Exploring the Mechanism of Salvianolic Acid B against Myocardial Ischemia-Reperfusion Injury Based on Network Pharmacology.

This study aimed to explore the mechanisms through which salvianolic acid B (Sal-B) exerts its effects during myocardial ischemia-reperfusion injury (MI/RI), aiming to demonstrate the potential pharmacological characteristics of Sal-B in the management of coronary heart disease. First, Sal-B-related targets and MI/RI-related genes were compiled from public databases. Subsequent functional enrichment analyses using the protein-protein interaction (PPI) network, gene ontology (GO), and the Kyoto Encyclopedia of Genes and Genomes (KEGG) predicted the core targets and approaches by which Sal-B counters MI/RI. Second, a Sal-B-treated MI/RI mouse model and oxygen-glucose deprivation/reoxygenation (OGD/R) H9C2 cell model were selected to verify the main targets of the network pharmacological prediction. An intersectional analysis between Sal-B and MI/RI targets identified 69 common targets, with a PPI network analysis highlighting caspase-3, c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (p38) as central targets. GO and KEGG enrichment analyses indicated remarkable enrichment of the apoptosis pathway among these targets, suggesting their utility in experimental studies in vivo. Experimental results demonstrated that Sal-B treatment not only mitigated myocardial infarction size following MI/RI injury in mice but also modulated the expression of key apoptotic regulators, including Bcl-2-Associated X (Bax), caspase-3, JNK, and p38, alongside enhancing the B-cell lymphoma-2 (Bcl-2) expression, thereby inhibiting myocardial tissue apoptosis. This study leveraged an integrative network pharmacology approach to predict Sal-B's potential targets in MI/RI treatment and verified the involvement of key target proteins within the predicted signaling pathways through both in vivo and in vitro experiments, offering a comprehensive insight into Sal-B's pharmacological mechanism in MI/RI management.

Salvianolic acid B exerts protective effects against Aß-induced neuroinflammation through the inhibition of NLRP3 inflammasome activation and switching of M1/M2 polarization.

BACKGROUND: Salvianolic acid B (SalB) is a bioactive extract of Salvia miltiorrhiza with the ability to ameliorate amyloid beta (Aß)-induced neuronal degeneration and neuroinflammation in Alzheimer's disease (AD). However, the underlying mechanisms of this action have not been elucidated. Herein, we aimed to investigate whether the neuroprotective effect of SalB is attributable to the modulation of microglial polarization and NLRP3 inflammasome-mediated neuroinflammation. METHODS: Based on the TMT-labeled proteomics analysis, immunofluorescence, western blot and quantitative reverse transcription polymerase chain reaction (qRT-PCR) were employed to investigate the effects of SalB on neuroinflammation in Aß1-42-stimulated BV2 microglia cells. RESULTS: At the proteomic level, a total of 6631 proteins were quantified, and of these, 104 were significantly influenced under Aß1-42 treatment. The expression of 36 Aß1-42-induced differentially expressed proteins were significantly recovered by SalB treatment (13 upregulated and 23 downregulated). NLRP3 was significantly recovered and was identified as one of the hub proteins. Consistent with the result of the proteomic analysis, western blot and qRT-PCR demonstrated that SalB reduced Aß1-42-induced NLRP3 upregulation at both the protein and mRNA levels. In addition, SalB significantly blocked M1 microglia polarization, enhanced M2 microglial polarization, and inhibited the production of caspase-1 and interleukin-1ß in BV2 microglia cells. CONCLUSION: our study demonstrated, for the first time, that the anti-inflammatory effects of SalB were mediated by the regulation of NLRP3 activation and promotion of microglial M2 polarization, indicating the potential of SalB as a novel therapeutic candidate for AD.

Salvianolic acid B combined with bone marrow mesenchymal stem cells piggybacked on HAMA hydrogel re-transplantation improves intervertebral disc degeneration.

Cell-based tissue engineering approaches have emerged as a realistic alternative for regenerative disc tissue repair. The multidirectional differentiation potential of bone marrow mesenchymal stem cells (BMSCs) to treat disc degeneration intervertebral disc degeneration has also become a viable option. We used 1% HAMA hydrogel as a carrier and co-encapsulated BMSCs and Salvianolic acid B (SalB) into the hydrogel to reduce the apoptosis of the transplanted cells. The protective effect of SalB on BMSCs was first verified in vitro using the CCK8 method, flow cytometry, and Western-Blotting, and the physical properties and biocompatibility of HAMA hydrogels were verified in vitro. The rat model was then established using the pinprick method and taken at 4 and 8 W, to examine the extent of disc degeneration by histology and immunohistochemistry, respectively. It was found that SalB could effectively reduce the apoptosis of BMSCs in vitro by activating the JAK2-STAT3 pathway. 1% HAMA hydrogels had larger pore size and better water retention, and the percentage of cell survival within the hydrogels was significantly higher after the addition of SalB to the HAMA hydrogels. In the in vivo setting, the HAMA + SalB + BMSCs group had a more pronounced delaying effect on the progression of disc degeneration compared to the other treatment groups. The method used in this study to encapsulate protective drugs with stem cells in a hydrogel for injection into the lesion has potential research value in the field of regenerative medicine.

