Wenguo Fan

Differential effects of melatonin on amyloid‐β peptide 25–35‐induced mitochondrial dysfunction in hippocampal neurons at different stages of culture

Abstract:  β‐Amyloid (Aβ) is strongly involved in the pathogenesis of Alzheimer’s disease (AD), and mitochondria play an important role in neurodegenerative disorders. To determine whether any different effect of melatonin on cultured neurons treated with Aβ in vitro and which may be produced through its different action on mitochondria at different stages of culture, we investigated the damage of cultured rat hippocampal neurons mitochondrial function induced by Aβ in young neurons [days in vitro 10 (DIV 10)] and senescent neurons (DIV 25) and the protective effect of melatonin. Rat hippocampal neurons were incubated with amyloid‐β peptide 25–35 (Aβ25‐35) alone or pretreatment with melatonin. Cell viability, mitochondrial membrane potential (Δψm), ATP and the activity of the respiratory chain complexes were measured. Data showed that Aβ25‐35 caused a reduction in Δψm, inhibited the activity of the respiratory chain complexes and led to ATP depletion, melatonin attenuated Aβ25‐35‐induced mitochondrial impairment in young neurons, whereas melatonin had no effect on Aβ25‐35‐induced mitochondrial damage in senescent neurons. These results demonstrate that melatonin has differential effect on Aβ25‐35‐induced mitochondrial dysfunction at different stages of culture and suggest that melatonin is useful for the prevention of AD, rather than treatment.

Carbon monoxide: A gas that modulates nociception

Carbon monoxide (CO) has been recognized to act as an atypical neurotransmitter or neuromodulator in the nervous system and to be involved in a wide variety of neuronal activities. Several lines of evidence suggest that CO may play a role through multiple mechanisms in nociception processing. Differential regulation of a family of CO‐generating enzymes, heme oxygenase (HO), contributes mainly to the complexity underlying the role of CO in nociception. This Mini‐Review describes the latest evidence for the role of CO during normal sensory transmission and pathological pain conditions and discusses potential cellular mechanisms by which CO is involved in pathological pain.

Melatonin influences proliferation and differentiation of rat dental papilla cells in vitro and dentine formation in vivo by altering mitochondrial activity

Melatonin mediates a variety of biological processes ranging from the control of circadian rhythms to immune regulation. Melatonin also influences bone formation and osteointegration of dental implants. However, the effects of melatonin on dentine formation have not been examined. This study investigated the effects of melatonin on the proliferation and differentiation of rat dental papilla cells (rDPCs) in vitro and dentine formation in vivo. We found that melatonin (0, 10−12, 10−10,10−8 m) induced a dose‐dependent reduction in rDPCs proliferation, increased alkaline phosphatase (ALP) activity, the expression of dentine sialoprotein (DSP), and mineralized matrix formation in vitro. In vivo melatonin (50 mg/kg, BW, i.p.) inhibited dentine formation. Melatonin (10−8 m) suppressed the activity of complex I and IV in the basal medium (OS−) and enhanced the activity of complex I and complex IV in osteogenic medium (OS+). These results demonstrate that melatonin suppresses the proliferation and promotes differentiation of rDPCs, the mechanisms of which may be related to activity of mitochondrial complex I and complex IV.

The role of nitric oxide in orofacial pain

Nitric oxide (NO) is a free radical gas that has been shown to be produced by nitric oxide synthase (NOS) in different cell types and recognized to act as a neurotransmitter or neuromodulator in the nervous system. NOS isoforms are expressed and/or can be induced in the related structures of trigeminal nerve system, in which the regulation of NOS biosynthesis at different levels of gene expression may allow for a fine control of NO production. Several lines of evidence suggest that NO may play a role through multiple mechanisms in orofacial pain processing. This report will review the latest evidence for the role of NO involved in orofacial pain and the potential cellular mechanisms are also discussed.

