[Effect of acetylalkannin from Arnebia euchroma on proliferation, migration, and invasion of human melanoma A375 cells].

This study aimed to explore the effect and mechanism of acetylalkannin from Arnebia euchroma on the proliferation, migration, and invasion of human melanoma A375 cells. A375 cells were divided into a blank group, and low-, medium-, and high-dose acetylalkannin groups(0.5, 1.0, and 2.0 µmol·L~(-1)). The MTT assay was used to detect cell proliferation. Cell scratch and transwell migration assays were used to detect cell migration ability, and the transwell invasion assay was used to detect cell invasion ability. Western blot was used to detect the protein expression of migration and invasion-related N-cadherin, vimentin, matrix metalloproteina-se-9(MMP-9), and Wnt/ß-catenin pathway-related Wnt1, Axin2, glycogen synthase kinase-3ß(GSK-3ß), phosphorylated GSK-3ß(p-GSK-3ß), ß-catenin, cell cycle protein D_1(cyclin D_1), and p21. Real-time fluorescence-based quantitative polymerase chain reaction(real-time PCR) was used to detect the mRNA expression of E-cadherin, matrix metalloproteinase-2(MMP-2), N-cadherin, vimentin, ß-catenin, snail-1, and CD44. MTT results showed that the cell inhibition rates in the acetylalkannin groups significantly increased as compared with that in the blank group(P<0.01). The results of cell scratch and transwell assays showed that compared with the blank group, the acetylalkannin groups showed reduced cell migration and invasion, and migration and invasion rates(P<0.05, P<0.01) and weakened horizontal and vertical migration and invasion abilities. Western blot results showed that compared with the blank group, the high-dose acetylalkannin group showed increased expression of Axin2 protein(P<0.05), and decreased expression of N-cadherin, vimentin, MMP-9, Wnt1, p-GSK-3ß, ß-catenin, cyclin D_1, and p21 proteins(P<0.05, P<0.01). The expression of GSK-3ß protein did not change significantly. PCR results showed that the overall trend of MMP-2, N-cadherin, vimentin, ß-catenin, snail-1, and CD44 mRNA expression was down-regulated(P<0.01), and the expression of E-cadherin mRNA increased(P<0.01). Acetylalkannin can inhibit the proliferation, migration, and invasion of human melanoma A375 cells, and its mechanism of action may be related to the regulation of Wnt/ß-catenin signaling pathway.

[Mechanism of Berberis atrocarpa anthocyanin against Alzheimer's disease based on network pharmacology and experimental verification].

This study aimed to explore the potential mechanism of Berberis atrocarpa Schneid. anthocyanin against Alzheimer's disease(AD) based on network pharmacology, molecular docking technology, and in vitro experiments. Databases were used to screen out the potential targets of the active components of B. atrocarpa and the targets related to AD. STRING database and Cytoscape 3.9.0 were adopted to construct a protein-protein interaction(PPI) network and carry out topological analysis of the common targets. Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment analyses were performed on the target using the DAVID 6.8 database. Molecular docking was conducted to the active components and targets related to the nuclear factor kappa B(NF-κB)/Toll-like receptor 4(TLR4) pathway. Finally, lipopolysaccharide(LPS) was used to induce BV2 cells to establish the model of AD neuroinflammation for in vitro experimental validation. In this study, 426 potential targets of active components of B. atrocarpa and 329 drug-disease common targets were obtained, and 14 key targets were screened out by PPI network. A total of 623 items and 112 items were obtained by GO functional enrichment analysis and KEGG pathway enrichment analysis, respectively. Molecular docking results showed that NF-κB, NF-κB inhibitor(IκB), TLR4, and myeloid differentiation primary response 88(MyD88) had good binding abilities to the active components, and malvidin-3-O-glucoside had the strongest binding ability. Compared with the model group, the concentration of nitric oxide(NO) decreased at different doses of malvidin-3-O-glucoside without affecting the cell survival rate. Meanwhile, malvidin-3-O-glucoside down-regulated the protein expressions of NF-κB, IκB, TLR4, and MyD88. This study uses network pharmacology and experimental verification to preliminarily reveal that B. atrocarpa anthocyanin can inhibit LPS-induced neuroinflammation by regulating the NF-κB/TLR4 signaling pathway, thereby achieving the effect against AD, which provides a theoretical basis for the study of its pharmacodynamic material basis and mechanism.

