Evaluation of reducing the south and reinforcing the north method on postmenopausal atherosclerosis disease based on 4 diagnostic objectifications: A prospective observational study.

Reducing the south and reinforcing the north method (RSRN) has a positive effect on atherosclerosis disease. However, there is a lack of objective standards based on the quantification of 4 diagnostic methods in evaluating the improvement or effectiveness of the treatment. This study aimed to explore the quantitative evaluation of the therapeutic effect of RSRN on postmenopausal atherosclerosis based on the 4 diagnostic methods. The observational prospective cohort study was conducted at Longhua hospital Shanghai University of traditional Chinese medicine. According to the inclusion criteria, 96 patients (disease group) and 38 healthy cases (control group) were selected, the pulse parameters were compared between the 2 groups to demonstrate the reliability and success of the disease model. Then 4 diagnostic information before and after RSRN treatment were collected and statistical analyzed by 1-way analysis of variance (ANOVA) (with Bonferroni correction). Furthermore, social network analysis was used to analyze the changes of symptoms, tongue, pulse, and complexion characteristics before and after treatment. There was a significant difference in pulse parameters between the disease group and the control group. The pulse parameters t1, h3, h3/h1, h4/h1, S, As, and w values in disease group were higher than those in control group, while the h5, h5/h1, and Ad values were lower than those in control group (P < .05). After the treatment of RSRN, the clinical symptoms of patients were greatly improved. The facial color indexes L, a, b values of the disease group at week 6 were different from those at week 0 (P < .05). The overall brightness and chroma of the patient's facial color were significantly improved. The patients had virtual string pulse at week 0, and mainly string I and string II at week 7. The pulse parameters t1, t5, w, w/t, h1, h5, h3/h1, and h5/h1 values at week 7 were different from those at weeks 0, 1, 2 (P < .05); the tongue image was mainly red and crimson, peeling or greasy fur at week 0, while at weeks 6, 7, mainly light red, or thin white tongue. The RSRN method can regulate the complexion, tongue and pulse condition, clinical symptoms of postmenopausal atherosclerosis.

Low phosphorus increases hepatic lipid deposition, oxidative stress and inflammatory response via Acetyl-CoA carboxylase-dependent manner in zebrafish liver cells.

Acetyl-CoA carboxylase (ACC) plays a regulatory role in both fatty acid synthesis and oxidation, controlling the process of lipid deposition in the liver. Given that existing studies have shown a close relationship between low phosphorus (P) and hepatic lipid deposition, this study was conducted to investigate whether ACC plays a crucial role in this relationship. Zebrafish liver cell line (ZFL) was incubated under low P medium (LP, P concentration: 0.77 mg/L) or adequate P medium (AP, P concentration: 35 mg/L) for 240 h. The results showed that, compared with AP-treated cells, LP-treated cells displayed elevated lipid accumulation, and reduced fatty acid ß-oxidation, ATP content, and mitochondrial mass. Furthermore, transcriptomics analysis revealed that LP-treated cells significantly increased lipid synthesis (Acetyl-CoA carboxylases (acc), Stearyl coenzyme A dehydrogenase (scd)) but decreased fatty acid ß-oxidation (Carnitine palmitoyltransferase I (cptI)) and (AMP-activated protein kinase (ampk)) mRNA levels compared to AP-treated cells. The phosphorylation of AMPK and ACC, and the protein expression of CPTI were significantly decreased in LP-treated cells compared with those in AP-treated cells. After 240 h of LP treatment, PF-05175157 (an ACC inhibitor) was supplemented in the LP treatment for an additional 12 h. PF-05175157-treated cells showed higher phosphorylation of ACC, higher protein expression of CPTI, and lower protein expression of FASN, lower TG content, enhanced fatty acid ß-oxidation, increased ATP content, and mitochondrial mass compared with LP-treated cells. PF-05175157 also relieved the LP-induced oxidative stress and inflammatory response. Overall, these findings suggest that ACC is a promising target for treating LP-induced elevation of lipid deposition in ZFL, and can alleviate oxidative stress and inflammatory response.

Exploration of the effect and mechanism of Scutellaria barbata D. Don in the treatment of ovarian cancer based on network pharmacology and in vitro experimental verification.

