Mechanism Of Jianpi Huazhuo Tiaozhi Granule In Treating Atherosclerosis Based On TMAO-Mediated ROS/TXNIP/NLRP3 Signaling Pathway

Objective:In this study,we took trimethylamine N-oxide（TMAO）and endothelial damage as the entry point,and investigated the modulating effect and specific regulatory mechanism of Jianpi Huazhuo Tiaozhi Granule（JHTG）on TMAO through in vivo and in vitro experimental studies to provide a reliable experimental basis for this formula to prevent and treat atherosclerosis（AS）,and to enrich the scientific connotation of treating AS from the spleen.Method:Chapter I Theoretical discussionThe classical literature on AS from the period of Huangdi Neijing to the present was collected and sorted out,and the theoretical understanding and clinical treatment experience of AS by major medical practitioners were summarized.By searching the Web of Science（WOS）database and using Cite Space software,we conducted a bibliometric and visual analysis of the studies related to the pathogenesis of AS in the past 5 years.The theoretical basis of JHTG and the relationship between the TMAO-mediated ROS/TXNIP/NLRP3 signaling pathway and AS are explained;the theory of"loss of spleen dispersal essence,internal growth of turbid evil,and unfavorable pulse pathways"in TCM is linked to the modern medical mechanism of gut microbiota and its metabolite TMAO and vascular endothelial damage.Chapter II Experimental ResearchExperiment 1 The mechanism of JHTG in treating AS based on UPLC-Q-TOF-MS combined with network pharmacologyThe main active ingredients of JHTG were screened by UPLC-Q-TOF-MS method;the corresponding targets were screened by BATMAN-TCM,Swiss Target Prediction and Uniport databases;the AS-related targets were collected from Gene Cards and OMIM database,and Venny platform was used to screen the main active ingredients of drugs and disease common targets.The Cytoscape 3.8.1 software and STRING database were used to construct the correlation network diagram and obtain the key targets,and the targets were entered into DAVID database for GO and KEGG enrichment analysis.Finally,the top six active ingredients were selected for molecular docking with the key targets in PPI.Experiment 2 Effect and mechanism of JHTG on atherosclerosis in Apo E^-/-miceThe AS animal model was established by feeding Apo E^-/-mice with high-fat chow,and the experiment was divided into control group,model group,JHTG low-dose group,JHTG medium-dose group,JHTG high-dose group,and atorvastatin group.The indexes were tested after 16 weeks of modeling in each group.The histomorphological changes of aortic root were observed by HE staining and oil red O staining,serum lipid level was detected by automatic biochemical analyzer,serum IL-1βand IL-18 level were detected by ELISA,serum TMAO level was detected by LC/MS,aortic ROS level were detected by fluorescent probe method,Western blot was used to detect the protein expression of TXNIP,ASC,NLRP3,Caspase-1 in aorta,RT-PCR was used to detect the m RNA expression of TXNIP,ASC,NLRP3 and Caspase-1in aorta.Experiment 3 Effect and mechanism of JHTG on the damage of HUVECs under TMAO stimulationThe changes in HUVECs viability after stimulation with different concentrations of TMAO（10μmol/L,25μmol/L,50μmol/L,100μmol/L,150μmol/L,200μmol/L,250μmol/L,and 300μmol/L）and the effects of different concentrations of JHTG-containing serum（2%,5%,10%,15%,20%）on the cell viability of HUVECs were detected by CCK-8 method,as well as to determine the induction concentration of TMAO and the intervention concentration of JHTG-containing serum.The experiments was divided into:blank control group,model group,JHTG group,atorvastatin group,ROS inhibitor group（NAC）and NLRP3 inhibitor group（MCC950）,and the cell morphology of each group was observed by inverted microscope,the cell viability of each group was detected by CCK-8 method,the apoptosis rate of each group was detected by flow cytometry,the levels of IL-1βand IL-18 in each group were detected by ELISA,the level of ROS in each group was detected by fluorescent probe staining,the protein expression of TXNIP and NLRP3 in each group was detected by immunofluorescence,and the protein expression of TXNIP,ASC,NLRP3 and Caspase-1 was detected by Western blot,and the m RNA expression of TXNIP,ASC,NLRP3 and Caspase-1was detected by RT-PCR.Results:Chapter I Theoretical discussionThis article summarizes the understanding of Chinese medicine on the name,location,etiology,pathogenesis and treatment of AS,and summarizes the treatment experience of some medical practitioners and related research on Chinese medicine.Keyword co-occurrence analysis by Cite Space V software revealed that inflammation,oxidative stress,micro RNA and gut microbiota are still the focus of research on the pathogenesis of AS.On this basis,our team’s view on the treatment of AS was introduced,namely,the method of"strengthening the spleen and benefiting the qi,dispersing essence and resolving turbidity",and the theoretical basis of using