Experimental Verification of Erchen Decoction Plus Huiyanzhuyu Decoction in the Treatment of Laryngeal Squamous Cell Carcinoma Based on Network Pharmacology.

BACKGROUND: The prescription of Chinese herbal medicine (CHM) consists of multiple herbs that exhibit synergistic effects due to the presence of multiple components targeting various pathways. In clinical practice, the combination of Erchen decoction and Huiyanzhuyu decoction (EHD) has shown promising outcomes in treating patients with laryngeal squamous cell carcinoma (LSCC). However, the underlying mechanism by which EHD exerts its therapeutic effects in LSCC remains unknown. METHODS: Online databases were utilized for the analysis and prediction of the active constituents, targets, and key pathways associated with EHD in the treatment of LSCC. The protein-protein interaction (PPI) network of common targets was constructed and visualized using Cytoscape 3.8.1 software. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to investigate the functional roles of core targets within the PPI network. Protein clustering was conducted utilizing the MCODE plug-in. The obtained results highlight the principal targets and pathways involved. Subsequently, clinical samples were collected to validate alterations in the levels of these main targets through Western blotting (WB) and immunohistochemistry (IHC). Furthermore, both in vivo and in vitro experiments were conducted to investigate the therapeutic effects of EHD on healing LSCC and elucidate its underlying mechanism. Additionally, to ensure experimental reliability and reproducibility, quality control measures utilizing HPLC were implemented for EHD herbal medicine. RESULTS: The retrieval and analysis of databases in EHD medicine and LSCC disease yielded a total of 116 overlapping targets. The MCODE plug-in methods were utilized to acquire 8 distinct protein clusters through protein clustering. The findings indicated that both the first and second clusters exhibited a size greater than 6 scores, with key genes PI3K and ErbB occupying central positions, while the third and fourth clusters were associated with proteins in the PI3K, STAT3, and Foxo pathways. GO functional analysis reported that these targets had associations mainly with the pathway of p53 mediated DNA damage and negative regulation of cell cycle in terms of biological function; the death-induced signaling complex in terms of cell function; transcription factor binding and protein kinase activity in terms of molecular function. The KEGG enrichment analysis demonstrated that these targets were correlated with several signaling pathways, including PI3K-Akt, FoxO, and ErbB2 signaling pathway. On one hand, we observed higher levels of key genes such as P-STAT3, P-PDK1, P-Akt, PI3K, and ErbB2 in LSCC tumor tissues compared to adjacent tissues. Conversely, FOXO3a expression was lower in LSCC tumor tissues. On the other hand, the key genes mentioned above were also highly expressed in both LSCC xenograft nude mice tumors and LSCC cell lines, while FOXO3a was underexpressed. In LSCC xenograft nude mice models, EHD treatment resulted in downregulation of P-STAT3, P-PDK1, PI3K, P-AKT, and ErbB2 protein levels but upregulated FOXO3a protein level. EHD also affected the levels of P-STAT3, P-PDK1, PI3K, P-AKT, FOXO3a, and ErbB2 proteins in vitro: it inhibited P-STAT3, P-AKT, and ErbB2, while promoting FOXO3a; however, it had no effect on PDK1 protein. In addition, HPLC identified twelve compounds accounting for more than 30% within EHD. The findings from this study can serve as valuable guidance for future experimental investigations. CONCLUSION: The possible mechanism of EHD medicine action on LSCC disease is speculated to be closely associated with the ErbB2/PI3K/AKT/FOXO3a signaling pathway.

Deciphering feedback regulation of prostaglandin F2α in blood stasis syndrome using nitrogen-doped porous transition metal carbides.

Blood stasis syndrome (BSS) has persistent health risks; however, its pathogenesis remains elusive. This obscurity may result in missed opportunities for early intervention, increased susceptibility to chronic diseases, and reduced accuracy and efficacy of treatments. Metabolomics, employing the matrix-assisted laser desorption/ionization (MALDI) strategy, presents distinct advantages in biomarker discovery and unraveling molecular mechanisms. Nonetheless, the challenge is to develop efficient matrices for high-sensitivity and high-throughput analysis of diverse potential biomarkers in complex biosamples. This work utilized nitrogen-doped porous transition metal carbides and nitrides (NP-MXene) as a MALDI matrix to delve into the molecular mechanisms underlying BSS pathogenesis. Structural optimization yielded heightened peak sensitivity (by 1.49-fold) and increased peak numbers (by 1.16-fold) in clinical biosamples. Validation with animal models and clinical serum biosamples revealed significant differences in metabolic fingerprints between BSS and control groups, achieving an overall diagnostic efficacy of 0.905 (95% CI, 0.76-0.979). Prostaglandin F2α was identified as a potential biomarker (diagnostics efficiency of 0.711, specificity = 0.7, sensitivity = 0.6), and pathway enrichment analysis disclosed disruptions in arachidonic acid metabolism in BSS. This innovative approach not only advances comprehension of BSS pathogenesis, but also provides valuable insights for personalized treatment and diagnostic precision.

Development of the Blood Stasis Questionnaire for Gynecological Diseases: An Analytical Cross-Sectional Study.

OBJECTIVES: Blood stasis is the slowing or stagnation of blood and can cause metabolic, musculoskeletal, and gynecological diseases. This study developed the Blood Stasis Questionnaire for gynecological disease (BSQ-GD) by extracting clinical indicators related to gynecological diseases using the Blood Stasis Questionnaires I and II (BSQ-I and II, respectively) and analyzed the clinical data of a cross-sectional study. PATIENTS AND METHODS: In total, 103 women aged between 25 and 65 years who met gynecological disease criteria were enrolled in this study. Blood stasis scores (BSS) were evaluated using the BSQ-II and categorized into BSS and non-BSS groups. To assess the reliability of BSQ-GD, the internal consistency coefficient was employed using Cronbach's α. Furthermore, correlation analyses were conducted for the clinical symptoms related to gynecological diseases, and the discriminant validity was confirmed by comparing the two groups. The prediction accuracy was determined using logistic regression and the cut-off value of the BSQ-GD was established via the sensitivity and specificity calculations. RESULTS: The BSQ-GD showed satisfactory internal consistency (Cronbach's α coefficient = 0.71) and validity, with significant differences in mean scores between blood stasis (22.30 ± 3.34) and non-blood stasis (14.93 ± 3.49) groups. The cut-off value of the BSQ-GD score was 19 points when the Youden index (73.45) and the concordance probability (0.75) were at their maximum. The area under the receiver operating characteristic curve was approximately 96%, and the sensitivity and specificity of the diagnostic accuracy according to the cut-off value are 80.95% and 92.50%, respectively. CONCLUSION: The BSQ-GD can be an appropriate instrument to estimate blood stasis in patients with gynecological diseases; its diagnostic sensitivity according to the cut-off value is high.