The mortality rate of ovarian cancer is the highest among gynecological cancers, posing a serious threat to women health and life. Scutellaria barbata D. Don (SBD) can effectively treat ovarian cancer. However, its mechanism of action is unclear. The aim of this study was to elucidate the mechanism of SBD in the treatment of ovarian cancer using network pharmacology, and to verify the experimental results using human ovarian cancer SKOV3 cells. The Herb and Disease Gene databases were searched to identify common targets of SBD and ovarian cancer. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, and Protein-Protein Interaction (PPI) network analyses were performed to identify the potential molecular mechanisms behind SBD. Finally, the molecular docking and main possible pathways were verified by experimental studies. Cell proliferation, the mRNA expression level of key genes and signaling pathway were all investigated and evaluated in vitro. A total of 29 bioactive ingredients and 137 common targets in SBD were found to inhibit ovarian cancer development. The active ingredients identified include quercetin, luteolin, and wogonin. Analysis of the PPI network showed that AKT1, VEGFA, JUN, TNF, and Caspase-3 shared centrality among all target genes. The results of the KEGG pathway analysis indicated that the cancer pathway, PI3K-Akt signaling pathway, and MAPK signaling pathways mediated the effects of SBD against ovarian cancer progression. Cell experiments showed that quercetin, luteolin, and wogonin inhibited the proliferation and clone formation of SKOV3 cells and regulated mRNA expression of 5 key genes by inhibiting PI3K/Akt signaling pathway. Our results demonstrate that SBD exerted anti-ovarian cancer effects through its key components quercetin, luteolin and wogonin. Mechanistically, its anti-cancer effects were mediated by inhibition of the PI3K/Akt signaling pathways. Therefore, SBD might be a candidate drug for ovarian cancer treatment.

Efficacy of the herbal pair, Radix Achyranthis Bidentatae and Eucommiae Cortex, in preventing glucocorticoid-induced osteoporosis in the zebrafish model.

OBJECTIVE: In traditional Chinese medicine, the herbal pair, Radix Achyranthis Bidentatae (RAB) and Eucommiae Cortex (EC), is widely used to treat osteoporosis. Herein, we determined whether this herbal pair can be used to ameliorate glucocorticoid (GC)-induced osteoporosis (GIOP) and find its optimal dosage in zebrafish. METHODS: The characteristics of the aqueous extract of RAB and EC were separately characterized using high-performance liquid chromatography. Osteoporosis was induced in 5-day post-fertilization zebrafish larvae by exposing them to 10 µmol/L dexamethasone (Dex) for 96 h. Seven combinations of different ratios of RAB and EC were co-administered. Treatment efficacy was determined by calculating zebrafish vertebral area and sum brightness, via alizarin red staining, and by detecting alkaline phosphatase (ALP) activity. Multiple regression analysis was conducted to test the optimal dosage ratio. RESULTS: According to the Chinese Pharmacopoeia (2015), ß-ecdysone (ß-Ecd) is a major bioactive marker in RAB extract, while pinoresinol diglucoside (PDG) is the major marker in EC extract. Both of ß-Ecd and PDG content values aligned with the Chinese Pharmacopoeia standards. Treatment with 10 µmol/L Dex reduced zebrafish vertebral area, sum brightness, and ALP activity, but RAB and EC attenuated these effects. Combining 50 µg/mL RAB and 50 µg/mL EC was optimal for preventing GIOP in zebrafish. Reverse transcription-quantitative polymerase chain reaction was used to evaluate the mRNA expression of osteogenesis-related genes. A treatment of 10 µmol/L Dex decreased runt-related transcription factor 2 (Runx2), osteogenic protein-1 (OP-1), bone γ-carboxyglutamic acid-containing protein (BGLAP), and ß-catenin levels. This effect was counteracted by RAB and EC co-treatment (P < 0.05). Additionally, the effect of using the two herbal extracts together was better than single-herb treatments separately. These results demonstrated that RAB and EC preserve osteoblast function in the presence of GC. The best mass ratio was 1:1. CONCLUSION: RAB and EC herbal pair could ameliorate GC-induced effects in zebrafish, with 1:1 as the optimal dosage ratio.

Effects of Reducing the South and Reinforcing the North Method on Inflammatory Injury Induced by Hyperlipidemia.

