| Purpose:In this thesis,the effect of subclinical hypothyroidism?SCH?on cognitive function was firstly studied.The RBANS was used to access the patients with SCH and to explore the factors that might influence cognitive function.Secondly,in order to clarify the mechanism and target of Jianpi Huatan Huoxue Decoction,its effects on spatial memory and brain function of rats with SCH were studied,and the synergistic effect between traditional Chinese medicine and Western medicine was observed at the same time.Material and method:The first part:54 SCH patients diagnosed from October 2014 to June 2016,and 30 cases of healthy control group were selected.The RBANS was measured in all patients and healthy controls,At the same time,the blood pressure,blood lipid and BDNF of SCH patients were measured,and the correlation between the measured index,the RBANS score,and the influencing factors were analyzed.The second part:110 SPF grade Wistar male rats were selected,after adaptive breeding for one week,11 rats were randomly selected as sham group,and subjected to sham thyroidectomy.For other 99 rats,the total thyroidectomy+L-T4 subcutaneous injection molding were performed.7 weeks later,by the detection of thyroid function,66 successfully modeled rats were randomly divided into 6 groups.With sham group,total 7 groups:?1?sham group;?2?model group;?3?Jianpi Huatan Huoxue Decoction in conventional dosage group?conventional dosage group?;?4?Jianpi Huatan Huoxue Decoction 3 times the conventional dosage group?high dosage group?;?5?levothyroxine group?L-T4 group?;?6?Jianpi Huatan Huoxue Decoction with conventional dosage and left thyroid hormone tablet group?conventional dosage+L-T4 group?;?7?Jianpi Huatan Huoxue Decoction 3 times of routine dosage+levothyroxine group?high dosage+L-T4 group?.Treatment factors:?1?sham group:5ml/kg/d 0.9%NS by gavage;?2?model group:5ml/kg/d 0.9%NS by gavage;?3?conventional dosage group:given Jianpi Huatan Huoxue Decoction 13.7g/kg/d by gavage;?4?high dosage group:given Jianpi Huatan Huoxue Decoction 3 times the dosage of 41.1g/kg/d by gavage;?5?L-T4 group:levothyroxine 5.0??/kg/d by gavage;?6?conventional dosage+L-T4 group:Jianpi Huatan Huoxue Decoction 13.7g/kg/d+levothyroxine 5.0??/kg/d by gavage;?7?high dosage+L-T4 group:Jianpi Huatan Huoxue Decoction 41.1g/kg/d and levothyroxine5.0??/kg/d by gavage.During intragastric administration,L-T4 was injected subcutaneously.The water maze test was performed 3 days before the sampling in the fourteenth week.On the fourth day,the rats were anesthetized,the serum and hippocampus were observed.Serum total T4?TT4?was detected by radioimmunoassay?RIA?and serum thyroid stimulating hormone?TSH?was detected by chemiluminescence immunoassay?ICMA?.The morphological changes of hippocampal CA1 region were observed by HE staining and Nissl staining under light microscope.The brain derived neurotrophic factor?BDNF?and T4 were detected by ELISA;MCT8,BDNF,TrkB,ERK,RSK and CREBmRNA expression in hippocampus of rats were detected by RT-PTC method;The expression of BDNF,CREB,ERK,MCT8,RSK and TrkB protein in hippocampus of rats were detected by Western blot method;BDNF/TrkB of Hippocampus tissue CA1 was marked by immunofluorescence staining method,and the number of double positive cells was counted.Results:1.case control study1.1 The RBANS was used to evaluate 54 cases of SCH and the normal group of 30cases,The results showed:the scores of the SCH group in the RBANS scale score,5dimensions of standard score?immediate memory,breadth of vision,speech function,attention,delayed memory?and 11 sub tests?vocabulary learning,story retelling,graphic copying,line orientation,semantic fluency test,digit span,encoding vocabulary,vocabulary memory and graphics memory,story recall?were lower than the normal group?P<0.05?.1.2 Correlation analysis showed that,in the SCH patients,TSH and BDNF levels and RBANS score and 5 dimension scores are highly correlated?P<0.05?,TSH and BDNF levels are the main influencing factors of cognitive function in patients with SCH