| Background: Animal traditional Chinese medicine resources are an important part of traditional Chinese medicine resources.They have a long history of application in our country.They have the characteristics of quick onset,strong effect,and clear curative effect.They play a unique role in the compatibility of traditional Chinese medicine compounds.Pangolin is the scales of pangolin(Manis pentadactyla Linnaeus),an animal of the dace carp family.It is one of the precious and fine traditional Chinese medicines commonly used in clinical practice.Efficacy such as reducing swelling and discharging pus,searching wind,and dredging collaterals has unique curative effect in TCM clinical and prescription composition,and its status is very important.However,due to resource constraints,in order to further strengthen the protection of pangolins,on June 5,2020,China’s State Forestry and Grassland Administration issued an announcement to make pangolins a national first-class protected animal.Subsequently,in the 2020 edition of "Chinese Pharmacopoeia"(Part One),pangolins were not continued to be included,so the contradiction between pangolins as a commonly used traditional Chinese medicine and their status as endangered wild animals further emerged.Guilingji,the earliest surviving traditional Chinese medicine compound preparation in my country,began in Jiajing in the Ming Dynasty.It has the effects of strengthening the body and nourishing the brain,strengthening the kidney,and invigorating qi,and increasing appetite.It is often used for kidney deficiency,weak yang,memory loss and other diseases.The prescription contains pangolin,a medicinal flavor derived from endangered and protected animals.Pangolins pass through twelve meridians,and the medicine is guided to its own meridian along with the air,which is an important part of the prescription.With the strengthening of national protection,the utilization of these medicinal materials will be gradually restricted.Therefore,it is urgent to find similar medicinal resources of pangolin to solve the contradiction between wildlife protection and resource shortage,and alternative research work on pangolin,an endangered medicinal material,is quite urgent and important.Amis: This topic takes the national confidential prescription GLJ and three alternative groups of GLJ-Z,GLJ-N,and GLJ-Y as the research object,adopts the rat model of cognitive dysfunction induced by D-galactose,and combines literature research and macro-efficacy indicators Multidisciplinary methods such as molecular pharmacological mechanism,metabolomics,and multivariate statistical analysis technology are used to investigate the comparative study of the pharmacodynamic effect of Guiling concentrated pangolin on improving aging cognitive dysfunction and the feasibility of substitution,in order to maintain the traditional famous and large species Validity of the tortoise set.Method: 1.Rat models of cognitive impairment induced by D-galactose were replicated,including normal blank group(Con),aging model group(Mod),Guilingji group(GLJ,75 mg/kg),Guilingji-pig hoof instead of pangolin group(GLJ-Z,75 mg/kg),Guilingji-cow hoof instead of pangolin group(GLJ-N,75 mg/kg),Guilingji-sheep hoof instead of pangolin group(GLJ-Y,75 mg/kg).2.Comprehensive screening of multiple pharmacodynamic indicators,including general condition observation,open field test,Morris water maze test and other three behavioral studies,combined with the detection of blood biochemical indicators,and the determination of histopathology of various organs,was used to investigate the comparison of the pharmacodynamic effects of Guilingji and the proposed replacement Guilingji on the improvement of D-galactose-induced cognitive impairment in rats,to avoid the onesidedness of single pharmacodynamic indicator screening,Try to reflect the systematic effect of improving cognitive impairment before and after the replacement of Guilingji.3.By measuring the changes of GSH-px,SOD,CAT,MDA and other biochemical indicators related to oxidative stress in rats,on the other hand,LC-MS method was used to determine six kinds of nerves in serum,including monoamine neurotransmitters(5-HT,NE,DA),amino acids and other neurotransmitters(Glu,GABA),cholinergic neurotransmitters(ACh),to explore the molecular pharmacological mechanism of Guilingji replacement before and after improving D-galactose-induced cognitive impairment in rats.4.Through the establishment of UPLC-Q-TOF-MS metabonomics method,explore the improvement of the overall metabolic profile of aging rats before and after the replacement of Guilingji,compare the different metabolites and metabolic pathways before and after the replacement,and comprehensively consider whether the replacement of Guilingji pangolin before and after the replacement of Guilingji is substitutable from the micro-metabolism level.Result: 1.The hair of the rats injected with D-galactose gradually turned yellow,thickened,and lost luster,showing signs of aging.In the Morris water maze test,the escape latency of the rats in the model group was significantly prolonged(P<0.01),suggesting that aging rats impaired memory consolidation.At the same time,in the space exploration experiment,we found that the number of times the rats entered the platform and the quadrant residence time also decreased,suggesting that the memory function of the rats in the model group was abnormal.The model build was successful.After the administration of Guilingji and the substitute Guilingji,it can shorten the escape latency of rats(P<0.01),increase the number of times rats enter the platform(P<0.01)and the dwelling time of the platform quadrant,and improve the rats to find the platform The trajectory of the movement shows that Guilingji and its three replacement groups can improve the cognitive dysfunction caused by D-galactose-induced aging.2.The results of the open field showed that the number of crossing grids(P<0.01)and the number of uprights(P<0.05)of the rats in the model group were significantly reduced,suggesting that the motor ability and exploration ability of the rats decreased.After giving Guilingji and other alternative groups,compared with the model group,the Guilingji group(P<0.01),the GLJ-Z group(P<0.01),the GLJ-N