Hongjun Luo

Urine formaldehyde level is inversely correlated to mini mental state examination scores in senile dementia

It is widely known that exogenous formaldehyde exposure induces human cognitive impairment and animal memory loss; and recent studies show that formaldehyde at pathological levels induces Aβ deposition and misfolding of tau protein to form globular amyloid-like aggregates. Endogenous formaldehyde may be a marker for progressive senile dementia. The aim of this study was to investigate the correlation of endogenous formaldehyde in urine of senile dementia and mini mental state examination (MMSE) scores. Formaldehyde level was analyzed by high-performance liquid chromatography (with fluorescence detection) in human urine from dementia patients (n=141), patients with hypertension (n=33) or diabetes (n=16) and healthy individuals (n=38), autopsy hippocampus samples from Alzheimers disease (AD) patients and brains of three types of AD animal model: namely, senescence accelerated mice (SAMP8), APP-transgenic mice and APP/PS1-transgenic mice. In a double-blind study, there was marked elevation of urine formaldehyde levels in patients (n=91) with dementia, and a slight increase in patients (n=50) with mild cognitive impairment. Urine formaldehyde level was inversely correlated with mini mental state examination scores (Rs=-0.441, p<0.0001). Furthermore, formaldehyde levels were significantly increased in the autopsy hippocampus from Alzheimers patients (n=4). In SAMP8 brains the formaldehyde level was significantly increased, suggesting that the endogenous formaldehyde is related to aging in mice. The brain formaldehyde level in APP/PS1-transgenic (n=8) mice at age of 3 months and APP-transgenic (n=8) mice at age of 6 months was increased (0.56 ± 0.02 mM), respectively, as compared with their respective age-matched controls, when these two types of AD-like animals, respectively, started to form Aβ deposits and memory loss obviously. According to the level of formaldehyde in the brain of the transgenic mice, we treated normal mice with formaldehyde (0.5mM, intraperitoneal administration) and observed the memory loss of the animal in Morris water maze trial. Cognitive impairments for the senile dementia are probably related to endogenous formaldehyde levels; and the mini mental state examination scores referred to the evaluation of urine formaldehyde level in dementia patients may be used as a non-invasive method for the investigation and diagnosis of senile dementia.

Tumor Tissue-Derived Formaldehyde and Acidic Microenvironment Synergistically Induce Bone Cancer Pain

Background There is current interest in understanding the molecular mechanisms of tumor-induced bone pain. Accumulated evidence shows that endogenous formaldehyde concentrations are elevated in the blood or urine of patients with breast, prostate or bladder cancer. These cancers are frequently associated with cancer pain especially after bone metastasis. It is well known that transient receptor potential vanilloid receptor 1 (TRPV1) participates in cancer pain. The present study aims to demonstrate that the tumor tissue-derived endogenous formaldehyde induces bone cancer pain via TRPV1 activation under tumor acidic environment. Methodology/Principal Findings Endogenous formaldehyde concentration increased significantly in the cultured breast cancer cell lines in vitro, in the bone marrow of breast MRMT-1 bone cancer pain model in rats and in tissues from breast cancer and lung cancer patients in vivo. Low concentrations (1∼5 mM) of formaldehyde induced pain responses in rat via TRPV1 and this pain response could be significantly enhanced by pH 6.0 (mimicking the acidic tumor microenvironment). Formaldehyde at low concentrations (1 mM to 100 mM) induced a concentration-dependent increase of [Ca2+]i in the freshly isolated rat dorsal root ganglion neurons and TRPV1-transfected CHO cells. Furthermore, electrophysiological experiments showed that low concentration formaldehyde-elicited TRPV1 currents could be significantly potentiated by low pH (6.0). TRPV1 antagonists and formaldehyde scavengers attenuated bone cancer pain responses. Conclusions/Significance Our data suggest that cancer tissues directly secrete endogenous formaldehyde, and this formaldehyde at low concentration induces metastatic bone cancer pain through TRPV1 activation especially under tumor acidic environment.

Aging-associated excess formaldehyde leads to spatial memory deficits

Recent studies show that formaldehyde participates in DNA demethylation/methylation cycle. Emerging evidence identifies that neuronal activity induces global DNA demethylation and re-methylation; and DNA methylation is a critical step for memory formation. These data suggest that endogenous formaldehyde may intrinsically link learning-responsive DNA methylation status and memory formation. Here, we report that during spatial memory formation process, spatial training induces an initial global DNA demethylation and subsequent re-methylation associated with hippocampal formaldehyde elevation then decline to baseline level in Sprague Dawley rats. Scavenging this elevated formaldehyde by formaldehyde-degrading enzyme (FDH), or enhancing DNA demethylation by a DNA demethylating agent, both led to spatial memory deficits by blocking DNA re-methylation in rats. Furthermore, we found that the normal adult rats intrahippocampally injected with excess formaldehyde can imitate the aged-related spatial memory deficits and global DNA methylation decline. These findings indicate that aging-associated excess formaldheyde contributes to cognitive decline during aging.

