In vivo bioluminescence imaging revealed the change of the time window of BDNF expression in the brain elicited by a single bout of exercise following repeated exercise.

Exercise increases the expression of brain-derived neurotrophic factor (BDNF) in the brain and contributes to cognitive and sensorimotor functions. This study aimed to elucidate how repeated exercise modifies BDNF expression elicited by a single bout of exercise in the brain using in vivo bioluminescence imaging (BLI). Bdnf-luciferase (Luc) mice with the firefly luciferase gene inserted at the translation start point of the Bdnf gene were used for BLI to monitor changes in BDNF expression in the brain. The treadmill exercise at a speed of 10 m/s for 60 min was repeated 5 days a week for 4 weeks. BLI in individual subjects was repeated four times: before the exercise intervention, on the first exercise day, and 14 and 28 days after the start of the intervention. Each BLI was performed after a single bout of exercise and monitored for 8 h after exercise. Repetitive BLI showed that the exercise regimen enhanced BDNF expression in the brain, specifically at 4-8 h after a single bout of exercise. Repeated exercise for 2 weeks accelerated the start of enhancement after a single bout of exercise, but not after 4 weeks of repeated exercise. This study showed that repeated exercise modulated the time window of exercise-enhanced BDNF expression, suggesting that repeated exercise could change the sensitivity of gene expression to a single bout of exercise. These findings can be attributed to the advantages of in vivo BLI, which allowed us to precisely measure the time course of BDNF expression after repeated exercise in individual subjects.

Analysis of the Anticipatory Behavior Formation Mechanism Induced by Methamphetamine Using a Single Hair.

While the suprachiasmatic nucleus (SCN) coordinates many daily rhythms, some circadian patterns of expression are controlled by SCN-independent systems. These include responses to daily methamphetamine (MAP) injections. Scheduled daily injections of MAP resulted in anticipatory activity, with an increase in locomotor activity immediately prior to the time of injection. The MAP-induced anticipatory behavior is associated with the induction and a phase advance in the expression rhythm of the clock gene Period1 (Per1). However, this unique formation mechanism of MAP-induced anticipatory behavior is not well understood. We recently developed a micro-photomultiplier tube (micro-PMT) system to detect a small amount of Per1 expression. In the present study, we used this system to measure the formation kinetics of MAP-induced anticipatory activity in a single whisker hair to reveal the underlying mechanism. Our results suggest that whisker hairs respond to daily MAP administration, and that Per1 expression is affected. We also found that elevated Per1 expression in a single whisker hair is associated with the occurrence of anticipatory behavior rhythm. The present results suggest that elevated Per1 expression in hairs might be a marker of anticipatory behavior formation.

Temporal dynamics of brain BDNF expression following a single bout of exercise: A bioluminescence imaging study.

Physical exercise increases brain-derived neurotrophic factor (BDNF) expression in the brain. However, the absence of non-invasive and repetitive monitoring of BDNF expression in the brains of living animals has limited the understanding of how BDNF expression changes after exercise. This study aimed to elucidate the temporal dynamics of BDNF expression in the brain after a single bout of exercise, using in vivo bioluminescence imaging. This study included Bdnf-Luc mice with a firefly Luciferase gene inserted at the translation start site of the mouse Bdnf gene. BDNF expression was evaluated based on the luminescence signal of the luciferase substrate administered to mice. Bioluminescence imaging was performed at 0, 1, 3, 6, 12, and 24 h after treadmill exercise (15 m/min for 1 h). Compared to the sedentary condition of each mouse, the luminescence signal increased by approximately 60 % between 1 and 3 h after exercise. The luminescence signal remained slightly increased by approximately 20 % even 6-24 h after exercise. This study is the first to demonstrate exercise-enhanced BDNF expression in the brains of living animals. These results provide evidence that a single bout of exercise transiently increases BDNF expression in the brain within a limited time window.

A real-time measurement system for gene expression rhythms from deep tissues of freely moving mice under light-dark conditions.

