Alternate-day fasting ameliorated anxiety-like behavior in high-fat diet-induced obese mice.

Alternate-day fasting (ADF) has been reported to reduce body weight, neuroinflammation, and oxidative stress damage. However, it is not known whether ADF affects obesity-induced anxiety-like behavior. Here, male C57BL/6 mice were given an alternate fasting and high-fat diet (HFD) or standard chow diet (SD) every other day for 16 or 5 weeks. After the intervention, the degree of anxiety of the mice was evaluated by the open field test (OFT) and the elevated plus maze (EPM) test. Pathological changes in the hippocampus, the expression of Sirt1 and its downstream protein monoamine oxidase A (MAO-A) in the hippocampus, and the expression of 5-hydroxytryptamine (5-HT) were detected. Compared with HFD-fed mice, HFD-fed mice subjected to ADF for 16 weeks had a lower body weight but more brown adipose tissue (BAT), less anxiety behavior, and less pathological damage in the hippocampus, and lower expression of Sirt1 and MAO-A protein and higher 5-HT levels in the hippocampus could be observed. In addition, we noted that long-term ADF intervention could cause anxiety-like behavior in SD mice. Next, we changed the intervention time to 5 weeks. The results showed that short-term ADF intervention could reduce the body weight and increase the BAT mass of SD mice, but it did not affect anxiety. These results indicated that long-term ADF ameliorated obesity-induced anxiety-like behavior and hippocampal damage, but caused anxiety in normal-weight mice. Short-term ADF did not produce adverse emotional reactions in normal-weight mice. Here, we might provide new ideas for the treatment of obesity-induced anxiety.

Calcitriol Suppressed Isoproterenol-induced Proliferation of Cardiac Fibroblasts via Integrin ß3/FAK/Akt Pathway.

OBJECTIVE: Cardiac fibroblasts (CFs) proliferation and extracellular matrix deposition are important features of cardiac fibrosis. Various studies have indicated that vitamin D displays an anti-fibrotic property in chronic heart diseases. This study explored the role of vitamin D in the growth of CFs via an integrin signaling pathway. METHODS: MTT and 5-ethynyl-2'-deoxyuridine assays were performed to determine cell viability. Western blotting was performed to detect the expression of proliferating cell nuclear antigen (PCNA) and integrin signaling pathway. The fibronectin was observed by ELISA. Immunohistochemical staining was employed to evaluate the expression of integrin ß3. RESULTS: The PCNA expression in the CFs was enhanced after isoproterenol (ISO) stimulation accompanied by an elevated expression of integrin beta-3 (ß3). The blockade of the integrin ß3 with a specific integrin ß3 antibody reduced the PCNA expression induced by the ISO. Decreasing the integrin ß3 by siRNA reduced the ISO-triggered phosphorylation of FAK and Akt. Both the FAK inhibitor and Akt inhibitor suppressed the PCNA expression induced by the ISO in the CFs. Calcitriol (CAL), an active form of vitamin D, attenuated the ISO-induced CFs proliferation by downregulating the integrin ß3 expression, and phosphorylation of FAK and Akt. Moreover, CAL reduced the increased levels of fibronectin and hydroxyproline in the CFs culture medium triggered by the ISO. The administration of calcitriol decreased the integrin ß3 expression in the ISO-induced myocardial injury model. CONCLUSION: These findings revealed a novel role for CAL in suppressing the CFs growth by the downregulation of the integrin ß3/FAK/Akt pathway.

Botulinum Toxin A Alleviates Persistent Erythema and Flushing in Patients with Erythema Telangiectasia Rosacea.

