| ObjectivePaeoniae Radix Rubra is the dry root of Paeonia lactiflora Pall.Or Paeonia veichi Lynch.Bitter taste,slight cold,belonging to liver meridian.It has the effect of clearing heat,cooling blood,removing blood stasis and relieving pain.It is used for heat entering blood points,spot caused by warming toxin,hematemesis and bleeding,red eye swelling and pain,liver depression and hypochondriac pain,dysmenorrhea due to amenorrhea,abdominal pain due to dysmenorrhea,tumbling injury,carbuncle and sore,etc.Total paeony glycosides has many pharmacological effects,such as antithrombotic,antioxidant,antitumor,anti endotoxin,protecting nervous system and heart.In this project,the extraction,purification and pharmacodynamic screening of total paeony glycosides,an effective component of Paeoniae Radix rubra,were studied.Using modern analytical instruments and methods,such as UPLC-QTOF-MS to study the chemical composition of total payony glycosides;using network pharmacology research methods,using big data resources of disease target database,Swiss Target Prediction 、 SEA and other network database platforms,revealing the target and signal pathway regulated by total payony glycosides in the treatment of liver cancer.Through H22 tumor bearing mice model and Hep G2 cell model in vitro,the pharmacological and pharmacodynamic studies of total paeony glycosides in vivo and in vitro were carried out to determine whether total paeony glycosides has anti-tumor effect;and the effects on total paeony from cell cycle,apoptosis,related genes,proteins and on different subtype activity and gene level of cytochrome P450 enzyme were studied.Through the changes of liver injury,oxidative stress and mitochondrial damage in serum of rats,to explore whether total paeony glycosides can cause liver toxicity.In this study,it is clear that total paeony glycosides has anti-tumor effect on liver cancer,and its mechanism of action is explained from multiple perspectives,providing experimental basis and theoretical basis for clinical application and further development and utilization of total paeony glycosides.Methods1.By L9(34)orthogonal test design,the ratio of liquid to material,extraction time and extraction times were taken as the factors,and the comprehensive scores of paeoniflorin,total paeony glycosides and extraction rate were taken as the evaluation indexes,the best extraction process of total paeony glycosides was selected by UV-vis and HPLC;The best purification process of total paeony glycosides was selected by UV-vis spectrophotometry and macroporous resin technology through static and dynamic adsorption and desorption tests.MTT method was used to compare the antitumor effects of Paeoniae Radix rubra and total paeony glycosides on Hep G2 cells in vitro.2.By using the technology of UPLC-QTOF-MS and the method of information comparison of reference materials,literature and relevant databases,the chemical composition of total paeony glycosides was studied.By using the method of molecular network research,the molecular ion visualization network diagram of total paeony glycosides was constructed,and the relevant information of mass spectrum was combined to fully characterize the composition of total paeony glycosides.3.The effect of total payony glycosides on Hep G2 cell proliferation was studied by MTT method.Through the establishment of H22 hepatoma bearing mice model,taking the tumor inhibition rate,organ index,tumor pathological section and the content of cytokines ALT,AST,Fas,Fas L,IL-2,IFN-γ,TNF-α in serum as the index,the pharmacodynamics of total paeony glycosides in vivo was studied.4.Using the network pharmacology research method,using big data resources such as swisstargetprediction,sea,tcmsp network database platform,mining the target gene information of total payonyglycides.Using drugbank,digenet,and TTD database,finding the related target of liver cancer,obtaining the biological target of the components of total payony glycosides regulating liver cancer.By using string database and other databases,the network map of protein interaction between targets is obtained,and the relationship between targets is discussed.Through the analysis of go gene function annotation of the target,it is clear that the components of total payony glycosides are involved in the regulation of biological process,cell composition and molecular function of liver cancer.By using