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Autophagy is believed to be essential for the maintenance of axonal homeostasis in neurons. However, whether autophagy is causally related to the axon degeneration in organophosphorus-induced delayed neuropathy (OPIDN) still remains unclear. This research was designed to investigate the role of autophagy in axon degeneration following tri-ortho-cresyl phosphate (TOCP) in an in vitro model. Differentiated wild-type and Atg7−/− neuro-2a (N2a) cells were treated with TOCP for 24 h. Axonal degeneration in N2a cells was quantitatively analyzed; the key molecules responsible for axon degeneration and its upstream signaling pathway were determined by Western blotting and real-time PCR. The results found that Atg7−/− cells exhibited a higher resistance to TOCP insult than wild-type cells. Further study revealed that TOCP caused a significant decrease in pro-survival factors NMNATs and SCG10 and a significant increase in pro-degenerative factor SARM1 in both cells. Notably, Atg7−/− cells presented a higher level of pro-survival factors and a lower level of pro-degenerative factors than wild-type cells in the same setting of TOCP administration. Moreover, DLK-MAPK pathway was activated following TOCP. Altogether, our results suggest that autophagy is able to affect TOCP-induced axonal injury via regulating the balance between pro-survival and pro-degenerative factors, providing a promising avenue for the potential therapy for OPIDN patients.

Recently, we read with great interest the article published in Cell by Yu et al, which reported that TAR DNA-binding protein 43 (TDP-43) overexpression stimulated the released of mitochondrial DNA (mtDNA) into the cytoplasm through mitochondrial permeability transition pore. Then, mtDNA bound to cyclic GMP-AMP synthase (cGAS) to activate the cGAS/STING pathway, which caused the inflammation of amyotrophic lateral sclerosis (ALS).

Tri-ortho-cresyl phosphate (TOCP) is a typical organophosphorus compound that can cause organophosphate-induced delayed neuropathy (OPIDN), which is pathologically characterized by axonal degeneration. Nowadays, mitochondrial dysfunction is regarded as a potential mechanism contributing to OPIDN progress. Mitophagy, a selective type of autophagy, is required to segregate damaged mitochondria from healthy mitochondrial networks and deliver them to lysosome for degradation. This research was designed to investigate the role of mitophagy in axon degeneration following TOCP administration in an in vitro model. Differentiated neuro2a (N2a) cells were divided into four groups and treated with 0, 5, 10, and 20 μM TOCP for 24 h, respectively. The critical proteins in PINK1-Parkin-dependent mitophagy including LC3, P62, PINK1, Parkin, mitochondrial proteins, and autophagic receptors were detected by immunoblotting and immunofluorescence. After TOCP treatment, increased level of ROS in N2a cells revealed a significant mitochondria damage. Meanwhile, it was observed that much more PINK1, Parkin, and LC3-II were translocated to the mitochondria. Furthermore, immunofluorescence analysis demonstrated that the co-localization of Parkin and LC3 was significantly increased. These results suggested that PINK1-Parkin dependent mitophagy pathway in N2a cells was activated by TOCP treatment. In addition, P62, a major autophagic receptor, was markedly accumulated on the mitochondria, which indicated that P62 might play a critical role in facilitating mitophagy under TOCP-induced axonal degeneration. Taken together, our results suggest that TOCP exposure resulted in the activation of PINK1-Parkin-dependent mitophagy in N2a cells. Mitophagy may act as a positively reactive mode in eliminating dysfunctional mitochondria and therefore protect neurons against TOCP neurotoxicity.

Tri-ortho-cresyl phosphate (TOCP) is an extensively used organophosphate in industry. It has been proven to lead to toxicity in different organ systems, especially in the nervous system. Neural stem cells (NSCs) play important roles in both embryonic and adult nervous systems. However, whether TOCP induces cytotoxicity in embryonic NSCs remains unclear. In this study, mouse NSCs were exposed to different concentrations of TOCP for 24 h. The results showed that TOCP led to impaired proliferation of NSCs and induced the autophagy of NSCs by increasing the generation of intracellular reactive oxygen species (ROS) and decreasing the phosphorylation of extracellular regulated protein kinase (ERK1/2). Melatonin has been reported to exert neuroprotective effects via various mechanisms. Therefore, we further investigate whether melatonin has potential protective effects against TOCP-induced cytotoxicity on NSCs. Our data showed that melatonin pretreatment attenuated TOCP-induced autophagy by suppressing oxidative stress and restoring ERK1/2 phosphorylation consistently. Taken together, the results indicated that TOCP induced the autophagy in mouse NSCs, and melatonin may effectively protect NSCs against TOCP-induced autophagy.

