Effects and mechanisms of Zhizi Chuanxiong herb pair against atherosclerosis: an integration of network pharmacology, molecular docking, and experimental validation.

BACKGROUND: The Zhizi Chuanxiong herb pair (ZCHP) can delay pathological progression of atherosclerosis (AS); however, its pharmacological mechanism remains unclear because of its complex components. The purpose of current study is to systematically investigate the anti-AS mechanism of ZCHP. METHODS: The databases of TCMSP, STITCH, SwissTargetPrediction, BATMAN-TCM, and ETCM were searched to predict the potential targets of ZCHP components. Disease targets associated with AS was retrieved from the GEO database. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway analyses were executed using DAVID 6.8. Molecular docking method was employed to evaluate the core target binding to blood components, and animal experiments were performed to test action mechanism. RESULTS: A ZCHP-components-targets-AS network was constructed by using Cytoscape, included 11 main components and 52 candidate targets. Crucial genes were shown in the protein-protein interaction network, including TNF, IL-1ß, IGF1, MMP9, COL1A1, CCR5, HMOX1, PTGS1, SELE, and SYK. KEGG enrichment illustrated that the NF-κB, Fc epsilon RI, and TNF signaling pathways were important for AS treatment. These results were validated by molecular docking. In ApoE-/- mice, ZCHP significantly reduced intima-media thickness, pulse wave velocity, plaque area, and serum lipid levels while increasing the difference between the end-diastolic and end-systolic diameters. Furthermore, ZCHP significantly decreased the mRNA and protein levels of TNF-α and IL-1ß, suppressed NF-κB activation, and inhibited the M1 macrophage polarization marker CD86 in ApoE-/- mice. CONCLUSION: This study combining network pharmacology, molecular biology, and animal experiments showed that ZCHP can alleviate AS by suppressing the TNF/NF-κB axis and M1 macrophage polarization.

Current status and emerging trends of cardiac metabolism from the past 20 years: A bibliometric study.

Background: Abnormal cardiac metabolism is a key factor in the development of cardiovascular diseases. Consequently, there has been considerable emphasis on researching and developing drugs that regulate metabolism. This study employed bibliometric methods to comprehensively and objectively analyze the relevant literature, offering insights into the knowledge dynamics in this field. Methods: The data source for this study was the Web of Science Core Collection (WoSCC), from which the collected data were imported into bibliometric software for analysis. Results: The United States was the leading contributor, accounting for 38.33 % of publications. The University of Washington and Damian J. Tyler were the most active institution and author, respectively. The American Journal of Physiology-Heart and Circulatory Physiology, Journal of Molecular and Cellular Cardiology, Cardiovascular Research, Circulation Research, and American Journal of Physiology-Endocrinology and Metabolism were highly influential journals that published numerous high-quality articles on cardiac metabolism. Common keywords in this research area included heart failure, insulin resistance, skeletal muscle, mitochondria, as well as topic words such as cardiac metabolism, fatty acid oxidation, glucose metabolism, and myocardial metabolism. Co-citation analysis has shown that research on heart failure and in vitro modeling of cardiovascular disease has gained prominence in recent years and making it a research hotspot. Conclusion: Research on cardiac metabolism is steadily growing, with a specific focus on heart failure and the interplay between mitochondrial dysfunction, insulin resistance, and cardiac metabolism. An emerging trend in this field involves the enhancement of maturation in human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) through the manipulation of cardiac metabolism.

A serum metabolomics study of vascular cognitive impairment patients based on Traditional Chinese medicine syndrome differentiation.

