (1.39 ± 0.30 vs 0.35 ± 0.12, P < 0.01), the expressions of nNOS (0.42 ± 0.08 vs 0.08 ± 0.01, P < 0.01) and eNOS (0.99 ± 0.24 vs 0.12 ± 0.08, P < 0.01) and the activity of superoxide dismutase (SOD) (ï¼»343.73 ± 58.57ï¼½ vs ï¼»153.50 ± 34.06ï¼½ U/mg prot, P < 0.01), but an increase in the malondialdehyde (MDA) level (ï¼»1.80 ± 0.31ï¼½ vs ï¼»3.25 ± 0.21ï¼½ nmol/mg prot, P < 0.01). In comparison with the Ri-ED group, the animals treated with ICAâ ¡ exhibited remarkably increased ICP/MAP (0.42 ± 0.06 vs 0.66 ± 0.07, P < 0.01), number of nNOS-positive nerve fibers (3.17 ± 1.72 vs 7.33 ± 1.75, P < 0.05), content of endothelial cells (0.35 ± 0.12 vs 1.07 ± 0.36, P < 0.01), and expressions of nNOS (0.08 ± 0.01 vs 0.16 ± 0.05, P < 0.05) and eNOS (0.12 ± 0.08 vs 0.86 ± 0.30, P < 0.01) in the corpus cavernosum, but a decreased level of MDA (ï¼»3.25 ± 0.21ï¼½ vs ï¼»2.17 ± 0.55ï¼½ nmol/mg prot, P < 0.05). In addition, ICAâ ¡ effectively reduced radiation injury-induced mortality of the rats. CONCLUSION: Icarisideâ ¡ can significantly improve ED and pathological changes and reduce mortality caused by radiation injury in rats, which may be related to its ability of improving radiation-induced oxidative stress.
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Disfunção Erétil , Lesões por Radiação , Humanos , Ratos , Masculino , Animais , Disfunção Erétil/tratamento farmacológico , Disfunção Erétil/etiologia , Disfunção Erétil/prevenção & controle , Células Endoteliais , Ratos Sprague-Dawley , Ereção Peniana/fisiologia , Pênis/patologiaRESUMO
OBJECTIVE: To investigate the effect and safety of low-intensity pulsed ultrasound (LIPUS) versus low-energy shock wave (LESW) in the treatment of neurogenic penile ED in male SD rats. METHODS: Twenty-four 12-week-old male SD rats were randomly divided into four groups of an equal number: sham operation, bilateral cavernous nerves injury (BCNI), LIPUS (300 mW /cm2, 3 times a week for 2 weeks), and LESW (300 strokes once, 3 times a week for 2 weeks). At 28 days after intervention, the erectile function of the rats was assessed by comparing the ratio of maximum intracavernous pressure to mean arterial pressure (ICPmax/MAP), and the histopathological changes in the corpus cavernosum of the penis were observed by immunohistochemistry, immunofluorescence staining, and Masson trichromatic staining. RESULTS: After treatment, the LIPUS and LESW groups, compared with the BCNI group, showed significantly increased ICPmax/MAP ratio (0.56 ± 0.13 and 0.55 ± 0.10 versus 0.35 ± 0.14, P = 0.017 and P = 0.013), improved smooth muscle/collagen value (0.08 ± 0.01 and 0.08 ± 0.02 versus 0.06 ± 0.02, P = 0.017 and P = 0.019), and elevated proportion of smooth muscle to cavernosum (0.20 ± 0.05 and 0.21 ± 0.03 versus 0.15 ± 0.02, P = 0.046 and P = 0.020), with no statistically significant difference between the LIPUS and LESW groups. No obvious adverse reactions were observed in the LIPUS or LESW group. CONCLUSIONS: Both LIPUS and LESW can effectively improve penile erectile function and repair histopathological injury in the animal model of neurogenic ED.
