Puerarin Alleviated Oxidative Damage And Mitochondrial Unfolded Protein Response Induced By Cadmium In Rat Neurocyte

Cadmium is a heavy metal widely found in the natural environment,it is easy to accumulate in the body for a long time and damage various tissues.Cadmium can cause brain damage and lead to memory decline,mental retardation.Oxidative stress is an important mechanism of cadmium neurotoxicity.Nuclear factor erythroid 2-related factor 2（Nrf2）may be the main regulator of redox homeostasis.Mitochondrial unfolded protein response（UPRmt）is mitochondrial stress reaction induced by external stimuli（for example,oxidative stress）and closely related to many diseases of the nervous system.Due to antioxidant or free radical inhibitor can effectively alleviate cadmium-induced organism damage,searching for safe and effective antioxidant drugs has become a research focus.Puerarin is an isoflavone glycoside,which exists in the root of pueraria.It is recognized as an antioxidant and can protect against nerve damage caused by cadmium,but whether Nrf2 signaling pathway and UPRmt are involved has not been reported.In this study,we used rat,rat adrenal pheochromocytoma（PC 12 cell line）and primary rat cerebral cortical neurons to explore the protective effect of puerarin in cadmium-induced rat neurocyte damage by relieving oxidative damage and UPRmt in vivo and in vitro.1.Puerarin alleviated oxidative damage and mitochondrial unfolded protein response induced by cadmium in cerebral cortices of ratsTwenty-four SD rats were prefed for 7 days,randomly grouped and treated in the following way,control group（Con）,puerarin group（Pur）,cadmium group（Cd）,cadmium and puerarin co-treatment group（Cd+Pur）.The rats in Pur group,Cd+Pur group underwent gavage of puerarin dissolved in sodium carboxymethyl cellulose at a dose of 200 mg/kg body weight daily,while the rats in the Con group and Cd group were given sodium carboxymethyl cellulose by gavage at the same dose and lasted for seven weeks.The rats in the Cd group,Cd+Pur group received drinking water containing 75 mg/L cadmium plus the gavage from the fourth weeks and lasted for four weeks.After the experiment,the cerebral cortices of rats were dissected and collected for subsequent experiments.Nissl staining and FJB fluorescence staining were used to observe the change of morphological structure and number of cerebral cortical neurons of rats.The activities of superoxide dismutase（SOD）,catalase（CAT）,and glutathione peroxidase（GSH-Px）,the contents of glutathione（GSH）and malondialdehyde（MDA）in cerebral cortices of rats were determined by colorimetry.The Nrf2 nuclear translocations of cerebral cortices of rats were observed by immunohistochemical.The expression levels of Nrf2 signaling pathway-and UPRmt-related proteins were detected by Western blot.The results showed as follows:①Compared with the Con group,the morphological structure of cerebral cortical neurons of rats in Cd group was fuzzy,the number of surviving neurons decreased,and the number of denatured and damaged neurons increased.Compared with the Cd group,pathological damage of cerebral cortical neurons of rats in Cd+Pur group was alleviated.②Compared with the Con group,the content of MDA was significantly increased（P<0.05）,the activities of SOD,CAT and GSH-Px and content of GSH were significantly decreased（P<0.05）,the nuclear translocation of Nrf2 was increased,the protein expressions of Nrf2 in the nuclear and HO-1,NQO1 in neurons were significantly increased（P<0.05）in cerebral cortices of rats in Cd group.Compared with the Cd group,the content of MDA was significantly decreased（P<0.05）,the activities of SOD,CAT and GSHPx and content of GSH were significantly increased（P<0.05）,the nuclear translocation of Nrf2 was reduced,the protein expressions of Nrf2 in the nuclear and HO-1,NQO1 in neurons were significantly decreased（P<0.05）in Cd+Pur group.