Mitochondrial dysfunction was considered as a critical event involved in acetaminophen (APAP)-induced acute liver injury. Mitophagy is a type of autophagy responsible for the selective removal of damaged mitochondria. However, the exact role and possible mechanism of mitophagy in APAP-induced hepatotoxicity remains largely unknown. In this study, C57/BL6 mice were used to establish a model of acute liver injury via intraperitoneal (i.p.) injection with different doses of APAP. Furthermore, autophagy intervention experiments were achieved by the administration of rapamycin (RAPA) or chloroquine (CQ) one hour prior to dosing 300 mg/kg APAP. The activity of serum enzymes and pathological changes of APAP-treated mice were evaluated, and the critical molecules in mitophagy and NLRP3 inflammasome pathway were determined by electron microscopy, immunoblot, immunofluorescence and real-time PCR. The results demonstrated that APAP overdose resulted in an activation of PINK1/Parkin-mediated mitophagy in mice liver. Moreover, the expression of the critical molecules in NF-kB and NLRP3 inflammasome signaling pathway were markedly increased by APAP. Our further investigation found that pretreatment with RAPA protected against APAP-induced hepatoxicity in mice. Notably, RAPA significantly inhibited the activation of NF-kB and NLRP3 inflammasome and the production of IL-1β in APAP-treated mice. By contrast, pretreatment with CQ further enhanced NLRP3 inflammasome signaling pathway. Taken together, these results indicated that activation of PINK1/Parkin-mediated mitophagy protects against APAP-induced acute liver injury in mice through inhibiting inflammasome activation. Therefore, mitophagy may represent a promising therapeutic target for APAP-induced liver injury.

Chronic exposure to carbon disulfide (CS 2 ) can induce polyneuropathy in occupational worker and experimental animals, but underlying mechanism for CS 2 neuropathy is currently unknown. In the present study, male Wistar rats were randomly divided into three experimental groups and one control group. The rats in experimental groups were treated with CS 2 by gavage at dosages of 200, 400 and 600 mg/kg/day respectively, six times per week for 6 weeks. The formation of autophagosomes and lysosomes in motor neurons of rat spinal cord was observed by transmission electron microscopy, the level of autophagy-related proteins, lysosome-associated membrane protein 1 (LAMP-1), and cathepsin B in spinal cord tissues was determined by Western blot analysis, and the activity of cathepsin B was measured by fluorescence assay. The results demonstrated that the number of lysosomes in motor neurons was markedly increased in CS 2 -treated rats. In the meantime, the administration of CS 2 significantly increased the level of microtubule-associated protein light chain 3-II (LC3-II), Atg1, UVRAG and LAMP-1 in rat spinal cord. Furthermore, the content and activity of cathepsin B in rat spinal cord also showed a significant elevation. Taken together, this study suggested that CS 2 intoxication was associated with the activation of lysosomal degradative machinery, which might play a protective role against CS 2 -induced neuronal damage.

The formation of pyrrole adducts might be responsible for peripheral nerve injury caused by n-hexane, but there is not an effective biomarker for monitoring occupational exposure of n-hexane. The current study was designed to investigate the changes of pyrrole adducts in serum and urine of rats exposed to 2,5-hexanedione (2,5-HD) and analyze the correlation between pyrrole adducts and 2,5-HD. Two groups of male Wistar rats (n = 8) were administered a single dose of 200 and 400 mg/kg 2,5-HD (i.p.), and another two groups (n = 8) were given daily dose of 200 and 400 mg/kg 2,5-HD (i.p.) for 5 days. Pyrrole adducts and 2,5-HD in serum and urine were determined, at different time points after dosing, using Ehrlich’s reagent and gas chromatography, respectively. The levels of pyrrole adducts in serum accumulated in a time-dependant manner after repeated exposure to 2,5-HD, while pyrrole adducts in urine, and 2,5-HD in serum and urine were kept stable. The half-life times (t 1/2) of 2,5-HD and pyrrole adducts in serum were 2.27 ± 0.28 and 25.3 ± 3.34 h, respectively. Furthermore, the levels of pyrrole adducts in urine were significantly correlated with the levels of 2,5-HD in serum (r = 0.736, P

