The Molecular Regulatory Mechanism Of Gandou Decoction On Copper Metabolism Pathway In The Hepatocytes Of Wilson’s Disease Model-tx Mouse

Hepatolenticular degeneration(HLD), also known as Wilson’s disease, is an inherited autosomal recessive disorder of copper balance disease. The etiological factor of WD is the mutations in ATP7 b gene, which maps to chromosome 13q14.3. ATP7 b gene mutations result in dyssynthesis of ceruloplasmin(CP) and impaired biliary excretion of copper in hepatocytes. Finally, excess toxic copperaccumulate predominantly in the liver, brain, kidney and comea, and caused cirrhosis,neurological/psychiatric symptoms and Kayser-Fleischer ring(K-F ring) in WD patients,critical case is fetal.WD is minority of neural genetic diseases which can be treated, while patie nts cannot tolerate the toxic side effects of metal complexing agents, and it serio usly affect the condition and prognosis of the WD patients. Since the 1970 s, th is topic unit has used TCM Gan Dou decoction to treat WD,and obtains a good curative effect. Although we have found GDD has a great effect in removing the intracellular copper, its exact mechanism is unclear. In this study, we plan to fi nd out the molecular regulatory mechanism of GDD on copper metabolism path way of Wilson’s disease model-TX mouse by immunof-luorescence and Western blot, and clarify its mechanism of improvement of clinical symptoms of WD pati ents at the molecular level.Objective:To detect copper metabolism pathway related proteins expression levels in TX mouse with GDD treatment. Explore its molecular targets and mechanism of regulating copper metabolism pathway.Methods:Atomic absorption method to detect the concentration of copper in the hepatocytes of TX mouses, which were treated with different concentrations of rabbit serum with TCM GDD for different times; atomic absorption method to detect the concentration of microelement in the hepatocytes of TX mouses, which were treated with differentconcentrations of rabbit serum with TCM GDD for 24h; flow cytometry was used to analyze the experssion of ATP7 b,CTR1,ATOX1,CCS,MT,COX17 in the hepatocytes of TX mouses after being treated with rabbit serum with TCM GDD; Western blot detect the expression of ATP7 b,CTR1,ATOX1,CCS,MT,COX17 in the hepatocytes of TX mouses after being treated with rabbit serum with TCM GDD; The intracellular location of ATP7 b was observed by immunofluorescence.Results:1. GDD regulated the copper metabolism pathway related proteins expression levels in the hepatocytes of TX mouse.(1) Atomic absorption method showed that GDD could reduce the concentration of copper in the hepatocytes of TX mouses in dose-dependent and time-dependen t manners.(2) GDD could reduce the concentration of copper and iron in the hepatocytes o f TX mouse, and increase the concentration of zinc in a dose-dependent manner.(3) Western blot and flow cytometry analysis indicated that GDD concentration-d ependently increases the expression of ATP7 b protein in the hepatocytes of TX mouse(P<0.01).(4) Western blot and flow cytometry analysis indicated that GDD concentration-d ependently increases the expression of ATOX1、CCS、COX17 in the hepatocyte s of TX mouse(P<0.01).(5) Western blot and flow cytometry analysis demonstrated that after being treate d with GDD the protein expression of MT and CTR1 in the hepatocytes of TX mouse were decreased(P<0.01).2. GDD regulated the intracellular location of the Cu ATPase ATP7 b in the hepatocytes of TX mouse.(1) Immunofluorescence analysis indicated that the ATP7 b protein in hepatocytes of DL mouse was predominantly in the TGN. When hepatocytes of DL mouse w ere transferred to 10 μM Cu Cl2 for 60 min, most of the ATP7 b was localized to the apical region. When Cu-treated cells were subsequently incubated in DME M with 10%FBS for 3 h, ATP7 b returned to the TGN region.(2) ATP7 b protei n in hepatocytes of TX mouse was neither localized in the TGN, nor in the api cal region. When hepatocytes of TX mouse were incubated in DMEM with 10%FBS for 3 h, ATP7 b could not return to the TGN region. GDD concentration-de pendently stimulated the translocation of ATP7 b protein from the cytoplasm to th e trans-Golgi network in the hepatocytes of TX mouse.Conclusion:Taking together, these findings suggest that GDD increases the copper excretion of TX mouse’s hepatocytes by up-regulating the expression of ATP7b、 ATOX1、CCS and COX17, and down-regulating the expression of CTR1 and MT. Meanwhile, GDD stimulate the translocation of ATP7 b protein from the apical domain to the trans-Golgi network in the hepatocytes of TX mouse. GDD reduces copper content of tissue cells through a multi-target, multi-channel regulation of copper metabolism pathway.