Efficacy of Fire-Needle Therapy in Improving Neurological Function Following Cerebral Infarction and Its Effect on Intestinal Flora Metabolites.

Objective: This study was to investigate the mechanism of action and clinical efficacy of fire-needle therapy in improving neurological function in patients with acute cerebral infarction (identified as a wind-phlegm-blood stasis syndrome in traditional Chinese medicine). Methods: We included patients diagnosed with acute cerebral infarction (wind-phlegm-blood stasis syndrome) admitted to the Encephalopathy and Acupuncture Center of the Second Affiliated Hospital of Tianjin University of Chinese Medicine. We randomly allocated them into the treatment and control groups, with 45 cases in each group. Acupuncture treatments that focused on regulating the mind and dredging the collaterals were used in the control group, while the treatment group additionally received fire-needle therapy. Our indicators included the National Institutes of Health Stroke Scale (NIHSS) scores, the Fugl-Meyer Assessment (FMA) scale, peripheral blood tumor necrosis factor-α (TNF-α), interleukin-17 (IL-17), hypersensitivity C-reactive protein (hs-CRP), and intestinal metabolites short-chain fatty acids (SCFAs). We measured these indicators before treatment and 14 days after treatment. Results: The post-treatment NIHSS scores of the two groups were significantly reduced (P < 0.05), and the treatment group showed a more significant decline in the score when compared to the control group (P < 0.05). The treatment group showing significant improvement in the domains of reflex activity, mobility, cooperative movement, and finger movement (P < 0.05). Both groups showed a significant decrease in the IL-17 and hs-CRP levels (P < 0.05), with the treatment group demonstrating a significant declining trend when compared to the control group (P < 0.05). The levels of acetic acid, propionic acid, butyric acid, and valeric acid all increased significantly in the two groups (P < 0.05), with acetic acid and butyric acid increasing significantly in the treatment group when compared to the control group (P < 0.05). Clinical efficacy rate: 78.6% of patients in the treatment group had an excellent rate, whereas it was 30.0% in the control group, and the difference was statistically significant (P < 0.001). Conclusion: Fire-needle therapy was effective in upregulating the SCFA content in patients with acute cerebral infarction (wind-phlegm-blood stasis syndrome), inhibiting the level of the inflammatory response, and improving the recovery of neurological functions. Clinical registration number: Registration website link: https://www.chictr.org.cn. Registration date: 2022/9/27. Registration number: ChiCTR2200064122.

Proteomics Analysis of Serum Reveals Potential Biomarkers for Heart Failure Patients with Phlegm-Blood Stasis Syndrome.

Heart failure (HF), a complex clinical syndrome, has become a global burden on health and economics around the world. Phlegm-blood stasis syndrome, one of the Traditional Chinese Medicine (TCM) syndrome differentiation, is the core pathogenesis dynamically throughout the occurrence, development, and prognosis of HF. Biomarkers having high sensitivity and specificity are highly demanded to facilitate the accurate differentiation of HF patients with phlegm-blood stasis syndrome. In the present study, serum samples were collected from 20 healthy controls and 40 HF patients (20 with and 20 without phlegm-blood stasis syndrome). We implemented data-independent acquisition mass spectrometry (DIA-MS) for discovery and parallel reaction monitoring (PRM) for validation of biomarkers for heart failure with phlegm-blood stasis syndrome. A total of 84 different proteins were found in the HF with phlegm-blood stasis syndrome (HF-TY) group compared with healthy controls. 37 candidate proteins were selected for the PRM assay, and five validated proteins with high sensitivity and specificity, including insulin-like growth factor-binding protein 4 (IGFBP4), ß-2-microglobulin (B2M), dystroglycan (DAG1), immunoglobulin J chain (JCHAIN), and kallikrein B1 (KLKB1), were considered potential biomarkers for heart failure patients with phlegm-blood stasis syndrome. Newly identified biomarkers might provide insights into the diagnosis and treatment of HF with TCM syndrome differentiation.

[Efficacy and mechanism of Xuefu Zhuyu Decoction on model rats of coronary heart disease with heart blood stasis syndrome based on metabolomics].

This study investigated the effects of Xuefu Zhuyu Decoction on myocardial metabolites in a rat model of coronary heart disease with heart blood stasis syndrome and explored the therapeutic mechanism of blood circulation-promoting and blood stasis-removing therapy. SD rats were randomly divided into a sham operation group, a model group, a Xuefu Zhuyu Decoction group(14.04 g·kg~(-1)), and a trimetazidine group(5.4 mg·kg~(-1)). The sham operation group underwent thread insertion without ligation, while the other groups underwent coronary artery left anterior descending branch ligation to induce a model of coronary heart disease with heart blood stasis syndrome. Three days after modeling, drug intervention was performed, and samples were taken after 14 days of intervention. General conditions were observed, and electrocardiogram and cardiac ultrasound indices were measured. Hematoxylin-eosin(HE) staining and Masson staining were used to observe tissue pathological morphology. The enzyme linked immunosorbent assay(ELISA) was used to measure the levels of triglyceride(TG) and total cholesterol(TC) in the serum. Ultra high performance liquid chromatography-quantitative exactive-mass spectrometry(UHPLC-QE-MS) technology was used to screen differential metabolites in myocardial tissue and conduct metabolic pathway enrichment analysis. The results showed that Xuefu Zhuyu Decoction significantly improved the general condition of the model rats, reduced heart rate and ST segment elevation in the electrocardiogram, increased left ventricular ejection fraction(LVEF) and left ventricular fractional shortening(LVFS), and decreased left ventricular internal diameter in diastole(LVIDd) and left ventricular internal diameter in systole(LVIDs). HE staining and Masson staining showed that Xuefu Zhuyu Decoction effectively alleviated myocardial tissue structural disorders, inflammatory cell infiltration, and collagen fiber deposition in the model rats. ELISA results showed that Xuefu Zhuyu Decoction effectively regulated serum TG and TC levels in the model rats. There were significant differences in the metabolic phenotypes of myocardial samples in each group. Fourteen differential metabolites were identified in the Xuefu Zhuyu Decoction group, involving five metabolic pathways, including arginine and proline metabolism, glycerophospholipid metabolism, aminoacyl-tRNA biosynthesis, ether lipid metabolism, and alanine, aspartate, and glutamate metabolism. Xuefu Zhuyu Decoction improved cardiac function and myocardial structural damage in the rat model of coronary heart disease with heart blood stasis syndrome, and its biological mechanism involved the regulation of lipid metabolism, choline metabolism, amino acid metabolism, energy metabolism, and protein synthesis pathways.

