| ObjectiveThe purpose of this study is to discuss the pathogenesis of AD from the perspective of deficiency of Pi and Shen,mutual accumulation of phlegm and blood stasis,and to clarify the theoretical basis of treating AD with Modified Shuyu Pill?MSP?.Using the methods of behavior,pathomorphology and molecular biology,effect and mechanism of MSP on learning and memory ability,hippocampal pathological injury,neuronal apoptosis and glial inflammatory reaction in AD model rats were discussed from the gene,PI3K/Akt/Bcl-2 and TLR4/My D88/NF-?B signal pathways.The research will provide theoretical and experimental basis for the study of AD treated by traditional Chinese medicine?TCM?.Methods1.Theoretical study: This paper systematically reviews and summarizes the knowledge of the ancient books of TCM and modern scholars on AD,discusses the pathogenesis of AD with deficiency of Pi and Shen,mutual accumulation of phlegm and blood stasis.From the point of view of group analysis,previous research results of the team and the mechanism of pharmacological effects of drugs,this paper expounds the theoretical basis of treating AD with MSP.2.Experimental study: A total of 48 SPF-grade male SD rats were injected into the lateral ventricle using A?1-42 oligomer under the stereotaxic locator to establish an AD animal model.After 7 days of modeling,30 successful rats were selected by water maze,and were randomly assigned into model group,western medicine group and TCM group,with 10 rats in each group.The western medicine group was given donepezil 0.45 mg·kg-1·d-1 intragastrically daily,TCM group was given MSP 10 g·kg-1·d-1 intragastrically daily,while the model group and sham operation group?only lateral ventricle injected normal saline 2 ?L?were given normal saline 10 m L·kg-1·d-1 intragastrically daily.The therapeutic course for all was 4 weeks.After 4 weeks of drug intervention,the spatial learning and memory ability of rats was measured by Morris water maze.After behavioral examination,the brain was perfused for pathological examination and immunofluorescence staining.Nissl staining?Nissl?was used to observe the Nissl body level of hippocampal neurons,Td T-mediated d UTP Nick-End Labeling?TUNEL?was used to detect the apoptosis of hippocampal neurons,and immunofluorescence staining was used to detect the expression of Iba1 and GFAP protein in hippocampal CA1 region of rats.The brain was decapitated on ice for Western Blot and real-time fluorescence quantitative PCR detection.Western Blot was used to detect the protein expression of PI3 K,Akt,p-Akt,Bcl-2,Bax,caspase-3,TLR4,My D88,NF-?B,IL-1? and TNF-? in hippocampus of rats.The expressions of PI3 K,Bcl-2,Bax,caspase-3,TLR4,My D88,NF-?B,IL-1?,TNF-? m RNA and mi R-34a-5p in hippocampus of rats were detected by real-time fluorescence quantitative PCR.Results1.Morris water maze test: On the 1st-5th day of locational navigation test,the escape latency of rats in each group was shortened gradually.On the 6th day,the escape latency of the model group was significantly longer than that of the sham operation group?P<0.01?,and the escape latency of the TCM group and the western medicine group was significantly shorter than that of the model group?P<0.01?.There was no significant difference between the TCM group and the western medicine group?P>0.05?.Space exploration test showed that the times of traversing platform and the stay time of the original platform in model group were significantly lower than those in sham operation group?P<0.01?.Compared with the model group,the times of traversing the platform and the stay time of the original platform in the TCM group and the western medicine group were significantly increased?P<0.01?,but there was no significant difference between the TCM group and the western medicine group?P>0.05?.2.Nissl staining: Nissl staining showed that the Nissl body was blue-purple and the nucleus was blue in the hippocampal CA1 region of rats in the sham operation group,and the number of Nissl bodies in the model group decreased and the staining was light compared with that in the sham operation group.Compared with the model group,the quantity of Nissl body in the western medicine group and the TCM group increased,and the staining was clearer.3.TUNEL staining: The apoptotic cells showed red fluorescence and the nucleus showed blue fluorescence.The number of apoptotic neurons in hippocampal CA1 area was low in the sham operation group.Compared with the sham operation group,the number of apoptotic neurons increased in the model group.Compared with the model group,the number of apoptotic neurons decreased in the western medicine group and the TCM group.The results showed that compared with sham operation group,the apoptosis rate in hippocampal CA1 region of rats increased significantly in the model group?P<0.01?.Compared with the model group,the apoptosis rate was significantly decreased in the TCM group and the western medicine group?P<0.01?.Compared with the western medicine group,the apoptosis rate of the TCM group decreased more obviously?P<0.01?.4.Immunofluorescence staining: Compared with the sham operation group,the expressions of Iba1 and GFAP in the model group were significantly increased?P<0.01?.Compared with the model group,the expressions of Iba1 and GFAP in the TCM group and the western medicine group were significantly decreased?P<0.05,P<0.01?.Compared with western medicine group,the expression of Iba1 and GFAP decreased significantly in the TCM group.5.Western blot detection: Compared with the sham operation group,the expressions of PI3 K,p-Akt and Bcl-2 in the model group were significantly decreased?P<0.01?,and the expressions of Bax,caspase-3,TLR4,My D88,NF-?Bp65,IL-1?,and TNF-? were significantly increased?P<0.01?.Compared with the model group,the expressions of PI3 K,p-Akt and Bcl-2 in the TCM group and the western medicine group were significantly increased?P<0.05,P<0.01?,and the expressions of Bax,caspase-3,TLR4,My D88,NF-?Bp65,IL-1?,and TNF-? were significantly decreased?P<0.05,P<0.01?.Compared with western medicine group,the expressions of PI3 K and Bcl-2were significantly increased in the TCM group?P<0.05,P<0.01?,and the expressions of caspase-3,TLR4,My D88 and TNF-? were significantly decreased?P<0.05,P<0.01?.However,there was no significant difference in the expression of Akt among the four groups?P>0 05?.6.Real-time fluorescence quantitative PCR detection: Compared with the sham operation group,the expressions of PI3 K and Bcl-2 in the model group were significantly decreased?P<0.01?,and the expressions of Bax,caspase-3,TLR4,My D88,NF-?Bp65,IL-1?,TNF-? and mi R-34a-5p were significantly increased?P<0.01?.Compared with the model group,the expressions of PI3 K and Bcl-2 in the TCM group and the western medicine group were significantly increased?P<0.01?,and the expressions of Bax,caspase-3,TLR4,My D88,NF-?Bp65,IL-1?,TNF-? and mi R-34a-5p were significantly decreased?P<0.05,P<0.01?.Compared with the western medicine group,the expressions of TLR4,My D88,IL-1? and TNF-? were significantly decreased in the TCM group?P<0.05,P<0.01?.Conclusion1.Deficiency of Pi and Shen and mutual accumulation of phlegm and blood stasis are the basic pathogenesis of AD.The treatment of AD with MSP,which is treated by invigorating the spleen,tonifying the kidney,removing phlegm and blood stasis,has rich theoretical basis.2.MSP can effectively improve the ability of spatial learning and memory and the pathological damage of hippocampus in AD model rats.3.MSP can regulate the expression of PI3K/Akt/Bcl-2 signaling pathway,and then inhibit the apoptosis of hippocampal neurons in AD model rats.4.MSP can regulate the expression of TLR4/MyD88/NF-?B signaling pathway,and then inhibit the glial inflammatory reaction in hippocampus of AD model rats.5.MSP can regulate the expression of mi R-34a-5p,then affect the signal of mi R-34a-5p/PI3K/Akt and mi R-34a-5p/TLR4,and playing a role of anti-AD. |
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