| 1.Purpose This topic consists of two parts:(1)Clinical trials: the effect of Huatan Quyu Recipe on the cognitive function of patients with Wilson’s disease with mild cognitive impairment,and on the plasma midbrain of patients with Wilson’s disease with mild cognitive impairment(2)Animal experiment: using Brd U incorporation method combined with immunofluorescence staining to investigate the effect of Huatan Quyu Recipe on the proliferation and regeneration of nerve cells in the hippocampal dentate gyrus of Wilson disease TX mice The differentiation of nerve cells into neurons and the effect of synaptic growth of new neurons.ELISA was used to investigate the effect of Huatan Quyu Recipe on the levels of BDNF and VEGF in the hippocampus and serum of Wilson disease TX mice.Through mutual verification of clinical research and animal experiments,explore the mechanism of Huatan Quyu Fang Gandouling on WD cognitive impairment,and open up new treatment directions and theoretical support for the research of WD with cognitive impairment in the field of traditional Chinese medicine.2 methods2.1 Clinical researchFrom December 2018 to November 2020,among the WD patients in our hospital,30 patients with WD with mild cognitive impairment who meet the criteria for inclusion and exclusion were selected as the experimental group,and 30 patients were recruited by age,gender,and education.Healthy people with similar levels were used as the control group.The two groups of subjects were tested with cognitive scale and BDNF and VEGF levels.Then,patients with WD with mild cognitive impairment were treated with copper excretion as the basis and given Gandouling for 40 days.The cognition after treatment was observed The scale score and the changes of BDNF and VEGF levels.2.2 Animal experimentThe mice used in this experiment are TX mice from the Jackson Laboratory Animal Center in the United States.They will be introduced when they are 4 months old and raised for 1 week.After adapting to the environment,they will be randomly divided into normal control group and Wilson disease.Model group,Gandouling group,positive control(Oxiracetam)group,15 rats in each group.Except for the normal control group,which used DL syngeneic control mice,the other groups used Wilson disease TX mice.The administration group was given Gandouling or Oxiracetam by gavage,and the normal control group and Wilson disease model group were given the same volume of normal saline daily for 28 consecutive days.HE staining was used to observe the histopathology of the hippocampus of TX mice with Wilson disease.Brd U incorporation method combined with immunofluorescence staining was used to explore the effect of Huatan Quyu Recipe on the proliferation of nerve cells in the hippocampus dentate gyrus of TX mice with Wilson disease and the transfer of new nerve cells to nerves.The effect of cell differentiation and synaptic growth of newborn neurons.ELISA was used to investigate the effect of Huatan Quyu Recipe on the levels of BDNF and VEGF in the hippocampus and serum of Wilson disease TX mice.3 results3.1 Clinical research results(1)The scores of various cognitive domains of the Mo CA scale for patients with WD with mild cognitive impairment: This study found that in the cognitive sub-items of the Mo CA scale,the WD with mild cognitive impairment group(experimental group)and normal The control group had no statistical significance in the comparison of naming and orientation(P>0.05).In the comparison of abstract ability,the two groups were statistically significant(P<0.05);in the comparison of visual space and executive ability,language ability,attention and calculation ability,and memory and delayed recall,there were significant differences between the two groups(P <0.01).The results indicate that the impairment of WD patients with MCI in the cognitive domain is mainly the impairment of visual space and executive ability,memory and delayed recall,attention and calculation,language ability and abstract ability.(2)The improvement of cognitive function by Huatan Quyu Recipe: After the treatment of Huatan Quyu Recipe,the MMSE and Mo CA scores of patients with WD with mild cognitive impairment increased,and the difference was statistically significant(P<0.01).