[Characteristic changes of blood stasis syndrome in rat model of steroid-induced femoral head necrosis based on the combination of disease, syndrome, and symptom].

The approach combining disease, syndrome, and symptom was employed to investigate the characteristic changes of blood stasis syndrome in a rat model of steroid-induced osteonecrosis of the femoral head(SONFH) during disease onset and progression. Seventy-two male SD rats were randomized into a healthy control group and a model group. The rat model of SONFH was established by injection of lipopolysaccharide(LPS) in the tail vein at a dose of 20 µg·kg~(-1)·d~(-1) on days 1 and 2 and gluteal intramuscular injection of methylprednisolone sodium succinate(MPS) at a dose of 40 mg·kg~(-1)·d~(-1) on days 3-5, while the healthy control group received an equal volume of saline. The mechanical pain test, tongue color RGB technique, gait detection, open field test, and inclined plane test were employed to assess hip pain, tongue color, limping, joint activity, and lower limb strength, respectively, at different time points within 21 weeks of modeling. At weeks 2, 4, 8, 12, 16, and 21 after modeling, histopathological changes of the femoral head were observed by hematoxylin-eosin(HE) staining and micro-CT scanning; four coagulation items were measured by rotational thromboelastometry; and enzyme-linked immunosorbent assay(ELISA) was employed to determine the levels of six blood lipids, vascular endothelial growth factor(VEGF), endothelin-1(ET-1), nitric oxide(NO), tissue-type plasminogen activator(t-PA), plasminogen activator inhibitor factor-1(PAI-1), bone gla protein(BGP), alkaline phosphatase(ALP), receptor activator of nuclear factor-κB(RANKL), osteoprotegerin(OPG), and tartrate-resistant acid phosphatase 5b(TRAP5b) in the serum, as well as the levels of 6-keto-prostaglandin 1α(6-keto-PGF1α) and thromboxane B2(TXB2) in the plasma. The results demonstrated that the pathological alterations in the SONFH rats were severer over time. The bone trabecular area ratio, adipocyte number, empty lacuna rate, bone mineral density(BMD), bone volume/tissue volume(BV/TV), trabecular thickness(Tb.Th), trabecular number(Tb.N), bone surface area/bone volume(BS/BV), and trabecular separation(Tb.Sp) all significantly increased or decreased over the modeling time after week 4. Compared with the healthy control group, the mechanical pain threshold, gait swing speed, stride, standing time, and walking cycle of SONFH rats changed significantly within 21 weeks after modeling, with the greatest difference observed 12 weeks after modeling. The time spent in the central zone, rearing score, and maximum tilt angle in the open field test of SONFH rats also changed significantly over the modeling time. Compared with the healthy control group, the R, G, and B values of the tongue color of the model rats decreased significantly, with the greatest difference observed 11 weeks after modeling. The levels of total cholesterol(TC), total triglycerides(TG), low-density lipoprotein-cholesterol(LDL-C), and apoprotein B(ApoB) in the SONFH rats changed significantly 4 and 8 weeks after modeling. The levels of VEGF, ET-1, NO, t-PA, PAI-1, 6-keto-PGF1α, TXB2, four coagulation items, and TXB2/6-keto-PGF1α ratio in the serum of SONFH rats changed significantly 4-16 weeks after modeling, with the greatest differences observed 12 weeks after modeling. The levels of BGP, TRAP5b, RANKL, OPG, and RANKL/OPG ratio in the serum of SONFH rats changed significantly 8-21 weeks after modeling. During the entire onset and progression of SONFH in rats, the blood stasis syndrome characteristics such as hyperalgesia, tongue color darkening, gait abnormalities, platelet, vascular, and coagulation dysfunctions were observed, which gradually worsened and then gradually alleviated in the disease course(2-21 weeks), with the most notable differences occurred around 12 weeks after modeling.

[Effect of Liangfang Wenjing Decoction on expression of key glycolytic enzymes in uterus and ovaries of rats with coagulating cold and blood stasis syndrome].

