Study On The Mechanism Of Ginseng And Dandelion In The Treatment Of Atherosclerotic Inflammation

Objective:Atherosclerosis(As)is a common cardiovascular and cerebrovascular disease.As is accompanied by varying degrees of inflammation and oxidative stress damage in the asymptomatic occult,ischemic,necrotic,and fibrotic stages,and some scholars have even proposed that As is a chronic vascular inflammatory disease.The pathogenesis of As is mainly characterized by asthenia in origin and asthenia in superficiality,and studies in Lingnan area have found that As is mostly heat-toxicity and blood stasis type.Methods of regulating,nourishing and clearing away heat-toxicity,we choose ginseng and dandelion,the "monarch" and "minister" herbs of Huayu Granules as the medicine pair,which can replenishing qi and detoxicating,and regulating the balance of qi,blood,yin and yang,so as to achieve the balance of yin and yang.This is consistent with the concept of regulating the imbalance in the number and function of different subgroups of pro-inflammatory/anti-inflammatory immune cells,thereby reducing inflammatory damage,improving tissue recovery,and inhibiting the progression of As plaques.Ginseng is a well-known traditional Chinese medicine that has been used for the treatment of As,such as ginseng decoction,Renshen Yangrong decoction,Shensong Yangxin capsule,etc.Ginsenosides are the most extensively studied chemical components of ginseng,while the mechanism of action for treating As are not yet clear enough,and it has not been used for the treatment of As in clinical practice.The research methods of Traditional Chinese Medicine need to based on the principle of holistic view,rather than just paid attention to its high-content components.And further studies are needed to explore if the combined use of saponins and non-saponins in ginseng may exert a better anti-As effect.Hence,in this study,in order to investigate the the mechanism and target of the anti-inflammatory effects of ginseng-dandelion drug pair for the treatment of As,an animal model of As was established with an ApoE-/-mouse fed with high-fat diets.It is assumed that the peptides in ginseng might exert anti-inflammatory activity in endothelial cells,and our previous studies have successfully established a model of BaP-induced inflammatory HUVECs.We attempt to separate bioactive peptides from the ginseng,and a BaP-induced HUVEC cell injury model was constructed to evaluate its protective activity and potential mechanism.Methods1.Study on the mechanism of ginseng-dandelion drug pair in the treatment of As based on network pharmacologyThe active components of ginseng and dandelion were identified using the TCMSP,HERB,TCM Database@Taiwan and TCMID databases,and the targets related to active compounds were predicted by the Swiss Target Prediction database;The CTD,NCBI gene and Genecards databases were used to obtain disease-related genes.The intersection targets of active components and As were obtained using the online platform of Bioinformatics through BioVenn diagram analysis.Furthermore,these overlapping genes were processed by the String database and the interaction results were imported into Cytoscape software to visualize and evaluate the topological characteristics of the networks.The“active ingredient-common target”network were also constructed for the intersection targets.GO and KEGG enrichment analysis of common target genes were performed using the DAVID database to obtain the related functions and pathways.2.The protective effects and potential mechanism of ginseng-dandelion drug pair on As by using a high fat diet(HFD)-fed ApoE-/-mouse model.Firestly,an ultra performance liquid chromatography coupled with time-of-fight mass spectrometry(UPLC/Q-TOFMS)was used to evaluate chemical constitution of ginseng-dandelion drug pair.Eight C57BL/6J male mice were randomly selected as the blank control group and given normal diet;Fourty male ApoE-/-mice(C57BL/6 strain)were randomly divide into 5 groups(n=8 per group):model,positive,high-dose,medium-dose,and low-dose ginseng-dandelion groups,mice were given high fat diet(HFD)feeding for 18 weeks.The positive drug control group was given a hypolipidemic drug,atorvastatin(5 mg/kg/day)orally,and the mice in ginseng-dandelion group were intragastrically administrated with ginseng-dandelion extracts at a dose of 8,4 and 2 g/(kg.d),respectively,and the model group was given the same amount of normal saline orally.After the last administration,all mice were fasted overnight of food but not water for 12 hours.Then the mice were anesthetized and killed.The automatic biochemical analyzer was used to detect the levels of serum total cholesterol(TC),triglyceride(TG),low-density lipoprotein cholesterol(LDL-C)and high-density lipoprotein cholesterol(HDL-C).Biomarkers of oxidative stress(CAT,SOD,and MDA)were analyzed using biochemical kits.Levels of TNF-α,IL-1β and IL-6 in the aortic tissues were detected using specific ELISA kits.Hematoxylin-eosin(H&E)and Oil Red O stainings were used to assess pathological morphological and lipid accumulation in the aortic tissue.The mRNA expression levels of MCP-1,ICAM-1 and VCAM-1 in each group was detected by qRT-PCR assay.The expressions of CD36 and iNOS in the aorta were detected by immunohistochemistry;The changes in the PI3K/Akt/mTOR and NLRP3 signaling pathway-related proteins were detected using western blotting.3.Mechanism of anti-inflammatory injury of non-saponin component from ginseng in HUVEC cellsThe oligopeptides were purified using membrane ultrafiltration,anion-exchange chromatography,gel filtration chromatography,and reverse