| ObjectiveCarbon disulfide (CS2) is an industrial organic solvent widely used in the manufacture of viscose fiber, cellophane, carbon tetrachloride etc. Occupational exposure to CS2 can cause damage to the nervous system of workers, mainly as peripheral neuropathy. However, its pathogenic mechanism remains unclear. CS2 poisoning can lead to oxidative stress, and more and more evidence of animal experiments and human studies indicate that oxidative stress is closely related to a variety of neurodegenerative diseases such as Parkinson’s disease. Therefore, we speculate that oxidative stress may relate to the pathogenic mechanism of CS2. Also, Keapl-Nrf2 signaling pathway is the major mechanism in the cellular defense against oxidative or electrophilic stress. In the present study we’d like to develop a neurotoxicological rat model subchronically exposed to CS2 by gavage and changes of Keapl-Nrf2 signaling pathway-associated protein expression are determined by Western blot. Then we can investigate whether it can cause changes in the signaling pathway or not and its possible role in the pathogenesis of CS2 to provide a theoretical basis for the future mechanism study.MethodsHealthy adult male Wistar rats weighing 300-330g, were randomly divided into control group,200,400 and 600mg/kg CS2 experimental groups (15 rats per group) after adaptive feeding for one week. CS2 was dissolved in corn oil and administered to rats in experimental groups at 2ml/kg by gavage for six consecutive weeks (six times per week), while the rats in control group were given the equivalent volume of corn oil by gavage. General state of rats was observed every day and particular attention was paid to the tremor phenomenon. The rats’body weight was weighed and recorded at the third day and the seventh day of every week. Gait score was conducted and recorded at the seventh day too. Rats were sacrificed after the last exposure with nerve tissues separated. The levels of Keapl-Nrf2 signaling pathway-associated proteins in the spinal cord and sciatic nerve were determined by Western blot.Results1. Impact of CS2 exposure on body weight of Rats:During the entire experimental period, body weights of rats in the control group increased steadily and increased by 28.69%compared with the initial weight at the end of the experiment. Compared with the control group, body weights of rats showed a slower increase in experimental groups and showed statistically significant differences from the forth week, the second week and the first week at exposure doses of 200,400 and 600mg/kg, respectively (P<0.05).2. Impact of CS2 exposure on gait of rats:From the beginning to the end of the experiment, there weren’t any obvious abnormalities of gait at dose of 200mg/kg. The gait score was 1 point. Abnormal gait changes began to appear from the forth week at dose of 400mg/kg. At the end of the experiment, one half of the rats got a score of 3 points, showing gross ataxia, abdomen or glide away, tumble etc. Abnormal gait started from the third week at dose of 600mg/kg and got a mean score of 3.33 points at the end of the experiment. About one third of the rats got 4 points, showing paralysis, unable to support weight and difficulties in moving ahead.3. Impact of CS2 exposure on the contents of Keapl-Nrf2 signaling pathway-associated proteins in the spinal cord:Under exposure to CS2, the content of Nrf2 in the spinal cord nuclear fraction was significantly increased. Compared with the control group, it was increased by 59.26% and 93.30% at exposure doses of 400 and 600mg/kg, respectively. The differences were statistically significant (.P<0.05). The content of cytosolic Nrf2 was decreased by 18.11% at dose of 600mg/kg compared with the control group (P<0.05). The level of Keap1 was reduced by 28.76% and 40.52% at exposure doses of 400 and 600mg/kg, respectively. The differences were statistically significant (P<0.05). The content of NQO1 showed an increasing trend, but there was significant change only under exposure to 600mg/kg (P<0.05). No significant change was observed in the level of p62 (P>0.05). Compared with p62, the content of p-p62 was increased by 21.52%,31.82% and 34.20% in three experimental groups, respectively. The differences were statistically significant (P<0.05).4. Impact of CS2 exposure on the contents of mTORC1 complex and Calcineurin in the spinal cord:Given CS2 treatment, the level of mTOR in the spinal cord was increased by 18.93%,23.42% and 32.82% in the three experimental groups, respectively, compared with the control group. The content of raptor was also increased at exposure doses of 400 and 600mg/kg by 23.83% and 27.95%, respectively. All the differences were statistically significant (P<0.05). There was no significant change in the content of Calcineurin (P>0.05).5. Impact of CS2 exposure on the contents of Keapl-Nrf2 signaling pathway-associated proteins in the sciatic nerve:After CS2 exposure, compared with the control group, the content of Nrf2 in total protein of the sciatic nerve showed significant increases by 60.15%,74.54% and 76.46% in the three experimental groups, respectively. The differences were statistically significant (P<0.05). The level of Keap1 experienced significant decrease only under exposure to 600mg/kg (P<0.05). Significant increases were also observed in the content of NQO1 by 79.52%,144.58% and 119.28% at three doses, respectively (P<0.05). The level of p62 was increased by 34.26%,71.32% and 75.89% at exposure doses of 200,400 and 600mg/kg, respectively and the content of p-p62 was increased by 91.53%,169.25% and 174.12% in the three experimental groups, respectively. All the differences were statistically significant (P<0.05).6. Impact of CS2 exposure on the contents of mTORC1 complex and Calcineurin in the sciatic nerve:Under exposure to CS2, there was no significant change in the level of mTOR in the sciatic nerve (P>0.05), while the content of raptor experienced significantly increases by 182.45% ,214.89% and 217.02% in the three experimental groups, respectively (P<0.05). The content of Calcineurin also increased significantly by 57.50%,83.79% and 85.92% in the three experimental groups, respectively (P<0.05).Conclusion1. Exposure to CS2 can activate the Keap1-Nrf2 signaling pathway, but whether sustained activation of Nrf2 is beneficial or detrimental to the organism is still need further research.2. CS2 maybe activated Keap1-Nrf2 signaling pathway in a p62-dependent manner.3. Phosphorylation of p62 may be associated with mTORCl complex and Calcineurin. |
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