| Objective:To investigate the effect of Huanglian Jiedu Decoction on atherosclerosis and the effect of Huanglian Jiedu Decoction on NF-κB inflammatory pathway.In order to provide more scientific molecular biological evidence for the clinical application of heat-clearing and detoxification to prevent atherosclerosis.Methods:1.Molecular docking:The molecular docking of Huanglian Jiedu decoction on inflammation-related factors(1)The TCMSP database was used to search drug components and their pharmacological properties.Keywords were "Scutellaria","Coptis","Coptis" and"gardenia".The results were screened using ADME(drug absorption,distribution,metabolism and excretion)model,and the screening conditions were set as oral bioavailability(OB)≥30%and drug-like drug(DL)≥ 0.18 to obtain the active ingredients of Coptis Jiadu Decoction.Gene targets associated with active ingredients were retrieved and saved using the UniProt database.(2)Screening of potential targets of Coronary heart Disease:In DisGeNet,Gene Cards,OMIM,DigSee and other databases,using "Coronary Artery Disease" as the key word,the potential targets of coronary heart disease were searched,and the related targets were obtained.(3)Molecular docking technology was used for research:core targets were selected,and 3D structures of target proteins were downloaded through AlphaFold and PDBe-KB databases.At the same time,the eligible active ingredients were screened and the 3D structures of these ingredients were downloaded from the TCMSP database.These two parts of data were imported into PyMOL and AutoDock Tools for pre-processing,and then AutoDock Vina was input for molecular docking and scoring,and the minimum binding energy of each pair of receptor-ligand was given.The results were statistically analyzed to study the binding activity between the active components of Huanglian Jiedu decoction and anti-CAD related target proteins.2.In vivo experiment:Experimental study of Huanglian Jiedu Decoction on high-fat miceFifty APOE-mice were divided into normal diet control group,high-fat diet(model group),Huanglian Jiedu decoction low(2.28g/kg),medium(4.55g/kg)and high-dose(9.1g/kg)groups,with 10 mice in each group.In addition to the normal diet control group,all the other groups were given high-fat diet.After high-fat feeding for 12 weeks,each dose group of Huanglian Jiedu Decoction was given intragastric administration for 12 weeks.After a total of 24 weeks,after the mice were killed under anesthesia,the aorta was separated for gross oil red O staining,HE staining,MASSON staining,and immunohistochemical detection of iNOS and Arg-1.Total cholesterol(Tc),triglyceride(TG),low density lipoprotein cholesterol(LDL-c)and high density lipoprotein cholesterol(HDL-c)were measured in serum of each group.The inflammatory factors(IL-6)in serum were detected by ELISA.Tumor necrosis factor α(TNF-α);Interleukin 1-B(IL-1β);Chemokine 12(CXCL12);Chemokine C-C conjunctin 2(CCL2);Intercellular adhesion molecule-1(VCAM-1)water(P<0.05).Real-time fluorescence quantification(qPCR)was used to detect the downstream genes of NF-κB inflammatory pathway:IL-6,TNF-α,IL-1β,iNOS(inducible nitric oxide synthase).Western Blotting was used to detect the expression of NF-κB inflammatory pathway related proteins:TLR4,MYD88,P-NF-κB p65,NF-κB p65,IKB α,TRAF6,iNOS,Arg-1.2.In vitro experiment:The drug-containing serum of Huanglian Jiedu Decoction alleviates the macrophage inflammation induced by lipopolysaccharide(LPS)(1)CCK8 experiment:Dose exploration was carried out on different concentrations of drug-containing serum of Huanglian Jiadu Decoction,and the concentrations were set as follows:1 μg/ml,5ug/ml,10ug/ml,20ug/ml,40ug/ml,80ug/ml,160ug/ml.(2)Uptake of fluorescent LDL:uptake of LDL by drug-containing serum in Huanglian Jiedu Decoction.(3)Intracellular ROS(Reactive Oxygen Species,reactive oxygen free radicals)level determination:the effect of drug-containing serum of Huanglian Jiedu Decoction on ROS oxidation level.