INTRODUCTION: The persistent erythema and flushing seen in some cases of rosacea do not respond effectively to, or may easily relapse after, oral medication or light-based therapies (laser or intense pulsed light). Intradermal botulinum toxin A (BTX-A) injection can be used to treat intractable erythema and flushing, but studies with large samples and long-term observation have not been conducted to determine its effectiveness and safety. The aim of this study is thus to investigate the effective duration and safety of intradermal BTX-A injection for intractable erythema and flushing. METHODS: Sixteen patients with rosacea with erythema telangiectasia were injected with BTX-A at 1-cm intervals between each point. Clinician Erythema Assessment (CEA) scores were obtained at baseline and 1 month after injection. Flushing assessment and survey using the Dermatological Quality of Life Index (DLQI) questionnaire were conducted at baseline and at 1, 3, and 6 months after injection. RESULTS: At 1 month after injection, CEA scores revealed significant improvements in erythema and flushing; the results of the questionnaire on flushing and DLQI indicated that the improvement of flushing usually lasted for 3-6 months, but the effect decreased significantly at 6 months, and individual patients needed another treatment. CONCLUSIONS: BTX-A significantly improves the symptoms and quality of life of patients with refractory rosacea with few adverse effects.

Low-dose alcohol ameliorated high fat diet-induced anxiety-related behavior via enhancing adiponectin expression and activating the Nrf2 pathway.

Long-term high-fat-diet (HFD)-induced obesity is associated with many comorbidities, such as cognitive impairment and anxiety, which are increasing public health burdens that have gained prevalence in adolescents. Although low-dose alcohol could attenuate the risk of cardiovascular disease, its mechanism on HFD-induced anxiety-related behavior remains not clear. The mice were divided into 4 groups, Control (Con), Alcohol (Alc), HFD and HFD + Alc groups. To verify the effects of low-dose alcohol on HFD-induced anxiety-related behavior, the mice were fed with HFD for 16 weeks. At the beginning of week 13, the HFD-fed mice were administered intragastrically with low-dose alcohol (0.8 g kg-1) for 4 weeks. After 4 weeks of oral administration, low-dose alcohol decreased body weight and Lee's index in HFD-induced obese mice. Moreover, low-dose alcohol alleviated the anxiety-related behaviors of obese mice in the open field test and the elevated plus maze test. The HFD-induced damage to the hippocampus was improved in hematoxylin-eosin staining assay in mice. In addition, low-dose alcohol also suppressed HFD-induced oxidative stress and increased HFD-suppressed adiponectin (APN) expression and nuclear factor erythroid 2-related factor 2 (Nrf2) activation in the hippocampus. Taken together, low-dose alcohol significantly ameliorates HFD-induced obesity, oxidative stress and anxiety-related behavior in mice, which might be related to APN upregulation, Nrf2 activation and related antioxidase expression including SOD1, HO-1, and catalase.

Progranulin protects against cerebral ischemia-reperfusion (I/R) injury by inhibiting necroptosis and oxidative stress.

Ischemic stroke is a leading cause of mortality and disability worldwide. Nevertheless, its molecular mechanisms have not yet been adequately illustrated. Progranulin (PGRN) is a secreted glycoprotein with pleiotropic functions. In the present study, we found that PGRN expression was markedly reduced in mice after stroke onset through middle cerebral artery occlusion (MCAO). We also showed that necroptosis was a mechanism underlying cerebral I/R injury. Importantly, PGRN knockdown in vivo significantly promoted the infarction volume and neurological deficits scores in mice after MCAO surgery. Necroptosis induced by MCAO was further accelerated by PGRN knockdown, as evidenced by the promoted expression of phosphorylated receptor-interacting protein (RIP) 1 kinase (RIPK1), RIPK3 and mixed lineage kinase domain-like (MLKL), which was accompanied with increased expression of cleaved Caspase-8 and Caspase-3. However, PGRN over-expression was neuroprotective. Additionally, PGRN-regulated ischemic stroke was related to ROS accumulation that MCAO-mice with PGRN knockdown exhibited severe oxidative stress, as proved by the aggravated malondialdehyde (MDA) and lipid peroxidation (LPO) contents, and the decreased superoxide dismutase (SOD) activity. However, PGRN over-expression in mice with cerebral ischemia showed anti-oxidative effects. Finally, PGRN was found to attenuate oxidative damage partly via its regulatory effects on necroptosis. Therefore, promoting PGRN expression could reduced cerebral I/R-induced brain injury by suppressing neroptosis and associated reactive oxygen species (ROS) production. These data elucidated that PGRN might provide an effective therapeutic treatment for ischemic stroke.

Diminished serum repetin levels in patients with schizophrenia and bipolar disorder.

