| The fresh rhizome of Curcuma Wenyujin Y.H.Chen et C.Ling is treated to obtain different decoction pieces:Pian-Jiang-Huang(PJH),Sheng-Ezhu(SEZ)and Cu-Ezhu(CEZ),by cutting,steaming and vinegar-boiling the rhizome respectively.These decoction pieces all have similar effects of promoting Qi,breaking blood stasis,and relieving pain.However,differences exist in their clinical applications.PJH specializes in breaking blood stasis,and promoting qi circulation;SEZ is best at removing qi stagnation and blood stasis to relieve pain;CEZ enters the liver meridian making its effect of breaking blood stasis,and relieving pain is the strongest of the three.The effects of different decoction pieces on promoting qi,relieving pain,and removing blood stasis are incrementally stronger,with CEZ at the top.Presently,the pharmacological mechanism of these different decoction pieces have not been fully explored.This study addresses the above by innovatively introducing modern frontier research technologies such as metabonomics,network pharmacology and molecular docking into the field of TCM processing research.Taking pharmacodynamic substances and endogenous metabolites as the entry point while leveraging HPLC,UPLC-Q/TOF-MS,the material basis changes of different decoction pieces of Curcuma wenyujin are identified in vivo and in vitro.Core targets are identified by combining results from molecular docking and network pharmacology.Metabolomics is used to evaluate the effects of different decoction pieces on endogenous metabolites and metabolic pathways.Correlation between effective ingredients,target proteins and metabolic pathways is evaluated comprehensively.This study will expound the processing mechanism of the different decoction pieces of Curcuma wenyujin,provide a theoretical reference for clinical use,and demonstrate a template to follow for research on the processing mechanism of traditional Chinese medicine.1.Study on the changes of material basis in vitro and in vivo of different decoction pieces of Curcuma WenyujinHPLC multi-component content determination of different decoction pieces of Curcuma Wenyujin indicated the average content of Curcumenol,(+)-Curdione,Gemacrone,Furanodiene,β-elemene and Curcumin in different decoction pieces had significant changes,where Curcumenol,Furanodiene,β-elemene,Curcumin in PJH,SEZ and CEZ decreased sequentially,(+)-Curdione decreased the most significantly;Curcumenol and Germacrone increased after steaming,but decreased after vinegar treatment.The contents of(+)-Curdione,Gemacrone and Furanodiene in three decoction pieces were higher than other chemical components.UPLC-Q/TOF-MS was used to further analyze the blood components of different decoction pieces of Curcuma Wenyujin.A total of 36 blood components of Curcuma Wenyujin were identified.The relative contents of blood components of different decoction pieces were also significantly different.The results showed that contents of(+)-curdione and Neocurdione increased sequentially in PJH,SEZ and CEZ.The contents of Curcumenol and Furanodiene were lower in PJH and SEZ,but higher in CEZ.The contents of Curcumol,Germacrone,β-elemene were higher in SEZ,but lower in PJH and CEZ.The above studies showed that the material bases of the different decoction pieces changed significantly in vivo and in vitro,mainly due to the changes in the proportions of each component.Among them,Curcumenol,(+)-Curdione,Gemacrone,Furanodiene and β-elemene were common components before and after blood injection,and their contents showed different trends in the different decoction pieces and their drug-containing plasma.The results showed that different processing methods changed the physical and chemical properties of the components and affected the absorption process of the components into blood.2.Study on the pharmacodynamics of different decoction pieces of Curcuma wenyujin for removing blood stasis and relieving painThe female rats were subcutaneously injected with estradiol benzoate and epinephrine hydrochloride every day,and treated with comprehensive stimulation(acousto-optic stimulation,restraint,tail clamping,ice water bath)to prepare the primary dysmenorrhea model of qi stagnation and blood stasis.To compare the effects of different decoction pieces of Wenyujin on removing blood stasis and relieving pain,The indexes such as torsion response,hemorheology,coagulation function,antiplatelet aggregation and pain related factors were observed.He staining was used to observe the pathological morphology of uterine tissue The results showed all groups significantly prolonged the writhing latency and reduced the writhing frequency of rats with dysmenorrhea due to qi stagnation and blood stasis,and the CEZ high-dose group had the