| Objectiven-Hexane is a widely used industrial organic solvent in processes for the extraction of edible oils,as raw materials for glue and paint-making,and as cleaning agents in the electronics manufacturing.Chronic n-hexane exposure could induce occupational poisoning accidents,characterized as a central-peripheral distal axonopathy.Initial clinical manifestations include weakness,paresthesia,and hypoesthesia,followed by progressive weekness and areflexia in the distal limbs.If severe,patients might develop sequelae of muscle wasting or even paralysis.The first case of n-hexane poisoning was discovered in Italy in shoemaking workers.Similar outbreaks of neuropathy were subsequently reported in Japan,the United States,Canada,South Korea,Brazil and other countries.There has been an increased number of reported cases of n-hexane induced peripheral neuropathy in Chinasince the first case was reported in 1970s.Metabolism studies have suggested that the axonopathy is mediated by its toxic metabolite,2,5-hexanedione(2,5-HD),but its molecular mechanism remains unclear.And there are few prevention and control measures to be proposed.Generally,with symptomatic treatment,it takes half a year or a year to restore health.Serious cases may even become permanently disabled.As n-hexane induced neuropathy mainly developed in young,it not only seriously damaged the patient's physical and mental health,but also the family and society.Therefore,the development of safe and effective drugs to prevent n-hexane induced axonal degeneration have a very important theoretical and practical significance.Garlic is a popular herbal product that has been applied for centuries for medicinal purposes.It has a variety of pharmacological activities such as anticancer,antimicrobial,antiatherosclerotic,antidiabetic,and immunomodulatory effects.Diallyl trisulfide(DATS)is the major phytochemical component contained in garlic and garlic products such as garlic essential oil.Previous studies have revealed that DATS has excellent antioxidant capacity and can attenuate many chemicals—induced oxidative damage in liver and also in nervous system.DATS also plays a regulatory role in a variety of P450 enzymes,which plays an important role in the metabolic activation of n-hexane.DATS may have the potential to prevent n-hexane induced neuropathy.Cystamine(Cys)is a linear aliphatic diamine composed of a disulfide bridge.It has been demonstrated that cystamine and its metabolite cysteamine exhibited protective effects in several models of neurodegenerative diseases including Huntington's disease,Parkinson's disease and amyotrophic lateral sclerosis(ALS).The exact mechanism by which cystamine operates the neuroprotective effects is controversial.It can inhibit caspase-3 and transglutaminase activity,act as an antioxidant,stimulate brain-derived neurotrophic factor(BDNF)secretion,and upregulate heat shock proteins(HSPs).These potentially involved mechanisms are also related to the pathway of toxicant induced neuropathy.In the present study,the protective effect of DATS and cystamine on n-hexane induced neuropathy was expored.The model of n-hexane peripheral nerve injury was established by oral administration of n-hexane and 2,5-HD,with DATS and cystamine co-treatment,And the neuroprotective mechanisms of DATS and cysteamine were discussed from the aspects of metabolism and molecular biology respectively.Finally,the combination of DATS and cystamine co-treatment effect was also evaluated.Methods1.DATS attenuated behavioral deficiency in n-hexane induced neurotoxiciy.72 Wistar rats,weighing 220?240 g were randomly divided into 6 groups,i.e.control group,n-hexane group,DATS group,and 3 DATS plus n-Hexane groups(n =12 in each group).Rats in n-hexane plus DATS groups were administrated with DATS(10,20 or 30 mg/kg/day,orally)combined with n-hexane(3 g/kg/day,orally)seven times per week for consecutive 8 weeks.The rats in n-hexane group were treated with corn oil and n-hexane(3 g/kg/day,orally)and rats in DATS group were treated with DATS(30 mg/kg/day,orally)and corn oil.The rats in control group received equal volume of vehicle.Forelimb and hind limb grip strength were measured every two weeks.Prior to the rats being sacrificed,gait scores were also obtained.Six rats randomly selected from n-hexane group and 3 DATS plus n-hexane groups were used to determine the long-term effect of DATS on n-hexane