Inhibition of the Type III Secretion System of Salmonella enterica Serovar Typhimurium via Treatment with Fraxetin.

The increasingly serious problem of bacterial drug resistance has led to the development of antivirulence agents. The Salmonella enterica serovar Typhimurium Salmonella pathogenicity island (SPI)-encoded type III secretion system (T3SS) and its effector proteins are important virulence factors for S. Typhimurium invasion and replication in host cells and for antivirulence drug screening. Fraxetin is isolated from Fraxinus spp. Extensive studies have reported its multiple pharmacological activities. However, it remains to be elucidated whether fraxetin affects the function of the S. Typhimurium T3SS. In this study, the anti-infection mechanism of fraxetin on S. Typhimurium and its T3SS was investigated. Fraxetin inhibited the S. Typhimurium invasion of HeLa cells without affecting the growth of bacteria in vitro. Further findings on the mechanism showed that fraxetin had an inhibitory effect on the S. Typhimurium T3SS by inhibiting the transcription of the pathogenesis-related SPI-1 transcriptional activator genes hilD, hilC, and rtsA. Animal experiments showed that fraxetin treatment protected mice against S. Typhimurium infection. Collectively, we provide the first demonstration that fraxetin may serve as an effective T3SS inhibitor for the development of treatments for Salmonella infection. IMPORTANCE The increasingly serious problem of bacterial antibiotic resistance limits the clinical application of antibiotics, which increases the need for the development of antivirulence agents. The type III secretion system (T3SS) plays a critical role in host cell invasion and pathogenesis of Salmonella and becomes a popular target for antivirulence agents screening. Our study found, for the first time, that fraxetin inhibited S. Typhimurium invasion by inhibiting the transcription of genes in a feed-forward regulatory loop. Further in vivo testing showed that fraxetin decreased bacterial burdens in the spleen and liver of S. Typhimurium-infected mice and improved survival outcomes in an in vivo mouse model of S. Typhimurium infection. Collectively, these results demonstrate that fraxetin inhibits S. Typhimurium infection by targeting the T3SS and may serve as a potential agent for the treatment of S. Typhimurium infection.

Melatonin Alleviates Neuronal Damage After Intracerebral Hemorrhage in Hyperglycemic Rats.

BACKGROUND: This study sought to investigate a novel effect of melatonin in reducing brain injury in an in vivo hyperglycemic intracerebral hemorrhage (ICH) model and further explore the mechanisms of protection. METHODS: Hyperglycemia ICH was induced in Sprague-Dawley rats by streptozocin injection followed by autologous blood injection into the striatum. A combined approach including RNA-specific depletion, electron microscopy, magnetic resonance, Western blots, and immunohistological staining was applied to quantify the brain injuries after ICH. RESULTS: Hyperglycemia resulted in enlarged hematoma volume, deteriorated brain edema, and aggravated neuronal mitochondria damage 3 days after ICH. Post-treatment with melatonin 2 hours after ICH dose-dependently improved neurological behavioral performance lasting out to 14 days after ICH. This improved neurological function was associated with enhanced structural and functional integrity of mitochondria. Mechanistic studies revealed that melatonin alleviated mitochondria damage in neurons via activating the PPARδ/PGC-1α pathway. Promisingly, melatonin treatment delayed until 6 hours after ICH still reduced brain edema and improved neurological functions. Melatonin supplementation reduces neuronal damage after hyperglycemic ICH by alleviating mitochondria damage in a PPARδ/PGC-1α-dependent manner. CONCLUSION: Melatonin may represent a therapeutic strategy with a wide therapeutic window to reduce brain damage and improve long-term recovery after ICH.

Concentration-dependent effects of the soy phytoestrogen genistein on the proteome of cultured cardiomyocytes.

The soy-derived phytoestrogen genistein (GEN) has received attention for its potential benefits on the cardiovascular system by providing direct protection to cardiomyocytes against pathophysiological stresses. Here, we employed a proteomic approach to study the concentration-dependent effects of GEN treatments on cardiomyocytes. Cultured HL-1 cardiomyocytes were treated with low (1µM) and high (50µM) concentrations of GEN. Proteins were pre-fractionated by sequential hydrophilic/hydrophobic extraction and both protein fractions from each treatment group were separated by 2D gel electrophoresis (2DE). Overall, approximately 2,700 spots were visualized on the 2D gels. Thirty-nine and 99 spots changed in volume relative to controls (p<0.05) following the low- and high-concentration GEN treatments, respectively. From these spots, 25 and 62 protein species were identified by ESI-MS/MS and Mascot database searching, respectively. Identified proteins were further categorized according to their functions and possible links to cardioprotection were discussed. MetaCore gene ontology analysis suggested that 1µM GEN significantly impacted the anti-apoptosis process, and that both the low and high concentrations of GEN influenced the glucose catabolic process and regulation of ATPase activity. This proteomics study provides the first global insight into the molecular events triggered by GEN treatment in cardiomyocytes.