This study aimed to investigate the relationship between coagulating cold and blood stasis syndrome and glycolysis, and observe the intervention effect of Liangfang Wenjing Decoction(LFWJD) on the expression of key glycolytic enzymes in the uterus and ovaries of rats with coagulating cold and blood stasis. The rat model of coagulating cold and blood stasis syndrome was established by ice-water bath. After modeling, the quantitative scoring of symptoms were performed, and according to the scoring results, the rats were randomly divided into a model group and LFWJD low-, medium-and high-dose groups(4.7, 9.4, 18.8 g·kg~(-1)·d~(-1)), with 10 in each group. Another 10 rats were selected as the blank group. After 4 weeks of continuous administration by gavage, the quantitative scoring of symptoms was repeated. Laser speckle flowgraphy was used to detect the changes of microcirculation in the ears and uterus of rats in each group. Hematoxylin-eosin(HE) staining was used to observe the pathological morphology of uterus and ovaries of rats in each group. The mRNA and protein expressions of pyruvate dehydrogenase kinase 1(PDK1), hexokinase 2(HK2) and lactate dehydrogenase A(LDHA) in the uterus and ovaries of rats were examined by real-time quantitative polymerase chain reaction(RT-qPCR) and Western blot, respectively. The rats in the model group showed signs of coagulating cold and blood stasis syndrome, such as curl-up, less movement, thickened veins under the tongue, and reduced blood perfusion in the microcirculation of the ears and uterus, and HE staining revealed a thinning of the endometrium with disorganized arrangement of epithelial cells and a decrease in the number of ovarian follicles. Compared with the model group, the treatment groups had alleviated coagulating cold and blood stasis, which was manifested as red tongue, reduced nail swelling, no blood stasis at the tail end as well as increased blood perfusion of the microcirculation in the ears and uterus(P<0.05 or P<0.01). Among the groups, the LFWJD medium-and high-dose groups had the most significant improvement in coagulating cold and blood stasis, with neatly arranged columnar epithelial cells in uterus, and the number of ovarian follicles was higher than that in the model group, especially mature follicles. The mRNA and protein expressions of PDK1, HK2, LDHA in uterus and ovaries were up-regulated in the model group(P<0.05 or P<0.01), while down-regulated in LFWJD medium-and high-dose groups(P<0.05 or P<0.01). The LFWJD low-dose group presented a decrease in the mRNA expressions of PDK1, HK2 and LDHA in uterus and ovaries as well as in the protein expressions of HK2 and LDHA in uterus and HK2 and PDK1 in ovaries(P<0.05 or P<0.01). The therapeutic mechanism of LFWJD against coagulating cold and blood stasis syndrome is related to the down-regulation of key glycolytic enzymes PDK1, HK2 and LDHA, and the inhibition of glycolytic activities in uterus and ovaries.

[Integrated strategy for biomarkers of stable coronary heart disease with phlegm and blood stasis syndrome based on RNA-seq and network pharmacology].

This study aimed to analyze the biological foundation and biomarkers of stable coronary heart disease(CHD) with phlegm and blood stasis(PBS) syndrome based on RNA-seq and network pharmacology. Peripheral blood nucleated cells from five CHD patients with PBS syndrome, five CHD patients with non-PBS syndrome, and five healthy adults were collected for RNA-seq. The specific targets of CHD with PBS syndrome were determined by differential gene expression analysis and Venn diagram analysis. The active ingredients of Danlou Tablets were screened out from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform, and the "component-target" prediction was completed through PubChem and SwissTargetPrediction. The "drug-ingredient-target-signaling pathway" network of Danlou Tablets against CHD with PBS syndrome was optimized by Cytoscape software. After the target biomarkers were identified, 90 participants were enrolled for diagnostic tests, and 30 CHD patients with PBS syndrome were included in before-and-after experiment to determine the therapeutic effect of Danlou Tablets on those targets. As revealed by RNA-seq and Venn diagram analysis, 200 specific genes were identified for CHD with PBS syndrome. A total of 1 118 potential therapeutic targets of Danlou Tablets were predicted through network pharmacology. Through integrated analysis of the two gene sets, 13 key targets of Danlou Tablets in the treatment of CHD with PBS syndrome were screened out, including CSF1, AKR1C2, PDGFRB, ARG1, CNR2, ALOX15B, ALDH1A1, CTSL, PLA2G7, LAP3, AKR1C3, IGFBP3, and CA1. They were presumably the biomarkers of CHD with PBS syndrome. The ELISA test further showed that CSF1 was significantly up-regulated in the peripheral blood of CHD patients with PBS syndrome, and was significantly down-regulated after Danlou Tablets intervention. CSF1 may be a biomarker for CHD with PBS syndrome, and it is positively correlated with the severity of the disease. The diagnostic cut-off of CSF1 for CHD with PBS syndrome was 286 pg·mL~(-1).

Data mining-based analysis on medication rules of Chinese herbal medicine treating headache with blood stasis syndrome.

Objective: The incidence of headaches with blood stasis syndrome has increased. Herein, we used scientific, statistical methods to explore the medication rules of Chinese herbal medicines (CHMs) to treat headaches with blood stasis syndrome and provide a scientific and reliable theoretical basis for clinical treatment. Methods: First, we retrieved studies related to CHMs used to treat headaches with blood stasis syndrome from the VIP, CNKI, Wanfang, and PubMed databases. We used Excel 2013 to establish a database and SPSS Modeler 18.0 and SPSS 25.0 to conduct frequency, association rule, and cluster analyses. Results: Based on the screening criteria, we retrieved 126 CHM prescriptions for headaches with blood stasis syndrome involving 149 herbs. The top three high-frequency herbs were Chuanxiong Rhizoma (Chuanxiong), Angelica Sinensis Radix (Danggui), and Carthami Flos (Honghua). Blood-activating and stasis-eliminating herbs were the most frequently used herb efficacy categories. The liver meridian represented the most frequently used herb meridian tropism. The properties and taste of herbs were mainly warm and bitter, respectively. We obtained 21 association rules and five new clusters. The Chuanxiong Rhizoma (Chuanxiong) and Angelica Sinensis Radix (Danggui) herb pair had the strongest correlation. Conclusion: We analyzed published CHM prescriptions for headaches with blood stasis syndrome and eliminated factors that did not reach an agreement, such as herb dosage. We used different data mining and analysis methods to ensure that the method and process were scientific and the conclusion was reliable, comprising a valuable reference for selecting herbs for the clinical treatment of headaches with blood stasis syndrome. The Xuefu Zhuyu Decoction (XFZYD) was the primary CHM prescription for headaches with blood stasis syndrome. Xiaoyao San (XYS) and Buyang Huanwu Decoction (BYHWD) might also be clinical references for treatment selection. Meridian-inducing and insect herbs might be used according to syndromes.

Exploring the mechanism of Taohong Siwu Decoction on the treatment of blood deficiency and blood stasis syndrome by gut microbiota combined with metabolomics.

