The Effects And Mechanism Of Defocused Low-Energy Shock Wave Acting On Diabetic Bladder Dysfunction Via Activating Adipose Tissue-Derived Stem Cells

BACKGROUNDiabetes Mellitus (DM) associated complications include diabetic kidney disease, nerve damage, pathological damage of retina and heart, etc. In its urologic complications, diabetic bladder dysfunction (DBD) is very common. DBD does not affect the lives fo DM patients, but their life quality are bad. To our knowledge, there are no effective therapy methods in the treatment of DBD. As a kind of mesenchymal stromal cells (MSCs), adipose tissue derived stem cells (ADSCs) possess the multi-potent differentiation ability which likes to bone marrow stromal cells (BMSCs). Compared to other MSCs (including BMSCs), ADSCs have the advantage of easy access and abundance in quantity. Therefore, ADSCs are now the most important promising materials in the application of tissue repair and regenerative medicine. Previous study reported when they injected ADSCs back to cavernous of the cavernous nerve injured rats, the erectile function of the rats could be improved. Defocused low-energy shock wave (DLSW) is also widely applied in regenerative medicine. We found DLSW could be applied in regenerative medicine by activating mesenchymal stem cells. However, the possible signaling pathways participated in this process remain unknown.OBJECTIVETo explore the effect and mechanism of defocused low-energy shock wave (DLSW) acting on diabetic bladder dysfunction via activating adipose tissue-derived stem cells (ADSCs).METHODS1. Cell experiment(1)Female Sprague-Dawley rats (8 weeks old) were under anaesthetic with pentobarbital and fat around the ovary was removed to a centrifuge tube filled with cold phosphate buffered saline (PBS). After digestion and centrifugation, ADSCs were harvested and seeded in specialized basal medium (RASMD-90011, Cyagen Biosciences Inc., Guangzhou, China) containing 10% fetal bovine serum (FBS),1% penicillin-streptomycin and 1% L-glutamine onto culture flasks and incubated in a humidified atmosphere containing 5% CO2 at 37?. Two days later, non-adherent cells were removed and fresh culture medium was added. The culture medium was changed every 3 days. Cells were passaged when they reached approximately 90% confluence.(2)DLSW treatment. Cultured rat ADSCs were treated by DLSW before each passage. The untreated ADSCs served as control. The shock wave probe was kept in contact with the culture flask containing adherent ADSCs by means of a water-filled cushion covered with common ultrasound gel. The cells were subjected to 800 impulses of DLSW at an energy flux density of 0.1 mJ/mm2 with a frequency of 120/min.(3)Cellular morphology of each passage was observed. ADSCs were stained with phalloidin after each DLSW.(4)ELISA assay. After each shock wave treatment, the culture medium was removed and replaced with 3 ml of specialized basal medium without FBS. Twenty-four hours later, the conditioned medium was harvested and centrifuged at 1200 rpm for 10 minutes. Then we recovered the supernatant, and stored them at-80? until use. The processed culture media (supernatants) from P1-P5 were collected. Untreated ADSCs conditioned media were collected at the same time and served as control. All of the supernatants were simultaneously subjected toELISA with nerve growth factor (NGF) commercial kit, vascular endothelial growth factor and CXC ligand 5 (CXCL5) com mercial kit.(5) Flow cytometry. The treated ADSCs (P4) were analyzed by flow cytometry for cell surface antigen expressions. All of the supernatants were simultaneously subjected to Flow cytometry was performed to analyze the ADSCs (passage 4) surface antigen expressions (CD34, Stro-1, OCT4, CD106, CD29 and CD49d).(6)Cell proliferation. To assess proliferation, treated ADSCs (P4) were treated with 10 ?M 5-ethynyl-2-deoxyuridine (EdU) overnight and were then incubated with the use of the Cell-Light EdU Apollo567 In Vitro Imaging Kit at room temperature. Nuclei were counterstained with DAPI. Images were captured by fluorescence microscope, and digital histomorphometric analysis was performed with Image-Pro Plus 6.0 software. Untreated ADSCs (P4) were treated in the same way and served as control. In addition, the expressions of proliferating cell nuclear antigen (PCNA) and Ki67 were analyzed by means of Western blot.(7)Cell migration. The expression of CXCR2 and the migrations of ADSCs in vitro were detected. Treated ADSCs (P4) were incubated with the primary antibody of CXCR2 over-night at 4? and were then exposed to Texas Red-conjugated goat anti-rabbit secondary antibody for 30 minutes. Nuclei were counterstained with DAPI. Images were captured by fluorescence microscope. In addition, the expressions of CXCR2 was also analyzed by means of Western blot.