Repetin (RPTN) protein is a member of S100 family and is known to be expressed in the normal epidermis. Here we show that RPTN is ubiquitously expressed in both mouse and human brain, with relatively high levels in choroid plexus, hippocampus and prefrontal cortex. To investigate the expression of RPTN in neuropsychiatric disorders, we determined serum levels of RPTN in patients with schizophrenia (n = 88) or bipolar disorder (n = 34) and in chronic psychostimulant users (n = 91). We also studied its expression in a mouse model of chronic unpredictable mild stress (CUMS). The results showed that serum RPTN levels were significantly diminished in patients with schizophrenia and bipolar disorder or in psychostimulant users, compared with healthy subjects (n = 115) or age-matched controls (n = 92) (p < 0.0001). In CUMS mice, RPTN expression in hippocampus and prefrontal cortex was reduced with progression of the CUMS procedure; the serum RPTN level remained unchanged. Since CUMS is a model for depression and methamphetamine (METH) abuse induced psychosis recapitulates many of the psychotic symptoms of schizophrenia, the results from this study may imply that RPTN plays a potential role in emotional and cognitive processing; its decrease in serum may indicate its involvement in the pathogenesis of schizophrenia and bipolar disorder.

Proteomic analysis of non-small cell lung cancer tissue interstitial fluids.

BACKGROUND: Non-small cell lung cancer (NSCLC) accounts for more than 80% of all lung cancers, and reliable biomarkers are desirable. The present investigation assesses our ability to identify tumor relevant proteins from NSCLC tissue interstitial fluid (TIF). METHODS: Paired TIF was collected from three NSCLC patients at the time of surgery, and resolved by two-dimensional gel electrophoresis and in-gel digestion for proteomic analysis. Differentially expressed spots were extracted from the two-dimensional gel and characterized by high-performance liquid chromatography-tandem mass spectrometry. Then, ELISA was used to verify the expression of peroxiredoxin 1 (PRDX1) in TIF of patients with NSCLC and benign lung disease. Finally, the relationship between expression of PRDX1 and clinicopathological features was determined. RESULTS: Comparative proteomic analysis showed 24 protein spots were differentially expressed with significant changes, including 11 upregulated proteins and 13 downregulated proteins. Of these, PRDX1 was selected for validation in TIF by Western blot and expression of PRDX1 was confirmed to be upregulated in tumor TIF. It was also demonstrated that PRDX1 was significantly elevated in 40 NSCLC patients with a mean level of 36.0 ng/mL compared to 6.26 ng/mL from 20 patients with benign lung disease. A significant correlation was found between the high level of PRDX1 expression and lymph node metastasis and tumor differentiation. CONCLUSIONS: PRDX1 might be correlated with lymph node metastasis and differentiation, and its elevated expression in TIF may be an adverse biomarker for patients with NSCLC. PRDX1 may be attributed to the malignant transformation of NSCLC, and attention should be paid to a possible target for therapy.

Uric acid-induced endoplasmic reticulum stress triggers phenotypic change in rat glomerular mesangial cells.

AIM: The aim of this study was to explore the contribution and the mechanism of uric acid (UA) to phenotypic change in rat glomerular mesangial cells. METHODS: Rat glomerular mesangial cells (HBZY-1) were exposed to UA (0.05 mmol/L to 0.4 mmol/L) for 24 h to 48 h. Subsequently, 4-phenyl butyric acid (4-PBA) (5 mg/dL) was added and 48 h incubation was performed. HBZY-1 cells exposed to UA (0.4 mmol/L) were incubated for 48 h. After incubation, the cells were examined under an inverted microscope and transmission electron microscope to observe their morphologies and the expressions of α-smooth muscle actin (α-SMA), transforming growth factor-ß1 (TGF-ß1), fibronectin (FN), glucose regulated protein 78 (GRP78), and the protein disulfide isomerase (PDI) proteins and mRNA in the HBZY-1 cells were measured by Western blot and reversed transcription-polymerase chain reaction. RESULTS: HBZY-1 cultured in UA showed evident morphological changes under transmission electron microscopy. The soluble UA stimulated the upregulation of the α-SMA, TGF-ß1 and FN mRNA and proteins in a concentration- and time-dependent manner. UA-induced endoplasmic reticulum (ER) stress, as evidenced by the upregulation of the mRNA and protein expressions of GRP78 and PDI. However, the upregulation was reverted by 4-PBA, an inhibitor of ER stress. CONCLUSIONS: Uric acid induces phenotypic change in HBZY-1 cells. ER stress plays a central role in UA-induced phenotypic transformation in vitro. 4-PBA may be beneficial in attenuating UA-induced glomerular injury.