Expression and colocalization of NADPH-diaphorase and heme oxygenase-2 in trigeminal ganglion and mesencephalic trigeminal nucleus of the rat

Carbon monoxide (CO) and nitric oxide (NO) are two endogenously produced gases that can function as second messenger molecules in the nervous system. The enzyme systems responsible for CO and NO biosynthesis are heme oxygenase (HO) and nitric oxide synthase (NOS), respectively. The present study was undertaken to examine the distribution of HO-2 and NOS of the trigeminal primary afferent neurons of the rat, located in the trigeminal ganglion (TG) and mesencephalic trigeminal nucleus (MTN), using histochemistry and immunohistochemistry. NADPH-d staining was found in most neurons in TG. The intensely NADPH-d-stained neurons were small- or medium-sized, while the large-sized neurons were less intensely stained. Immunocytochemistry for HO-2 revealed that almost all neurons in TG expressed HO-2, but they did not appear cell size-specific pattern. NADPH-d and HO-2 positive neurons appeared the same pattern, which was NADPH-d activity and HO-2 expression progressively declined from the caudal to rostral part of the MTN. A double staining revealed that the colocalization of NADPH-d/HO-2 neurons was 97.3% in TG and 97.6% in MTN. The remarkable parallels between NADPH-d and HO-2 suggest that NO and CO are likely neurotransmitters and mediate the orofacial nociception and sensory feedback of the masticatory reflex arc together.

Involvement of NOS/NO in the development of chronic dental inflammatory pain in rats.

Nitric oxide (NO) is believed to be an important messenger molecule in nociceptive transmission. To assess the possible roles of NO in trigeminal sensory system, we examined the distribution and density of histochemical staining for nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d), a marker for nitric oxide synthase (NOS), and immunohistochemical staining for c-Fos, a neuronal activity marker, in the trigeminal ganglion (TG) and trigeminal nucleus caudalis (Vc) following pulp exposure (PX) injured rats. The neurons innervating injured tooth in TG were labeled by the retrograde transport of fluoro-gold (FG). Teeth were processed for H&E staining. We found that NADPH-d activity increased significantly in the TG and Vc following PX pretreatment (7-28 days, especially in 21-28 days). Such changes were closely corresponding to the pattern of c-Fos detected by immunocytochemistry. The results demonstrate that PX-induced chronic pulpal inflammation results in significant alterations in the TG cells and in the Vc, and such changes may underlie the observed NADPH-d activity. It suggests that NOS/NO may play an active role in both peripheral and central processing of nociceptive information following chronic tooth inflammation.

The heme oxygenase system and oral diseases.

Oral Diseases (2011) 17, 252-257 Heme oxygenase (HO) system catabolizes heme into three products: carbon monoxide (CO), biliverdin/bilirubin and free iron, which consists of three forms identified to date: the oxidative stress-inducible protein HO-1 and the constitutive isozymes HO-2 and HO-3. HO has been involved in many physiological and pathophysiological processes, ranging from Alzheimers disease to cancer. The interest in HO system by scientists and clinicians involved with the oral and maxillofacial region is fairly recent, and few papers currently cited on HO relate to diseases in this anatomical area. This review will focus on the current understanding of the physiological significance of HO-1 induction and its possible roles in the oral diseases studied to date. The implications for possible therapeutic manipulation of HO are also discussed.

