Study On The Protective Effects Of Thioredoxin Inducers On Formaldehyde-and Morphine-induced Toxicity

Thioredoxin-1(Trx-1) is a12-KDa multifunctional protein with a redox-active disulfide/dithiol within its active site sequence,-Cys-Gly-Pro-Cys-. Trx-1has various biological activities, including the regulation of cellular redox balance, the activation of transcription factors, the inhibition of cellular apoptosis and the modulation of inflammation. Trx-1is induced in response to various stresses, such as viral infections, hydrogen peroxide, ultraviolet and x-ray irradiation, mitogens, phorbol myristate acetate and ischemic reperfusion. It has been reported that Trx-1acts as a neurotrophic co-factor which augments the effect of nerve growth factor on neuronal differentiation and regeneration. Trx-1transgenic mice display elongated life span and resistance against ischemic injury, diabetes and toxicity caused by environmental stressors. Trx-1is also reported as a modulator of endoplasmic reticulum (ER) stress. Recently, many studies have used Trx-1inducers, such as geranylgeranylacetone (GGA), sulforaphane, neurotropin and temocapril, for therapeutic purposes. Among them, the protective effects of GGA are attracting more attention. GGA is an acyclic polyisoprenoid and widely used as an anti-ulcer drug in clinic. Many studies suggest that GGA up-regulates Trx-1expression in various cells. GGA can exert cytoprotective effects against oxidative stress in the gastric mucosa, liver and heart. It also has been reported that GGA is a lipid-soluble reagent and can easily pass the blood-brain barrier to exert neuroprotective effects. A body of evidence indicates that Trx-1provides cytoprotection against oxidative stress and modulation of Trx-1expression may represent a novel strategy for therapeutic intervention. A view that the non-cytotoxic pharmacological inducers of Trx-1are beneficial for neurogeneration and protection against oxidative stress-associated neural diseases has been presented. Therefore, the present study intends to find non-toxic Trx-1inducers from the natural medicine resources in Yunnan province to treat diseases.Formaldehyde (FA) is a member of the aldehyde family. It is a common environmental pollutant found in household products, exhaust gas, cigarette smoke, and many other medical and industrial products. Recent reports have shown that there was a relationship between indoor FA concentrations and the sick building syndrome. FA has various toxic effects on the central nervous system and has been classified as "probable neurotoxicity". Long-term exposure to FA may cause neurotoxicity and result in neurodegenerative disorders. FA may cause a viariety of morphological changes in the brain of rat and result in behavior and memory disorders. Considering its serious threats to human health, FA has raised concern about public health. To prevent against FA-induced neurotoxic effects, the mechanisms on the noxious effects of FA should be elucidated.Endoplasmic reticulum (ER) is the site of synthesis and folding of proteins and has crucial functions involving modulation of intracellular calcium homeostasis and glycosylation. When ER function is disturbed and unfolded or misfolded proteins are accumulated in ER, response to ER stress is activated. The ER contains high levels of protein chaperones including the glucose regulated proteins (GRPs), which are ER molecular chaperones studied mostly. While ER stress occurs, the protein level of GRP78is up-regulated to increase the folding capacity of ER. Low-level ER stress activates anti-apoptotic ER chaperone protein, whereas severe ER stress causes cell death by activation of JNK, C/EBP homologous protein (CHOP) and caspase-12. ER stress is a contributory factor in neuronal death. Recent studies have suggested the effects of FA on protein misfolding and aggregation. As a cross-linking agent, FA reacts with thiol and amino groups, leads to protein polymerization. FA interacts with β-amyloid and produces cross-linked neurotoxic amyloid-like complexes. FA leads to tau aggregation, which in turn results in apoptosis of human neuroblastoma cells and hippocampal cells. This raises a question whether ER stress-mediated pathway is involved in neuronal death caused by FA.Morphine abuse is a serious clinical and social problem in world. Repeated use of morphine would cause the development of dependence, which limits its usage for chronic pain. Morphine dependence is a compulsive pattern of drug seeking and drug taking, resulting from the positive reinforcement of the rewarding effects of drug taking and the negative reinforcement of withdrawal syndrome that accompany cessation of drug taking. Although it has been known that anatomical substrates including prefrontal cortex, amygdale, ventral tegmental area, nucleus accumbens and hippocampus are related with morphine dependence, the cellular and molecular mechanisms underlying these remain largely unknown. Furthermore, prolonged exposure to opioid drugs can result in pathological changes in the liver in nearly100%of cases. Drug addicts suffered from acute hepatitis in44%, chronic hepatitis in34%and liver damage in16%. Lately, clinical studies also suggest that opiate addicts are at increased risk for progressive renal failure. Renal diseases in opiate addicts are associated with nephritic syndrome, acute glomerulonephritis and interstitial nephritis. The acute or chronic administration of opioid drugs can cause oxidative damage and cellular apoptosis in liver and kidney, and hence result in hepatic and renal damage. Considering the role of liver and kidney in metabolism and detoxification, the liver and kidney toxicity caused by opioid drugs has been