Huayu Qutan Recipe promotes lipophagy and cholesterol efflux through the mTORC1/TFEB/ABCA1-SCARB1 signal axis.

This study aims to investigate the mechanism of the anti-atherosclerosis effect of Huayu Qutan Recipe (HYQT) on the inhibition of foam cell formation. In vivo, the mice were randomly divided into three groups: CTRL group, MOD group and HYQT group. The HYQT group received HYQT oral administration twice a day (20.54 g/kg/d), and the plaque formation in ApoE-/- mice was observed using haematoxylin-eosin (HE) staining and oil red O (ORO) staining. The co-localization of aortic macrophages and lipid droplets (LDs) was examined using fluorescent labelling of CD11b and BODIPY fluorescence probe. In vitro, RAW 264.7 cells were exposed to 50 µg/mL ox-LDL for 48 h and then treated with HYQT for 24 h. The accumulation of LDs was evaluated using ORO and BODIPY. Cell viability was assessed using the CCK-8 assay. The co-localization of LC3b and BODIPY was detected via immunofluorescence and fluorescence probe. LysoTracker Red and BODIPY 493/503 were used as markers for lysosomes and LDs, respectively. Autophagosome formation were observed via transmission electron microscopy. The levels of LC3A/B II/LC3A/B I, p-mTOR/mTOR, p-4EBP1/4EBP1, p-P70S6K/P70S6K and TFEB protein level were examined via western blotting, while SQSTM1/p62, Beclin1, ABCA1, ABCG1 and SCARB1 were examined via qRT-PCR and western blotting. The nuclear translocation of TFEB was detected using immunofluorescence. The components of HYQT medicated serum were determined using Q-Orbitrap high-resolution MS analysis. Molecular docking was employed to identify the components of HYQT medicated serum responsible for the mTOR signalling pathway. The mechanism of taurine was illustrated. HYQT has a remarkable effect on atherosclerotic plaque formation and blood lipid level in ApoE-/- mice. HYQT decreased the co-localization of CD11b and BODIPY. HYQT (10% medicated serum) reduced the LDs accumulation in RAW 264.7 cells. HYQT and RAPA (rapamycin, a mTOR inhibitor) could promote cholesterol efflux, while chloroquine (CQ, an autophagy inhibitor) weakened the effect of HYQT. Moreover, MHY1485 (a mTOR agonist) also mitigated the effects of HYQT by reduced cholesterol efflux. qRT-PCR and WB results suggested that HYQT improved the expression of the proteins ABCA1, ABCG1 and SCARB1.HYQT regulates ABCA1 and SCARB1 protein depending on the mTORC1/TFEB signalling pathway. However, the activation of ABCG1 does not depend on this pathway. Q-Orbitrap high-resolution MS analysis results demonstrated that seven core compounds have good binding ability to the mTOR protein. Taurine may play an important role in the mechanism regulation. HYQT may reduce cardiovascular risk by promoting cholesterol efflux and degrading macrophage-derived foam cell formation. It has been observed that HYQT and ox-LDL regulate lipophagy through the mTOR/TFEB signalling pathway, rather than the mTOR/4EBP1/P70S6K pathway. Additionally, HYQT is found to regulate cholesterol efflux through the mTORC1/TFEB/ABCA1-SCARB1 signal axis, while taurine plays a significant role in lipophagy.

Hepatocyte steatosis activates macrophage inflammatory response accelerating atherosclerosis development. / è‚ç»†èƒžè„‚è‚ªå˜æ€§å½±å“å·¨å™¬ç»†èƒžç‚Žç—‡ååº”åŠ é€ŸåŠ¨è„‰ç²¥æ ·ç¡¬åŒ–å½¢æˆ.

