Study On The Regulation And Mechanism Of Gandouling On Neuroinflammation In Wilson Disease Model Mice And Inflammatory Response Of Copper Loaded Microglia Based On Nf-?B Signal Pathway

ObjectiveWilson's disease(WD),also known as hepatolenticular degeneration,is a hereditary disorder of copper metabolism.Neuroinflammation caused by copper deposition can aggravate the damage of nervous system.Chinese medicine has certain advantages in treating this disease.Many years of clinical practice found that gandouling(GDL)can effectively relieve the symptoms of WD with nervous system.The objective of this study is to elucidate the molecular mechanism of GDL in improving wilson's disease.We used the Wilson disease model TX mice and the BV-2 copper loaded model based on the NF-?B signaling pathway.Combined with in vivo and in vitro experiments to explore the effect of GDL on WD neuroinflammation.To provide theoretical support and new ideas for the treatment of WD with Chinese MedicineMethodsIn vivo experiments:A total of 50 toxic milk mouse were assigned randomly into five groups:model group,Gandouling high dose group(GDL-H),Gandouling medium dose group(GDL-M),Gandouling low dose group(GDL-L)and Penicillium group.10 wild-type mice as normal group.After 4 weeks of intervention,immunofluorescence staining was used to detect the expression of Iba1 and GFAP protein in striatum of mice.Western blot was used to detect the expression of p-p65,P-I?B?,TNF-?,IL-1 ?,IL-6,COX-2,iNOS and Caspase-3 in striatum of mice.RT-PCR was used to detect the expression of TNF-??IL-1??IL-6?COX-2?iNOS mRNA in each group.In vitro experiments:BV-2 cells were divided into four groups:normal group,model group,Gandouling group and curcumin group.After 24-hourcopper load intervention and drug treatment.We used inverted microscope to observe the cell morphology of each group.Western blot was used to detect the expression of p-I?B?,p-p65 and the level of TNF-?,IL-1?,IL-6 were detect by ELISA.TNF-?,IL-1?,IL-6mRNA expression were detected by RT-PCRResultsIn vivo experiments1)Effect of GDL on the expression of Iba1 and GFAP in striatum:Compared with the normal group,the expressions of Iba1 and GFAP in the striatum of the model group significantly increased(P<0.01).Compared with the model group,the expression of Iba1 in penicillamine,GDL-M and GDL-H groups significantly decreased(P < 0.05 ? P < 0.01).Compared with penicillamine group,the expression of Iba1 decreased in the GDL-M and GDL-H group(P<0.05),and the expression of GFAP decreased in the GDL-H group(P < 0.01).Compared with GDL-L group,the expression of Iba1 decreased in the GDL-M and GDL-H dose GDL group(P<0.01)and the expression of GFAP decreased in the GDL-H group(P < 0.05).Compared with GDL-M group,the expression of GFAP decreased in the GDL-H group(P<0.05).2)Effect of GDL on the expression of p-I ? B ? and p-p65 in striatum:Compared with the normal control group,the expression of p-I?B? and p-p65 increased in the model group(P<0.01).Compared with the model group,the expression of p-I?B? and p-p65 decreased in each dose group of GDL and penicillamine group.Compared with GDL-L group,the expression of P-I?B? and p-p65 decreased in the GDL-M and GDL-H group(P<0.05,P<0.01).Compared with GDL-M group,the expression of p-I?B? decreased in the GDL-H group(P<0.01).3)Effects of GDL on the expression of TNF-?,IL-1 ?,IL-6,COX-2,iNOS protein in striatum:Compared with the normal group,the expression ofTNF-?,IL-1?,IL-6,COX-2 and iNOS in the model group increased(P<0.01).Compared with the model group,the expression of TNF-? and IL-1? decreased in GDL group and penicillamine group(P<0.05,P<0.01).The expression of COX-2 and iNOS decreased in GDL-M group,GDL-H group,GDL-H group and penicillamine group(P<0.01).Compared with GDL-L group,the expression of TNF-? decreased in the GDL-H group(P<0.01)and the expression of COX-2,iNOS decreased in the GDL-M,GDL-H