toxicokinetics.Blood samples were collected from the jugular vein every 2 h after n-hexane exposure.Free 2,5-HD and pyrrole adducts in serum were detected and analysed with DAS 3.0 for the toxicokinetics parameters.Tissue pyrrole adduts were also measured.Total protein extract was obtained from liver and detected the CYP1A1?CYP1A2?CYP2B1/2 and CYP2E1 expression by western blot,which relate to n-hexane metabolism.The liver microsomal fractions were also prepared and measured the activities of CYP1A1?CYP1A2,CYP2B1/2 and CYP2E1.2.Cystamine attenuated behavioral deficiency in 2,5-HD induced neurotoxiciy.50 Wistar rats were randomly divided into 5 groups(n = 10),that is,control group,2,5-HD group,cystamine group(60 mg/kg bw),and 2 Cys/2,5-HD groups(30 and 60 mg/kg bw).The rats in 2,5-HD group and Cys/2,5-HD groups were treated with 300 mg/kg bw 2,5-HD orally.The animals in other two groups received equal volume of vehicle.Cys was administrated to rats in Cys group and Cys/2,5-HD groups by intraperitoneal injection,while animals in other two groups received equal volume of sterile saline.All groups of animals were treated for continuous 6 weeks(6 times per week).Gaits scores and accelerating rota-rod were measured every week.Luxol fast blue were used to evaluated the sciatic nerve damage.Serum BDNF level were detected by Elisa.BDNF mRNA level in spinal card were investigated by RT-PCR.BDNF,PI3K,p-PI3K,Akt,p-Akt and Hsp-70 were detected by western blot.3.Combined effect of DATS and cystamine in n-hexane induced neuropathy.40 Wistar rats were randomly divided into 4 groups,i.e.n-hexane group,DATS group,cystamine group and combined intervention group(n = 10 in each group).Rats in DATS group and combined intervention group were administrated with DATS(1 30 mg/kg/day,orally).Cystamine was administrated to rats in Cystamine group and combined intervention group by intraperitoneal injection(60 mg/kg/day,i.p.).Each group were treated with n-hexane(3 g/kg/day,orally).Accelerating rota-rod and tail euroelectrophysiologic parameters were measured every two weeks.Results1.DATS attenuated behavioral deficiency in n-hexane induced neurotoxiciy.1.1 Effect of DATS on n-hexane induced peripheral neuropathyn-Hexane exposure led to significant peripheral neuropathy,shown by the decrease of grip strength and increase of gait scores.Before n-hexane exposure,all rats exhibited normal,unaffected grip strength.By the end of 8 weeks' exposure,rats exposed to n-hexane exhibited a significantly grip strength lost compared with the control group,which was significantly suppressed by DATS(20 and 30 mg/kg)co-treatment.Parallelly,20 and 30 mg/kg DATS also inhibited the increase of gait scores induced by n-hexane.1.2 DATS co-treatment led to the decrease of serum concentration of 2,5-HD and pyrrole adductsCompared with the rats in n-hexane group,the AUC0-t value of 2,5-HD in rats of DATS(10,20 and 30 mg/kg)groups were decreased by 3.3%,17.1%and 37.4%(P<0.05),respectively,while the AUC0-t of pyrrole adducts were reduced by 6.9%,7.0%and 26.5%(P<0.05),respectively.The Cmax and Cmin of 2,5-HD and pyrrole adducts were also suppressed by the DATS co-treatment.1.3 Tissue distribution of pyrrole adductsThe nervous system had higher adducts levels after adjusting by protein concentration,compared with the pyrrole adducts levels in the liver and the kidney.DATS(20 and 30 mg/kg)co-administrate led to a significant decline of the pyrrole adducts levels in the brain,spinal cord and sciatic nerves.1.4 Effects of DATS and n-hexane on the protein levels and activities of hepatic cytochrome P450s in rats.n-Hexane exposure resulted in the dramatic increase of the activities of CYP1A1,CYP1A2,CYP2B1 and CYP2E1.Compared with the control group,the activities of CYP1A1,CYP1A2,CYP2B1 and CYP2E1 in n-hexane group were increased by 113%,64%,274%and 79%,respectively(P<0.05).In DATS group,the activities of CYP2E1 were decrease by 47%,while the activities of CYP1A1 and CYP2B1 were increased by 44%and 114%.The changes of protein levels of the above four phase I enzymes were paralleled with those of the activities.The protein level of CYP2E1 showed a dose-dependent decrease in rats treated with 10,20 and 30 mg/kg DATS.The expression changes of CYP2B1 induced by n-hexane were suppressed by mega dose DATS co-treatment.The expression of CYP1A1 was upregulated after DATS co-treatment but CYP1A2 was not changed