OBJECTIVES: To investigate the mechanism of comorbidity between non-alcoholic fatty liver disease (NAFLD) and atherosclerosis (AS) based on metabolomics and network pharmacology. METHODS: Six ApoE－/－ mice were fed with a high-fat diet for 16 weeks as a comorbid model of NAFLD and AS (model group). Normal diet was given to 6 wildtype C57BL/6J mice (control group). Serum samples were taken from both groups for a non-targeted metabolomics assay to identify differential metabolites. Network pharmacology was applied to explore the possible mechanistic effects of differential metabolites on AS and NAFLD. An in vitro comorbid cell model was constructed using NCTC1469 cells and RAW264.7 macrophage. Cellular lipid accumulation, cell viability, morphology and function of mitochondria were detected with oil red O staining, CCK-8 assay, transmission electron microscopy and JC-1 staining, respectively. RESULTS: A total of 85 differential metabolites associated with comorbidity of NAFLD and AS were identified. The top 20 differential metabolites were subjected to network pharmacology analysis, which showed that the core targets of differential metabolites related to AS and NAFLD were STAT3, EGFR, MAPK14, PPARG, NFKB1, PTGS2, ESR1, PPARA, PTPN1 and SCD. The Kyoto Encyclopedia of Genes and Genomes showed the top 10 signaling pathways were PPAR signaling pathway, AGE-RAGE signaling pathway in diabetic complications, alcoholic liver disease, prolactin signaling pathway, insulin resistance, TNF signaling pathway, hepatitis B, the relax in signaling pathway, IL-17 signaling pathway and NAFLD. Experimental validation showed that lipid metabolism-related genes PPARG, PPARA, PTPN1, and SCD were significantly changed in hepatocyte models, and steatotic hepatocytes affected the expression of macrophage inflammation-related genes STAT3, NFKB1 and PTGS2; steatotic hepatocytes promoted the formation of foam cells and exacerbated the accumulation of lipids in foam cells; the disrupted morphology, impaired function, and increased reactive oxygen species production were observed in steatotic hepatocyte mitochondria, while the formation of foam cells aggravated mitochondrial damage. CONCLUSIONS: Abnormal lipid metabolism and inflammatory response are distinctive features of comorbid AS and NAFLD. Hepatocyte steatosis causes mitochondrial damage, which leads to mitochondrial dysfunction, increased reactive oxygen species and activation of macrophage inflammatory response, resulting in the acceleration of AS development.

[Mechanism of Didang Decoction in prevention of anti-atherosclerosis and hyperlipidemia by HPLC-Q-TOF-MS/MS and network pharmacology based on theory of "nutrients return to heart and fat accumulates in channels"].

Atherosclerosis(AS) is caused by impaired lipid metabolism, which deposits lipids in the intima, causes vascular fibrosis and calcification, and then leads to stiffening of the vascular wall. Hyperlipidemia(HLP) is one of the key risk factors for AS. Based on the theory of "nutrients return to the heart and fat accumulates in the channels", it is believed that the excess fat returning to the heart in the vessels is the key pathogenic factor of AS. The accumulation of fat in the vessels over time and the blood stasis are the pathological mechanisms leading to the development of HLP and AS, and "turbid phlegm and fat" and "blood stasis" are the pathological products of the progression of HLP into AS. Didang Decoction(DDD) is a potent prescription effective in activating blood circulation, removing blood stasis, resolving turbidity, lowering lipids, and dredging blood vessels, with the functions of dispelling stasis to promote regeneration, which has certain effects in the treatment of atherosclerotic diseases. This study employed high-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry(HPLC-Q-TOF-MS/MS) to screen the main blood components of DDD, explored the targets and mechanisms of DDD against AS and HLP with network pharmacology, and verified the network pharmacological results by in vitro experiments. A total of 231 blood components of DDD were obtained, including 157 compounds with a composite score >60. There were 903 predicted targets obtained from SwissTargetPrediction and 279 disease targets from GeneCards, OMIM, and DisGeNET, and 79 potential target genes of DDD against AS and HLP were obtained by intersection. Gene Ontology(GO) analysis suggested that DDD presumably exerted regulation through biological processes such as cholesterol metabolism and inflammatory response, and Kyoto Encyclopedia of Genes and Genomes(KEGG) analysis suggested that signaling pathways included lipid and atherosclerosis, insulin resistance, chemo-carcinogenesis-receptor activation, and AGE-RAGE signaling pathways in diabetic complications. In vitro experiments showed that DDD could reduce free fatty acid-induced lipid accumulation and cholesterol ester content in L02 cells and improve cellular activity, which might be related to the up-regulation of the expression of PPARα, LPL, PPARG, VEGFA, CETP, CYP1A1, and CYP3A4, and the down-regulation of the expression of TNF-α and IL-6. DDD may play a role in preventing and treating AS and HLP by improving lipid metabolism and inflammatory response, and inhibiting apoptosis with multi-component, multi-target, and multi-pathway characteristics.