Benefits of Curcumin in the Vasculature: A Therapeutic Candidate for Vascular Remodeling in Arterial Hypertension and Pulmonary Arterial Hypertension?

Growing evidence suggests that hypertension is one of the leading causes of cardiovascular morbidity and mortality since uncontrolled high blood pressure increases the risk of myocardial infarction, aortic dissection, hemorrhagic stroke, and chronic kidney disease. Impaired vascular homeostasis plays a critical role in the development of hypertension-induced vascular remodeling. Abnormal behaviors of vascular cells are not only a pathological hallmark of hypertensive vascular remodeling, but also an important pathological basis for maintaining reduced vascular compliance in hypertension. Targeting vascular remodeling represents a novel therapeutic approach in hypertension and its cardiovascular complications. Phytochemicals are emerging as candidates with therapeutic effects on numerous pathologies, including hypertension. An increasing number of studies have found that curcumin, a polyphenolic compound derived from dietary spice turmeric, holds a broad spectrum of pharmacological actions, such as antiplatelet, anticancer, anti-inflammatory, antioxidant, and antiangiogenic effects. Curcumin has been shown to prevent or treat vascular remodeling in hypertensive rodents by modulating various signaling pathways. In the present review, we attempt to focus on the current findings and molecular mechanisms of curcumin in the treatment of hypertensive vascular remodeling. In particular, adverse and inconsistent effects of curcumin, as well as some favorable pharmacokinetics or pharmacodynamics profiles in arterial hypertension will be discussed. Moreover, the recent progress in the preparation of nano-curcumins and their therapeutic potential in hypertension will be briefly recapped. The future research directions and challenges of curcumin in hypertension-related vascular remodeling are also proposed. It is foreseeable that curcumin is likely to be a therapeutic agent for hypertension and vascular remodeling going forwards.

[Overview of systematic reviews on Shenmai Injection in prevention and treatment of diseases].

To summarize and evaluate the efficacy and safety of Shenmai Injection in the treatment of viral myocarditis, shock, pulmonary heart disease, coronary heart disease, neutropenia and tumor chemotherapy, so as to provide supportive evidences for clinical rational use of Shenmai Injection. By searching literatures about studies on the systematic reviews on Shenmai Injection in treatment of viral myocarditis, shock, pulmonary heart disease, coronary heart disease, neutropenia and tumor chemotherapy from the main Chinese and English databases. Primary efficacy and safety outcome measures were selected for comparative analysis and summary, and the appraisal tool of AMSTAR 2 was used to evaluate the included studies.A total of 36 systematic reviews(published from 2005 to 2020) were included, involving viral myocarditis, shock, pulmonary heart disease, malignant tumor and coronary heart disease. The number of cases included in each type of the above diseases was 3 840, 2 484, 12 702, 28 036 and 27 082, respectively. The comparison results showed that, Shenmai Injection combined with conventional/western medicine treatment groups had better efficacy than conventional/western medicine groups alone in the prevention and treatment of the above five diseases. The main adverse reactions of Shenmai Injection reported in the included studies were facial flushing, rash, palpitation, etc., but the incidence was low and the general symptoms were mild, so no special treatment was needed. Therefore, the application of Shenmai Injection on the basis of conventional treatment or western medicine treatment had better prevention and treatment efficacy of the diseases. It was suggested that more multi-center and larger sample-size randomized controlled trials should be carried out in the future, and the relevant reporting standards should be strictly followed in systematic reviews, so as to improve the scientificity and transparency of the study.

Azospirillum oleiclasticum sp. nov, a nitrogen-fixing and heavy oil degrading bacterium isolated from an oil production mixture of Yumen Oilfield.

Two nitrogen-fixing and heavy oil degrading strains, designated RWY-5-1-1T and ROY-1-1-2, were isolated from an oil production mixture from Yumen Oilfield in China. The 16S rRNA gene sequence showed they belong to Azospirillum and have less than 96.1 % pairwise similarity with each species in this genus. The average nucleotide identity and digital DNA-DNA hybridization values between them and other type strains of Azospirillum species were less than 75.69 % and 22.0 %, respectively, both below the species delineation threshold. Pan-genomic analysis showed that the novel isolate RWY-5-1-1T shared 2145 core gene families with other type strains in Azospirillum, and the number of strain-specific gene families was 1623, almost two times more than the number known from other species. Furthermore, genes related to nitrogenase, hydrocarbon degradation and biosurfactant production were found in the isolates' genomes. Also, this strain was capable of reducing acetylene to ethylene at a rate of 22nmol ethylene h-1 (108 cells) and degrading heavy oil at a rate of 36.2 %. The major fatty acids and polar lipids were summed feature 8 (C18:1ω7c/C18:1ω6c), and phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylcholine. Furthermore, a combination of phenotypic, chemotaxonomic, phylogenetic and genotypic data clearly indicated that strains RWY-5-1-1T and ROY-1-1-2 represent a novel species, for which the name Azospirillum oleiclasticum sp. nov. is proposed. The type strain is RWY-5-1-1T (=CGMCC 1.13426T =KCTC 72259 T). Azospirillum novel strains with the ability of heavy oil degradation associated with the promotion of plant growth has never been reported to date.