KEGG database toenrich and analyze the signal pathway of the target,the target and signal pathway regulated by total payony glycides in the treatment of liver cancer were revealed from big data.5.By using flow cytometry and annexin V-FITC / PI double staining method,the effect of total payony glycides on Hep G2 cell apoptosis was studied.By using PI single staining method,the effect of total payonyglycides on Hep G2 cell cycle was studied.6.Real time quantitative(q RT-PCR)was used to detect the expression of related genes in Hep G2 cells after the effect of total payony glycides on Hep G2 cells.Western blot was used to detect the expression of related proteins in Hep G2 cells after the effect of total payony glycides on Hep G2 cells,and the mechanism of total payony glycides on Hepatocarcinoma was explained from gene and protein levels.7.The activities of CYP1A2,CYP3A4,CYP2D6,CYP2C9 and CYP2E1 in rat liver were detected by ELISA.The expression of CYP1A2,cyp3a2,cyp2d1,CYP2C11 and CYP2E1 m RNA was detected by real-time fluorescence quantitative q RT-PCR.The effect of total payony glycides on different subtypes of cytochrome P450 enzyme was investigated.By detecting the activities of CYP1A2,CYP3A4,CYP2D6,CYP2C9 and CYP2E1 in rat liver,and detecting the expression of CYP1A2,CYP3A2,CYP2D1,CYP2C11 and CYP2E1 m RNA by real-time fluorescence quantitative(q RT-PCR),the effect of total paeony glycosides on different subtypes of CYP450 was investigated.8.The changes of ALT,AST,ALP and γ-GT in serum of rats were detected by ELISA to explore the damage degree of total paeony glycosides on liver tissue.The changes of SOD,MDA and GSH in serum of rats were detected by ELISA to explore the total paeony the degree of oxidative damage of glycosides to liver tissue was studied.The content of Na +-K +-ATPase in serum of rats was detected by ELISA to explore the degree of damage of total paeony glycosides to liver mitochondria.Results1.The best extraction process of total paeony glycosides is 8 times of 30%ethanol,2 times of reflux extraction,2 hours each time;the best purification process of total paeony glycosides is HPD-700 macroporous adsorption resin,the concentration of crude drug is 0.1g/ml,the resin is 0.3g/ml(original medicine / wet resin),2BV water is used to remove impurities,5BV 30% ethanol is used to elute,so total paeony is obtained chemical composition of total paeony glycosides.The inhibition rates of Paeoniae Radix rubra and total paeony glycosides on Hep G2 cells were 69.66% and 66.00%,respectively.2.By using UHPLC-Q-TOF-MS technology and positive ion detection mode,paeoniflorin,benzoylpaeoniflorin,albiflorin,oxypaeoniflorin were determined by reference,literature and relevant database information;the comparison inferred the albiflorin isomer,calycosin-7-glucoside,mudanpioside E,galileaeoniflorin or isomer.The molecular visualization network of total paeony glycides was constructed.Five molecular network component clusters were found,one of which was paeoniflorin and its related compounds.3.Based on TCM-SP、SEA and Swiss Target,139 potential target proteins were identified.A total of 5727 cancer related targets were found by using drugbank,disgenet and TTD databases.By using Venny software to map the target proteins of total payony glycosides and potential targets of liver cancer,a total of 99 cross targets were obtained,including IL-2,Bcl-2,TNF,PTEN,etc.Through the network diagram of protein interaction between the targets,the relationship between the targets is clear,and through the go gene function annotation analysis of the targets,the total payony glycosides is clear in the aspect of biological process,the components of glycosides are mainly enriched in the positive regulation of cell proliferation,negative regulation of apoptosis,MAPK cascade,negative regulation of inflammatory response,etc.;in the aspect of cell composition,they are mainly enriched in mitochondria,extracellular space,death inducing signal complex,etc.;in the aspect of molecular function,they are mainly enriched in the regulation activity of nitric oxide synthetase,protein serine/threonine Phosphatase activity,growth factor activity and other protease activities.The results of KEGG pathway enrichment analysis showed that there were 84 pathways involved in the process of total payony glycosides acting on hepatoma cells,mainly including MAPK,PI3 K / Akt,VEGF,tumor necrosis