Occupational exposure to carbon disulfide (CS2) exhibits central nervous systems toxicity. But the mechanism is unclear. The present study was designed to investigate the relationship between the CNS damage and cognitive dysfunction caused by CS2, and eventually reveal the possible oxidative-related mechanism of hippocampus pathological changes in CS2 exposed rats. Male Wistar rats were administrated with CS2 at dosage of 200, 400 and 600 mg/kg for consecutive 20 days, respectively. Cognitive performances were evaluated by Morris water maze tests. Thionin and immunohistochemical analysis were used to investigate the hippocampal neuron damage, and the expression of apoptosis related proteins (cleaved-caspase 3, Bax and Bcl-2) were detected to explore the possible mechanisms of neuronal loss. Oxidative stress parameters were checked by commercial assay kits. Rats exposed to CS2 displayed cognitive dysfunction manifested as decreased spatial learning ability and memory lesion. Pathological changes and significant neuron loss were observed in hippocampus, especially in CA1 and CA3 sub-regions. Mitochondria-dependent apoptosis pathway was implicated in the CS2-induced neuronal loss which was demonstrated by the up-regulation of cleaved-caspase 3 and Bax accompanied with down-regulation of Bcl-2. Furthermore, extensive oxidative stress induced by CS2 was also revealed by the measurement of ROS, RNS, MDA, GSH&GSSG and antioxidant enzymes (CAT, T-SOD, and GSH-Px). Our study suggested that oxidative stress mediated hippocampal neuron apoptosis might play an important role in CS2 induced CNS damage and cognitive dysfunction.

Oxidative stress is associated with the pathogenesis of carbon disulfide (CS2) induced polyneuropathy. The nuclear factor erythroid 2-related factor 2 (Nrf2) plays a critical role in protecting cells against oxidative stress. However, whether there exists a Nrf2-mediated antioxidative machinery in CS2-induced neuropathy has not been elucidated. In the present study, male wistar rats were randomly divided into three experimental groups and one control group. The rats in experimental groups were treated with CS2 by gavage at dosages of 200, 400 and 600mg/kg/day respectively, six times per week for 6 weeks. Nrf2-keap1 antioxidative pathway and p62-related kinase signaling in rat nerve tissues was examined by western blotting and real-time PCR. The results demonstrated that CS2 treatment resulted in Nrf2 translocation from the cytosol to the nucleus in rat spinal cords. In the meantime, the expression of antioxidative enzymes such as NAD(P)H quinone oxidoreductase-1, heme oxygenase-1, and glutamate-cysteine ligase was significantly increased. Furthermore, CS2 treatment increased the level of p62 and its phosphorylation status, while decreased the level of keap1. In addition, CS2 also lead to the activation of CAMKK2 and ULK1 kinase signaling in rat spinal cords and sciatic nerves. Taken together, our results indicated that CS2 intoxication was associated with the activation of Nrf2-ARE antioxidative machinery, which might play a protective role against CS2-induced neuronal damage.

A few organophosphorus compounds (OPs) can cause toxic neuropathy known as organophosphorus ester-induced delayed neuropathy (OPIDN). Although the incidents of OPIDN have been documented for over a century, its molecular mechanisms underlying the axonopathy are still unclear. Recently, increasing evidences suggest that proteases are closely associated with OPIDN. Herein, we have summarized the roles of calcium-dependent cysteine proteases (calpains) in OPIDN. The activation of calpains should be an early molecular event during the onset and development of OPIDN. However, the understanding of the mechanism underlying the disruption of Ca(2+) homeostasis and the activation of calpain by neurotoxic OPs is still limited. Therefore, a better understanding of molecular mechanisms that can prevent the disturbance in cellular Ca(2+) homeostasis can facilitate to establish the novel therapeutic strategies for OPIDN.

Accumulating evidences support the important roles of sterol regulatory element-binding protein-1 (SREBP-1) activation in ethanol-induced fatty liver, but the underlying mechanisms for its activation are not fully understood. Recent studies have demonstrated that phosphatidylinositol 3 kinase (PI3K)/Akt pathway activation could enhance SREBP-1 activity. The current study was designed to investigate the potential roles of PI3K/Akt pathway in acute ethanol-induced fatty liver in mice. In the first experiment, mice were treated with ethanol (2.5 or 5 g/kg bw) or isocaloric/isovolumetric maltose-dextrin solution, and sacrificed at several time points after ethanol exposure. As expected, ethanol dose-dependently increased the hepatic triglyceride (TG) levels and the protein levels of the mature form of SREBP-1 (n-SREBP-1). The phosphorylation of Akt and glycogen synthase kinase-3β (GSK-3β) was significantly increased in mice treated with ethanol (5 g/kg bw), while the protein levels of PI3K-p85 were significantly reduced. To confirm the roles of PI3K/Akt pathway, mice were then pretreated with wortmannin (0.7 or 1.4 mg/kg bw), a specific PI3K/Akt pathway inhibitor, before exposure to ethanol. Interestingly, a dual effect of wortmannin was observed. Low dose of wortmannin significantly reduced the hepatic TG levels, while high dose of wortmannin aggravated ethanol-induced fatty liver. The ratio of LC3II/LC3I of wortmannin (1.4 mg/kg bw) group mice was significantly increased, while the p62 protein level was significantly decreased compared to those of ethanol group, which indicated that wortmannin (1.4 mg/kg bw) might suppress the lipid degradation by autophagy. These results supported the hypothesis that PI3K/Akt activation might be involved in acute ethanol-induced fatty liver, and PI3K/Akt inhibitors might have therapeutic potential for the treatment of ethanol-induced fatty liver.

