The study on interacting factors and functions of GASA6 in Jatropha curcas L.

BACKGROUND: The gibberellic acid-stimulated Arabidopsis (GASA) gene encodes a class of cysteine-rich functional proteins and is ubiquitous in plants. Most GASA proteins are influence the signal transmission of plant hormones and regulate plant growth and development, however, their function in Jatropha curcas is still unknown. RESULTS: In this study, we cloned JcGASA6, a member of the GASA family, from J. curcas. The JcGASA6 protein has a GASA-conserved domain and is located in the tonoplast. The three-dimensional structure of the JcGASA6 protein is highly consistent with the antibacterial protein Snakin-1. Additionally, the results of the yeast one-hybrid (Y1H) assay showed that JcGASA6 was activated by JcERF1, JcPYL9, and JcFLX. The results of the Y2H assay showed that both JcCNR8 and JcSIZ1 could interact with JcGASA6 in the nucleus. The expression of JcGASA6 increased continuously during male flower development, and the overexpression of JcGASA6 was associated with filament elongation of the stamens in tobacco. CONCLUSION: JcGASA6, a member of the GASA family in J. curcas, play an important role in growth regulation and floral development (especially in male flower). It is also involved in the signal transduction of hormones, such as ABA, ET, GA, BR, and SA. Also, JcGASA6 is a potential antimicrobial protein determined by its three-dimensional structure.

Establishment of protoplasts transient expression system in Pinellia ternata (Thunb.) Breit.

OBJECTIVE: In this study, we established an efficient and rapid transient expression system in the protoplasts of Pinellia ternata (Thunb.) Breit. (P. ternata). RESULTS: The protoplasts of P. ternata were prepared from plant leaves as the source material by digesting them with the combination of 20 g·l-1 cellulase and 15 g·l-1 macerozyme for 6 h. Based on the screening of PEG concentration, the conditions for PEG-mediated protoplast transformation were improved, and the highest transformation efficiency was found for 40% PEG 4000. Furthermore, we used the subcellular protein localization technique in P. ternata protoplasts to allow further validation of transient expression system. CONCLUSIONS: We present the method that can be applicable for studying both gene verification and expression in P. ternata protoplasts, thus allowing for engineering the improved varieties of P. ternata through molecular plant breeding techniques. This method can also be widely applicable for analyzing protein interactions, detecting promoter activity, for somatic cell fusion in plant breeding, as well as for other related studies.

Transcriptome revealing the dual regulatory mechanism of ethylene on the rhynchophylline and isorhynchophylline in Uncaria rhynchophylla.

Rhynchophylline (RIN) and isorhynchophylline (IRN) are extracted from Uncaria rhynchophylla, which are used to treat Alzheimer's disease. However, the massive accumulation of RIN and IRN in U. rhynchophylla requires exogenous stimulation. Ethylene is a potential stimulant for RIN and IRN biosynthesis, but there is no study on the role of ethylene in RIN or IRN synthesis. This study investigated the regulation of ethylene in RIN and IRN biosynthesis in U. rhynchophylla. An increase in the content of RIN and IRN was observed that could be attributed to the release of ethylene from 18 mM ethephon, while ethylene released from 36 mM ethephon reduced the content of RIN and IRN. The transcriptome and weighted gene co-expression network analysis indicated the up-regulation of seven key enzyme genes related to the RIN/IRN biosynthesis pathway and starch/sucrose metabolism pathway favored RIN/IRN synthesis. In comparison, the down-regulation of these seven key enzyme genes contributed to the reduction of RIN/IRN. Moreover, the inhibition of photosynthesis is associated with a reduction in RIN/IRN. Photosynthesis was restrained owing to the down-regulation of Lhcb1 and Lhcb6 after 36 mM ethephon treatment and further prevented supply of primary metabolites (such as α-D-glucose) for RIN/IRN synthesis. However, uninterrupted photosynthesis ensured a normal supply of primary metabolites at 18 mM ethephon treatment. AP2/ERF1, bHLH1, and bHLH2 may positively regulate the RIN/IRN accumulation, while NAC1 may play a negative regulatory role. Our results construct the potential bidirectional model for ethylene regulation on RIN/IRN synthesis and provide novel insight into the ethylene-mediated regulation of the metabolism of terpenoid indole alkaloids.

