Study On The Calming Heart Effective Components And Mechanisms Of Poria Cocos

1 OBJECTIVEPoria cocos is the dry sclerotia of Poria cocos(Schw.)wolf.Poria has the effect of strengthening the spleen,diluting water and calming the heart,it is widely used in clinical practice,and is known as the theory of“Ten Drugs and Nine Poria”.At present,there are many studies on the effects of Poria on strengthening the spleen,diluting water,but there are few reports on its core effect of calming the heart,and the calming heart active substances and mechanisms of Poria are not clear.This study is guided by the theory of traditional Chinese medicine,TCM symptoms animal model was established to explore the calming heart effect of Poria and its pharmacodynamic components,modern analytical techniques combined with multi-omics analysis were used to explore the potential calming heart effect components of Poria based on the molecular,intestinal micro-ecology and metabolomics.This study aimed to provide a scientific basis for the interpretation of the material basis and mechanism of Poria’s calming heart effect,and it also lays the foundation for the clinical needs and product development.2 METHODS AND RESULTS2.1 Pharmacodynamic study on calming heart of PoriaThe rat model of restlessness syndrome was established by“chronic sleep deprivation”,and the calming heart pharmacodynamic effect of Poria was evaluated by measuring behavioral indexes such as open field test,hypothalamic neurotransmitters and pathological sections;The results showed that aqueous extract(PCD),water-soluble polysaccharides(PCWP)and triterpenes(PCT)of Poria could reduce the distance,speed,duration of movement and times of crossing the central area in the open field test of restless rats(P<0.05 and P<0.01),improve the rest duration(P<0.01),and could relieve the anxiety behavior of rats,increase the levels of 5-hydroxytryptamine,dopamine,norepinephrine,andγ-aminobutyric acid(P<0.05 and P<0.01)in the hypothalamus,reduce the levels of serum cholecystokinin(CCK),improve the content of serum amylase(P<0.01),promote neuronal cell proliferation and improve neuronal cell morphology.PCWP and PCT could also significantly reduce the content of substance P(P<0.05 and P<0.01),PCD could also reduce the content of substance P to some extent.2.2 Chemical constituents analysis of water extract and effective fraction of PoriaTriterpenoids and total triterpenes in the aqueous extract of Poria were analyzed by LC/MS/MS.Based on the mass spectral information obtained by UPLC-Q-TOF-MS,a total of 62 triterpenoids were identified in the aqueous extracts,a total of 65 triterpenoids were identified in the total triterpenes according to the standard and relevant literature,62 of which were the same.At the same time,the molecular weight of water-soluble polysaccharides of Poria was determined by size exclusion chromatography(SEC)coupled to multi-angle laser light scattering(MALLS)and Refractive index detector(RID)and it showed that the water-soluble polysaccharides consisted of three different molecular weight segments,the weight-averaged molecular weights molecular weights of them are 4.87×10~3 KD、4.62×10~2KD、7.36 KD respectively.2.3 Studies on the serum pharmacochemistry and intracerebral constituents of total tripenes of PoriaBase on the method of serum pharmacochemistry,UPLC-Q-TOF-MS was used to explore the blood components of PCT in normal rats and restless rats.The results showed that there were 33 blood components of Poria triterpenoids in normal rats.There were 31 blood components in the rats with restlessness syndrome,30 of these components are the same blood components.The results indicated that the blood components of Poria triterpenoids in normal rats and rats with restlessness syndrome were basically the same.These components may be the potential active ingredients of Poria.The results also showed that there were many triterpenoids that did not enter the bloodstream.It was speculated that Poria might also exert its tranquilizing effect by affecting intestinal flora.On the basis of the study of serum pharmacochemistry,we further investigated the brain-entering constituents of Poria total triterpenes in normal rats and rats with restlessness syndrome.It was found that the components of Poria triterpenes into the brain of normal rats and rats with restlessness syndrome were consistent.A total of seven components were found entered into the brain:Polyporenic acid C,Dehydrotumulosic acid,Poricoic acid B,Poriacosone B,Poricoic acid A,Dehydropachymic acid and Pachymic acid.These components may be potential calm heart core components of Poria.2.4 Gut microbiota study of calming heart effects of Poria16S rDNA sequencing was used to explore the effect of Poria on intestinal flora in rats with restlessness syndrome.The results showed that theαlpha-diversity index(Shannon,Simpson,ACE,Chao1 index)of intestinal flora in rats with restlessness syndrome was significantly decreased when compared with normal rats,indicating that the diversity and abundance of intestinal flora in model rats were decreased;PCD could significantly extract Shannon index and Simpson index of rats with restlessness syndrome.PCWP could significantly increase ACE index and Shannon index of rats with restlessness syndrome,and PCT have no significant effect on intestinal diversity index in rats with