Halorussus amylolyticus sp. nov., isolated from an inland salt lake.

A halophilic archaeal strain, YC93T, was isolated from Yuncheng salt lake in Shanxi Province, China. Cells were pleomorphic rods, stained Gram-negative and formed light-red-pigmented colonies on agar plates. Strain YC93T was able to grow at 25­50 °C (optimum 37 °C), with 1.4­4.8 M NaCl (optimum 2.0 M), with 0­1.0 M MgCl2 (optimum 0.05 M) and at pH 6.0­9.5 (optimum pH 7.0). Cells lysed in distilled water and the minimal NaCl concentration to prevent cell lysis was 8 % (w/v). 16S rRNA gene sequence analysis revealed that strain YC93T had two dissimilar 16S rRNA genes both of which were phylogenetically related to those of the two recognized members of the genus Halorussus (93.0­95.3 % similarity). The rpoB' gene of strain YC93T was phylogenetically related to the corresponding gene of Halorussus rarus TBN4T (91.3 % similarity) and Halorussus ruber YC25T (90.5 %). The major polar lipids were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate and five glycolipids chromatographically identical to those of Halorussus rarus CGMCC 1.10122T. The DNA G+C content of strain YC93T was 64.6 mol%. The phenotypic, chemotaxonomic and phylogenetic properties suggested that strain YC93T represents a novel species of the genus Halorussus, for which the name Halorussus amylolyticus sp. nov. is proposed. The type strain is YC93T ( = CGMCC 1.12126T = JCM 18367T).

The mechanism of Shenlong Jianji treatment of idiopathic pulmonary fibrosis inhibits fibroblast-to-myofibroblast transformation via the TGF-ß1/smads signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Shenlong Jianji (SLJJ) is a Chinese herbal compound composed of traditional medicines for supplementing Qi, nourishing Yin, promoting blood circulation, and removing obstruction in channels. It is widely used to treat idiopathic pulmonary fibrosis (IPF) in China. However, the underlying mechanism of SLJJ remains unclear. AIM OF THIS STUDY: To elucidate the efficacy and mechanisms of SLJJ in the treatment of IPF through in vivo and in vitro experiments. MATERIAL AND METHODS: 84 Wistar rats were randomly and equally divided into 7 groups: the control group (CTRL), the sham operation group (SHAM), the model group (IPF), the low dose of SLJJ group (L-SLJJ), the middle dose of SLJJ group (M-SLJJ), the high dose of SLJJ group (H-SLJJ), and the pirfenidone group (PFD). The rats in the CTRL, SHAM, and IPF groups were given normal saline each time for 28 days; the SLJJ groups were given Shenlong Jianji (9 g kg-1·d-1, 18 g kg-1·d-1, 36 g kg-1·d-1), and pirfenidone was administered as a sequential dose. After 28 days, the general condition of the rats was evaluated, and samples were collected. The lung coefficient was measured. The pathological changes of lung in each group were observed by H&E staining and Masson staining. α-SMA, collagen 1, and E-cadherin proteins were detected by immunohistochemistry. α-SMA, collagen 1, vimentin, E-cadherin, N-cadherin, TGF-ß1, smad2, and smad3 proteins were detected by WB in vivo.In vitro, A scratch test was used to assess the ratio of cell migration. α-SMA, vimentin, E-cadherin, and N-cadherin protein levels were evaluated by a cellular immunofluorescence assay. TGF-ß1/smads signaling pathway was detected by WB. HPLC-Q-TOF/MS analysis was used to identify the active compounds in the SLJJ. Molecular docking determined the free binding energy of the compound with the TGF-ß1 protein. RESULTS: SLJJ significantly improved the respiratory symptoms, heart rate, mental state, and food intake of IPF group rats and decreased the lung coefficient. In the IPF group, inflammatory cells were infiltrated, and the thickened alveoli wall and alveoli collapse were shown, while significantly alleviating pathological changes in the SLJJ and PFD groups. Masson staining showed that SLJJ and PFD decreased the collagen expression. Immunohistochemical results showed that the expressions of α-SMA, collagen 1, and N-cadherin decreased in the SLJJ and PFD groups, while E-cadherin increased significantly compared with the IPF group. SLJJ regulates TGF-ß1/smads signaling pathway proteins in vivo. SLJJ decreased the ratio of migration in HFL-1 cells; SLJJ reduced the fluorescence intensity of α-SMA, vimentin, and N-cadherin and increased the fluorescence intensity of E-cadherin in primary rat lung (PRL) fibroblast cells and HFL-1 cells. WB results showed that SLJJ significantly down-regulated α-SMA, Vimentin, N-cadherin, TGF-ß1, smad2, and p-smad2/3 proteins expression and up-regulated E-cadherin protein expression in vitro, whereas SRI-011381 (a TGF-ß1 agonist) antagonized the effects of SLJJ. CONCLUSION: SLJJ inhibits idiopathic pulmonary fibrosis. The TGF- ß1/Smads signaling pathway can be the target of SLJJ, which inhibits fibroblast-to-myofibroblast transformation and is expected to be a new drug for the treatment of IPF.

