| Objective:1.To evaluate the efficacy and safety of Guanmaitong granules（GMTG）in the treatment of stable angina pectoris（SAP）patients with phlegm-blood stasis syndrome through clinical study.2.To explore the potential mechanisms of GMTG in the treatment of atherosclerosis（AS）through network pharmacology and provide heoretical basis for experimental study.3.To observe the effect of GMTG on plaque and inflammatory immune response in AS model conducted by Apo E-/-mice through experimental study.The related pathways were also verified to clarify the mechanisms of GMTG on AS.Methods:1.Clinical study:SAP patients with phlegm-blood stasis syndrome who met the inclusion criteria from July 2021 to July 2022 in the outpatient department of the Affiliated Hospital of Shandong University of Traditional Chinese Medicine were included and randomly divided into control group and experimental group.Taking the healthy subjects in the same period as the healthy control group.The control group was treated with western medicine,the experimental group was treated with GMTG and western medicine.The intervention period was 8 weeks.The healthy control group was without any special intervention.Observe the changes of TCM syndrome score and curative effect,angina pectoris curative effect,nitroglycerin stopping rate and electrocardiogram curative effect before and after treatment in the control group and the experimental group.The levels of blood lipid and serum Toll-like receptor 4（TLR4）,NOD-like receptor protein 3（NLRP3）,interleukin-18（IL-18）and interleukin-1β（IL-1β）were detected.2.Network Pharmacology study:Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform（TCMSP）,HERB and Gene Cards databases were used to screen the potential targets of GMTG in the treatment of AS.The protein-protein interaction（PPI）network was constructed by Metascape online analysis tool.Gene Ontology（GO）biological process（BP）enrichment analysis,Kyoto Encyclopedia of Genes and Genomes（KEGG）pathway enrichment analysis and molecular complex detection（MCODE）analysis were performed to explore the mechanisms of GMTG in the treatment of AS.3.Experimental study:48 Apo E-/-mice and 18 C57BL/6J mice were fed with normal diet for one week.C57BL/6J mice continued to be fed with normal diet,while Apo E-/-mice were fed with high-fat diet to establish AS model.After sixteen weeks of continuous feeding,3 Apo E-/-mice and 3 C57BL/6J mice were randomly selected to verify the model.After successful modeling,the remaining Apo E-/-mice were randomly divided into model group（MOD）,Guanmaitong granule group（GMTG）and atorvastatin group（ATO）,with15 mice in each group.The remaining 15 C57BL/6J mice were used as normal control group（NC）.The NC group was fed with normal diet,and 0.9%normal saline 0.2ml/20g was intragastrically administered every day.The MOD group,GMTG group and ATO group were fed with high fat diet,and 0.9%normal saline,GMTG and atorvastatin0.2ml/20g were given by gavage every day.After 8 weeks of intervention,the levels of total cholesterol（TC）,triglyceride（TG）,low density lipoprotein cholesterol（LDL-C）and high density lipoprotein cholesterol（HDL-C）in serum were examined by removing the eyeball and separating serum.The levels of serum interleukin 6（IL-6）,tumor necrosis factor-α（TNF-α）,IL-18,IL-1βand oxidized low density lipoprotein（ox-LDL）were detected by enzyme linked immunosorbent assay（ELISA）.The thoracic aorta was taken for oil red staining;hematoxylin-eosin staining（HE）,oil red staining and Movat staining were used to quantitatively analyze the contents of lipids,collagen fibers and foam cells in the plaque.The expression of TLR4,Myeloid differentiation factor 88（My D88）,Nuclear factor kappa-B（NF-κB）,NLRP3 and Cysteinyl aspartate specific proteinase（caspase-1）in the plaque was detected by immunohistochemical staining.The expression of TLR4,My D88,NF-κB,NLRP3 and caspase-1 protein in aortic tissue was detected by Western blot（WB）.The expression of TLR4,My D88,NF-κB,NLRP3 and caspase-1 m RNA in aorta was detected by q PCR.Results:1.The results of clinical study showed that compared with the control group,the TCM syndrome scores in the experimental group were significantly reduced after treatment,and the efficacy of TCM syndromes and angina pectoris was significantly improved（P<0.05）.From the curative effect of single symptom,the symptoms of chest pain,chest tightness,palpitation,shortness of breath,phlegm,thick and greasy tongue coating in the experimental group and the control group were improved to varying degrees,and the improvement of the experimental group was better than that of the control group（P<0.05）.There was no significant difference in nitroglycerin stop rate and electrocardiogram curative effect between the two groups（P>0.05）.After treatment,the levels of serum TC,TG and LDL-C in the experimental group decreased significantly,and the level of HDL-C increased significantly.After treatment,the levels of serum TC and LDL-C in the control group decreased significantly,and the level of HDL-C increased significantly.The regulation of blood lipid in the experimental group was better than that in the control group（P<0.05）.The levels of serum TLR4,NLRP3,IL-18 and IL-1βin the two groups were significantly decreased after treatment,and the decrease in the experimental group was more obvious（P<0.05）.There was no significant difference in the levels of red blood cell（RBC）,white blood cell（WBC）,platelet（PLT）,hemoglobin（HGB）,alanine transaminase（ALT）,aspartate transaminase（AST）,creatinine（Cr）and blood urea nitrogen（BUN）（P>0.05）.There were no adverse events in the two groups during the treatment.2.The results of network pharmacology analysis showed that GMTG may play a therapeutic role in AS by regulating 337 potential targets,and its mechanism may regulate multiple biological processes,such