ABSTRACT
Gut microbiota dysbiosis is an avenue for the promotion of atherosclerosis (AS) and this effect is mediated partly via the circulating microbial metabolites. More microbial metabolites related to AS vascular inflammation, and the mechanisms involved need to be clarified urgently. Paeonol (Pae) is an active compound isolated from Paeonia suffruticoas Andr. with anti-AS inflammation effect. However, considering the low oral bioavailability of Pae, it is worth exploring the mechanism by which Pae reduces the harmful metabolites of the gut microbiota to alleviate AS. In this study, ApoE-/- mice were fed a high-fat diet (HFD) to establish an AS model. AS mice were administrated with Pae (200 or 400 mg·kg-1) by oral gavage and fecal microbiota transplantation (FMT) was conducted. 16S rDNA sequencing was performed to investigate the composition of the gut microbiota, while metabolomics analysis was used to identify the metabolites in serum and cecal contents. The results indicated that Pae significantly improved AS by regulating gut microbiota composition and microbiota metabolic profile in AS mice. We also identified α-hydroxyisobutyric acid (HIBA) as a harmful microbial metabolite reduced by Pae. HIBA supplementation in drinking water promoted AS inflammation in AS mice. Furthermore, vascular endothelial cells (VECs) were cultured and stimulated by HIBA. We verified that HIBA stimulation increased intracellular ROS levels, thereby inducing VEC inflammation via the TXNIP/NLRP3 pathway. In sum, Pae reduces the production of the microbial metabolite HIBA, thus alleviating the ROS/TXNIP/NLRP3 pathway-mediated endothelial inflammation in AS. Our study innovatively confirms the mechanism by which Pae reduces the harmful metabolites of gut microbiota to alleviate AS and proposes HIBA as a potential biomarker for AS clinical judgment.
Subject(s)
Animals , Mice , Atherosclerosis/drug therapy , Diet, High-Fat , Endothelial Cells , Inflammation/drug therapy , Mice, Inbred C57BL , NLR Family, Pyrin Domain-Containing 3 Protein/genetics , Reactive Oxygen SpeciesABSTRACT
ObjectiveTo investigate whether the effects of paeonol （Pae） on angiotensin Ⅱ （AngⅡ）-induced senescence in vascular smooth muscle cells （VSMCs） were related to angiotensinogen of silencing regulatory information factor 6 （SIRT6）/adenosine diphosphate ribose polymerase 1 （PARP1） signaling pathway in VSMCs. MethodThe model of VSMC-stress aging induced by AngⅡ （100 nmol·L-1） was established. The rats were divided into normal group， model group， low， medium， and high-concentration Pae groups （30， 60， 120 μmol·L-1）. The positive rate of cell senescence was detected by SA-β-Gal staining， the ability of cell proliferation was detected by the cell counting kit-8 （CCK-8） method， the expression of SIRT6， PARP1， p16， p21， p53， proliferating cell nuclear antigen （PCNA）， deoxyribonucleic acid （DNA）-damaged protein γ-H2AX was detected by Western blot， and VSMC proliferation was detected by EdU staining. The silenced VSMCs were prepared by siRNA-SIRT6 transfection， and the protein expressions of SIRT6， PARP1， p16， and γ-H2AX in VSMCs silenced by SIRT6 were observed. ResultThe results of SA-β-Gal staining showed that the senescence positive rate of SA-β-Gal staining in the model group was higher than that in the normal group （P<0.01）， and the positive rate of SA-β-Gal staining in the Pae group was significantly lower than that in the model group （P<0.05， P<0.01）. The results of Western blot showed that as compared with the normal group， the expression of PCNA， SIRT6， and PARP1 in the model group was down-regulated， and the expression of aging-related proteins p16， p21， p53， and γ-H2AX was up-regulated in the model group （P<0.05， P<0.01）. Compared with the model group， Pae promoted the protein expression of PCNA， SIRT6， and PARP1 and inhibited the protein expression of p16， p21， p53， and γ-H2AX in a dose-dependent manner （P<0.05， P<0.01）. The results of EdU staining showed that the number of EdU positive cells in the model group was lower than that in the normal group （P<0.01）， and the number of EdU positive cells in Pae groups was significantly higher than that in the model group （P<0.05， P<0.01）. After SIRT6 silencing， the effects of Pae on promoting SIRT6 and PARP1 and inhibiting P16 were reversed （P<0.05， P<0.01）. In addition， the addition of SIRT6 inhibitor （IN-1） promoted the occurrence of cell senescence induced by AngⅡ （P<0.05， P<0.01）. ConclusionPae can effectively inhibit the aging of VSMCs， and its mechanism may be related to the regulation of SIRT6/PARP1 signal pathway.