Objective: Vascular cognitive impairment (VCI) accounts for approximately 50%-70% of all dementia cases and poses a significant burden on existing medical systems. Identifying an optimal strategy for preventing VCI and developing efficient symptomatic treatments remains a significant challenge. Syndrome differentiation represents a fundamental approach for personalized diagnosis and treatment in Traditional Chinese Medicine (TCM) and aligns with the principles of precision medicine. The objective of this study was to elucidate the metabolic characteristics of VCI based on TCM syndrome differentiation, thus providing novel insights into the diagnosis and treatment of VCI. Methods: A 2-year cross-sectional cognitive survey was conducted in four communities in Beijing between September 2020 and November 2022. The syndrome differentiation of participants was based on the Kidney-Yang Deficiency Syndrome Scale (KYDSS), which was originally developed by Delphi expert consultation. The identification of serum metabolites was performed by Ultra performance liquid chromatography (UPLC) analysis coupled with an electrospray ionization quadruple time-of-flight mass spectrometer (ESI-QTOF MS). Multivariate, univariate, and pathway analyses were used to investigate metabolic changes. Logistic regression models were also used to construct metabolite panels that were capable of discerning distinct groups. Phospholipase A2 (PLA2) levels were measured by a commercial ELISA kit. Results: A total of 2,337 residents completed the survey, and the prevalence of VCI was 9.84%. Of the patients with VCI, those with Kidney-Yang deficiency syndrome (VCIS) accounted for 70.87% of cases and exhibited more severe cognitive impairments. A total of 80 participants were included in metabolomics study, including 30 with VCIS, 20 without Kidney-Yang deficiency syndrome (VCINS), and 30 healthy control participants (C). Ultimately, 45 differential metabolites were identified when comparing the VCIS group with group C, 65 differential metabolites between the VCINS group and group C, and 27 differential metabolites between the VCIS group and the VCINS group. The downregulation of phosphatidylethanolamine (PE), and phosphatidylcholine (PC) along with the upregulation of lysophosphatidylethanolamine (LPE), lysophosphatidylcholine (LPC), phosphatidic acid (PA) and phospholipase A2 (PLA2) can be considered as the general metabolic characteristics associated with VCI. Dysfunction of glycerophospholipids, particularly LPEs and PCs, was identified as a key metabolic characteristic of VCIS. In particular Glycerophospho-N-Arachidonoyl Ethanolamine (GP-NArE) was discovered for the first time in VCI patients and is considered to represent a potential biomarker for VCIS. The upregulation of PLA2 expression was implicated in the induction of alterations in glycerophospholipid metabolism in both VCIS and VCINS. Moreover, robust diagnostic models were established based on these metabolites, achieving high AUC values of 0.9322, 0.9550, and 0.9450, respectively. Conclusion: These findings contribute valuable information relating to the intricate relationship between metabolic disorders in VCI, neurodegeneration and vascular/neuroinflammation. Our findings also provide a TCM perspective for the precise diagnosis and treatment of VCI in the context of precision medicine.

Mapping current status and emerging trends in NETosis: A bibliometric study.

BACKGROUND: NETosis is a critical innate immune mechanism of neutrophils that contributes to the accelerated progression of autoimmune diseases, thrombosis, cancer, and coronavirus disease 2019 (COVID-19). This study qualitatively and quantitatively analyzed the relevant literature by bibliometric methods in order to provide a more comprehensive and objective view of the knowledge dynamics in the field. METHODS: The literature on NETosis was downloaded from the Web of Science Core Collection, analyzed with VOSviewer, CiteSpace, and Microsoft for co-authorship, co-occurrence, and co-citation analysis. RESULTS: In the field of NETosis, the United States was the most influential countries. Harvard University was the most active institutions. Mariana J. Kaplan and Brinkmann V were, respectively, the most prolific and most co-cited authors. Frontiers in Immunology, Journal of Immunology, Plos One, Blood, Science, Journal of Cell Biology, and Nature Medicine were the most influential journals. The top 15 keywords are associated with immunological and NETosis formation mechanisms. The keywords with the strongest burst detection were mainly related to COVID-19 (coronavirus, ACE2, SARS coronavirus, cytokine storm, pneumonia, neutrophil to lymphocyte ratio), and cancer (circulating tumor cell). CONCLUSION: Research on NETosis is currently booming. The mechanism of NETosis and its role in innate immunity, autoimmune diseases, especially systemic lupus erythematosus and rheumatoid arthritis, and thrombosis are the focus of research in the field of NETosis. A future study will concentrate on the function of NETosis in COVID-19 and recurrent metastasis of cancer.

Whole genome methylation combined with RNA-seq reveals the protective effects of Gualou-Xiebai herb pair in foam cells through DNA methylation mediated PI3K-AKT signaling pathway.