Tri-ortho-cresyl phosphate (TOCP) can cause toxic neuropathy known as organophosphate-induced delayed neuropathy (OPIDN), which is pathologically characterized by the swollen axon containing aggregations of neurofilaments, microtubules, and multivesicular vesicles. Autophagy is a self-degradative process which plays a housekeeping role in removing misfolded proteins and damaged organelles. The current study was designed to investigate the possible roles of autophagy in the pathogenesis of OPIDN. Adult hens were treated with a dose of 750 mg/kg TOCP by gavage, or injected subcutaneously with 60 mg/kg phenylmethanesulfonyl fluoride (PMSF) dissolved in DMSO 24 h earlier and subsequently treated with TOCP, then sacrificed on the time-points of 0, 1, 5, 10, and 21 days after dosing of TOCP respectively. The levels of beclin-1 and μ-calpain in tibial nerves and spinal cords were determined by immunoblotting. The results showed that in both tissues TOCP increased the expression of μ-calpain while decreased that of beclin-1. When given before TOCP administration, PMSF pretreatment could protect hens against the delayed neuropathy. In the meantime, pretreatment with PMSF reduced calpain expression below basal and increased beclin-1 expression above basal in tibial nerve, whereas it simply returned calpain and beclin-1 expression to their basal levels in spinal cord. In conclusion, the intoxication of TOCP was associated with a significant change of beclin-1 in hen nervous tissues, which suggested that disruption of autophagy-regulated machinery in neurons might be involved in the pathogenesis of OPIDN.

Tri-ortho-cresyl phosphate (TOCP) can cause a type of neurotoxicity known as organophosphate-induced delayed neuropathy (OPIDN). The characteristic axonal swelling containing aggregations of neurofilaments, microtubules, and multivesicular vesicles is consistent with a disturbance of axonal transport. We hypothesized that there existed a disturbance of molecular motor in the pathogenesis of OPIDN. In the present study, adult hens were treated with a dosage of 750 mg/kg TOCP by gavage, or pretreated 24h earlier with phenylmethanesulfonyl fluoride (PMSF) and subsequently with TOCP, then sacrificed on the time-points of 0, 1, 5, 10, and 21 days after dosing of TOCP, respectively. The level of kinesin-1, dynein, and dynactin in spinal cords and cerebral cortexes of hens was determined. Immunoblotting analysis showed a progressive decline of dynein and dynactin in spinal cords after dosing TOCP. Furthermore, a significant reduction in dynactin and dynein was observed in cerebral cortexes at several time-points post dosing TOCP. In contrast, no significant changes of kinesin-1 were observed throughout the period of experiment. When given before TOCP administration, PMSF could inhibit TOCP-induced motor protein disruption, while it protected hens against the delayed neuropathy. In conclusion, the reduction of the motor proteins, dynein and dynactin, might be associated with the disruption of retrograde neuronal axonal transport in OPIDN.

Neurofilaments (NFs), the most abundant cytoskeletal components in large neurons and myelinated axons, are the targets of n-hexane-induced neuropathy, in which a specific loss of NFs protein has been frequently observed. However, the precise mechanisms regulating NFs contents are not well understood. The aim of this study was to elucidate the role of ubiquitin-proteasome system (UPS) in NFs degradation. We first demonstrated that the E3 ligase carboxyl-terminus of Hsc70 interacting protein (CHIP), originally identified as a co-chaperone of Hsc70, directly interacted with NFs medium chain (NF-M) and then enhanced NF-M ubiquitination and degradation after 2,5-hexanedione (HD) treatment. Consistent with this result, the application of proteasome inhibitor MG132 partly reversed HD-induced decrease of NF-M. Finally, we found that other components of UPS system (e.g. ubiquitin-activating enzyme E1, CHIP and proteasome) were significantly increased in sciatic nerve of HD-intoxicated rats. In conclusion, this study indicated that the CHIP ubiquitin ligase complex interacted with and repressed NFs by targeting NFs for ubiquitin-mediated proteolysis, which led to reduction of NFs contents in HD-induced neuropathy.