Salvianolic acid B ameliorates non-alcoholic fatty liver disease by inhibiting hepatic lipid accumulation and NLRP3 inflammasome in ob/ob mice.

Non-alcoholic fatty liver disease (NAFLD) has high occurrence in the global world, which poses serious threats to human health. Salvianolic acid B (SalB), an extract of the root of Salvia miltiorrhiza, has the protective effect on metabolic homeostasis. However, the mechanism is still unknown. In this study, we used ob/ob mice, a model of NAFLD, to explore the hepatoprotective effects of SalB. The results showed that SalB significantly reduced the body weights and liver weights, and ameliorated plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG), hepatic free fatty acid (FFA), total cholesterol (TC) levels, and hepatic TG and TC levels in ob/ob mice. SalB reduced the number of lipid droplets and inhibited hepatic lipogenesis by regulating peroxisome proliferator-activated receptor gamma (PPARγ), fatty acid synthase (FASN), stearoyl-Co A desaturase 1 (SCD1), and cluster of differentiation 36 (CD36). Compared to ob/ob mice, the lower expressions of the pro-inflammatory cytokines, such as interleukin-1ß (IL-1ß), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and F4/80, were observed after SalB treatment. Importantly, SalB treatment inhibited the activation of NLRP3 inflammasome and reduced the severity of liver inflammation. Our findings suggested that SalB improved NAFLD pathology in ob/ob mice by reducing hepatic lipid accumulation and NLRP3 inflammasome activation, which might be the potential hepatoprotective mechanism of SalB.

Salvianolic acid B improves the survival rate, acute kidney dysfunction, inflammation and NETosis-mediated antibacterial action in a crush syndrome rat model.

Crush syndrome (CS) is a potentially lethal condition characterized by muscle cell damage resulting from decompression following compression. Patients with CS can develop cardiac failure, kidney dysfunction, shock, systemic inflammation and sepsis. Salvianolic acid B (SalB) has cardiac and kidney protective effects and anti-oxidative, anti-inflammatory, anti-apoptotic and anti-bacterial properties. The present study aimed to demonstrate the survival benefit of SalB in the CS rat model, which comprised anesthetized rats with bilateral hindlimb compression by a rubber tourniquet for 5 h. The rats examined were randomly divided into four groups: i) Sham; ii) sham treated with SalB; iii) CS rat model without treatment; and iv) CS rat model treated with SalB. Under continuous monitoring and recording of arterial blood pressures, blood and tissue samples were collected for biochemical analyses at designated timepoints before and after reperfusion. SalB administration improved the survival rate, kidney function (by treating shock and metabolic acidosis) and inflammation (by reducing mitochondrial dysfunction and endothelial damage). Reduced incidence of cardiac failure due to hyperkalemia was associated with reduced muscle injury via the prevention of mitochondrial dysfunction. Additionally, indirect antibacterial action by the neutrophil extracellular trap system (NETs) was observed. SalB administration to the CS rat model led to a substantial improvement in survival following CS by decreasing kidney and cardiac dysfunctions, inflammation, and endothelial dysfunction by improving the mitochondrial function and through antibacterial effects via NETs.

Thermodynamic Modeling of Solvent-Assisted Lipid Bilayer Formation Process.

The solvent-assisted lipid bilayer (SALB) formation method provides a simple and efficient, microfluidic-based strategy to fabricate supported lipid bilayers (SLBs) with rich compositional diversity on a wide range of solid supports. While various studies have been performed to characterize SLBs formed using the SALB method, relatively limited work has been carried out to understand the underlying mechanisms of SALB formation under various experimental conditions. Through thermodynamic modeling, we studied the experimental parameters that affect the SALB formation process, including substrate surface properties, initial lipid concentration, and temperature. It was found that all the parameters are critically important to successfully form high-quality SLBs. The model also helps to identify the range of parameter space within which conformal, homogeneous SLBs can be fabricated, and provides mechanistic guidance to optimize experimental conditions for lipid membrane-related applications.