(4)After the intervention of macrophages with the drug-containing serum of Huanglian Jiedu Decoction for 24 hours,the cell supernatant was collected and the human umbilical vein endothelial cells(HUVEC)were interfered with for 24 hours.The experiment of cell migration and invasion was used to investigate whether the drug-containing serum of Huanglian Jiedu decoction affected the migration and adhesion of endothelial cells.(5)The expression of proinflammatory phenotype marker CD86 and anti-inflammatory phenotype marker CD206 in macrophages were investigated by fluorescence assay.(6)ELISA was used to detect inflammatory factors in the cell supernatant:interleukin-6(IL-6);Tumor necrosis factor a(TNF-α);Interleukin 1-B(IL-1β);Intercellular adhesion molecule-1(VCAM-1).The downstream genes of NF-κB inflammatory pathway,IL-6,TNF-α,IL-1β,iNOS,were detected by qPCR.Western Blotting was used to detect the expression of NF-κB inflammatory pathway related proteins:TLR4,MYD88,P-NF-κB p65,NF-κB p65,IKBα,TRAF6,iNOS,Arg-1.Mechanism study:The drug-containing serum of Huangyan Jiedu Decoction promoted MCPIP1(monocyte chemoattractant inducer protein 1)to mediate TRAF6(tumor necrosis factor receptor-related factor 6)ubiquitination to regulate NF-κB signaling pathwayThe genes related to NF-κB pathway were detected by qPCR:TLR4,MYD88,NFκB p65,IKBα,TRAF6.The effect of drug-containing serum of Huanglian Jiedu Decoction on the expression intensity of NF-κB p65 in cell nucleus was detected by fluorescence assay.The cell nucleus and cytoplasm were separated and NF-κB p65 protein levels in the nucleus and cytoplasm were detected.siRNA-MCPIP1 and SIRRNA were transfected into 293T cells,and the expression intensity of NF-κB p65 in the nucleus was detected by fluorescence.The expression levels of MCPIP,TRAF6,NF-κB p65 were detected by WB.The plasmids of HA-MCPIP1 and HA-Ub were transfected into 293T cells,and magnetic beads containing TRAF6 antibodies were co-immunoprecipitated to detect the ubiquitination of TRAF6 by MCPIP1.HA-Ub plasmid was transfected into 293T cells,LPS and drug-containing serum were administered,and magnetic beads containing TRAF6 antibody were co-immunoprecipitated to detect the ubiquitination of TRAF6.The plasmids of HA-MCPIP1 and HA-Ub were transfected into 293T cells,followed by the intervention of LPS and the drug-containing serum of coprecipitation of magnetic beads containing TRAF6 antibodies to detect the ubiquitination of TRAF6.Results:1.Molecular docking of genes related to NF-κB inflammatory pathway by Huanglian Jiedu Decoction(1)Collection of active ingredients and targets of Huanglian Jiadu Decoction:96 active ingredients were obtained from the TCMSP database using oral bioavailability OB≥30%and drug-like drug DL≥0.18 as screening conditions,including 29 Scutellaria baicalensis,15 Coptis coptidis,37 Phellodendri Phellodendri and 15 gardenia gardenia.(2)Molecular docking test of effective ingredients of Huanglian Jiedu Decoctiondisease target protein:The active ingredients of Huanglian Jiedu decoction and the targets IL-1B,TNF-α,IL-10 and Arg-1 were selected to conduct molecular docking simulation test.The scoring results of AutoDock Vina showed that 96.20%of the component-target pairs were up to standard.Among them,85.75%can reach ≤-6.0kcal/mol and 41.77%can reach ≤-7.0kcal/mol.This showed that the prediction results were reliable and the affinity between the effective components and the target proteins was strong.2.In vivo experiment:Experimental study of Huanglian Jiedu Decoction on high-fat mice(1)aorta gross oil red O staining showed that after the intervention of different doses of Huanglian Jiedu decoction,the formation of whole-body plaques in APOE mice was significantly reduced.(2)HE staining showed that different doses of Huanglian Jiedu decoction reduced foam cell infiltration and cholesterol crystallization,and the high dose of Huanglian Jiedu decoction group was the most obvious.