Alterations of mitochondrial dynamics, inflammation and mineralization potential of lipopolysaccharide-induced human dental pulp cells after exposure to N-acetyl cysteine, Biodentine or ProRoot MTA.

AIM: To investigate the effects of N-acetyl cysteine (NAC), Biodentine, ProRoot MTA and their combinations, on cell viability, mitochondrial reactive oxygen species (mtROS) production, mineralization and on the expression of genes related to inflammatory cytokine production, mitochondrial dynamics and cell apoptosis of lipopolysaccharide (LPS)-induced human dental pulp cells (hDPCs). METHODOLOGY: Isolated hDPCs were exposed to 20 µg mL-1 of Escherichia coli (E. coli) LPS for 24 h, before the experiment, except for the control group. Eight experimental groups were assigned: (i) control (hDPCs cultured in regular medium), (ii) +LPS (hDPCs cultured in LPS medium throughout the experiment), (iii) -LPS/Media, (iv) -LPS/BD, (v) -LPS/MTA, (vi) -LPS/NAC, (vii) -LPS/BD + NAC and (viii) -LPS/MTA + NAC. Cell viability was measured using Alamar blue assay at 24 and 48 h. Production of mtROS was evaluated at 6 and 24 h by MitoSOX Red and MitoTracker Green. The expressions of IL-6, TNF-α, Bcl-2, Bax, Mfn-2 and Drp-1 genes were investigated at 6 h using reverse transcriptase-polymerase chain reaction (RT-PCR). For differentiation potential, cells were cultured in the osteogenic differentiation media and stained using Alizarin red assay at 14 and 21 days. The Kruskal-Wallis test, Mann-Whitney U test and one-way anova were performed for statistical analysis. RESULTS: NAC was associated with significantly greater LPS-induced hDPC viability (P < 0.05). Both Biodentine and MTA extracts promoted cell survival, whereas the combination of NAC to these material extracts significantly increased the number of viable cells at 24 h (P < 0.05). Biodentine, MTA or NAC did not alter the mtROS level (P > 0.05). NAC supplementation to the MTA extract significantly reduced the level of IL-6 and TNF-α expression (P < 0.05). Regarding mitochondrial dynamics, the use of NAC alone promoted significant Mfn-2/Drp-1 expression (P < 0.05). Most of the groups exhibited a level of Bcl-2/Bax gene expression similar to that of the control group. The increases in mineralization productions were observed in most of the groups, except the LPS group (P < 0.05). CONCLUSIONS: The antioxidant effect of NAC was not evident under the LPS-induced condition in DPC in vitro. NAC combined either with Biodentine or MTA improved LPS-induced hDPCs survival at 24 h. The combination of NAC with MTA promoted mineralization.

Liraglutide preserves intracellular calcium handling in isolated murine myocytes exposed to oxidative stress.

In ischemic/reperfusion (I/R) injured hearts, severe oxidative stress occurs and is associated with intracellular calcium (Ca(2+)) overload. Glucagon-Like Peptide-1 (GLP-1) analogues have been shown to exert cardioprotection in I/R heart. However, there is little information regarding the effects of GLP-1 analogue on the intracellular Ca(2+) regulation in the presence of oxidative stress. Therefore, we investigated the effects of GLP-1 analogue, (liraglutide, 10 microM) applied before or after hydrogen peroxide (H(2)O(2), 50 microM) treatment on intracellular Ca(2+) regulation in isolated cardiomyocytes. We hypothesized that liraglutide can attenuate intracellular Ca(2+) overload in cardiomyocytes under H(2)O(2)-induced cardiomyocyte injury. Cardiomyocytes were isolated from the hearts of male Wistar rats. Isolated cardiomyocytes were loaded with Fura-2/AM and fluorescence intensity was recorded. Intracellular Ca(2+) transient decay rate, intracellular Ca(2+) transient amplitude and intracellular diastolic Ca(2+) levels were recorded before and after treatment with liraglutide. In H(2)O(2) induced severe oxidative stressed cardiomyocytes (which mimic cardiac I/R) injury, liraglutide given prior to or after H(2)O(2) administration effectively increased both intracellular Ca(2+) transient amplitude and intracellular Ca(2+) transient decay rate, without altering the intracellular diastolic Ca(2+) level. Liraglutide attenuated intracellular Ca(2+) overload in H(2)O(2)-induced cardiomyocyte injury and may be responsible for cardioprotection during cardiac I/R injury by preserving physiological levels of calcium handling during the systolic and diastolic phases of myocyte activation.

