Beneficial Effects of Ethyl Pyruvate through Inhibiting High-Mobility Group Box 1 Expression and TLR4/NF-κB Pathway after Traumatic Brain Injury in the Rat

Ethyl pyruvate (EP) has demonstrated neuroprotective effects against acute brain injury through its anti-inflammatory action. The nuclear protein high-mobility group box 1 (HMGB1) can activate inflammatory pathways when released from dying cells. This study was designed to investigate the protective effects of EP against secondary brain injury in rats after Traumatic Brain Injury (TBI). Adult male rats were randomly divided into three groups: (1) Sham + vehicle group, (2) TBI + vehicle group, and (3) TBI + EP group (n = 30 per group). Right parietal cortical contusion was made by using a weight-dropping TBI method. In TBI + EP group, EP was administered intraperitoneally at a dosage of 75 mg/kg at 5 min, 1 and 6 h after TBI. Brain samples were harvested at 24 h after TBI. We found that EP treatment markedly inhibited the expressions of HMGB1 and TLR4, NF-κB DNA binding activity and inflammatory mediators, such as IL-1β, TNF-α and IL-6. Also, EP treatment significantly ameliorated beam walking performance, brain edema, and cortical apoptotic cell death. These results suggest that the protective effects of EP may be mediated by the reduction of HMGB1/TLR4/NF-κB-mediated inflammatory response in the injured rat brain

Suppression of JAK2/STAT3 signaling reduces end-to-end arterial anastomosis induced cell proliferation in common carotid arteries of rats.

BACKGROUND: JAK2/STAT3 pathway was reported to play an essential role in the neointima formation after vascular intima injury. However, little is known regarding this pathway to the whole layer injury after end-to-end arterial anastomosis (AA). Here, we investigated the role of JAK2/STAT3 pathway in common carotid arterial (CCA) anastomosis-induced cell proliferation, phenotypic change of vascular smooth muscle cells (VSMCs) and re-endothelialization. METHODS: CCAs of adult male Wistar rats were resected at 3, 7, 14, and 30 days after end-to-end CCA anastomosis. Activation of JAK2/STAT3 pathway was detected by Western blotting and Immunofluorescence, and expression of proliferating cell nuclear antigen (PCNA) was detected by Q-PCR and Western blotting. Under the treatment with AG490 (a JAK2 inhibitor), protein levels of JAK2, STAT3 and PCNA, morphological changes of artery, phenotypic change of VSMCs, and re-endothelialization were measured by Western blotting, H&E, Q-PCR, and Evans blue staining respectively. RESULTS: The protein levels of p-JAK2, p-STAT3, and PCNA were up-regulated, peaked on the 7(th) day in the vessel wall after AA. AG490 down-regulated the levels of p-JAK2, p-STAT3, and PCNA on the 7(th)-day-group, resulting in reduced vessel wall proliferation on the 7(th) and 14(th) day after AA. Besides, AG490 switched the phenotypic change of VSMCs after AA representing inhibited mRNA levels of synthetic phase markers (osteopoitin and SMemb) and up-regulated contractile phase markers (ASMA, SM2 and SM22α). Furthermore, AG490 did not affect the re-endothelialization process on all indicated time points after AA (the 3(rd), 7(th), 14(th), and 30(th) day). CONCLUSION: Our study indicated that JAK2/STAT3 signaling pathway played an important role on cell proliferation of the injured vessel wall, and probably a promising target for the exploration of drugs increasing the patency or reducing the vascular narrowness after AA

Phenotypic modulation of VSMCs by AG490 after AA procedure.

<p>The mRNA levels of ASMA, SM2, and SM22α was lowered after AA, while that of osteopoitin and SMemb were raised markedly and peaked at 7^th day after AA. At the 14^th day time point, expression of SM2 and SM22α began to increase but still lower than Sham group, and expression of osteopoitin and SMemb decreased to lower levels. AG490 down-regulated the osteopoitin and SMemb expression, and had a trend to increase the ASMA and SM2 mRNA levels. All the results were normalized to Sham. n = 4 anastomoses. (^* p<0.05, ^** p<0.01 versus AA+AG490 respectively).</p

activation features of JAK2/STAT3 in the vessel wall after AA.

<p><b>A–F</b>: p-STAT3 positive cells (red) in Sham group and AA group determined by immunofluorecence (Samples: 7 days after AA; Arrows indicated the typical positive cells. n = 3 anastomoses. Bar = 100 µm). <b>G–I</b>: Representative bands of target proteins in Sham group and AA groups at 3, 7, and 14 days after surgery (n = 4 anastomoses). The analysis showed the expression p-JAK2 and p-STAT3 increased and peaked at 7 days after the AA procedure (^** p<0.01 versus Sham group).</p

AG490 inhibited the up-regulation of p-JAK2, p-STAT3 and PCNA, and attenuated cell proliferation after AA.

