| Background and ObjectiveAtherosclerosis（AS）and its related cardiovascular diseases have become a significant public health concern.Therefore,safe and effective preventive strategies for AS are in urgent need to reduce the morbidity and mortality of cardiovascular diseases.Changing lifestyle,especially dietary strategy,is currently considered to be an effective way to prevent AS.Many epidemiological studies demonstrated that increasing the intake of vegetables,fruits,teas and other polyphenols,flavonoids,and dietary fiber enriched food would reduce the cardiovascular disease risk in adults.Thus,exploring the role of certain natural plants or their extracts in preventing AS has attracted increasing attention in recent years.Ligustrum robustum（LR）is an inexpensive and wildly accepted tea-like plant with a long history of drinking in the Southwest of China.Our previous studies indicated that LR has multiple beneficial effects,including lowering lipid level,reducing weight and protecting vascular endothelial cells,further suggesting that LR is an excellent candidate in AS prevention.However,few studies have been reported regarding the effects and mechanisms of LR on AS.LR is rich in phenols,flavonoids,polysaccharides,and phenylpropanoid glycoside,which are natural products that have been demonstrated to have low bioavailability,yet their health-improving effects could be exerted by modulation of gut microbiota.Gut microbiota and its metabolites,such as trimethylamine n-oxide（TMAO）,bile acids（BAs）and short-chain fatty acids（SCFAs）,have been reported to associate with the development of AS by interfering with lipid metabolism,inflammatory response and endothelial cell dysfunction.Collectively,those pieces of evidence support that diet supplementation regulating gut microbiota would be a practical approach for the prevention of AS.In addition to utilizing the intestinal microbes,the LR components in serum could also be transported to target organs through blood circulation,where the biological potentials could be employed.Our previous studies indicated that LR could prevent and control obesity by modulating gut microbiota in human subjects and mice models,while its ability to decrease serum TMAO levels in obese women with a higher TMAO baseline was also described.However,the components of LR in serum and their mechanisms in AS regulation are still unclear,and thus the effect of LR on AS prevention,its gut microbiota related mechanism,as well as and components of LR in serum are investigated in this study.MethodsPart 1To evaluate the effects of LR on regulating gut microbiota and reducing serum TMAO levels in mice treated with the high-choline diet,C57BL/6J mice were divided into a normal diet group,high-choline diet group and low-,medium-and high-dose LR intervention groups.After 17 weeks,the mice were sacrificed and the serum,cecal contents,liver,spleen and kidney samples of mice were collected.The body weight,organ indexes,the serum AST,ALP,TP,ALT,ALB,GGT,Scr and BU levels,and the pathological changes in liver,kidney and spleen were recorded to evaluate the effect of LR on the organ functions in high-choline dieted mice.The abundance of previously reported AS associated bacteria,such as Firmicutes,Bacteroidetes,Actinobacteria,Lactobacillus,Bifidobacterium,Akkermansia muciniphila and Prevotella in the cecal contents were determined by RT-PCR,while LC-MS method was applied to determine the level of TMAO and TMA in serum.To evaluate the prevention effect of LR on AS in high-choline dieted mice,Apoe-/-mice were randomly divided into the normal dieted group,high-choline dieted group,LR supplementation group,and drug intervention group（broad-spectrum antibiotic intervention group）.After 17 weeks of intervention,the serum and aorta samples were collected.The serum TMAO levels were evaluated by LC-MS,and the areas of atherosclerotic plaque in aortic root slides and middle aortic artery were evaluated by oil red O staining.The contents of macrophages and smooth muscle cells in the plaques of each mouse were determined by immunohistochemical analysis.Luminex was employed to determine the levels of ICAM-1,IL-1α,IL-1β,TNF-α,IL-2,IL-6,IL-10 and IFN-γin mice serum,and the contents of ICAM-1 and VCAM-1 in aortic root slides were determined by immunohistochemical analysis as well.Part 2To explore