injury?P<0.01?.2 animal experiment results2.1 Model evaluationAt the eighth week of the experiment,according to the mean value of the serum TSH level of the sham operation group,the 90%confidence interval:0.014?IU/mL-0.02?IU/mL;The 90%confidence interval of the mean value of serum T4 level:46.57ng/ml-63.27ng/ml.As a standard to evaluate the TSH and TT4 model,66 rats of subclinical hypothyroidism were successfully modeled,the success rate is 66.67%.2.2 Therapeutic index2.2.1 Morris water maze results?1?escape latency:total 11 rats in each group were tested for 12 times.In the 12 test sites,t1,t3,t6,t9,t12 test points were selected for comparison between groups.Compared with the sham group,escape latency of the rats in the model group was obviously prolonged in the 12test points?P<0.01?;Compared with the model group,the rats in the conventional dosage group had shorter escape latency in the 12 test sites?P<0.05?;high dosage group and L-T4group were compared with conventional dosage group,the escape latency of the high dosage group had shorter escape latency at t1,t3 and t12 test point?P<0.01 or P<0.05?,and L-T4group had shorter escape latency only at t1 and t3 test point?P<0.01?;Compared with the conventional dosage group,conventional dosage+L-T4 group had shorter escape latency at t1,t3 and t6 test point?P<0.01 or P<0.05?;Compared with high dosage group,high dosage+L-T4 group had shorter escape latency at t1,t3 and t12 test point?P<0.01 or P<0.05?.?2?platform crossing times:Compared with sham group,the platform crossing times of the model group rats was significantly decreased?P<0.01?;Compared with the model group,the platform crossing times of the control group rats was increased?P<0.05?;Compared with the control group,the platform crossing times of the high dosage group rats and L-T4 group rats increased significantly?P<0.05?;Compared with the control group,the platform crossing times of the conventional dosage+L-T4 group increased significantly?P<0.01?;Compared with the high dosage group,the platform crossing times of the high dosage+L-T4group rats increased?P<0.05?.2.2.2 staining results of HE and Nissl in Hippocampal CA1 regionIn the sham group,HE staining showed that the cell bodies of the neurons were in the shape of vertebral body,spindle or round shape,which were distributed in multiple layers and arranged closely and uniformly.The nucleus is big and round,located in the central cell body,12 nucleolus in the nucleus were clearly visible,less cytoplasm and nuclear staining shallow,nuclear envelope was clear.Nissl staining showed clear cell body contour,tightly layered arrangement,structural integrity.Blue stained Nissl bodies were visible in the cytoplasm,Nissl body was big,and of large number.Compared with the sham group,in the model group,the distribution of HE neurons was scattered,the arrangement was not close,the morphology of some neurons was not complete,and the space between cells increased.Nissl staining showed that the cell bodies were scattered and sparse,and the gap between them increased.The number of Nissl bodies was small and the number was significantly less?P<0.01?;Compared with the model group,the HE staining in the conventional dosage group showed that the cell bodies of neurons were layered,and the arrangement of the neurons was significantly improved.Nissl staining showed that the number of Nissl bodies was higher than that of model group?P<0.05?;Compared with the conventional dosage group,in the high dosage group and the L-T4group,HE staining showed that the neuronal cell body was layered distribution,the arrangement of compactness is good,and there is no significant difference in cell morphology.Nissl staining also showed that the number of Nissl bodies significantly increased?P<0.01?;Compared with the conventional dosage group,the HE staining in the conventional dosage+L-T4 group showed that the cell bodies of the neurons were stratified and arranged closely.Nissl staining showed that the number of Nissl bodies was close to that of the sham group,which was better