DNA methylation, including aberrant hypomethylation and hypermethylation, plays a significant role in atherosclerosis (AS); therefore, targeting the unbalanced methylation in AS is a potential treatment strategy. Gualou-xiebai herb pair (GXHP), a classic herb combination, have been used for the treatment of atherosclerotic-associated diseases in traditional Chinese medicine. However, the effects and underlying mechanism of GXHP on AS remain nebulous. In this study, the CCK-8 method was applied to determine the non-toxic treatment concentrations for GXHP. The formation of foam cells played a critical role in AS, so the foam cells model was established after RAW264.7 cells were treated with ox-LDL. The contents of total cholesterol (TC) and free cholesterol (FC) were determined by Gas chromatography-mass spectrometry (GC-MS). Enzyme-linked immunosorbent assay (ELISA) was used to check the expressions of inflammatory factors including IL-1ß, TNF-α, and VCAM-1. Methyl-capture sequencing (MC-seq) and RNA-seq were applied to observe the changes in genome-wide DNA methylation and gene expression, respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) were performed to analyze differentially methylated genes (DMGs) and differentially expressed genes (DEGs). The targeted signaling pathway was selected and verified using western blotting (WB). The results showed that the lipids and inflammatory factors in foam cells significantly increased. GXHP significantly reduced the expression of TC, FC, and inflammatory factors. MC-seq and RNA-seq showed that GXHP not only corrected the aberrant DNA hypermethylation, but also DNA hypomethylation, thus restored the aberrant DEGs in foam cells induced by ox-LDL. GXHP treatment may target the PI3K-Akt signaling pathway. GXHP reduced the protein levels of phosphorylated(p)-PI3K and p-AKT in foam cells. Our data suggest that treatment with GXHP showed protective effects against AS through the inhibition of DNA methylation mediated PI3K-AKT signaling pathway, suggesting GXHP as a novel methylation-based agent.

Tetramethylpyrazine and Paeoniflorin Synergistically Attenuate Cholesterol Efflux in Macrophage Cells via Enhancing ABCA1 and ABCG1 Expression.

The formation of foam cells is a characteristic of the occurrence and development of atherosclerosis. ATP-binding cassette subfamily A1 and G1 (ABCA1 and ABCG1) and scavenger receptor B1 (SR-B1) play critical roles in promoting intracellular cholesterol efflux to high-density lipoprotein (HDL) or apolipoprotein A1 (apoA1). We attempted to test the effect of the tetramethylpyrazine-paeoniflorin pair (TP) on cholesterol outflow in foam cells derived from macrophages. In this study, RAW264.7 macrophages were treated with 80 mg/L oxidized low-density lipoprotein (ox-LDL) for 24 h to obtain foam cells. Then they were intervened with TP (tetramethylpyrazine 40 ug/ml plus paeoniflorin 80 ug/ml) for additional 24 h. The distribution of cholesterol in foam cells was evaluated by oil red O staining. The contents of total cholesterol (TC) and free cholesterol (FC) were assessed with commercial kits. Fluorescent imaging was observed with a fluorescent inverted microscope. The capacity of cholesterol efflux was measured with a fluorescent plate reader, and the transcript and protein levels of ABCA1, ABCG1, and SR-B1 were detected by Western blot and quantitative polymerase chain reactions (Q-PCRs). Cytokines in the medium were detected by ELISA and adjusted by total cellular proteins. The results showed that TP decreased ox-LDL-induced cholesterol deposition and foam cell formation by promoting cholesterol efflux to apoA1, which was related to the upregulation of ABCA1 and ABCG1. Moreover, TP decreased the secretion of ox-LDL-induced tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1ß), and monocyte chemotactic protein-1 (MCP-1), an important profoam cell cytokine in atherosclerosis.

A bibliometric analysis of DNA methylation in cardiovascular diseases from 2001 to 2021.

BACKGROUND: DNA methylation is a dynamically reversible form of epigenetics. Dynamic regulation plays an important role in cardiovascular diseases (CVDs). However, there have been few bibliometric studies in this field. We aimed to visualize the research results and hotspots of DNA methylation in CVDs using a bibliometric analysis to provide a scientific direction for future research. METHODS: Publications related to DNA methylation in CVDs from January 1, 2001, to September 15, 2021, were searched and confirmed from the Web of Science Core Collection. CiteSpace 5.7 and VOSviewer 1.6.15 were used for bibliometric and knowledge-map analyses. RESULTS: A total of 2617 publications were included in 912 academic journals by 15,584 authors from 963 institutions from 85 countries/regions. Among them, the United States of America, China, and England were the top 3 countries contributing to the field of DNA methylation. Harvard University, Columbia University, and University of Cambridge were the top 3 contributing institutions in terms of publications and were closely linked. PLoS One was the most published and co-cited journal. Baccarelli Andrea A published the most content, while Barker DJP had the highest frequency of co-citations. The keyword cluster focused on the mechanism, methyl-containing substance, exposure/risk factor, and biomarker. In terms of research hotspots, references with strong bursts, which are still ongoing, recently included "epigenetic clock" (2017-2021), "obesity, smoking, aging, and DNA methylation" (2017-2021), and "biomarker and epigenome-wide association study" (2019-2021). CONCLUSIONS: We used bibliometric and visual methods to identify research hotspots and trends in DNA methylation in CVDs. Epigenetic clocks, biomarkers, environmental exposure, and lifestyle may become the focus and frontier of future research.