(8)The signaling pathway activated in the process of DLSW activating ADSCs. The expression of Mitogen-activated protein kinases (MAPK) pathway, Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway, phosphoinositide 3-kinase (PI-3K)/Akt signaling pathway or nuclear factor-kappa B (NF-?B) pathway was also detected by means of Western blot. To confirm the activation of the three pathways by DLSW, another group ADSCs were cultured in medium with inhibitors of the three pathways (U0126, SB202190, SP600125, LY294002 and pyrrolidine dithiocarbamate) before each DLSW (up to P4) and the untreated ADSCs served as control group. The secretion and proliferation of the two groups were detected.2. Animal experiment(1) Experiment design.Forty female SD rats (8 weeks old) were radonly divided into two groups: Control group (N group),10 rats were fed with normal food (n=10); Diabetic group, 30 rats were fed with high fat diet (HFD) for 1 month, and then they accepted intraperitoneal injections of streptozotozin (STZ,30mg/kg) twice. Three months after STZ application, we harvested adipose tissue via laparotomy and isolated ADSCs. Cultured ADSCs were labeled with 10?M EdU and injected back into the rats via tail vein injection.Then, we randomly divide the 30 diabetic rats into three groups:DM control group (DM group, n=10):the rats received PBS 0.5ml via tail vein injection;ADSC group (n=10):the rats received injection of EdU-labeled autologous ADSCs (3×106) in 0.5ml PBS via tail vein;ADSC+SW group (n=10):the rats received EdU-labeled autologous ADSCs (3×106) in 0.5ml PBS via tail vein injection, followed by shocked with DLSW.One month after ADSCs injection and DLSW therapy, the conscious cystometry of all rats in all groups was examined. And then, we killed all rats for histology examination.(2) Conscious cystometry.(3) Quantification of EdU-labeled cells and apoptotic cells in blader tissue.The number of EdU-labeled ADSCs in each bladder tissue was calculated by the number of EdU-labeled ADSCs/the number of DAPI-labeled ADSCs.In bladder tissues, we analyzed the quantification of apoptotic cells using TUNEL kit. Then, the pictures of apoptotic cell nuclei were recovered by fluorescence microscopy, and were analyzed by Image-Pro Plus 6.0 sofeware.(4)Immunofluorescence and Immunohistochemical stainPrimary antibodied included mouse anti-smooth muscle actin (SMA), rabbit anti-collagen ? (Col ?), mouse anti-stromal cell-derived factor-1 (SDF-1) and rabbit anti-endothelial nitric oxide synthase (eNOS).3. Statistical analysisData were analyzed with SPSS 19.0 software. The continuous data were analyzed with 1-way ANOVA. To evaluate the repairmen of bladder function in all groups, Chi-Square test was performed with Fisher's Exact Test. Data are giver as the mean (standard deviation), and statistical significance was considered at P<0.05..RESULTS1. Cell experiment(1) No significant morphological change was observed between treated and untreated ADSCs.(2) The expressions of CD34, Stro-1, OCT4, CD106, CD29 and CD49d showed no significant differences (P> 0.05) between the treated and untreated ADSCs. DLSW did not change the expression profile of ADSCs surface antigens.(3) DLSW promoted secretions of ADSCs. The treated ADSCs secreted more VEGF, BDNF, NGF and CXCL5 than untreated ADSCs (P< 0.05).(4) DLSW promoted proliferation of ADSCs. The treated ADSCs demonstrated higher expression levels of PCNA (P< 0.05) and Ki67 (P< 0.05) than untreated ADSCs. There were more EdU-positive cells (P< 0.05) in treated ADSCs group.(5) DLSW promoted migration of ADSCs in vitro and in vivo. In the cell migration assay, there were significantly more migration cells (P<0.05) in treated ADSCs (P4) than untreated ADSCs (P4).(6) ADSCs responded to DLSW by significant activation of MAPK pathway, PI-3K/Akt signaling pathway and NF-?B signaling pathway (P< 0.05). The inhibitors blocked these activities as well as the secretion and proliferation of treated ADSCs.2. Animal experiment(1) In the assessment of voiding function,70% of the rats in ADSC+SW group manifested as normal bladder funciton, significantly (P< 0.05) more than the rats without DLSW therapy (40%).(2) Tracking of EdU-labeled cells. In ADSC+SW group, there were more EdU-positive cells than that in ADSC group (P>0.05).(3) Quantification of apoptosis. In the bladder tissue, we mainly found apoptotic cells in the mucosal layer and muscular layer. compared with N group, there are significantly more apoptotic cells in DM group (P<0.05). In the ADSC group and ADSC+SW group, the number of apoptotic cells significantly decreased in mucosal layer (P<0.05).(4) Quantification of vascularity. A dense and continuous network of Col ?-positive capillaries was showed below the urotheliem in the bladder tissues of N group rats. In the rats of DM group, the expression of Col ? was decresed and the continuity.of the capillaries network lost. In ADSC and ADSC+SW group, this capillaries network was improved.CONCLUSIONSOverall, this study showed that DLSW enhanced the secretion arid proliferation of ADSCs and promoted the activation of MAPK pathway PI-3K/Akt signaling pathway and NF-?B signaling pathway without changing the nature of ADSCs. Otherwise, DLSW could enhance the effect of ADSCs to improve the repair process of DBD in type ? diabetic rat model.