Age-related formaldehyde interferes with DNA methyltransferase function, causing memory loss in Alzheimer's disease

Hippocampus-related topographic amnesia is the most common symptom of memory disorders in Alzheimers disease (AD) patients. Recent studies have revealed that experience-mediated DNA methylation, which is regulated by enzymes with DNA methyltransferase (DNMT) activity, is required for the formation of recent memory as well as the maintenance of remote memory. Notably, overexpression of DNMT3a in the hippocampus can reverse spatial memory deficits in aged mice. However, a decline in global DNA methylation was found in the autopsied hippocampi of patients with AD. Exactly, what endogenous factors that affect DNA methylation still remain to be elucidated. Here, we report a marked increase in endogenous formaldehyde levels is associated with a decline in global DNA methylation in the autopsied hippocampus from AD patients. In vitro and in vivo results show that formaldehyde in excess of normal physiological levels reduced global DNA methylation by interfering DNMTs. Interestingly, intrahippocampal injection of excess formaldehyde before spatial learning in healthy adult rats can mimic the learning difficulty of early stage of AD. Moreover, injection of excess formaldehyde after spatial learning can mimic the loss of remote spatial memory observed in late stage of AD. These findings suggest that aging-associated formaldehyde contributes to topographic amnesia in AD patients.

Inhibition of semicarbazide-sensitive amine oxidase attenuates myocardial ischemia–reperfusion injury in an in vivo rat model

AIMS This study tested the hypothesis that the inhibition of semicarbazide-sensitive amine oxidase (SSAO) after ischemia could attenuate myocardial ischemia-reperfusion (I/R) injury. MAIN METHODS Anesthetized male Sprague-Dawley rats underwent myocardial I/R injury. Saline, semicarbazide (SCZ, 30 mg/kg), hydralazine (HYD, 10mg/kg), or LJP 1207 (30 mg/kg) was administered intraperitoneally 3 min before reperfusion. After 30 min of ischemia and 180 min of reperfusion, the myocardial infarct size was determined using nitroblue tetrazolium staining. Myocardial myeloperoxidase activity was determined through biochemical assay. HE staining was used for histopathological evaluation. Myocardial SSAO activity was assayed with high performance liquid chromatography analysis. Additionally, the endothelial expression of P-selectin was evaluated using immunohistochemistry after 30 min of ischemia and 20 min of reperfusion. KEY FINDINGS Myocardial SSAO activity was increased in myocardial I/R injury. Administration of SCZ, HYD, or LJP 1207 reduced the myocardial infarct size and decreased leukocyte infiltration and endothelial P-selectin expression in myocardial I/R injury in vivo. SIGNIFICANCE These data suggest that myocardial I/R injury up-regulates myocardial SSAO activity, and the inhibition of SSAO prior to reperfusion is able to attenuate acute myocardial I/R injury.

In vitro and in vivo study of hydralazine, a potential anti-angiogenic agent.

Hydralazine (HYD), an old routine clinical anti-hypertension drug, is rarely used in clinic nowadays. Since the strategy of repositioning old drugs was put forward, HYD has been reported to possess various biological activities, including antitumor efficacy and reducing intra-tumor microvessel. Here, we investigated that whether HYD had the ability of anti-angiogeneis and its underlying mechanism. Cells proliferation, wound-healing, Transwell migration and invasion, tube formation and rat aortic ring assays in vitro and chicken chorioallantoic membrane (CAM) model in vivo were designed to investigated HYDs anti-angiogenic effect. Levels of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) were assessed by enzyme-linked immune sorbent assay (ELISA). Hepatocellular carcinoma (HCC) mice model was used to evaluate HYDs effect on tumor growth and microvessel density. Our results showed that HYD not only inhibited human umbilical vascular endothelial cell (HUVEC) proliferation, wound-healing, Transwell migration and invasion and tube formation, but also suppressed the microvessel outgrowth of rat aortic ring in vitro and the neovascularzation of CAM in vivo. Furthermore, we demonstrated that HYD attenuated tumor angiogenesis and tumor growth. In the co-culture system of Transwell migration, the secretion of VEGF and bFGF was reduced by HYD respectively. In sum, our data indicate that HYD has the pharmacological effect of ant-angiogenesis by interference with VEGF and bFGF signaling pathways in endothelial cells. These findings suggest that HYD might be a promising angiogenesis inhibitor and a potential effective therapeutic agent for cancer therapy.

SSAO inhibitors suppress hepatocellular tumor growth in mice.

Vascular adhesion protein-1 (VAP-1) is both an endothelial adhesion molecule involved in leukocytes emigration, and an oxidase belonging to the family of semicarbazide-sensitive amine oxidases (SSAOs). The enzyme activity of VAP-1 plays an important role in the migration of myeloid-derived suppressor cells (MDSCs) into tumor site, and SSAO inhibitors can block the function of VAP-1. The effects of SSAO inhibitors on leukocyte infiltration and tumor progression were evaluated in H22 hepatocellular carcinoma-bearing C57BL/6 mice. Tumor weight and volume were measured after SSAO inhibitor treatment. Then, MDSCs recruitment and neo-angiogenesis were determined using immunostaining. SSAO inhibitors significantly blocked the catalytic activity of VAP-1 in tumor, attenuated tumor progression, and reduced neo-angiogenesis. CD11b(+) and Gr-1(+) MDSCs, which normally infiltrate into tumors, were significantly diminished in tumor-bearing mice treated with SSAO inhibitors. The present study demonstrated that SSAO inhibitors might have an anti-tumor effect on hepatocellular carcinoma by inhibiting recruitment of CD11b(+) and Gr-1(+) cells and hindering angiogenesis, which could be attributed to impairing the catalytic activity of VAP-1.