Fibroblast growth factor 21 (FGF21) therapy attenuates left ventricular dysfunction and metabolic disturbance by improving FGF21 sensitivity, cardiac mitochondrial redox homoeostasis and structural changes in pre-diabetic rats.

AIMS: Fibroblast growth factor 21 (FGF21) acts as a metabolic regulator and exerts cardioprotective effects. However, the effects of long-term FGF21 administration on the heart under the FGF21-resistant condition in obese, insulin-resistant rats have not been investigated. We hypothesized that long-term FGF21 administration reduces FGF21 resistance and insulin resistance and attenuates cardiac dysfunction in obese, insulin-resistant rats. METHODS: Eighteen rats were fed on either a normal diet (n = 6) or a high-fat diet (HFD; n = 12) for 12 weeks. Then, rats in the HFD group were divided into two subgroups (n = 6 per subgroup) and received either the vehicle (HFV) or recombinant human FGF21 (rhFGF21, 0.1 mg kg(-1)  day(-1) ; HFF) injected intraperitoneally for 28 days. The metabolic parameters, inflammation, malondialdehyde (MDA), heart rate variability (HRV), left ventricular (LV) function, cardiac mitochondrial redox homoeostasis, cardiac mitochondrial fatty acid ß-oxidation (FAO) and anti-apoptotic signalling pathways were determined. RESULTS: HFV rats had increased dyslipidaemia, insulin resistance, plasma FGF21 levels, TNF-α, adiponectin and MDA, depressed HRV, and impaired LV and mitochondrial function. HFV rats also had decreased cardiac Bcl-2, cardiac PGC-1α and CPT-1 protein expression. However, FGF21 restored metabolic parameters, decreased TNF-α and MDA, increased serum adiponectin, and improved HRV, cardiac mitochondrial and LV function in HFF rats. Moreover, HFF rats had increased cardiac Bcl-2, cardiac PGC-1α and CPT-1 protein expression. CONCLUSION: Long-term FGF21 therapy attenuates FGF21 resistance and insulin resistance and exerts cardioprotection by improving cardiometabolic regulation via activating anti-apoptotic and cardiac mitochondrial FAO signalling pathways in obese, insulin-resistant rats.

Blockade of mitochondrial calcium uniporter prevents cardiac mitochondrial dysfunction caused by iron overload.

AIM: Iron overload in the heart can lead to iron-overload cardiomyopathy and cardiac arrhythmia. In the past decades, growing evidence has suggested that cardiac mitochondrial dysfunction is associated with the development of cardiac dysfunction and lethal arrhythmias. Despite these facts, the effect of iron overload on cardiac mitochondrial function is still unclear. In this study, we determined the effects of iron overload on the cardiac mitochondrial function and the routes of cardiac mitochondrial iron uptake. We tested the hypothesis that iron overload can lead to cardiac mitochondrial dysfunction and that mitochondrial calcium uniporter (MCU) plays a major role for cardiac mitochondrial iron uptake under iron-overload condition. Cardiac mitochondrial function was assessed via the determination of mitochondrial swelling, mitochondrial reactive oxygen species (ROS) production and mitochondrial membrane potential changes. METHODS: Isolated cardiac mitochondria from male Wistar rats were used in this study. To determine the routes for cardiac mitochondrial iron uptake, isolated mitochondria were exposed to MCU blocker (Ru360), mitochondrial permeability transition pore (mPTP) blocker (cyclosporin A) and an iron chelator (deferoxamine). RESULTS: We found that (i) iron overload caused cardiac mitochondrial dysfunction, indicated by increased ROS production, mitochondrial membrane depolarization and mitochondrial swelling; and (ii) only MCU blocker completely protected cardiac mitochondrial dysfunction caused by iron overload. CONCLUSIONS: These findings strongly suggest that MCU could be the major route for iron uptake into cardiac mitochondria. The inhibition of MCU could be the novel pharmacological intervention for preventing iron-overload cardiomyopathy.

Decreased jaw bone density and osteoblastic insulin signaling in a model of obesity.