Mitochondrial dysfunction in long-term neuronal cultures mimics changes with aging

Summary Background Aging is a highly complex process that affects various tissues and systems in the body. Senescent changes are relatively more prevalent and severe in the postmitotic cells. Mitochondria play an important role in the aging process. Recently, cell cultures have been widely used as an in vitro model to study aging. The present study was designed to investigate mitochondrial dysfunction associated with aging in a long-term cell culture system. Material/Methods Rat hippocampal neurons were maintained in culture in serum-free medium for 30 days in vitro (DIV). The morphology and development of hippocampal neurons was observed by phase contrast microscope. The levels of cellular senescence were evaluated by cytochemical staining of senescence-associated β-galactosidase (SA-β-Gal) at DIV 5, 10, 15, 20, 25 and 30. In addition, we investigated the changes in mitochondrial membrane potential (Δψm) and intracellular reactive oxygen species (ROS) generation of hippocampal neurons by flow cytometry at different ages. Results The proportion of the senescent cells steadily increased with age in neuron cultures. Δψm decreased gradually with age in long-term culture, while ROS generation increased. Conclusions This study indicates an age-related decrease in mitochondrial function in long-term hippocampal neuronal culture and suggests that DIV 25 neurons could possibly serve as a platform for the future study of anti-aging from the perspective of mitochondrial function.

The rs391957 variant cis-regulating oncogene GRP78 expression contributes to the risk of hepatocellular carcinoma.

Glucose-regulated protein 78 (GRP78) is one of the most important responders to disease-related stress. We assessed the association of the promoter polymorphisms of GRP78 with risk of hepatocellular carcinoma (HCC) and GRP78 expression in a Chinese population. We examined 1007 patients undergoing diagnostic HCC and 810 unrelated healthy controls. Mechanisms by which the GRP78 promoter polymorphism modulates HCC risk and GRP78 levels were analyzed. The promoter haplotype and diplotype carrying rs391957 (-415bp) allele G and genotype GG was strongly associated with HCC risk. Luciferase reporter assays indicated that the promoter carrying rs391957 allele G (haplotype GCCd) showed increased activity in HepG2 cells and Hela cells. rs391957 was also shown to increase the affinity of the transcriptional activator Ets-2, the resistance to apoptosis, as well as cell instability in stressful microenvironment. Furthermore, compared with allele A, rs391957 allele G was associated with higher levels of GRP78 mRNA and protein in HCC tissues. These findings provided new insights into the pathogenesis of HCC and an unexpected effect of the interaction between rs391957 and Ets-2 on hepatocarcinogenesis, and especially supported the hypothesis that stress-related and evolutionarily conserved genetic variant(s) influencing transcriptional regulation could predict susceptibilities.

An Intronic Polymorphism in GRP78 Improves Chemotherapeutic Prediction in Non-small Cell Lung Cancer

BACKGROUND Glucose-regulated protein 78 (GRP78) is involved in not only the progression of non-small cell lung cancer (NSCLC) but also chemotherapeutic effects. We hypothesized that an intronic polymorphism (rs430397G>A) in GRP78 affects survival among patients with NSCLC treated with platinum-based chemotherapy. METHODS Blood samples of patients with advanced NSCLC (IIIB/IV) were maintained in our specimen bank between 2001 and 2006. Genomic DNA was genotyped for rs430397. Associations between rs430397 and platinum-based treatment response, overall survival (OS), NSCLC-related survival, progression-free survival (PFS), and relapses were evaluated. GRP78 RNA and protein in NSCLC tissues were tested by real-time polymerase chain reaction and immunohistochemistry. RESULTS The AA genotype is significantly associated with platinum-based chemoresistance (P = .019) and NSCLC-related death (P = .022). OS, NSCLC-related survival, and PFS of the AA genotype group are decreased compared with the GG and AG genotype groups (log-rank P < .05, respectively). The AA group showed a higher prevalence of early NSCLC relapses than the AG and GG group (P = .030). In addition, the AA genotype showed a significantly increased risk for OS (hazard ratio, 1.95) and PFS (hazard ratio, 1.80) compared with the GG group. Functional analysis showed that NSCLC tissues with genotype AA have higher GRP78 RNA and protein expression compared with those carrying GG at rs430397. CONCLUSIONS The rs430397 AA genotype of GRP78 is associated with reduced survival and higher prevalence of early relapses in patients with advanced NSCLC treated with platinum-based chemotherapy.