A Bibliometric Analysis of Research on the Links Between Gut Microbiota and Atherosclerosis.

Background: Emerging evidence has linked gut microbiota (GM) and its related metabolites to atherosclerosis (AS). This study aimed to analyze the evolution of GM in AS in the past decades, and provide valuable insights in this field. Methods: Web of Science Core Collection (WoSCC) was applied to retrieve the publications related to GM in AS from their inception until 2 December 2021, and the data was analyzed in Microsoft Excel, Scimago Graphica, CiteSpace, and VOSviewer. Results: In total, 560 documents were extracted from the WoSCC databases. The publications have shown rapid growth since 2008. China and Cleveland Clin were the most prolific country and institution, respectively. The journal with the most publications is Nutrients, and Nature was the most co-cited journal. Among 3556 related authors, Hazen, Stanley L., Tang, W. H. Wilson, and Wang, Zeneng were the top 3 contributing authors in this field. Aside from "gut microbiota," "atherosclerosis," the terms "TMAO," "metabolite," "obesity," and "phosphatidylcholine" were frequently occurred in the abstract and title of articles. Burst detection of keywords indicated that "metabolic syndrome," "acid," and "bile acid" were hot topics in recent years. According to the co-citation analysis of references, the research focus in this area has changed over time, and recent researches focus on choline, hypertension, butyrate, and berberine. Conclusion: Our study showed that the researches of GM in AS have been flourishing, and the content themes were constantly deepened. Human GM is critical to atherosclerotic diseases, and this hot topic is still worthy of more focus in the future.

Hypermethylation Effects of Yiqihuoxue Decoction in Diabetic Atherosclerosis Using Genome-Wide DNA Methylation Analyses.

PURPOSE: To investigate if a traditional Chinese medicine formulation, called "Yiqihuoxue" (YQHX), could improve diabetic atherosclerosis (DA) and explore potential mechanisms based on DNA methylation. METHODS: Apolipoprotein E-knockout mice were administered streptozotocin (50 mg/d, i.p.) for 5 days and fed a high-fat diet for 16 weeks. Mice were divided randomly into DA model, rosiglitazone, as well as low-, medium-, and high-dose YQHX groups. Ten healthy C57BL/6J mice were the control group. Serum levels of fasting insulin, blood glucose, homeostasis model-insulin resistance index (HOMA-IR), serum lipids, and inflammatory factors were analyzed after the final treatment. Aorta tissues were collected for staining (hematoxylin and eosin, and Oil red O). Genomic DNA was extracted for methyl-capture sequencing (MC-seq). Kyoto Encyclopedia of Genes and Genomes (KEGG) and Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) databases were used to analyze differentially methylated genes. Pyrosequencing was used to verify MC-seq data. RESULTS: Low-dose and high-dose YQHX could reduce the HOMA-IR (P < 0.05). Low-dose YQHX reduced expression of total cholesterol (TC), low-density lipoprotein-cholesterol (LDL-C), TNF-α, andI L-6 in serum compared with that in the model group (P < 0.05). Medium-dose YQHX decoction inhibited the expression level of TNF-α (P < 0.05). High-dose YQHX decreased the expression level of IL-6 (P < 0.05). Staining also showed the anti-atherosclerosis effects of YQHX (P < 0.05). MC-seq revealed many abnormally hypermethylated and hypomethylated genes in DA mice compared with those in the control group. KEGG database analysis showed that the hypermethylated genes induced by YQHX treatment were related to pathways in cancer, Hippo signaling, and mitogen activated protein kinase. The network analysis suggested that the hypermethylated genes epidermal growth factor receptor(Egfr) and phosphoinositide-3-kinase regulatory subunit 1(Pik3r1) induced by YQHX treatment had important roles in DA. Pyrosequencing revealed that YQHX treatment increased methylation of AKT1, Nr1h3 and Fabp4 significantly (P < 0.05). CONCLUSION: YQHX decoction had positive treatment effects against DA, because it could regulate aberrant hypomethylation of DNA.