③Compared with the Con group,the protein expressions of UPRmt-related proteins（LonPl,HSP60,CLpP and HtrA2）in cerebral cortical mitochondria of rats were significantly increased（P<0.05）in Cd group.Compared with the Cd group,the protein expressions of LonP1,HSP60,CLpP and HtrA2 in cerebral cortical mitochondria of rats were significantly decreased（P<0.05）in Cd+Pur group.These results suggest that Pur alleviated Cd-induced oxidative damage by reducing the level of lipid peroxidation,increasing the level of antioxidant and inhibiting the activation of Nrf2 signaling pathway in cerebral cortices of rats,and it also alleviated UPRmt.Therefore,Pur alleviated Cd-induced cerebral cortical damage of rats.2.Puerarin alleviated oxidative damage and mitochondrial unfolded protein response induced by cadmium in rat neurocytePC 12 cells and primary neurons were treated as follows for 12 h:Con group（Con）,puerarin group（Pur,100 μM Pur）,cadmium group（Cd,10 μM CdCl2）,cadmium and puerarin co-treatment group（Cd+Pur,100 μM Pur+10 μM CdCl2）,or small interference control group（Si NC,20 nM/60 nM Si NC）,Nrf2 small interference group（Si Nrf2,20 nM/60 nM Si Nrf2）,small interference control and cadmium co-treatment group（Si NC+Cd,20 nM/60 nM Si NC+10 μM CdCl2）,Nrf2 small interference and cadmium co-treatment group（20 nM/60 nM Si Nrf2+10 μM CdCl2）.The viability of neurocyte was detected by CCK-8 kit.ROS level was detected by DCFH-D A probe.The transcription levels of oxidative damage-related genes were detected by qRT-PCR.Nrf2 nuclear translocation was detected by immunofluorescence.The expressions of Nrf2 signaling pathway-and UPRmt-related proteins were detected by Western blot.The results showed as follows:①Compared with the Con group,the viability of neurocyte in Cd group was significantly decreased（P<0.05）.Compared with the Cd group,the viability of neurocyte in Cd+Pur group was significantly increased（P<0.05）.②Compared with the Con group,the ROS level and the transcription levels of SOD,GCLC,GCLM in Cd group were significantly increased（P<0.05）.Compared with the Cd group,the ROS level and the transcription levels of SOD,GCLC,GCLM were significantly decreased（P<0.05）in Cd+Pur group.③Compared with the Con group,the protein expressions of Nrf2 in the nuclear and HO-1,NQO1 in neurocyte treated with cadmium（2.5 μM,5μM,10μM,20μM）for 12 h or 10 μM cadmium for different time（3 h,6 h,12 h,24 h）were significantly increased（P<0.05）.④Compared with Si NC group,the protein expressions of Nrf2 in the nuclear and HO-1,NQO1 in neurocyte were significantly increased（P<0.05）,the viability of neurocyte was significantly decreased（P<0.05）in Si NC+Cd group.Compared with Si NC+Cd group,the protein expressions of Nrf2 in the nuclear and HO-1,NQO1 in neurocyte were significantly decreased（P<0.05）,the viability of neurocyte was significantly increased（P<0.05）in Si Nrf2+Cd group.⑤Compared with the Con group,Nrf2 nuclear translocation was increased,the protein expressions of Nrf2 in the nuclear and HO-1,NQO1 in neurocyte were significantly increased（P<0.05）in Cd group.Compared with the Cd group,the Nrf2 nuclear translocation was reduced,the protein expressions of Nrf2 in the nuclear and HO-1,NQO1 in neurocyte were significantly decreased（P<0.05）in Cd+Pur group.⑥Compared with the Con group,the protein expressions of UPRmt-related proteins（LonP1,HSP60,CLpP and HtrA2）in mitochondria of neurocyte were significantly increased（P<0.05）in Cd group.Compared with the Cd group,the protein expressions of LonP1,HSP60,CLpP and HtrA2 in mitochondria of neurocyte were significantly decreased（P<0.05）in Cd+Pur group.The results indicate that activation of Nrf2 signaling pathway can participate in Cd-induced oxidative stress,and exacerbate Cd-induced rat neurocyte injury.Moreover,Pur alleviated Cd-induced oxidative damage by reducing ROS accumulation and antioxidant compensation,inhibiting the activation of Nrf2 signaling pathway in rat neurocyte,and it also alleviated UPRmt.Therefore,Pur alleviated rat neurocyte damage induced by Cd.In conclusion,activation of Nrf2 signaling pathway can participate in Cd-induced oxidative stress,and exacerbate Cd-induced rat neurocyte injury.Moreover,Pur alleviated Cd-induced oxidative damage by reducing ROS accumulation and antioxidant compensation,reducing the level of lipid peroxidation and inhibiting the activation of Nrf2 signaling pathway in rat neurocyte,and it also alleviated UPRmt.Therefore,Pur alleviated rat neurocyte damage induced by Cd.