Zixue Powder attenuates septic thrombosis via reducing neutrophil extracellular trap through blocking platelet STING activation.

ETHNOPHARMACOLOGICAL RELEVANCE: Microthrombosis is commonly seen in sepsis and COVID-19. Zixue Powder (ZXP) is a traditional Chinese herbal formula with the potential to treat microvascular and infectious diseases. However, the role and mechanism of ZXP in sepsis-associated thrombosis remain unclear. AIM OF THE STUDY: Investigating the therapeutic effectiveness and underlying mechanisms of ZXP in septic thrombosis. MATERIALS AND METHODS: ZXP's compositions were examined with UPLC-QTOF-MS. The efficacy of ZXP on sepsis-induced thrombosis was assessed through various methods: liver tissue pathology was examined using hematoxylin-eosin staining, platelet count was determined by a blood cell analyzer, and an enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of serum tissue factor (TF), thromboxane B2 (TXB2), D-Dimer, and plasminogen activator inhibitor-1 (PAI-1). Neutrophil extracellular traps (NETs) were localized and expressed in liver tissues by immunofluorescence, and the number of NETs in peripheral blood was evaluated by ELISA, which measured the quantity of cf-DNA and MPO-DNA in serum. Platelet P-selectin expression and platelet-neutrophil aggregation were measured by flow cytometry, and plasma P-selectin expression was measured by ELISA. Furthermore, the mechanism of the stimulator of interferon genes (STING) signaling pathway in ZXP's anti-sepsis thrombosis effect was investigated using the STING agonist, Western blot experiments, and immunoprecipitation experiments. RESULTS: UPLC-QTOF-MS identified 40 chemical compositions of ZXP. Administration of ZXP resulted in significant improvements in liver thrombosis, platelet counts, and levels of TXB2, TF, PAI-1, and D-Dimer in septic rats. Moreover, ZXP inhibited NETs formation in both liver tissue and peripheral blood. Additionally, ZXP decreased the levels of P-selectin in both platelets and plasma, as well as the formation of platelet-neutrophil aggregates, thereby suppressing P-selectin-mediated NETs release. Immunoprecipitation and immunofluorescence staining experiments revealed that ZXP attenuated P-selectin secretion by inhibiting STING-mediated assembly of platelet soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) complex, ultimately preventing inhibition of NETs formation. CONCLUSION: Our study showed that ZXP effectively mitigates platelet granule secretion primarily through modulation of the STING pathway, consequently impeding NET-associated thrombosis in sepsis. These findings offer valuable insights for future research on the development and application of ZXP.

Impact of Lactiplantibacillus plantarum and casein fortification on angiotensin converting enzyme inhibitory peptides in yogurt: identification and in silico analysis.

In this study, the effects of Lactiplantibacillus plantarum M11 (Lb. plantarum M11) in conjunction with sodium caseinate on the characteristics and angiotensin converting enzyme (ACE) inhibitory activity of yogurt were investigated. ACE inhibitory peptides (ACEIPs) in yogurt were identified by nano-LC-MS/MS and potential ACEIPs were predicted by in silico and molecular docking methods. The results showed that the ACE-inhibitory activity of yogurt was significantly enhanced (p < 0.05), while maintaining the quality characteristics of the yogurt. Thirteen ACEIPs in the improved yogurt (883 + M11-CS group) were identified, which were more abundant than the other yogurt groups (control 883 group, 883 + M11 group and 883-CS group). Two novel peptides with potential ACE inhibitory activity, YPFPGPIH and NILRFF, were screened. The two peptides showed PeptideRanker scores above 0.8, small molecular weight and strong hydrophobicity, and were non-toxic after prediction. Molecular docking results showed that binding energies with ACE were -9.4 kcal mol-1 and -10.7 kcal mol-1, respectively, and could bind to the active site of ACE. These results indicated that yogurt with Lb. plantarum M11 and sodium caseinate has the potential to be utilized as a functional food with antihypertensive properties. The combination of ACEIP-producing strains and casein fortification could be an effective method to promote the release of ACEIPs from yogurt.