(3)In terms of total cholesterol(Tc),high-dose Huanglian Jiedu decoction could significantly reduce the level of total cholesterol in APOE-mice(P<0.05).In terms of triglyceride(TG),high-dose Huanglian Jiedu decoction could significantly reduce the level of total cholesterol in APOE-mice(P<0.05).In terms of low density lipoprotein cholesterol(LDL-c),high-dose Huanglian Jiedu decoction could significantly reduce the level of low density lipoprotein cholesterol in APOE-mice(P<0.05).In terms of high-density lipoprotein cholesterol(HDL-c),each dose of Huanglian Jiedu decoction did not significantly increase the level of HDL-c(P>0.05).(4)Immunohistochemical results showed that different doses of Huang Lian Jiedu decoction significantly increased the expression of anti-inflammatory factor Arg-1(Arginase-1)(P<0.05),and decreased the expression of pro-inflammatory factor iNOS(inducible nitric oxide synthase)(P<0.05).(5)ELISA experiment showed that:compared with model group,medium and high dose Huanglian Jiedu decoction significantly decreased the level of inflammatory factor interleukin-6(IL-6)(P<0.05);High-dose Huanglian Jiedu decoction significantly decreased the level of tumor necrosis factor α(TNF-α)(P<0.05);High-dose Huanglian Jiedu decoction significantly decreased the level of interleukin 1-B(IL-1β)(P<0.05);High-dose Huanglian Jiedu decoction significantly decreased the level of chemokine 12(CXCL12)(P<0.05);High-dose Huanglian Jiedu decoction significantly decreased the level of chemokine C-C conjunctin 2(CCL2)(P<0.05);High dose of Huanglian Jiedu decoction significantly decreased the level of intercellular adhesion molecule-1(VCAM-1)(P<0.05).(6)qPCR experiment showed that compared with model group,in terms of IL-6 mRNA transcription level,medium and high dose Huanglian Jiedu decoction group could significantly reduce IL-6 mRNA transcription level in APOE-mouse aortic tissue(P<0.05).In terms of IL-1β mRNA transcription level,medium and high dose Huanglian Jiedu decoction group could significantly reduce the IL-1 β mRNA transcription level in APOE-mouse aortic tissue(P<0.05).In terms of TNF-α mRNA transcription level,low,medium and high doses of Huanglian Jiedu decoction group could significantly reduce the TNF-α mRNA transcription level in APOE-mouse aortic tissue(P<0.05).In terms of iNOS mRNA transcription level,low,medium and high doses of Huanglian Jiedu decoction group could significantly reduce the iNOS mRNA transcription level in APOE-mouse aortic tissue(P<0.05).(7)Western Blotting showed that:compared with model group,low,medium and high dose Huanglian Jiedu decoction group could significantly reduce the expression level of TLR4 protein in APOE-mouse aorta tissue(P<0.05);Medium and high dose Huanglian Jiedu decoction group could significantly reduce the expression level of MYD88 protein in APOE-mouse aorta tissue(P<0.05).High-dose Huanglian Jiedu decoction group could significantly reduce the P-NF-κB p65/NF-κB p65 ratio of APOE-mouse aortic tissue(P<0.05).Medium,high-dose Huanglian Jiedu decoction group could significantly increase the expression level of IKB α protein in APOE-mouse aorta tissue(P<0.05).In addition,high dose of Huanglian Jiedu decoction group could significantly reduce iNOS protein expression level in APOE-mouse aorta tissue(P<0.05).Low,medium and high doses of Huanglian Jiedu decoction group could significantly increase the expression level of Arg-1 protein in APOE-mouse aorta tissue(P<0.05).3.In vitro experiment:The drug-containing serum of Huanglian Jiedu Decoction alleviates the macrophage inflammation induced by lipopolysaccharide(LPS)CCK8 experiment showed that the cell activity was not significantly decreased when the concentration of Huanglian Jiedu Decoction was lower than 160μg/ml,while the cell activity was significantly decreased when the concentration of Huanglian Jiedu Decoction was 160μg/ml.The difference was statistically significant(P<0.05).Therefore,20μg/ml,40μg/ml and 80μg/ml were selected as the low,medium and high dose concentrations of Coptis Jiedu decoction in this study.Fluorescence assay of LDL uptake showed that compared with LPS group,40μg/ml and 80μg/ml drug-containing serum of Huanglian Jiedu Decoction significantly increased LDL uptake(P<0.05).Intracellular reactive oxygen species(ROS)fluorescence assay showed that 40μg/ml and 80μg/ml drug-containing serum