BACKGROUND: Taohong Siwu Decoction (THSWD) is a prescription which included in the "List of Ancient Classic Prescriptions (First Batch)" issued by the National Administration of Traditional Chinese Medicine (TCM) and the National Medical Products Administration of the People's Republic of China. THSWD is effective and widely applied clinically for many diseases caused by blood deficiency and stasis syndrome in TCM, such as primary dysmenorrhea, menopausal syndrome, coronary heart disease, angina pectoris, and diabetes. METHODS: The TCM model of blood deficiency and blood stasis syndrome was prepared by ice water bath combined with cyclophosphamide, and the rats were randomly divided into control group, blood deficiency, and blood stasis model group, positive group, and THSWD treatment group. Pharmacodynamics measured the blood routine, blood coagulation, and other related indexes in rats. UHPLC-MS technology was used to analyze the changes in the fingerprints of metabolites in the plasma of rats with blood deficiency and blood stasis syndrome, and combined with mass spectrometry information and public database retrieval, to find potential biomarkers for screening metabolites. At the same time, 16S rDNA sequencing technology was used to identify intestinal flora, and statistical analysis was used to find differences in strain diversity between groups. RESULTS: THSWD administration can significantly improve the physical signs, blood routine, and hematopoietic factors caused by the blood deficiency and blood stasis syndrome model, and improve the symptoms of blood deficiency. The results of the general pharmacological studies showed THSWD groups improved changes in blood plasma viscosity and coagulation-related factors caused by modeling, and improved coagulation function significantly. The metabolomic analysis found that compared to the model group, THSWD exerted better effects on ß-alanine, taurine, L-tyrosine, L-arginine, Eugenol, sodium deoxycholate, and deethylatrazine. Twenty-three potential differential metabolites showed intervention effects, mainly involved in eight metabolic pathways, including amino acid metabolism, taurine and hypotaurine metabolism, vitamin metabolism, and nucleotide metabolism. Gut microbiota data showed that, compared to the control group, the relative abundance and value of Firmicutes and Bacteroidota of the blood deficiency and blood stasis model group was significantly reduced, while the relative abundance of Actinobacteria, Spirochaetota, Proteobacteria, Campilobacterota, and other pathogenic bacteria was significantly increased. Following THSWD intervention, the abundance of beneficial bacteria increased, and the abundance of pathogenic bacteria decreased. Correlation analysis between the gut microbiota and differential metabolites showed that the two are closely related. THSWD affected the host blood system through mutual adjustment of these two factors, and improved blood deficiency and blood stasis syndrome in rats. CONCLUSION: The blood deficiency and blood stasis syndrome model of TCM disease caused by ice bath combined with cyclophosphamide lead to changes in the pharmacology, metabolomics, and gut microbiota. The intervention of THSWD can improve the symptoms caused by blood deficiency and blood stasis. The mechanism is mainly through the regulation of platelet function and amino acid metabolism.

To explore the regulatory effect of Buyang Huanwu Decoction on cerebral infarction based on quantitative proteomics.

Buyang Huanwu Decoction (BYHW) contains chemical components such as ligustrazine, oxypaeoniflora, chlorogenic acid, and others. To explore the neuroprotective effect and potential target protein of BYHW in cerebral infarction (CI). A double-blind, randomized controlled trial was established and patients with CI were divided into the BYHW group (n = 35) and the control group (n = 30). To evaluate the efficacy by TCM syndrome score and clinical indicators, and to explore the changes of serum proteins by proteomics technology, so as to explore the mechanism of BYHW and potential target proteins. The study found that compared with the control group, the TCM syndrome score, including Deficiency of Vital Energy (DVE), Blood Stasis (BS), and NIHSS in the BYHW group decreased significantly (p < 0.05), and the Barthel Index (BI) score was significantly higher. A total of 99 differential regulatory proteins were identified by proteomics, which act on lipids and atherosclerosis, complement and coagulation cascade, and TNF-α signaling pathway. In addition, Elisa verified the results of proteomics and found that BYHW can reduce the neurological impairments focus on IL-1ß, IL-6, TNF-α, MCP-1, MMP-9, and PAI-1. Significance: In this study, quantitative proteomics was used in combination with liquid chromatography-mass spectrometry (LC-MS/MS) to study the therapeutic effect of BYHW on cerebral infarction (CI) and potential changes in serum proteomics. In addition, the public proteomics database was used for bioinformatics analysis, and Elisa experiment verified the results of proteomics, further clarifying the potential protection mechanism of BYHW on CI.

Mechanism investigation of Shi-Xiao-San in treating blood stasis syndrome based on network pharmacology, molecular docking and in vitro/vivo pharmacological validation.

ETHNOPHARMACOLOGICAL RELEVANCE: Shixiao San (SXS) is a traditional Chinese formula that has been widely used in clinical practice to treat blood stasis syndromes, such as hyperlipidemia, atherosclerotic, thrombosis and coronary heart disease. However, the effectiveness and mechanism of SXS have not been studied in detail yet. AIM OF THE STUDY: Current study aimed to identify the compounds in SXS, evaluate the formula efficacies using network pharmacology, molecular docking, and verify the pharmacological effects by in vivo and in vitro experiments. MATERIALS AND METHODS: The compounds in SXS were analyzed using UPLC-QTOF-MS. Potential target genes for identified compounds were obtained from three databases. DAVID database was used to perform GO and KEGG pathway enrichment analyses. PPI network was constructed to screen core targets. Molecular docking was used to examine interactions between active compounds and potential targets. The mechanism was also verified by model of acute blood stasis rats and human umbilical vein cells. RESULTS: In total, 45 compounds were identified from SXS. Among the detected phytochemicals, quercetin, isorhamnetin, kaempferol, D-catechin, naringenin and amentoflavone were identified as the active constituents. SXS is primarily involved in the modulation of hypoxic state, vascular regulation, and inflammation response, according to GO and KGG pathway enrichment analysis. A network of protein-protein interactions (PPIs) was constructed and five core targets were identified as VEGFA, AKT1, EGFR, PTGS2, and MMP9. Molecular docking simulation revealed good binding affinity of the five putative targets with the corresponding compounds. SXS reduced HIF-1α and COX-2 levels and increased the eNOS expression levels in hypoxic HUVECs. SXS can reduce the whole blood viscosity in adrenaline induced acute blood stasis rats and relieve blood stasis. CONCLUSIONS: SXS removes blood stasis might through VEGFA/AKT/eNOS/COX-2 pathway and flavonoids are the main active components in the formula.

Psoriasis complicated with metabolic disorder is associated with traditional Chinese medicine syndrome types: a hospital-based retrospective case-control study.

