Zhezhu Gao

Autophagic deficiency is related to steroidogenic decline in aged rat Leydig cells.

Late-onset hypogonadism (LOH) is closely related to secondary androgen deficiency in aged males, but the mechanism remains unclear. In this study, we found that reduced testosterone production in aged rat Leydig cells is associated with decreased autophagic activity. Primary rat Leydig cells and the TM3 mouse Leydig cell line were used to study the effect of autophagic deficiency on Leydig cell testosterone production. In Leydig cells from young and aged rats, treatment with wortmannin, an autophagy inhibitor, inhibited luteinising hormone (LH)-stimulated steroidogenic acute regulatory (StAR) protein expression and decreased testosterone production. In contrast, treatment with rapamycin, an autophagy activator, enhanced LH-stimulated steroidogenesis in Leydig cells from aged, but not young, rats. Intracellular reactive oxygen species (ROS) levels were increased in both young and aged Leydig cells treated with wortmannin but decreased only in aged Leydig cells treated with rapamycin. Furthermore, an increased level of ROS, induced by H(2)O(2), resulted in LH-stimulated steroidogenic inhibition. Finally, knockdown of Beclin 1 decreased LH-stimulated StAR expression and testosterone production in TM3 mouse Leydig cells, which were associated with increased intracellular ROS level. These results suggested that autophagic deficiency is related to steroidogenic decline in aged rat Leydig cells, which might be influenced by intracellular ROS levels.

Therapeutic Potential of Adipose‐Derived Stem Cells‐Based Micro‐Tissues in a Rat Model of Postprostatectomy Erectile Dysfunction

INTRODUCTION Stem cells (SCs) show significant benefits in the treatment of postprostatectomy erectile dysfunction (ED). However, the low retention rate of the traditional single-cell strategy at the injection sites limits its therapeutic potential. AIM This study aims to investigate the feasibility and mechanism of adipose-derived stem cells (ADSCs)-based micro-tissues (MTs) in the treatment of ED in a rat model of bilateral cavernous nerves (CNs) injury. METHODS ADSCs labeled with 5-ethynyl-2-deoxyuridine (EdU) were used to generate MTs with hanging drop method. 10 Sprague-Dawley (SD) rats underwent sham surgery and intracavernous (IC) injection of phosphate buffer solution (PBS) (the sham group). Another 70 rats underwent bilateral CN crush and were then treated with PBS (n = 10, the crush group), dissociated ADSCs (n = 30, the ADSCs group), and MTs (n = 30, the MTs group), respectively. At day 1, 3, 7, 14 (n = 5), and 28 (n = 10) postsurgery, specimens were harvested for histology. At day 28, 10 rats in each group were examined for erectile function before tissue harvest. MAIN OUTCOME MEASURES Light microscopy of the dynamic aggregation of the MT, immunohistologic examination of the MTs, the retention and distribution of EdU + ADSCs in the corpus cavernosum (CC), and the penis histological analyses of collagen content, Western blot of functional proteins in MTs, intracavernous pressure recording on CN electrostimulation. RESULTS Three-day-old MTs became stable and expressed nerve growth factor, vascular endothelial growth factor, C-X-C chemokine receptor type 4, Wnt5a, and collagen IV. More EdU + ADSCs retained in the CC in the MTs group than that in the ADSCs group. IC injection of MTs resulted in significant restoration of the erectile function and histopathological changes compared with the ADSCs group. CONCLUSION IC-injected MTs resulted in a better restoration of erectile function than traditional single-cell strategy. The underlying mechanisms of recovery appear to involve enhanced cellular retention in the penis and upregulation of some paracrine factors.

Icariin Ameliorates Streptozotocin-Induced Diabetic Retinopathy in Vitro and in Vivo

This study investigated the effect of Icariin (ICA) supplementation on diabetic retinopathy (DR) in a streptozotocin-induced diabetic rat model system. Fifty Sprague Dawley rats were randomly distributed into a control group and a streptozotocin-induced diabetes group. Diabetic rats were randomly divided into two groups; one group received ICA 5 mg/kg/day for 12 weeks by oral gavage; the other group received saline gavage as a placebo. Retinal morphological changes, endothelial markers (RECA), collagen IV (Col-IV), vascular endothelial growth factor (VEGF), and neuropathic changes (Thy-1 and Brn3a expression) of the retinal ganglion cells (RGCs) were investigated. The effects of ICA at various concentrations (0, 101, 102, 103 nmol/mL) on neurite growth were investigated also in retinal ganglion cells (RGC) cultured from both diabetic and normal animals. Numerous pathological changes (deceased expression of RECA, VEGF, Thy-1, and Brn3a as well as decreased Collagen IV and Müller cell content) were noted in the retinal vessels of diabetic rats; these changes were attenuated in diabetic animals that received ICA. ICA enhanced neurite growth in RGC from both normal rats and diabetic rats in a dose dependent fashion. ICA may be useful in the treatment of diabetic retinopathy. Further investigations are indicated.