Comparative transcriptome analysis revealed the molecular mechanism of the effect of light intensity on the accumulation of rhynchophylline and isorhynchophylline in Uncaria rhynchophylla.

Rhynchophylline (RIN) and isorhynchophylline (IRN), the main medicinal components in plant Uncaria rhynchophylla, have potential effects on Alzheimer's disease. Understanding the influence of environmental factors, especially light intensity, on the production of these active ingredients will help to improve cultivation techniques. Compared with the 100% light intensity (CK), the contents of RIN and IRN in U. rhynchophylla leaves significantly increased at 20% light intensity (HS) after 7 and 21 days. Short-term shading (21d) changed some morphological indicators of U. rhynchophylla, but did not affect its biomass. Transcriptome profile analysis was performed on data from two groups (7 and 21 days) of CK and HS samples and yielded 79,817 unigenes with an average length of 1023 bp. Concurrently, 2391 and 2136 differentially expressed genes were identified in the transcriptome data for, respectively, 7 and 21 days of shade treatment. Notably, unigenes known to be involved upstream in the biosynthesis of RIN and IRN, such as G8O, IO, 7-DLGT, LAMT, TDC, and STR, were mostly upregulated. In addition, 1065 putative transcription factors (TFs) were identified and grouped into 55 TF families; 26 TFs showed differential expression in the shade treatment after 7 and 21 days. HY5 and PIFs, two important TFs of the light signaling pathway, also showed differential expression. This study provides insight into how gene expression was affected by light intensity during RIN and IRN accumulation in U. rhynchophylla. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-022-01142-2.

Ginsenoside Rg1 promoted the wound healing in diabetic foot ulcers via miR-489-3p/Sirt1 axis.

PURPOSE: Diabetic foot ulcers (DFUs) are common complications of high severity for diabetes. Ginsenoside Rg1 (Rg1) has the potential for diabetes and cardiovascular diseases therapy. This research aimed at exploring the regulation of Rg1 on DFUs treatment and the underlying mechanism. METHODS: Human umbilical vein endothelial cells (HUVECs) incubated with high-glucose culture medium were established for induction of diabetes model. The MTT assay, Annexin V/PI assay and oxidative stress detection were carried out on high-glucose-induced HUVECs. Dual-luciferase reporter assay was performed to prove the interaction of miR-489-3p and Sirt1. DFUs model was established to determine the efficiency of Rg1 and miR-489-3p in wound closure of DFUs in vivo. RESULTS: Rg1 promoted cell proliferation, migration and angiogenesis, and reduced cell apoptosis in high-glucose-induced HUVECs. Knockdown of miR-489-3p alleviated the high-glucose-induced damage to HUVECs, while overexpression of miR-489-3p attenuated the protection effects of Rg1. Overexpression Sirt1 promoted wound healing in DFUs and Sirt1 was a direct target of miR-489-3p. In addition, animal experiments demonstrated that Rg1 promoted wound closure by regulating miR-489-3p/Sirt1 axis. CONCLUSIONS: Rg1 alleviated the DFUs by increasing Sirt1 expression via miR-489-3p downregulation and promoting activation of PI3K/AKT/eNOS signaling.

Efficacy of 50 Hz electromagnetic fields on human epidermal stem cell transplantation seeded in collagen sponge scaffolds for wound healing in a murine model.