of Huanglian Jiedu Decoction significantly decreased the content of ROS(P<0.05).The experiment of cell migration and invasion showed that the human umbilical vein endothelial cells(HUVEC)were interfered with by collecting cell supernatant after the treatment of macrophages with the drug-containing serum of Huanglian Jiedu Decoction for 24 hours.Compared with LPS group,the migration and invasion rate of HUVEC cells were slowed down by different concentrations of drug-containing serum of Huanglian Jiedu Decoction,among which 40μg/ml and 80μg/ml drug-containing serum of Huanglian Jiedu decoction were significant(P<0.05).The fluorescence experiment showed that,compared with LPS group,different concentrations of drug-containing serum of Huanglian Jiedu decoction decreased the fluorescence expression of CD86.The fluorescence expression intensity of CD206 was enhanced.ELISA experiment showed that compared with LPS group,high-dose Huanglian Jiedu decoction group(HLJDD-80μg/ml)could significantly reduce the level of IL-6 in macrophages(P<0.05).Medium(HLJDD-40μg/ml)and high-dose Huanglian Jiedu decoction group(HLJDD-80 μg/ml)could significantly reduce the level of IL-1 β in macrophages(P<0.05).High-dose Huanglian Jiedu decoction group(HLJDD-80 μg/ml)could significantly reduce the level of TNF-α in macrophages(P<0.05).The level of VCAM-1 in serum containing Huanglian Jiedu decoction was not decreased in different dose groups(P>0.05).The detection of downstream target gene(qPCR)of NF-κB pathway in macrophages was as follows:Compared with LPS group,the transcription level of IL-6 mRNA in high-dose(HLJDD-80μg/ml)Huanglian Jiedu decoction group was significantly decreased(P<0.05).The mRNA transcription level of IL-1β was significantly decreased(P<0.05).The mRNA transcription level of TNF-α was significantly decreased(P<0.05).The mRNA transcription level of iNOS was significantly decreased(P<0.05).Western Blotting detection of NF-κB pathway related proteins in the drug-containing serum of Huanglian Jiedu Decoction:Compared with LPS group,low,medium and high doses of drug-containing serum of Huanglian Jiedu decoction could significantly reduce TLR4 protein expression level in macrophages(P<0.05);Medium,high dose of drug-containing serum of Huanglian Jiedu decoction could significantly reduce the expression level of MYD88 protein in macrophages(P<0.05);The proportion of P-NF-κB p65/NF-κB p65 in low(HLJD-20μg/ml),medium(HLJD-40μg/ml)and high dose(HLJD-80μg/ml)groups was significantly decreased(P<0.05).IKBα level in low(HLJD-20μg/ml),medium(HLJD-40μg/ml)and high dose(HLJD-80μg/ml)groups was significantly increased(P<0.05);iNOS levels in low(HLJD-20μg/ml),medium(HLJD-40μg/ml)and high dose(HLJD-80μg/ml)groups were significantly decreased(P<0.05).Arg-1 levels in low(HLJD-20μg/ml),medium(HLJD-40μg/ml)and high dose(HLJD-80μg/ml)groups were significantly increased(P<0.05).Mechanism study:The drug-containing serum of Huangyan Jiedu Decoction promoted MCPIP1(monocyte chemoattractant inducer protein 1)to mediate TRAF6(tumor necrosis factor receptor-related factor 6)ubiquitination to regulate NF-κB signaling pathwayqPCR detection of NF-κB pathway related genes in the serum of Huanglian Jiedu Decoction:Compared with LPS group,the mrna transcription levels of TLR4 and MyD88in the serum of Huanglian Jiedu Decoction decreased;However,there was no reduction in TRAF6,IKBα,NF-κB p65 mrna transcription levels.The expression intensity of NF-κB p65 in the nucleus was decreased by the drug-containing serum of Huanglian Jiedu DecoctionThe fluorescence assay showed that compared with LPS group,the expression intensity of NF-κB p65 in the nucleus was significantly increased(P<0.05),and the expression intensity of NF-κB p65 in the nucleus was significantly decreased after the intervention of Huanglian Jiedu Decoction drug-containing serum(P<0.05).The expression of NF-κB p65 in nucleus and cytoplasm was decreased in the serum containing Huanglian Jiedu decoctionThe protein levels of NF-κB p65 in the nucleus and cytoplasm were detected.Compared with LPS group,the expression levels of