Tri-ortho-cresyl phosphate (TOCP), an organophosphorus ester, is capable of producing organophosphorus ester-induced delayed neuropathy (OPIDN) in human being and sensitive animals. In the present study, adult hens were treated with TOCP by gavage at single dosage of 750 mg/kg, and sacrificed by decapitation on the corresponding time points of 1, 5, 10, and 21 day post-dosing, respectively. The tibial nerves were dissected, homogenized, and centrifuged at 100,000 × g . The level of neurofilaments protein in both pellet and supernatant fractions was determined. Western blot analysis showed a nearly depletion of NF-M and a dramatic decrease of NF-L in both fractions of tibial nerves. These changes were observed within 24 h of TOCP administration and then followed by an obvious recovery. In contrast, a progressive reduction in NF-H was observed in tibial nerves of TCOP-treated hens throughout the period of experiment. With the reduction of NF-L level, the rate of NF-L degradation demonstrated a significant increase in both fractions of tibial nerves. Furthermore, the expression of μ-calpain in tibial nerves was increased following TOCP. Taken together, these results demonstrated that NFs changes occurred much earlier than the clinical appearance of ataxia in TOCP-induced delayed neuropathy, indicating that disruption of NF homeostasis in peripheral nerves might be an early molecular event in the development of OPIDN.

Chronic exposure to carbon disulfide (CS2) can induce polyneuropathy in occupational worker and experimental animals, but underlying mechanism for CS2 neurotoxicity is currently unknown. In the present study, male Wistar rats were randomly divided into two experimental groups and one control group. The rats in two experimental groups were treated with CS2 by gavage at dosages of 300 and 500 mg/kg per day, respectively, five times per week for 12 weeks. The contents of neurofilament triplet proteins (NF-H, NF-M, NF-L) and two calpain isoforms (m-calpain and u-calpain) in sciatic nerves were determined by immunoblotting. In the meantime, the mRNA levels of NF-H, NF-M and NF-L in spinal cords were quantified by reverse transcriptase-polymerase chain reaction, and the total activity of calpains in sciatic nerves was measured by fluorescence assay. Results showed that the contents of NF-M and NF-L in CS2-treated rats sciatic nerves increased significantly except NF-M in low dose group. The contents and activity of m-calpain and u-calpain in sciatic nerve also demonstrated a significant elevation. Furthermore, the levels of mRNA expression of NFH, NFM and NFL genes were up-regulated consistently in spinal cords of treated rats. These findings suggested that CS2 intoxication was associated with the disruption of neurofilaments homeostasis and activiation of calpains in rat sciatic nerves, which might be involved in the development of CS2-induced peripheral neuropathy.

To investigate the reversibility of the neuropathy induced by 2,5-HD, adult male rats were administered at a dosage of 400 mg/kg/day 2,5-HD (five times per week) for 2, 4, and 8 weeks, respectively. After stopping HD exposure, half of 8-week treated animals were allowed to naturally recover for 16 weeks. The relative levels of NF-H, NF-M, and NF-L in spinal cords and sciatic nerves of rats were determined by immunoblotting during the HD neuropathy. The results showed that NFs content in nerve tissues demonstrated a progressive decline as the intoxication continued. Furthermore, after a recovery of 16 weeks, the levels of three NF subunits in spinal cords of treated rats returned to normal while those in sciatic nerves displayed an inconsistent reversal. Among them, the level of NF-H in sciatic nerves returned to normal completely, and NF-L also showed a significant improvement, whereas NF-M did not demonstrate an obvious reversal. These findings suggest that HD-induced NFs decline is at least partially irreversible within the time frame of this study, which might be associated with the incomplete recovery of neurological dysfunctions of HD-treated rats.

To investigate the mechanism of carbon disulfide-induced neuropathy, male wistar rats were administrated by gavage at dosage of 300 or 500 mg/kg carbon disulfide, five times per week for 12 weeks. By the end of the exposure, the animals produced a slight or moderate level of neurological deficits, respectively. Cerebrums of carbon disulfide-intoxicated rats and their age-matched controls were Triton-extracted and centrifuged at a high speed (100,000 x g) to yield a pellet fraction of NF polymer and a corresponding supernatant fraction, which presumably contained mobile monomer. Then, the contents of six cytoskeletal protein (NF-L, NF-M, NF-H, alpha-tubulin, beta-tubulin, and beta-actin) in both fractions were determined by immunoblotting. Results showed that the contents of the three neurofilament subunits in the pellet and the supernatant fraction decreased significantly regardless of dose levels (P0.01). As for microtubule proteins, in the pellet fraction of cerebrum, the levels of alpha-tubulin and beta-tubulin demonstrated some inconsistent changes. However, in the supernatant fractions, the content of alpha-tubulin and beta-tubulin increased significantly in both two dose groups (P0.01). In comparison to neurofilament and tubulin proteins, the content of beta-actin changed less markedly, only the supernatant fraction of the high dose group displayed significant increase (P0.01), but the others remained unaffected. These findings suggested that the changes of cytoskeleton protein contents in rat cerebrum were associated with the intoxication of carbon disulfide, which might be involved in the development of carbon disulfide neurotoxicity.

