| ObjectiveWilson’s disease(WD),also known as Hepatolenticular degeneration,is a disorder of copper metabolism,and the neuroinflammation caused by abnormal accumulation of copper can further aggravate the neurological damage of this disease.The neuroinflammation caused by abnormal copper accumulation can further aggravate the neurological damage of this disease.Chinese medicine has certain advantages in treating WD,and years of clinical practice research found that the Chinese medicine hepatomycin can effectively improve the cognitive impairment of WD.To further elaborate its mechanism of action in improving the cognitive impairment of WD,this study was based on the Nrf2/NLRP3 signaling pathway-mediated neuroinflammation.MethodsIn vivo experiment:Exploring the regulation and mechanism of Gandouling on cognitive function in Wilson disease model mice based on Nrf2/NLRP3 signaling pathway:In this experiment,50 TX mice were randomly divided into model group,Gandouling low,medium and high dose group,penicillamine group,and 10 wild-type mice with the same genetic background.After 6 weeks of intervention,the cognitive function of the mice was observed using Morris water maze;the damage of hippocampal neurons was observed by HE and Nissl staining;the apoptosis of hippocampal cells was observed by TUNEL staining;the damage of neuronal cells was observed by electron microscopy;the Nrf2,NLRP3,HO-1 and IL-1β proteins were detected by Western Blot in the hippocampal tissues of the mice in each group;RT-qPCR to detect Nrf2,NLRP3,TNF-α,IL-1β,IL-6 mRNA expression in hippocampal tissues;ELISA to detect TNF-α,IL-1β,IL-6,SOD,GSH content in hippocampal tissues.In vitro experiments:To explore the regulation and mechanism of the inflammatory response of Gandouling on copper-loaded BV-2 cell model in vitro based on Nrf2/NLRP3 signaling pathway:BV-2 cells were used in this experiment and divided into 5 groups according to different interventions:normal control group,copper-loaded group,Gandouling group,ML385 group(Nrf2 inhibitor),Gandouling+ML385 group.After copper loading and 24 hours of intervention,the cell morphology of each group was observed by inverted microscope,and the protein expression of Nrf2,NLRP3,HO-1 and IL-1β in each group was detected by Western Blot,and the TNF-α,IL-1β and IL-6 contents of cell supernatant in each group were detected by ELISA;the expression of Nrf2,NLRP3,TNF-α,IL-1β and IL-6 mRNA in each group was detected by RT-qPCR.ResultsIn vivo experiments1)Effects of Gandouling on cognitive function in TX mice:Morris water maze:during acquired spatial learning and memory training,the escape latency of the model group was longer than that of the control group from days 2-5(P<0.05).From day 2 to day 6,the latency periods of the Gandouling and model groups were significantly different.On day 6,there was a difference between the Gandouling medium and high dose groups and the model group,and no difference between the Gandouling low dose group with the model group.TX mice had decreased memory recall and were on the platform less often.The frequency of platform crossing was significantly higher in the Gandouling high dose group than in the TX mice after the intervention(P<0.05).2)Effects of Gandouling on histopathology and ultrastructure of hippocampus in TX mice:① HE and Nissl staining:hippocampal neurons in the control group were clearly arranged and regular,with clear nuclei,undamaged nuclear membranes,and no contraction was seen.Compared with the control group,the neurons in CA1 and CA3 areas of hippocampus in the model group were loosely arranged and disordered,with edema,vacuolation,degeneration and necrosis of cytosol,and irregular arrangement,deep staining and solid contraction of nuclei.In addition,the hippocampal histopathological changes were significantly reduced in each dose group of Gandouling(P<0.05).Nissl staining in the control group showed abundant Nissl vesicles in the CA1 and CA3 regions of the hippocampus.In the model group,the number of Nissl vesicles in the CA1 and CA3 regions of the hippocampus was reduced,and the number of Nisin vesicles increased after Gandouling treatment(P<0.05).3)Effects of Gandouling on key proteins of Nrf2/NLRP3 signaling pathway in hippocampal tissue of TX mice:NLRP3/IL-1β expression was increased and Nrf2/HO-1 expression was decreased in the model group compared with the normal control group(P<0.05);NLRP3/IL-1β expression was decreased and Nrf2/HO-1 expression was increased in all dose groups of Gandouling compared with the model group(P<0.05).4)Effects of Gandouling on the expression of inflammatory factors TNF-α,IL-1β,IL-6,SOD and GSH in hippocampal tissue of TX mice:compared with the normal control group,the expression of inflammatory factors TNF-α,IL-1β and IL-6 was increased and the expression of SOD and GSH was decreased in the model group(P<0.05);compared with the model group,the expression of TNF-α,IL-1β and IL-6 in The expression of TNF-α,IL-1β and IL-6 were decreased in all dose groups of Gandouling and penicillamine group,and the expression of SOD and GSH were increased(P<0.05).5)Effects of Gandouling on TNF-α,IL-1β and IL-6 mRNA expression in hippocampal tissue of TX mice:compared with normal control group,TNF-α,IL-1β and IL-6 mRNA expression was higher in the model group(P<0.05);compared with the model group,TNF-α,IL-1β and IL-6 mRNA expression was decreased in each dose group of Gandouling and penicillamine group(P<0.05).In vitro experiments1)Effects of Gandouling on key proteins of Nrf2/NLRP3 signaling pathway in copper-loaded BV-2 cells:NLRP3/IL-1β expression was increased and Nrf2/HO-1 expression was decreased in the model group compared with the normal control group(P<0.05);NLRP3/IL-1β expression was decreased and Nrf2/HO-1 expression was increased in all dose groups of Gandouling compared with the model group(P<0.05).(P<0.05).2)Effects of Gandouling on inflammatory factors TNF-α,IL-1β and IL-6 mRNA in copper-loaded BV-2 cells:compared with the normal control group,the relative expression of TNF-α,IL-1β and IL-6 mRNA in the model group was significantly higher than that in the normal control group(P<0.05);compared with the model group,the expression in the Gandouling group and Gandouling+ML385 group was reduced(P<0.05).3)Effects of Gandouling on inflammatory factors TNF-α,IL-1β and IL-6 in copper-loaded BV-2 cells:compared with the normal control group,the expression of TNF-α,IL-1β and IL-6 in the model group increased(P<0.05);compared with the model group,the expression in the Gandouling and Gandouling+ML385 groups decreased(P<0.05).Conclusions1)There was neuroinflammation in the hippocampus of WD model TX mice,mainly manifested by the activation of microglia.the expression of NLRP3/IL-1β,a key protein of Nrf2/NLRP3 signaling pathway,was elevated and the expression of Nrf2/HO-1 was decreased.The inflammatory factors TNF-α,IL-1β,IL-6 content and mRNA were highly expressed,and apoptosis was observed in brain tissue cells.2)Gandouling can play an inhibitory role in neuroinflammation and reduce apoptosis in hippocampal brain tissue cells by improving the expression of key proteins of Nrf2/NLRP3 signaling pathway,reducing inflammatory factors TNF-α,IL-1β,IL-6 content and mRNA expression,and inhibiting microglia activation.3)Copper-loaded BV-2 cell model,Nrf2/NrRP3 signaling pathway expression,elevated expression of key protein NLRP3/IL-1β,decreased expression of Nrf2/HO-1,elevated content and mRNA expression of inflammatory factors TNF-α,IL-1β,IL-6.4)Gandouling drug serum can decrease the expression of NLRP3 protein and decrease the expression of inflammatory factors TNF-α,IL-1β,IL-6 content and mRNA by elevating the expression of Nrf2/HO-1. |
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