ABSTRACT
OBJECTIVE:To establish the method for simultaneous determination of alantolactone,isoalantolactone,gallic acid,emodin,aloe-emodine,rhein,physcion and chrysophanol in Liuwei nengxiao pills.METHODS:HPLC method was adopted.The determination was performed on Diamonsil C18 with mobile phase consisted of methanol-acetonitrile-0.1％ glacial acetic acid (gradient elution) at the flow rate of 1.0 mL/min.The detection wavelengths were set at 254 nm (alantolactone,isoalantolactone,emodin,aloe-emodine,rhein,physcion and chrysophanol),270 nm (gallic acid).The column temperature was 25 ℃,and sample size was 10 μ L.RESULTS:The linear ranges of alantolactone,isoalantolactone,gallic acid,emodin,aloe-emodine,rhein,physcion,chrysosphanol were 0.121-3.63 μg(r=0.999 9),0.122-3.66 μg(r=0.999 9),0.219-6.57 μg(r=0.999 9),0.016 4-0.492 μg(r=0.999 7),0.017 3-0.519 μg(r=0.999 9),0.015 3-0.459 μg(r=0.999 9),0.007 2-0.216 μg(r=0.999 9),0.016 2-0.486(r=0.999 9).The limits of quantification were 0.41,0.26,0.35,0.13,0.17,0.14,0.15,0.13 ng;limits of detection were 0.12,0.08,0.11,0.04,0.05,0.04,0.05,0.04 ng.RSDs of precision,stability and reproducibility tests were all lower than 2.0％.The recoveries were 98.05％-102.46％ (RSD=1.75 ％,n=6),98.55％-102.89％ (RSD=1.91％,n=6),98.53 ％-102.34％ (RSD=1.66％,n=6),101.71％-103.41％ (RSD =0.57 ％,n=6),101.04％-103.01％ (RSD=0.69％,n=6),101.63％-102.75％ (RSD=0.39 ％,n=6),96.94％-101.11％ (RSD=1.61％,n=6),98.06％-99.10％ (RSD=0.40％,n=6).CONCLUSIONS:The method is accurate,simple and suitable for simultaneous determination of 8 components in Liuwei nengxiao pills.
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AIM: To establish the methods of identifing and determining Yiganjian Tables. METHODS: Herba Halenia,extract of Radix et Rhizoma Glycyrrhizale,Radix Astragali were identified by TLC.The content of 1-hydroxy-3,4,5-trimethoxyxanthon in Herba Halenia was determined by HPLC on Hypersil-ODS2 column with the mobile phase of methanol-0.2%H_3PO_4(55∶45).The detective wavelength was set at 243 nm and monoammonium glycyrrhizinate was determinad by HPLC on Hypersil-ODS2 column with the mobile phase of acetonitrile-2% aceacid(60∶40).The detective wavelength was set at 250 nm. RESULTS: The linear range of 1-hydroxy-3,4,5-trimethoxyxanthon was from 0.173 2 to 0.866 0 ?g.The average recovery was 100.56%,RSD was 2.7%(n=9).The linear range of monoammonium glycyrrhizinate was from 0.55 to 2.75 ?g.The average recovery was 98.29%,RSD was 1.7%(n=9). CONCLUSION: The method is simple,accurate and sensitive,so it can be used for the quality control of Yiganjian Tablets.
ABSTRACT
Objective: To establish the quality standards for Jingzhuchongchao Tablets (cordyceps, Rhodiolae rosea Radix Astragali. Fruetus Canarii Radix Ginseng, Radix Ophiopogonis etc.) Methods: Radix Ginseng,Radix Ophiopogonis were identified by TLC. AstragalosideIV was determined by TLC scanning. Results: Radix Ginseng, Radix Ophiopogonis could be detected by TLC. Astragaloside IV showed a good linear relationship at a range of 0.5～2.5 ?g, r= 0.9999.The average recovery was 98%, and RSD was 1.35. Conclusion: The established methods are simple, feasible and reproducible. This study provide a method for the quality control of Jingzhuchonggcao Tablets.