Salvianolic acid B ameliorates atherosclerosis via inhibiting YAP/TAZ/JNK signaling pathway in endothelial cells and pericytes.

Atherosclerosis (AS) is a chronic disease of the arterial wall where both innate and adaptive immunoinflammatory mechanisms are involved. Inflammation plays an important role in the pathological process of atherosclerosis at various stages. Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ, also known as WWTR1) behave as a novel drug target against atherosclerosis. Therefore, the mechanism relationship of YAP/TAZ, inflammation and AS was explored in this study. Experiments demonstrated that serine dephosphorylation and nuclear translocation of YAP was increased in ECs and pericytes induced by oxidative low-density lipoprotein (ox-LDL), while the inhibition of YAP degraded the expression of downstream inflammatory factors. The expression of YAP/TAZ and inflammation proteins (JNK, NF-κB and TNF-α) in ECs and pericytes was suppressed through the application of Sal-B. Besides, Sal-B protects ECs and pericytes from oxidative stress and apoptosis. In vivo, Sal-B reduced en face and aortic root sinus lesions size, and decreased the expression of inflammation related factors (IL-6, IL-1ß, TNF-α) and ox-LDL in serum sample of ApoE-/- mice fed a high fat diet. Therefore, our work provides a potential therapeutic strategy of using Sal-B to attenuate the development of atherosclerosis, the anti-atherosclerosis effects of Sal-B is related to regulate YAP/TAZ/JNK signaling pathway.

Salvianolic Acid B Improves Chronic Mild Stress-Induced Depressive Behaviors in Rats: Involvement of AMPK/SIRT1 Signaling Pathway.

INTRODUCTION: Depression is one of the most common neuropsychiatric illnesses which leads to a huge social and economic burden on modern society. So, it is necessary to develop an effective and safe pharmacological intervention for depression. Accumulating evidence has shown that adenosine monophosphate-activated protein kinase/sirtuin 1 (AMPK/SIRT1) signaling pathway plays a pivotal role in the development of depression. Our present study aimed to investigate the antidepressant effect and possible mechanisms of salvianolic acid B (SalB) in a chronic mild stress (CMS)-induced depression model in rats. MATERIALS AND METHODS: The rats were randomly divided into three groups: control group with no stressor, CMS group and CMS+SalB (30 mg/kg/d) group. After administration for 28 consecutive days, the behavior tests were performed. The rats were sacrificed after behavior tests, and the brain tissues were collected for biochemical analysis. RESULTS: It was observed that the administration of SalB for 28 consecutive days successfully corrected the depressive-like behaviors in CMS-treated rats. SalB could effectively reduce the gene expression of pro-inflammatory cytokines such as interleukin-6 (IL-6), interleukin-1ß (IL-1ß) and tumor necrosis factor α (TNF-α), as well as nuclear factor-kappa B (NF-κB) p65 protein. In addition, inhibitor of NF-κB (IκB) protein expression was significantly increased after the administration of SalB. Moreover, SalB could effectively decrease protein expression of oxidative stress markers such as 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA) and increase the activity of catalase (CAT). SalB treatment also reversed CMS-induced inhibition of Nrf2 signaling pathway, along with increasing the mRNA expression of NAD(P)H:quinone oxidoreductase (NQO-1) and heme oxygenase 1 (HO-1). Regarding the endoplasmic reticulum (ER) stress markers, the protein expressions of C/EBP-homologous protein (CHOP) and glucose-regulated protein 78 kD (GRP78) were also significantly reduced after SalB administration. Furthermore, the supplementation of SalB could effectively activate the AMPK/SIRT1 signaling pathway, which indicated significant increase in pAMPK/AMPK ratio and SIRT1 protein expression. CONCLUSION: Our study demonstrated that SalB relieved CMS-induced depressive-like state through the mitigation of inflammatory status, oxidative stress, and the activation of AMPK/SIRT1 signaling pathway.

Effect of Toll-Like Receptor 4/Myeloid Differentiation Factor 88 Inhibition by Salvianolic Acid B on Neuropathic Pain After Spinal Cord Injury in Mice.

