| Objective:To expound the theoretical basis of the Bushen Yangxin Huatan Anshen therapy and its representative prescription Qiangji Decoction to prevent and treat amnesia by reviewing and sorting out the classic literature on amnesia,so as to provide theoretical support for the clinical application of Qiangji Decoction to prevent and treat amnesia.The model of cognitive impairment were established by subcutaneous injection of D-galactose into the back and neck.After the mice were being treated with Qiangji Decoction,the learning and memory,neurodegenerative changes and mitochondrial function of the mice were detected.In addition,the molecular mechanism of Qiangji Decoction improving D-galactose induced cognitive impairment and mitochondrial dysfunction was explored through the mitochondrial biogenesis mediated by AMPK/SIRT1/PGC-1? signaling pathway,so as to provide a scientific basis for Qiangji Decoction to prevent and treat amnesia.Method:1.Theoretical discussion The mechanism of the heart,kidney-brain-spirit axis in the pathogenesis of amnesia was studied by retrieving the literatures about amnesia in the Chinese Medical Classic,and the theoretical basis of the Bushen Yangxin Huatan Anshen therapy and its representative prescription,Qiangji Decoction,for preventing and treating amnesia was investigated.2.Experimental study After the acclimation,the eighty C57BL/6 mice were randomly assigned to the following 4 different groups,namely,Control group(Control),D-galactose group(D-gal),Metformin group(Met),Qiangji Decoction group(QJD).The model of cognitive impairment were established by subcutaneous injection of D-gal into the back and neck.The mice in control group were administered with 0.9 % saline by subcutaneous injection for 8 weeks,while the other groups was administered with D-gal(100 mg/kg/d).From the 5th to the 8th week,the Met group was given metformin(0.2 g/kg/d),the QJD-treated groups were treated with QJD extract(24.96 g/kg/d),and the Control group and the D-gal group were received the same amount normal saline by oral administration(20 m L/kg/d).After treatment,the Morris water maze(MWM)test and New object recognition(NOR)test were employed to evaluate the cognitive function of mice in each group.HE staining and Fluoro-Jade B(FJB)staining were used to observe the pathological damage and neuronal degeneration in the hippocampus of mice.The hippocampal neurons of mice were labeled with Nissl staining and the number of neurons in hippocampal area was calculated.TUNEL staining was employed to mark the number of apoptotic hippocampal neurons of mice,and immunofluorescence(IF)was used to detect the expression levels of apoptotic proteins(Bcl-2 and Bax)in hippocampal neurons of mice.Kits were used to detect the expression levels of ROS,MDA,SOD,GSH-px and CAT in the hippocampus,the relative expression levels of ROS were analyzed by flow cytometry.Western Blot was used to detect the protein expression levels of Bcl-2,Bax,Caspase-3and Cleaved Caspase-3 in the hippocampus.Transmission electron microscope(TEM)was used to observe the mitochondrial morphology of hippocampal neurons,and the length and area of mitochondria was calculated by software Image J.The mitochondrial membrane potential(MMP)of hippocampal neurons was detected by JC-1 fluorescent probe and flow cytometry,and the red-green fluorescence ratio was calculated.Kits were used to detect the contents of mitochondrial ATP,mitochondrial respiratory complexes I,III and IV in hippocampal tissue;Real Time-PCR was used to detect the copy number of mt DNA and the m RNA expression levels of PGC-1?,NRF1,NRF2 and TFAM in the hippocampal tissue.Western Blot was used to detect the protein expression level of AMPK?,p-AMPK?