The Study Of Rutin Activating PI3K/AKT Pathway On Alleviating Acrylamide-induced Frontal Cortex Neuronal Injury In Rats

Objective:Acrylamide（ACR）is a recognized chemical toxic substance,which is widely stored in the environments of industrial production and scientific research laboratories.It has neurotoxicity on humans and experimental animals,mainly manifested as ataxia,skeletal muscle weakness and other motor dysfunction.Studies have shown that the main pathological features of ACR neurotoxicity include neuronal loss,synaptic damage,Nissl body reduction,and axonal swelling and degeneration.Telencephalic frontal cortex is the site of advanced motor center and it is very important to manage the autonomic movement of the body.It is of great practical significance to study the damage of ACR to the frontal cortex neurons and the protective measures.In recent years,more and more studies confirmed that oxidative stress might be one of the important mechanisms of ACR-induced neurotoxicity and involved in ACR-induced apoptosis.Rutin（Rut）is a natural flavonoid compound with a variety of pharmacological activities,such as antioxidant,anti-inflammatory,and neuroprotective.Previous study has shown that Rut can reduce ACR-induced PC12 cell death,and its protective effect may be related to antioxidant effects.It has been reported that PI3K/AKT signaling pathway is an important pathway to regulate cell survival,oxidation resistance and anti-apoptosis in some mechanisms of oxidative stress-induced neurotoxicity.Whether Rut can reduce ACR-induced injury to rat frontal cortex neurons by activating PI3K/AKT signaling pathway has not been reported yet.Therefore,in this experiment,the rat model of ACR poisoning was established,and Rut was given for intervention protection.Then,the PI3K/AKT signaling pathway inhibitor LY294002 was intraperitoneally injected to block the pathway,and the specific effect of PI3K/AKT signaling pathway was observed.The purpose of this study was to explore the protective effect of Rut on ACR-induced rat frontal cortex neuron injury,and whether its protective mechanism is related to the activation of PI3K/AKT signaling pathway,so as to screen new alternative drugs and find therapeutic targets for ACR-induced nerve injury.Method:1.Animal grouping and administration mode:36 adult male SD rats were randomly divided into 4 groups:（1）Control group:0.5%CMC-Na+dd H₂O（2）ACR group:0.5%CMC-Na+20 mg/kg ACR（3）ACR+Rut group:20 mg/kg ACR+200 mg/kg Rut（4）ACR+Rut+LY294002 group:20 mg/kg ACR+200 mg/kg Rut+1.2 mg/kg LY294002The rats were treated with intraperitoneal injection of LY294002 inhibitor in the morning for 10 min,followed by gavage with 0.5%CMC-Na or Rut in the afternoon,and gavage with dd H₂O or ACR in the afternoon for continuous 21 days.The frontal cortex of the rats was taken on the 22nd day for HE staining,Nissl staining,TUNEL staining,immunofluorescence staining,biochemical analysis of oxidative stress and Western blot.2.Neurobehavioral test:Behavioral tests were performed on days 0,7,14,and 21 of modeling.Gait scores were used to evaluate the changes of gait.Hind limb distance measurement to assess changes in hindlimb support.3.Protective effects of Rut on ACR-induced neuronal injury in the frontal cortex of rats:The morphological changes of frontal cortex cells were observed by HE staining and Nissl staining.The apoptotic neurons in the frontal cortex were detected by TUNEL and Neu N immunofluorescence double labeling staining.The expression levels of Neu N and GAP-43 in the frontal cortex were detected by Western blot.4.The possible mechanisms of protective effect of Rut on ACR-induced neuronal injury in rat frontal cortex:The immunofluorescence and Western blot were used to detect the expression of apoptosis-related proteins（Bcl-2,Bax,casepase-3 and cleaved caspase-3）.The biochemical analysis kit was used to detect the expression levels of SOD,GSH and LDH in the frontal cortex of rats.Western blot was used to detect the expressions of PI3K/AKT signaling pathway related proteins（PI3K,p-PI3K,AKT,p-AKT）and oxidative stress-related proteins（Nrf2,HO-1）.5.Statistical analysis:Statistical analysis was performed on the experimental data using SPSS 23.0 and Graph Pad Prism 8.0 software.One-way analysis of variance（One-way ANOVA）was used to analyze the difference among multiple groups,and all data were expressed as mean standard deviation（（?）