| Objective:In this study,network pharmacology and a rat model of blood stasis syndrome due to stagnation of the circulation of vital energy and cold accumulation were used to study the effect of Total flavonoids of Elaeagnus Rhamnoides?L.?A.Nelson on vascular endothelial injury.The potential targets and signaling pathways of quercetin,isorhamnetin,kaempferine and other components against vascular endothelial injury were analyzed by means of network pharmacology.At the same time,a rat model of blood stasis syndrome due to stagnation of the circulation of vital energy and cold accumulation was established by using an adrenaline and ice water bath to detect the endothelium secretion function,blood rheology,endothelial histopathology and endothelial cell damage molecular markers of total flavonoids of sea buckthorn in this model rat.We further validated the predicted targets of network pharmacology and explored the mechanism of total flavonoids from Elaeagnus Rhamnoides?L.? A.Nelson on vascular endothelial injury in model rats of blood stasis syndrome due to stagnation of the circulation of vital energy and cold accumulation.Methods:1.Network pharmacology analysis?1?The molecular structure of the chemical constituents contained in seabuckthorn was obtained through literature review and TCMSP database query.These compounds were then analyzed for oral bioavailability and drug-like,and structural identification was performed to screen for flavonoids that met the screening conditions.A ChemMapper server was used to perform predictive analysis of potential targets for the above compounds.?2?In this study,the OMIM database was searched for disease-related protein or gene information,and MAS 3.0 was used to obtain proteins that directly interacted with the disease target.Finally,a network of diseases associated with endothelial injury was constructed using Cystoscope software.The predicted targets were compared with the relevant targets in the disease network.?3?Import target target protein information into MAS 3.0 database to obtain target pathway information.Pathway annotations and GO enrichment analysis using this database-available pathway,and pathway analysis combined with the KEGG database.?4?Establish a“compound-target-pathway”network model using Cystoscope3.3.0 to map the interaction network of flavonoids molecules and target proteins in seabuckthorn.The network analyzer plug-in was used to analyze the “compound-target-pathway”network map of TFE in the treatment of vascular endothelial injury.2.Effect of TFE on model rats of blood stasis syndrome due to stagnation of the circulation of vital energy and cold accumulation?1?In this experiment,a rat model of blood stasis syndrome due to stagnation of the circulation of vital energy and cold accumulation was established using an adrenergic ice bath,and the effects of TFE on the relevant indexes of the model rats were evaluated.The experimental animals were randomly divided into 7 groups: blank control group,model control group,aspirin group,compound Danshen tablet group,TFE high dose group,TFE middle dose group and low dose of TFE group.Continuous gavage administration for 15 days,once daily.From day 10 to day 14 of the experiment,all rats except the blank control group were injected subcutaneously with epinephrine three times daily.From the 13th to the 14th day of the experiment,after each injection of epinephrine hydrochloride,all rats were treated with an ice-water bath.In this way,a rat model of blood stasis syndrome due to stagnation of the circulation of vital energy and cold accumulation was reproduced.After the last dose,blood was taken from the abdominal aorta,anticoagulated with a 3.8%sodium citrate solution?1:9?,and some blood samples were taken to measure WBV.The remaining plasma was used to determine the PT,APTT,and FIB contents.?2?The levels of TXB2,6-keto-PGF1?,vWF and TM in plasma were detected by ELISA.At the same time,histopathological examination of the thoracic aorta was performed.In addition,qRT-PCR method was used to determine the expression levels of PI3K,MAPK1,Bcl-2,and Caspase-3 genes in thoracic aorta.The results of network pharmacology prediction and the potential mechanism of TFE protection of vascular endothelial cells were analyzed.Results1.Network pharmacology analysis of total flavonoids from seabuckthorn in the treatment of endothelial injuryUsing databases such as ChemMapper,KEGG,and MAS 3.0,and Cytoscape software,seven flavonoids of quercetin,isorhamnetin,kaempferine,pelargonidin,epicatechin,catechin,and AC1LDVZ in seabuckthorn are used to target Point prediction,constructed a seabuckthorn"compound-target-pathway"network map.The relationship between seven compounds,35 target proteins,and 44 pathways of action and vascular endothelial injury was visually presented.The analysis shows that MAPK1,PIK3R1,PIK3R2,and PIK3CG are the most node nodes in the network,suggesting that they may be the core target of TFE treatment of vascular endothelial injury.The VEGF signaling pathway is the most significant pathway,suggesting that VEGF signaling pathway may be the core role of TFE in the treatment of endothelial injury.2.Effects of total flavonoids of Hippophae