the mechanism of the LR-regulated serum TMAO level,the gene and protein expression of FMO3 in livers of C57BL/6J and Apoe-/-mice treated by the high-choline diet were determined by RT-q PCR and Western blot,respectively.The evaluation of FMO3 activity of each mouse was performed using LC-MS in vitro,while the same method was utilized to exam the TMA-lyse activity of cecal contents to explore the role of gut microbiota in reducing serum TMAO level of LR.The compositional changes of mouse microbial community were assessed by 16S r RNA gene sequencing,and the Spearman rank correlation was used to explore the correlation between TMA or TMAO levels and gut microbiota.Part 3In order to explore the role of gut microbiota in regulating lipid metabolism of LR,cholesterol levels in the liver and serum of C57BL/6J and Apoe-/-mice on the high-choline diet and their total bile acid levels in the liver,gallbladder,small intestinal contents,serum and feces were determined by ELISA or LC-MS/MS.Additionally,the gene expression of Hmgr,Sr-b1 in the liver and Npc1L1 and Abcg5/8 in the ileum were determined by RT-q PCR.The gene and protein expression levels of bile acid synthetase CYP7A1,CYP8B1,CYP27A1 and CYP7B1 were determined RT-q PCR and Western blot,respectively,and the expression level of the upstream regulators Fxr and Shp in the liver and Fxr and Fgf15 in the ileum,were also determined by RT-q PCR,as well as the levels of bile acid transporter（Bsep,Mrp2,Mrp3 and Mrp4）and reabsorption related genes（Ntcp,Oatp1 and Oatp2）in the liver,and bile acid transporters（Asbt,Ostαand Ostβ）in the ileum.Bile acid compositions in the serum and feces were determined by LC-MS/MS,and Spearman rank correlation was used to describe the correlation between fecal BAs levels and gut microbiota,while GC-MS analyzed the levels of short-chain fatty acids in feces.Part 4In order to explore the effects of LR components in the serum on prevention and treatment of AS,UPLC-Q-TOF-MS was used to describe the components of LR in the serum,while network pharmacological analysis was used to construct a"component-target-pathway"network,the potential LR involved pathways were analyzed by the GO and KEGG databases,and TMAO constructed endothelial inflammation model in vitro.After treatment,the protein expression levels of ICAM-1,MCP-1,JNK,p-JNK,Jun,p-Jun,c-FOS and p-c-FOS were detected by Western blot,while the effect of LR serum components on endothelial cell inflammation and the role of JNK/AP-1 signaling pathway were investigated with a variety inhibitors and agonists of JNK,Jun and c-fos.ResultsPart 1After a 17-week treatment,the bodyweight of choline-fed mice was significantly increased and decreased in medium and high dose LR-supplemented mice（P?0.05）,respectively.In contrast,the different groups have no significant difference in the organ index,serum levels of aspartate aminotransferase（AST）,alkaline phosphatase（ALP）,total protein（TP）,alanine aminotransferase（ALT）,albumin（ALB）,glutamyl transpeptidase（GGT）,serum creatinine（Scr）and blood urea（BU）.HE stains showed that the livers and kidneys of high-choline dieted mice only have mild pathological changes,while no apparent changes were observed regarding the pathology of the livers,kidneys and spleens among other groups.Regarding the bacterial composition,the abundance of Firmicutes,Bacteroidetes and Akkermansia muciniphila were unchanged among all groups（P?0.05）.However,the mice supplemented with medium and high doses of LR showed increased abundance of Actinobacteria and Bifidobacterium（P?0.05）compared with high-choline dieted mice.Meanwhile,the abundance of Prevotella was decreased after LR administration（P=0.006）.The high-choline dieted C57BL/6J mice had higher serum TMA and TMAO levels than those in chow dieted mice（P?0.0001）,while daily oral administration of medium dose or high dose of LR appears to significantly inhibit the increase in serum TMA and TMAO levels compared to the choline-fed water-treated C57BL/6J mice.Consistent with the results of high-choline dieted C57BL/6J mice,the serum TMA and TMAO levels were higher in high-choline dieted Apoe-/-mice than those chow dieted Apoe-/-mice（P?0.0001）,agreeing to the results of LR or broad-spectrum antibiotics treatment,which has significantly decreased serum TMA and TMAO contents in high-choline dieted Apoe-/-mice（P?0.05）.After 