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Disfunção Erétil , Animais , Disfunção Erétil/terapia , Humanos , Masculino , Pênis , Ratos , Ratos Sprague-Dawley , Ondas UltrassônicasRESUMO
OBJECTIVE: To determine the expression of tyrosine protein kinase Met (c-MET), epidermal growth factor receptor (EGFR), and human epidermal growth factor receptor 2 (HER-2) in cases with gastric adenocarcinoma (GA). METHODS: The positive rates of the c-MET, EGFR, and HER-2 proteins between cancerous tissues and normal tissues sampled from 87 patients with GA were compared. The patients were assigned to different subgroups according to their clinicopathological characteristics and analyzed. Then the relationship between the above three indexes and the positive expression of Ki-67 were analyzed. In addition, the patients were assigned to positive and negative groups based on the situation of c-MET, EGFR and HER-2 proteins, and followed up for three years. These groups were compared in terms of recurrence-free survival, overall survival, and risks factors of prognosis. RESULTS: The positive rates of c-MET, EGFR and HER-2 proteins in GA tissues were all higher than those in corresponding non-tumor tissues (all P<0.001), and the positive rates of them were greatly different in subgroups with different differentiation, invasion depth, TNM stage, lymph node metastasis (LNM), distant metastasis and presence of tumor thrombus (all P<0.05) and were positively correlated with the expression of Ki-67 protein (P<0.05). Moreover, the survival analysis results revealed lower recurrence-free survival and overall survival rates in groups with negative expression of c-MET, EGFR, and HER-2 than those in groups with positive expression of them (both P<0.001). Furthermore, the positive EGFR was an independent prognostic factor affecting the survival of patients with GA. CONCLUSION: The expression of c-MET, EGFR and HER-2 proteins is correlated with clinical characteristics of patients with GA, and patients with positive expression of them face a higher recurrence rate. Additionally, EGFR protein may affect patients' survival.
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OBJECTIVE: To clarify the effects of cellular self-aggregation of adipose-derived stem cells (ADSCs) on erectile function (EF). METHODS: A model of neurogenic erectile dysfunction was performed using bilateral cavernous nerve crush injury in rats. ADSCs suspensions (1 × 106/0.2 ml), were administered via intracavernous injection (ICI) after being allowed to shelve for 0 minute (ICI 0) or 60 minutes (ICI 60) in vitro, as well as cell aggregates isolated from ICI 60 (ICI A). The caudal vein injection group (CVI 60) was used to evaluate whether cell self-aggregation was beneficial to EF when introduced into the peripheral circulation. One day after the transplantation, the distribution of cells was observed. EF and histopathological changes were evaluated after 4 weeks. RESULTS: Approximately 85% of ADSCs self-aggregated into cell clusters at 60 minutes. The ICI 60 had more significant improvements in EF and more visualized ADSCs retained in the corpus cavernosum (CC) than ICI 0 and CVI 60 (P <.05), but no significant difference between ICI 60 and ICI A. In the CVI 60 group, the cell clusters formed by self-aggregation could hardly reach the CC and were mostly found in lung tissue. Immunofluorescence staining showed increased the content of expressing biomarkers of smooth muscle, nerve within the CC tissue in the ICI groups when compared to the CVI group. CONCLUSION: ADSCs self-aggregation before ICI may be an influential factor in the treatment of neurogenic erectile dysfunction. Its potential mechanism may be through improving cell retention in the CC.