The material basis of bitter constituents in Carbonized Typhae Pollen, based on the integration strategy of constituent analysis, taste sensing system and molecular docking.

The discovery of bitter constituents is of great significance to the exploration of medicinal substances for they have potential physiological effects. Carbonized Typhae Pollen (CTP), which is a typical example of carbonized Traditional Chinese Medicine (TCM), has a bitter taste and hemostatic effect after carbonized processing. The objective of this study is to elucidate the material basis of bitter constituents in CTP. Firstly, the constituents of CTP extracts with 7 different solvents were characterized by UPLC-Q-TOF-MS. Then, multivariate statistical analysis was used to visualize the CTP extracts from 7 solvents. A total of 37 constituents were tentatively identified and 17 constituents were considered as the key constituents in differentiating 7 different solvent extracts. Subsequently, the bitter evaluation of extracts from different polar parts was investigated by using an electronic tongue. As a result, the order of bitterness of the extracts was as follows: ethanol > methanol > water > n-butyl alcohol > petroleum ether > butyl acetate > isopropanol. There were statistically significant differences in the bitter degree of extracts. By correlation analysis of bitter information and chemical constituents with partial least squares regression (PLSR), 8 potential bitterness constituents were discovered, including phenylalanine, valine, chlorogenic acid, isoquercitrin, palmitic acid, citric acid, quercetin-3-O-(2-α-L-rhamnosyl)-rutinoside, and typhaneoside. Additionally, molecular docking analysis was conducted to reveal the interaction of these constituents with the bitter taste receptor. The docking result showed that these constituents could be embedded well into the active pocket of T2R46 and had significant affinity interactions with critical amino acid residues by forming hydrogen bonds. This study provided a reliable theoretical basis for future research on biological activity of bitterness substances.

Identification and In Silico Analysis of ACE-Inhibitory Peptides Derived from Milk Fermented by Lacticaseibacillus paracasei.

Inhibition of angiotensin I-converting enzyme (ACE) activity is an effective way to treat hypertension. In the present study, the ability to produce ACE-inhibitory peptides during fermentation of skimmed milk by the Lacticaseibacillus paracasei M3 strain was evaluated, and the inhibitory mechanism and stability were studied by bioinformatics analysis. The results showed that the ACE inhibition activity of fermented milk was 71.94 ± 1.39%. After digestion with gastric juice and pancreatic juice, the ACE inhibitory activities of the fermented milk were 78.40 ± 1.93 and 74.96 ± 1.73%, respectively. After the fermented milk was purified using ultrafiltration and gel chromatography, 11 peptides from milk proteins were identified and sequenced by Nano LC-MS/MS. Molecular docking displayed that peptide PWIQPK had a high affinity, with ACE showing a binding energy of -6.10 kcal/mol. Hydrogen bonds were formed between PWIQPK and Glu384 in the S1 active pocket of ACE and Asp358. In addition, van der Waals forces were observed. In silico proteolysis suggested that PWIQPK could resist the digestion of pepsin and trypsin, indicating that it is relatively stable in the digestive tract. All results indicate that milk fermented by L. paracasei M3 has the potential to be used as a functional food having antihypertensive effects.

[Effect of Qishen Yiqi droplet on adenylate contents in myocardium of cor pulmonale rats].

Cor pulmonale rat models were induced by a single intraperitoneal injection of monocrotaline(MCT), and the sham group received a single intraperitioneal injection of normal saline. After the model rats received intragastric administration of Qishen Yiqi droplet(QS) for 6 weeks, the contents of adenylate(ATP, ADP and AMP) in right myocardial tissues were measured by HPLC, and then the metabolism changes in myocardium of cor pulmonale rats with QS were investigated. The results showed that ATP, ADP, and AMP were well separated, with a good linearity within a certain range of concentration; and the recovery rates were within the range of 90%-108%. As compared with model group, the level of ATP was significantly elevated in high-dose treatment group; ADP contents showed an increasing trend and AMP contents showed a decreasing trend, indicating that QS could significantly improve energy metabolism system in myocardium. By using the HPLC, a qualitative and quantitative analysis method was given for the determination of ATP, ADP and AMP contents in myocardium, providing a method for energy metabolism measurement in biological samples.