-Thr172,SIRT1,PGC-1?,NRF1,NRF2 and TFAM in the hippocampal tissue.Results:1.The results of experiment one During the training period of the Morris water maze(MWM)test,the escape latency of mice in each group did not change significantly from the 1st to 3th day(P>0.05),but from the 4th to 5th day,the escape latency in D-gal group was significantly increased in comparison with the control group(P<0.01),while the increased escape latency was reduced by the Met and QJD(P<0.05 or P<0.01).During the testing period,compared with the control group,the escape latency of the D-gal group was significantly increased(P<0.01),while the times of crossing the platform and the time of staying in target quadrant were significantly decreased(P<0.01).Compared with the D-gal group,the escape latency of mice in the Met group and the QJD group were decreased(P<0.01),while the times of crossing the platform and the time of staying in target quadrant were increased(P<0.05).In addition,the average swimming speed of mice in each group did not change significantly,and the difference was not statistically significant(P>0.05).During the training period of the novel object recognition(NOR)test,the time of exploring familiar object in each group did not change significantly,and the difference was not statistically significant(P>0.05).During the test period,compared with the control group,the time of exploring novel object and novel recognition index(NOR)in the D-gal group were significantly reduced(P<0.01).Compared with the D-gal group,the time of exploring novel object and novel recognition index(NOR)in the Met group and the QJD group were increased(P<0.05 or P<0.01).2.The results of experiment two(1)The expression levels of ROS,MDA,SOD,GSH-px and CAT in hippocampus.Compared with the control group,the contents of ROS and MDA in the hippocampus of the D-gal group were significantly increased(P<0.01),while the activities of SOD,GSH-px and CAT were significantly decreased(P<0.01).Compared with the D-gal group,the contents of ROS and MDA in the hippocampus of the Met group and QJD group were decreased(P<0.01),while the activities of SOD,GSH-px and CAT were increased(P<0.01 or P<0.05).(2)The results of HE staining The neurons in the CA1 and CA3 regions of the hippocampus in the control group were closely arranged,and the cells were evenly stained.Moreover,the outline of cells in the hippocampal CA1 and CA3 regions was clear,and the cytoplasm was plump.In the D-gal group,the neurons in the hippocampal CA1 and CA3 regions were irregularly arranged,and the increased intercellular space,disordered cell morphology,and hyperchromatic or pyknotic nuclei could be seen in the cells.However,the above mentioned phenomenon was reversed by QJD and Met.(3)The results of FJB staining and Tunel staining Compared with the control group,the number of FJB-labeled degenerative neurons and Tunel-labeled apoptotic neurons in the hippocampal CA1 and CA3 regions of the D-gal group were significantly increased(P<0.01).The number of FJB-labeled degenerative neurons and Tunel-labeled apoptotic neurons in hippocampal CA1 and CA3 regions of the Met group and QJD group were decreased(P<0.05 or P<0.01).(4)The results of Nissl staining The neurons in the hippocampal CA1 and CA3 regions of the control group were arranged regularly,the morphology and nuclear structure of cell was clear and completely.Moreover,there was no obvious dyed Nissl body in the cytoplasm.The neurons in the hippocampal CA1 and CA3 regions of the D-gal group were disordered,the intercellular space was increased,and the Nissl bodies in the cytoplasm were decreased.The neurons in the hippocampal CA1 and CA3 regions of the Met group and the QJD group were slightly arranged,the intercellular space was reduced,and the dyed Nissl bodies in the cytoplasm were significantly reduced.Compared with the control group,the number of neurons in the hippocampal CA1 area and CA3 area of the D-gal group was significantly reduced(P<0.01),while the number of neurons in the hippocampal CA1 area and CA3 area of the Met group and QJD group was higher than that in the D-gal group(P<0.01 or P <0.05).(5)The results of Immunofluorescence Compared with the control group,the fluorescence intensity of Bcl-2 in the hippocampal CA1 and CA3 regions in the D-gal group was significantly decreased(P<0.01),while the fluorescence intensity of Bax was significantly increased(P<0.01).Compared with the D-gal group,the fluorescence intensity of Bcl-2 in the hippocampal CA1 and CA3 regions in the Met group and QJD group were increased(P<0.01 or P<0.05),while the fluorescence intensity of Bax was decreased(P<0.01 or P<0.05).