BACKGROUND:Adipose-derived stem cells have been considered as a promising therapy for erectile dysfunction. However, the therapeutic efficacy of adipose-derived stem cell-based therapy requires improvement.OBJECTIVE:To determine whether the inhibition of phosphodiesterase type 5 in adipose-derived stem cells would improve stem cell therapy for rats with diabetes-induced erectile dysfunction.MATERIALS AND METHODS:A phosphodiesterase type 5 siRNA was incorporated into lentiviral vectors and transduced into adipose-derived stem cells. The mRNA and protein levels of phosphodiesterase type 5 were evaluated. Three days after transduction, the adipose-derived stem cell supernatant was collected to determinate the levels of insulin-like growth factor 1 and vascular endothelial growth factor. Streptozotocin-induced diabetic rat models were established and used for comparative analysis of 1- and 2-week treatment regimens with intracavernosal injection of adipose-derived stem cells or Lv-siPDE5-modified adipose-derived stem cells.RESULTS:Lv-siPDE5-ADSCs secreted more insulin-like growth factor 1 and vascular endothelial growth factor in supernatants than unmodified adipose-derived stem cells. Preconditioned Adipose-derived stem cells-treated diabetic rats showed consistently superior erectile function when compared with non-preconditioned adipose-derived stem cells after 2 weeks of treatment. Lv-siPDE5-ADSCs provided additional benefits in recovery of cavernous structures with rapid effects (1 week) when compared to plain adipose-derived stem cells. These features were associated with the significantly increased levels of insulin-like growth factor 1 and vascular endothelial growth factor in Lv-siPDE5-ADSC-treated diabetic rats.CONCLUSIONS:Adipose-derived stem cell therapy could serve as an alternate approach for diabetes-induced erectile dysfunction, albeit with a long onset period. In vitro preconditioning of adipose-derived stem cells could accelerate the functional and structural recovery in vivo, indicating that preconditioning by inhibition of phosphodiesterase type 5 may improve adipose-derived stem cells therapy following diabetes-induced erectile dysfunction.

To investigate the effects of melatonin on rats with hyperhomocysteinemia (Hhcy)-related erectile dysfunction (HED), and the potential mechanisms via modulating oxidative stress and apoptosis. The rat model of hyperhomocysteinemia was induced by methionine-rich diet (7% methionine). Twelve rats fed a standard diet were regarded as a control group. Twenty-four rats were fed a methionine-rich diet. After a month, the rats with Hhcy were administered daily melatonin (n=12) in a dose of 10mg/kg or vehicle (n=12) intraperitoneally for 4 weeks. Rats as control group (n=12) were injected vehicle intraperitoneally for 4 weeks. Then, cavernous nerve electrostimulation was used to evaluate the erectile function of all rats. Total homocysteine (Hcy) levels in plasma were determined with ELISA. Fluorescent probe, ELISA and western blotting were performed to determine the levels of oxidative stress and related signaling pathway. TUNEL assay, RT-PCR and western blotting were performed to detect apoptosis. Compared with the control group and the melatonin-treated group, the HED group showed (1) lower erectile function: lower intracavernosal pressure (ICP)/mean arterial pressure(MAP) ratio and lower total ICP (area under curve, AUC); (2) higher levels of total Hcy in plasma (3) higher levels of ROS and MDA, but lower levels of SOD activity; (4)higher expression levels of Erk1/2/Nrf2/HO1 signaling pathway than the control group, but lower levels than melatonin-treated group; (5) higher levels of apoptosis proved by the expression levels of Bax, Bcl-2, caspase 3 and apoptosis index.