<p><b>A</b>: AG490 down-regulated the activation of JAK2/STAT3 in the AA+AG490 group on the 7^th day (n = 4 anastomoses). <b>B–D</b>: mRNA levels of PCNA were up-regulated after AA, and inhibited markedly after treating with AG490 on the 7^th day. Protein levels of PCNA were increased and peaked at the 7^th day after AA, and down-regulated in AA+AG490 group (n = 4 anastomoses). <b>E–F:</b> Morphometry analysis of CCA on the 7^th and 14^th day. Quantification analysis results were illustrated by the outside radius (R_o)/inside radius (R_i) ratio. Bar = 500 µm. n = 3 anastomoses. (^* p<0.05 versus AA+AG490 group (7^th day); ^** p<0.01 versus Sham group; ## p<0.01, §§ p<0.01; NS: no significant difference).</p

Re-endothelialization assessment on AA site at different experimental time points.

<p>Quantification analysis of the blue staining area demonstrated heavy staining in and around the AA sites, which peaked at the 3^rd day, and regressed to normal level at the 30^th day after AA. No statistic difference was found between the AA+DMSO and AA+AG490 groups at all indicated time points. n = 3 anastomoses.</p

DNA-Conjugated Quantum Dot Nanoprobe for High-Sensitivity Fluorescent Detection of DNA and micro-RNA

Herein, we report a convenient approach to developing quantum dots (QDs)-based nanosensors for DNA and micro-RNA (miRNA) detection. The DNA-QDs conjugate was prepared by a ligand-exchange method. Thiol-labeled ssDNA is directly attached to the QD surface, leading to highly water-dispersible nanoconjugates. The DNA-QDs conjugate has the advantages of the excellent optical properties of QDs and well-controlled recognition properties of DNA and can be used as a nanoprobe to construct a nanosensor for nucleic acid detection. With the addition of a target nucleic acid sequence, the fluorescence intensity of QDs was quenched by an organic quencher (BHQ₂) via Förster resonance energy transfer. This nanosensor can detect as low as 1 fM DNA and 10 fM miRNA. Moreover, the QDs-based nanosensor exhibited excellent selectivity. It not only can effectively distinguish single-base-mismatched and random nucleic sequences but also can recognize pre-miRNA and mature miRNA. Therefore, the nanosensor has high application potential for disease diagnosis and biological analysis

Target-Induced Conjunction of Split Aptamer Fragments and Assembly with a Water-Soluble Conjugated Polymer for Improved Protein Detection

Rapid and sensitive detection of proteins is crucial to biomedical research as well as clinical diagnosis. However, so far, most detection methods rely on antibody-based assays and are usually laborious and time-consuming, with poor sensitivity. Herein, we developed a simple and sensitive fluorescence-based strategy for protein detection by using split aptamer fragments and a water-soluble polycationic polymer (poly­{[9,9-bis­(6′-(<i>N</i>,<i>N</i>,<i>N</i>-diethylmethylammonium)­hexyl)-2,7-fluorenylene ethynylene]-alt-<i>co</i>-[2,5-bis­(3′-(<i>N</i>,<i>N</i>,<i>N</i>-diethylmethylammonium)-1′-oxapropyl)-1,4-phenylene] tetraiodide} (PFEP)). The thrombin-binding DNA aptamer was split into two fragments for target recognition. The PFEP with high fluorescence emission was used as energy donor to amplify the signal of dye-labeled DNA probe. In the absence of target, three DNA/PFEP complexes were formed via strong electrostatic interactions, resulting in efficient Föster resonance energy transfer (FRET) between two fluorophores. While the presence of target induces a conjunction of two split aptamer fragments to form G-quadruplex, and subsequent assemble with PFEP leading to the formation of G-quadruplex/thrombin/PFEP complex. The distance between the PFEP and dye increased due to protein’s large size, leading to a remarkable decrease of the FRET signal. Compared with the intact aptamer, the use of shorter split aptamer fragments increases the possibility of forming G-quadruplex upon target. Thus, the rate of change of FRET signal before and after the addition of target improved significantly and a higher sensitivity (limit of detection (LOD) = 2 nM) was obtained. This strategy is superior in that it is rapid, has low cost and homogeneous detection, and does not need heating to avoid an unfavorable secondary structure of DNA probe. With further efforts, this method could be extended to a universal way for simple and sensitive detection of a variety of biomolecules