Chronic exposure to n-hexane induces peripheral-central axonopathy, mediated by its metabolite 2,5-hexanedione (2,5-HD), in occupational workers and experimental animals, but the underlying mechanism is still unclear. In the current study, we investigated the effects of 2,5-HD on middle-molecular-weight neurofilament (NF-M) axonal transport using live-cell imaging technique in cultured rat dorsal root ganglia (DRG) cells. PA-GFP-NF-M plasmid was transfected into DRG neurons and live-cell imaging was performed to observe the slow axonal transport of NF-M. The levels of cytoskeleton and motor proteins in DRG cells were detected by Western-blot and the concentration of ATP was determined using an ATP Assay Kit. The results showed that 2,5-HD administration resulted in a decrease of NF-M axonal transport and a reduction of three neurofilament subunits levels in DRG cells. Furthermore, 2,5-HD exposure significantly decreased ATP contents and the protein levels of kinesin heavy chain (KHC). These findings indicated that 2,5-HD reduced slow axonal transport, neurofilaments cargoes, motor proteins and ATP energy in rat DRG cells, which may contribute to 2,5-HD-induced neurotoxicity.

Autophagy is a highly conserved cellular self-degradative process that plays a housekeeping role in removing aggregated proteins and damaged organelles. Our recent work has found that tri-ortho-cresyl phosphate (TOCP), a neuropathic organophosphate (OP), decreased the level of beclin 1 (a key molecule in the process of autophagy) in hen nerve tissues (Song et al., 2012). However, the role of autophagy in the pathogenesis of organophosphorus ester-induced delayed neuropathy (OPIDN) remains unclear. Here, we investigated whether dysfunctional autophagy was associated with the initiation and development of TOCP-induced delayed neuropathy. Adult hens were given a single dose of 750mg/kg TOCP (p.o.) and sacrificed on days 1, 5, 10, and 21 after dosing, respectively. The formation of autophagosomes in spinal cord motor neurons was observed by transmission electron microscopy, the level of autophagy-related proteins in hen spinal cords and tibial nerves was determined by Western blot analysis. The results demonstrated that the number of autophagosomes was markedly increased in the myelinated and unmyelinated axons of hen spinal cords after TOCP exposure. In the meantime, the level of two molecular markers for autophagy, microtubule-associated protein light chain-3 (LC3) and p62/SQSTM1 in hen nerve tissues was significantly decreased and increased, respectively. Furthermore, a marked reduction in autophagy-regulated proteins including ULK 1, AMBRA 1, ATG 5, ATG 7, ATG 12 and VPS34 expression was also observed. Our results suggested that the administration of TOCP resulted in a significant inhibition of autophagy activity in neurons, which might be associated with the pathogenesis of OPIDN.

Background Diallyl disulfide (DADS) is a garlic-derived organosulfur compound. The current study is designed to evaluate the protective effects of DADS against ethanol-induced oxidative stress, and to explore the underlying mechanisms by examining the HO-1/Nrf-2 pathway. Methods We investigated whether or not DADS could activate the HO-1 in normal human liver cell LO2, and then evaluated the protective effects of DADS against ethanol-induced damage in LO2 cells and in acute ethanol-intoxicated mice. The biochemical parameters were measured using commercial kits. HO-1 mRNA level was determined by RT-PCR. Histopathology and immunofluorescence assay were performed with routine methods. Protein levels were measured by western blot. Results DADS significantly increased the mRNA and protein levels of HO-1, stimulated the nuclear translocation of Nrf-2 and increased the phosphorylation of MAPK in LO2 cells. The nuclear translocation of Nrf-2 was abrogated by MAPK inhibitors. DADS significantly suppressed ethanol-induced elevation of lactate dehydrogenase (LDH) and aspartate transaminase (AST) activities, decrease of glutathione (GSH) level, increase of malondialdehyde (MDA) levels, and apoptosis of LO2 cells, which were all blocked by ZnPPIX. In mice, DADS effectively suppressed acute ethanol-induced elevation of aminotransferase activities, and improved liver histopathological changes, which might be associated with HO-1 activation. Conclusion These results demonstrate that DADS could induce the activation of HO-1/Nrf-2 pathway, which may contribute to the protective effects of DADS against ethanol-induced liver injury. General significance DADS may be beneficial for the prevention and treatment of ALD due to significant activation of HO-1/Nrf-2 pathway.