BACKGROUND: Spinal cord injury (SCI) is a common type of injury, and about half of patients affected by SCI will suffer from neuropathic pain within a year after injury. However, the treatment effect of neuropathic pain is far from satisfactory. Our study attempted to reveal whether salvianolic acid B (SalB) could relieve the neuropathic pain caused by SCI in mice by inhibiting the Toll-like receptor 4 (TLR4)/Myeloid differentiation factor 88 (MyD88) pathway. METHODS: The mice were randomly divided into a sham group, model group, high-dose treatment group, and low-dose treatment group. The high- and low-dose groups received varying doses of SalB after modeling. RESULTS: The increase of pain sensitivity was evaluated by detecting paw withdrawal mechanical threshold and withdrawal thermal latency. Messenger RNA and protein expression levels of TLR4 and myD88 were detected by using quantitative reverse-transcription polymerase chain reaction and western blot, respectively. Compared with the model group, there was a significant reduction in paw withdrawal mechanical threshold and withdrawal thermal latency after SalB treatment. CONCLUSIONS: SalB reduced the release of tumor necrosis factor-α and substance P by inhibiting the TLR4/MyD88 pathway in the SCI mouse model. This not only resulted in lower pain, but also contributed to long-term relief of mechanical hyperalgesia.

Salvianolic acid B remits LPS-induced injury by up-regulating miR-142-3p in MH7A cells.

BACKGROUND: Rheumatoid arthritis (RA) is a common inflammatory disease, which significantly reduces the quality of life and increases the risk of cardiovascular and cerebrovascular diseases. The present work studied the therapeutic potency of Salvianolic acid B (Sal-B) for RA and revealed one of the possible underlying mechanisms. METHODS: Human rheumatoid fibroblast-like synoviocytes (MH7 A) were treated with Sal-B before, during or after lipopolysaccharide (LPS) stimulation. CCK-8 assay, Annexin V-FITC/PI double-staining, RT-qPCR, Western blotting and ELISA were carried out to measure the changes of cell viability, apoptosis, and the release of pro-inflammatory cytokines. Next, the involvement of miR-142-3p and related signaling pathways in Sal-B-mediated protection was studied. RESULTS: Sal-B (10 µM) treatment significantly ameliorated LPS injury to MH7 A cells, as cell viability was increased, expression of p53 and p21 was repressed, apoptosis was inhibited, and the release of MCP-1, IL-6 and TNF-α was reduced. However, Sal-B (10 µM) treated alone has no impacts on MH7 A cells in the abovementioned aspects. miR-142-3p was down-regulated by LPS stimulation, while was up-regulated by treatment of Sal-B. Rescue assay results showed that Sal-B did not remit LPS injury when miR-142-3p was silenced. And also, the inhibitory effects of Sal-B on NF-κB and JNK pathways were abolished by miR-142-3p silence. CONCLUSION: Sal-B could protect against and reverse LPS-induced injury in MH7 A cells, showing anti-apoptotic and anti-inflammatory capacities. The anti-RA potential of Sal-B might be via up-regulating miR-142-3p, and subsequently modulating NF-κB and JNK pathways.

Chemogenetic inactivation of the dorsal hippocampus and medial prefrontal cortex, individually and concurrently, impairs object recognition and spatial memory consolidation in female mice.

The dorsal hippocampus (DH) and medial prefrontal cortex (mPFC) are brain regions essential for processing and storing episodic memory. In rodents, the DH has a well-established role in supporting the consolidation of episodic-like memory in tasks such as object recognition and object placement. However, the role of the mPFC in the consolidation of episodic-like memory tasks remains controversial. Therefore, the present study examined involvement of the DH and mPFC, alone and in combination, in object and spatial recognition memory consolidation in ovariectomized female mice. To this end, we utilized two types of inhibitory Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) to inactivate the DH alone, the mPFC alone, or both brain regions concurrently immediately after object training to assess the role of each region in the consolidation of object recognition and spatial memories. Our results using single and multiplexed DREADDS suggest that excitatory activity in the DH and mPFC, alone or in combination, is required for the successful consolidation of object recognition and spatial memories. Together, these studies provide critical insight into how the DH and mPFC work in concert to facilitate memory consolidation in female mice.

Salvianolic acid B as an anti-emphysema agent I: In vitro stimulation of lung cell proliferation and migration, and protection against lung cell death, and in vivo lung STAT3 activation and VEGF elevation.