rhamnoides on blood rheology?WBV?in model rats of blood stasis syndrome due to stagnation of the circulation of vital energy and cold accumulation The results showed that compared with the blank control group,WBV increased significantly in the model group at the shear rates of 1 s-1,5 s-1,50 s-1,100 s-1 and 200s-1?P<0.01?.After TFE intervention,WBV can be reduced.Compared with the model group,WBV at all shear rates in the TFE-treated group?high and middle doses?was significantly decreased?P<0.05 or P<0.01?.Compared with the model group,the positive control group,aspirin group and FFDSP group were significantly decreased?P<0.05 or P<0.01?.The TFE concentration significantly inhibited the WBV increase caused by adrenaline plus ice water bath,but the effect was not significant in the experimental concentration range.There was no significant difference in WBV between TFE-L,TFE-M and TFE-H groups.3.Effects of total flavonoids of Hippophae rhamnoides on blood coagulation?PT,APTT and FIB?and secretion of vascular endothelial cells in model rats of blood stasis syndrome due to stagnation of the circulation of vital energy and cold accumulationThe results showed that compared with the blank control group,the PT and APTT of the model control group rats were shortened and the FIB level was decreased?P<0.01?.After TFE intervention,TFE?0.24,0.48 g/kg?significantly prolonged PT,APTT?P<0.05 or P<0.01?and decreased FIB content?P<0.01?.With the increase of concentration,the effect of total flavonoids of Hippophae rhamnoides on PT,APTT shortening and FIB content increase in model rats gradually increased.The difference between TFE-L group and TFE-H group was statistically significant?P<0.01?.The results showed that there was a significant difference in the content of TXB2 and 6-keto-PGF1?in the model group compared with the blank group?P<0.01?.Treatment with high doses of TFE could effectively counteract the increase in TXB2levels?P<0.05?,treatment with medium and high doses of TFE could significantly reduce 6-keto-PGF1?levels?P<0.01?.4.Effects of TFE on endothelial injury of aorta in model rats of blood stasis syndrome due to stagnation of the circulation of vital energy and cold accumulationHistopathological examination of the thoracic aorta showed that compared with the model group,the aortic intima of the normal group was smooth and the smooth muscle cells of the media were arranged neatly without smooth muscle cell proliferation.In the model group,the endothelium-membrane aorta endothelium was detached or even ruptured,and the elastic fibers proliferated and the smooth muscle cells were arranged irregularly.The loss of endothelial cells in the positive and TFE treatment groups was mild,and the endothelial layer was clear and complete.The results of vWF and TM showed that the plasma vWF level in blood stasis model group was significantly higher than that in blank control group?P<0.01?.The plasma TM level in model group was significantly higher than that in blank control group ?P<0.01?.Compared with the model group,TFE groups significantly down-regulated plasma vWF levels and TM levels?P<0.01?.The qRT-PCR analysis confirmed the trend of expression of four genes that were treated with TFE.Compared with the blank control group,PI3K and MAPK1 expression in the model control group had an upward trend,but there was no significant difference?P>0.05?;Bcl-2 expression was significantly decreased?P<0.05?;Caspase-3 expression was significantly increased?P<0.01?.In addition,there was no significant difference in the expression of MAPK1 between the experimental groups?P>0.05?.Compared with the model control group,the expression of PI3K and Bcl-2 increased significantly?P<0.05 or P<0.01?,and the expression of Caspase-3 decreased significantly?P<0.05 or P<0.01?.Conclusion:1.The results of network pharmacology showed that MAPK1,PIK3R1,PIK3R2,and PIK3CG may be the core targets for the treatment of vascular endothelial injury by TFE.VEGF signaling pathway may be the central role of TFE in the treatment of endothelial injury.2.TFE can significantly improve the hemorheology,coagulation function and vascular endothelial cell secretion in model rats of blood stasis syndrome due to stagnation of the circulation of vital energy and cold accumulation,and have the effect of promoting blood circulation to remove blood stasis.3.TFE can significantly down-regulate serum TXB2 levels in rats with blood stasis syndrome and up-regulate plasma 6-keto-prostaglandin F1?levels,resulting in a decrease in the ratio between the two?T/K?,indicating that TFE's antithrombotic effects and participation in the regulation of TXA2 and PGI2 is related.4.TFE can significantly reduce the damage of vascular endothelial cells caused by epinephrine hydrochloride combined with ice water bath treatment,and reduce plasma vWF and TM content.TFE may inhibit the apoptosis of vascular endothelial cells through activation of PI3K/Bcl-2/Caspase-3 pathway,and reduce the degree of damage of vascular endothelial cells under pathological conditions caused by modeling methods,thereby protecting and maintaining the integrity of the vascular endothelium. |
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