(6)The results of Western Blot Compared with the control group,the protein expression level of Bcl-2 in the hippocampus of the D-gal group was significantly decreased(P<0.01),while the protein expression levels of Bax and Cleaved Caspase-3 were significantly increased(P<0.01).Compared with the D-gal group,the protein expression levels of Bcl-2 in the hippocampus of the Met group and the QJD group were increased(P<0.05),while the protein expression levels of Bax and Cleaved Caspase-3 were decreased(P<0.01 or P<0.05).In addition,the protein expression level of Caspase-3 in the hippocampus in each group did not change significantly,and the difference was not statistically significant(P>0.05).3.The results of experiment three(1)The results of TEM Compared with the control group,the mitochondria of the hippocampal neurons in the D-gal group were swollen and fragmented.In addition,there was broken mitochondrial ridges in hippocampal neurons.While,these phenomenon were reversed by Met and QJD.Compared with the control group,the length and area of mitochondria in the hippocampus of the D-gal group were significantly reduced(P<0.01).Compared with the D-gal group,the length and area of mitochondria in the hippocampus of the Met group and QJD group were increased(P<0.05 or P<0.01).(2)The results of mitochondrial membrane potential(MMP)in hippocampal tissue.Compared with the control group,the mitochondrial membrane potential(MMP)of the hippocampal tissue in the D-gal group was significantly decreased(P<0.01).Compared with the D-gal group,the mitochondrial membrane potential(MMP)of the hippocampal tissue in the Met group and the QJD group were increased(P<0.05 or P<0.01).(3)The expression levels of mitochondrial ATP and mitochondrial respiration complexes I,III and IV in the hippocampal tissue.Compared with the control group,the expression levels of mitochondrial ATP and mitochondrial respiration complexes I,III and IV in the hippocampal tissue of the D-gal group were significantly decreased(P<0.01).Compared with the D-gal group,the expressions of mitochondrial ATP and mitochondrial respiratory complexes I,III and IV in the Met group and the QJD group were increased(P<0.05 or P<0.01).(4)The results of mt DNA copy number in hippocampal tissue.Compared with the control group,the copy number of mt DNA in the hippocampal tissue of the D-gal group was significantly decreased(P<0.01).Compared with the D-gal group,the copy number of mt DNA in the hippocampal tissue of the Met group and the QJD group were increased(P<0.05).4.The results of experiment four Compared with the control group,the m RNA and protein expression levels of PGC-1?,NRF1,NRF2 and TFAM in the hippocampus of the D-gal group were significantly decreased(P<0.01).Compared with the D-gal group,the m RNA and protein expression levels of PGC-1?,NRF1,NRF2 and TFAM in the hippocampus of the Met group and the QJD group were increased(P<0.01 or P<0.05).Compared with the control group,the protein expression levels of p-AMPK?-Thr172 and SIRT1 in the hippocampus of the D-gal group were significantly decreased(P<0.01).Compared with the D-gal group,the protein expression levels of p-AMPK?-Thr172 and SIRT1 in the hippocampus of the Met group and the QJD group were increased(P<0.01 or P<0.05).In addition,the protein expression levels of AMPK? in the hippocampus of each group did not change significantly,and the difference was not statistically significant(P>0.05).Conclusion:(1)The onset of amnesia is related to the imbalance of the heart,kidney-brain-spirit axis,and disharmony between the heart and kidney is the important pathogenesis of amnesia.The Bushen Yangxin Huatan Anshen therapy is a representative method for preventing and treating amnesia.Qiangji Decoction has the effect of Bushen Yangxin Huatan Anshen,and is an effective prescription for preventing and treating amnesia.(2)Qiangji Decoction could alleviate the learning and memory impairment in D-gal-induced mice.(3)Qiangji Decoction could alleviate the neurodegeneration and neuronal apoptosis in D-gal-induced mice via inhibiting hippocampal oxidative stress.(4)Qiangji Decoction could alleviate the mitochondrial dysfunction in D-gal-induced mice by promoting mitochondrial biogenesis,and its mechanism may be related to the activation of AMPK/SIRT1/PGC-1?signaling pathway. |
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