Triptolide promotes generation of FoxP3+ T regulatory cells in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Triptolide (TPT), a component of the Chinese herb Triptergium wilfordii, has potent immunosuppressive and anti-inflammatory activity and is used clinically in recipients of kidney transplantation. AIM OF THE STUDY: This work aimed to investigate the effect of TPT on the differentiation of regulatory T lymphocytes (Tregs) from CD4+ cells in rats. MATERIALS AND METHODS: MACS-purified rat CD4+ cells were costimulated with anti-CD3 and anti-CD28 in the presence of TGF-beta to induce the expression of FoxP3, which was detected by flow cytometry. TPT and cyclosporine A (CsA) were separately added into the cultures to observe the effect on the expression of FoxP3. Kidney transplantation was performed in rats that either received no treatment or were treated with TPT after transplantation. RESULTS: TPT treatment enhanced the expression of FoxP3 in CD4+ cells, whereas CsA inhibited the FoxP3 expression. In the rat kidney transplantation model, the recipient rats treated with TPT survived longer than the control rats (18-19.83 vs 6.83 days, P<0.05). Meanwhile, the FoxP3+ T cells in the spleens of treated rats were higher than those from the untreated rats (12.4% vs 4.7%, P<0.05). CONCLUSIONS: These data suggest that TPT may promote the differentiation of CD4+ cells to FoxP3+ Tregs. This would be at least one of the pathways responsible for the immunosuppressive activity of TPT.

Losartan, an Angiotensin type I receptor, restores erectile function by downregulation of cavernous renin-angiotensin system in streptozocin-induced diabetic rats.

INTRODUCTION: The high incidence of erectile dysfunction (ED) in diabetes highlights the need for good treatment strategies. Recent evidence indicates that blockade of the angiotensin type I receptor (AT1) may reverse ED from various diseases. AIM: To explore the role of cavernous renin-angiotensin system (RAS) in the pathogenesis of diabetic ED and the role of losartan in the treatment of diabetic ED. METHODS: The AT1 blocker (ARB) losartan (30 mg/kg/d) was administered to rats with streptozocin (65 mg/kg)-induced diabetes. Erectile function, cavernous structure, and tissue gene and protein expression of RAS in the corpora cavernosa were studied. MAIN OUTCOME MEASURE: We sought to determine the changes of cavernous RAS in the condition of diabetes and after treatment with losartan. RESULTS: RAS components (angiotensinogen, [pro]renin receptor, angiotensin-converting enzyme [ACE], and AT1) were expressed in cavernosal tissue. In diabetic rats, RAS components were upregulated, resulting in the increased concentration of angiotensin II (Ang II) in the corpora. A positive feedback loop for Ang II formation in cavernosum was also identified, which could contribute to overactivity of cavernous RAS in diabetic rats. Administration of losartan blocked the effect of Ang II, downregulated the expression of AT1 and Ang II generated locally, and partially restored erectile function (losartan-treated group revealed an improved intracavernous pressure/mean systemic arterial pressure ratio as compared with the diabetic group (0.480 +/- 0.031 vs. 0.329 +/- 0.020, P < 0.01). However, losartan could not elevate the reduced smooth muscle/collagen ratio in diabetic rats. CONCLUSIONS: The cavernous RAS plays a role in modulating erectile function in corpora cavernosa and is involved in the pathogenesis of diabetic ED. ARB can restore diabetic ED through downregulating cavernous RAS.

Differential expression of neurotrophins in penises of streptozotocin-induced diabetic rats.

To explore the mechanism of diabetic erectile dysfunction, we studied the distribution of neurotrophins in the penises of diabetic rats, including nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4). Male Sprague-Dawley rats were injected with 65 mg/kg streptozotocin to induce diabetes mellitus (DM). The control rats were raised as age-matched control. Eight weeks later, the intercavernous pressure (ICP) of the rats was measured after electrostimulation and before sacrifice. Each peeled penis was divided into 2 parts, one for immunohistochemistry and the other for Western blot analysis. The ICP of the DM group rats was significantly decreased as compared to the vehicle control rats. There were significantly more NGF-positive neurons in the penises of the diabetic rats than in those of the control rats, while the opposite results were observed for BDNF-positive neurons. In the Western blot analysis, the proteins of NGF, NT-3, and NT-4 were all increased, while that of BDNF was decreased in diabetic rats. This is the first study revealing the expression of NT-4 protein in cavernous tissue. The abnormal level of these 4 neurotrophins in cavernous tissue may be one of the factors of the pathogenesis of diabetic ED. The increase of neurotrophins may reflect the degree of cavernous tissue denervation and may represent a compensatory mechanism. The lesion of the retrograde axonal transport of the nerves caused by hyperglycemia may be related to this phenomenon.

Effects of coffee and caffeine on bladder dysfunction in streptozotocin-induced diabetic rats.