AGE-Breaker ALT-711 Plus Insulin Could Restore Erectile Function in Streptozocin-Induced Type 1 Diabetic Rats

INTRODUCTION The interaction between advanced glycation end-products (AGEs) and its receptors for AGEs (RAGEs) elicits oxidative stress and mediates the development of erectile dysfunction (ED). ALT-711, an AGE cross-link breaker, has the therapeutic potential for ED but has been less intensively investigated. AIM The aim of this study was to investigate the effects of an AGEs breaker 3-phenacyl-4,5-dimethylthiazolium chloride (ALT-711) plus insulin on erectile function in streptozocin (STZ)-induced type 1 diabetic rats. METHODS Fifty 8-week-old Sprague-Dawley rats were randomly distributed into five groups: normal control (C), diabetic (D), insulin-treated diabetic (D + I), ALT-711-treated diabetic (D + ALT-711) and insulin plus ALT-711-treated diabetic (D + I + ALT-711) rats. Diabetes was induced by a single intraperitoneal injection of STZ. Eight weeks after induction of diabetes, ALT-711 was administered by intraperitoneal injection. Two to six units of intermediate-acting insulin were utilized to achieve normal levels of glycemic control. After treatment for 6 weeks, erectile function was determined via measurement of intracavernous pressures (ICPs) following electrostimulation of the cavernous nerve. The deposition of AGEs, expression of RAGEs, superoxide dismutase activity, and lipid peroxidation were measured. We also evaluated penile histological changes such as smooth muscle contents, endothelial cells contents, and apoptotic activity. MAIN OUTCOME MEASURES The main outcome measures were the ratio of ICP/mean arterial pressure (MAP), penile endothelial cells, smooth muscle cells, neuronal nitric oxide synthase, AGE and RAGE expression, malondialdehyde concentration, SOD activity, and apoptosis index. RESULTS Diabetic rats demonstrated significantly reduced ICP/MAP ratio, penile endothelial cells, smooth muscles cells, increased AGEs and RAGE expression, and increased apoptosis. Insulin and ALT-711 monotherapy partially restored erectile function and histological changes. However, the combination therapy group showed erectile parameters and components similar to those in C. ALT-711-treated group demonstrated less deposition of AGEs and lower expression of RAGE than those in insulin-treated group. CONCLUSION These results suggest that although insulin can effectively control glycemic levels, it does not completely alter the pathological changes in erectile tissues. Better efficacy could be expected with tight glycemic control plus ALT-711, an AGEs cross-link breaker. The combination therapy might have the potential to eliminate metabolic memory by down-regulating the AGEs-RAGE oxidative stress axis.

Studies on the mechanism of testicular dysfunction in the early stage of a streptozotocin induced diabetic rat model

Streptozotocin (STZ) induced diabetic model has been widely used to study the effects of diabetes mellitus (DM) on male infertility, but it remains unclear whether the responses in this model are due to hyperglycemia or STZ per se. This study was designed to investigate the mechanism of STZ on testicular dysfunction. In the present study, sperm characteristics, serum testosterone, steroidogenic enzymes (StAR and 3β-HSD), and the vimentin apical extension of sertoli cells decreased significantly in the STZ group compared with those in the normal controls (p<0.05), while Johnsens score, testicular lipid peroxidation, spermatogenic cell apoptosis, and the expressions of NF-κB and Wnt4 significantly increased (p<0.05). Insulin replacement mainly restored the decreased serum testosterone and steroidogenic enzymes, but not other parameters. The results indicated that spermatogenic dysfunction in the early stage of STZ-induced diabetic rats was due to direct STZ cytotoxicity to sertoli cells, which could be regulated by Wnt4 and NF-κB, while steroidogenic dysfunction might be a direct or indirect consequence of insulin deficiency. The results suggested that STZ-induced diabetic model, at least in the early stage, is not suitable to study the diabetes-related spermatogenic dysfunction.

