Study On Improving Deep Vein Thrombosis Effect And Mechanism Of Mai Luo Shutong Pill Based On NF-κB

Objective:To study the potential active ingredients of Mailuo Shutong Pill（MLSTP）in the treatment of deep vein thrombosis（DVT）and the mechanism of MLSTP in intervening DVT through NF-κB pathway based on network pharmacology,DVT rat model and inflammatory injury of Human umbilical vein endothelial cells（HUVEC）.Methods:1.Network pharmacology:The corresponding compounds and targets of 12 traditional Chinese medicines in the compound were collected through TCMSP and TCMID databases.Then,the disease targets of DVT were integrated from Drugband database,Genecard database and OMIM database.After screening the common targets of traditional Chinese medicine and disease,the biological interaction network of"traditional Chinese medicine-molecule-disease-target"for MLSTP intervention in DVT was constructed to predict the effective compounds of MLSTP.Import common targets on the Metascape platform for gene ontology and KEGG enrichment analysis.2.In vivo experiment:SD rats aged eight months were randomly divided into blank control group(Control,distilled water 10 m L·Kg^-1),sham operation group(Sham,distilled water 10 m L·Kg^-1),model control group(Model,distilled water 10 m L·Kg^-1),MLSTP low dosage(ML,1.5g·Kg^-1),MLSTP medium dosage(MM,3 g·Kg^-1),MLSTP high dosage(MH,6 g·Kg^-1),low molecular weight heparin sodium injection group(LMWH,1230IU·Kg^-1),MLSTP and LMWH combined administration group(LMWH+MM,LMWH 1230IU·Kg^-1+MLSTP medium dosage 3g·Kg^-1),MLSTP and Ammonium pyrrolidinedithiocarbamate（PDTC）combined administration group(MM+PDTC,MLSTP medium dosage 3g·Kg^-1+PDTC 100 mg·Kg^-1).Inferior vena cava ligation-induced DVT rat model which was constructed on the seventh day,fasting for 12 hours.After normal administration for 1 hour,the rats was anesthetized with isoflurane,and sacrificed by taking blood from the abdominal aorta.Then,we weighed the thrombus,and measured the blood indexes such as APTT,PT,TT,FIB,D-dimer,NO,ET-1,IL-1β,and TNFα.m RNAs of endothelial cell adhesion factor and NF-κB pathway-related factors were detected by RT-PCR.Western Blot was used to detect the expression levels of proteins in the NF-κB pathway.HE staining and IHC staining were used to observe the infiltration of inflammatory cells,and observing the expression of MPO in the thrombus tissue by IF.3.In vitro experiments:LPS induced HUVEC cells inflammation.Firstiy,the CCK-8 cell proliferation-toxicity assay was used to screen the optimal model-induced concentration of LPS,the optimal administration concentration of MLSTP-containing serum and LMWH.Then,Setting up the control group（Control）,the model group（LPS）,the MLSTP-containing serum group（MLSTP）,the PDTC group(PD,100μmol·L^-1),the MLSTP and PDTC combined administration group（MMP）,and the LMWH group（LM）.When the cells grew to about 70%,after PBS cleaning,LPS and complete culture medium containing various therapeutic drugs were added at the same time and cultured for 24 hours.Drug intervention was carried out while inducing cell inflammation.The cell culture medium was collected for v WF content determination.After washing with PBS,Western blot was used to detect the protein expression of NF-κB P65,p-NF-κB P65,IκBa,p-IκBa and IKKβin the NF-κB pathway.Results:1.Network pharmacology predicted that there were 51 active compounds of MLSTP to interfere with DVT,and 150 disease targets for the treatment of DVT.The degree value screened out the active ingredients including quercetin,β-sitosterol,formic acid,kaempferol,ursolic acid and so on.KEGG enrichment analysis showed that the active mechanism of MLSTP in the treatment of DVT included cancer pathway,IL-17 pathway,AGE-RAGE signaling pathway,HIF-1 signaling pathway,rheumatoid arthritis,JAK-STAT signaling pathway,complement and coagulation cascade,NF-κB signaling pathway,etc.Under the guidance of the theory of"Tiaogan Qishu Huazhuo",the hypothesis was proposed that the NF-κB pathway is the main mechanism of MLSTP’s intervention in DVT.2.Both MLSTP and LMWH+MM groups could significantly reduce the weight of inferior vena cava thrombosis in DVT rats,improve the function of blood coagulation,and improve the levels of vascular tone regulators NO and ET-1.The plasma levels of IL-1βand TNF-αthat activate the NF-κB signaling pathway were significantly decreased.3.MLSTP and combined administration group（MLSTP and NF-κB pathway inhibitor Ammonium pyrrolidinedithiocarbamate（PDTC））could significantly inhibit the protein expression of NF-κB pathway in the LPS-induced endothelial cell,and significantly reduce the m RNA and protein expressions of NF-κB pathway-related factors in the thrombus.Inflammatory cells which expressed p-NF-κB P65 were reduced and the protein expression of MPO was decreased.Conclusions:1.The combined administration（MLSTP and LMWH）and MLSTP could improve the burden of thrombosis,blood hypercoagulation,endothelial dysfunction,and inflammation in DVT rats.And MLSTP could significantly improve the intervention effect of LMWH on the coagulation system.It embodied the characteristics of complementary advantages of integrated traditional Chinese and Western medicine.2.MLSTP could reduce the thrombus inflammation and endothelial cell activation through NF-κB pathway.The research would provide the basic research fundation for the clinical medication of MLSTP.