factor,cancer center carbon metabolism and other signaling pathways.The results of KEGG pathway enrichment analysis showed that there were 84 pathways involved in the process of total payony glycosides acting on hepatoma cells,mainly including MAPK,PI3K/Akt,VEGF,tumor necrosis factor,cancer center carbon metabolism and other signaling pathways.4.The results of in vitro pharmacodynamic experiment showed that the inhibitory rates of total payony glycosides(1mg / ml)on Hep G2 cells were 66.00%,72.52% and 74.10% at 24 h,36h and 48 h respectively,and IC50 values were 0.71,0.57 and 0.52mg/ml respectively.The results of in vivo pharmacodynamic experiment showed that the inhibitory rates of cyclophosphamide group and total payony glycosides(low,medium and high dose groups)were respectively 57.61%,23.06%,36.64% and 48.13% respectively.Except for the low dose group of total payonyglycides,the inhibition of tumor was more than 30%(the tumor weight inhibition rate of traditional Chinese medicine was more than 30%,and the evaluation drug had the inhibition effect),which met the relevant requirements.5.Compared with the model group,the content of ALT,AST,Fas,Fas L and IL-2 in the serum of the high and middle dose groups of total paeony glycosides were significantly lower,and the content of IFN-γ and TNF-α in the serum of the high and middle dose groups were significantly higher(P<0.01 or P<0.05).6.Total payony glycosides acted on Hep G2 cells for 36 hours,the apoptosis rate of Hep G2 cells in the low,middle and high dose groups was 11.7%,16.9% and 22.9%,respectively.Compared with the model group,the G2/ M phase ratio of Hep G2 cells in the middle dose group decreased.7.Total payony glycosides acted on Hep G2 cells for 36 hours,compared with the model group,the levels of Akt,Bcl-2,PI3 K,MEK,ERK m RNA and protein in Hep G2 cells of total payony glycosides group decreased in different degrees,with significant differences(P<0.01),PTEN and Bax m RNA increased in different degrees,with significant differences(P<0.01).9.Compared with the blank group,the CYP1A2 activities ofrats from thetotal payony glycosides group were significantly enhanced,with asignificant difference(p<0.01),while the CYP3A4,CYP2D6,CYP2C9 and CYP2E1 activi-ties were not significantly changed.Compared with the blank group,the m RNA levels of CYP1A2 was significantly increased in rats from the total payony glycosides group(p<0.01),while the m RNA levels of CYP3A2,CYP2D1,CYP2C11 and CYP2E1 were not significantly changed.10.Compared with the blank group,the serum levels of ALT,AST,ALP,γ-GT、SOD、MDA、GSH 及 Na+-K+-ATPase in the total glycosides of red peony root group did not change significantly Conclusion1.The extraction and purification method of total payony glycosides is optimizedin this paper.The process is simple and easy to operate,which lays a certain foundation for the follow-up study of the efficacy of total payony glycosides.2.By using UHPLC-Q-TOF-MS technology and building the chemical composition network of total payony glycosides,it can fully characterize the composition of total payony glycosides and show the relationship between the components,which lays the experimental foundation for its development,utilization and pharmacodynamic research.3.Through the analysis of protein network interaction,gene function annotation and target regulatory pathway,the rapid and systematic analysis of the anti-tumor mechanism of total payony glycosides was realized.The antitumor effect of glycosides may be related to PI3K/Akt,MAPK and other signaling pathways.4.Total payony glycosides can inhibit the proliferation of Hep G2 hepatoma cells by inducing apoptosis of Hep G2 cells,reducing the proportion of G2/M phase cells and disturbing the normal cell division.5.Total payony glycosides can reduce the degree of liver injury and have a certain protective effect on the liver.6.The anti-tumor effect of total payonyglycides may be related to theinhibition of Hep G2 cell proliferation,induction of apoptosis and regulation of PI3K/Akt and MEK/ERK signaling pathways.7.Total payonyglycides may play an anti-tumor role by inducing the activities of CYPl A2,the subtypes of CYP450.8.Within a certain dose range and a certain administration time,total payonyglycides does not affect the function of liver and has no hepatotoxicity,so it is worth further study. |
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