Previous studies have demonstrated that decreased bone mass results from either the impairment of osteoblastic insulin signaling or obesity. Our previous study revealed that 12-week high-fat-diet (HFD) consumption caused obesity as well as peripheral and brain insulin resistance. However, the osteoblastic insulin resistance induced by HFD has not been elucidated. Therefore, we hypothesized that 12-week HFD rats exhibited not only peripheral insulin resistance but also osteoblastic insulin resistance, which leads to decreased jawbone quality. We found that the jawbones of rats fed a 12-week HFD exhibited increased osteoporosis. The osteoblastic cells isolated from HFD-fed rats exhibited the impairment of osteoblastic insulin signaling as well as reduction of cell proliferation and survival. In conclusion, this study demonstrated that insulin resistance induced by 12-week HFD impaired osteoblastic insulin signaling, osteoblast proliferation, and osteoblast survival and resulted in osteoporosis in the jawbone.

NaV 1.8, but not NaV 1.9, is upregulated in the inflamed dental pulp tissue of human primary teeth.

AIM: To investigate alterations in Na(V) 1.8 and Na(V) 1.9 expression within inflamed dental pulp tissue of human primary teeth. METHODOLOGY: Dental pulp tissue obtained from both normal and inflamed pulps in primary teeth as well as pulps from normal and inflamed permanent teeth was used. The quantity of Na(V) 1.8 and Na(V) 1.9 expression in the dental pulp tissue was investigated using Western blot analysis. General neuron marker (PGP9.5) was used to quantify for neural density, and an increase in metalloproteinase-9 was used to indicate pulpal inflammation in inflamed teeth. Statistically significant differences for each determined parameter between normal and inflamed teeth of both primary and permanent teeth were tested using the Mann-Whitney rank sum test. RESULTS: There was no significant difference in neural density of normal and inflamed dental pulp tissue, although degrees of inflammation were increased in the inflamed dental pulp of both permanent and primary teeth (P < 0.05). Na(V) 1.8 and Na(V) 1.9 expression in inflamed pulps of permanent teeth increased significantly compared with normal permanent teeth (P < 0.05). However, only Na(V) 1.8 expression was increased significantly in the inflamed dental pulp of primary teeth (P < 0.05). CONCLUSIONS: Na(V) 1.8 alone may be the therapeutic target for treatment of painful pulpitis in primary teeth.

Granulocyte colony-stimulating factor stabilizes cardiac electrophysiology and decreases infarct size during cardiac ischaemic/reperfusion in swine.

AIM: Effects of granulocyte colony-stimulating factor (G-CSF) on cardiac electrophysiology during ischaemic/reperfusion (I/R) period are unclear. We hypothesized that G-CSF stabilizes cardiac electrophysiology during I/R injury by prolonging the effective refractory period (ERP), increasing the ventricular fibrillation threshold (VFT) and decreasing the defibrillation threshold (DFT), and that the cardioprotection of G-CSF is via preventing cardiac mitochondrial dysfunction. METHODS: In intact-heart protocol, pigs were infused with either G-CSF or vehicle (n = 7 each group) without I/R induction. In I/R protocol, pigs were infused with G-CSF (0.33 µg kg(-1 ) min(-1) ) or vehicle (n = 8 each group) for 30 min prior to a 45-min left anterior descending artery occlusion and at reperfusion. Diastolic pacing threshold (DPT), ERP, VFT and DFT were determined in all pigs before and during I/R period. Rat's isolated cardiac mitochondria were used to test the protective effect of G-CSF (100 nm) in H(2) O(2) -induced mitochondrial oxidative damage. RESULTS: Neither G-CSF nor vehicle altered any parameter in intact-heart pigs. During ischaemic period, G-CSF significantly increased the DPT, ERP and VFT without altering the DFT. During reperfusion, G-CSF continued to increase the DPT without altering other parameters. The infarct size was significantly decreased in the G-CSF group, compared to the vehicle. G-CSF could also prevent cardiac mitochondrial swelling, decrease ROS production, and prevent mitochondrial membrane depolarization. CONCLUSION: G-CSF increases the DPT, ERP and VFT and reduces the infarct size, thus stabilizing the myocardial electrophysiology, and preventing fatal arrhythmia during I/R. The protective mechanism could be via its effect in preventing cardiac mitochondrial dysfunction.

Effect of sildenafil citrate on the cardiovascular system.