Clock gene expression in most organs of the living body exhibits a diurnal rhythm synchronized with the external 24 h light-dark (LD) cycle via circadian pacemaker suprachiasmatic nucleus (SCN). Disturbances in clock gene expression due to desynchronization of clock gene expression of the external LD cycle are risk factors for developing various diseases. Measuring the in vivo clock genes expression rhythm for a long duration under LD conditions can greatly contribute to understand the pathogenic mechanism of the disease caused by the disturbance of the biological rhythm. However, it is presently difficult to continuously measure gene expression for a long duration under LD conditions. In present study, we succeeded in measuring Period1 (Per1) gene expression under LD conditions using ultraviolet (UV) light with filter cut the visible light range. In addition, we succeeded in measuring the kinetic change of liver Per1 gene expression during the process of desynchronization of behavioral rhythm from the LD cycle by chronic administration of methamphetamine (MAP). In the future, by using this system to measure clock gene expression rhythms of brain tissues such as SCN and peripheral tissues under LD conditions, it could contribute to understand the onset mechanism of diseases induced by the desynchronization mechanism of biological rhythm to the LD cycle.

The usefulness of measuring n-butyric acid concentration as a new indicator of blood decomposition in forensic autopsy.

In forensic medicine, although various alcohols have been reported as indicators of decomposition in collected blood, no studies have examined short-chain fatty acids as indicators. In this study, the blood n-butyric acid concentration was quantified, and the association between n-butyric acid and decomposition was investigated to determine whether the detection of n-butyric acid could be a new indicator of decomposition. Among the forensic autopsies performed from 2016 to 2018 in our laboratory, the cases were divided into decomposed (n = 20) and non-decomposed (n = 20) groups based on macroscopic findings. Blood samples collected at the time of autopsy were derivatized with 3-nitrophenylhydrazine hydrochloride after solid-phase extraction. The n-butyric acid concentration was measured using liquid chromatography-tandem mass spectrometry. In addition, ethanol and n-propanol were measured using a gas chromatography-flame ionization detector. There was a significant difference (p < 0.01) in the concentrations of n-butyric acid between the decomposed and non-decomposed groups (0.343 ± 0.259 [0.030-0.973] and 0.003 ± 0.002 [0.001-0.007] mg/mL, respectively). In the decomposed group, n-butyric acid was detected at high concentrations, even in cases where n-propanol was low. These results suggest that n-butyric acid is more likely to be an indicator of blood decomposition than n-propanol.

Poly(ADP-ribose) Polymerase (PARP) is Critically Involved in Liver Ischemia/Reperfusion-injury.

BACKGROUND: Poly(ADP-ribose) polymerase (PARP) is a DNA-repairing enzyme activated by extreme genomic stress, and therefore is potently activated in the remnant liver suffering from ischemia after surgical resection. However, the impact of PARP on post-ischemic liver injury has not been elucidated yet. MATERIALS AND METHODS: We investigated the impact of PARP on murine hepatocyte/liver injury induced by hypoxia/ischemia, respectively. RESULTS: PJ34, a specific inhibitor of PARP, markedly protected against hypoxia/reoxygenation (H/R)-induced cell death, though z-VAD-fmk, a pan-caspase inhibitor similarly showed the protective effect. PJ34 did not affect H/R-induced caspase activity or caspase-mediated cell death. z-VAD-fmk also did not affect the production of PAR (i.e., PARP activity). Therefore, PARP- and caspase-mediated cell death occurred in a mechanism independent of each other in H/R. H/R immediately induced activation of PARP and cell death afterwards, both of which were suppressed by PJ34 or Trolox, an antioxidant. This suggests that H/R-induced cell death occurred redox-dependently through PARP activation. H/R and OS induced nuclear translocation of apoptosis inducing factor (AIF, a marker of parthanatos) and RIP1-RIP3 interaction (a marker of necroptosis), both of which were suppressed by PJ34. H/R induced PARP-mediated parthanatos and necroptosis redox-dependently. In mouse experiments, PJ34 significantly reduced serum levels of AST, ALT & LDH and areas of hepatic necrosis after liver ischemia/reperfusion, similar to z-VAD-fmk or Trolox. CONCLUSION: PARP, activated by ischemic damage and/or oxidative stress, may play a critical role in post-ischemic liver injury by inducing programmed necrosis (parthanatos and necroptosis). PARP inhibition may be one of the promising strategies against post-ischemic liver injury.