than that of other treatment groups?P<0.01?.Compared with the high dosage group,the HE staining of the high dosage+L-T4 group showed that the distribution of the neuronal cell body was laminar and arranged closely.Nissl staining showed that the number of Nissl bodies was close to that of sham group,which was better than that of other treatment groups?P<0.01?.2.2.3 Serum TT4 levels and TSH levelsTT4 results:there was no difference in serum TT4 level between the groups?P>0.05?.TSH results:Compared with sham operation group,the serum TSH level of model group was significantly higher?P<0.01?;Compared with the model group,the serum TSH level of the conventional dosage group decreased?P<0.05?;Compared with the conventional dosage group,the serum TSH level in the high dosage group and the L-T4 group was significantly lower?P<0.01?;Between conventional dosage+L-T4 group and conventional dosage group,there was no significant difference in serum TSH level?P>0.05?;Comparing the high dosage+L-T4 group and the high dosage group,there was no significant difference in serum TSH level?P>0.05?.2.2.4 Expression of TT4 and MCT8 protein in hippocampus?1?T4:Compared with sham group,the level of TT4 in hippocampus of the model group was significantly lower?P<0.01?;Compared with the model group,the levels of TT4 in the hippocampus of the conventional dosage group increased?P<0.05?;In the high dosage group+L-T4 group,compared with the control group,the levels of TT4 in the hippocampus of the high dosage group increased?P<0.05?,and the levels of TT4 in hippocampus of L-T4 group were significantly higher?P<0.01?;Compared with the control group,the levels of TT4 in the hippocampus of the conventional dosage+L-T4 group were significantly higher than those in the conventional dosage group?P<0.01?;Compared with the high dosage group,the TT4 level in the high dosage+L-T4 group increased significantly?P<0.01?.?2?MCT8 protein:Compared with sham group,hippocampus MCT8 mRNA integral optical density value and protein gray value of the model group increased significantly?P<0.01?;Compared with the model group,there was no statistical significance in MCT8 mRNA integral optical density of the conventional dosage group?P>0.05?;In the high dosage group and L-T4 group,compared with the conventional dosage group,the value of MCT8 mRNA integral optical density and the gray value of hippocampus increased significantly?P<0.01?;Compared with the conventional dosage group,the MCT8 mRNA integral optical density value and the gray value of the conventional dosage+L-T4 group were significantly higher?P<0.01?;Compared with the high dosage group,hippocampus MCT8 mRNA integral light density value and protein gray value of the high+L-T4 group increased significantly?P<0.01?.2.2.4 Results of BDNF levels in hippocampus.Compared with sham group,the level of BDNF in hippocampus of model group was significantly lower?P<0.01?;Compared with the model group,the levels of BDNF in the hippocampus of the conventional dosage group were significantly higher?P<0.01?;Comparing the high dosage group,the L-T4 group with the conventional dosage group,the BDNF level in the three groups was not significantly different?P<0.01?;Compared with the conventional dosage group,the levels of BDNF in the hippocampus of the conventional dosage+L-T4 group increased significantly?P<0.01?;Between the high dosage+L-T4 group and high dosage group,the BDNF level in hippocampus of high dosage+L-T4 group was significantly higher?P<0.01?.2.2.5 Expression of key proteins in BDNF-ERK/RSK-CREB pathway in hippocampus:?1?WB results:Compared with sham group,the gray value of BDNF,TrKB,ERK1,RSK,CREB protein in model group decreased significantly?P<0.01?;Compared with the model group,the gray value of BDNF,TrKB,ERK1,RSK,CREB protein in the hippocampus of the conventional dosage group increased significantly?P<0.01?;Comparing The high dosage group,L-T4 group and conventional dosage group,the gray value of BDNF,TrKB,ERK1,RSK protein in the hippocampus of the high dosage group increased?P<0.01or