The Protective Effect And Mechanism Of Rutin On Acrylamide-induced Sciatic Nerve Injury In Rats

Acrylamide（ACR）is a water-soluble olefin and is widely used as an important chemical raw material for industrial synthesis of polyacrylamide.ACR has reproductive toxicity,embryo toxicity,developmental toxicity,neurotoxicity and so on.One investigation has shown that experimental animals exposed to low-concentration ACR environment for a long time will experience ataxia,numbness of limbs and skeletal muscle weakness.Decreasing in the speed of the peripheral nerve conduction signal,the decrease in the expression levels of myelin-related protein and the injury of peripheral nerve endings are the main pathological manifestations in ACR model.Rutin（Rut）belongs to flavonoids and mainly exists in a variety of plants and it has anti-oxidation,anti-cancer,cell protection,vascular protection and neuroprotective effect.In Clinical,Rut is mainly used to treat cerebral edema,hypertensive encephalopathy,cerebral hemorrhage,acute glomerulonephritis and other diseases.Studies have shown that Rut can significantly reduce the oxidative damage of PC12 cells caused by ACR.However,the effect of Rut on peripheral nerve damage caused by ACR is still uncertain.Therefore,this study intends to select L_4-6spinal cord,bilateral sciatic nerves and bilateral gastrocnemius muscles of rats as the research objects,aiming to explore whether Rut could protect the sciatic nerve damage caused by ACR poisoning in rats and its possible protective mechanism.Methods:1.Sixty SD male rats were randomly divided into control group（control）,ACR group（ACR）,Rut low（Rut-L+ACR）medium（Rut-M+ACR）and high（Rut-H+ACR）dose group.There are 12 rats in each group.The rats were given 100,200 and 400 mg/kg Rut suspensions and 20 mg/kg ACR solutions by intragastric administration for 21consecutive days.During this time,body weight of rats was recorded.2.Neurobehavioral tests:The gait score and hindlimb distance were measured once a week for each group.The gait score is used to evaluate the motor function of the rat,and the hindlimb distance measurement is used to evaluate the support of the hindlimb in rats.3.L_4-6 spinal anterior horn motor neurons were observed by HE staining and Nissl staining.The changes of sciatic nerve were observed by HE staining and Luxol Fast Blue staining,the morphology and structure of the end plates was observed by gold chloride staining.4.Immunohistochemistry and Western blot were used to detect changes of Ch AT and ACh E proteins in L_4-6 spinal cord,MBP protein in sciatic nerve and ACh E protein in the end plates.5.The microplate method was used to detect the expression levels of SOD,GSH and LDH in L_4-6 spinal cord and sciatic nerve.Western blot was used to detect the expression of P-ERK/ERK and Nrf2 proteins in L_4-6 spinal cord,sciatic nerve and end plates.6.HE staining was used to observe the morphological structure of the liver and kidney tissues in each group,in order to evaluate the safety of Rut dose.Results:1.During the period of intragastric administration,compared with the control group,the body weight of the rats in ACR group decreased since 15^th day（P<0.05）.2.The neurobehavioral results showed that ACR poisoning can cause abnormal gait in rats and weaken the support of the hind limbs,Rut can alleviate neurobehavioral abnormalities in rats.3.The results of HE staining and Nissl staining in L_4-6 spinal anterior horn motor neurons showed that ACR would cause uneven neuronal staining,shrink cell bodies,and reduce the number of Nissl bodies（P<0.01）.The neurons in the Rut dose group were uniformly stained,the morphology of the neuron cell bodies was regular,the structure was complete and clear and the number of Nissl bodies increased（P<0.01）.The results of HE staining and Luxol fast blue staining of the sciatic nerve showed that the ACR group was lightly stained,the nucleus was sparsely distributed,and the myelin lipid content was reduced.The Rut dose group stained evenly,the cell structure was intact,the arrangement was orderly and the myelin lipid content was increased.The results of gold chloride staining of the gastrocnemius motor endplate showed that ACR poisoning would cause the end of the gastrocnemius motor endplate to become thinner and reduce the attachment area of the motor endplate.The Rut-M+ACR group increased the attachment area of the motor endplate.4.The results of immunohistochemistry and Western blot showed that ACR infection caused a decreasing in Ch AT protein expression in L_4-6 spinal cord（P<0.01）and an increasing in ACh E protein expression（P<0.01）.The Rut dose group increased the expression level of Ch AT protein（P<0.05）and decreased the expression level of ACh E protein（P<0.01）,indicating that ACR would reduce the amount of neurotransmitter acetylcholine synthesized by motor neurons,and Rut could increase the synthesis of acetylcholine.So Rut has a protective effect on neuronal damage caused by ACR.The expression of MBP protein in the sciatic nerve of the ACR group decreased（P<0.01）and the expression of MBP protein in the Rut dose group increased（P<0.001）.This result indicates that Rut has a protective effect on the decrease in the expression of MBP protein in the sciatic nerve of rats caused by ACR.Western blot result of ACh E protein showed that ACh E protein expression increases in ACR group（P<0.01）and ACh E protein expression decreases in Rut dose group（P<0.01）.The result indicates that Rut can reduce decomposition amount of acetylcholine in the gastrocnemius motor endplate and it has a protective effect.5.The oxidative stress results of L_4-6spinal cord and sciatic nerve tissue showed that expression levels of SOD and GSH in the ACR group were significantly decreased（P<0.001）and expression level of LDH was significantly increased（P<0.01）.The expression levels of SOD and GSH in Rut group were increased（P<0.01）and LDH was decreased（P<0.01）.These results showed that Rut can reduce the oxidative stress.Western blot results showed that the expression of P-ERK/ERK protein and Nrf2 protein in the L_4-6 spinal cord,sciatic nerve and gastrocnemius motor endplate of rats in each group decreased（P<0.01）and the P-ERK/ERK protein and Nrf2 protein in Rut group.This result indicates that Rut may play a protective effect on ACR-induced sciatic nerve damage in rats through the ERK/Nrf2 signaling pathway.6.The results of HE staining of liver and kidney showed that the hepatocyte cords of rats in each group were arranged neatly,the structure of liver lobules was intact,and the cytoplasm and nucleus were normal;the structure of glomeruli and renal tubules of rats in each group was clear,no casts and particles were seen in the renal tubules,and the ratio of balloons was moderate.It shows that the dose of Rut has no toxic effect on liver and kidney tissues and it has security.Conclusions:1.ACR could cause sciatic nerve injury and Rut can alleviate the sciatic nerve injury in rats caused by ACR.It has a protective effect on the sciatic nerve injury.2.This mechanism may be related to the antioxidant effect of Rut,and Rut may exert its antioxidant effect through ERK/Nrf2 signaling pathway.