Inhibition of Cellular and Animal Inflammatory Disease Models by NF-κB Inhibitor DHMEQ.

General inflammatory diseases include skin inflammation, rheumatoid arthritis, inflammatory bowel diseases, sepsis, arteriosclerosis, and asthma. Although these diseases have been extensively studied, most of them are still difficult to treat. Meanwhile, NF-κB is a transcription factor promoting the expression of many inflammatory mediators. NF-κB is likely to be involved in the mechanism of most inflammatory diseases. We discovered a specific NF-κB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), about 20 years ago by molecular design from a natural product. It directly binds to and inactivates NF-κB components. It has been widely used to suppress cellular and animal inflammatory disease models and was shown to be potent in vivo anti-inflammatory activity without any toxicity. We have prepared ointment of DHMEQ for the treatment of severe skin inflammation. It inhibited inflammatory cytokine expressions and lowered the clinical score in mouse models of atopic dermatitis. Intraperitoneal (IP) administration of DHMEQ ameliorated various disease models of inflammation, such as rheumatoid arthritis, sepsis, and also graft rejection. It has been suggested that inflammatory cells in the peritoneal cavity would be important for most peripheral inflammation. In the present review, we describe the synthesis, mechanism of action, and cellular and in vivo anti-inflammatory activities and discuss the clinical use of DHMEQ for inflammatory diseases.

Period1 gene expression in the olfactory bulb and liver of freely moving streptozotocin-treated diabetic mouse.

Clock genes express circadian rhythms in most organs. These rhythms are organized throughout the whole body, regulated by the suprachiasmatic nucleus (SCN) in the brain. Disturbance of these clock gene expression rhythms is a risk factor for diseases such as obesity. In the present study, to explore the role of clock genes in developing diabetes, we examined the effect of streptozotocin (STZ)-induced high glucose on Period1 (Per1) gene expression rhythm in the liver and the olfactory bub (OB) in the brain. We found a drastic increase of Per1 expression in both tissues after STZ injection while blood glucose content was low. After a rapid expression peak, Per1 expression showed no rhythm. Associated with an increase of glucose content, behavior became arrhythmic. Finally, we succeeded in detecting an increase of Per1 expression in mice hair follicles on day 1 after STZ administration, before the onset of symptoms. These results show that elevated Per1 expression by STZ plays an important role in the aggravation of diabetes.

Stability of d-luciferin for bioluminescence to detect gene expression in freely moving mice for long durations.

Circadian disturbance of clock gene expression is a risk factor for diseases such as obesity, cancer, and sleep disorders. To study these diseases, it is necessary to monitor and analyze the expression rhythm of clock genes in the whole body for a long duration. The bioluminescent reporter enzyme firefly luciferase and its substrate d-luciferin have been used to generate optical signals from tissues in vivo with high sensitivity. However, little information is known about the stability of d-luciferin to detect gene expression in living animals for a long duration. In the present study, we examined the stability of a luciferin solution over 21 days. l-Luciferin, which is synthesized using racemization of d-luciferin, was at high concentrations after 21 days. In addition, we showed that bioluminescence of Period1 (Per1) expression in the liver was significantly decreased compared with the day 1 solution, although locomotor activity rhythm was not affected. These results showed that d-luciferin should be applied to the mouse within, at most, 7 days to detect bioluminescence of Per1 gene expression rhythm in vivo.

Double recording system of Period1 gene expression rhythm in the olfactory bulb and liver in freely moving mouse.