group(P<0.05,P<0.01).Compared with GDL-M group,the expression of TNF-? decreased in the GDL-H group(P<0.05).4)Effects of GDL on the expression of TNF-?,IL-1 ?,IL-6,COX-2,iNOS mRNA in striatum:The expression of TNF-?,IL-1?,IL-6,COX-2,iNOS mRNA in the model group was higher than that in the normal group(P<0.01).Compared with model group,TNF-?,IL-1? and IL-6 mRNA decreased in GDL each group and Penicillium group(P<0.05,P<0.01),and COX-2,iNOS mRNA expression decreased in GDL groups(P<0.05 ? P<0.01).Compared with GDL-L group,the expression of TNF-?,IL-1?,IL-6,COX-2 mRNA decreased in the GDL-M and GDL-H group(P<0.05,P<0.01).5)Western blot detection of caspase-3 protein in striatum:Compared with the normal group,the expression of Caspase-3 increased in the model group(P<0.01).Compared with the model group,the expression of Caspase-3decreased in GDL group and penicillamine group(P<0.01).Compared with penicillamine group,the expression of Caspase-3 in GDL high dose group significantly decreased(P < 0.05).Compared with GDL-L group,the expression of caspase-3 decreased in the GDL-M and GDL-H group(P<0.01).In vitro experiments1)Effect of GDL on the morphology of copper loaded BV-2 cells:In the normal group,most of the BV-2 cells were adherent growth,a few of them were suspended,the form of BV-2 cells was long and thin,with branching process.Model group: a small part adheres growth,most of them aresuspended.some of them are agglomerated,cell volume slightly increases,branching process decreases,shortens and changes.Compared with the model group,suspension cells decreased in groups of GDL and Curcumin.between the normal control group and the model group.2)Effect of GDL of p-I?B? and p-p65 in BV-2 cells:Compared with the normal group,the expression of p-I?B? in the model group significantly increased(P<0.01).Compared with the model group,the expression of p-I?B? and p-p65 in GDL group and curcumin group decreased(P<0.01).3)Effects of GDL on TNF-?,IL-1?,IL-6 mRNA of copper loaded BV-2 cells:RT-PCR analysis show a higher expression level of TNF-??IL-1??IL-6mRNA in model group than in normal group(P<0.01).Compared with the model group,the relative expression levels of TNF-??IL-1??IL-6 mRNA in GDL group and Curcumin group were down-regulated(P < 0.01).There is no significant difference between GDL group and Curcumin group in expression levels of TNF-??IL-1??IL-6 mRNA.4)Effects of GDL on TNF-?,IL-1 ?,IL-6 in copper loaded BV-2cells:Compared with the normal group,the expression of TNF-?,IL-1?,IL-6 increased in the model group(P<0.01).Compared with the model group,the expression of TNF-?,IL-1?,IL-6 decreased in GDL group and Curcumin group(P<0.01).Compared with curcumin group,the level of TNF-?,IL-6 in GDL group was lower than that in curcumin group(P<0.05).Conclusions1)There is neuroinflammation in the striatum area of TX mice.It is mainly manifested by the activation of microglia and astrocytes with NF-?B signal pathway activation.Up regulation of phosphorylation expression of P65 and I?B?.The expression of TNF-?,IL-1?,IL-6,iNOS,COX-2 mRNA and protein were increased.Neuroinflammation is accompanied by increased apoptosis of nerve cells.2)GDL can reduce the expression of TNF-??IL-1??IL-6?iNOS?COX-2 mRNAand protein by reducing the phosphorylation of I?B? and p65 in NF-?B signaling pathway.GDL can inhibit the activation of microglia and astrocytes?It play the role of inhibiting neuroinflammation,and reduce the apoptosis of striatum brain cells.With the increase of concentration,the effect of GDL is more obvious3)In copper loaded BV-2 model,NF-?B signaling pathway was activated,were up-regulated in phosphorylation,and inflammatory factors TNF-?,IL-1?,IL-6 mRNA and protein levels were up regulated.4)GDL drug serum can reduce the phosphorylation of I?B? and p65,to inhibit NF-?B signal pathway.It can reduce the expression of TNF-?,IL-1?,IL-6 mRNA and protein,and improve neuroinflammation.