Emphysema causes progressive and life-threatening alveolar structural destruction/loss, yet remains irreversible and incurable to date. Impaired vascular endothelial growth factor (VEGF) signaling has been proposed as a new pathogenic mechanism, and if so, VEGF recovery may enable reversal of emphysema. Thus, we hypothesized that salvianolic acid B (Sal-B), a polyphenol in traditional Chinese herbal danshen, is an alveolar structural recovery agent for emphysema by virtue of VEGF stimulation/elevation via activation of Janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3), as stimulating lung cell proliferation and migration, and protecting against lung cell death. Using in vitro human lung microvascular endothelial (HMVEC-L) and alveolar epithelial (A549) cell systems, Sal-B was examined for 1) stimulation of cell proliferation by the MTT and BrdU assays; 2) promotion of cell migration by the scratch wound closure assay; 3) protection against emphysema-like induced cell death by the trypan blue exclusion and flow cytometry assays; and 4) mechanistic involvement of JAK2/STAT3/VEGF in these activities. Sal-B was also spray-dosed to the lungs of healthy rats for two weeks to verify the lung's STAT3 activation and VEGF elevation by western blot, as well as the absence of functional and morphological abnormalities. All the in vitro cell-based activities were concentration-dependent. At 25 µM, Sal-B 1) stimulated cell proliferation by 1.4-2.6-fold; 2) promoted migratory cell wound closure by 1.5-1.7-fold; and 3) protected against cell death induced with H2O2 (oxidative stress) and SU5416 (VEGF receptor blockade) by 49-86%. JAK2 and STAT3 inhibitors and VEGF receptor antagonist each opposed these Sal-B's activities by over 65%, suggesting the mechanistic involvement of JAK2/STAT3 activation and VEGF stimulation/elevation. In rats, Sal-B at 0.2 mg/kg enabled 1.9 and 1.5-fold increased STAT3 phosphorylation and VEGF elevation in the lungs, respectively, while causing no functional and morphological abnormalities. Hence, Sal-B was projected to be a new class of anti-emphysema agent capable of reversing alveolar structural destruction/loss via JAK2/STAT3/VEGF-dependent stimulation of lung cell proliferation and migration, and inhibition of induced lung cell death.

Salvianolic acid B as an anti-emphysema agent II: In vivo reversal activities in two rat models of emphysema.

Emphysema progressively destroys alveolar structures, leading to disability and death, yet remains irreversible and incurable to date. Impaired vascular endothelial growth factor (VEGF) signaling is an emerging pathogenic mechanism, thereby proposing a hypothesis that VEGF stimulation/elevation enables recovery from alveolar structural destruction and loss of emphysema. Our previous in vitro study identified that salvianolic acid B (Sal-B), a polyphenol of traditional Chinese herbal danshen, stimulated lung cell proliferation and migration, and protected against induced lung cell death, by virtue of signal transducer and activator of transcription 3 (STAT3) activation and VEGF stimulation/elevation. Thus, this study examined Sal-B for in vivo therapeutic reversal of established emphysema in two rat models. Emphysema was induced with porcine pancreatic elastase (PPE) and cigarette smoke extract (CSE), and established by day 21. Sal-B was then spray-dosed to the lung three times weekly for three weeks. Functional treadmill exercise endurance; morphological airspace enlargement and alveolar destruction; apoptosis, cell proliferation and tissue matrix proteins; phosphorylated STAT3 (pSTAT3) and VEGF expressions; neutrophil accumulation; and lipid peroxidation were determined. In both models, Sal-B at 0.2 mg/kg significantly reversed impaired exercise endurance by 80 and 64%; airspace enlargement [mean linear intercept (MLI)] by 56 and 67%; and alveolar destructive index (%DI) by 63 and 66%, respectively. Induced apoptosis activity [cleaved caspase-3] was normalized by 94 and 82%; and cell proliferation activity [proliferative cell nuclear antigen (PCNA)] was stimulated by 1.6 and 2.1-fold. In the PPE-induced model, Sal-B reduced induction of lung's matrix metalloproteinase (MMP)-9 and MMP-2 activities by 59 and 94%, respectively, and restored pSTAT3 and VEGF expressions to the healthy lung levels, while leaving neutrophil accumulation unchecked [myeloperoxidase (MPO) activity]. In the CSE-induced model, Sal-B elevated pSTAT3 and VEGF expressions both by 1.8-fold over the healthy lung levels, and normalized induced lipid peroxidation [malondialdehyde (MDA) activity] by 68%. These results provide an in vivo proof-of-concept for Sal-B as one of the first anti-emphysema agents enabling reversal of alveolar structural destruction and loss via local lung treatment by virtue of its STAT3 activation and VEGF stimulation.

Extracellular SalB Contributes to Intrinsic Cephalosporin Resistance and Cell Envelope Integrity in Enterococcus faecalis.