Identification of key genes for atherosclerosis in different arterial beds.

Atherosclerosis (AS) is the pathologic basis of various cardiovascular and cerebrovascular events, with a high degree of heterogeneity among different arterial beds. However, mechanistic differences between arterial beds remain unexplored. The aim of this study was to explore key genes and potential mechanistic differences between AS in different arterial beds through bioinformatics analysis. Carotid atherosclerosis (CAS), femoral atherosclerosis (FAS), infrapopliteal atherosclerosis (IPAS), abdominal aortic atherosclerosis (AAS), and AS-specific differentially expressed genes (DEGs) were screened from the GSE100927 and GSE57691 datasets. Immune infiltration analysis was used to identify AS immune cell infiltration differences. Unsupervised cluster analysis of AS samples from different regions based on macrophage polarization gene expression profiles. Weighted gene co-expression network analysis (WGCNA) was performed to identify the most relevant module genes with AS. Hub genes were then screened by LASSO regression, SVM-REF, and single-gene differential analysis, and a nomogram was constructed to predict the risk of AS development. The results showed that differential expression analysis identified 5, 4, 121, and 62 CAS, FAS, IPAS, AAS-specific DEGs, and 42 AS-common DEGs, respectively. Immune infiltration analysis demonstrated that the degree of macrophage and mast cell enrichment differed significantly in different regions of AS. The CAS, FAS, IPAS, and AAS could be distinguished into two different biologically functional and stable molecular clusters based on macrophage polarization gene expression profiles, especially for cardiomyopathy and glycolipid metabolic processes. Hub genes for 6 AS (ADAP2, CSF3R, FABP5, ITGAX, MYOC, and SPP1), 4 IPAS (CLECL1, DIO2, F2RL2, and GUCY1A2), and 3 AAS (RPL21, RPL26, and RPL10A) were obtained based on module gene, gender stratification, machine learning algorithms, and single-gene difference analysis, respectively, and these genes were effective in differentiating between different regions of AS. This study demonstrates that there are similarities and heterogeneities in the pathogenesis of AS between different arterial beds.

DiDang decoction improves mitochondrial function and lipid metabolism via the HIF-1 signaling pathway to treat atherosclerosis and hyperlipidemia.