JHTG for the prevention and treatment of AS,which deeply links the mechanisms of gut microbiota dysbiosis,TMAO production and vascular endothelial damage with the theory of"loss of dispersing essence in the spleen,internal growth of turbidity,and adverse pulse pathways"in Chinese medicine,laying a solid theoretical basis for the subsequent experimental studies.Chapter II Experimental ResearchExperiment 1 The mechanism of JHTG in treating AS based on UPLC-Q-TOF-MS combined with network pharmacologyIn this study,a total of 39 main active ingredients of JHTG were screened,including nuciferine,9,10-dihydroxystearic acid,alisol G,（9Z,11E,13S）-13-hydroxyoctadeca-9,11-dienoic acid,phenylalanine and9,10-dihydroxy-12-octadecenoic acid,etc.The main active ingredients and disease co-acting targets were 599,and 33 targets such as STAT3,AKT1,SRC,GRB2,MAPK3 and JUN were identified as key targets in the network by Cyto NCA topology analysis.GO function enrichment analysis showed 4188biological processes,and KEGG pathway enrichment screening showed 198pathways,including lipid and atherosclerosis,PI3K-AKT signaling pathway,MAPK signaling pathway,calcium ion signaling pathway,NOD-like receptor signaling pathway and so on.The results of molecular docking show that it has good binding with JHTG.Experiment 2 Effect and mechanism of JHTG on atherosclerosis in Apo E^-/-mice1.Compared with the control group,the model group showed the most significant aortic plaque deposition,lumen narrowing,and laque area increased significantly（P<0.01）.Serum TC,TG,LDL-C,IL-1β,IL-18 and TMAO levels were significantly increased（P<0.01）,and HDL-C levels were significantly decreased（P<0.01）;The level of ROS in aorta were significantly increased（P<0.01）;The protein and m RNA expression levels of TXNIP,NLRP3,ASC,Caspase-1 in aorta were significantly increased（P<0.01）.2.Compared with the model group,the low-dose group,medium-dose group and high-dose groups,and atorvastatin group can significantly reduce the aortic plaque area,serum TC,TG,LDL-C,IL-1β,IL-18,TMAO levels,and aortic ROS levels（P<0.05,P<0.01）and significantly increased serum HDL-C levels（P<0.01）.Compared with the model group,the middle and high dose of JHTG,and atorvastatin group can significantly reduce the protein levels of TXNIP,NLRP3,and ASC（P<0.05,P<0.01）,the high-dose group of JHTG and atorvastatin group can significantly reduce the protein level of Caspase-1（P<0.01）.Compared with the model group,the low-dose,medium-dose and high-dose groups of JHTG,and atorvastatin group can significantly reduce the m RNA levels of TXNIP,NLRP3,ASC m RNA levels（P<0.05,P<0.01）,the middle-dose of JHTG,high-dose of JHTG,and atorvastatin group can significantly reduce the m RNA levels of Caspase-1（P<0.05,P<0.01）.Experiment 3 Effect and mechanism of JHTG on the damage of HUVECs under TMAO stimulation1.The effects of different concentrations of TMAO and JHTG-containing serum on the cell viability of HUVECs were examined by the CCK-8 method,and the results showed that 300μmol/L TMAO induction for 24h was suitable for establishing the HUVECs damage model,and 5%JHTG-containing serum was a suitable concentration for experimental intervention.2.Compared with the blank control group,the cell morphology and structure of the model group changed and the number of cells decreased.The cell viability was significantly decreased（P<0.01）and the apoptosis rate,IL-1β,IL-18 and ROS levels were significantly increased in the model group（P<0.01）;The protein and m RNA expression levels of TXNIP,ASC,NLRP3,Caspase-1 were significantly increased（P<0.01）。3.Compared with the model group,the cell morphology and structure of JHTG group,atorvastatin group,ROS inhibitor group and NLRP3 inhibitor group were improved,and the number of cells increased.Compared with the model group,the JHTG group,atorvastatin group,ROS inhibitor group and NLRP3 inhibitor group can significantly improve cell viability（P<0.05,P<0.01）and decreased the apoptosis rate,IL-1β,IL-18 and ROS level（P<0.05,P<0.01）.Compared with the model group,the JHTG group,atorvastatin group,ROS inhibitor group and NLRP3 inhibitor group can significantly reduce the protein levels of TXNIP,NLRP3,ASC,Caspase-1（P<0.05,P<0.01）.Compared with the model group,the JHTG group,atorvastatin group,ROS inhibitor group and NLRP3 inhibitor group can significantly reduce the m RNA levels of TXNIP,NLRP3,ASC,Caspase-1（P<0.05,P<0.01）.Conclusion:1.The results of UPLC-Q-TOF-MS combined with network pharmacology show that the 39 active components of JHTG may play a therapeutic role in AS by inhibiting the activation of NLRP3 inflammasome.2.JHTG was able to reduce blood lipid levels in Apo E^-/-mice,reduce the production of TMAO,inhibit the ROS/TXNIP/NLRP3 pathway,improve the inflammation levels and delay the formation of AS.3.JHTG can alleviate endothelial cells damage by inhibiting TMAO-induced activation of the ROS/TXNIP/NLRP3 signaling pathway and decreasing the expression of downstream inflammatory cytokines IL-1βand IL-18.