Enterococci are major causes of hospital-acquired infections. Intrinsic resistance to cephalosporins is a universal trait among clinically relevant enterococci. Cephalosporin resistance enables enterococci to proliferate to high densities in the intestines of patients undergoing cephalosporin treatment, a precursor to the emergence of infection. However, the genetic and biochemical mechanisms of intrinsic cephalosporin resistance in enterococci are not well understood. A two-component signal transduction system, CroR/S, is required for cephalosporin resistance in enterococci. Although the CroR/S regulon is not well defined, one gene reported to be CroR dependent in Enterococcus faecalis JH2-2 encodes an extracellular putative peptidoglycan hydrolase, SalB. To test the hypothesis that SalB is responsible for CroR-dependent cephalosporin resistance, we examined ΔsalB mutants in multiple genetic lineages of E. faecalis, revealing that SalB is required not only for intrinsic cephalosporin resistance but also for maintenance of cell envelope integrity in the absence of antibiotic stress. The N-terminal signal sequence is necessary for SalB secretion, and secretion is required for SalB to promote cephalosporin resistance. Functional dissection revealed that the C-terminal SCP domain of SalB is essential for biological activity and identified three residues within the SCP domain that are required for the stability and function of SalB. Additionally, we found that in contrast to what is seen in E. faecalis JH2-2, SalB is not regulated by the CroR/S two-component system in E. faecalis OG1, suggesting diversity in the CroR/S regulon among distinct lineages of E. faecalis IMPORTANCE Resistance to cephalosporins is universal among clinically relevant enterococci, enabling enterococcal proliferation to high densities in the intestines of patients undergoing cephalosporin treatment, a precursor to the emergence of infection. Disabling cephalosporin resistance could therefore reduce the incidence of enterococcal infections. However, the genetic and biochemical mechanisms of cephalosporin resistance are not well understood. The significance of this work is the identification of a novel extracellular factor (SalB) that promotes cephalosporin resistance in E. faecalis, which could potentially serve as a target for therapeutics that impair enterococcal cephalosporin resistance. Additionally, our work highlights the importance of the C-terminal SCP domain of SalB, including several conserved residues within the SCP domain, for the ability of SalB to promote cephalosporin resistance.

Expression analysis of ROS producing and scavenging enzyme-encoding genes in rubber tree infected by Pseudocercospora ulei.

South American Leaf Blight (SALB), caused by the ascomycete Pseudocercospora ulei, is responsible for the low productivity of rubber trees in Latin America and is a serious threat to rubber plantations in Asia and Africa, where the rubber trees are derived from highly susceptible clones. Three contrasted genotypes were chosen for their levels of resistance to the pathogen: FX2784 (totally resistant), MDF180 (partially resistant) and PB314 (susceptible). Array analyses were previously performed to identify genes differentially expressed in resistant and susceptible genotypes. Twenty-one genes were selected for further gene expression analysis in non-inoculated and inoculated genotypes from 24 to 216 h post infection (hpi). These genes are involved in ROS production (HbRBOHA, HbRBOHB, HbRBOHC, HbRBOHD), ROS-scavenging systems (cytoplasmic and chloroplastic HbCuZnSOD, HbMnSOD, HbCAT, HbAPX1, HbAPX2, HbMDHAR, HbGCL1, HbGCL2, HbOASTL, HbGPX, HbDHAR), and leaf senescence (HbCASP, HbPCYST, HbWRKY2, HbPLY, HbKAT2). First, a genotype-dependent level of expression was observed. The genes HbRBOHA, HbCuZnSOD cyto, HbCAT, HbGCL and HbWRKY2 were constitutively expressed at lower levels in the MDF180 genotype than in the FX2784 and PB314 genotypes. Conversely, the levels of expression of HbDHAR, HbGPX and HbPCYST were higher in the older, non-inoculated leaves of MDF180. Lower production of ROS and efficient regeneration of reduced ascorbate ensure a balanced redox intracellular state in this genotype. Second, inoculation of the leaves induced few modifications in the expression level of the studied genes. In the MDF180 partially resistant genotype, an increase in the expression level of HbRBOHB, HbRBOHD 48 hpi and a decrease in the expression level of HbDHAR 216 hpi were observed. In the FX2784 totally resistant genotype, an increase in the expression level of HbRBOHD and HbCuZnSOD cyto and a decrease in HbCAT were observed 48 hpi. This transitory variation could be associated with the oxidative burst classically observed in hypersensitive response (HR). The increase in the synthesis of reduced glutathione in this genotype could ensure redox balance and consequently cell homeostasis. In the PB314 susceptible genotype, HbROHC, HbCuZnSOD chloro was up-regulated 216 hpi concomitantly with a decrease in the expression level of HbCAT, consequently causing an accumulation of H2O2 and programmed cell death. The level of expression of a transcription factor, HbWRKY2, was also modulated by the P. ulei infection with early transient up-regulation in the FX2784 totally resistant genotype and permanent up-regulation in the MDF180 partially resistant genotype. These results complement studies on genetic determinism of SALB resistance and a recent publication on Hevea glutathione reductase gene.