Clock genes express circadian rhythms in most organs. These rhythms are organized throughout the whole body, regulated by the suprachiasmatic nucleus (SCN) in the brain. Disturbance of these clock gene expression rhythms is a risk factor for diseases such as obesity and cancer. To understand the mechanism of regulating clock gene expression rhythms in vivo, multiple real time recording systems are required. In the present study, we developed a double recording system of Period1 expression rhythm in peripheral tissue (liver) and the brain. In peripheral tissue, quantification of gene expression in a steadily moving target was achieved by using a photomultiplier tube (PMT) attached to a tissue contact optical sensor (TCS). Using this technique, we were able to analyze circadian rhythms of clock gene expression over a prolonged period in the liver and olfactory bub (OB) of the brain. The present double recording system has no effect on behavioral activity or rhythm. Our novel system thus successfully quantifies clock gene expression in deep areas of the body in freely moving mice for a period sufficient to analyze circadian dynamics. In addition, our double recording system can be widely applied to many areas of biomedical research, as well as applications beyond medicine.

Sample preparation method with ultrafiltration for whole blood thiosulfate measurement.

Quantitative analysis of thiosulfate is useful for diagnosing hydrogen sulfide poisoning. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) enables more rapid and sensitive measurements than previous methodologies. As simple measurements of blood thiosulfate concentration are affected by the blood matrix, blood is used as the solvent to prepare the standard solution for calibration curve generation. Thus, a large amount of blood devoid of thiosulfate is required. We developed a preparation method by incorporating an ultrafiltration step to overcome this limitation and generate a calibration curve using a standard solution prepared with pure water. We used this improved method to investigate the stability of thiosulfate in refrigerated samples. To compare the effects of refrigeration, blood samples were prepared using the following two methods: one sample was treated with a 50-kDa exclusion ultrafiltration membrane and the other was not treated. The samples were stored at 4 °C, and then measured at 0, 3, 6, 24, 48, and 96 h. The incorporation of the ultrafiltration step in the measurement procedure enabled the quantification of thiosulfate, by plotting a calibration curve using a standard of pure water; it did not require a blood standard. Additionally, the reduction in whole blood thiosulfate concentration was within 10% during 2 days of refrigeration. Thus, the need for a large amount of blood to prepare the standard solution was resolved by the ultrafiltration step in test sample preparation. This method is useful to measure thiosulfate concentration and is not hindered by sample refrigeration for a few days.

Mouse period1 gene expression recording from olfactory bulb under free moving conditions with a portable optic fibre device.

Because the disruption of circadian clock gene is a risk factor in many diseases such as obesity and cancer, it is important to monitor and analyzed the expression of the rhythm of the clock gene throughout the body over a long period of time. Although we previously reported on a new gene expression analysis system tracking a target position on the body surface of freely moving mice, the experimental apparatus required a large space. We have therefore developed an in vivo recording system using a portable photomultiplier tube (PMT) system attached to an optical fibre. Directly connecting the target area with the device, we could easily measure the photon counts in a very small space. However, little information is known about the characteristics of optical fibres when exposed to twisting/looping in association with a moving mouse and the effect of the surface of optical fibre. In the present study, we report on the characteristics of optical fibres to detect gene expression rhythm in freely moving mice. Using this portable optical device directly connected with a target area, we were able to measure the circadian rhythm of clock gene expression over a prolonged period in freely moving mice in a small space.

Cellular and molecular mechanisms of liver regeneration: Proliferation, growth, death and protection of hepatocytes.