Arsenic concentration in peripheral blood is correlated with efficacy of a Traditional Chinese Medicine regimen containing realgar for the treatment of myelodysplastic syndrome.

OBJECTIVE: To explore the relationship between the efficacy of realgar for the treatment of myelodysplastic syndromes with multilineage dysplasia (MDS-MLD) and arsenic concentration in the peripheral blood of patients. METHODS: In this prospective study, a total of 50 MDS-MLD patients were treated with traditional Chinese drugs containing realgar for 3 months in Xiyuan Hospital from March 2018 to January 2019. Routine blood examination as well as liver and kidney function were monitored before and after treatment. The concentration of arsenic in the peripheral blood was measured using an atomic fluorescence spectrometer after treatment. The correlation between clinical effect and arsenic concentration was analyzed by Spearman's method. RESULTS: The treatment response rate was 54%. Two patients (4% ) achieved complete remission, 50% (25 of 50) showed hematologic improvement, and 23 patients had stable disease (23% ). No disease progression was observed. Arsenic concentration in the peripheral blood ranged from 14.60 to 85.96 µg/L. Clinical efficacy was positively correlated with arsenic concentration (P < 0.05). The incidence of mild adverse reactions was 16%. CONCLUSION: A relatively high concentration of arsenic in the peripheral blood may improve the clinical efficacy of realgar in MDS-MLD patients.

DNA Methylation in Atherosclerosis: A New Perspective.

Atherosclerotic cardiovascular diseases, in which atherosclerosis (AS) is the main pathologic basis, are currently the primary diseases leading to human deaths. Emerging evidence showed that DNA methylation, which could affect the transcription and expression of critical regulatory genes, has key roles in AS. Aberrant DNA methylation including aberrant hypomethylation and hypermethylation plays key roles in endothelial-cell dysfunction, macrophage inflammation, abnormal proliferation of vascular smooth muscle cells, plaque rupture, and thrombosis in AS. Chinese herbal medicines, including single compounds and formulations, showed light on the treatment of AS through regulating the aberrant DNA methylation in AS. Targeting the aberrant DNA methylation may be one of the most important treatment strategies in the cure and prevention of AS. In this review, we focus on the relationship between DNA methylation and AS, as well as the beneficial effects of Chinese herbal medicines on DNA methylation in AS.

Clinical Response to Traditional Chinese Herbs Containing Realgar (As2S2) is Related to DNA Methylation Patterns in Bone Marrow DNA from Patients with Myelodysplastic Syndrome with Multilineage Dysplasia.

PURPOSE: DNA methylation is known to play an important role in myelodysplastic syndrome (MDS). We previously showed that Chinese herbs (CHs) containing realgar (As2S2) were effective at treating MDS with multilineage dysplasia (MDS-MLD). We tested whether the response to CH treatment was related to changes in DNA methylation in MDS-MLD. PATIENTS AND METHODS: First, the Illumina methylation 850K array BeadChip assay was used to assess the pretreatment methylation status in bone marrow cells from eight MDS-MLD patients and 3 healthy donors. The eight MDS-MLD patients were then treated with CHs for six months, the arsenic concentration was measured following treatment. The patients were subsequently divided into "effective" and "ineffective" treatment response groups and the DNA methylation patterns of the two groups were compared. Finally, the BeadChip data were validated by pyrosequencing. RESULTS: Five of the eight MDS-MLD patients showed hematological improvement (effective-treatment group), while three showed disease progression (ineffective-treatment group) (positive response rate: 62.5%). The arsenic concentrations in the patients ranged from 26.60 to 64.16 µg/L (median 48.4 µg/L) and were not significantly different between the two groups (p = 0.27). Compared with the healthy controls, three genes were hypomethylated and 110 were hypermethylated in the ineffective-treatment group. However, in the group showing hematological improvement, 102 genes were markedly hypomethylated and 87 hypermethylated. The effective-treatment group had a higher proportion of hypomethylated sites than the ineffective-treatment group (53.9% vs 2.6%, respectively; chi-square test) (p < 0.0001). Two hypermethylated and two hypomethylated genes were selected for validation by pyrosequencing (all p < 0.05). CONCLUSION: MDS-MLD patients may present different DNA methylation subtypes. CHs containing realgar may be effective for treating MDS-MLD patients with the hypomethylation subtype.