Reversal of muscle atrophy by Zhimu and Huangbai herb pair via activation of IGF-1/Akt and autophagy signal in cancer cachexia.

PURPOSE: Muscle atrophy is the prominent clinical feature of cancer-induced cachexia. Zhimu and Huangbai herb pair (ZBHP) has been used since ancient China times and have been phytochemically investigated for constituents that might cause anti-cancer, diabetes, and their complication. In this study, the effects and mechanisms of ZBHP on reversal of muscle atrophy were explored. METHODS: C57BL/6 mice implanted with colon-26 adenocarcinoma were chosen to develop cancer cachexia for evaluating the effects of ZBHP on reversal of muscle atrophy. The body weight, survival time, inflammatory cytokines, and pathological changes of muscle were monitored. In addition, IGF-1/Akt and autophagy pathway members were analyzed to interpret the mechanism of drug response. RESULTS: The function and morphology of skeletal muscle in cachexia model were significantly disturbed, and the survival time was shortened. Consistently, inflammatory cytokines and muscle atrophy-related atrogin-1, MuRF1, and FOXO3 were significantly increased, and IGF-1/Akt and autophagy signal pathways were depressed. Treatment with ZBHP significantly alleviated tumor-free body weight reduction and cachexia-induced changes in cytokines and prolonged survival. ZBHP treatment not only inhibited the muscle atrophy-related genes but also activated the IGF-1/Akt and autophagy signal pathways to facilitate the protein synthesis. CONCLUSIONS: The results revealed that ZBHP treatment could inhibit the muscle atrophy induced by cancer cachexia and prolong the survival time, and ZBHP may be of value as a pharmacological alternative in treatment of cancer cachexia.

Protective effect of Suxiao jiuxin pill, a traditional Chinese medicine, against acute myocardial ischemia in dogs.

BACKGROUND: The purpose of this study was to investigate the effect of Suxiao Jiuxin Pill (SX), a traditional Chinese medicine, on acute myocardial ischemia induced by coronary occlusion in anesthetized dogs. METHODS: Acute myocardial ischemia model was established by ligating the left anterior descending artery to reduce flow by 90 %. Adult mongrel dogs were randomly divided into six groups: model, SX high dose, SX middle dose, SX low dose, Isosorbide dinitrate (ISD) and Sham groups. Adult mongrel dogs were anesthetized and instrumented for measurements of heart rate (HR), mean arterial pressure (MAP), left ventricular systolic pressure (LVSP), left ventricular dP/dt, coronary blood flow (CBF), myocardial blood flow (MBF), coronary vascular resistance (CVR), and epicardial electrocardiogram (EECG). After administration with SX, changes in hemodynamics were recorded. Serum enzymes and blood gas analysis were also detected. RESULTS: SX has a dose-dependent effect on the reduction of infarct size. Besides, SX exerted a notable inhibition on the elevation of serum creatine kinase MB (CK-MB), lactate dehydrogenase (LDH), malondialdehyde (MDA), and elevation in the superoxide dismutase (SOD) activity. SX also showed a capacity to recover myocardial function by significantly reducing MAP, CVR, LVSP, left ventricular systolic pressure (LVEDP), systolic blood pressure (SBP), diastolic blood pressure (SDP), and increasing CBF and myocardial blood flow (MBF). In addition, SX high dose group markedly reduced total mV of ST segment elevation (Σ-ST), total number of sites with this degree of ST segment elevation (N-ST) and oxygen extraction ratio (O2 Extr). CONCLUSION: SX can improve hemodynamic and myocardial oxygen metabolism, reduce the degree and scope of myocardial ischemia, and hence exert notable anti-anginal ischaemic effect.

The efficacy of Jing Wan Hong ointment for nerve injury diabetic foot ulcer and its mechanisms.