Hydrogen Sulfide Prevents Elastin Loss and Attenuates Calcification Induced by High Glucose in Smooth Muscle Cells through Suppression of Stat3/Cathepsin S Signaling Pathway.

Vascular calcification can be enhanced by hyperglycemia. Elastin loss in tunica media promotes the osteogenic transformation of smooth muscle cells (SMCs) and involves arterial medial calcification (AMC) that is associated with a high incidence of cardiovascular risk in patients with type 2 diabetes. Here, we tested whether hydrogen sulfide (H2S), an endogenous gaseous mediator, can prevent elastin loss and attenuate calcification induced by high glucose in SMCs. Calcification was induced by high glucose (4500 mg/L) in human aortic SMCs (HASMCs) under the condition of calcifying medium containing 10 mM ß-glycerophosphate (ß-GP). The experiments showed that NaHS (an H2S donor, 100 µM) mitigated the calcification of HASMCs treated with high glucose by decreasing calcium and phosphorus levels, calcium deposition and ALP activity and inhibited osteogenic transformation by increasing SMα-actin and SM22α, two phenotypic markers of smooth muscle cells, and decreasing core binding factor α-1 (Cbfα-1), a key factor in bone formation, protein expressions in HASMCs. Moreover, NaHS administration inhibited the activation of Stat3, cathepsin S (CAS) activity and its expression, but increased the level of elastin protein. Pharmacological inhibition or gene silencing Stat3 not only reversed elastin loss, but also attenuated CAS expression. Inhibition of CAS alleviated, while CAS overexpression exacerbated, elastin loss. Interestingly, overexpression of wild type (WT)-Stat3, but not its mutant C259S, elevated CAS protein expression and reduced elastin level. Moreover, NaHS induced S-sulfhydration in WT, but not in the C259S Stat3. These data suggest that H2S may directly regulate Cys259 residue in Stat3 and then impair its signaling function. Our data indicate that H2S may attenuate vascular calcification by upregulating elastin level through the inhibition of Stat3/CAS signaling.

[Cloning and functional identification of a new NADPH-cytochrome P450 reductase in Andrographis paniculata].

Andrographolide is a main active ingredient in traditional Chinese medicine Andrographis paniculata，with a variety of pharmacological activity，widely used in clinical practice. However its biosynthetic pathway has not been resolved. Cytochrome P450 reductase provides electrons for CYP450 and plays an important role in the CYP450 catalytic process. In this study，the coding sequence of A. paniculata CPR was screened and cloned by homologous alignment，named ApCPR4. The ApCPR4 protein was obtained by prokaryotic expression. After isolation and purification，the enzyme activity was identified in vitro. The results showed that ApCPR4 could reduce the cytochrome c and ferricyanide in NADPH-dependent manner. In order to verify its in vivo function，ApCPR4 and CYP76AH1 were co-transformed into yeast engineering bacteria. The results showed that ApCPR4 could help CYP76AH1 catalyze the formation of rustols in yeast. Real-time quantitative PCR results showed that the expression of ApCPR4 increased gradually in leaves treated with methyl jasmonate (MeJA). The expression pattern was consistent with the trend of induction and accumulation of andrographolide by MeJA，suggesting that ApCPR4 was associated with biosynthesis of andrographolide.

[Rapid analysis of chemical components in Zizyphi Spinosae Semen by UHPLC-LTQ-Orbitrap-MS].