Soluble vascular adhesion protein-1: Decreased activity in the plasma of trauma victims and predictive marker for severity of traumatic brain injury

BACKGROUND This study done was to investigate the clinical significance of soluble vascular adhesion protein-1 (sVAP-1) activity in trauma patients with different patterns. METHODS 96 patients with consecutive trauma ≥15 years who were admitted to emergency department of the Second Affiliated Hospital, Shantou University Medical College, China, between January 2007 and December 2009 were enrolled in this study. Plasma was collected at admission. Injury-severity score (ISS) and Glasgow Coma Scale (GCS) were used to determine the patient conditions. sVAP-1 activity was determined by using the high performance liquid chromatographic (HPLC) system. RESULTS Mean sVAP-1 activity in trauma patients was significantly lower than that of controls (P<0.0001), and the level was negatively correlated with circulating leucocytes and neutrophils (P<0.0001). There was a significant correlation between lower sVAP-1 activity and injury patterns. However, plasma sVAP-1 activity increased significantly in accordance with the severity of traumatic brain injury (TBI), and the patients with sVAP-1 value above 8.61 nmol/ml/h have much higher mortality rate (25.0%) than patients with sVAP-1 value lower than 8.61 nmol/ml/h (0.0%) (P=0.011). CONCLUSIONS Trauma patients had a decreased sVAP-1 activity. However, isolated TBI patients with higher activity of sVAP-1 at admission were more likely to have a poor outcome.

Benzylamine and methylamine, substrates of semicarbazide-sensitive amine oxidase, attenuate inflammatory response induced by lipopolysaccharide.

Current evidence indicates that semicarbazide-sensitive amine oxidase (SSAO) substrates possess insulin-mimic effect, which was thought to play an anti-inflammatory role. The purpose of the present study was to determine whether SSAO substrates benzylamine (BZA) and methylamine (MA) attenuate inflammatory response induced by lipopolysaccharide (LPS). BALB/c mice peritoneal macrophages (PMs) that express SSAO and RAW264.7 mouse macrophages that do not express SSAO were used in vitro studies. Experimental mice were given BZA or MA through intraperitoneal injection before LPS challenge. The results showed that BZA or MA treatment significantly reduced LPS-induced pro-inflammatory mediators (nitric oxide, TNF-α) production, the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2, and glucose consumption in murine PMs, but not in RAW264.7 cell line. The metabolites of BZA or MA catalyzed by SSAO, hydrogen peroxide, formaldehyde, and benzaldehyde could also significantly decrease LPS-induced nitric oxide and TNF-α production, iNOS and COX-2 expression, and glucose consumption in vitro. In addition, BZA or MA administration could significantly decrease plasma pro-inflammatory mediators and the expression of iNOS and COX-2 in liver and lung, and could also attenuate LPS-induced transient hyperglycemia and chronic hypoglycemia. These findings indicated that substrates of SSAO might be involved in the anti-inflammatory effects. The metabolites of BZA and MA catalyzed by SSAO might be responsible for the anti-inflammatory effects. Moreover, BZA or MA administration could be useful for normalization of glucose disposal during endotoxemia.

Renal tubular epithelial cells injury induced by mannitol and its potential mechanism

Abstract Administration of mannitol with high dose could induce extensive isometric renal proximal tubular vacuolization and acute renal failure in clinic. We previously demonstrated that mannitol-induced human kidney tubular epithelial cell (HK-2) injury. The objective of our present work was to further study the cytotoxicity of mannitol in HK-2 cells and its potential mechanism. Cell viability was assessed by an MTT method. Cell morphological changes were observed. Furthermore, levels of malondialdehyde (MDA) and glutathione (GSH) were measured. Flow cytometry was performed to determine cell apoptosis by using Annexin V-FITC and PI. In addition, the F-actin of cells was labeled by FITC-Phalloidin for observation of cytoskeleton. The MTT assay displayed that the cell viability decreased significantly in a dose- and time-dependent manner. The morphological changes were observed, including cell membrane rapture and cell detachment. The GSH concentration in HK-2 cells decreased dramatically in mannitol treatment group, while MDA content increased significantly. The results of flow cytometry indicated that apoptotic percentages of HK-2 cells increased in 250 mmol/L mannitol treatment group. After treatment with 250 mmol/L mannitol for 48 h, HK-2 cells showed disorganization of cytoskeleton and even exhibited a totally destroyed cytoskeleton. Therefore, high dose of mannitol has a toxic effect on renal tubular epithelial cells, which might be attributed to oxidative stress, destroyed cellular cytoskeleton and subsequent cell apoptosis.