Previously, we have demonstrated that human tissue kallikrein 1 (hKLK1) improves age-related erectile dysfunction (ED). Autophagy has been implicated in age-related diseases, including ED. However, the molecular mechanisms underlying hKLK1-mediated amelioration of age-related ED via regulation of autophagy remains unknown. To explore the potential mechanism, male wild-type Sprague-Dawley rats (WTR) and transgenic rats harboring human KLK1 (TGR) were bred till 4 or 18 months of age and divided into three groups: young WTR (yWTR) as the control group, aged WTR (aWTR) group, and aged TGR (aTGR) group. The erectile function of each rat was evaluated using cavernous nerve electrostimulation. The ratio of intracavernous pressure/mean arterial pressure (ICP/MAP) and total ICP were also measured. Western blotting, immunohistochemistry, and transmission electron microscopy were performed to detect the levels of autophagy. The expression levels of related signaling pathways were determined by western blotting and immunohistochemistry. We found that hKLK1 improved the impaired erectile function of aged rats. Compared to the yWTR and aTGR groups, the aWTR group showed reduced smooth muscle/collagen ratio, fewer autophagosomes, and lower expression of Beclin 1 and LC3-II, which indicate impaired smooth muscle function and low level of autophagy in the smooth muscle cells. Moreover, the PI3K/Akt/mTOR signaling pathway, which is considered to be a negative regulator of autophagy, was upregulated in the aWTR group. hKLK1 may partially restore erectile function in aged transgenic rats by upregulating protective autophagy via the PI3K/Akt/mTOR pathway. These observations indicate that hKLK1 is a potential gene therapy candidate for age-related ED.

Considering 35%–90% of patients with diabetes mellitus (DM) suffer erectile dysfunction (ED), which starts about 10–15 years earlier than in the population without DM. Moreover, these patients showed a poor response to the first-line oral phosphodiesterase type 5 inhibitors. Thus, a novel treatment method is urgently needed. We tried to demonstrate whether melatonin could improve erectile function in diabetic rats and the underlying mechanisms. Type 1 DM was induced by using streptozotocin. 8 weeks later, we conducted an apomorphine test to confirm diabetes mellitus erectile dysfunction (DMED). Only rats with DMED (n=20) were administered melatonin (n=10) in a dose of 10mg/kg or vehicle (n=10) intraperitoneally each day for 4 weeks, rats as control group (n=10) were fed in the same condition for 4 weeks. Then, cavernous nerve electrostimulation was used to evaluate the erectile function of all rats. Histologic and molecular alterations of the corpus cavernosum also were analyzed. Furthermore, corpus cavernosum smooth muscle cells of rats were cultured under different conditions: normal glucose (NG), high glucose (HG), high glucose + melatonin (HG+M), high glucose + melatonin + melatonin receptor antagonist luzindole(HG+M+L), and related molecular biological parameter were detected.

SummaryAdipose‐derived stem cells (ADSCs) have recently been considered as a promising therapy for erectile dysfunction (ED). However, the mechanism of ADSC‐based therapy is unclear. Insulin‐like growth factor‐1 (IGF‐1), basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF) secreted by ADSCs were assessed in vitro. Sixteen 24‐month‐old male Sprague–Dawley rats were used for comparative analysis of 2‐week treatment with labeled ADSCs or PBS. Eight additional 5‐month‐old rats were used as a young rat group. At 2 weeks post‐transplantation, all rats were analyzed for erectile function, cavernous IGF‐1, bFGF and VEGF levels, and penile histology. Conditioned medium and co‐culture systems were used in cell experiments to detect how growth factors act on corpus cavernosum smooth muscle cells (CCSMCs) under oxidative stress conditions via crystal violet staining and immunofluorescence staining. We found that ADSCs secreted significantly higher IGF‐1, bFGF, and VEGF levels in culture medium compared with basal medium. Compared with young rats, untreated aged rats had significantly lower Max ICP/MAP and ADSC treatment significantly increased the ratio. Immunofluorescence staining demonstrated a small number of labeled ADSCs in the corpus cavernosum. The untreated aged rats showed significantly decreased cavernous IGF‐1, bFGF, and VEGF levels and significantly decreased contents of cavernous smooth muscle and endothelium compared with young rats. ADSC treatment partially normalized these alterations. In cell experiments, the groups receiving growth factor neutralizing antibody separately or combined had significantly decreased numbers of CCSMCs compared with control groups. These results indicated that ADSC treatment may improve aging‐related ED partially through the secretion of IGF‐1, bFGF, and VEGF.