To explore the possible efficacy of electromagnetic fields (EMF) for skin tissue engineering, effects of EMF exposure on epidermal stem cells (ESC) seeded in collagen sponge scaffolds for wound healing in a murine model were investigated. The wound models of a full-thickness defect established with 36 7 ∼ 8-week-old nude mice were randomly divided into three groups: a control group, an ESC-only group, and an ESC with EMF exposure group (frequency of 50 Hz, magnetic induction of 5 mT, 60 min per day for 20 days). ESC were separated from human foreskin and cultured in vitro, and then transplanted with collagen sponge scaffolds as a delivery vehicle to wounds of the ESC-only group, and ESC with EMF exposure group was exposed to EMF after ESC transplantation. Effects of EMF on morphological changes and expression of ß1 integrin in regenerated skins were observed. Wound healing rates and healing times were collected to evaluate the efficacy of repairment. Results showed that human ESC were successfully transplanted to nude mice, which facilitated the formation of intact skin on nude mice. In contrast to other groups, the wound healing of ESC with EMF exposure group was the fastest (P < 0.05), the structure of regenerated skins was more mature, and it contained more continuity in the number of viable cell layers and rich hair follicles' structure. These results suggest that the use of 50 Hz EMF as a non-invasive treatment can accelerate wound healing of ESC transplantation, and restore structural integrity of regenerated skin. Bioelectromagnetics. 38:204-212,2017. © 2017 Wiley Periodicals, Inc.

[Vasodilation of quercetin on rat renal artery and the relationship with L-type voltage-gated Ca2+ channels and protein kinase C].

To investigate the diastolic function of quercetin on rat renal artery in vitro and its mechanism, the tension of rat renal artery was recorded by multi myograph system, and the L-type voltage-gated Ca2+ channels (LVGC) current was recorded by whole-cell patch clamp technique. Quercetin produced relaxation effect on rat renal artery pre-contracted by 60 mmol/L KCl or 1 × 10-5 mol/L phenylephrine, and the maximal diastolic percentage was  (84.53 ± 7.35)% or (76.42 ± 4.63)%. There was no statistical difference in the maximal diastolic percentage between endothelium-intact and endothelium-denuded groups. Pre-incubation of protein kinase C (PKC) inhibitor C6303 inhibited the maximal diastolic amplitude induced by quercetin. The peak current density of LVGC in rat renal artery vascular smooth muscle cells (VSMCs) was (23.17 ± 1.33) pA/pF. Quercetin (10 µmol/L) inhibited the peak current to (10.46 ± 1.35) pA/pF, and the inhibition percentage was 54.86%. C6303 (1 µmol/L) partially reversed the inhibitory effect of quercetin, and the inhibition percentage was 62.08% (P < 0.05). These results suggest that quercetin can relax rat renal artery in vitro in a concentration-dependent and endothelium-independent manner. The vasodilation of quercetin may be related to inhibition of LVGC current and activation of PKC.

[Mechanisms underlying rat coronary arterial vasoconstriction induced by blockade of inward rectifier potassium channels with BaCl2].

In order to explore the mechanisms underlying the vasoconstriction induced by blockade of inward rectifier K+ channels (Kir) with BaCl2, myogenic tone of isolated rat coronary artery (RCA) was recorded with wire myograph. The dependence of BaCl2- induced contraction on intracellular Ca2+ ([Ca2+]i) release and extracellular Ca2+ ([Ca2+]o) influx was studied by Ca2+ deprivation and restoration. The mechanisms underlying BaCl2-induced RCA contraction were investigated with specific inhibitors. BaCl2 (0.1-1.0 mmol/L) contracted isolated RCA in a concentration-dependent manner and the maximal contraction was (5.69 ± 1.07) mN, nearly equal to contraction induced by 60 mmol/L KCl. The contractions induced by BaCl2 in Ca2+-free solution and by followed restoration of 2.5 mmol/L Ca2+ accounted for (35.44 ± 6.72)% and (64.56 ± 5.94)%, respectively. Calcium channel blocker nifedipine (0.3 µmol/L), cyclooxygenase inhibitor indomethacin (100 µmol/L), ERK1/2 inhibitor PD98059 (10 µmol/L) and chloride channel blocker niflumic acid (100 µmol/L) pretreatment depressed the BaCl2-induced maximal contraction by (87.82 ± 5.43)% (P < 0.01), (73.23 ± 5.47)% (P < 0.01), (75.69 ± 7.94)% (P < 0.01) and (83.24 ± 7.69)% (P < 0.01), respectively. These results demonstrate that BaCl2 induces vasoconstriction in RCA by enhancing both [Ca2+]i release and [Ca2+]o influx, and suggest that increase of prostanoids synthesis, activation of calcium channels and chloride channels, as well as ERK1/2 pathway may be involved in this process.