Activation of VEGF and ERK1/2 and improvement of urethral function by adipose-derived stem cells in a rat stress urinary incontinence model.

OBJECTIVE To investigate the injected autologous adipose-derived stem cells (ADSCs) in improving stress urinary incontinence in a rodent model of parturition-related stress incontinence and the possible mechanism. METHODS The 40 rats were developed stress urinary incontinence models by postpartum balloon dilation of the vagina for 4 hours followed by bilateral ovariectomy. ADSCs were isolated from the peri-ovarian fat and labeled with thymidine analog 5-ethynyl-2-deoxyuridine (EdU). Twenty stress urinary incontinence rats received peri-urethral injection of phosphate-buffered saline as the negative controls and the other 20 stress urinary incontinence rats received peri-urethral injection of EdU-labeled ADSCc. Twenty control rats underwent sham ovariectomy without balloon dilation and served as positive controls. Four weeks later, voiding function was assessed by cystometry. Urethral histologic examination (Masson trichrome stain, picrosirius red stain, Hart elastin stain, Gordon and Sweet stain, and immunohistochemical stain) and Western blot were performed on urethral tissues. RESULTS Both leak point pressure and bladder capacity were significantly increased in ADSC-treated rats, compared to the balloon-injured ovariectomized rats. Histologic examination revealed normalized appearance of the fibromuscular structure of the urethra as well as increased peri-urethral blood vessel density in ADSC-treated rats. On Western blot, vascular endothelial growth factor and P-extracellular signal-regulated kinases (ERKs)1/2 protein was expressed at a higher rate in tissues from ADSC-treated rats compared to phosphate-buffered saline-treated rats. CONCLUSION Peri-urethral injection of ADSCs is associated with more normal urinary function and urethral structure in rats with parturition-related incontinence. The activation of vascular endothelial growth factor and ERK1/2 may be responsible for the paracrine effects from ADSCs.

Antioxidant icariside II combined with insulin restores erectile function in streptozotocin-induced type 1 diabetic rats

Erectile dysfunction (ED) worsens in patients with diabetes mellitus (DM) despite good control of blood glucose level with insulin. Recent studies imply that diabetic vascular stresses (e.g. oxidative stress) persist in spite of glucose normalization, which is defined as metabolic memory. Studies suggest that the interaction between advanced glycation end products (AGEs) and their receptor (RAGE) mediates the development of metabolic memory. To investigate the effects of the antioxidant icariside II plus insulin on erectile function in streptozotocin (STZ)‐ induced type 1 diabetic rats. Fifty 8‐week‐old Sprague‐Dawley rats were randomly distributed into five groups: normal control, diabetic, insulin‐treated diabetic, icariside II‐treated diabetic, and insulin plus icariside II‐treated diabetic. Diabetes was induced by a single intraperitoneal injection of STZ. Eight weeks after induction of diabetes, icariside II was administered by gastric lavage once a day (5 mg/kg) for 6 weeks; and 2–6 units of intermediate‐acting insulin were given to maintain normal glycemia for 6 weeks. The main outcome measures were the ratio of intracavernous pressure (ICP) to mean arterial pressure (MAP); histology of penile endothelial cells and smooth muscle cells; neural nitric oxide synthase, AGEs and RAGE expression; malondialdehyde concentration; superoxide dismutase activity; and apoptosis index. Diabetic rats demonstrated a significantly lower ICP/MAP ratio, reduced penile endothelial cells, reduced smooth muscle cells, increased AGEs and RAGE, and increased apoptosis. Insulin and icariside II monotherapy partially restored erectile function and histological changes. However, the combination therapy group showed significantly better erectile parameters, cytological components and biochemistry, similar to those in the normal control group. These results suggest that, although insulin can effectively control glycemic levels, it does not completely alter the pathological changes in erectile tissues. Better efficacy could be expected with tight glycemic control plus the antioxidant icariside II. The proposed combination therapy might have the potential to eliminate metabolic memory by down‐regulating the AGEs‐RAGE‐oxidative stress axis.

Icariside II ameliorates diabetic nephropathy in streptozotocin-induced diabetic rats.

