Puerarin Alleviates Cadmium-Induced Neurotoxicity Mediated By Mitochondrial Damage In Rat Cerebral Cortical Neurons

Cadmium(Cd)is a common heavy-metal pollutant in the environment.It can cause damage to multiple systems and organs in humans and animals,and the brain is one of the critical target organs of Cd toxicity.Cd can damage the blood-brain barrier and enter the brain,and its neurotoxic mechanisms are mainly manifested as oxidative stress,apoptosis,and autophagy.Mitochondria are platforms for intracellular signal cross-linking,and Cd-induced oxidative stress,apoptosis,and autophagy of neuronal cells are closely related to mitochondrial damage.Therefore,targeted repair of damaged mitochondria may alleviate the neurotoxicity of Cd.Puerarin(Pur)is a plant isoflavone extracted from the Chinese medical herb kudzu root,which can penetrate the blood-brain barrier and play a neuroprotective role in various neurological diseases.However,whether Pur can alleviate the neurotoxicity of Cd remains unclear.In this study,SD rats were used to systematically explore the protective effects of Pur against Cd-induced neurotoxicity mediated by mitochondrial damage in rat cerebral cortical neurons in vitro and in vivo.This provides a theoretical basis for the clinical use of Pur to alleviate the neurotoxicity of Cd,and also provides an example of the benefit of Chinese herbal extracts to prevent and treat Cd poisoning in production practice.1.In vivo studiesTwenty-four 6-week-old male SD rats were randomly divided into four groups:control group,Pur group,Cd group,and Cd+Pur group.The Pur group and Cd+Pur group received Pur(200 mg/kg body weight)daily by gavage,while the control group and Cd group received the same volume of 0.5%carboxymethylcellulose sodium by gavage.Fourteen days later,the Cd and Cd+Pur groups were free to drink Cd water(50 mg/L)plus gavage for another 90 days.The daily Cd consumption was assessed based on the amount of water intake of rats.The Cd concentrations in urine,feces,serum,and cerebral cortices,the histopathological and ultrastructural changes of cerebral cortices,and the parameters of oxidative stress,mitochondrial apoptosis pathway,and mitophagy were detected.The results showed as follows:(1)During the exposure,the average daily Cd consumption of rats in the Cd group and Cd+Pur group were 3.64 and 3.71 mg/kg body weight,respectively.Administration of Pur significantly reduced the Cd levels in the cerebral cortices and serum,and increased the Cd levels in urine and feces of Cd-exposed rats(P<0.05).Moreover,Pur alleviated Cd-induced histopathological and ultrastructural damage in rat cerebral cortices.(2)Pur significantly inhibited Cd-induced increase in CAT activity and GSH and MDA levels in rat cerebral cortices(P<0.05),but had no significant effect on SOD activity(P>0.05).Moreover,Pur inhibited Cd-induced apoptosis in rat cerebral cortices,and significantly inhibited Cd-induced increase in the ratio of Bax/Bcl-2,the cleavage of caspase-9/3 and PARP1(P<0.05),as well as nuclear translocation of AIF(P<0.05).(3)Pur inhibited Cd-induced mitophagosomes formation in rat cerebral cortices,and significantly inhibited Cd-induced decrease in the protein expression of mitochondrial marker HSP60 and COX Ⅳ and the increase in the protein expression of PINK1,Parkin,Nix,and LC3 Ⅱ on mitochondria(P<0.05).In summary,Pur alleviated Cd-induced rat cerebral cortical injury by reducing the accumulation of Cd in the cerebral cortices and promoting Cd excretion,inhibiting oxidative stress,the activation of mitochondrial apoptosis pathway,and mitophagy.2.In vitro studiesThe model of primary rat cerebral cortical neurons was established,and neurons were treated with 10 μM Cd for different time periods(0,3,6,12,and 24 h).Neurons were pretreated with varying concentrations of Pur(0,50,100,200 μM)for 1 h or transfected with PINK1 siRNA/Nix siRNA before exposure to 10 μM Cd for 12 h.Neurons were pretreated with 100 μM Pur or 1 μM Cyclosporine A(CsA)for 1 h before exposure to 10 μM Cd for 12 h.The cell viability,the parameters of oxidative stress,apoptosis,and mitophagy were detected.The results showed as follows:(1)Pur(50,100,200 μM)had no significant effect on neuronal viability(P>0.05).Pur(50,100 μM)significantly inhibited Cd-induced decrease in neuronal viability(P<0.05).Pur(100 μM)alleviated Cd-induced mitochondrial superoxide overproduction,and significantly inhibited Cd-induced increase in CAT activity and GSH and MDA levels(P<0.05).(2)Pur(100 μM)inhibited Cd-induced neuronal apoptosis,nuclear and mitochondrial ultrastructural damage,mitochondrial membrane potential reduction,and significantly inhibited Cd-induced increase in the ratio of Bax/Bcl-2,the cleavage of caspase-9/3 and PARP1(P<0.05),as well as nuclear translocation of AIF(P<0.05).(3)After treatment with Cd for 12 and 24 h,the expression of HSP60 and COX Ⅳproteins decreased significantly(P<0.05),the number of mitophagosomes increased,and the expression of PINK1,Parkin,and Nix proteins increased significantly(P<0.05).Moreover,Cd promoted the co-localization of Parkin and TOMM20.Knockdown of PINK1 significantly inhibited Cd-induced increase in the protein expression of PINK1 and mitochondrial Parkin and LC3 II(P<0.05),and blocked the co-localization of PINK 1 and TOMM20.Knockdown of Nix significantly inhibited Cd-induced increase in the protein expression of Nix and mitochondrial LC3 II(P<0.05),and blocked the co-localization of Nix and TOMM20.CsA inhibited Cd-induced mitophagosomes formation in neurons.Administration of CsA or knockdown of PINK1 significantly promoted a Cd-induced decrease in neuronal viability and the activation of caspase-9/3(P<0.05),and promoted neuronal apoptosis induced by Cd.Pur inhibited Cd-induced mitophagosomes formation,significantly inhibited Cd-induced decrease in the protein expression of HSP60 and COX IV and the increase in the protein expression of mitochondrial PINK1,Parkin,and Nix(P<0.05),and blocked the co-localization of LC3 and TOMM20.In summary,Cd induced mitophagy by activating PINK1/Parkin and Nix pathways in rat cerebral cortical neurons,and mitophagy can promote neuronal survival by inhibiting Cd-induced apoptosis.Administration of Pur alleviated Cd-induced mitochondrial damage and its mediated oxidative stress,apoptosis,and mitophagy,and promoted neuronal survival.