High-throughput screening suggests glutathione synthetase as an anti-tumor target of polydatin using human proteome chip.

Polydatin (PD) is a bio-active ingredient with known anti-tumor effects. However, its specific protein targets yet have not been systematically screened, and the molecular anti-tumor mechanism is still unclear. Here, proteomic-chip was efficiently used to screen potential targets of PD. First, we investigated through animal experiment and proteomics studies, and found that polydatin play an important role in tumor cells. Then, the red-green fluorescent of polydatin was compared comprehensively to screen its targets on chip, followed by bioinformatics analysis. Glutathione synthetase (GSS) was selected as candidate research target. After a series of molecular biological experiments GSS was confirmed a target protein for PD in vitro. Moreover, we also found that PD can significantly inhibit the activity of GSS in vitro and live cells. Our findings reveal that PD could be a selective small-molecule GSS enzyme activity inhibitor and GSS could be a potential therapeutic target in cancer.

Arsenic Disulfide Promoted Hypomethylation by Increasing DNA Methyltransferases Expression in Myelodysplastic Syndrome.

BACKGROUND: Previous studies have shown that DNA methylation plays a significant role in myelodysplastic syndrome (MDS). In addition to hypermethylation, aberrant hypomethylation can result in the transcriptional activation of oncogenes in cancer, including MDS. Therefore, drugs targeting DNA hypomethylation are needed for the treatment of MDS. This study aimed to investigate whether As2S2 promoted hypomethylation by increasing DNA methyltransferases (DNMTs) expression in MDS. PATIENTS AND METHODS: Ten bone marrow samples from MDS patients and 3 healthy donors were obtained for the examination of the DNA methylation with a Human Methylation 850K BeadChip. The mRNA expressions for the DNMTs in the ten MDS patients and 3 controls were compared by Q-PCR. Then, the MDS cell line SKM-1 was treated with As2S2. After 2 days of treatment, Human Methylation 850K BeadChip was applied to analyze the changes of gene methylation status in the cells. Q-PCR and Western blot were taken to test the changes of mRNA and protein expressions for DNMTs in SKM-1 cells after treatment. RESULTS: Five hundred ninety-two abnormally hypomethylated genes were found in MDS patients compared to those in controls by Human Methylation 850K. The mRNA expressions of DNMTs (DNMT1, DNMT3a and DNMT3b) in MDS patients were significantly lower than those in healthy individuals. The IC50 value of As2S2 for SKM-1 cells was 4.97 µmol/L.Treatment with As2S2 at 2 µmoL/L resulted in significant alterations in the methylation levels at 1718 sites in SKM-1 cells compared to those in the controls. Hypermethylation was observed in 1625 sites (94.58%), corresponding to 975 genes, compared to those in the controls. Finally, the expression levels of DNMTs (DNMT1, DNMT3a, and DNMT3b) significantly increased in SKM-1 cells treated with As2S2 at 2 µmoL/L and 4 µmoL/L. CONCLUSION: These data show a potential clinical application of As2S2 as an innovative hypermethylation agent in MDS.

Traditional Chinese Medicine Containing Arsenic Treated MDS Patients Effectively through Regulating Aberrant Hypomethylation.

Aberrant hypermethylation and hypomethylation both play important roles in myelodysplastic syndrome (MDS). Hypomethylating agents targeting hypermethylation have been employed for the MDS treatment, but the treatment effect is limited. Novel drugs for DNA hypomethylation-targeted therapy may be needed to improve clinic efficacy for the treatment of MDS. Chinese medicine (CM) herbs have been used to treat MDS for many years in our hospital. However, the long-term treatment effect and mechanism remain unclear. In this study, all 135 patients received CM treatment for at least 36 months. The response rates for CM treatment were 81.53% (106/130) for hematological improvement in 130 MDS-RCMD patients and 80% (4/5) for bone marrow CR in 5 MDS-RAEB patients, respectively. The Human Methylation 850K BeadChip showed that 115 genes (50.88%) were aberrantly hypomethylated in 5 MDS patients compared with 3 healthy individuals. GO-analysis showed that these hypomethylated genes participated in many cancer-related biological functions and pathways. Furthermore, 60 genes were hypermethylated and the protein expression level of DNMT1 was significantly increased in the 5 MDS patients after 6 months of CM treatment. Our study suggests that CM can improve aberrant hypomethylation by increasing DNMT1 expression in MDS. The data support the clinical application of CM herbs containing arsenic as an innovative hypermethylation-inducing regimen for the treatment of MDS.