Jing Wan Hong ointment contains 30 kinds of Chinese herbs, with functions of activating blood circulation to disperse blood stasis, clearing heat, eliminating dampness, and reducing swelling by detoxification. Therefore, Jing Wan Hong ointment may facilitate the healing of ulcers. The aim of this study was to evaluate the efficacy and mechanisms of Jing Wan Hong ointment for healing diabetic foot ulceration in Wistar rats induced by streptozotocin and sciatic nerve damage. The results showed that Jing Wan Hong ointment had a marked effect on foot ulcers in diabetic rats induced by initial nerve injury. These effects were manifested by reducing the foot ulcer size and Wagner grade after seven days of treatment. The diabetic rats with foot ulcers were almost healed after 21 days of treatment. Moreover, the mechanisms of this effect seem to be dependent on increased expression of PDGF mRNA, but there was no influence on the expression of TGF-ß, VEGF, and FLT-1 mRNA.

Reversal of muscle atrophy by Zhimu-Huangbai herb-pair via Akt/mTOR/FoxO3 signal pathway in streptozotocin-induced diabetic mice.

Skeletal muscle atrophy is one of the serious complications of diabetes. Zhimu-Huangbai herb-pair (ZB) is widely used in Chinese traditional medicine formulas for treating Xiaoke (known as diabetes) and its complications. However, the effect of ZB on reversal of muscle atrophy and the underlying mechanisms remain unknown. In this research, we investigated the effect and possible mechanisms of ZB on skeletal muscle atrophy in diabetic mice. Animal model of diabetic muscle atrophy was developed by high fat diet (HFD) feeding plus streptozotocin (STZ) injection. After oral adminstration of ZB for 6 weeks, the effects of ZB on reversal of muscle atrophy and the underlying mechanisms were evaluated by biochemical, histological and western blot methods. The skeletal muscle weight, strength, and cross-sectional area of diabetic mice were significantly increased by ZB treatment. Biochemical results showed that ZB treatment reduced the serum glucose level, and elevated the serum insulin-like growth factor 1 (IGF-1) and insulin levels significantly compared with untreated diabetic group. The western blot results showed that ZB activated the mTOR signal pathway, shown as increased phosphorylations (p-) of Akt, mTOR, Raptor, S6K1 and reduced Foxo3 expression compared with the model group. ZB could reverse muscle atrophy in diabetic mice. This may be through activation of mTOR signaling pathway that promotes protein synthesis, and inactivation foxo3 protein that inhibits protein degradation. These findings suggested that ZB may be considered as a potential candidate drug in treatment of diabetic muscle atrophy.

Suxiaojiuxin pill enhances atherosclerotic plaque stability by modulating the MMPs/TIMPs balance in ApoE-deficient mice.

: Suxiaojiuxin pill (SX) is a famous Chinese formulated product, which has been used to treat coronary heart disease and angina pectoris in China. This study was carried out to investigate the effect and possible mechanism of SX on the stability of atherosclerotic plaque in ApoE-deficient mice. ApoE-/- mice of 6-8 weeks old were fed with high-fat diet for developing artherosclerosis. After oral administration of SX for 8 weeks, histopathology of aortic plaque was performed by Sudan III and hematoxylin-eosin staining, and muscle protein was detected by Western blotting (WB). The mRNA and proteins associated with aortic plaque stability were detected by reverse transcription-polymerase chain reaction and WB, respectively. SX treatment could not only reduce serum triglyceride level and plaque area but also increase fibrous cap thickness and collagen content compared with the model group. WB results showed that SX could increase α-smooth muscle actin, tissue inhibitor of metalloproteinase 1 (TIMP-1), and TIMP-2 protein expression, whereas decrease matrix metalloproteinase 2 (MMP-2) and MMP-9 protein expression. Moreover, SX could upregulate the expression of α-smooth muscle actin mRNA and downregulate the expression of vascular endothelial growth factor mRNA. These results showed that SX could enhance atherosclerotic plaque stability in ApoE-deficient mice. The mechanism may be associated with modulating the MMPs/TIMPs balance.

The efficacy of betulinic acid in triple-negative breast cancer.