Pharmacokinetic study of salvianolic acid D after oral and intravenous administration in rats.

A sensitive, specific and rapid LC-MS method was developed and validated for the determination of salvianolic acid D (SalD) in rat plasma. This method used a single quadrupole mass spectrometer with an electrospray ionization (ESI) source. A single ion monitoring scanning (SIM) mode was employed. It showed good linearity over the concentration range from 3.3 to 666.7 ng/mL for the determination of SalD. The R.S.D.% of intra-day and inter-day precision values were no more than 7.69%, and the accuracy was within 91%-104% at all quality control levels. This LC-MS method was applied to the pharmacokinetic study of SalD in rats. A two-compartmental model analysis was employed. The plasma concentrations at 2 min (C 2min) were 5756.06±719.61, 11,073.01±1783.46 and 21,077.58±5581.97 µg/L for 0.25, 0.5 and 1 mg/kg intravenous injection, respectively. The peak plasma concentration (C max) was 333.08±61.21 µg/L for 4 mg/kg oral administration. The area under curve (AUC0-t ) was 14,384.379±8443.184, 22,813.369±11,860.823, 46,406.122±27,592.645 and 8201.740±4711.961 µg/L·h for intravenous injection (0.25, 0.5 and 1 mg/kg) and oral administration (4 mg/kg), respectively. The bioavailability of SalD was calculated to be 4.159%±0.517%.

Anti-pulmonary fibrotic activity of salvianolic acid B was screened by a novel method based on the cyto-biophysical properties.

Various methods have been used to evaluate anti-fibrotic activity of drugs. However, most of them are complicated, labor-intensive and lack of efficiency. This study was intended to develop a rapid method for anti-fibrotic drugs screening based on biophysical properties. A549 cells in vitro were stimulated with transforming growth factor-ß1 (TGF-ß1), and fibrogenesis was confirmed by conventional immunological assays. Meanwhile, the alterations of cyto-biophysical properties including morphology, roughness and stiffness were measured utilizing atomic force microscopy (AFM). It was found that fibrogenesis was accompanied with changes of cellular biophysical properties. TGF-ß1-stimulated A549 cells became remarkably longer, rougher and stiffer than the control. Then, the effect of N-acetyl-L-cysteine (NAC) as a positive drug on ameliorating fibrogenesis in TGF-ß1-stimulated A549 cells was verified respectively by immunological and biophysical markers. The result of Principal Component Analysis showed that stiffness was a leading index among all biophysical markers during fibrogenesis. Salvianolic acid B (SalB), a natural anti-oxidant, was detected by AFM to protect TGF-ß1-stimulated A549 cells against stiffening. Then, SalB treatment was provided in preventive mode on a rat model of bleomycin (BLM) -induced pulmonary fibrosis. The results showed that SalB treatment significantly ameliorated BLM-induced histological alterations, blocked collagen accumulations and reduced α-SMA expression in lung tissues. All these results revealed the anti-pulmonary fibrotic activity of SalB. Detection of cyto-biophysical properties were therefore recommended as a rapid method for anti-pulmonary fibrotic drugs screening.

Salvianolic acid B protects against acetaminophen hepatotoxicity by inducing Nrf2 and phase II detoxification gene expression via activation of the PI3K and PKC signaling pathways.

Acetaminophen (APAP) is used drugs worldwide for treating pain and fever. However, APAP overdose is the principal cause of acute liver failure in Western countries. Salvianolic acid B (SalB), a major water-soluble compound extracted from Radix Salvia miltiorrhiza, has well-known antioxidant and anti-inflammatory actions. We aimed to evaluate the ability of SalB to protect against APAP-induced acute hepatotoxicity by inducing nuclear factor-erythroid-2-related factor 2 (Nrf2) expression. SalB pretreatment ameliorated acute liver injury caused by APAP, as indicated by blood aspartate transaminase levels and histological findings. Moreover, SalB pretreatment increased the expression of Nrf2, Heme oxygenase-1 (HO-1) and glutamate-l-cysteine ligase catalytic subunit (GCLC). Furthermore, the HO-1 inhibitor zinc protoporphyrin and the GCLC inhibitor buthionine sulfoximine reversed the protective effect of SalB. Additionally, siRNA-mediated depletion of Nrf2 reduced the induction of HO-1 and GCLC by SalB, and SalB pretreatment activated the phosphatidylinositol-3-kinase (PI3K) and protein kinase C (PKC) signaling pathways. Both inhibitors (PI3K and PKC) blocked the protective effect of SalB against APAP-induced cell death, abolishing the SalB-induced Nrf2 activation and decreasing HO-1 and GCLC expression. These results indicated that SalB induces Nrf2, HO-1 and GCLC expression via activation of the PI3K and PKC pathways, thereby protecting against APAP-induced liver injury.