phase high-performance liquid chromatography.HUVEC cells in the logarithmic growth phase were randomly divided into four groups:Con group,BaP group,GSH group and GPs group.HUVEC cells were treated with a complete medium containing 10 nM BaP for 12 h to establish a cell model in all groups except for the Con group.After that,the cells in the Con and Bap groups were incubated in complete medium without any treatment.The cells in the GSH group were cultured in complete medium supplemented with 10 μM GSH for 24 h.The cells in the GPs groups were cultured in complete medium supplemented with 50 μM GPs(GP-1,GP-2 and GP-3).The CCK-8 assay was used to detect cell viability and screen the oligopeptides that provides the best protective effect against BaP-induced damage.The level of intracellular ROS was detected by the DCFH-DA method.The levels of IL-6,IL-1β,IL-18 and NO were measured using the commercial ELISA kits.Cell apoptosis was detected by flow cytometry.The expression levels of Bax,Bcl-2,Cyt c,APAF-1,pro-caspase 9,NLRP3,ASC,Caspase 1,p-PI3K,p-Akt,AhR and CYP1A1 were detected by western blot.The mRNA expression levels of AhR and CYP1A1 were detected by qRT-PCR.In order to determine whether GP-2 can reduce the secretion of inflammatory factors by reducing the activation of the PI3K/Akt pathway,the PI3K inhibitor LY294002 was employed.The group assignments were as follows:Con group,BaP group,and LY294002 group.HUVEC cells were treated with a complete medium containing 10 nM BaP for 12 h to establish a cell model in all groups except for the Con group.The cells in the LY294002 group were cultured in complete medium supplemented with 20 μM LY294002 for 24 h.The suppressive effect of LY294002 on HUVEC was confirmed by western blot analysis.Cytokine concentrations in the supernatants were measured using ELISA kits for human IL-1β,IL-6,and IL-18.Furthermore,to determine whether GP-2 can reduce the secretion of inflammatory factors by reducing the activation of the AhR,the siRNA transfection technology and inhibitors(CH223 191)to interfere the expression of AhR was used.The group assignments were as follows:Con,si-Con,BaP,si-AhR or AhR inhibitor(CH223191).Western blot analysis confirmed the suppressive effect of si-AhR on HUVEC.Cytokine concentrations in the supernatants were measured using ELISA kits for human IL-1β,IL-6,and IL-18.Results:1.1Study on the mechanism of ginseng-dandelion drug pair in the treatment of As based on network pharmacologyVia network pharmacology analysis,a total of 18 key active ingredients from ginseng and 47 active ingredients from dandelion were regarded to be effective on As.The Swiss Target Prediction databases were searched to acquire 1813 targets for the potential active ingredients.About 36363 atherosclerosis targets were obtained,and 1027 common targets were obtained after intersection with herbal targets.KEGG enrichment analysis suggested that multiple signaling pathways including PI3K-Akt,MAPK,Ras,Rap1,HIF-1 were significantly enriched.2.The protective effects and potential mechanism of ginseng-dandelion drug pair on As by using a high fat diet(HFD)-fed ApoE-/-mouse model.The animal research results show that the ginseng and dandelion extraction supplementation can effectively reduce the body weight of mice,decrease the levels of serum total cholesterol(TC),triglyceride(TG)and low-density lipoprotein cholesterol(LDL-C),increase the HDL-C levels,remarkably enhances the antioxidant enzyme activities of SOD and CAT,efficiently reduces the MDA content in serum.GD also significantly decreased the release of pro-inflammatory factors,including IL-6,TNF-α and IL-1β in the mouse aortic tissues.GD alleviated pathological damages in the aortic tissues.The aortic atherosclerotic plaque area was significantly decreased.GD also significantly decreased mRNA expression of MCP-1,ICAM-1 and VCAM-1 in aortic tissues of ApoEmice.The protein levels of iNOS and CD36 were effectively decreased.It can effectively inhibit the expression of PI3K/Akt/mTOR and NLRP3signaling pathway-related proteins in As mice.3.Mechanism of anti-inflammatory injury of non-saponin component from ginseng in HUVEC cellsThree ginseng oligopeptides,designated GP-1,GP-2 and GP-3,have been isolated and purified from ginseng,among which the GP-2(Phe-Thr-Glu-Gly-Pro)showed significant protective activity against BaP-induced HUVEC injury.GP-2 suppressed the release of ROS.IL-18.IL-1β and NO in BaP-induced HUVECs.GP-2 also significantly decreased BaP-induced apoptosis in HUVEC cells by down-regulating the protein expression of Bax,Cyt-c,APAF-1,and pro-caspase 9,and up-regulating the protein expression of Bcl-2.GP-2 decreased protein expression of NLRP3 inflammasome related proteins(NLRP3,ASC and Caspase 1),Furthermore,the inhibition of AhR expression with AhR-specific siRNA or AhR antagonist CH223191 significantly reduced BaP-induced IL-1β and IL-18 levels,and GP-2 significantly downregulated the mRNA and protein expression levels of AHR and CYP1A1 in HUVEC cells.Importantly,the PI3K/Akt pathway inhibitor LY294002 further enhanced the secretion of IL-1β and IL-6 in BaP-induced HUVEC cells,and GP-2 can significantly increase the phosphorylation levels of PI3K and Akt.ConclusionThe combination of ginseng and dandelion alleviated As lesions in a dose-dependent manner by inhibit the activation of NLRP3 and PI3K/Akt/mTOR pathway.GP-2,the non-saponin component of ginseng,contribute to the repair of damaged endothelial cells function through multiple pathways such as AHR,NLRP3 and PI3K/Akt.In short,the combined use of saponins and non-saponins in ginseng can exert better anti-As effect.