significantly.Meanwhile,DATS co-treatment also significantly inhibited the increases of the activities of CYP2E1 and CYP2B1 and increased the activity of CYP1A1.2.Cystamine attenuated behavioral deficiency in 2,5-HD induced neurotoxiciy.2.1 Effects of cystamine on 2,5-HD induced neurobehavioral changes Gait ChangesRats in control group showed normal gait(gait score:1.00±0.00),while the rats in 2,5-HD group showed obvious motor deficits(gait score:3.70 ± 0.48)at the end of the 6 weeks.Interestingly,cystamine co-treatment profoundly suppressed 2,5-HD-induced motor deficits.Compared with rats in 2,5-HD group,the cystamine co-treated animals were able to walk normally although they moved slowly than the rats in control group.The gait scores of the rats in 30 and 60 mg/kg co-treatment groups were increased to 2.80±0.63 and 2.3±0.48,respectively,with significant difference compared to that of the 2,5-HD group(P<0.01).Accelerating Rota-rodThe performance of rats in control and cystamine groups rose steadily over the whole test period.The latency to fall of the 2,5-HD intoxicated rats was significantly decreased compared to control rats since the 1st week(P<0.01).Compared with rats in 2,5-HD group,the duration of stay was significantly prolonged since the 5th week in low dose cystamine group and the 2nd week in high dose cystamine group,respectively.In the last week of test,rats in 2,5-HD group could not stay on the rod;however,the rats in cystamine group were able to stay on the rod for 32.0 ± 11.8 s and 43.0 ± 8.5 s in the lower cystamine group and higher cystamine group,respectively..2.2 Cystamine co-treatment increased the expression of BDNFCompared with 2,5-HD group,serum BDNF of the rats in 30 and 60 mg/kg co-treatment groups were increased by 18.1%(P<0.05)and 38.9%(P<0.01).The level of BDNF mRNA in spinal cord were 2.1 and 2.7 times as much as that in 2,5-HD group.Spinal cord precursor-BDNF expressions were also increased by 8.2%and 29.3%(P<0.01),while mature-BDNF expressions were were increased by 107.2%(P<0.05)and 300.5%(P<0.01).2.3 Cystamine co-treatment led to the activation of PI3K/Akt signaling pathway and increase expression of Hsp-70Compared with those of the control group rats,the ratio of p-PI3K/t-PI3K in rats of 2,5-HD group was decreased by 34.7%(P<0.01),while the ratio of p-Akt/t-Akt was decreased by 32.2%(P<0.01).Compared with those of the 2,5-HD group rats,the ratio of p-PI3K/t-PI3Kand p-Akt/t-Akt in cystamine and co-treated group was significantly increased.The protein level of Hsp-70 was not significantly affected by 2,5-HD compared with control group.Cystamine treated alone significantly increased Hsp-70 expression.Compared with the 2,5-HD group,the protein levels of Hsp-70 in cystamine co-treated group was increased by 21.1%and 39.7%(P<0.01),respectively.3.Combined effect of DATS and cystamine in n-hexane induced neuropathyThe latency to fall of rats in the DATS group was significantly different compared to control rats since the 6th week,while latency in the cystamine and the combined intervention group was significantly different since the 4th week.Compared to rats in the DATS group and cystamine group,rats in the combined intervention group showed better performance since the 4th and 6th week,respectively.In the last week of test,latency of rats in the combined intervention group increased by 95.2%and 60.0%to that of DATS and cystamine rats.The tail NCV of rats in the DATS,cystamine and combined intervention group group were significantly different compared to control rats since the 6th,6th and 4th week,respectively.Compared to that in the DATS group and cystamine group,NCV in the combined intervention group showed better performance since the 4th week.In the last week of test,tail NCV of rats in the combined intervention group increased by 26.3%and 22.5%to that of DATS and cystamine rats.Conclusions1.DATS protected the rats from n-hexane-induced neurotoxicity,which might be attributed to the modulation of P450 enzymes that decreased the internal 2,5-HD and pyrrole adducts.2.Cystamine could attenuate behavioral deficiency induced by 2,5-HD and the mechanism might be related to the increase in the BDNF and Hsp-70 expression,together whith activation of the PI3K/Akt signaling pathway.3.Combined intervention with DATS and cystamine is more efficient to attenuate n-hexane neurotoxicity. |
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