Liver regeneration is an important and necessary process that the liver depends on for recovery from injury. The regeneration process consists of a complex network of cells and organs, including liver cells (parenchymal and non-parenchymal cells) and extrahepatic organs (thyroid, adrenal glands, pancreas, duodenum, spleen, and autonomic nervous system). The regeneration process of a normal, healthy liver depends mainly on hepatocyte proliferation, growth, and programmed cell death. Cell proliferation and growth are regulated in a cooperative manner by interleukin (IL)-6/janus kinase (Jak)/signal transducers and activators of transcription-3 (STAT3), and phosphoinositide 3-kinase (PI3-K)/phosphoinositide-dependent protein kinase 1 (PDK1)/Akt pathways. The IL-6/Jak/STAT3 pathway regulates hepatocyte proliferation and protects against cell death and oxidative stress. The PI3-K/PDK1/Akt pathway is primarily responsible for the regulation of cell size, sending mitotic signals in addition to pro-survival, antiapoptotic and antioxidative signals. Though programmed cell death may interfere with liver regeneration in a pathological situation, it seems to play an important role during the termination phase, even in a normal, healthy liver regeneration. However, further study is needed to fully elucidate the mechanisms regulating the processes of liver regeneration with regard to cell-to-cell and organ-to-organ networks at the molecular and cellular levels.

Extracts of bilberry (Vaccinium myrtillus L.) fruits improve liver steatosis and injury in mice by preventing lipid accumulation and cell death.

Bilberry has been reported to have anti-oxidant and anti-inflammatory properties. We studied the effect of bilberry (Vaccinium myrtillus L.) fruits extracts (BEs) on the pathogenesis caused by lipid accumulation in fatty liver and non-alcoholic steatohepatitis (NASH). 5 µg/ml of BEs was enough to suppress lipid accumulation in the fatty liver model of the mouse hepatic AML12 cells. BEs increased cell viability and anti-oxidant capacity, presumably by activating (phosphorylating) Akt/STAT3 and inducing MnSOD/catalase. BEs also significantly reduced Rubicon and induced p62/SQSTM1, possibly contributing to reduce cellular lipids (lipophagy). When the mice were fed supplemented with BEs (5% or 10%, w/w), hepatic steatosis, injury, and hypercholesterolemia/hyperglycemia were significantly improved. Furthermore, histological and cytokine studies indicated that BEs possibly suppress hepatic inflammation (hepatitis) and fibrosis. Therefore, BEs improved liver steatosis and injury, and potentially suppress fibrosis by suppressing inflammatory response, which therefore may prevent the progression of fatty liver to NASH.

Activation of caspase-3 during Chlamydia trachomatis-induced apoptosis at a late stage.

The obligate intracellular bacterium Chlamydia trachomatis activates the host cell apoptosis pathway at a late stage of its developmental cycle. However, whether caspase-3, which is a key enzyme of apoptosis, is activated in Chlamydia-infected cells remains unknown. Here, we established HEp-2 cells stably expressing cFluc-DEVD, which is a caspase-3 substrate sequence inserted into cyclic firefly luciferase, and then monitored the dynamics of caspase-3 activity in cells infected with Chlamydia. Transfected cells without infection showed a significant increase in luciferase activity due to stimulation with staurosporine, an inducer of apoptosis. Activation was significantly blocked by addition of caspase inhibitor z-VAD-fmk. Furthermore, as expected, Chlamydia infection caused a significant increase in luciferase activation at 36-48 h postinfection with a contrastive decrease at 24 h postinfection, which is already well known. Such activation caused by the infection was much stronger when the amount of bacteria was increased. Thus, caspase-3 activation was accurately monitored by the luciferase activity in HEp-2 cells constitutively expressing the cFluc-DEVD probe. Furthermore, our data showed that C. trachomatis activates caspase-3 in host cells at a late stage of infection.

Photo-Activatable Akt Probe: A New Tool to Study the Akt-Dependent Physiopathology of Cancer Cells.