Effect of Qinghuang Powder () Combined with Bupi Yishen Decoction () in Treating Patients with Refractory Cytopenia with Multilineage Dysplasia through Regulating DNA Methylation.

OBJECTIVE: To explore the effect of Qinghuang Powder (QHP,()combined with Bupi Yishen Decoction (BPYS, ) on myelodysplastic syndromes (MDS) patients with refractory cytopenia with multilineage dysplasia (RCMD) and determine the change of DNA methylation in MDS-RCMD patients after the treatment of Chinese medicine formula. METHODS: All 308 MDS-RCMD patients were treated with QHP combined with BPYS for 2 months at least, absolute neutrophil count (ANC), hemoglobin (Hb), platelets (PLT), primitive bone marrow cells and chromosome karyotype were chosen as the main evaluation indexes to analyze the treatment effect according to criteria from the MDS International Working Group. Then 43 bone marrow samples from 15 MDS-RCMD patients and 28 healthy donors were obtained for the examination of DNA methylation. Gene Ontology (GO) and Pathway analysis were applied to analyze the methylation data. RESULTS: The overall MDS response rate to QHP was 61.68% (190/360) including hematologic improvement-neutrophil (HI-N) or hematologic improvement-erythroid (HI-E) or hematologic improvement-platelet (HI-P). Patients with anemia had a better response rate than patients with neutropenia or thrombocypenia (55.88% vs 31.54% or 55.88% vs. 36.9%). The DNA methylation microarray analysis disclosed that 4,257 hypermethylated genes were demethylated upon the treatment with QHP and BPYS. GO analysis and Pathway analysis showed that these demethylated genes were involved in a lot of tumor-related pathways and functions. CONCLUSIONS: QHP combined with BPYS could effectively treat MDS-RCMD patients through hematologic improvement (HI-N, HI-P or HI-E) and PLT and RBC transfusion independence due to the demethylation, thereby providing another choice for the treatment of patients with MDS-RCMD.

Effects of Zhizi Chuanxiong Capsule () on the Abnormal Methylation in Rabbits with Atherosclerosis.

OBJECTIVE: To investigate the effects of Zhizi Chuanxiong Capsule (ZCC, ) on abnormal DNA methylation in a rabbit model of atherosclerosis (AS). METHODS: After 1 week of adaptive feeding, 48 New Zealand white rabbits were randomly divided into 4 groups: a control group (n=12) fed with normal diet for 22 weeks; a model group (n=12) fed with high fat diet for 14 weeks followed by 8 weeks of normal diet feeding; a low-dose ZCC group (n=12) fed with high fat diet and low-dose ZCC for 14 weeks, followed by 8 weeks of normal diet and low-dose drug; a high-dose ZCC group (n=12) fed with high fat diet and high-dose drug for 14 weeks, followed by 8 weeks of normal diet and high-dose drug. After 22 weeks of feeding, blood samples were taken from the rabbit ear vein, and the genomic DNA was extracted for methylation immunoprecipitation sequencing (Medip-seq). The aorta tissues were collected for hematoxylin-eosin (HE) staining. RESULTS: Eight rabbits died during the feeding process. HE staining showed that the size of the lipid deposition on vessel wall and atherosclerotic plaque formation were reduced in both low- and high-dose group. The Medip-seq results showed that there were 146 abnormally methylated genes (including both hypermethylated gene and hypomethylated genes) in the model group, compared with the control group. Gene Ontology (GO) and Pathway analysis showed that these abnormally methylated genes were found to be involved in multiple AS-related functions and pathways, such as protein kinase C activity, cholesterol transport, mitogen-activated protein kinase (MAPK) signaling pathway, peroxisome proliferater-activated receptor signaling pathway, vascular smooth muscle contraction, inflammation and so on. The abnormal methylated genes in AS model group were altered in both low- and high-dose groups: low-dose ZCC could change 72 of the 146 abnormally methylated genes, high-dose ZCC could change 71. Through GO and Pathway analysis, these altered methylated genes were involved in protein kinase C activity, inflammatory pathway, MAPK signaling pathway, vascular endothelial growth factor signaling pathway, etc. CONCLUSION: ZCC could treat AS through regulating the abnormal hypermethylated and hypomethylated genes in AS rabbit model.