Activation of caspase-12 at early stage contributes to cardiomyocyte apoptosis in trauma-induced secondary cardiac injury.

Trauma-induced secondary cardiac injury (TISCI) is associated with increased adverse cardiac events and death. We have previously reported that TISCI results in myocardial apoptosis and secondary cardiac dysfunction. However, the underlying mechanism is unclear. To identify the time course of trauma-induced cardiomyocyte apoptosis and possible apoptotic pathway, traumatic rat models were built with Noble-Collip drum. Meanwhile, normal rat cardiomyocytes were cultured with traumatic plasma (TP) for 48 h. Cardiomyocyte apoptosis, cardiac function and the apoptosis related enzymes, including caspase-3, -8, -9, and -12, were determined. The results showed that there was no direct injury of rat hearts immediately after trauma. However, compared with hearts from the sham rats, hearts isolated from traumatic rats exhibited reduced +dP/dTmax and -dP/dTmax 24 h after trauma. In traumatic rats, myocardial apoptotic index and caspase-3 activity obviously increased 6 h after trauma, and achieved the maximal value 12 h after trauma. The activity and expression of caspase-12, an endoplasmic reticulum (ER) stress-specific caspase, elevated markedly 3 h after trauma and reached its peak 6 h after trauma. Otherwise, caspase-8 (extrinsic apoptotic pathway) and caspase-9 (intrinsic apoptotic pathway) in the myocardial tissue of traumatic rats were activated 24 h after trauma. Meanwhile, incubation of normal rat cardiomyocytes with TP increased caspase-12 activity at 6 h, caspase-3 activity at 12 h, caspase-8 and -9 activities at 24 h, respectively. TP-induced cardiomyocyte apoptosis was virtually abolished by Z-ATAD-FMK (a caspase-12 specific inhibitor). In addition, there was a significant negative correlation between myocardial caspase-12 activity and trauma-induced secondary cardiac dysfunction. Our present study demonstrated that caspase-12 is firstly activated and plays an important role in TISCI rats. Inhibition of caspase-12 mediated apoptosis may be a novel strategy in ameliorating posttraumatic cardiomyocyte apoptosis and secondary cardiac injury.

Epigallocatechin-3-gallate protects HUVECs from PM2.5-induced oxidative stress injury by activating critical antioxidant pathways.

Endothelial dysfunction and oxidative stress likely play roles in PM2.5-induced harmful effects. Epigallocatechin-3-gallate (EGCG), the major polyphenolic constituent of green tea, is a potent antioxidant that exerts protective effects on cardiovascular diseases (CVDs) in part by scavenging free radicals. The exposure to ambient fine particulate matter (PM2.5) is responsible for certain CVDs. The aim of the present study was to investigate whether EGCG could also inhibit PM2.5-induced oxidative stress by activating the nuclear factor E2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway in human umbilical vein endothelial cells (HUVECs). PM2.5 (200 µg/mL) increased both cell death and intracellular ROS levels significantly, whereas EGCG (50-400 µM) inhibited these effects in a concentration-dependent manner. Western blotting and PCR demonstrated that EGCG increased Nrf2 and HO-1 expression in HUVECs that had been exposed to PM2.5. PD98059 (a selective inhibitor of extracellular signal regulated kinase [ERK]-1/2) and SB203580 (a selective inhibitor of p38 MAPK), but not SP600125 (a selective inhibitor of c-jun N-terminal kinase [JNK]), attenuated the EGCG-induced Nrf2 and HO-1 expression. In addition, silencing Nrf2 abolished EGCG-induced Nrf2 and HO-1 upregulation and enhancement of cell viability. The present study suggests that EGCG protects HUVECs from PM2.5-induced oxidative stress injury by upregulating Nrf2/HO-1 via activation of the p38 MAPK and the ERK1/2 signaling pathways.