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Agregação Celular , Disfunção Erétil/terapia , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/fisiologia , Tecido Adiposo/citologia , Animais , Modelos Animais de Doenças , Disfunção Erétil/etiologia , Disfunção Erétil/fisiopatologia , Injeções Intravenosas , Masculino , Músculo Liso/patologia , Óxido Nítrico Sintase Tipo I/metabolismo , Ereção Peniana , Pênis/inervação , Pênis/patologia , Traumatismos dos Nervos Periféricos/complicações , Nervos Periféricos/enzimologia , RatosRESUMO
BACKGROUND: Traditional tissue engineering methods to fabricate urinary tract patch have some drawbacks such as compromised cell viability and uneven cell distribution within scaffold. In this study, we combined three-dimensional (3D) bioprinting and tissue engineering method to form a tissue-engineered urinary tract patch, which could be employed for the application on Beagles urinary tract defect mode to verify its effectiveness on urinary tract reconstruction. METHODS: Human adipose-derived stem cells (hADSCs) were dropped into smooth muscle differentiation medium to generate induced microtissues (ID-MTs), flow cytometry was utilized to detect the positive percentage for CD44, CD105, CD45, and CD34 of hADSCs. Expression of vascular endothelial growth factor A (VEGFA) and tumor necrosis factor-stimulated gene-6 (TSG-6) in hADSCs and MTs were identified by Western blotting. Then the ID-MTs were employed for 3D bioprinting. The bioprinted structure was encapsulated by transplantation into the subcutaneous tissue of nude mice for 1âweek. After retrieval of the encapsulated structure, hematoxylin and eosin and Masson's trichrome staining were performed to demonstrate the morphology and reveal collagen and smooth muscle fibers, integral optical density (IOD) and area of interest were calculated for further semi-quantitative analysis. Immunofluorescent double staining of CD31 and α-smooth muscle actin (α-SMA) were used to reveal vascularization of the encapsulated structure. Immunohistochemistry was performed to evaluate the expression of interleukin-2 (IL-2), α-SMA, and smoothelin of the MTs in the implanted structure. Afterward, the encapsulated structure was seeded with human urothelial cells. Immunofluorescent staining of cytokeratins AE1/AE3 was applied to inspect the morphology of seeded encapsulated structure. RESULTS: The semi-quantitative assay showed that the relative protein expression of VEGFA was 0.355â±â0.038 in the hADSCs vs. 0.649â±â0.150 in the MTs (tâ=â3.291, Pâ=â0.030), while TSG-6 expression was 0.492â±â0.092 in the hADSCs vs. 1.256â±â0.401 in the MTs (tâ=â3.216, Pâ=â0.032). The semi-quantitative analysis showed that the mean IOD of IL-2 in the MT group was 7.67â±â1.26, while 12.6â±â4.79 in the hADSCs group, but semi-quantitative analysis showed that there was no statistical significance in the difference between the two groups (tâ=â1.724, Pâ=â0.16). The semi-quantitative analysis showed that IOD was 71.7â±â14.2 in non-induced MTs (NI-MTs) vs. 35.7â±â11.4 in ID-MTs for collagen fibers (tâ=â3.428, Pâ=â0.027) and 12.8â±â1.9 in NI-MTs vs. 30.6â±â8.9 in ID-MTs for smooth muscle fibers (tâ=â3.369, Pâ=â0.028); furthermore, the mean IOD was 0.0613â±â0.0172 in ID-MTs vs. 0.0017â±â0.0009 in NI-MTs for α-SMA (tâ=â5.994, Pâ=â0.027), while 0.0355â±â0.0128 in ID-MTs vs. 0.0035â±â0.0022 in NI-MTs for smoothelin (tâ=â4.268, Pâ=â0.013), which indicate that 3D bioprinted structure containing ID-MTs could mimic the smooth muscle layer of native urinary tract. After encapsulation of the urinary tract patch for additional cell adhesion, urothelial cells were seeded onto the encapsulated structures, and a monolayer urothelial cell was observed. CONCLUSION: Through 3D bioprinting and tissue engineering methods, we provided a promising way to fabricate tissue-engineered urinary tract patch for further investigation.
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Molécula-1 de Adesão Celular Endotelial a Plaquetas/análise , Impressão Tridimensional , Engenharia Tecidual/métodos , Sistema Urinário/citologia , Actinas/análise , Animais , Moléculas de Adesão Celular/análise , Células Cultivadas , Cães , Imunofluorescência , Humanos , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Endogâmicos BALB C , Fator A de Crescimento do Endotélio Vascular/análiseRESUMO
[This corrects the article on p. 534 in vol. 8, PMID: 28790931.].