This study aimed to analyze the chemical components of Zizyphi Spinosae Semen by using ultra high performance liquid chromatography-linear ion trap/orbitrap high resolution mass spectrometry (UHPLC-LTQ-Orbitrap-MS). The chromatographic separation of the components was achieved on a Waters Acquity UPLC BEH-C18 column (2.1 mm×50 mm, 1.7 µm), with acetonitrile-water (0.1% formic acid) as mobile phase at a flow rate of 0.3 mL·min⁻¹. Electrospray ionization-(ESI) source was applied and operated in negative ion mode. The parent ion list (PIL)-MS2 and high energy collision dissociation (HCD) techniques were used to identify triterpene saponins in Zizyphi Spinosae Semen. Through analysis of the mass spectrometry data and literature data, 24 compounds and 4 unknown compounds were identified and inferred, including 21 flavonoids, 5 saponins and 2 triterpenoids. The use of UHPLC-LTQ-Orbitrap-MS technology provide an efficient and rapid analytical method for the qualitative analysis and quality control of the chemical components in Zizyphi Spinosae Semen. It can also provide reference data for the research on the material basis of pharmacodynamics and the resources development and utilization.

[Overexpression and isolation of CYP76AH1 in Salvia miltiorrhiza hairy roots].

Protein complexes are involved in the synthesis of multiple secondary metabolites in plants, and their separation is essential to elucidate plant secondary metabolism and improve in vitro catalytic efficiency. In this study, the transgenic hairy roots of CYP76AH1, a key enzyme of tanshinone synthesis pathway, was constructed and the transgenic hairy roots of Danshen overexpressing CYP76AH1 protein were screened by Western blotting and used as a tissue culture material for the subsequent extraction of protein complex in tanshinone synthesis pathway. By optimizing the type and concentration of the detergent in the protein extraction buffer, the buffer containing 0.5% Triton X-100 was selected as the best extraction buffer, and a relatively large amount of soluble CYP76AH1 protein was isolated. This study lays the foundation for the further separation and purification of protein complexes interacting with CYP76AH1, and provides the idea for deep analysis of tanshinone metabolic pathway.

[Effect of turmeric volatile oil on proliferation and apoptosis of human skin SCC A431 cells].

To investigate the effect of turmeric volatile oil (TVO) on the apoptosis and proliferation of human skin SCC A431 cells, A431 cells were incubated with different concentrations (5-80 mg•L⁻¹) of TVO in vitro．The proliferation and cell cycle were assessed by CCK8 assay. The change of morphology was observed with inverted microscope. Apoptosis was evaluated with AO/EB double staining and flow cytometry (FCM); cell cycle was analyzed with FCM ．Western blot method was used to detect caspase-3 and caspase-9 protein expression. Results indicated that TVO has significant inhibitory effects on the growth of A431 cells in a dose dependent relationship, the difference between groups has statistically significant (P<0.05). TVO group compared with control group, concentrations in cells shrivel and broken phenomenon, cell apoptosis rate increased, and a dose dependent and increased the expression of caspase-3 and caspase-9. The experiment results suggested that TVO could restrain skin squamous carcinoma A431 cells proliferation, and induce its apoptosis. The mechanism may be related to increase the expression of caspase-3 and caspase-9.

Tanshinone IIA Attenuates Chronic Pancreatitis-Induced Pain in Rats via Downregulation of HMGB1 and TRL4 Expression in the Spinal Cord.