most significant effect.Each group significantly improved the high-viscous blood state in rats induced by stagnation and blood stasis dysmenorrhea,wherein the PJH high-dose group has the best effect.The effect on the four items of coagulation shows that PJH high-dose group prolonged APTT,TT,and FIB more significantly.In terms of anti-platelet aggregation,each group had significant anti-platelet effects.Among them,the antiplatelet effect of the CEZ high-dosage group was the most obvious.The regulation of pain-related factors:each administration group regulated the pain-related factors to different degrees.Among them,high-dose of CEZ group increased PGE2,6-keto-PGFla,NO,β-EP,and decreased PGF2a,TXB2,Ca2+,IL-6,and TNF-α more significantly.Uterine pathomorphological observation showed that the uterine tissue morphology of each administration group was improved compared with the model group.Among them,the improvement effect of the CEZ high-dose group was the most obvious.The different decoction pieces of Curcuma wenyujin could significantly improve the hemorheology,coagulation,torsion,antiplatelet effect,pain related factors and uterine pathomorphology of the rats with qi stagnation and blood stasis dysmorrhea.The improvement effect of the CEZ high-dose group was the most obvious.The above studies showed that the different decoction pieces of Curcuma wenyujin had therapeutic effects on primary dysmenorrhea of rats with qi stagnation and blood stasis.Among them,the high-dose group of CEZ had more obvious effects on the improvement of torsion response,platelet aggregation,pain-related factors and uterine pathological morphology.3.Metabonomics of different decoction pieces of Curcuma wenyujinMetabonomics method was used to study the mechanism of different decoction pieces of Curcuma wenyujin on removing blood stasis and relieving pain from an overall perspective.As a result of the study,12 endogenous metabolites were identified in plasma samples of each group,including phenylalanine,tryptophan,hemolytic phosphatidylcholine,glutamic acid,phosphatidylcholine,etc.The results of pathway enrichment showed that the main metabolic pathways in plasma samples of each group involved different metabolites such as glutathione metabolism,phenylalanine metabolism,glycerolipid metabolism,tryptophan metabolism,etc.22 endogenous metabolites were identified in urine samples of each group.These metabolites mainly included lysophosphatidylcholine,phenylalanine,phosphatidylcholine,L-tyrosine,L-cysteine,phosphatidylcholine,steroid hormone,etc.The enrichment results of pathways showed that the main metabolic pathways involved in the different metabolites of each group included cysteine and methionine Metabolism of amino acids,biosynthesis of steroid hormones,metabolism of arginine and proline,metabolism of tyrosine,metabolism of glycerophospholipids,etc.28 endogenous metabolites were identified from fecal samples of each group.These metabolites mainly included endogenous differential metabolites,including arachidonic acid,phenylalanine,phosphatidylcholine,phosphatidylethanolamine,lysophosphatidylcholine,L-tyrosine,prostaglandin G2,prostaglandin D2,sphingomyelin,and so on.The results of pathway enrichment showed that the main metabolic pathways involved in the differential metabolites of fecal samples in each group included arachidonic acid metabolism,tyrosine metabolism,Glycerophosphatidyl metabolism,etc.The different decoction pieces can alleviate dysmenorrhea by regulating endogenous differential metabolites.Among them,PJH has obvious effects on sterol biosynthesis and arachidonic acid metabolism pathway regulation,and the endogenous metabolites of CEZ are more significant in the regulation of glycerolipid metabolism,phenylalanine metabolism,tyrosine metabolism,glutathione metabolism,arginine and proline metabolism,cysteine and methionine metabolism,and tryptophan metabolism pathways.Through the above studies,the abnormal metabolism of qi stagnation and blood stasis dysmenorrhea model rats and the therapeutic effect of the different decoction pieces on qi stagnation and blood stasis dysmenorrhea were closely related to lipid metabolism and amino acid metabolism.There were significant differences in the types and regulation of endogenous metabolites in platelet aggregation related metabolic pathways among the different decoction pieces.The regulation effect of CEZ was more significant.4.Prediction and analysis of molecular mechanism network of different decoction pieces of Curcuma WenyujinThe molecular mechanism of different decoction pieces of Curcuma Wenyujin in the treatment of primary dysmenorrhea by multi-component,multi-target and multi-channel was elucidated by Network pharmacology combined with