Akt is commonly overexpressed and activated in cancer cells and plays a pivotal role in cell survival, protection, and chemoresistance. Therefore, Akt is one of the target molecules in understanding characters of cancer cells and developing anticancer drugs. Here we examined whether a newly developed photo-activatable Akt (PA-Akt) probe, based on a light-inducible protein interaction module of plant cryptochrome2 (CRY2) and cryptochrome-interacting basic helix-loop-helix (CIB1), can regulate Akt-associated cell functions. By illuminating blue light to the cells stably transfected with PA-Akt probe, CRY2-Akt (a fusion protein of CRY2 and Akt) underwent a structural change and interacted with Myr-CIBN (myristoylated N-terminal portion of CIB1), anchoring it at the cell membrane. Western blot analysis revealed that S473 and T308 of the Akt of probe-Akt were sequentially phosphorylated by intermittent and continuous light illumination. Endogenous Akt and GSK-3ß, one of the main downstream signals of Akt, were also phosphorylated, depending on light intensity. These facts indicate that photo-activation of probe-Akt can activate endogenous Akt and its downstream signals. The photo-activated Akt conferred protection against nutritional deprivation and H2O2 stresses to the cells significantly. Using the newly developed PA-Akt probe, endogenous Akt was activated easily, transiently, and repeatedly. This probe will be a unique tool in studying Akt-associated specific cellular functions in cancer cells and developing anticancer drugs.

Detection of Necroptosis in Ligand-Mediated and Hypoxia-Induced Injury of Hepatocytes Using a Novel Optic Probe-Detecting Receptor-Interacting Protein (RIP)1/RIP3 Binding.

Liver injury is often observed in various pathological conditions including posthepatectomy state and cancer chemotherapy. It occurs mainly as a consequence of the combined necrotic and apoptotic types of cell death. In order to study liver/hepatocyte injury by the necrotic type of cell death, we studied signal-regulated necrosis (necroptosis) by developing a new optic probe for detecting receptor-interacting protein kinase 1 (RIP)/RIP3 binding, an essential process for necroptosis induction. In the mouse hepatocyte cell line, TIB-73 cells, TNF-α/cycloheximide (T/C) induced RIP1/3 binding only when caspase activity was suppressed by the caspase-specific inhibitor z-VAD-fmk (zVAD). T/C/zVAD-induced RIP1/3 binding was inhibited by necrostatin-1 (Nec-1), an allosteric inhibitor of RIP1. The reduced cell survival by T/C/zVAD was improved by Nec-1. These facts indicate that T/C induces necroptosis of hepatocytes when the apoptotic pathway is inhibited/unavailable. FasL also induced cell death, which was only partially inhibited by zVAD, indicating the possible involvement of necroptosis rather than apoptosis. FasL activated caspase 3 and, similarly, induced RIP1/3 binding when the caspases were inactivated. Interestingly, FasL-induced RIP1/3 binding was significantly suppressed by the antioxidants Trolox and N-acetyl cysteine (NAC), suggesting the involvement of reactive oxygen species (ROS) in FasL-induced necroptotic cellular processes. H2O2, by itself, induced RIP1/3 binding that was suppressed by Nec-1, but not by zVAD. Hypoxia induced RIP1/3 binding after reoxygenation, which was suppressed by Nec-1 or by the antioxidants. Cell death induced by hypoxia/reoxygenation (H/R) was also improved by Nec-1. Similar to H2O2, H/R did not require caspase inhibition for RIP1/3 binding, suggesting the involvement of a caspase-independent mechanism for non-ligand-induced and/or redox-mediated necroptosis. These data indicate that ROS can induce necroptosis and mediate the FasL- and hypoxia-induced necroptosis via a molecular mechanism that differs from a conventional caspase-dependent pathway. In conclusion, necroptosis is potentially involved in liver/hepatocyte injury induced by oxidative stress and FasL in the absence of apoptosis.

Type XVII collagen coordinates proliferation in the interfollicular epidermis.

Type XVII collagen (COL17) is a transmembrane protein located at the epidermal basement membrane zone. COL17 deficiency results in premature hair aging phenotypes and in junctional epidermolysis bullosa. Here, we show that COL17 plays a central role in regulating interfollicular epidermis (IFE) proliferation. Loss of COL17 leads to transient IFE hypertrophy in neonatal mice owing to aberrant Wnt signaling. The replenishment of COL17 in the neonatal epidermis of COL17-null mice reverses the proliferative IFE phenotype and the altered Wnt signaling. Physical aging abolishes membranous COL17 in IFE basal cells because of inactive atypical protein kinase C signaling and also induces epidermal hyperproliferation. The overexpression of human COL17 in aged mouse epidermis suppresses IFE hypertrophy. These findings demonstrate that COL17 governs IFE proliferation of neonatal and aged skin in distinct ways. Our study indicates that COL17 could be an important target of anti-aging strategies in the skin.