P<0.05?,but the gray value of CREB protein in the hippocampus was not statistically significant?P>0.05?,Similarly,the gray values of TrKB,ERK1,and RSK proteinin the hippocampus of L-T4 group were significantly higher?P<0.01?,but there was no significant difference in the gray value of CREB protein in hippocampus?P>0.05?;Compared with the conventional dosage group,BDNF group,TrKB,ERK1,RSK,CREB protein in the hippocampus of the conventional dosage+L-T4 group decreased significantly?P<0.01?;Compared with the high dosage group,the gray value of BDNF,TrKB,ERK1,RSK,CREB protein in the hippocampus of high dosage+L-T4 group decreased significantly?P<0.01?.?2?RT-PCR results:Compared with sham group,integral optical density values of BDNF mRNA,TrKB mRNA,ERK1/2 mRNA,RSK mRNA,CREB mRNA in hippocampus of the model group were significantly reduced?P<0.01?;Compared with the model group,integral optical density values of BDNF mRNA,TrKB mRNA,ERK1/2 mRNA,RSK mRNA,CREB mRNA in the hippocampus of the conventional dosage group increased significantly?P<0.01?;Comparing The high dosage group,L-T4 group and conventional dosage group,integral optical density of BDNF mRNA,TrKB mRNA,RSK mRNA in the hippocampus of high dosage group increased?P<0.01 or P<0.05?,integral optical density values of BDNF mRNA,ERK1/2 mRNA,RSK mRNA,CREB mRNA in hippocampuswere of L-T4 group increased?P<0.01 or P<0.05?;Compared with the conventional dosage group,integral optical density values of BDNF mRNA,TrKB mRNA,ERK1/2 mRNA,RSK mRNA,mRNA CREB in the hippocampus of the conventional dosage+L-T4 group were significantly higher?P<0.01?;Compared with the high dosage group,integral optical density values of BDNF,TrKB,ERK1/2 mRNA,RSK mRNA,CREB mRNA in the hippocampus of high dosage+L-T4 group were significantly higher?P<0.01?.2.5 Hippocampus CA1 BDNF/TrkB fluorescence double immunostaining resultsFluorescence microscopy:After staining,the nucleus DAPI was blue under the excitation of 345nm wavelength,BDNF was green under the excitation of 494nm wavelength,TrkB was red under the excitation of 650nm wavelength;After fusion imaging,the co expression site of BDNF and TrkB was orange yellow;The orange expression of the sham operation group was the most abundant;The model group cells expressed less orange;In the treatment group,the expression of the high dosage group was most rich.Compared with sham operation group,the BDNF/TrkB double expression positive cells in hippocampus CA1 area of the model group decreased significantly?P<0.01?;Compared with the model group,the BDNF/TrkB double expression positive cells in the hippocampal CA1 area of the conventional dosage group increased,but there was no significant difference?P<0.05?;Comparedthe high dosage group and the L-T4 group with the conventional dosage group,the high dosage+L-T4 group had the highest number of double expression positive cells?P<0.01?;Comparing the control group,the high dosage group and the L-T4group,there was no significant difference in the number of the BDNF/TrkB double expression positive cells in the hippocampal CA1 region?P>0.05?;Compared with high dosage group,in the hippocampus CA1 area,BDNF/TrkB double expression positive cells of the high dosage+L-T4 group was increased?P<0.05?;Compared with the conventional dosage group,the number of BDNF/TrkB positive cells in the hippocampal CA1 area of the high dosage+L-T4 group was increased?P<0.05?.Conclusion:1.SCH patients have a wide range of cognitive impairment,serum TSH,BDNF levels are significant factors affecting the cognitive function of SCH;2 Jianpi Huatan Huoxue decoction has obvious effect on the recovery of SCH rat thyroid function,spatial learning and memory ability.The mechanism may be related to the increase of the number of CA1 cells and the levels of TT4 and BDNF in the hippocampal area and the increase of TT4 and BDNF levels in the hippocampus,and the regulation of the signal transduction pathway of BDNF-ERK/RSK-CREB.Meanwhile,the Jianpi Huatan Huoxue decoction has synergistic effect with L-T4. |
PDF Full Text Request