BACKGROUND: Chronic pancreatitis (CP) is a long-standing inflammation of the exocrine pancreas, which typically results in severe and constant abdominal pain. Previous studies on the mechanisms underlying CP-induced pain have primarily focused on the peripheral nociceptive system. A role for a central mechanism in the mediation or modulation of abdominal pain is largely unknown. Tanshinone IIA (TSN IIA), an active component of the traditional Chinese medicine Danshen, exhibits anti-inflammatory properties via downregulation of the expression of high-mobility group protein B1 (HMGB1), a late proinflammatory cytokine. HMGB1 binds and activates toll-like receptor 4 (TLR4) to induce spinal astrocyte activation and proinflammatory cytokine release in neuropathic pain. OBJECTIVE: In this study, we investigated the effect of TSN IIA on pain responses in rats with trinitrobenzene sulfonic acid (TNBS)-induced CP. The roles of central mechanisms in the mediation or modulation of CP were also investigated. STUDY DESIGN: A randomized, double-blind, placebo-controlled animal trial. METHODS: CP was induced in rats by intrapancreatic infusion of trinitrobenzene sulfonic acid (TNBS). Pancreatic histopathological changes were characterized with semi-quantitative scores. The abdomen nociceptive behaviors were assessed with von Frey filaments. The effects of intraperitoneally administered TSN IIA on CP-induced mechanical allodynia were tested. The spinal protein expression of HMGB1 was determined by western blot. The spinal mRNA and protein expression of proinflammatory cytokines IL-1ß, TNF-α, and IL-6 were determined by RT-PCR and western blot, respectively. The spinal expression of the HMGB1 receptor TRL4 and the astrocyte activation marker glial fibrillary acidic protein (GFAP) were determined by western blot or immunohistological staining after intraperitoneal injection of TSN IIA or intrathecal administration of a neutralizing anti-HMGB1 antibody. RESULTS: TNBS infusion resulted in pancreatic histopathological changes of chronic pancreatitis and mechanical allodynia in rats. TSN IIA significantly attenuated TNBS-induced mechanical allodynia in a dose-dependent manner. TNBS significantly increased the spinal expression of HMGB1 and proinflammatory cytokines IL-1ß, TNF-α, and IL-6. These TNBS-induced changes were significantly inhibited by TSN IIA in a dose-dependent manner. Furthermore, TSN IIA, but not the neutralizing anti-HMGB1 antibody, significantly inhibited TNBS-induced spinal TLR4 and GFAP expression. LIMITATIONS: In addition to TLR4, HMGB1 can also bind to toll-like receptor-2 (TLR2) and the receptor for advanced glycation end products (RAGE). Additional studies are warranted to ascertain whether HMGB1 contributes to CP-induced pain through activation of these receptors. CONCLUSIONS: Our results suggest that spinal HMGB1 contributes to the development of CP-induced pain and can potentially be a therapeutic target. TSN IIA attenuates CP-induced pain via downregulation of spinal HMGB1 and TRL4 expression. Therefore, TSN IIA may be a potential anti-nociceptive drug for the treatment of CP-induced pain.

Comparing the actions of the three flavone ingredients in choerospondias axillaris on arrhythmias induced by aconitine / 中国中药杂志

<p><b>OBJECTIVE</b>To compare the actions of the three flavone ingredients in choerospondias axillaris on arrhythmias Induced by aconitine.</p><p><b>METHOD</b>Langendorff perfuse was applied in the experiment, the antiarrhythmic action was to study by using aconitine on the the isolated heart; The antiarrhythmic action of the three flavone ingredients in choerospondias axillaris was to study by using i.v. aconitine in rat to induce arrhythmias.</p><p><b>RESULT</b>Compared with the NS group, sample 1 and sample 2 both significantly prolonged the beginning time of VF of isolated heart and increased the dosage of aconitine, sample 3 reduced the beginning time of VF of isolated heart and decreased the dosage of aconitine, sample 1 and sample 2 both greatly prolonged the beginning time of VE, VT, VF, HA; sample 3 greatly reduced the beginning time of VT,VF. The actions of the three samples were in a concentration-dependent way.</p><p><b>CONCLUSION</b>Sample 1 and sample 2 both resisted the occurrence of arrhythmias induced by aconitine, sample 3 markedly promoted the occurrence of arrhythmias induced by aconitine.</p>

The effect of Baicalin on gene expression profile in rat brain of focal cerebral ischemia / 中国中药杂志

<p><b>OBJECTIVE</b>To explore the difference of genes expression profiles between focal cerebral ischemia tissue and that treated with Baicalin using cDNA microarray.</p><p><b>METHOD</b>The total RNAs were isolated from rat brains of sham-operation, vehicle (focal cerebral ischemia of rat brain) and baicalin-treated groups. mRNAs were reversely transcribed to cDNA with incorporation of fluorescent dUTP (Cy5 or Cy3 dUTP) to prepare hybridization probes. The PCR products of 4096 genes were spotted on the chip after a serial of treatment. The mixed probes were hybridized to the cDNA microarray. Axon Genepix 4000B and GenePixPro 3.0 software were used to scan and analyze the fluorescent signals.</p><p><b>RESULT</b>The expressions of 199 and 12 genes were found up-regulated and down-regulated, respectively, in the vehicle group compared with the sham-operation one. But the numbers of genes whose expressions were up-regulated and down-regulated were 89 and 88, respectively, when comparing the gene expression in the Baicalin-treated rat brain with that in the vehicle group. Moreover, one down-regulated and three up-regulated genes in the vehicle group were up-regulated and down-regulated in the Baicalin-treated group, respectively. Expressions of three up-regulated genes in the vehicle group were further reinforced in the Baicalin-treatment group.</p><p><b>CONCLUSION</b>Multiple pathways and nodes may be involved in the pharmacological effect of Baicalin on brain ischemia.</p>