PURPOSE: The treatment of triple-negative breast cancer remains a daunting challenge with the standard-of-care treatments eventually failing due to acquired drug resistance, toxic side effects and the presence of a deregulated immune response. New treatments for overcoming these drawbacks include the use of plant extracts. STUDY DESIGN: In this study, the efficacy of betulinic acid, a naturally abundant phytochemical exhibiting anti-inflammatory and anti-proliferative activity, has been evaluated for the treatment of triple-negative breast cancer MDA-MB-231 and MDA-MB-468 cell lines. Furthermore, the ability of betulinic acid to inhibit angiogenesis was also determined. RESULTS: Here, we report that betulinic acid was able to inhibit the inflammatory response, inhibit angiogenesis and cause cell cycle arrest ultimately causing apoptosis in triple-negative breast cancer cells. Our findings support that the identification of naturally occurring anti-tumour compounds may provide a chemotherapeutic approach for the treatment of triple-negative breast cancer. CONCLUSION: Overall, our results provide a molecular basis for the ability of betulinic acid to mediate apoptosis, suppress inflammation and inhibit angiogenesis in triple-negative breast cancer cell lines.

Baicalin regulates neuronal fate decision in neural stem/progenitor cells and stimulates hippocampal neurogenesis in adult rats.

BACKGROUND: Recent studies revealed that baicalin, a flavonoid compound derived from the root of Scutellaria baicalensis Georgi, could promote neuron differentiation of NSPCs after commencing the differentiation process in vitro. However, this may not be the most efficacious strategy to determinate cell fate. Here, we have investigated whether baicalin can influence early events of neuron generation and stimulate adult neurogenesis. RESULTS: Transient exposure of NSPCs to baicalin during proliferation could activate Mash1 to alter the differential fate and increase the proportion of cells expressing neuronal markers. Seven days after, rats were exposed to transient cerebral ischemia, they were treated for 3 weeks with baicalin, BrdU labeling study showed that exposure to baicalin increased the number of newly generated cells in hippocampus, BrdU/NeuN double staining analysis indicated that baicalin could promote new neuron production after cerebral ischemia. Additionally, Morris water maze test showed that delayed postischemic treatment with baicalin improved cognitive impairment. CONCLUSIONS: These results identify the existence of a single molecule, baicalin, which can specify the neuronal fate of multipotent NSPCs and stimulate neurogenesis, making it a promising candidate for developing clinically relevant strategies to manipulate neuronal fate of NSPCs for brain repair.

Direct stimulation of adult neural stem/progenitor cells in vitro and neurogenesis in vivo by salvianolic acid B.

BACKGROUND: Small molecules have been shown to modulate the neurogenesis processes. In search for new therapeutic drugs, the herbs used in traditional medicines for neurogenesis are promising candidates. METHODOLOGY AND PRINCIPAL FINDINGS: We selected a total of 45 natural compounds from Traditional Chinese herbal medicines which are extensively used in China to treat stroke clinically, and tested their proliferation-inducing activities on neural stem/progenitor cells (NSPCs). The screening results showed that salvianolic acid B (Sal B) displayed marked effects on the induction of proliferation of NSPCs. We further demonstrated that Sal B promoted NSPCs proliferation in dose- and time-dependent manners. To explore the molecular mechanism, PI3K/Akt, MEK/ERK and Notch signaling pathways were investigated. Cell proliferation assay demonstrated that Ly294002 (PI3K/Akt inhibitor), but neither U0126 (ERK inhibitor) nor DAPT (Notch inhibitor) inhibited the Sal B-induced proliferation of cells. Western Blotting results showed that stimulation of NSPCs with Sal B enhanced the phosphorylation of Akt, and Ly294002 abolished this effect, confirming the role of Akt in Sal B mediated proliferation of NSPCs. Rats exposed to transient cerebral ischemia were treated for 4 weeks with Sal B from the 7th day after stroke. BrdU incorporation assay results showed that exposure Sal B could maintain the proliferation of NSPCs after cerebral ischemia. Morris water maze test showed that delayed post-ischemic treatment with Sal B improved cognitive impairment after stroke in rats. SIGNIFICANCE: Sal B could maintain the NSPCs self-renew and promote proliferation, which was mediated by PI3K/Akt signal pathway. And delayed post-ischemic treatment with Sal B improved cognitive impairment after stroke in rats. These findings suggested that Sal B may act as a potential drug in treatment of brain injury or neurodegenerative diseases.