Fifty-Hertz electromagnetic fields facilitate the induction of rat bone mesenchymal stromal cells to differentiate into functional neurons.

BACKGROUND AIMS: Research results have shown that bone mesenchymal stromal cells (BMSC) can different into neural cells. Electromagnetic fields (EMF) play a role in regulating cell proliferation and differentiation, but the mechanisms behind this are unknown. In the present study, we explored the efficacy of EMF on the induction of rat BMSC differentiation into neurons in vitro. METHODS: First, rat BMSC were induced in a nerve cell culture environment and divided into three groups: an EMF induction treatment group (frequency of 50 Hz, magnetic induction of 5 mT, 60 min per day for 12 days), an induction-only group and a control group. Second, we observed cell phenotypes in a confocal microscope, tested gene expression through the use of reverse transcriptase-polymerase chain reaction, and detected postsynaptic currents by means of a cell patch-clamp. We analyzed the cell cycles and the portion of cells expressing neural cell markers with the use of flow cytometry. RESULTS: The results indicated that EMF can facilitate BMSC differentiation into neural cells, which expressed neuronal-specific markers and genes; they formed synaptic junctions and pulsed excitatory postsynaptic currents. At the same time, the G0-G1 phase ratio recorded by means of flow cytometry gradually decreased under the EMF treatment, whereas there was an increase of S-phase ratio, and the portion of cells expressing neuronal-specific markers increased. CONCLUSIONS: Given that a noninvasive treatment of 50-Hz EMF could significantly facilitate BMSC to differentiate into functional neurons, EMF appears to be a promising clinical option for stem cell transplantation therapies to combat central nervous system diseases.

[Involvement of heme oxygenase in PM2.5-toxicity in human umbilical vein endothelial cells].

OBJECTIVE: To investigate the involvement of heme oxygenase (HO-1) in PM2.5 induced toxic responses in human umbilical vein endothelial cells (HUVECs). METHODS: The experiment groups are as follows: (1) control group; (2) PM2.5 groups: the cells were cultured with various concentrations of PM2.5 (200, 400, 800 µg/ml) for 24 h and 400 µg/ml was chosen for the main study; (3) PM2.5+Trion group: the cells were pre-treated by 10 µmol/L Trion [a scavenger of reactive oxygen species(ROS)] for 1 h before PM2.5 (400 µg/ml) treatment for 24 h; (4) PM2.5+ZnPP group: the cells were pretreated by HO-1 inhibitor ZnPP (10 µmol/L) for 1 h before treatment with PM2.5 (400 µg/ml) for 24 h. MTT assay was used to detect cell viability. Reverse transcription polymerase chain reaction (RT-PCR) and indirect immunofluorescence assay were used to determine the mRNA and protein expressions of HO-1. Fluorescence labeling probe method was used to measure intracellular ROS level and flow cytometry was used for cell apoptosis. Colorimetric assay was used to detect intracellular caspase-3 activity. RESULTS: Compared with control, PM2.5 significantly decreased cell viability, increased intracellular ROS, cell apoptosis and caspase-3 activity (all P < 0.05), these effects were significantly attenuated in PM2.5+Tiron group while enhanced in PM2.5+ZnPP group (all P < 0.05 vs. PM2.5 group). PM2.5 upregulated HO-1 mRNA and protein expressions in HUVECs which was downregulated in both PM2.5+Tiron group and PM2.5+ZnPP group. CONCLUSION: PM2.5 could induce oxidative injury through increasing ROS production via modulating HO-1 mRNA and protein expressions, the injury could be aggravated with inhibition of the activity of HO-1 suggesting a potential protective role of HO-1 against PM2.5 induced oxidative stress in HUVECs.