±s）,where P<0.05 indicated that the difference was statistically significant.Result:1.Rut inhibits ACR-induced body weight loss in ratsWithin 7 days of modeling,the body weight gains of rats in each group were about the same.On the 15th day of modeling,compared with the Control group,the body weight of ACR group was decreased（P<0.05）.Compared with the ACR group,the body weight of the ACR+Rut group was increased（P<0.05）,and the body weight of rats treated with LY294002 inhibitor was decreased compared with that of the ACR+Rut group（P<0.01）.2.Rut can improve ACR-induced motor dysfunction in rats（1）Measurement of gait:On the 7th day of modeling,there was no significant change in the gait of rats in each group.Compared with the Control group,the ACR group had an increased gait score since the 14th day of modeling（P<0.01）;Compared with the ACR group,the gait score of the ACR+Rut group was decreased（P<0.01）,and the gait score of rats treated with LY294002 inhibitor was increased compared with that of the ACR+Rut group（P<0.05）.（2）Measurement of hind limb distance:On the seventh day of modeling,there was no significant change in the hind limb distance of rats in each group.Compared with the Control group,the hind limb distance in the ACR group was widened since the 14th day of modeling（P<0.01）.Compared with the ACR group,the hind limb distance between ACR+Rut groups was decreased（P<0.01）,and hind limb distance between rats treated with LY294002 inhibitor was wider than that of ACR+Rut group（P<0.05）.3.Rut has a protective effect on ACR-induced neuronal injury in the frontal cortex of rats（1）The results of HE staining and Nissl staining:Compared with the Control group,the frontal cortex cells in the ACR group and ACR+Rut+LY294002 group were decreased in size and karyopycnosis,and the Nissl body staining area was decreased（P<0.001）.However,under the protection of Rut intervention,the cell morphology in the ACR+Rut group was not significantly abnormal,and the Nissl body staining area was increased compared with that in the ACR group（P<0.01）.（2）TUNEL and Neu N immunofluorescence double labeling staining results:TUNEL labeled apoptotic cells,Neu N labeled neurons,and TUNEL and Neu N co-located,suggesting that neurons might be apoptotic.Compared with the Control group,the number of TUNEL positive cells in the ACR group was increased（P<0.001）.The number of TUNEL positive cells in the ACR+Rut group was decreased as compared with that in the ACR group（P<0.001）,while the number of TUNEL positive cells was increased after treatment with LY294002 inhibitor as compared with that in the ACR+Rut group（P<0.05）.（3）Western blot results of Neu N and GAP-43 in rat frontal cortex:Compared with the Control group,the expression levels of Neu N and gap-43 in ACR group were decreased（P<0.001）.Compared with the ACR group,the expression levels of Neu N and GAP-43 in the ACR+Rut group were increased（P<0.01）,while after treatment with the LY294002inhibitor,the expression levels of Neu N and GAP-43 were decreased compared with those in the ACR+Rut group（P<0.01）4.Rut inhibits ACR-induced apoptosis in rat frontal cortex cellsImmunofluorescence and Western blot results showed that,compared with the Control group,the ratio of Bcl-2 to Bax was decreased in the ACR group（P<0.001）,and the expression levels of caspase-3 and cleaved caspase-3 were increased in the ACR group（P<0.05）.Compared with ACR group,the ratio of Bcl-2 to Bax in ACR+Rut group was increased（P<0.001）,and the expression levels of caspase-3 and cleaved caspase-3 were decreased（P<0.05）.Compared with ACR+Rut group,the ratio of Bcl-2 to Bax was decreased（P<0.001）and the expression levels of caspase-3 and cleaved caspase-3 were increased（P<0.05）in ACR+Rut+LY294002 group.5.Rut reduces ACR-induced oxidative stress in the frontal cortex of ratsCompared with the Control group,the ACR group had higher LDH levels（P<0.001）,lower SOD and GSH levels（P<0.001）.Compared with ACR group,ACR+Rut group had lower LDH levels（P<0.001）,and higher SOD and GSH levels（P<0.001）.Compared with ACR+Rut group,the LDH level of ACR+Rut+LY294002 group was significantly increased（P<0.001）,and the SOD and GSH level were decreased（P<0.001）.6.Rut can activate PI3K/AKT signaling pathway in frontal cortex of rats exposed to ACRWestern blot results showed that the expression levels of p-PI3K,p-AKT,Nrf2,and HO-1 in the ACR group were lower than those in the Control group（P<0.01）.Compared with the ACR group,the expression levels of p-PI3K,p-AKT,Nrf2,and HO-1 in the ACR+Rut group were increased（P<0.01）,while the expression levels of p-PI3K,p-AKT,Nrf2,and HO-1 were decreased after treatment with the LY294002 inhibitor as compared with that in the ACR+Rut group（P<0.01）.Conclusion:Rut has a protective effect on ACR-induced neuronal injury in the frontal cortex of rats,which may be related to the activation of PI3K/AKT signaling pathway by Rut,up-regulation of the expression levels of Nrf2and HO-1,and alleviation of oxidative stress,thereby inhibiting ACR-induced neuronal apoptosis.