ETHNOPHARMACOLOGICAL RELEVANCE: DiDang Decoction (DDD) is a traditional classical prescription that has been used to treat atherosclerosis (AS) and hyperlipidemia (HLP) in China. Nevertheless, the underlying mechanism of DDD remains unclear. AIM OF THE STUDY: To validate the mechanism of DDD in AS and HLP based on network pharmacology and in vitro experiments. MATERIALS AND METHODS: The chemical components of DDD were obtained from the Traditional Chinese Medicine System Pharmacology Database and Analysis Platform (TCMSP) database and literature mining, and the disease targets of AS and HLP were obtained from the Gencards, OMIM, and DisGeNET databases. The intersection genes were imported into the STRING database to construct protein-protein interaction (PPI) network, and the DAVID database was used for gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Combined with the results of KEGG pathway analysis, the HIF-1 signaling pathway was selected for further in vitro experiments. RESULTS: The results showed that network pharmacology predicted 112 targets related to DDD treatment of AS and HLP, and the top 10 related pathways are: Lipid and atherosclerosis, AGE-RAGE signaling pathway in diabetic complications, Chemical carcinogenesis - receptor activation, Pathways in cancer, Proteoglycans in cancer, Fluid shear stress and atherosclerosis, HIF-1 signaling pathway, Alcoholic liver disease, PPAR signaling pathway, and Coronavirus disease-COVID-19. In vitro experiments showed that DDD effectively reduced lipid accumulation in FFA-treated L02 cells; DDD attenuated mitochondrial damage and reduced ROS content; DDD inhibited ferroptosis and apoptosis; DDD up-regulated the expression of HIF-1α, Glutathione Peroxidase 4(GPX4), and Bcl2 proteins, and down-regulated expression of Bax protein. CONCLUSION: DDD exerts therapeutic effects on AS and HLP through multiple targets and pathways, and improves mitochondrial function, reduces ROS content, inhibits ferroptosis and apoptosis by activating the HIF-1 signaling pathway, which provides reliable theoretical and experimental support for DDD treatment of AS and HLP.

Transcriptome Sequencing of lncRNAs, circRNAs, miRNAs, mRNAs, and Interaction Network Constructing in Acute Exacerbation of Idiopathic Pulmonary Fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) patients who suffer from acute exacerbation of idiopathic pulmonary fibrosis (AE-IPF) are at increased risk of respiratory deterioration and death. Non-coding RNAs (ncRNAs) play a vital role in AE-IPF, but studies of crosstalk between transcripts of IPF based on Traditional Chinese Medicine (TCM) syndrome type are relatively few. The construction of long non-coding RNAs (lncRNA)/circular RNAs (circRNA)-microRNAs (miRNA)-mRNA interaction networks can promote understanding RNA interaction in different syndrome types of AE-IPF. The study aimed to identify the difference in RNA transcription expression between IPF patients with "lung heat and collateral stasis (LHCS)" and "lung deficiency with collateral stasis (LDCS)" syndromes, further to construct the potential RNA networks. METHODS: Five IPF patients with LHCS and five IPF patients with LDCS were recruited in this study to perform RNA sequencing and miRNA sequencing. Further analysis was carried out on the differential expression profiles of lncRNAs, circRNAs, miRNAs, and mRNAs among patients with LHCS and LDCS. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed. The lncRNA/circRNA-miRNA-mRNA competing endogenous RNAs (ceRNAs) network was constructed, and the key regulatory molecules were analyzed. RESULTS: For LHCS and LDCS, we identified 69 lncRNAs, 150 circRNAs, 27 miRNAs, and 56 mRNAs. Differential expression analysis through GO and KEGG highlights that differentially expressed mRNAs have significant associations with pathways such as tight junction and Hepatitis C. Within the ceRNA network, all nodes have a direct or indirect association with LHCS progression. The hsa-miR-150-5p core sub-network is composed of 1 lncRNA, 6 circRNAs, 1 miRNA, and 5 mRNAs. From the ceRNA sub-network analysis, NR_120628/hsa-miR-150-5p/E2F3 and hsa-circ-0053515/hsa-miR-150-5p/E2F3 emerged as the pivotal ceRNA pairs. CONCLUSIONS: This study highlights that the NR_120628/hsa-miR-150-5p/E2F3 and hsa-circ-0053515/hsa-miR-150-5p/E2F3 axes could be central in the regulation of LHCS, providing valuable insights into potential directions for subsequent research on LHCS. TRIAL REGISTRATION: Chinese clinical trial registry (CHiCTR23007405). Registered on July 27, 2023. https://www.chictr.org.cn/.