AIM: To explore the effects and mechanisms of caffeine and coffee on bladder dysfunction in streptozotocin-induced diabetic rats. METHODS: Sprague-Dawley male rats were divided randomly into 4 groups: control, diabetes mellitus (DM), DM with coffee treatment, and DM with caffeine treatment. The diabetic rat was induced by intraperitoneal injection of streptozotocin (60 mg/kg). After 7 weeks of treatment with coffee and caffeine, cystometrogram, contractile responses to electrical field stimulation (EFS) and acetylcholine (ACh), and cyclic AMP (cAMP) concentration of the bladder body and base were measured. RESULTS: The bladder weight, volume threshold for micturition and post-void residual volume (PVR) in the diabetic rats were significantly higher compared to those in the control animals. Coffee or caffeine treatment significantly reduced the bladder weight, bladder capacity and PVR in the diabetic rats. DM caused significant decreases in cAMP concentration of the bladder and coffee and caffeine caused upregulation of cAMP content in the diabetic bladder. In addition, coffee and caffeine tended to normalize the altered detrusor contractile responses to EFS and ACh in the diabetic rats. CONCLUSION: These results indicate that caffeine and coffee may have beneficial effects on bladder dysfunction in the early stage of diabetes by increasing cAMP content in the lower urinary tract, recovering the micturition reflex and improving the detrusor contractility.

[Study on prevention of catheter associated urinary tract infection by using JUS long-acting antibacterial material].

OBJECTIVE: To observe the effect of reducing the incidence of CAUTI by spraying the long-acting antibacterial material JUS on the surface of catheter and urethral orifice. METHODS: Sixty male patients, aged from 68 to 79, with indwelling catheter after TURP were divided randomly into two groups (control group and treated group), each consisting of 30 patients. For the control group, their urethral orifice was treated conventionally twice a day; while for the treated group, in addition to the conventional treatment of their urethral orifice, the catheter and their urethral orifice were sprayed with the long-acting antibacterial material JUS twice a day. RESULT: The number of cases of urinary tract infection in the treated group during catheterization was evidently less than those of the control group (P < 0.01), so the difference was of remarkable significance. CONCLUSION: The long-acting antibacterial material, after spraying on the wall of catheter and urethral orifice of the patients with indwelling catheter, may form a layer of physically antibacterial molecular film to prevent the formation of a bacterial biological film and effectively reduce the incidence of CAUTI.

[Omission factors in detecting early stage prostate cancer by TRUS needle biopsy].

OBJECTIVE: To analyze and reduce the omission factors in detecting early stage prostate cancer by TRUS needle biopsy, and to improve the diagnosis of the disease. METHODS: A total of 80 benign prostatic hyperplasia patients suspected of prostatic carcinoma underwent TRUS sextant biopsies. The pathological results being negative, the patients received transurethral resection of the prostate (TURP). RESULTS: After TURP, 25 cases were pathologically diagnosed as prostate cancer, with an omission rate of 31.25% (25/80). Of the diagnosed cancer patients, 10 were treated by radical perineum prostatectomy, 8 by surgical castration, and 7 by medical castration. CONCLUSION: Some tumors may fail to be detected by TRUS needle biopsy. Serial or multi-core needle biopsies can decrease the omission rate in the diagnosis of organ confined cancer.

[Effect of FK506 on the cavernous nerve regeneration after injury].

OBJECTIVE: To investigate the effect of FK506 on the cavernous nerve regeneration after injury and to discuss its possible action mechanisms. METHODS: Fifty-four male adult Sprague-Dawley rats were randomized into three groups: Group 1 (sham control), Group 2 (unilateral cavernous nerve ablation), and Group 3 (unilateral cavernous nerve ablation with subsequent injection of FK506). Electrostimulation of the cavernous nerve was performed at 1 and 3 months after surgical injury. The intracavernous pressure was continuously detected and the rats were followed by nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) staining to identify NOS in the penile nerve fibers of the penile shaft. RESULTS: At 1 month, the number of NOS-positive nerve fibers significantly decreased with no statistical difference among the three groups except the sham controls (P > 0.05). At 3 months, electrostimulation revealed greater maximal intracavernous pressure in Group 3 than in Group 2 (P < 0.01). Furthermore, the number of NOS-positive nerve fibers showed a significant increase (P < 0.01), but not in Group 2 (P > 0.05). CONCLUSION: FK506 injection enhances the regeneration of cavernous nerves after injury and the recovery of erectile function in rats.

[Progress of erectile in the treatment dysfunction following peripheral nerve injury].

Trauma and surgery sometimes cause erectile dysfunction because of injury to pelvic nerves and cavernous nerves of the penis. The ideal treatment is to make the injured nerves regenerate themselves and erectile function recover completely. This paper reviews the recent advances in the studies of erigentes regeneration and plerosis following injury in order to find the best method to enhance erigentes regeneration.