Sildenafil citrate is a drug commonly used to manage erectile dysfunction. It is designated chemically as 1-[[3-(6,7-dihydro-1-methyl-7-oxo-3-propyl-1H -pyrazolo[4,3-d]pyrimidin-5-yl)-4 ethoxyphenyl] sulfonyl]-4-methylpiperazine citrate (C22H30N6(O4)S). It is a highly selective inhibitor of cyclic guanine monophosphate-specific phosphodiesterase type 5. In late March through mid-November 1998, the US Food and Drug Administration (FDA) published a report on 130 confirmed deaths among men (mean age, 64 years) who received prescriptions for sildenafil citrate, a period during which >6 million outpatient prescriptions (representing about 50 million tablets) were dispensed. The US FDA recently reported that significant cardiovascular events, including sudden cardiac death, have occurred in men with erectile dysfunction who were taking sildenafil citrate. These reports have raised concerns that sildenafil citrate may increase the risk of cardiovascular events, particularly fatal arrhythmias, in patients with cardiovascular disease. In the past few years, the cardiac electrophysiological effects of sildenafil citrate have been investigated extensively in both animal and clinical studies. According to extensive data available to date, sildenafil citrate has been shown to pose minimal cardiovascular risks to healthy people taking this drug. Some precautions are needed for patients with cardiovascular diseases. However, the only absolute contraindication for sildenafil citrate is the concurrent use of nitrates. This article is intended to review sildenafil citrate's cardiovascular effects, as well as current debates about its arrhythmogenic effects.

Effect of sildenafil citrate on the cardiovascular system

Sildenafil citrate is a drug commonly used to manage erectile dysfunction. It is designated chemically as 1-[[3-(6,7-dihydro-1-methyl-7-oxo-3-propyl-1H -pyrazolo[4,3-d]pyrimidin-5-yl)-4 ethoxyphenyl] sulfonyl]-4-methylpiperazine citrate (C22H30N6O4 S). It is a highly selective inhibitor of cyclic guanine monophosphate-specific phosphodiesterase type 5. In late March through mid-November 1998, the US Food and Drug Administration (FDA) published a report on 130 confirmed deaths among men (mean age, 64 years) who received prescriptions for sildenafil citrate, a period during which >6 million outpatient prescriptions (representing about 50 million tablets) were dispensed. The US FDA recently reported that significant cardiovascular events, including sudden cardiac death, have occurred in men with erectile dysfunction who were taking sildenafil citrate. These reports have raised concerns that sildenafil citrate may increase the risk of cardiovascular events, particularly fatal arrhythmias, in patients with cardiovascular disease. In the past few years, the cardiac electrophysiological effects of sildenafil citrate have been investigated extensively in both animal and clinical studies. According to extensive data available to date, sildenafil citrate has been shown to pose minimal cardiovascular risks to healthy people taking this drug. Some precautions are needed for patients with cardiovascular diseases. However, the only absolute contraindication for sildenafil citrate is the concurrent use of nitrates. This article is intended to review sildenafil citrate's cardiovascular effects, as well as current debates about its arrhythmogenic effects.

The effects of noxious dental heating on the jaw-opening reflex and trigeminal Fos expression in the ferret.

Previous studies have established that the activation of peripheral nociceptors alters the central processing of nociceptive stimuli. In this study, we examined whether noxious heating of the dental pulp enhances the nociceptive jaw-opening reflex (JOR) and the expression of the immediate early gene c-fos in chloral hydrate/pentobarbital-anesthetized ferrets. We hypothesized that the application of noxious heat to the dental pulp, a procedure that evokes a preferential activation of pulpal C-fibers, will enhance JOR responses to electrical stimulation of the tooth pulp and that this enhanced response will be associated with the expression of Fos protein in discrete regions of the trigeminal nucleus. Consistent with our predictions, we observed that noxious heat conditioning enhanced the JOR as indicated by an increase in the magnitude of the signal averaged digastric electromyogram response evoked by electrical stimuli applied to either a heat-conditioned maxillary canine or the contralateral nonconditioned canine. The enhancement in JOR responses was independent of temporal summation of the electrical stimulus for test stimuli delivered at either 1.0 or 0.1 Hz. Sensitization of the JOR was associated with an increase in the number of immunohistochemically identified Fos-positive nuclei in trigeminal caudalis (Vc) and the transition zone between trigeminal interpolaris and caudalis (Vi/Vc) ipsilateral to the site of stimulation compared with sham stimulated animals. These findings suggest that neuronal populations in Vc and Vi/Vc play a role in the enhanced reflex responses to tooth pulp stimulation and may contribute to the pain and hyperalgesia associated with a symptomatic pulpitis.