restlessness syndrome.At the level of phylum,class,order,family and genus,Poria could regulate the dysbiosis of the gut microbiota of restless rats,at the genus level,all of them could regulate the relative abundance of Lactobacillus,Roseburia,Fusicatenibacter,PCD could also regulate the genus Ruminococcus and Colidextribacter,PCWP could regulate Prevotellaceae＿NK3B31＿group and Rikenellaceae＿RC9＿gut＿group,and PCT could regulate the relative abundance of Ruminococcus.These results showed that the effects of different parts of Poria in regulating intestinal flora in restless rats have similarities and differences.2.5 Fecal metabonomics study of calming heart effects of PoriaThe fecal metabolomics method was used to study the biomarkers and mechanism of calming heart effect of Poria,32 fecal biomarkers related to the calming heart effect of PCD,38 biomarkers related to the calming heart effect of PCWP and 25 biomarkers related to calming heart effect of PCT were screened out.Based on the potential biomarkers,metabolic pathways were enriched,and the same metabolic pathways acting by PCD,PCWP and PCT were vitamin B6 metabolism and glycerophospholipid metabolism;Sphingolipid metabolism is also the common metabolic pathway of PCWP and PCT;Tyrosine metabolism is also the common metabolic pathway of PCD and PCT;The unique metabolic pathways of PCD were:valine,leucine and isoleucine biosynthesis,drug metabolism-cytochrome P450,valine,leucine and isoleucine degradation,aminoacyl-t RNA biosynthesis;The unique metabolic pathways of PCWP were taurine and low taurine metabolism and ether lipid metabolism.Spearman’s correlation analysis was used to analyze the relationship between intestinal flora and fecal metabolomics,the results showed that there was a close correlation between intestinal flora and fecal metabolomics.At the same time,the fecal metabolites pyridoxine and L-isoleucine may be the effectors that play a tranquilizing effect,and whether other fecal metabolites may be tranquilizing effectors need to be further analyzed.2.6 Fecal metabonomics study of calming heart effects of PoriaThe serum biomarkers and pathways were studied by serum metabolomics,12 serum biomarkers related to the calming heart effects of PCD,26 serum biomarkers related the calming heart effects of PCWP and 27 serum biomarkers related the calming heart effects of PCT were screened out.Based on the potential biomarkers screened,metabolic pathways were enriched,and the metabolic pathways acting by PCD,PCWP and PCT were taurine and hypotaurine metabolism and primary bile acid biosynthesis;The common metabolic pathways of PCWP and PCT were purine metabolism,arachidonic acid metabolism,phenylalanine metabolism,linoleic acid metabolism,α-linolenic acid metabolism,sphingolipid metabolism and glycerophospholipid metabolism;The common metabolic pathways of PCD and PCT were tyrosine metabolism;The unique metabolic pathways of PCD were:valine,leucine and isoleucine biosynthesis,aminoacyl-t RNA biosynthesis,cysteine and methionine metabolism,glycine,serine and threonine metabolism;The unique metabolic pathway of PCT is tyrosine metabolism.Spearman’s correlation analysis was used to analyze the relationship between serum and fecal metabolomics,it was also found that fecal metabolomics was closely related to serum metabolomics.2.7 Effects of Poria on"TNF-α/NF-κB"signaling pathway in rats with restlessnessSerum levels of interleukin-1β(IL-1β),interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)were measured by enzyme-linked immunosorbent assay;The expressions of TNF-α,TNF-R1,TRADD,p-NFκB p65,p-IκBα,p-IKKα/βprotein in hippocampus were detected by Western blot.Compared with the model group,the levels of serum IL-1β,IL-6,TNF-αand the expression of the above proteins in each treatment group were also variably reduced.LPS-induced inflammatory mediators in BV2 microglial cells were used to identify the effects of Dehydrotumulosic acid,Poricoic acid A,Dehydropachymic acid and Pachymic acid.It is suggested that the PCD,PCWP and PCT may play a tranquilizing effect by the regulation of the TNF-α/NFκB signaling pathway.Spearman’s correlation analysis was used to analyze the relationship between intestinal flora and inflammatory factors,the results showed that the intestinal flora was closely associated with inflammatory factors.The results showed that Dehydrotumulosic acid,Poricoic acid A,Dehydropachymic acid and Pachymic acid had no effect on the viability of BV2 cells under normal conditions,but could inhibit the excessive activation and proliferation of BV2 cells induced by LPS,and alleviate the inflammatory response.3 CONCLUSIONThe triterpenes and water-soluble polysaccharides are the material basis of Poria,Dehydrotumulosic acid、Poricoic acid A,Dehydropachymic acid and Pachymic acid were the core calming heart effect components of Poria,the fecal metabolites pyridoxine and L-isoleucine might be the potential core calming heart effect components.Poria could calm heart by modulating the composition of gut microbiota,acting on other relevant metabolic pathways and inhibiting the TNF-α/NF-κB signaling pathway.This study primarily elucidates the calming heart material basis and mechanisms of Poria,and provide the scientific evidence for perfecting the quality evaluation system,clinical application and further product development of Poria.