Delayed Chronic Acidic Postconditioning Improves Poststroke Motor Functional Recovery and Brain Tissue Repair by Activating Proton-Sensing TDAG8.

Acidic postconditioning by transient CO2 inhalation applied within minutes after reperfusion has neuroprotective effects in the acute phase of stroke. However, the effects of delayed chronic acidic postconditioning (DCAPC) initiated during the subacute phase of stroke or other acute brain injuries are unknown. Mice received daily DCAPC by inhaling 5%/10%/20% CO2 for various durations (three cycles of 10- or 20-min CO2 inhalation/10-min break) at days 3-7, 7-21, or 3-21 after photothrombotic stroke. Grid-walk, cylinder, and gait tests were used to assess motor function. DCAPC with all CO2 concentrations significantly promoted motor functional recovery, even when DCAPC was delayed for 3-7 days. DCAPC enhanced the puncta density of GAP-43 (a marker of axon growth and regeneration) and synaptophysin (a marker of synaptogenesis) and reduced the amoeboid microglia number, glial scar thickness and mRNA expression of CD16 and CD32 (markers of proinflammatory M1 microglia) compared with those of the stroke group. Cerebral blood flow (CBF) increased in response to DCAPC. Furthermore, the mRNA expression of TDAG8 (a proton-activated G-protein-coupled receptor) was increased during the subacute phase of stroke, while DCAPC effects were blocked by systemic knockout of TDAG8, except for those on CBF. DCAPC reproduced the benefits by re-expressing TDAG8 in the peri-infarct cortex of TDAG8-/- mice infected with HBAAV2/9-CMV-TDAG8-3flag-ZsGreen. Taken together, we first showed that DCAPC promoted functional recovery and brain tissue repair after stroke with a wide therapeutic time window of at least 7 days after stroke. Brain-derived TDAG8 is a direct target of DCAPC that induces neuroreparative effects.

[Effect of ginsenoside on fine particulate matter induced oxidative injury in human endothelial cells].

OBJECTIVE: To explore the mechanism of fine particulate matter (PM(2.5)) induced endothelial injury and the efficacy and mechanism of ginsenoside Rg1 on the inhibition of endothelium injuries in human endothelial cells exposure to PM(2.5). METHODS: Human umbilical vein endothelial cells (HUVECs) were stimulated with various concentrations PM(2.5) (0.1, 0.2, 0.4, 0.8 mg/ml) and PM(2.5) at concentration 0.8 mg/ml induced significant endothelial injury and was chosen for the main study in the presence or absence of Rg1 (0.04 mg/ml). After 24 h treatment, cell growth A value was detected through MTT, intracellular reactive oxygen species (ROS) level through fluorescence labeling probe method and HO-1, Nrf2 mRNA expression was detected by RT-PCR. RESULTS: The cell A value was significantly lower while the ROS fluorescence gray value and the average optical density ratio of HO-1 were significantly higher in PM(2.5) group than in the control group (all P < 0.05). The average optical density ratio of Nrf2 was similar between PM(2.5) group and control group (P > 0.05). The A value and the average optical density ratio of HO-1 were significantly higher while the ROS fluorescence gray value was significantly lower in co-treated PM(2.5) (0.8 mg/ml) + Rg1 (0.04 mg/ml) group than in the PM(2.5) (0.8 mg/ml) group (all P < 0.05). CONCLUSION: PM(2.5) could induce human endothelial cells injury by increasing oxidative stress which could be attenuated by ginsenoside Rg1.

Nab-paclitaxel plus S-1 versus oxaliplatin plus S-1 as first-line treatment in advanced gastric cancer: results of a multicenter, randomized, phase III trial (GAPSO study).