Inflammation is the pathophysiological basis of hyperlipidemia-related disease (HRD). Reducing the south and reinforcing the north method (RSRN) has a positive effect on HRD. However, the pharmacological mechanisms of RSRN are still unclear in the treatment of HRD. We obtained RSRN compounds from the Traditional Chinese Medicine Systems Pharmacology (TCMSP) and identified potential targets of these compounds through target fishing based on the TCMSP databases. Next, we identified the HRD targets by using multiple databases. Then, the overlapping genes between the RSRN potential targets and the HRD targets were used to establish a protein-protein interaction (PPI) network, and we further analyzed their interactions and identified the major hub genes in this network. Subsequently, the Metascape database was utilized to conduct the enrichment of Gene Ontology biological processes (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. A total of 187 potential active components and 106 related core targets were obtained and identified overall. Then after the Metascape enrichment analysis, a total of 148 KEGG pathways were screened, which were mainly associated with AGE-RAGE signaling pathway, PI3K-Akt signaling pathway, TNF signaling pathway, and NF-kappa B signaling pathway. Furthermore, 34 hub genes, such as AKT1, NF-κBp65(RELA), IκBα(CHUK), MAPK8, and MAPK14, CCND1, were considered potential therapeutic targets. Furthermore, evaluations of protein levels of NF-κBp65, IκBα, TNF-α, IL-1 ß, and IL-6 were performed for experimental validation. RSRN can reduce the expression of NF-κBp65 protein, increase the level of IκBα protein, and reduce the protein levels of TNF-α, IL-1ß, and IL-6 in ovariectomized rats. The results indicate that the mechanism of RSRN against inflammation may be related to AKT1, NF-κBp65, IκBα, MAPK8, and MAPK14, as well as TNF, NF-kappa B, PI3K-Akt signaling pathways.

Glutamine Anabolism Plays a Critical Role in Pancreatic Cancer by Coupling Carbon and Nitrogen Metabolism.

Glutamine is thought to play an important role in cancer cells by being deaminated via glutaminolysis to α-ketoglutarate (aKG) to fuel the tricarboxylic acid (TCA) cycle. Supporting this notion, aKG supplementation can restore growth/survival of glutamine-deprived cells. However, pancreatic cancers are often poorly vascularized and limited in glutamine supply, in alignment with recent concerns on the significance of glutaminolysis in pancreatic cancer. Here, we show that aKG-mediated rescue of glutamine-deprived pancreatic ductal carcinoma (PDAC) cells requires glutamate ammonia ligase (GLUL), the enzyme responsible for de novo glutamine synthesis. GLUL-deficient PDAC cells are capable of the TCA cycle but defective in aKG-coupled glutamine biosynthesis and subsequent nitrogen anabolic processes. Importantly, GLUL expression is elevated in pancreatic cancer patient samples and in mouse PDAC models. GLUL ablation suppresses the development of KrasG12D-driven murine PDAC. Therefore, GLUL-mediated glutamine biosynthesis couples the TCA cycle with nitrogen anabolism and plays a critical role in PDAC.

Beta-Ecdysone Protects Mouse Osteoblasts from Glucocorticoid-Induced Apoptosis In Vitro.

Glucocorticoid-induced osteoporosis is a common form of secondary osteoporosis. Glucocorticoids affect both bone formation and resorption, and prolonged glucocorticoid exposure can suppress osteoblast activities. beta-Ecdysone, found in many plants, is involved in protein synthesis, carbohydrate and lipid metabolism, and immunologic modulation. Here, we evaluated the effects of beta-ecdysone on osteoblast viability by assessing apoptosis following treatment with excess glucocorticoids. Mouse bone marrow stromal cells were induced to differentiate and grow into osteoblasts, and then treated with 10 µM glucocorticoid and 10, 1, or 0.1 µM beta-ecdysone. The expression levels of osteoblast growth and differentiation factors (runt-related transcription factor 2, osteogenic protein-1, and alkaline phosphatase), apoptosis-related genes (transformation-related protein 53, ataxia telangiectasia mutated protein, caspase-3, and caspase-8), and Akt1 and phospho-Akt (Thr308) were then assessed via alkaline phosphatase staining, acridine orange-propidium iodide staining, annexin V/PI apoptosis assay, real-time RT-PCR, and Western blot analyses. Notably, treatment with 10 µM glucocorticoid resulted in reduced osteoblast viability and the specific activity of alkaline phosphatase as well as reduced runt-related transcription factor 2, osteogenic protein-1, and alkaline phosphatase mRNA expression in vitro, indicating that glucocorticoid inhibited osteogenic differentiation. Moreover, glucocorticoid treatment yielded increased transformation-related protein 53, ataxia telangiectasia mutated protein, caspase-3, and caspase-8 expression and decreased Akt1 and phospho-Akt levels, indicating glucocorticoid-induced apoptosis. Meanwhile, beta-ecdysone inhibited glucocorticoid function, preserving the expression of Akt1 and phospho-Akt and reducing the expression of transformation-related protein 53, ataxia telangiectasia mutated protein, caspase-3, and caspase-8. Thus, beta-ecdysone prevented glucocorticoid-induced osteoblast apoptosis in vitro. These data highlight the potential for beta-ecdysone as a treatment for preventing the effects of glucocorticoid on bone growth.