OBJECTIVE: To explore the distribution law of traditional Chinese medicine (TCM) syndrome types in patients with psoriasis vulgaris complicated by metabolic disorders based on the same pathogenic factors as blood-heat and blood-stasis in the pathogenesis of psoriasis and metabolic disorders and to further analyze the correlation between adiponectin and the distribution law. METHODS: From 1 January 2018 to 31 December 2019, patients diagnosed with psoriasis in the inpatient or outpatient department of Dermatology Ward of Shanghai Yueyang Hospital and normal participants who underwent physical examination in the physical examination center over the same period were retrospectively reviewed. Demographic data, medical history, metabolic disorder indices, and TCM syndrome indices of psoriasis patients and healthy volunteers were evaluated. RESULTS: We included 307 patients with psoriasis and 613 healthy controls. On analyzing past medical history, the proportion of overweight and obesity and the comorbidity of diabetes in the psoriasis group (53.42 and 14.66%) were significantly higher than in the control group (43.88 and 7.67%, respectively; p < .05). The abnormal rates of triglyceride (34.20%), high-density lipoprotein cholesterol (50.49%), and HbA1c (18.57%) levels in the psoriasis group were higher than those in the normal control group (26.75, 17.13, and 12.56%, respectively). Overall, the incidence of metabolic disorders in psoriasis patients (267/307, 86.97%) was higher than that in the normal controls (484/613, 78.96%). Among the different syndrome types, the blood-stasis group had significantly higher rates of hypertension, diabetes, and abnormal glycosylated hemoglobin (46.07, 19.10, and 24.72%, respectively) than those of the control group (27.57, 7.67, and 12.56%; p < .05). Patients with blood stasis syndrome had the highest metabolic disorder comorbidity rate (93.26%) and lowest adiponectin level (p < .05). CONCLUSIONS: TCM syndrome differentiation of psoriasis, especially the diagnosis of blood-stasis syndrome, prompts the early screening of patients with metabolic comorbidities. For patients with psoriasis with metabolic disorder, TCM for promoting blood circulation and removing blood stasis can be compatibly applied without contraindications. TRIAL REGISTRATION: The trial was registered at ClinicalTrials.gov (Trial ID: NCT03942185).

Label-free based quantitative proteomics analysis to explore the molecular mechanism of gynecological cold coagulation and blood stasis syndrome.

Cold coagulation and blood stasis (CCBS) syndrome is one of the common traditional Chinese medicine (TCM) syndromes of gynecological diseases. However, the molecular mechanism of CCBS syndrome is still unclear. Thus, there is a need to reveal the occurrence and regulation mechanism of CCBS syndrome, in order to provide a theoretical basis for the treatment of CCBS syndrome in gynecological diseases. The plasma proteins in primary dysmenorrhea (PD) patients with CCBS syndrome, endometriosis (EMS) patients with CCBS syndrome, and healthy women were screened using Label-free quantitative proteomics. Based on the TCM theory of "same TCM syndrome in different diseases," the differentially expressed proteins (DEPs) identified in each group were subjected to intersection mapping to obtain common DEPs in CCBS syndrome. The DEPs of gynecological CCBS syndrome in the intersection part were again cross-mapped with the DEPs of gynecological CCBS syndrome obtained by the research group according to the TCM theory of "different TCM syndromes in same disease" theory in the early stage, so as to obtain the DEPs of gynecological CCBS syndrome that were shared by the two parts. The common DEPs were subjected to bioinformatics analysis, and were verified by enzyme-linked immunosorbent assay (ELISA). A total of 67 common DEPs were identified in CCBS syndrome, of which 33 DEPs were upregulated and 34 DEPs were downregulated. The functional classification of DEPs involved in metabolic process, energy production and conversion, immune system process, antioxidant activity, response to stimulus, and biological adhesion. The subcellular location mainly located in the cytoplasm, nucleus, and extracellular. Gene ontology (GO) enrichment analysis showed that the upregulated DEPs mainly concentrated in lipid transport, cell migration, and inflammatory reaction, and the downregulated DEPs mostly related to cell junction, metabolism, and energy response. Protein domain enrichment analysis and clustering analysis revealed that the DEPs mainly related to cell proliferation and differentiation, cell morphology, metabolism, and immunity. The Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis clustering analysis showed that the upregulated DEPs were involved in inflammation and oxidative damage, while the downregulated DEPs were involved in inflammation, cell adhesion, cell apoptosis, and metabolism. The results of ELISA showed significantly increased levels of Cell surface glycoprotein MUC18 (MCAM) and Apolipoprotein C1 (APOC1), and significantly decreased levels of Vasodilator-stimulated phosphoprotein (VASP), Fatty acid-binding protein 5 (FABP5), and Vinculin (VCL) in patients with CCBS syndrome compared with healthy women. We speculated that cold evil may affect the immune process, inflammatory response, metabolic process, energy production and conversion, oxidative damage, endothelial cell dysfunction, and other differential proteins expression to cause CCBS syndrome in gynecological diseases.

[Mechanism of Danggui Sini Decoction in improving kidney injury caused by blood stasis syndrome based on metabolomics and network pharmacology].

This article analyzed the mechanism of Danggui Sini Decoction(DSD) in improving kidney injury caused by blood stasis syndrome(BSS) in rats. Firstly, 32 female SD rats were randomly divided into the following four groups: a normal group and a BSS group, both receiving an equal amount of distilled water by gavage; a normal+DSD group and a BSS+DSD group, both receiving 5.103 g·kg~(-1) DSD orally for a total of 14 days. Daily cold water bath was given to establish the BSS model, and on the 14th day, BSS rats were subcutaneously injected with 0.8 mg·kg~(-1) adrenaline. Normal rats were subjected to the water bath at 37 ℃ and injected with an equal volume of distilled water. After the experiment, 24-hour urine, serum, and kidney samples were collected for metabolomic analysis, biochemical measurements, and hematoxylin-eosin(HE) staining. The study then employed ~1H-NMR metabolomic technology to reveal the metabolic network regulated by DSD in improving BSS-induced kidney injury and used network pharmacology to preliminarily elucidate the key targets of the effectiveness of DSD. Pathological and biochemical analysis showed that DSD intervention significantly reduced inflammation and abnormal levels of blood creatinine, blood urea nitrogen, and urine protein in the kidneys. Metabolomic analysis indicated that DSD attenuated BSS-induced kidney injury primarily by regulating 10 differential metabolites and three major metabolic pathways(taurine and hypotaurine metabolism, citrate cycle, and acetaldehyde and dicarboxylic acid metabolism). Network pharmacology analysis suggested that the protective effect of DSD against BSS-induced kidney injury might be related to two key genes, ATP citrate lyase(ACLY) and nitric oxide synthase 2(NOS2), and two main metabolic pathways, i.e., arginine biosynthesis, and arginine and proline metabolism. This study, from the perspective of network regulation, provides initial insights and evidence into the mechanism of DSD in improving kidney injury induced by BSS, offering a basis for further investigation into the molecular mechanisms underlying its efficacy.