Modified method of sutureless intrascleral posterior chamber intraocular lens fixation without capsular support.

PURPOSE: This study reports the clinical results of sutureless intrascleral fixation of posterior chamber intraocular lenses (PC IOLs) in eyes without capsular support.
 METHODS: Thirty patients without capsular support who underwent standard 3-piece foldable PC IOL implantation using the sutureless intrascleral fixation technique were studied retrospectively. Folded PC IOLs were injected through corneal incisions, with 2 haptics piercing out from 2 corresponding scleral punctures, which were 1.5 mm from the limbus and exactly 180° to each other, and then 2 haptics piercing respectively were placed in 2 corresponding limbus-parallel intrascleral tunnels. All patients were evaluated for preoperative and postoperative status and complications.
 RESULTS: During the follow-up period, the visual acuity of all 30 patients significantly improved, with that of 21 patients improving by more than 0.6. The intraocular pressures of all patients were stable. All PC IOLs were stable and centered. Optical coherence tomography revealed that the haptics were located within the tunnel. Other complications, such as IOL dislocation, endophthalmitis, and retinal detachment, were not detected.
 CONCLUSION: Sutureless intrascleral fixation is safe and effective for the implantation of PC IOLs in eyes without capsular support from a variety of causes.

Cardioprotective effect of calcitriol on myocardial injury induced by isoproterenol in rats.

BACKGROUND: Calcitriol (CAL), an active form of vitamin D, plays a vital role in controlling cardiac hypertrophy and heart failure. The aim of the present study is to explore the effects of CAL and to elucidate its underlying mechanisms on myocardial injury induced by isoproterenol (ISO). METHODS: Myocardial impairment was induced by the subcutaneous injection of ISO in adult male Sprague-Dawley rats, and the therapeutic effect of CAL was assessed. Biometric and echocardiographic parameters, interstitial fibrosis, oxidant-antioxidant status, and protein expression relevant to the mitochondrial apoptosis pathway were then measured. RESULTS: Calcitriol treatment improved the cardiac injury resulting from excessive ISO stimulation, as supported by the suppression of the development of myocardial hypertrophy, interstitial fibrosis, and H2O2 level in heart tissue. The decreased superoxide dismutase and catalase activities induced by ISO were also improved by CAL. Finally, the administration of CAL downregulated the protein expression of Bax and caspase-9. CONCLUSIONS: This study provides evidence that CAL ameliorated cardiac hypertrophy, interstitial fibrosis, and oxidative stress in ISO-induced cardiac injury and might play a vital cardioprotective role in such injuries.

Gut bacterial translocation is associated with microinflammation in end-stage renal disease patients.

AIM: To investigate whether gut bacteria translocation occurs in end-stage renal disease patients and contributes to microinflammation in end-stage renal disease (ESRD). METHODS: The subjects were divided into two groups: nondialysed ESRD patients (n = 30) and healthy controls (n = 10). Blood samples from all participants were subjected to bacterial 16S ribosomal DNA amplification and DNA pyrosequencing to determine the presence of bacteria, and the alteration of gut microbiomes were examined with the same methods. High-sensitive C-reactive protein and interleukin-6 were detected. Plasma D-lactate was tested for gut permeability. RESULTS: Bacterial DNAs were detected in the blood of 20% (6/30) of the ESRD patients. All the observed genera in blood (Klebsiella spp, Proteus spp, Escherichia spp, Enterobacter spp, and Pseudomonas spp) were overgrown in the guts of the ESRD patients. Plasma D-lactate, High-sensitive C-reactive protein, and interleukin-6 levels were significantly higher in patients with bacterial DNA than those without. The control group showed the same results as that of patients without bacterial DNA. CONCLUSION: Bacterial translocation occurs in ESRD patients and is associated with microinflammation in end stage renal disease.