17 weeks,we observed that the choline-diet remarkably promoted the atherosclerotic lesion in Apoe-/-mice（P?0.05）while the atherosclerotic lesion areas were significantly decreased with LR and broad-spectrum antibiotics administration（P?0.05）,suggesting that a AS mice model was successfully constructed.Furthermore,IHC histological staining confirmed that LR decreased the relative contents of macrophages and induced an increase in the relative contents of collagen after LR supplementation（P?0.05）,which further validated the AS model.Comparing to the high-choline dieted mice,the serum levels of ICAM-1,IL-1α,IL-1β,TNF-αand IFN-γin the LR treatment group were significantly decreased（P?0.05）.Similarly,the relative contents of ICAM-1 and VCAM-1 of aortic root slides were significantly decreased after LR supplementation（P?0.05）.Part 2LR did not affect the gene and protein expression levels of FMO3 in both C57BL/6J and Apoe-/-mice（P?0.05）,yet its activity was unchanged after LR treatment（P?0.05）.Moreover,in vitro analysis showed that the synthesis of TMA from choline with the cecal contents of LR-treated mice was lower than that of sterile water treated mice（P?0.001）,indicating that the decrease in TMAO caused by LR was at least partly associated with gut microbiota rather than the hepatic FMO3.The alpha diversity did not change after LR supplementation,implying LR treatment did not affect the cecal microbiota richness and diversity.However,principal coordinates analysis（PCA）of microbial taxa in Apoe-/-mice revealed distinct clusters,meaning that LR supplementation induced significant rearrangements in microbial composition,which is not observed in C57BL/6J mice.Analysis of 16S RNA sequencing also demonstrated that LR altered the bacterial composition in both C57BL/6J mice and Apoe-/-mice,especially for the abundance of Actinobacteria,which was increased with the LR treatment in both mice at the phylum level（P?0.05）.At the genus level,the relative abundances of Anaeroplasma,Bacteroides,Bifidobacterium,Caldicoprobacter,Candidatus＿Arthromitus,Candidatus＿Saccharimonas,Desulfovibrio,Eisenbergiella,Rikenellaceae＿RC9＿gut＿group and Ruminiclostridium＿6 were significantly increased in LR-treated C57BL/6J mice（P?0.05）while,the relative abundances of Lachnospiraceae＿NK4A136＿group,Prevotellaceae＿UCG-001and uncultured＿Bacteroidales＿bacterium were significantly decreased in LR-treated C57BL/6J mice（P?0.05）.Similarly,LR supplementation also increased the relative abundances of Allobaculum,Bifidobacterium,Clostridium＿sensu＿stricto＿1,Flavobacteriu,Parasutterella,Rikenellaceae＿RC9＿gut＿group,Coriobacteriaceae＿UCG-002,Sphingomonas and Staphylococcus（P?0.05）in high-choline Apoe-/-dited mice,yet the relative abundances of Ambiguous＿taxa,Butyricicoccus,Butyricimonas,GCA-900066575,Helicobacter,Intestinimonas,Odoribacter,Oscillibacter,Lachnospiraceae＿FCS020＿group,Ruminiclostridium and UBA1819（P?0.05）.Furthermore,we found some genera were significantly correlated with serum TMA and TMAO using spearman correlation analysis.Among those,the relative abundances of Oscillibacter,Alistipes and Preprevellaceae＿UCG-001 were positively correlated with serum TMA and TMAO levels（P?0.05）,while the negative correlation with serum TMA and TMAO levels（P?0.05）.was found in the relative abundances of Candidatus arthromitus and Bacteroides.Part 3Although a high-choline diet did not affect hepatic cholesterol concentrations（P?0.05）,LR supplementation could reduce this value in both C57BL/6J mice and Apoe-/-mice.In addition to the hepatic level,LR also decreased serum cholesterol levels and increased the fecal cholesterol levels in a high-choline fed Apoe-/-mice（P?0.05）.The hepatic m RNA expression levels of Hmgr were similar between H2O-treated and LR-treated mice（P?0.05）,while the m RNA levels in the liver cholesterol transporter Sr-b1,were significantly increased after LR administration（P?0.05）.Compared to choline-fed mice,mice supplemented with LR had a significantly lower Npc1L1 m RNA level and higher Abcg8 m RNA level in the ileum（P?0.05）as well.This study also showed that LR significantly inhibit choline-induced reduction of BAs concentrations in small intestine contents and feces（P?0.05）,but has no effect on those in liver,gall bladder and serum of the C57BL/6J mice and Apoe-/-mice（P?0.05）.The results of RT-q PCR