The aim of this study was to investigate the underlying mechanism of androgen deficiency inducing corporal fibrosis, thereby causing erectile dysfunction (ED). Forty 12-week-old healthy male rats were divided randomly into four groups: normal control group (Control); castration group (Castration); the other 20 rats were castrated followed by testosterone (T) (orally) each day: castration + 10mg/kg T group (Castration + 10T) and castration + 20 mg/kg T group (Castration + 20T). After 8 weeks' treatment, the main outcome measures were the following: serum levels of T; the ratios of intracavernous pressure (ICP) to mean arterial pressure (MAP); histologic changes in penile smooth muscle cells; the Smad and non-Smad pathways; and extracellular matrix (ECM) protein deposition. Castration group showed lower level of T and ratio of ICP/MAP, reduced ratio of penile smooth muscle cells/collagen, increased extracellular matrix protein deposition, and a higher expression of the Smad and non-Smad pathways. Castration + 10T partially preserved erectile function and histology stabilisation. However, the Castration + 20T group showed significantly better erectile function and molecular changes. Better efficacy could be expected with ART of adequate dose. Androgen deficiency induces corporal fibrosis through activation of the Smad and non-Smad pathways, and accumulation of ECM proteins.

This prospective randomized clinical study is aimed to evidence the reproductive impairment of frequent scrotal heat exposure. A total of 20 normozoospermic subjects were randomly divided into two groups to undergo testicular warming in a 43 °C water bath 10 times, for 30 min each time; the subjects in group 1 underwent testicular warming for 10 consecutive days and those in group 2 once every 3 days. Sperm chromatin structure assay (SCSA), sperm mitochondrial membrane potential (MMP), apoptosis, and seminal plasma-soluble Fas (sFas) were analyzed before treatment and every 2 weeks after, for a total of 10 times. In group 1, some critical proteins involved in heat stress, hypoxia, structure, and function of sperm mitochondria and flagella were evaluated before hyperthermia and 2, 6, 10, and 16 weeks after hyperthermia. Both groups showed a reversible increase in the proportion of spermatozoa with a disrupted MMP (both p < 0.05 when the minimums were compared with baseline levels, the same below), sperm apoptosis (both p < 0.01) and high DNA stainability (both p < 0.05). The sFas concentration in both groups showed no obvious changes except one: the value at week 2 was significantly increased over baseline in group 1 (p = 0.036). The level of Bcl-2 decreased significantly at weeks 6 and 10 (p = 0.017 and 0.05, respectively) and recovered to baseline at week 16. Proteins involved in heat stress and mitochondria functions were up-regulated, whereas in flagella structure and function was down-regulated (all p < 0.05). This study demonstrated that transient and frequent scrotal hyperthermia severely and reversibly damaged spermatogenesis, consecutive heat exposure had more serious effects than intermittent exposure, whereas intermittent exposure led to a later recovery of sperm damage.

Inflammatory bowel disease (IBD) arises when intestinal immune homeostasis is broken, the maintenance of such homeostasis is principally controlled by cross talk between commensal bacteria, mucosal immune cells and intestinal epithelial cells (IECs). IECs can prevent the contact between luminal bacteria with immune cells through the formation of a physical barrier and the expression of antimicrobial peptides to maintain intestinal immune homeostasis. During Colitis the IECs can express increased ANXA1, which is important for regeneration of intestinal mucosa and function as a potent anti-inflammatory protein. Natural Killer (NK) cells can also suppress the progression of colitis. It is uncertain about the effect of the cross-talk between injured IECs and recruited NK cells during colitis. In this study, the expression of ANXA1 in IECS from DSS treated mice was increased, and more NK cells were recruited to intestinal mucosa. In addition, the expression of NKG2A was upregulated when co-cultured with NK cells. The results further proved that overexpression of NKG2A in NK cells was important for inhibiting the recruitment and activity of neutrophils to alleviate DSS-induced colitis. Here, we provide a new anti-inflammation mechanism about ANXA1 secreted from injured IECs, where ANXA1 can stimulate the expression of NKG2A in NK cells that affect the recruitment and activity of neutrophils necessary for pathology of colitis.

