Study On The Mechanism Of Qifuyin Improving Cognitive Impairment In D-gal Mice

Objective Based on ultra high performance liquid chromatography quadrupole time of flight tandem mass spectrometry(UPLC-Q-TOF-MS),the prototype blood and brain components in qifuyin,a traditional Chinese medicine compound,were screened to clarify the potential pharmacodynamic material basis of qifuyin for improving cognitive impairment;The effect of qifuyin on endogenous metabolites in D-galactose(D-gal)model mice was studied by metabonomics;By observing the changes of energy metabolism autophagy of hippocampal neurons,the mechanism of qifuyin improving cognitive impairment in D-gal model mice was preliminarily discussed.Methods Positive and negative ion monitoring method was adopted,and UPLC-Q-TOF-MS was used for full scanning,and spectrum matching was carried out by using msdial and database.The plasma and cerebrospinal fluid samples of Qifuyin rats were analyzed and identified quickly to clarify the chemical components of Qifuyin prototype into blood and brain.Morris water maze test and new object recognition test are being used to assess Qifuyin’s influence on brain impairment in D-gal model mice.Through UPLC-Q-TOF-MS metabolomics method,the effect of qifuyin on plasma metabolism of D-gal model mice was studied.Using multivariate statistical analysis method,the plasma metabolic profile of different groups of mice was analyzed,the different metabolites and their focusing pathways were identified,and the metabolites and metabolic pathways of qifuyin in improving cognitive impairment of D-gal model mice were clarified.Blank mice,D-gal model mice,piracetam mice,and qifuyin mice were classified into four groups.The learning and memory abilities of mice were investigate using the Morris water maze method,a new item identification approach,and a dark avoidance test.HE and Nishtensite staining were used to study the morphology of neurons and nishtensite in the hippocampus;The expression of the autophagy marker protein LC3 in hippocampal neurons was identified using immunofluorescence;ATP concentration in the hippocampus was assessed using an ELISA kit;The expressions of p-AMPK,p-m TOR,p-ULK1,p-Beclin1,LC3 and P62 in the hippocampus of rats were determined by immunohistochemistry,Western Blot and RT-q PCR.Results UPLC-Q-TOF-MS method was established for the analysis of Qifuyin blood and brain components.A total of 53 prototype blood and brain components were identified,including15 components,including flavonoids,triterpenoid saponins,organic acids,sugar esters and so on.This study basically clarified the blood and brain components of Qifuyin prototype,and provided experimental basis for the study of chemical components of Qifu Yin in vivo.The latency of the water maze decreased after Qifuyin administration,the latency of the water maze decreased,the times of crossing the target platform risen,and the residence time of the target quadrant increased.The object recognition index increases.The results of metabolomics showed that qifuyin had an obvious callback trend to the abnormal differential metabolites of D-gal model mice,in which 18 metabolites such as 1-phosphate sphingosine,L-tryptophan,L-phenylalanine were identified,the pathways involved are phenylalanine,tyrosine and tryptophan biosynthesis,phenylalanine metabolism,and tryptophan metabolism.The outcomes of a multivariate statistical analysis of differential metabolites revealed substantial differences between the groups.The results indicated that the improvement of cognitive function by Qifuyin may be related to the improvement of brain energy metabolism and the increase of autophagy level.The behavioral results revealed that the D-gal model group’s escape latency seemed to be longer than the blank group’s,the times of crossing the platform and residence time in the effective area were shorter,the object recognition index was lower inside the new object recognition test,the escape latency was shorter,and the number of errors was high in the dark avoidance test,and the object recognition index was less in the new object recognition test.(P<0.01);The number of times traveling over the target platform and the duration spent in the effective area rose,the recognition index increased,the avoidance delay lengthened,and the error rate dropped in the piracetam and Qifuyin groups.(P<0.01,P<0.05).HE staining revealed that,compared with the blank group,the neurons in the hippocampus of D-gal model group were disordered,with obvious cell shrinkage,the cells shrank,some neurons lost and nuclear pyknosis was obvious;Compared with D-gal model group,piracetam group and Qifuyin group had regular arrangement of neurons,more neurons,clear nucleoli and no obvious nuclear pyknosis.The layout of hippocampal neurons in the model group was not in order,and the quantity of nissl in the cells was reduced,as compared to the blank group,as revealed by Nissl staining.The neurons in the hippocampus area of the piracetam and Qifuyin groups were more abundant and the number of nistensite was increased as compared to the D-gal model group.Autophagy in the cytoplasm of neurons in the D-gal model group decreased as compared to the blank group,according to LC3 immunofluorescence.In contrast to the D-gal model group;Compared with the D-gal model group,the phenomenon of neuronal autophagy in piracetam and qifuyin groups increased.The results of immunohistochemistry,ELISA,Western blot and RT-q PCR revealed that compared with the control group,the expressions of ATP,p-AMPK,p-ULK1,LC3A/B,and pBeclin1 in the hippocampus of D-gal model group dropped(P<0.01),and the expressions of pm TOR and P62 enhanced(P<0.01);Compared with the D-gal model group,the expressions of ATP,p-AMPK,p-ULK1,LC3A/B and p-Beclin1 in the hippocampus of piracetam group and qifuyin group increased(P<0.01),and the expressions of p-m TOR and P62 diminished(P<0.01,P<0.05)Conclusions After intragastric administration of qifuyin,a total of 53 prototype blood components and 15 brain components were identified.These components can regulate brain energy metabolism and improve neuronal damage,so as to play a neuroprotective role.A total of 18 differential metabolites were detected in plasma using metabolomic methods,and KEGG pathway analysis may regulate phenylalanine,tyrosine and tryptophan biosynthesis,phenylalanine metabolism,sphingolipid metabolism,tryptophan Metabolism and other pathways improve the body’s energy metabolism and enhance autophagy to play a neuroprotective role.Qifuyin improves cognitive impairment by enhancing brain energy metabolism and promoting autophagy of hippocampal neurons.This experiment preliminarily discussed the components of qifuyin into the blood and brain after administration and the mechanism of improving cognitive impairment in Dgal model mice,it lays an experimental foundation and theoretical basis for the clinical use of Seven Blessing Drinks and the development of anti-cognitive dysfunction drugs.