Background: This study aimed to investigate the superiority of nab-paclitaxel plus S-1 (AS) over oxaliplatin plus S-1 (SOX) in patients with advanced gastric cancer (AGC). Methods: In this multicenter, randomized, phase III superiority trial, eligible patients with unresectable, locally advanced gastric adenocarcinoma were recruited and randomly assigned (1:1) to receive AS (nab-paclitaxel 260 mg/m2 on day 1 or 130 mg/m2 on days 1 and 8; oral S-1 40-60 mg twice daily for 14 days) or SOX (130 mg/m2 oxaliplatin on day 1; oral S-1 40-60 mg twice daily for 14 days) every 3 weeks for up to six cycles. The primary endpoint was progression-free survival (PFS), and the secondary endpoints were overall survival, objective response rate, and safety. Results: Owing to slow enrolment, an unplanned interim analysis was performed, resulting in the early termination of the study on 31 December 2021 (data cutoff). Between March 2019 and March 2021, 97 patients (AS, n = 48; SOX, n = 49) were treated and evaluated for efficacy and safety of AS and SOX. As of the data cutoff, the median follow-up was 23.13 months [95% confidence interval (CI), 13.39-32.87]. The median PFS was 9.03 months (95% CI, 6.50-11.56) in the AS group and 5.07 months (95% CI, 4.33-5.81) in the SOX group, demonstrating a better PFS tendency following AS treatment than SOX treatment (hazard ratio = 0.59; 95% CI, 0.37-0.94; p = 0.03). The most common grade 3 or worse adverse events were anemia, neutropenia, and leukopenia in both groups, with a higher incidence of thrombocytopenia in the SOX group. Conclusion: Although this study was terminated early, the results demonstrated a better PFS tendency in patients with AGC who were treated with AS than in those treated with SOX, with controllable toxicities. Trial registration: Clinical Trials.gov identifiers: NCT03801668. Registered January 11, 2019.

Monophosphoryl lipid A-induced delayed preconditioning in rat small intestine is mediated by calcitonin gene-related peptide.

BACKGROUND: Protective effects of ischemic preconditioning in rat small intestine have been shown to be related to the release of calcitonin gene-related peptide. AIMS: The purpose of the present study was to explore whether monophosphoryl lipid A participated in the protective process of the delayed ischemic preconditioning in the peripheral vascular bed (the anse intestinalis of rat), and whether endogenous calcitonin gene-related peptide is a mediator in this process. METHODS: Intestinal ischemia was induced by occlusion of the superior mesenteric artery for 30 min, followed by reperfusion for 60 min. The intestinal lesions were evaluated by the measurement of serum lactate dehydrogenase, myeloperoxidase levels, and small intestine tissue contents of malondialdehyde. In addition, calcitonin gene-related peptide in plasma and superior mesenteric vein effluent were also examined. RESULTS: Pretreatment with monophosphoryl lipid A (500 µg/kg. i.p.) 24 h prior to ischemia-reperfusion significantly alleviated the intestinal tissue histology lesions, decreased serum levels of lactate dehydrogenase, myeloperoxidase, and reduced tissue content of malondialdehyde. Moreover, monophosphoryl lipid A markedly increased plasma concentrations of calcitonin gene-related peptide. Pretreatment with capsaicin, which specifically depletes the neurotransmitter content of sensory nerves or calcitonin gene-related peptide-(8-37), a selective calcitonin gene-related peptide receptor antagonist, inhibited the increased calcitonin gene-related peptide release and subsequently abrogated the protection by monophosphoryl lipid A. CONCLUSIONS: In conclusion, monophosphoryl lipid A pharmacologically mimics delayed preconditioning and the protective effects are related to the stimulation of calcitonin gene-related peptide release in rat small intestine.

Endothelial dysfunction induced by antibodies against angiotensin AT1 receptor in immunized rats.