Purpose To investigate the therapeutic effects and potential mechanisms of icariside II (ICA II) on reversing diabetic nephropathy in streptozotocin (STZ)-induced type I diabetic rats. Methods Newborn male Sprague Dawley rats were labeled with thymidine analog 5-ethynyl-2-deoxyuridine (EdU) for tracking endogenous label retaining progenitor cells (LRCs). At age of 8 weeks, 48 rats were randomly divided into three groups: normal control group (n=16), diabetes mellitus group (DM; n=16), and diabetes mellitus plus ICA II therapy group (DM+ICA II, n=16). Eight weeks induced for diabetes with STZ, rats in DM group and DM+ICA II group were treated with vehicle or ICA II (5 mg/kg/day) for another 8 weeks, respectively. Then, blood creatinine, 24-hour urine protein, blood urea nitrogen, and glycosylated hemoglobin were measured, as well as the expression of von Willebrand factor, malondialdehyde, transforming growth factor-β/drosophila mothers against decapentaplegic protein/connective tissue growth factor (TGF-β/Smad/CTGF) signaling, marker of proliferation Ki-67, and EdU+ LRCs in renal tissues. Results Increased levels of creatinine, 24-hour urine protein, and blood urea nitrogen and remarkably decreased proportion of normal glomeruli and increased proportions of I, IIa, IIb, and III glomeruli were observed in diabetic rats, while ICA II could reverse these changes. Interestingly, ICA II could significantly downregulate the levels of malondialdehyde and TGF-β/Smad/CTGF signaling and increase the expression of von Willebrand factor, Ki-67, and EdU+ LRCs in the kidney. Conclusion ICA II treatment could ameliorate diabetic nephropathy in STZ-induced diabetic rats by increasing endothelial cell contents, downregulating TGF-β/Smad/CTGF signaling pathway and oxidative stress level, and promoting cell proliferation both in kidney cortex and medulla. These beneficial effects appear to be mediated by its antioxidant capacity and recruitment of endogenous EdU+ progenitor cells into the kidney tissue.

Icariside II prevents high-glucose-induced injury on human cavernous endothelial cells through Akt-eNOS signaling pathway

Dysfunction of human cavernous endothelial cells (HCECs) is a common pathological alteration caused by elevated high blood glucose levels associated with diabetes. To explore the protective effects of Icariside II (ICA II) on human cavernous endothelial cells, HCECs were isolated from non‐diabetic human donors, cultured under high glucose (HG) conditions and treated with ICA II. The cell apoptosis and proliferation, expression of Ki67 and Erk1/2, antioxidant capacity, and expression of Akt and eNOS were examined. Changes in cell apoptosis and proliferation indicated that HG treatment inhibited HCEC proliferation with lower percentage of Ki67‐positive cells and lower expression and phosphorylation of Erk1/2. Furthermore, the total antioxidant capacity (T‐AOC) of HCECs was reduced under HG conditions. In line with these findings, both expression and phosphorylation of Akt as well as eNOS was down regulated after HG treatment. The reduction in proliferative capacity, p‐Erk1/2, p‐Akt, and p‐eNOS were partially prevented by ICA II in a concentration‐dependent manner. The protective effects of ICA II rescued HCEC from injury and dysfunction induced by HG in vitro. ICA II may be a candidate for prevention of the development of diabetic erectile dysfunction.

Prophylactic protective effects and its potential mechanisms of icarisideii on streptozotocin induced spermatogenic dysfunction

The aim of this study was to investigate the effects of IcarisideII(ICAII) on the prevention of streptozotocin (STZ) induced spermatogenic dysfunction. Forty male Sprague-Dawley rats received intraperitoneal injection of STZ (55 mg/kg) and were equally randomized to gavage feeding of vehicle (the vehicle group) or ICAII (0.5, 1.5 or 4.5 mg/kg/day, respectively). Ten normal rats received vehicle and served as control. Four weeks later, sperm parameters, histopathological changes, testicular lipid peroxidation, antioxidant enzyme activities, and apoptosis index (AI) were evaluated. Results showed that ICAII treatment resulted in a significant recovery of sperm parameters and histopathological changes relative to the vehicle group (p < 0.05). In the vehicle group, antioxidant enzyme activities and the expression of Sertoli cell Vimentin filaments obviously decreased, while lipid peroxidation and AI significantly increased as compared with the control group (p < 0.05). Following ICAII treatment, corrective effects on these items towards normal levels were observed. The results suggested that ICAII has beneficial effect on the preservation of spermatogenic function in the STZ-induced diabetic rats. The mechanisms might be related to its improvement of antioxidant enzyme activities, preservation of the protein expression and apical extensions of Vimentin filaments, and anti-apoptosis capability.