group(P<0.01)and the GLJ-Y(P<0.05)can significantly increase the number of crossing grids and the number of crossing grids of rats.In the results of pathological slices,both Guilingji and the replaced Guilingji can improve the pathological changes in the brain,liver,kidney,prostate and other parts of aging rats.The three replacement groups of hippocampal pathological slices can improve the pathological damage of the hippocampus;the replacement of GLJ-Z has a significant effect on improving kidney lesions;the liver pathological slices have the same effect as the original formula.The closest,followed by pig’s hooves,and bovine’s hooves were less effective;in prostate tissue sections,the effect of GLJ-Z group was better than that of the other two replacement groups.3.The activities of serum oxidative stress indicators GSH-Px(P<0.05),SOD(P<0.05),and CAT(P<0.05)in the model group were significantly reduced,and the level of lipid peroxide MDA was significantly increased(P<0.05),indicating that the rats in the model group had signs of aging.Compared with the model group,the levels of GSH-Px(P<0.05),SOD(P<0.05),and CAT(P<0.05)in serum of rats in Guilingji group were significantly increased,and lipid peroxide MDA(P<0.05)0.05)level decreased significantly;the level of CAT(P<0.05)in the serum of rats in the GLJ-Z group was significantly increased,the level of MDA was significantly decreased(P<0.05),and the levels of GSH-Px and SOD were also relatively increased,but not statistically significant;the levels of GSH-Px(P<0.05)and CAT(P<0.05)in the serum of rats in the GLJ-N group were significantly increased,and the level of MDA was significantly decreased(P<0.05),The level of SOD was also relatively increased,but there was no statistical significance;the levels of GSH-Px(P<0.05),SOD(P<0.05),and CAT(P<0.05)in the serum of rats in the GLJ-Y group were significantly The level of MDA(P<0.05)decreased significantly.In terms of neurotransmitters,compared with the rats in the blank group,the serum neurotransmitter levels of the rats in the model group changed significantly,among which the cholinergic neurotransmitter Ach(P<0.05),the monoamine neurotransmitter NE,DA(P<0.05)and the levels of amino acid neurotransmitters Glu and GABA decreased significantly(P<0.05),indicating that the neurotransmitters in the model rats were relatively disordered.After giving Guilingji and its replacement Guilingji,the neurotransmitters in all aspects were relatively adjusted.Among them,compared with the model group,Guiling set can significantly recall the content of Ach(P<0.05),the content of DA and NE(P<0.05),and the content of Glu(P<0.05),and can significantly improve the content of Glu The ratio of neurotransmitters to GABA;GLJ-Z group can significantly regulate the content of 5-HT(P<0.05),and the other 5 neurotransmitters have a callback effect,but there is no significant difference;GLJ-N group can significantly restore the content of Ach(P<0.05),NE(P<0.05)and Glu(P<0.05),and can significantly improve the ratio of Glu to GABA;The GLJ-Y group can significantly restore the content of Ach(P<0.05),NE(P<0.05)and Glu(P<0.05),and can significantly improve the ratio of Glu and GABA.4.Through serum metabolomics technology,24 potential differential metabolites related to aging were identified.The metabolic pathways related to aging mainly include amino acid metabolism and lipid metabolism,including 7 amino acid metabolic pathways:phenylalanine tyrosine and tryptophan biosynthesis,taurine and hypotaurine metabolism,phenylalanine metabolism,arginine synthesis,cysteine and methionine metabolism,arginine and proline metabolism,tryptophan metabolism and Metabolic pathways such as glycerophospholipid metabolism in fatty acid metabolism.Guilingji can significantly recall 17 differential metabolites in model rats,including 10 metabolites such as amino acids,2 nucleoside metabolites,Lipid compound and flavonoid metabolites,1 organic substance,and taurine and hypotaurine.Four metabolic pathways are acid metabolism,arginine synthesis,cysteine and methionine metabolism,arginine,and proline metabolism.The GLJ-Z group could significantly recall 8 differential metabolites,including 4 amino acid metabolites,2 flavonoid metabolites,1 nucleoside metabolite and 1 lipid metabolite,through taurine Acid and hypotaurine metabolism,arginine synthesis two metabolic pathways to improve metabolic disorders in rats.GLJ-N group can significantly recall 10 differential metabolites,including 5 amino acid metabolites,2 lipid metabolites and flavonoid metabolites,1 organic matter,through taurine and secondary cattle The three metabolic pathways of sulfonic acid metabolism,arginine synthesis,cysteine and methionine metabolism can improve the body’s metabolic disorders.The GLJ-Y group can significantly recall 5 differential metabolites,including 3 amino acid components,1flavonoid and 1 lipid metabolite,through the metabolism of taurine and hypotaurine,and the synthesis of arginine Metabolic pathways to improve metabolic disorders in rats.Conclusion: From the perspective of activity and immune function,the effect of improving activity after the replacement of GLJ-Z and GLJ-N was the closest to the original formula,and the effect of improving immune function of GLJ-Z group is close to the original formula.From the perspective of spatial learning and memory ability,Guilingji can significantly improve the spatial learning and memory dysfunction of aging rats.GLJ-Z group is the closest to the original formula,and the other two replacement groups are the second.From the perspective of the molecular pharmacological mechanism of oxidative stress and neurotransmitter,the replaced GLJ-N and GLJ-Y showed similar effects,while GLJ-Z took second place.From the perspective of metabolites in vivo,the GLJ-Z group and GLJ-N group can relatively recall about 50% of the differential metabolites,while the GLJ-Y group can only recall about 30% of the differential metabolites of the original formula.Therefore,the three turtle age groups after replacement can improve the cognitive function increase caused by D-galactose-induced aging to varying degrees,but the replacement effect is different at different level. |
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