Primer reporte de susceptibilidad del clon de caucho natural FX-3864 a Microcyclus ulei en la altillanura colombiana / First report of susceptibility of natural rubber clone FX-3864 a Microcyclus ulei en la altillanuera colombiana

El mal suramericano de las hojas (SALB), enfermedad endémica del caucho (Hevea brasiliensis), es causado por Microcyclus ulei (forma imperfecta Fusicladium macrosporum) y constituye el principal limitante del cultivo en América, área donde el microorganismo patógeno es endémico. En forma semejante al de otros cultivos agrícolas, el manejo de esta enfermedad está condicionado a la disponibilidad de resistencia genética en el hospedero. En razón de su productividad y condición de resistencia genética, el clon FX 3864 ha sido ampliamente plantado en zonas con diferente potencialidad epidémica a la incidencia del SALB en Colombia, particularmente las denominadas de “no escape” a la enfermedad. Durante el 2010, plantaciones con el clon FX 3864 en fase productiva presentaron síntomas de SALB en zonas de escape ubicadas en la altillanura colombiana (departamento del Meta). En parcelas trampa ubicadas en áreas aledañas a los cultivos se estableció que la severidad promedio de la enfermedad alcanzó niveles de 5,78% en este clon. Verificada la causalidad de la enfermedad mediante observaciones al microscopio se procedió a confirmar el origen del material sobre el cual se desarrollaban las lesiones, utilizando marcadores moleculares (4 microsatélites específicos). Los resultados de la prueba permitieron confirmar la susceptibilidad del hasta hace poco resistente clon FX 3864 al SALB en Colombia. Se sugiere tomar en consideración la nueva condición de este clon y, en concordancia, reorientar los programas de fomento del cultivo advirtiendo a los agricultores sobre los riesgos potenciales de ocurrencia de la enfermedad en las nuevas áreas programadas.

South American Leaf Blight (SALB), caused by Microcyclus ulei (anamorph Fusicladium macrosporum), is an endemic major disease of the rubber tree (Hevea brasiliensis) in America. As well as in other crop systems, its management on rubber plantations relies on plant genetic resistance availability, among other means. FX 3864 is a rubber tree clone widely planted in Colombia due to its production capability and disease resistance. During 2010 SALB symptoms developed in commercial crops at the Meta region of Colombia. Crop traps located nearby the plantations showed mean disease severity levels of 5.78%. Once the causal organism was microscopically confirmed as responsible for the diseased tissue, their origin was characterized by molecular means using 4 microsatellites specific to the rubber tree. The procedure confirmed that FX 3864 was the clone of origin of the leaf tissue. SALB occurring over FX 3864 implies the need to redirect crop disease management measures to be followed on the new development areas of rubber cultivation, warning growers about potential hazards of disease incidence.

Investigation on the effect of SaIB on bone marrow-derived mesenchymal stem cells apoptosis induced by hypoxia and serum deprivation / 国际中医中药杂志

ObjectiveTo investigate the effect of SalB on bone marrow-derived mesenchymal stem cells (BMSCs) apoptosis induced by hypoxia and serum deprivation (hypoxia/SD) in the vitro. Methods BMSCs were cultured in the vitro and randomly divided into control group, hypoxia/SD group and SalB group.SalB group was composed by four groups and were pretreated by complete medium with 0.1、 1、 10、 100 mg/L SalB for 1 hour. And after that they were washed with phosphate buffer for 2 times, added by IMDM with 0.1、1、 10、 100 mg/L SalB and cultured with hypoxia/SD group together in the same condition of hypoxia/SD for 6hours. The control group was cultured for 6 hours in the condition of aerobic and enough serum. Apoptosis was detected by Hoechst33342 staining with inverted phase contrast, fluorescence microscope and Annexin V/PI dual-color flow cytometry. Results Significant apoptosis of BMSCs was induced by hypoxia/SD in the vitro.The early apoptosis of BMSCs induced by hypoxia/SD was significantly decreased by SalB of 0.1、 1、 10 mg/L(P＜0.05) . Conclusion0.1、 1、 10 mg/L SalB can decrease the early apoptosis of BMSCs induced by hypoxia/SD.