absorbed more and more attention in the therapy. In addition, there are few efficacious interventions to treat morphine dependence.Oxidative stress, apoptosis and the down-regulation of neurotrophic factors are involved in formaldehyde-and morphine-induced toxicities. Trx-1has been shown to have antioxidant, anti-apoptosis and neurotrophic activities. Based on the association between the biological activities of Trx-1and the mechanisms underlying formaldehyde-and morphine-induced toxicities, two questions are proposed:whether Trx-1inducers could protect neural cells against formaldehyde-toxicities; whether Trx-1inducers could protect against morphine addiction and morphine-induced liver and kidney toxicity. Thus, the present study was designed to examine these questions.The main results were as follows:(1) Many products of panax notoginseng saponins (PNS) are commercially available and widely applied for the clinical medicine in China. PNS are composed of panaxadiol saponins and panaxatriol saponins (PTS). PTS was obtained from Kunming Pharmaceutical Corporation. Ginsenoside Rgl, R1and Re are the main components of PTS (about80%), and the content of ginsenoside Rgl is up to60%. Trx-1expression in PC12cells was significantly elevated in response to PTS in a dose-dependent and time-dependent manner. Furthermore, western blot analysis clearly showed up-regulation of Trx-1expression in the cerebral cortex, striatum and hippocampus of mice treated with PTS compared with those treated with saline only. Ginsenoside Rgl is the main component of PTS. In this study, we found that ginsenoside Rgl induced Trx-1expression in PC12cells in a dose-dependent manner.(2) It has been widely reported that endoplasmic reticulum stress is a contributory factor in neuron death. In this study, FA treatment significantly increased the expression of GRP78. CHOP expression was increased after FA treatment12h and pro-caspase-12level was decreased after FA treatment24h. These data suggest that FA induces neurotoxicity partly via an ER stress-mediated pathway.(3) It has been reported that FA causes oxidative stress as a general toxic effect. Trx-1is up-regulated by oxidative stress and acts as an antioxidant. Trx-1was induced by FA in PC12cells after exposure to FA for12h. However, Trx-1expression was decreased after FA treatment24h in a dose-dependent manner. Furthermore, we found that50nM of Trx-1siRNA significantly reduced Trx-1expression. The inhibition of Trx-1expression by siRNA increased the susceptibility to FA-induced toxicity. These results suggest that suppression of Trx-1could contribute to FA-induced toxicity.(4) The over-expression of Trx-1is related to low ratio of cell death in response to oxidative stress. We previously reported that over-expression of Trx-1suppressed ER stress. Preliminary experiments demonstrated that ginsenoside Rgl had no significant effect on the viability of PC12cells. Pretreatment with ginsenoside Rgl prevented PC12cells against FA-induced neurotoxicity and rescued FA-induced down-regulation of tyrosine hydroxylase expression. In the present study, we also found that ginsenoside Rgl reversed the FA-induced down-regulation of Trx-1expression. The enhanced level of GRP78expression in response to FA was suppressed by pretreatment of PC12cells with ginsenoside Rgl. Furthermore, the pretreatment with ginsenoside Rgl inhibited FA-induced CHOP increase and the decrease of pro-caspase-12. These data suggest that Trx-1inducer has protective role against FA-induced neurotoxicity.(5) After repeated morphine treatments, Trx-1and heat shock protein70(Hsp70) expression were induced in the ventral tegmental area, nucleus accumbens, prefrontal cortex and hippocampus which are associated with drug addiction, suggesting that cellular stress responses are activated by morphine. Trx-1and Hsp70expression were up-regulated by GGA in the ventral tegmental area, nucleus accumbens, prefrontal cortex and hippocampus of mice. GGA reduced morphine-induced motor activity and suppressed conditioned place preference. In addiction, GGA significantly attenuated the morphine-naloxone-induced withdrawal signs including jumping, rearing and forepaw tremor. Furthermore, the activation of cAMP-responsive element-binding protein and the expression of ΔFosB and cyclin-dependent kinase5were decreased in the nucleus accumbens by GGA treatment after morphine withdrawal. In the nucleus accumbens, GGA enhanced morphine-induced expression of Trx-1and Hsp70after morphine withdrawal. These results suggest that strengthening the expression of Trx-1and Hsp70in brain by using non-cytotoxic pharmacological inducers may provide a novel therapeutic strategy for morphine dependence. GGA could be a safe and novel therapeutic agent for morphine dependence.(6) Morphine induced apoptosis in liver and kidney through mitochondria-mediated apoptosis pathway, but not ER-mediated pathway. The activation of caspase-9and caspase-3were attenuated by the pretreatment of GGA. In addition, the morphine-induced increase of malondialchehyche level was suppressed by GGA. Furthermore, GGA enhanced morphine-induced expression of Trx-1and Hsp70in liver and kidney. The results of this study suggest that GGA could be a safe and novel therapeutic agent for morphine-induced liver and kidney toxicity.In conclusion, we demonstrated that FA caused neurotoxicity partly via ER pathway. Ginsenoside Rg1induced Trx-1expression and protected against FA-induced neurotoxicity. In addition, GGA, a Trx-1inducer, protected against morphine addiction and morphine-induced liver and kidney toxicity. These findings may provide theoretical basis and a new target for the prevention of formaldehyde-and morphine-induced toxicities.