SummaryBackgroundAllergic asthma is characterized by inflammation and airway remodelling. Airway remodelling with excessive deposition of extracellular matrix (ECM) and larger smooth muscle mass are correlated with increased airway responsiveness and asthma severity. Calpain is a family of calcium‐dependent endopeptidases, which plays an important role in ECM remodelling. However, the role of calpain in airway smooth muscle remodelling remains unknown.ObjectiveTo investigate the role of calpain in asthmatic airway remodelling as well as the underlying mechanism.MethodsThe mouse asthma model was made by ovalbumin sensitization and challenge. Calpain conditional knockout mice were studied in the model. Airway smooth muscle cells (ASMCs) were isolated from smooth muscle bundles in airway of rats. Cytokines IL‐4, IL‐5, TNF‐α, and TGF‐β1, and serum from patients with asthma were selected to treated ASMCs. Collagen‐I synthesis, cell proliferation, and phosphorylation of Akt in ASMCs were analysed.ResultsInhibition of calpain using calpain knockout mice attenuated airway smooth muscle remodelling in mouse asthma models. Cytokines IL‐4, IL‐5, TNF‐α, and TGF‐β1, and serum from patients with asthma increased collagen‐I synthesis, cell proliferation, and phosphorylation of Akt in ASMCs, which were blocked by the calpain inhibitor MDL28170. Moreover, MDL28170 reduced cytokine‐induced increases in Rictor protein, which is the most important component of mammalian target of rapamycin complex 2 (mTORC2). Blockage of the mTORC2 signal pathway prevented cytokine‐induced phosphorylation of Akt, collagen‐I synthesis, and cell proliferation of ASMCs and attenuated airway smooth muscle remodelling in mouse asthma models.Conclusions and Clinical RelevanceOur results indicate that calpain mediates cytokine‐induced collagen‐I synthesis and proliferation of ASMCs via the mTORC2/Akt signalling pathway, thereby regulating airway smooth muscle remodelling in asthma.

The study investigated the effects of adipose tissue-derived stem cells (ADSCs) modified with penile neuronal nitric oxide synthase (PnNOS) gene on intracellular calcium concentration in rat corpus cavernosum smooth muscle cells (CCSMCs). ADSCs and CCSMCs of Sprague-Dawley (SD) rats were isolated and cultured in vitro respectively. The rat PnNOS gene was transferred into the ADSCs mediated by a recombinant adenovirus vector. The expression of the PnNOS gene was detected. At the same time, the concentration of nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) was assayed. After coculturing with the CCSMCs of SD rats, which were isolated and expanded ex vivo, the cGMP and NO levels of ADSCs and CCSMCs were measured. Intracellular calcium concentration ([Ca(2+) ]i ) in rat CCSMCs was measured with Fluo-3/AM by flow cytometer after cocultured with ADSCs overexpressing PnNOS gene. The mRNA and protein expression of PnNOS gene mediated by recombinant adenovirus vector significantly overexpressed and lasted at least 2 weeks. Meanwhile, the concentration of NO and cGMP in ADSCs was greatly increased. The concentration of cGMP was significantly increased, and [Ca(2+) ]i was obviously decreased in CCSMCs compared with the control groups (P < 0.05) after cocultured with ADSCs for 3 days. These findings demonstrated that ADSCs overexpressing PnNOS gene might decrease [Ca(2+) ]i in CCSMCs by up-regulating NO-cGMP signalling pathway.