as inflammatory response,positive regulation of cell migration,positive regulation of cytokine production,response to molecule of bacterial origin,response to inorganic substance and response to wounding.The main pathways involved include pathways in cancer,lipid and atherosclerosis.The lipid and atherosclerosis pathway is not only significantly enriched,but also has the same clustering tree with the Toll-like receptor signaling pathway and the NOD-like receptor signaling pathway,suggesting that the Toll-like receptor signaling pathway and the NOD-like receptor signaling pathway may be related to AS.The results of MCODE analysis also showed that modules such as pathways in cancer,signaling by interleukins,cytokine signaling in immune system and Toll-like receptor signaling related to My D88 were also related to inflammation and immune response,which were roughly consistent with the results of enrichment analysis.The TLR4/NF-κB,NLRP3/Caspase-1 and downstream IL-1β,IL-6,TNF-αwere found in the results of enrichment analysis,suggesting that the inflammatory immune response mediated by TLR4 and NLRP3 may play an important role in the treatment of AS by GMTG.3.The results of the experimental study showed that the AS model of Apo E-/-mice could be successfully established by feeding high-fat diet for sixteen weeks.The results of ELISA showed that compared with the NC group,the serum levels of IL-18,IL-1β,IL-6 and TNF-αin the MOD group were significantly increased（P<0.05）.Compared with MOD group,the levels of IL-18,IL-1β,IL-6 and TNF-αin GMTG group and ATO group were significantly decreased（P<0.05）.There was no significant difference between GMTG group and ATO group（P>0.05）.Compared with NC group,the levels of TC,TG,LDL-C and ox-LDL in MOD group were significantly increased,and the level of HDL-C was significantly decreased（P<0.05）.Compared with MOD group,the levels of TC,TG,LDL-C and ox-LDL in GMTG group were significantly decreased,and the level of HDL-C was significantly increased（P<0.05）.The levels of TC,LDL-C and ox-LDL in ATO group were significantly decreased（P<0.05）,and there was no significant difference in TG and HDL-C（P>0.05）.The HDL-C level in the GMTG group was higher than that in the ATO group（P<0.05）,and the difference was statistically significant.There was no significant difference in TC,TG,LDL-C and ox-LDL between GMTG group and ATO group（P>0.05）.The results of oil red staining of aortic root showed that there was no plaque formation in the aortic root of NC group,while AS plaque formation and lipid deposition were obvious in MOD group,GMTG group and ATO group.Compared with MOD group,the lipid deposition area of plaques in GMTG group and ATO group was reduced to varying degrees（P<0.05）.There was no significant difference between GMTG group and ATO group（P>0.05）.The results of Movat staining showed that there was no obvious plaque formation in NC group,and the changes of plaque composition were observed in MOD group,GMTG group and ATO group.Compared with the MOD group,the content of collagen fibers in the plaques of the GMTG group and the ATO group increased,and the content of foam cells decreased（P<0.05）.There was no significant difference in the content of collagen fibers and foam cells between GMTG group and ATO group（P>0.05）.The results of immunohistochemical staining showed that compared with NC group,the expression of TLR4,My D88,NF-κB,NLRP3 and caspase-1 protein in aortic tissue of MOD group was significantly increased（P<0.05）.Compared with MOD group,the expression of TLR4,My D88,NF-κB,NLRP3 and caspase-1 protein in GMTG group and ATO group were significantly decreased（P<0.05）.Compared with the ATO group,the NLRP3 protein in the GMTG group decreased more significantly,and the difference was statistically significant（P<0.05）.There was no significant difference in TLR4,My D88,NF-κB and caspase-1 proteins between GMTG group and ATO group（P>0.05）.The results of WB showed that compared with NC group,the expression of TLR4,My D88,NF-κB,NLRP3 and Cleaved caspase-1 protein in aortic tissue of MOD group was significantly increased（P<0.05）.Compared with MOD group,the expression of TLR4,My D88,NF-κB,NLRP3 and Cleaved caspase-1 protein in GMTG group and ATO group were significantly decreased（P<0.05）.The expression of NF-κB protein in ATO group was significantly decreased（P<0.05）.There was no significant difference in the expression of TLR4,My D88,NLRP3 and Cleaved caspase-1 between GMTG group and ATO group（P>0.05）.PCR results showed that compared with NC group,the expression of TLR4,My D88,NF-κB,NLRP3 and caspase-1 m RNA in aorta tissue of MOD group was significantly increased（P<0.05）.Compared with MOD group,the expressions of TLR4,My D88,NF-κB,NLRP3 and caspase-1 m RNA in GMTG group and ATO group were significantly decreased（P<0.05）.The expression of TLR4 m RNA in GMTG group was significantly decreased,and the expression of caspase-1 m RNA in ATO group was significantly decreased（P<0.05）.There was no significant difference in the expression of My D88,NF-κB and NLRP3 m RNA between GMTG group and ATO group（P>0.05）..Conclusion:1.GMTG combined with western medicine can effectively reduce the TCM syndrome score of SAP patients with phlegm-stasis blood syndrome,improve the TCM syndromes and angina pectoris symptoms,without obvious adverse events.It also has the effect of regulating lipid and inhibiting inflammatory immune response through regulating lipid metabolism and reducing serum TLR4,NLRP3,IL-1βand IL-18 levels.2.GMTG can effectively improve the lipid metabolism of AS mice,promote plaque remodeling,increase plaque stability,and may improve the inflammatory immune response of AS mice by inhibiting the expression of TLR4/My D88/NF-κB pathway and the activation of NLRP3 inflammasome. |
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