Protective Effect And Mechanism Of Lycium Ruthenicum Polyphenols Against Acrylamide-induced Neurotoxicity

Objective Acrylamide(ACR)is a white crystalline chemical widely used in oil extraction,water purification,and paper industry cases.ACR is a neurotoxic,genotoxic,reproductive toxic,and potentially carcinogenic substance.Previous studies have shown that oxidative stress is the main mechanism of in vivo toxicity induced by ACR.Therefore,inhibition of ACR-induced oxidative stress is an important way to prevent and control its toxicity.Polyphenols,widely found in dark fruits and vegetables,have strong antioxidant and anti-apoptotic effects.Lycium ruthenicum Murr.is distributed in northwest China and is important in traditional ethnic medicine.Modern pharmacological studies have shown that the anti-apoptosis,anti-free radical,and cognitive enhancement effects of Lycium ruthenicum Murr.are mainly due to its large amount of polyphenols,of which Lycium ruthenicum polyphenols(LRP)is the main component.Our previous studies also found that LRP can significantly improve the redox levels of cells and the liver through its good antioxidant and anti-apoptotic effects.Therefore,this study aims to establish a model of ACR-induced oxidative stress nerve damage in SD rats and evaluate the effects of LRP on the behavior,neurons in the substantia nigra and striatum,and oxidative stress in rats.To explore the protective effect of LRP on oxidative stress injury of rat striatum induced by ACR and its related mechanism.To further reveal polyphenols’ action and protective mechanism,a deeper understanding of their application potential in prevention and treatment is needed.Methods SD rats(6-8 weeks old,180-220 g)were used to develop an ACRinduced oxidative stress model in SD rats.Fifty-four SD rats were divided into six groups of nine rats each: Control group(saline),ACR group(40 mg/kg/d),and intervention group with a low,medium and high dose of LRP(50,100,200 mg/kg/d)+ ACR(40 mg/kg/d)and LRP control group(200 mg/kg/d)(all administered).First,pre-protection of each dose of LRP lasted for 5 days,and ACR was poisoned on the6 th day.At the same time,each dose of LRP or normal saline was given intragastric administration 1 hour before poisoning for 14 days.1.To investigate the effect of LRP on the substantial nigra-striatal of ACRinduced rats: behavioral experiments were performed on rats using gait scoring,open field experiments,and tape removal experiments.DA levels in the striatum and substantia nigra and their metabolites DOPAC and HVA were measured using ELISA.2.The effect of LRP on ACR-induced blood-brain barrier and DNA damage in the striatum of rats was investigated: S100β and ALB levels in serum were measured by ELISA,and ALB protein levels in the striatum were measured by Western blot to assess blood-brain barrier damage.We examined γ-H2 AX levels,a marker for DNA damage,by Western blot analysis.3.To investigate the protective effect of LRP on the striatum in different brain regions and at different time points in rats: the expression levels of Nrf2 and the downstream proteins GCLC and GCLM in different brain regions and at different time points were detected by Western blot.4.To investigate the protective effects of LRP on ACR oxidative stress-induced damage in the brain striatum: biochemical kits were used to determine the levels of GSH,SOD and ROS in the rat striatum and the expression levels of the antioxidant proteins Nrf2,GCLC and GCLM,as well as the apoptotic proteins JNK/P38 and caspase3 in the nucleus accumbens,were detected by Western blot.The TUNEL method was used to check the degree of apoptosis in striatal cells.Results 1.ACR induced impaired behavioral performance in SD rats,and the gait impairment,exploratory behavior and motor impairment were alleviated after LRP pretreatment.The results suggest that LRP can alleviate ACR-induced behavioral impairment.2.ACR resulted in significant impairment of TH neurons in the striatum and substantia nigra in SD rats.Using immunohistochemical staining,we found that THpositive neurons in the striatum and substantia nigra of rats were significantly upregulated following LRP intervention(P < 0.05).At the same time,Western blot results verified that TH protein levels were significantly increased(P < 0.05).After LRP pretreatment,DA and its metabolites DOPAC and HVA levels were significantly increased after LRP pretreatment(P < 0.05).The results suggest that LRP can alleviate ACR-induced dopamine neuron deficiency in rats.3.ACR induced striatal blood-brain barrier damage in SD rats,and the serum S100β was significantly decreased,and striatal ALB protein expression was significantly decreased after LRP pretreatment(P < 0.05).ACR induced striatal DNA damage in SD rats,the striatal XRCC1 level was increased,and the γ-H2 AX level was significantly decreased after LRP pretreatment(P < 0.05).The results suggest that LRP can attenuate ACR-induced blood-brain barrier damage and DNA damage in rats through the blood-brain barrier.4.LRP induced Nrf2 expression in the striatum and substantia nigra of SD rats and promoted the expression of downstream Nrf2 proteins such as GCLC and GCLM.LRP increased Nrf2 protein expression in the striatum of SD rats in a time-dependent manner and promoted the expression of downstream Nrf2 proteins such as GCLC and GCLM(P <0.05).The results suggest that LRP can activate Nrf2-related pathways to exert antioxidant effects.5.ACR induced oxidative stress in the striatum of SD rats,and striatal GSH and SOD levels increased after LRP administration.ROS levels decreased,Nrf2 protein in the testes increased,and downstream proteins GCLC and GCLM increased to varying degrees(P < 0.05).Meanwhile,the expression of JNK/P-JNK,P38/P-P38 and Caspase3 was decreased.The results of the TUNEL assay further verified that the apoptosis of striatal cells was significantly increased after ACR induction(P < 0.05).In contrast,the apoptosis of striatal cells was significantly reduced after LRP pretreatment(P < 0.05).The results suggest that LRP can inhibit ACR-induced oxidative stress injury and apoptosis through the Nrf2 and JNK/P38 pathways.Conclusion 1.LRP restores behavioral damage and protects against ACRinduced striatal neurotoxicity in rats through its good antioxidant effect.2.LRP can have a protective effect on ACR-induced degeneration of striatal and substantia nigra TH neurons.3.LRP can cross the blood-brain barrier and attenuate ACR-induced striatal blood-brain barrier and oxidative DNA damage.4.LRP exerts protective effects against ACR-induced neurotoxicity by activating the Nrf2 signaling pathway and inhibiting the JNK/P38 apoptosis signaling pathway.