Involvement of oxidative stress associated with glutathione depletion and p38 mitogen-activated protein kinase activation in arsenic disulfide-induced differentiation in HL-60 cells.

Arsenic disulfide (As2S2) has been traditionally used to treat certain types of leukemia. However, a detailed mechanism of action of As2S2 is not sufficiently documented. The effects of As2S2 on HL-60 cells were therefore investigated by focusing on proliferation, differentiation, generation of reactive oxygen species (ROS), intracellular glutathione (GSH) depletion and activation of p38 mitogen-activated protein kinase (MAPK). As2S2 at 0.5-8 µM induced cell differentiation based on an increment in CD11b expression, nitroblue tetrazolium (NBT)-positive cells and cell size change. A transient increase in ROS level along with intracellular GSH level was also observed. p38 MAPK activation gradually increased after ROS generation and was sustained during cell differentiation. Decreased CD11b expression was accompanied by p38 MAPK activation, and a p38 MAPK inhibitor restored CD11b expression. The results suggest that moderate levels of oxidative stress induced by As2S2 correlate with HL-60 cell differentiation. Suppression of p38 MAPK can augment the efficacy of As2S2 to induce HL-60 cell differentiation.

[Effect of treatment for hypocellular myelodysplastic syndromes by a low dose qinghuang powder combined with Chinese drugs for Shen supplementing and Pi invigorating: a clinical observation].

OBJECTIVE: To observe the clinical efficacy of a low dose Qinghuang Powder (QP) combined with Chinese drugs for Shen supplementing and Pi invigorating (CDSSPI) in treatment of hypocellular myelodysplastic syndromes (hypo-MDS). METHODS: Totally 33 hypo-MDS patients enrolled in this study came from outpatient clinics between November 2011 and December 2012. A self-control method was used in this study. Patients took QP (0.4 g per day) combined with CDSSPI (one dose per day), and Stanozolol Tablet (2 mg each time, three times per day), 3 months as one therapeutic course, a total of 2 courses. The clinical efficacy was evaluated timely at the end of each therapeutic course. The venous blood was withdrawn before treatment, at month 3 and 6 after treatment. Changes of neutrophils (ANC), hemoglobin (Hb), and platelet (PLT) were mainly observed. RESULTS: Totally 31 patients in this study finished the treatment. Three months after treatment ANC, Hb, and PLT increased more than before treatment (P < 0.05). Six months after treatment Hb and PLT increased (P < 0.01, P < 0.05), but with no statistical difference in ANC (P > 0.05). Hb increased higher at month 6 after treatment than at month 3 after treatment (P < 0.01), but with no statistical difference in ANC or PLT (P > 0.05). After 3-month treatment the number of hematologic progress, stability, disease progression were: 13 cases (41.9%), 15 cases (48.4%), and 3 cases (9.7%), respectively; after 6-month treatment the number of hematologic improvement, stability, and disease progression were: 18 cases (58.1%), 7 cases (22.6%), 6 cases (19.3%), respectively. There was no significant difference between 3-month efficacy and 6-month efficacy (P > 0.05). There was no correlation between the efficacy and ages of hypo-MDS patients or the efficacy and courses of hypo-MDS patients (P > 0.05). CONCLUSIONS: A low dose QP combined with CDSSPI showed confirmative efficacy in treatment of hypo-MDS. But the efficacy had little correlation with ages and courses of hypo-MDS patients.

[Progress of cytogenetic detection in myelodysplastic syndromes].

In recent years, significant progresses have been got in study on pathogenesis, treatment and prognosis of myelodysplastic syndromes (MDS), especially on use of new technology, that has great importance for cytogenetics of MDS. Recently, the progress of cytogenetic detection in MDS is very remarkable. Based on the metaphase cytogenetics (MC) method, prognostic significance of cytogenetics in MDS was clarified gradually. For example, people have known the prognostic significance of 12 p-, 11 q-, +21, t(11(q23)), although these genetic abnormalities are rare in the MDS. In addition, chromosome mutation emerged in the process of MDS may indicate the poor prognosis. On the other hand, with the use of SNP-A and aCGH in the study of genetics, MDS cytogenetic abnormality detection rate has been further improved and can reach to 78%. At the same time, some of MDS patients with the "normal karyotype" detected by MC have new hidden aberrations through the SNP or CGH detection, and these patients have a poorer prognosis. In this review, the advances of study on cytogenetic detection for MDS based on MC and SNP-A or aCGH methods are summarized.