NF-κB p65 in the nucleus and cytoplasm were significantly decreased.In summary,the present study found that Huanglian Jiaduo Decoction can significantly reduce the protein expression levels of TLR4,MYD88,TRAF6 and IKBα,and can reduce the phosphorylation level of NF-κB p65 and its ability to enter the nucleus.The mRNA transcription levels of TRAF6,IKBα,and NF-κB p65 were not decreased.Therefore,we hypothesized that Huanglian Jiadutang regulates the phosphorylation of NF-κB p65 by affecting the protein post-translational modification of TRAF6,thereby reducing the activation level of NF-κB signaling pathway and reducing the entry of NF-κB p65 into the nucleus.(4)MCPIP1 mediates the ubiquitination of TRAF6,promotes the protein degradation of TRAF6,and then affects the activity of NF-κB p65Fluorescence experiments showed that compared with siRNA control,the expression of NF-κB p65 in the nucleus increased after MCPIP1 was silenced(siRNA MCPIP1),indicating that MCPIP1 could affect the expression of NF-κB p65 in the nucleus.Compared with the siRNA control group,the protein content of MCPIP1 in the SIRNA-MCPIP1 group was decreased,indicating that MCPIP1 knockdown was successful.The protein expression of TRAF6 and NF-κB p65 increased,indicating that MCPIP1 negatively regulates the expression of TRAF6 and NF-κB p65.The results of immunocoprecipitate experiment showed that overexpression of MCPIP1 could increase TRAF6 ubiquitination and thus increase TRAF6 protein degradation in cells.In summary,a positive and negative experiment was used to fully demonstrate that MCPIP1 mediates TRAF6 ubiquitination,promotes TRAF6 protein degradation,and then affects the activity of NF-κB p65.(5)Huanglian Jiedu Decoction enhanced the ubiquitination of TRAF6 and promoted the protein degradation of TRAF6Chx tracking experiment showed that compared with LPS group,Huanglian Jiedu decoction enhanced the protein degradation of TRAF6.These results suggest that the enhancement of TRAF6 protein degradation by Huanglian Jiadu Decoction is closely related to ubiquitination pathway.The co-immunoprecipitation experiment showed that compared with LPS group,Huanglianjiadu decoction promoted the ubiquitination of TRAF6 and enhanced the degradation of TRAF6 protein.(6)Huanglian Jiedu Decoction promoted the expression of MCPIP1 and enhanced the ubiquitination degradation of TRAF6Compared with the overexpression of MCPIP1 group or the intervention of Huanglian Jiedu decoction alone,the expression level of MCPIP1 in the overexpression of MCPIP1 combined with Huanglian Jiedu decoction group was significantly increased,and the expression of TRAF6 was significantly down-regulated.The results of immunoprecipitation experiment showed that the ubiquitination of TRAF6 was more significant after the intervention of Huanglian Jiedu Decoction on the basis of overexpression of MCPIP1 plasmid.The experiment confirmed that Huanglian Jiedu Decoction promoted the expression of MCPIP1 and enhanced the ubiquitination degradation of TRAF6.Conclusion:1.Huanglian Jiedu Decoction can reduce aortic plaque formation,improve blood lipid level,reduce serum inflammation level,reduce the expression of inflammatory target genes downstream of NF-κB pathway,and inhibit the related proteins of NF-κB pathway in high-fat diet mice.2.Huanglian Jiedu Decoction can reduce the uptake of LDL in macrophages,reduce the level of inflammation in cells,reduce the expression of inflammatory target genes downstream of NF-κB pathway,and inhibit the related proteins of NF-κB pathway.3.MCPIP1 mediates the ubiquitination of TRAF6,promotes the protein degradation of TRAF6,and then affects the activity of NF-κB p65.4.Huanglian Jiedu Decoction promoted the expression of MCPIP1,enhanced the ubiquitination degradation of TRAF6,regulated the activity of NF-κB p65,thereby reducing the contact between NF-κB into the nucleus and inflammatory factors,thereby reducing the transcription and expression of inflammatory factors,alleviating the inflammatory response and thus stabilizing AS plaques. |
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