Gut bacterial translocation contributes to microinflammation in experimental uremia.

BACKGROUND: Microinflammation frequently develops in chronic uremia with pathological intestinal changes. However, the relationship between gut bacterial translocation and microinflammation in uremia has not been widely investigated. AIM: This study aimed to investigate whether gut microbiome dysbiosis and translocation occurred in experimental uremia, and whether they consequently contributed to microinflammation. METHODS: Forty rats underwent surgical renal mass 5/6 ablation. The surviving (uremic group, n = 21) and healthy (sham group, n = 20) rats were used in the experiment. Postoperative blood, livers, spleens, and mesenteric lymph nodes (MLNs) were subjected to bacterial 16S ribosomal DNA amplification to determine if bacteria were present. Bacterial genomic DNA samples from the MLNs and colon were amplified with specific primers designed by the 16S rRNA sequence of the species obtained from blood, livers, and spleens. Pyrosequencing was used to analyze the colonic microbiome of each subject. Intestinal permeability to (99m)Tc-DTPA, plasma hs-CRP, and IL-6 were measured. RESULTS: Bacterial DNA in extraintestinal sites and altered colonic microbiomes were detected in some rats in the uremic group. Bacterial genomic DNA in MLNs and colon were obtained by primers specific for bacterial species observed from blood, livers, and spleens of identical individuals. Intestinal permeability, plasma hs-CRP, and IL-6 levels were statistically higher in the uremic group compared with the sham group. Plasma hs-CRP and IL-6 were significantly higher in uremic rats with bacterial DNA in their blood than in those without. CONCLUSIONS: Gut microbiome dysbiosis occurs and bacteria translocate to the systemic and lymph circulation, thereby contributing to microinflammation in experimental uremia.

Podocyte injury and overexpression of vascular endothelial growth factor and transforming growth factor-beta 1 in adriamycin-induced nephropathy in rats.

The aim of this study is to investigate the expression of nephrin, vascular endothelial growth factor (VEGF), transforming growth factor-beta 1 (TGF-ß1), and podocyte number in adriamycin (ADR)-induced nephropathy. A total of 60 male Sprague-Dawley rats were randomly divided into the control group and the ADR nephropathy group. The nephropathy was induced by tail-vein injection of ADR (4 mg/kg) twice at a 14-day interval. The expression levels of nephrin, VEGF, and TGF-ß1 in glomeruli were assessed by immunohistochemistry and western blotting. The podocyte number was also evaluated after anti-Wilms' tumor-1 (WT1) immunohistochemical staining. In addition, the urinary protein content, biochemical parameters in serum samples and glomerular sclerosis index (SI) were compared between groups. In the ADR nephropathy group, the expression levels of nephrin was significantly decreased with the fusion of podocyte foot processes at 6 weeks after the first ADR injection, which was associated with a marked proteinuria. A decrease in podocyte number and an increase in SI with the overexpression of both VEGF and TGF-ß1 were also observed in the glomeruli at 10 weeks after the first ADR injection. This was associated with focal segmental glomerulosclerosis (FSGS). The study data suggest that podocyte injury and decreased nephrin, as well as increased VEGF and TGF-ß1, may contribute to the development of proteinuria and FSGS in ADR-induced nephropathy in rats.

A mutation in the cdh23 gene causes age-related hearing loss in Cdh23(nmf308/nmf308) mice.