AIM: To investigate the association between autoantibodies against angiotensin AT1 receptor (AT1-AAs) and endothelial dysfunction in vivo. METHODS: Rat models with AT1 receptor antibodies (AT1-Abs) were established by active immunization for nine months. Lactate dehydrogenase (LDH) activity was regarded as an indicator of cell necrotic death. Endothelin-1 (ET-1) in the sera of rats was determined and endothelium-dependent vasodilatation was detected in isolated thoracic aorta. Endothelial intercellular adhesion molecule-1 (ICAM-1) expression in aorta endothelium was assessed using confocal microscopy. Coronary artery endothelial ultrastructure was observed. RESULTS: IgGs in the immunized group significantly increased the LDH activity (0.84±0.17 vs 0.39±0.12, P<0.01 vs vehicle group IgGs)in incubated human umbilical vein endothelial cells through AT1 receptor. Higher content of ET-1 occurred in the immunized rats than that of the vehicle group, and reached two peaks at month 3 (27±4 ng/L, P<0.01) and month 7 (35±5 ng/L, P<0.01), respectively. In addition, aortic endothelium-dependent vasodilatation was attenuated; endothelial ICAM-1 level was markedly increased and cardiac capillary endothelium was damaged following immunization. CONCLUSION: Our study demonstrated that AT1-Abs contributed to endothelial dysfunction in vivo, which was a potential mechanism through which the antibodies play vital roles in related diseases.

Bone marrow stromal cells-derived exosomes target DAB2IP to induce microglial cell autophagy, a new strategy for neural stem cell transplantation in brain injury.

Bone marrow stromal cells (MSCs) are a useful source of stem cells for the treatment of various brain injury diseases due to their abundant supply and fewer ethical problems compared with transplant treatment. However, the clinical application of MSCs is limited due to allograft rejection and immunosuppression in the process of MSCs transplantation. According to previous studies, microglial cell autophagy occurs following co-culture with MSCs. In the present study, exosomes were obtained from MSCs and subsequently characterized using transmission electron microscopy, atomic force microscopy and dynamic light scattering particle size analysis. The type of microRNAs (miRs) found in the exosomes was then analyzed via gene chip. The results demonstrated that microglial cell autophagy could be induced by exosomes. This mechanism was therefore investigated further via reverse transcription-quantitative PCR, western blotting and luciferase assays. These results demonstrated that exosomes from MSCs could induce microglial cell autophagy through the miR-32-mediated regulation of disabled homolog 2-interacting protein, thus providing a theoretical basis for the clinical application of miRs in MSCs.

Delayed metformin treatment improves functional recovery following traumatic brain injury via central AMPK-dependent brain tissue repair.

Accumulating evidence suggests that chronic metformin posttreatment offers potent neuroreparative effects against acute brain injury. However, in previous studies, metformin was not initially administered beyond 24 h postinjury, and the effects of delayed metformin treatment in traumatic brain injury (TBI) and other types of acute brain injury and the related mechanisms are unclear. To test this, male C57BL/6 mice received once daily metformin treatment (20, 50 or 100 mg/kg/d, i.p.) at day 1-14, day 1-2, day 1-10, day 3-10, day 5-12 or day 5-28 after cryogenic TBI (cTBI). The results showed that 100 mg/kg/d metformin administered at day 1-14 postinjury significantly promoted motor functional recovery in the beam walking and gait tests and reduced the infarct volume. Metformin (100 mg/kg/d) administered at day 1-10 or day 3-10 but not day 1-2 or day 5-12 after cTBI significantly improved motor functional outcomes at day 7 and 14, and reduced the infarct volume at day 14. Interestingly, the therapeutic time window was further expanded when the duration of metformin treatment starting at day 5 postinjury was extended to 2 weeks. Furthermore, compared with cTBI, the administration of metformin at day 3-10 or day 5-28 after cTBI significantly elevated the expression of phosphorylated adenosine monophosphate-activated protein kinase (AMPK) and growth associated protein 43 (an axonal regeneration marker) and the number of vascular branch points and decreased the area of glial scar and the number of amoeboid microglia in the peri-infarct area at day 14 or 28 postinjury. The above beneficial effects of metformin were blocked by the intracerebroventricular injection of the AMPK inhibitor compound C (40 µg/mouse/d). Our data provide the first evidence that metformin has a wide therapeutic time window for at least 5 days after cTBI, during which it can improve functional recovery by promoting tissue repair and inhibiting glial scar formation and microglial activation in a central AMPK-dependent manner.