(3)Effect of Huatan Quyu Recipe on plasma BDNF and VEGF levels in patients with WD with mild cognitive impairment: Compared with the normal control group,the plasma BDNF and VEGF levels in Wilson patients with mild cognitive impairment were significantly reduced(P < 0.01);The BDNF and VEGF levels of the patients increased after the treatment of Huatan Quyu Decoction,and the difference was statistically significant(P < 0.05);the BDNF and VEGF levels of the patients after treatment were still lower than the normal control group,and the difference was statistically significant Academic significance(P<0.01).3.2 Animal experiments(1)HE staining of pathological sections of mouse hippocampus: Compared with DL mice,the hippocampus of Wilson disease model group showed obvious changes in cell death characteristics.The degree of neuronal necrosis in the hippocampus of the mice in the Gandouling group and the Oxiracetam group was lighter than that of the model group,and the pathological changes of the Gandouling group were lighter than that of the Oxiracetam group.(2)The effect of Huatan Quyu Recipe on the proliferation of nerve cells in the hippocampal dentate gyrus of Wilson disease TX mice: Compared with the normal control group,the Brd U positive cells of Wilson disease model group,Gandouling group and Oxiracetam group The number of Brd U positive cells was significantly increased in Gandouling group and Oxiracetam group compared with Wilson’s disease model group(P < 0.01),and Gandouling group and Oxiracetam There was no statistically significant difference in the Tan group compared with each other(P=0.31>0.05).(3)The effect of Huatan Quyu Recipe on the differentiation of nerve cells in the hippocampal dentate gyrus of Wilson disease TX mice: Compared with the normal control group,the number of Neu N positive cells in the Wilson disease model group was significantly reduced(P<0.01).The number of Neu N-positive cells in the Ling group decreased(P<0.05),and the number of Neu N-positive cells in the Oxiracetam group was not significantly different from that in the normal control group(P>0.05).Compared with the Wilson disease model group,the number of Neu N positive cells in Gandouling group and Oxiracetam group was significantly increased(P < 0.01),and Gandouling group and Oxiracetam group were compared with each other,the difference No statistical significance(P>0.05).(4)The effect of Huatan Quyu Recipe on the synaptic growth of newborn neurons in the hippocampal dentate gyrus of Wilson disease TX mice: Compared with the normal control group,the number of MAP2 positive cells in the Wilson disease model group was significantly reduced(P<0.01),The number of MAP2 positive cells in the Gandouling group decreased(P<0.05),and the number of MAP2 positive cells in the Oxiracetam group was not significantly different from that in the normal control group(P>0.05).Compared with the Wilson disease model group,the Gandouling group and the Oxiracetam group showed a significant increase in the number of MAP2 positive cells(P<0.01),and the Gandouling group and the Oxiracetam group were compared with each other in pairs.No statistical significance(P>0.05).(5)The effect of Huatan Quyu Recipe on the hippocampus and serum BDNF and VEGF levels of Wilson disease TX mice: This study shows that compared with the normal control group,the Wilson disease model group TX mice hippocampus and serum BDNF and VEGF levels Significantly reduced(P<0.01),indicating that copper ions can inhibit the expression of BDNF and VEGF in TX mice with Wilson disease.Compared with the Wilson disease model group,the Gandouling group and Oxiracetam group showed a significant increase in hippocampal tissue and serum BDNF(P<0.01),and the level of VEGF in hippocampus and serum increased(P<0.05).There was no statistically significant difference between the Douling group and the Oxiracetam group(P>0.05),which indicates that both Gandouling and Oxiracetam can promote the hippocampus and serum of TX mice The levels of BDNF and VEGF,and the efficacy of the two are equivalent.4 Conclusion1.Patients with WD with mild cognitive impairment have extensive cognitive impairment,which is mainly impaired in five cognitive sub-items: visual space and executive ability,memory and delayed recall,attention and calculation ability,language ability and abstract ability Mainly.2.The levels of plasma BDNF and VEGF in Wilson patients with mild cognitive impairment were significantly lower than those in healthy controls,suggesting that BDNF and VEGF may be serological reference indicators for evaluating cognitive function in Wilson disease patients.3.The phlegm and blood stasis decoction Gandouling may improve the cognitive function of Wilson patients with mild cognitive impairment by increasing the levels of BDNF and VEGF.4.Huatan Quyu Recipe may increase the levels of BDNF and VEGF in the hippocampus and serum of Wilson’s disease TX mice,thereby promoting the proliferation of hippocampal dentate gyrus nerve cells,the differentiation of new nerve cells into neurons,and the synaptic growth of new neurons,Play a neuroprotective effect. |
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