Cadherin 23 (CDH23) is an important constituent of the hair cell tip link in the organ of Corti. Mutations in cdh23 are associated with age-related hearing loss (AHL). In this study, we proposed that the Cdh23(nmf308/nmf308) mice with progressive hair cell loss had specific morphological changes and suffered a base to apex gradient and age-related hearing loss, and that mutations in cdh23 were linked to AHL. The Cdh23(nmf308/nmf308) mice produced by the N-nitrosourea (ENU) mutagenesis program were used as an animal model to study AHL and progressive hair cell loss. RT-PCR was performed to confirm the cdh23 mutation in Cdh23(nmf308/nmf308) mice and genetic analysis was used to map the specific mutation site. Distortion product otoacoustic emission (DPOAE) assay and acoustic brainstem evoked response (ABR) threshold analysis were carried out to evaluate the AHL. Cochlear histology was examined with scanning electron microscope (SEM) and transmission electron microscope (TEM), as well as the nuclear labeling by propidium iodide staining; terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay and caspase-3 activities were examined to evaluate cell apoptosis. Genetic mapping identified the candidate gene linking AHL in Cdh23(nmf308/nmf308) mice as cdh23. A mutation in exon3 (63 T>C) was screened as compared with the sequence of the same position of the gene from B6 (+/+) mice. The cochleae outer hair cells were reduced from 5-10% at one month to 100% at three months in the basal region. DPOAE and ABR exhibited an increasing threshold at high frequencies (≥16kHz) from one month of age. Morphological and cellular analysis showed that Cdh23(nmf308/nmf308) mice exhibited a time course of histological alterations and cell apoptosis of outer hair cells. Our results suggest that the cdh23 mutation may be harmful to the stereociliary tip link and cause the hair cell apoptosis. Due to the same cdh23 mutations in human subjects with presbycusis (Petit et al., 2001; Zheng et al., 2005), the Cdh23(nmf308/nmf308) mouse is an excellent animal model for investigating the mechanisms involved in human AHL.

[Podocyte injury and expression of nephrin and VEGF in rat nephrosis model induced by adriamycin].

OBJECTIVE: To investigate podocyte injury and the expression of nephrin and VEGF in rat nephrosis model induced by adriamycin. METHODS: The rat adriamycin induced nephrosis model was established, while the biochemical indicators in blood and urine were measured and the pathological changes of the renal tissue were evaluated by light microscope and electron microscope. The podocyte number was counted, and the expression levels of nephrin, VEGF were examined at different time by means of immunohistochemistry. RESULTS: After second injected with adriamycin,the model group nephrin presented a weak signal in the end of the first week (P < 0.05), and the expression of VEGF started to increase at the end of the eighth week (P < 0.05). The podocyte number decreased at the end of the eighth week (P < 0.05). The expression of nephrin and the number of podocyte were negatively correlated with the 24-hour urine protein, blood urea nitrogen and serum creatinine; while the expression of VEGF was positively correlated with the 24-hour urine protein, blood urea nitrogen and serum creatinine. CONCLUSION: The decrease of nephrin expression and the change of its distribution might be the significant factors resulting in considerable proteinuria. VEGF participated in the process of proteinuria and glomerular sclerosis in the development of rat adriamycin nephrosis.

Encystation--survival of Blastocystis hominis in immunocompetent mice abdomen cavity.

Human Blastocystis hominis were isolated from diarrhea patients' feces and cultured in vitro. Then the cultures were inoculated intraperitoneally to laboratory mice. The B. hominis in living mice were collected and inoculated again to healthy mice. The B. hominis showed dose-dependent pathogenicity in the primary inoculation. No pathogenicity was observed in the secondary inoculation. The protozoan existed in the living mice abdomen cavity for more than 6 months and the cyst was the only form. These results showed that encystation enable the parasite to avoid the immune attack in competent host and simultaneously decrease the pathogenicity to host. Intraperitoneal inoculation to laboratory mice is a good method to maintain and propagate B. hominis. This is also a good model to study the interaction of B. hominis and immune system.

[Investigation of progesterone receptor on human sperm plasma membrane].

OBJECTIVES: To investigate the localization and positive percentage of progesterone receptor (PR) on the human sperm surface. METHODS: After in vitro capacitation, progesterone binding sites on the sperm were quantitatively analyzed by fluorescence microscopy and flow cytometry using fluorescein isothiocyanate-labeled bovine serum albumin-progesterone complex (P-BSA-FITC). RESULTS: The spermatozoa stained by P-BSA-FITC mainly showed two labeling patterns, with the green fluorescence on the whole acrosomal region or the equatorial acrosomal region only and the stainless postacrosomal and tail regions. The percentage of progesterone-binding sperm was (30.2 +/- 2.4)%. CONCLUSIONS: There is selective expression of PR on the human sperm acrosome surface.