Study on the regular pattern of "disease-syndrome-medicine" of different diseases with the same treatment for blood stasis syndrome based on complex network similarity analysis / 国际中医中药杂志

Objective:To reveal the regular pattern characteristics of different diseases with the same treatment in the most common diseases with blood stasis syndrome; To provide reference for the clinical treatment of blood stasis syndrome and the development of new drugs.Methods:RCTs of blood stasis syndrome were retrieved from CNKI, Chongqing VIP, Wanfang Data, SinoMed, and China Medical Journal Full-text Database from the establishment of the databases to December 31, 2022. Diseases, accompanied symptoms, prescriptions and medicines were extracted. The diseases with the highest frequency among the three disease systems with the highest frequency were collected, and their medication characteristics and prescription rules were analyzed using frequency statistics and association rules Apriori algorithm. The core prescriptions of blood stasis syndrome of three kinds of diseases were excavated and their network similarity was analyzed.Results:A total of 2 052 articles were included. Stable coronary heart disease, ischemic stroke and DN were more common diseases with blood stasis syndrome. The common drugs for the three diseases were Chuanxiong Rhizoma, Carthami Flos, Persicae Semen, Angelicae Sinensis Radix. The core prescription of stable coronary heart disease was Persicae Semen- Carthami Flos- Chuanxiong Rhizoma- Angelicae Sinensis Radix- Paeoniae Radix Rubra; the core prescription of ischemic stroke is Buyang Huanwu Decoction; the core prescription of DN was Persicae Semen- Carthami Flos- Chuanxiong Rhizoma- Angelicae Sinensis Radix- Cornus Officinalis- Dioscoreae Rhizoma- Astragali Radix. The similarity between stable coronary heart disease and ischemic stroke core prescription network was 0.35, the similarity between ischemic stroke and DN core prescription network was 0.29, and the similarity between stable coronary heart disease and DN core prescription network was 0.26. Conclusions:The theory of "different diseases with the same treatment" can profoundly guide clinical practice. The core medicines of blood stasis syndrome are Persicae Semen, Carthami Flos, Angelicae Sinensis Radix, and Chuanxiong Rhizoma. On this basis, combined with different diseases and syndromes to make changes of adding and subtracting.

Differential expression profiles analysis of DNA methylation between “disease” and “syndrome” in coronary heart disease-induced unstable angina patientswith Qi deficiency and blood stasis syndrome / 数字中医药（英文）

Objective@#To explore the differential expression profiles of DNA methylation sites/regions and potential molecular mechanisms in the peripheral blood of coronary heart disease (CHD)-induced unstable angina pectoris patients with or without Qi deficiency and blood stasis syndrome, and to provide scientific evidence for the conbination of disease and syndrome.@*Methods@#According to the pre-determined inclusion and exclusion criteria, the study subjects were enrolled and divided into two groups namely CHD-induced unstable angina group (G group) and healthy control group (J group) to conduct “disease” analysis, while G group was further divided into Qi deficiency and blood stasis syndrome group (case group) and non-Qi deficiency blood stasis syndrome group (control group) to perform “syndrome” analysis. The general data and clinical information of the study subjects were collected. The peripheral venous blood was extracted on an empty stomach, and the Illumina Infinium MethylationEPIC BeadChip (850K methylation chip) was used to detect the differential expressionprofiles of DNA methylation in each group, ChAMP software (V 2.14.0) was used for the differential methylation data analysis, with a threshold of the adjusted P value (adj.P.val) < 0.01. Gene Ontology (GO) and Kyoto Encyclopedia of Genomes (KEGG) were employed for the functional and pathway enrichment analyses of related mapped genes.@*Results@#A total of 263 differentially methylated CpG positions (DMPs) were screened out between G and J groups, including 191 hypermethylated positions such as cg05845204 and cg08906898, and 72 hypomethylated positions such as cg26919182 and cg13149459. These positions were mainly mapped to 148 genes encompassing RNA binding motif protein 39 (RBM39), acetyl-CoA acyltransferase 2 (ACAA2), protein phosphatase 1 regulatory subunit 12B (PPP1R12B), and the dual-specificity tyrosine phosphorylation-regulated kinase 2 (DYRK2). GO functional enrichment analysis revealed that the genes of the DMPs were primarily enriched in protein localization to chromosomes, regulation of cell morphogenesis, negative regulation of calcium-mediated signals, etc. KEGG pathway analysis suggested that the genes were mainly enriched in fatty acid metabolism and endocytosis pathways. In addition, a total of 23 differential methylation regions (DMRs) were identified, with overlapping genes such as transmembrane protein 232 (TMEM232), ribosomal protein large P1 (RPLP1), peroxisomal biogenesis factor 10 (PEX10), and forkhead box N3 (FOXN3) recognized. It was found that GO functions were mainly enriched in the negative regulation of Ras protein signal transduction, small GTPase-mediated signal transduction, negative regulation, etc. A total of 1 703 differential methylation sites were screened out between case and control groups, including 444 increased methylation positions such as cg05573767 and 1 259 decreased methylationpositions such as cg19938535, and cg03893872. These positions were mapped to 1 108 genes such as ribosomal protein S6 kinase A2 (RPS6KA2), leucine rich repeat containing 16A (LRRC16A), and hedgehog acyltransferase (HHAT). According to the GO functional enrichment analysis, the genes relating to the DMPs were mainly enriched in biological functions such as transmembrane receptor protein serine/threonine kinase signaling pathway and axonogenesis. The KEGG pathway enrichment analysis suggested the involvement of Rap1 signaling pathway, adenosine 5’-monophosphate-activated protein kinase (AMPK) signaling pathway, etc. A total of 21 DMRs were identified, including 22 overlapping genes such as mucin 4 (MUC4), three prime repair exonuclease 1 (TREX1), and LIM homeobox 6 (LHX6). GO analysis demonstrated that the genes primarily participated in molecular functions such as positive regulation of transmembrane transport, regulation of fatty acid metabolism, and copper ion binding.@*Conclusion@#This study reveals the methylation patterns of DMPs and DMRs in patients with Qi deficiency and blood stasis syndrome caused by CHD-induced unstable angina pectoris. Potential epigenetic regulation of fatty acid metabolism, Rap1 signaling, and other molecular functions are involved in the development of CHD between the "disease" and "syndrome".