Relevance of FXR-p62/SQSTM1 pathway for survival and protection of mouse hepatocytes and liver, especially with steatosis.

BACKGROUND: Liver injury and regeneration involve complicated processes and are affected by various physio-pathological conditions. Surgically, severe liver injury after surgical resection often leads to fatal liver failure, especially with some underlying pathological conditions such as steatosis. Therefore, protection from the injury of hepatocytes and liver is a serious concern in various clinical settings. METHODS: We studied the effects of the farnesoid X receptor (FXR) on cell survival and steatosis in mouse hepatocytes (AML12 mouse liver cells) and investigated their molecular mechanisms. We next studied whether or not FXR improves liver injury, regeneration and steatosis in a mouse model of partial hepatectomy (PH) with steatosis. RESULTS: An FXR-specific agonist, GW4064, induced expressions of the p62/SQSTM1 gene and protein in AML12 mouse liver cells. Because we previously reported p62/SQSTM1 as a key molecule for antioxidation and cell survival in hepatocytes, we next examined the activation of nuclear factor erythroid 2-related factor-2 (Nrf2) and induction of the antioxidant molecules by GW4064. GW4064 activated Nrf2 and subsequently induced antioxidant molecules (Nrf2, catalase, HO-1, and thioredoxin). We also examined expressions of pro-survival and cell protective molecules associated with p62/SQSTM1. Expectedly, GW4064 induced phosphorylation of Akt, expression of the anti-apoptotic molecules (Bcl-xL and Bcl-2), and reduced harmful hepatic molecules (Fas ligand and Fas). GW4064 promoted hepatocyte survival, which was cancelled by p62/SQSTM1 siRNA. These findings suggest the potential relevance of the FXR-p62/SQSTM1 pathway for the survival and protection of hepatocytes. Furthermore, GW4064 induced the expression of small heterodimer partners (SHP) and suppressed liver X receptor (LXR)-induced steatosis in hepatocytes, expecting the in vivo protective effect of FXR on liver injury especially with steatosis. In the hepatectomy model of db/db mice with fatty liver, pre-treatment by GW4064 significantly reduced post-PH liver injury (serum levels of LDH, AST & ALT and histological study) and improved steatosis. The key molecules, p62/SQSTM1, Nrf2 and SHP were upregulated in fatty liver tissue by GW4064 treatment. CONCLUSIONS: The present study is the first to demonstrate the relevance of FXR-p62/SQSTM1 and -SHP in the protection against injury of hepatocytes and post-PH liver, especially with steatosis.

Development for the measurement of serum thiosulfate using LC-MS/MS in forensic diagnosis of H2S poisoning.

Thiosulfate measurement is crucial to diagnosis of hydrogen sulfide (H2S) poisoning in forensic toxicology. Although GC-MS method is currently regarded as a standard thiosulfate measurement, it requires complicated sample preparation prior to analysis. This study presents a simple, rapid, and highly sensitive method for the quantitative analysis of serum thiosulfate by using liquid chromatography-tandem mass spectrometry (LC-MS/MS). This method is based on selected reaction monitoring and has high sensitivity with a lower quantification limit of 0.5µM. Precision and accuracy of this method meet the basic requirements for quantitative analysis (intra- and inter-day tests have a relative standard deviation of ⩽10.4%; range of analytical recovery is 94.3-102.6%). On the measurements of serum thiosulfate by our developed method, a thiosulfate concentration as 57.5µM was detected clearly in the H2S poisoning case comparing to the non poisoning case in which only a trace amount of thiosulfate was observed.