Occupational exposure to n-hexane produces a central–peripheral distal axonopathy, which is characterized by giant axonal swellings filled with neurofilaments (NFs). To investigate the change of NFs degradation and their possible role in n-hexane neuropathy, adult male Wistar rats were administered intraperitoneally at a dosage of 400 mg/kg/day 2,5-hexanedione (2,5-HD) for 4 weeks. The time course of low-molecular-weight neurofilament (NF-L) degradation and autophagy-related protein in rat sciatic nerves and spinal cords was determined by Western blotting. The results demonstrated that the administration of 2,5-HD inhibited NF-L degradation to an undetectable level in sciatic nerves. Furthermore, a significant reduction of NF-L degradation in spinal cords was observed in the early stage of 2,5-HD exposure. In the meantime, 2,5-HD significantly decreased the level of Beclin-1, a key autophagy-regulated protein in sciatic nerves of rats while increased the level of P62, a selective substrate of autophagy degrading pathway, which indicated a dysfunctional autophagy in rat nerve tissues. Collectively, our findings suggested that the inhibition of autophagy by 2,5-HD might be responsible for the reduction of NF-L degradation in rat sciatic nerves, and involved in the pathogenesis of 2,5-HD-induced axonopathy.

Garlic oil (GO) has been shown to partially attenuate ethanol-induced fatty liver, but the underlying mechanisms remain unclear. The current study was designed to evaluate the protective effects of GO against ethanol-induced steatosis in vitro and in vivo, and to explore potential mechanisms by investigating the sterol regulatory element binding protein-1c (SREBP-1c), peroxisome proliferators-activated receptor-α (PPAR-α), cytochrome P4502E1 (CYP2E1), and etc. In the in vitro study, human normal cell LO2 was exposed to 100 mM ethanol in the presence or absence of GO for 24 h. We found that ethanol increased the protein levels of n-SREBP-1c and CYP2E1, but decreased the protein levels of PPAR-α, which was significantly attenuated by GO co-treatment. In the in vivo study, male Kun-Ming mice were pretreated with single dose of GO (50-200 mg/kg body weight) at 2 h before ethanol (4.8 g/kg body weight) exposure. The changes of n-SREBP-1c, PPAR-α and CYP2E1 were paralleled well to those of in vitro study. Furthermore, GO significantly reduced the protein levels of fatty acid synthase (FAS), and suppressed ethanol-induced hepatic mitochondrial dysfunction. These results suggested that GO had the potential to ameliorate alcoholic steatosis which might be related to its modulation on SREBP-1c, PPAR-α, and CYP2E1.

BACKGROUND: Inconsistent results were obtained for the lipid-regulating effects of garlic in clinical trials. With increasing interest in complementary medicine for hyperlipoidemia, it is important to explore the real effects of garlic. This meta- analysis was performed to investigate the influence of garlic on serum lipid parameters. RESULTS: A total of 26 studies were included into meta-analysis. Overall, garlic was superior to placebo in reducing serum total cholesterol (TC) and triglyceride (TG) levels. Compared with the placebo groups, serum TC and TG levels in the garlic group were reduced by 0.28 (95% CI, − 0.45, − 0.11) mmol L−1 (P = 0.001) and 0.13 (95% CI, − 0.20, − 0.06) mmol L−1 (P < 0.001), respectively. The effects of garlic were more striking in subjects with long-term intervention and higher baseline TC levels. Garlic powder and aged garlic extract were more effective in reducing serum TC levels, while garlic oil was more effective in lowering serum TG levels. In contrast, garlic did not influence other lipid parameters, including low-density lipoprotein cholesterol, high-density lipoprotein cholesterol (HDL-C), apolipoprotein B, and TC/HDL-C ratio. CONCLUSION: Garlic could reduce serum TC and TG levels, and garlic therapy should benefit patients with risk of cardiovascular diseases. Copyright © 2012 Society of Chemical Industry

Tri-ortho-cresyl phosphate (TOCP) is an organophosphorus ester, which can cause a type of neurotoxicity known as organophosphate-induced delayed neuropathy (OPIDN). Our recent study has shown that the enhanced degradation of neurofilament (NF) in peripheral nerve of hens is an early event of TOCP-induced OPIDN (Song et al., 2009). The main objective of this investigation is to study the effect of TOCP administration on NF content and NF degradation when OPIDN is blocked by pretreatment with phenylmethylsulfonyl fluoride (PMSF). The hens were pretreated 24h earlier with PMSF and subsequently treated with a single dosage of 750 mg/kg TOCP, then sacrificed on the corresponding time points of 0, 1, 5, 10, and 21 days after dosing TOCP, respectively. The tibial nerves were dissected, homogenized, and centrifuged at 100,000 x g. The level of NF triplet protein in both pellet and supernatant fractions of tibial nerves was determined. Western blotting analysis showed a significant increase of three NF subunits in hens treated with PMSF and TOCP compared with the control. These changes were observed within 24h of PMSF administration and then followed by an obvious recovery. Furthermore, accompanied with the increase of NF content, a significant decline in NF-L degradation rate was observed in both fractions of tibial nerves. Taken together, these results demonstrated the pretreatment with PMSF could inhibit TOCP-induced NF degradation while it protected hens against the development of OPIDN, which suggested the inhibition of NF-associated protease in peripheral nerves might be an underlying protective mechanism of PMSF against OPIDN.