and Western blot suggested that LR did not affect the m RNA expression and protein expression of CYP7A1,CYP8B1,CYP27A1 and CYP7B1 gene in the liver（P?0.05）,and the m RNA levels of upstream genes Fxr and Shp in the liver were also unchanged with LR treatment in both C57BL/6J mice and Apoe-/-mice（P?0.05）.Similarly,no significant difference was observed after LR supplementation（P?0.05）regarding the bile acid transporters in the liver（Bsep,Mrp2,Mrp3 and Mrp4）.In contrast,m RNA levels in the ileum of the bile acid transporters（Ostαand Ostβ）were significantly lower in mice supplemented with LR（P?0.05）,with no changes in the expression of Asbt（P?0.05）.In addition to the transporters,the the hepatic m RNA expression levels of proteins related to bile acid reabsorption,including Ntcp in both mice,Oatp2 in C57BL/6J mice and Oatp1 in Apoe-/-mice were also significantly decreased（P?0.05）with LR supplementation.Notbaly,significantly increased total BA and unconjugated BA levels in feces were observed in LR-treated mice compared with water-treated mice（P?0.05）.As revealed by the LC-MS/MS results,fecal levels ofω-muricholic acid（ω-MAC）and deoxycholic acid（DCA）were evaluated and the serum levels of chenodeoxycholic acid（CDCA）and DCA were decreased in LR-treated C57BL/6J mice（P?0.05）.Similarly,the fecal levels of cholic acid（CA）and DCA were evaluated while the serum levels of CDCA and ursodeoxycholic acid（UDCA）were decreased in LR-treated Apoe-/-mice（P?0.05）.However,SCFAs such as acetic acid,propionic acid,isobutyric acid,butyric acid,isovaleric acid,valeric acid and caproic acid,were not affected by LR after 17 weeks of dietary intervention（P?0.05）.Regarding the intestinal flora,analysis of the relationships between the fecal concentrations of BA and the relative abundance of cecal microbiota using the Spearman correlations showed that the fecal DCA in both mice were positively significant correlated with Bifidobacterium and Bacteroides.Part 4In combination of the chemical compositions of LR,163 relevant components were identified in the serum,including 72 prototype components and 91 metabolites.Using the network pharmacology method,a comprehensive network of‘components of LR in the serum-target-pathway-AS’was constructed,indicating that the serum components of LR have an anti-AS potential through anti-inflammatory pathways.To investigate the cytotoxicity of TMAO on endothelial cells,EAhy.926 cells were treated with TMAO in different concentrations at different time points（24 h,48 h and 72 h）,showing that the endothelial cells are able to tolerate 0-4,000μM TMAO（P?0.05）.The 24 h treatment of 600μM TMAO gives a significant increase of ICAM-1 or MCP-1 protein expression in endothelial cells（P?0.05）,meaning that TMAO triggers inflammation in vitro.Compared with the normal control,the ICAM-1 protein expression levels,p-JNK/JNK,p-Jun/Jun and p-c-FOS/c-FOS value were increased in the blank serum pretreatment group after TMAO treatment（P?0.05）,and the pretreatment with LR serum or/and inhibitors of JNK,Jun or c-FOS shows a markedly inhibitory effect on TMAO-induced ICAM-1 protein expression,which were reversed after pretreatment with agonists of JNK,Jun or c-FOS.ConclusionThis study shows the gut microbiota modulated LR attenuation of high-choline induced AS development was accompanied by decreased serum TMAO,increased bile acids excretion,reduced bile acid reabsorption and the improvement of lipid metabolism disorder.Regarding the microbiota-independent aspect,the LR components in serum may inhibit the inflammatory injury of endothelial cells through the JNK/AP-1 pathway,thus playing a role in the prevention of AS.Innovation Points1.This study firstly reported that LR had an effect of preventing atherosclerosis validated by animal experiments.2.In this study,it was found that the effect of LR on preventing AS may be related to the pathway of‘Gut microbiota-TMAO-cholesterol metabolism’,which has not been reported before.3.Using network pharmacology and serum pharmacology techniques,this study innovatively analyzed the LR components in the serum and the potential mechanism of preventing AS.These studies not only provided a reference for the further utilization of LR,but also provide the foundation for exploring natural substances to prevent AS. |
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