It has been acknowledged that oxidative stress, resulting in the apoptosis of dopaminergic neurons, is a key mechanism in the pathogenesis of Parkinson's disease (PD). Puerarin, extracted from the root of pueraria lobata, has been clinically used for ischemic heart disease and cerebrovascular diseases as an oxygen free radical scavenger. In this study, we aimed to explore the effect of puerarin on dopaminergic cell degeneration in vitro and in vivo and its possible underlying mechanisms. In SH-SY5Y cells, the reduction of cell viability, apoptosis rate and average DCFH-DA fluorescence intensity of puerarin-treated (0, 10, 50, 100 and 150 μM) cells were significantly lower than control group. In rotenone-based rodent models, puerarin treatment for 7 days ameliorated apomorphine-induced rotations significantly in Pue-50 and Pue-100 group by 45.65% and 53.06% in the first week, by 44.60% and 48.45% in the second week. Moreover, compared to control group, puerarin increased tyrosine hydroxylase (TH) expression in the substantia nigra by 85.52% and 84.26% in Pue-50 group and Pue-100 group, and upregulated the vesicular monoamine transporter 2 (VMAT2) by 41.24% in Pue-50 group and 35.20% in Pue-100 group, and decreased ubiquitin expression by 47.55% in Pue-50 group and 69.15% in Pue-100 group. These data indicated that puerarin alleviated the oxidative stress and apoptosis in a PD cellular model, protected the dopaminergic neurons against rotenone toxicity and decreased the abnormal protein overexpressing in PD animal models. These findings suggest that puerarin may develop into a neuroprotective alternative for patients with PD.

Diabetic erectile dysfunction (ED) is a common complication in diabetes mellitus, and the efficacy of first-line therapies is not satisfactory. Recent studies revealed that corporal apoptosis was responsible for the nonresponsiveness of severe ED to phosphodiesterase type 5 inhibitors. Mitofusin 2 (Mfn2) is a versatile protein, regulating mitochondrial morphology and playing an important role in apoptosis. Several studies showed that expression of Mfn2 was decreased in STZ-induced diabetic rats' kidney, myocardium and retina, which was associated with diabetic nephropathy, cardiomyopathy and retinopathy respectively. In this study, our aim was to explore the expression of Mfn2 and apoptosis in diabetic rats' penes. We found that erectile function (ICP/MAP) elicited by electrical stimulation of cavernous nerve was markedly impaired in diabetic rats compared with the normal rats. The mRNA and protein levels of Mfn2 were found to be significantly reduced in diabetic rats' penile tissues. Compared with normal rats, the content of smooth muscle and B-cell lymphoma 2 (Bcl-2)/Bcl-2-associated X protein (Bax) ratio were dramatically decreased, and penile apoptotic index and expression of activated-caspase-3 were dramatically increased in diabetic rats. This data indicated that repression of Mfn2 in diabetic rats' penes might be associated with excessive apoptosis in diabetes-induced severe ED.

Benzo(a)pyrene (BaP), a ubiquitous environmental pollutant known to cause many diseases including atherosclerosis, induces a dose-dependent reduction in the levels of the inducible Hsp70. To explore the mechanism underlying the reduction of Hsp70, we measured the levels of Hsp70, cytoplasmic and nuclear heat shock factor 1 (HSF1) in porcine aortic endothelial cells using Western blot, and then further characterized the binding ability of HSF1 and heat shock element (HSE) by electrophoretic mobility shift assay. We found that when porcine aortic endothelial cells were treated by 0.1–10 μM of BaP for 24 h, there was a significant reduction of Hsp70, cytoplasmic and nuclear HSF1 and the binding rate of HSF1 and HSE at 5, 10 μM of BaP but less effective at lower concentrations. The effect of BaP on the Hsp70 expression level was markedly attenuated by co-treatment with phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC). Staurosporine (STP), an inhibitor of PKC, blocked the effect of PMA treatment in combination with BaP. These results suggest that BaP might inhibit Hsp70 levels by reducing the expression of HSF1 and decreasing binding of HSF1 and HSE via PKC-dependent signaling pathways that might be involved in the regulation of Hsp70 gene expression under BaP.