To investigate the mechanisms and biomarker of the neuropathy induced by 2,5-hexanedione (HD), male Wistar rats were administrated HD at dosage of 200 or 400mg/kg for 8 weeks (five-times per week). All rats were sacrificed after 8 weeks of treatment and the cerebrum cortex (CC), spinal cord (SC) and sciatic nerves (SN) were dissected, homogenized and used for the determination of cytoskeletal proteins by western blotting. The levels of neurofilaments (NFs) subunits (NF-L, NF-M and NF-H) in nerve tissues of 200 and 400mg/kg HD rats significantly decreased in both the supernatant and pellet fractions. Furthermore, significant negative correlations between NFs levels and gait abnormality were observed. As for microtubule (MT) and microfilament (MF) proteins, the levels of alpha-tubulin, beta-tubulin and beta-actin in the supernatant and pellet fraction of SN significantly decreased in 200 and 400mg/kg HD rats and correlated negatively with gait abnormality. However, the contents of MT and MF proteins in CC and SC were inconsistently affected and had no significant correlation with gait abnormality. The levels of NF-L and NF-H in serum significantly increased, while NF-M, alpha-tubulin, beta-tubulin and beta-actin contents remain unchanged. A significant positive correlation (R=0.9427, P

To investigate the mechanisms of the axonopathy induced by 2,5-hexanedione (2,5-HD), male Wistar rats were administered at a dosage of 400 mg/kg/day 2,5-HD (five times per week). The rats produced a slightly, moderately, or severely abnormal neurological changes, respectively, after 2, 4, or 8 weeks of treatment. The cerebrums were Triton-extracted and ultracentrifuged to yield a pellet fraction and a corresponding supernatant fraction. The relative levels of six cytoskeletal proteins (NF-L, NF-M, NF-H, alpha-tubulin, beta-tubulin, and beta-actin) in both fractions were determined by immunoblotting. The results showed that NFs content in HD-treated rats demonstrated a progressive decline as the intoxication of HD continued. As for microtubule proteins, the levels of alpha-tubulin and beta-tubulin demonstrated some inconsistent changes. The content of alpha-tubulin kept unchangeable, while the content of beta-tubulin increased significantly at the late stage of HD exposure. Furthermore, the content of beta-actin in both fractions remained unaffected throughout the study. These findings suggest that HD intoxication resulted in a progressive decline of NFs, which was highly correlated with the development of HD-induced neuropathy.

Allyl chloride (AC) is widely used in industries as raw material and has been reported to produce occupational peripheral neuropathies in man chronically exposure to it. Although many studies have been done addressing to it, the mechanisms still remain unclear. To elucidate the molecular mechanism of neuropathy induced by AC, we measured the contents of glutathione (GSH), Bcl-2, Bax, cytochrome c (CytC) and Caspase-3 in a time-dependent manner by biochemical and quantitative immunoblotting techniques in rats' cerebrum and spinal cord after 3, 6, 9 and 12 weeks of AC intoxication. The results showed that the levels of Bcl-2 of cerebrum and spinal cord significantly (P

To investigate the mechanism of carbon disulfide-induced neuropathy, male Wistar rats were randomly divided into two experimental groups and one control group. The rats in two experimental groups were treated with carbon disulfide by gavage at dosages of 300 and 500 mg/kg/day, respectively, five times per week for 12 weeks. Spinal cords of carbon disulfide-intoxicated rats and their age-matched controls were Triton-extracted and ultracentrifuged to yield a pellet fraction of neurofilament (NF) polymer and a corresponding supernatant fraction. Then, the contents of NF triplet proteins (NF-H, NF-M, NF-L) and two calpain isoforms (m-calpain and mu-calpain) in both fractions were determined by immunoblotting. In the meantime, the mRNA levels of NF-H, NF-M, and NF-L in spinal cords were quantified using reverse transcriptase-polymerase chain reaction. Results showed that in the pellet fraction, the contents of three NF subunits in both treated groups decreased significantly except NF-L in low dose group. In the supernatant fraction, the pattern of NFs alteration varied according to dose-levels. Compared to controls, three neurofilmant subunits in the high dose group displayed significant reduction consistently. However, in the low dose group, they remained unaffected. As for calpains, the contents of mu-calpain in both fractions increased significantly regardless of carbon disulfide dose-levels. Meanwhile, m-calpain demonstrated a significant decline in the supernatant fraction, and remained unchangeable in the pellet fraction compared to the control group. Furthermore, the levels of mRNA expression of NF-H, NF-M, and NF-L genes were elevated consistently in CS(2)-treated groups. These findings suggested that carbon disulfide intoxication was associated with obvious alterations of NFs content in rat spinal cord, which might be involved in the development of carbon disulfide neurotoxicity.