Derivation of Self-inhibitory Helical Peptides to Target Rho-kinase Dimerization in Cerebrovascular Malformation: Structural Bioinformatics Analysis and Peptide Binding Assay.

Rho-kinase dimerization is essential for its kinase activity and biological function; disruption of the dimerization has recently been established as a new and promising therapeutics strategy for cerebrovascular malformation (CM). Based on Rho-kinase dimer crystal structure we herein combined in silico analysis and in vitro assay to rationally derive self-inhibitory peptides from the dimerization interface. Three peptides namely Hlp1, Hlp2 and Hlp3 were successfully designed that have potential capability to rebind at the dimerization domain of Rho-kinase. Molecular dynamics (MD) simulations revealed that these peptides are helically structured when bound to Rho-kinase, but exhibit partially intrinsic disorder in unbound state. Binding free energy (BFE) analysis suggested that the peptides have a satisfactory energetic profile to interact with Rho-kinase. The computational findings were then substantiated by fluorescence anisotropy assays, conforming that the helical peptides can bind tightly to Rho-kinase with affinity KD at micromolar level. These designed peptides are considered as lead molecular entities that can be further modified and optimized to obtain more potent peptidomimetics as self-competitors to disrupt Rho-kinase dimerization in CM.

Mannotriose regulates learning and memory signal transduction in the hippocampus.

Rehmannia is a commonly used Chinese herb, which improves learning and memory. However, the crucial components of the signal transduction pathway associated with this effect remain elusive. Pri-mary hippocampal neurons were cultured in vitro, insulted with high-concentration (1 × 10(-4) mol/L) cor-ticosterone, and treated with 1 × 10(-4) mol/L mannotriose. Thiazolyl blue tetrazolium bromide assay and western blot analysis showed that hippocampal neuron survival rates and protein levels of glucocorti-coid receptor, serum and glucocorticoid-regulated protein kinase, and brain-derived neurotrophic factor were all dramatically decreased after high-concentration corticosterone-induced injury. This effect was reversed by mannotriose, to a similar level as RU38486 and donepezil. Our findings indicate that mannotriose could protect hippocampal neurons from high-concentration corticosterone-induced injury. The mechanism by which this occurred was associated with levels of glucocorticoid receptor protein, serum and glucocorticoid-regulated protein kinase, and brain-derived neurotrophic factor.

[Effect of acupuncture on contents of beta-endorphin in the plasma and hypothalamus in rats with stress-induced gastric mucosal injury].

OBJECTIVE: To explore the mechanism of acupuncture in preventing and treating stress-induced gastric mucosal injury from the view of brain-gut axis. METHODS: Forty male Wister rats were randomly divided into normal control, model 1, treatment, model 2 and prevention groups (n=8). Gastric mucosa injury model was established by intragastric perfusion of dehydrated alcohol (1.0 mL/rat). Rats of the treatment group were treated with acupuncture after modeling, while those of the prevention group treated first, followed by modeling. The time of modeling in model 1 group and model 2 group was simultaneous with that of the treatment group and prevention group respectively. Acupuncture was applied to "Zusanli"(ST 36), "Zhongwan" (CV 12) and "Neiguan" (PC 6) for 20 min, once daily for 5 days. Before sampling the tissues, 10% charcoal suspension was intragastric perfused 1 h for analyzing the rate of gastrointestinal propulsion(distance from the upper end of the charcoal powder to the cardia/total length of the cardia to the anus x 100%). Gastric mucosal ulcer index was measured by using Guth's method. The contents of beta-endorphin(beta-EP)in plasma and hypothalamus were determined by enzyme-linked immunosorbent assay. RESULTS: Compared with the normal control group, the gastrointestinal propulsion rates were decreased considerably in the two model groups (P < 0.05), while the gastric mucosal ulcer indexes and the contents of beta-EP in both plasma and hypothalamus were increased significantly in the model groups (P < 0.05, P < 0.01). Compared with the corresponding model groups, the gastrointestinal propulsion rate was increased remarkably in the prevention group (P < 0.05), and the gastric mucosal ulcer indexes and the contents of plasma beta-EP level were decreased obviously in both treatment and prevention groups (P < 0.05, P < 0.01). The contents of hypothalamic beta-EP were increased further in the later two groups (P < 0.01). CONCLUSION: Acupuncture of ST 36, CV 12 and PC 6 can promote the repair of gastric mucosal injury and improve gastrointestinal function, which may be related to its effects in reducing plasma beta-EP and upregulating hypothalamic beta-EP level. Acupuncture also has a better effect in preventing gastric mucosal injury.