Exploring biological connotation of blood stasis syndrome of rheumatoid arthritis and establishment of improved animal models based on syndrome-symptom mapping / 药学学报

Blood stasis syndrome is one of the core clinical syndrome of rheumatoid arthritis (RA), but the biological connotation of this syndrome is not clear, and there is a lack of disease improved animal models that match the characteristics of this disease and syndrome. The aim of this study was to screen the candidate biomarker gene set of blood stasis syndrome of RA, reveal the biological connotation of this syndrome, and explore and evaluate the preparation method of the improved animal model based on the characteristics of "disease-syndrome-symptom". The study was approved by the ethics committee of Guang'anmen Hospital, Chinese Academy of Traditional Chinese Medicine (No. 2019-073-KY-01) and the First Affiliated Hospital of Tianjin University of Traditional Chinese Medicine (No. TYLL2021[K]018), and the study subjects gave their informed consent. Animal welfare and experimental procedures followed the regulations of the Experimental Animal Ethics Committee of the Chinese Academy of Traditional Chinese Medicine (No. IBTCMCACMS21-2207-01). The whole blood samples were collected clinically from RA patients with blood stasis syndrome (3 cases) or other syndromes (7 types, 3 cases/type), and healthy volunteers (4 cases), and then transcriptome sequencing, KEGG, gene set enrichment analysis (GSEA) and weighted correlation network analysis (WGCNA) analysis were performed. 126 pivotal genes were screened, and their functional annotation results were significantly enriched in "immune-inflammation" related pathways and lipid metabolism regulation (sphingolipids, ether lipid metabolism and steroid biosynthesis). Syndrome-symptom mapping of hub gene set to the TCM primary and secondary symptoms, Western phenotypic symptoms and pathological links showed that joint tingling, abnormal joint morphology, petechiae and abnormal blood circulation are representative of blood stasis syndrome of RA. The results of the improved animal model showed that the rats in the collagen-induced arthritis + adrenaline hydrochloride (CIA+Adr) 3 model group had increased blood rheology, coagulation, platelet function and endothelial function abnormalities compared with the CIA-alone model group, suggesting that the rats with blood stasis syndrome of RA may be in a state of "blood stasis". The results of the study can help to advance the objective study of the evidence of blood stasis syndrome in RA, and provide new ideas for the establishment of an animal model that reflects the clinical characteristics of the disease and syndrome.

Mechanisms of Yangxin Tongmai Formula for blood stasis syndrome in coronary heart disease rats based on untargeted plasma metabolomics and intestinal flora 16S rRNA sequencing / 数字中医药（英文）

@#【Objective】  To investigate the correlations between intestinal flora, plasma metabolites, and blood stasis syndrome in coronary heart disease (CHD), and the mechanisms of Yangxin Tongmai Formula (养心通脉方, YXTMF) for blood stasis syndrome in CHD rats. 【Methods】  A total of 18 specific pathogen free (SPF) male Sqrague-Dawley (SD) rats were used to establish CHD rat models with blood stasis syndrome, which were then randomized into model, YXTMF, and atorvastatin calcium (AVT) groups, with six rats in each group, and were intervened through gavage for two weeks. Subsequently, additional six rats that received normal diet were included as normal group. The pathological changes in the CHD rat models were identified by hematoxylin-eosin (HE) staining. The electrocardiogram, hemodynamics, and lipid profiles of the rats were detected as well. The untargeted plasma metabolomics of rats were analyzed by liquid chromotography-tandem mass spectrometry (LC-MS/MS), their ileal mucosal flora by 16S rRNA sequencing, and the correlation between the two results were also analyzed. 【Results】  The whole blood viscosity, total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-C) of rats in the model group increased compared with those in the control group (P < 0.05). In the model group, the proliferation of endothelial cells in the coronary artery of rats was damaged, with quite a few vacuolated pathological changes observed. However, the endothelial lesions in the coronary artery of rats were alleviated in the intervention groups (YXTMF and AVT groups). With the use of  LC-MS/MS, a total of 33 potential endogenous metabolites were identified in plasma, among which 1-methylhistidine, N-acetylhistamine, progesterone, and deoxycorticosterone were expected to be the differential metabolites in CHD rats with blood stasis syndrome. The 16S rRNA sequencing results showed that improved diversity and abundance of intestinal flora were observed in the YXTMF group. The correlation analysis suggested that Hydrogenophaga, Limnohabitans, and Polaromonas, which were highly related to the formation of blood stasis syndrome in CHD patients, were positively correlated with plasma metabolites such as 5-hydroxyindole, N-acetylhistamine, and progesterone (P < 0.01), but were negatively correlated with plasma metabolites such as L-arginine, homoarginine, and Boc-beta-cyano-L-alanine (P < 0.01). After YXTMF intervention, Lactobacillus, Corynebacterium, and Candidatus Nitrososphaera were positively correlated with plasma metabolites such as Boc-β-cyano-L-alanine, stachydrine, and naringenin (P < 0.05), while negatively correlated with 5-hydroxyindole, N-acetylhistamine, and oleoylethanolamide (P < 0.05). 【Conclusion】  YXTMF could alleviate blood stasis syndrome in CHD rats through improving their plasma metabolisms achieved by regulating the intestinal flora.