molecular docking.Furthermore,the target proteins and related pathways of different decoction pieces of Curcuma Wenyujin were predicted by network pharmacology method in conjunction with relevant online databases.Based current study as well as previous test results and literature basis,5 effective core components,10 key targets and the 20 most important pathways were predicted,Among them,the main pathways related to the enrichment of the core targets of blood components in Curcuma Wenyujin include calcium signaling pathway,MAPK signaling pathway,cAMP signaling pathway and PI3K/AKT signaling pathway,all of which are related to antiplatelet aggregation,indicating that different decoction pieces of Curcuma Wenyujin may play the role of removing blood stasis and relieving pain through The anti-aggregation of blood platelet pathway.Molecular docking method was used to score the effective blood entry core components and core targets,and to verify the network prediction key targets of Curcuma Wenyujin in the treatment of primary dysmenorrhea.The results showed that 5 core components(Germacrone,Curcumenol,(+)-curdione,Furanodiene,β-elemene)and 10 core targets(APP,PIK3CA,MAPK1,ADRA2a5 ADRA2C,ADRA2B,MAPK3,CCR5,GCGR,STAT3)could spontaneously bind.The binding energies of CCR5 and MAPK1 suggesting that CCR5 and MAPK1 are the key targets in the treatment of primary dysmenorrhea by Curcuma Wenyujin,and both of them are important targets of anti-platelet aggregation related pathway,which indicates that the molecular docking results are consistent with the network pharmacology screening results,further verifying the reliability of network pharmacological prediction targets.The above studies showed that the main pathways for enrichment of core targets related to the core components screened by network pharmacology combined with molecular docking,including calcium signaling pathway,MAPK signaling pathway,cAMP signaling pathway,and PI3K/AKT signaling pathway which associated with anti-platelet aggregation.The results showed that the different decoction pieces of Curcuma Wenyujin had the effect of removing blood stasis and relieving pain through the way of anti-platelet aggregation.5.Study on in vivo and in vitro validation of different decoction pieces of Curcuma Wenyujin to remove stasis and relieve painThe in vitro anti-platelet aggregation experiment further verified the inhibitory effect of the 5 components screened by molecular docking on platelet aggregation in vitro.Studies have shown that these 5 components(Germacrone,Curcumenol,(+)-curdione,Furanodiene,β-elemene)inhibit platelet aggregation in vitro.The results show that β-elemene,(+)-Curdione,Furanodiene,and Germacrone have strong anti-platelet aggregation activity and are dosage-dependent.β-elemene has the strongest inhibitory effect on platelet aggregation.This is consistent with the prediction results of network pharmacology and molecular docking.Due to the solubility of Curcumenol,its anti-platelet aggregation activity cannot be determined.The results are consistent with the predicted results of network pharmacology and molecular docking.Western blotting method confirmed that Curcuma Wenyujin can effectively regulate the MAPK signaling pathway and PI3K/AKT signaling pathway,and reduce the protein expression levels of ERK,JNK,MAPK,PI3K,AKT and CCR5 related to these pathways,thus inhibiting platelet aggregation to achieve stasis analgesic purpose in the treatment of primary dysmenorrhea.It further verified the reliability of network pharmacology and molecular docking prediction.In summary,UPLC-Q/TOF-MS Combined with pharmacodynamics were used to identify the core components absorbed into the blood,network pharmacology combined with molecular docking was used to screen the core targets,and metabonomics was used to comprehensively evaluate the correlation between effective components,target proteins and metabolic pathways.At a holistic,as well as at the tissue and molecular level,the paper elucidates that target proteins,pain related factors,endogenous metabolites and metabolic pathways are regulated by the different decoction pieces of Curcuma Wenyujin in multiple ways,which lead to the different changes of the effect of removing blood stasis and relieving pain,and reveal the bioactive components and molecular mechanism of action of the different decoction pieces of Curcuma Wenyujin as well as the characteristics of multi-component,multi-target and multi-channel treatment of primary dysmorrhea with qi stagnation and blood stasis.Consequently,this research provides a theoretical basis for the development and clinical application of different decoction pieces of Curcuma Wenyujin. |
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