Platelet-rich plasma promotes peripheral nerve regeneration after sciatic nerve injury.

The effect of platelet-rich plasma on nerve regeneration remains controversial. In this study, we established a rabbit model of sciatic nerve small-gap defects with preserved epineurium and then filled the gaps with platelet-rich plasma. Twenty-eight rabbits were divided into the following groups (7 rabbits/group): model, low-concentration PRP (2.5-3.5-fold concentration of whole blood platelets), medium-concentration PRP (4.5-6.5-fold concentration of whole blood platelets), and high-concentration PRP (7.5-8.5-fold concentration of whole blood platelets). Electrophysiological and histomorphometrical assessments and proteomics analysis were used to evaluate regeneration of the sciatic nerve. Our results showed that platelet-rich plasma containing 4.5-6.5- and 7.5-8.5-fold concentrations of whole blood platelets promoted repair of sciatic nerve injury. Proteomics analysis was performed to investigate the possible mechanism by which platelet-rich plasma promoted nerve regeneration. Proteomics analysis showed that after sciatic nerve injury, platelet-rich plasma increased the expression of integrin subunit ß-8 (ITGB8), which participates in angiogenesis, and differentially expressed proteins were mainly enriched in focal adhesion pathways. Additionally, two key proteins, ribosomal protein S27a (RSP27a) and ubiquilin 1 (UBQLN1), which were selected after protein-protein interaction analysis, are involved in the regulation of ubiquitin levels in vivo. These data suggest that platelet-rich plasma promotes peripheral nerve regeneration after sciatic nerve injury by affecting angiogenesis and intracellular ubiquitin levels.

Activation of HIF-1α/VEGF-A pathway by deferoxamine ameliorates retinal hypoxia in a rat subarachnoid hemorrhage model.

BACKGROUND AND PURPOSE: Subarachnoid hemorrhage (SAH) is associated with sustained vasoconstriction in retinal vessels and vasoconstriction leads to retinal ischemia and hypoxia. Our previous finding also revealed the changes in hypoxia-related elements in the retina after SAH, further lending weight to the hypothesis that retinal vasospasm and hypoxia after SAH. Deferoxamine is a high-affinity iron chelator with reported neuroprotective effects against stroke. Here, we aimed to explore the effects of deferoxamine on retinal hypoxia after SAH. METHODS: SAH was established and deferoxamine was injected intraperitoneally for 3 days in the treatment group. To detect retinal new vessels, platelet endothelial cell adhesion molecule (CD31) was labeled by immunofluorescence and immunohistochemistry. Furthermore, the effects of deferoxamine on the expression of vascular endothelial growth factor A (VEGF-A) and hypoxia-inducible factor-1α (HIF-1α) were revealed by western blot analysis. RESULTS: The immunofluorescence and immunohistochemical staining of CD31 revealed a marked increase in new vessels in the retinal ganglion cell layer after deferoxamine treatment. By western blot analysis, HIF-1α and VEGF-A increased gradually in the first day and then rebounded to a new level on day 7. A deferoxamine-induced increase in HIF-1α/VEGF-A expression was also confirmed by western blot. CONCLUSIONS: Our findings suggest that modulating the application of deferoxamine may offer therapeutic approaches to alleviate retinal complications after SAH.

Inhibition of TRPA1 Attenuates Oxidative Stress-induced Damage After Traumatic Brain Injury via the ERK/AKT Signaling Pathway.

Neuron apoptosis is a feature of secondary injury after traumatic brain injury (TBI). Evidence implies that excess calcium (Ca2+) ions and reactive oxidative species (ROS) play critical roles in apoptosis. In reaction to increased ROS, the anti-oxidative master transcription factor, Transient receptor potential Ankyrin 1 (TRPA1) allows Ca2+ ions to enter cells. However, the effect of TBI on the expression of TRPA1 and the role of TRPA1 in TBI are unclear. In the present study, TBI in the mouse brain was simulated using the weight-drop model. The process of neuronal oxidative stress was simulated in HT22 neuronal cells by treatment with hydrogen peroxide. We found that TRPA1 was significantly upregulated in neurons at 24 h after TBI. Neuronal apoptosis was increased in the in vivo and in vitro models; however, this increase was reduced by the functional inhibition of TRPA1 in both models. After TBI, TRPA1 was upregulated via nuclear factor, erythroid 2 like 2 (Nrf2) in neurons. TRPA1-mediated neuronal apoptosis after TBI might be achieved in part through the CaMKII/AKT/ERK signaling pathway. To sum up, TBI-triggered TRPA1 upregulation in neurons is mediated by Nrf2 and the functional blockade of TRPA1 attenuates neuronal apoptosis and improves neuronal dysfunction, partially mediated through the activation of the calcium/calmodulin dependent protein kinase II (CaMKII) extracellular regulated kinase (ERK)/protein kinase B (AKT) signaling pathway. Our results suggest that functional blockade of TRPA1 might be a promising therapeutic intervention related to ROS and Nrf2 in TBI.

Expression of FOXO transcription factors in the brain following traumatic brain injury.

Traumatic brain injury (TBI) is a substantial clinical and social problem worldwide, causing high morbidity and mortality along with significant economic and medical costs. Forkhead box O transcription factors (FOXOs) have been found to play a critical role in the regulation of cell functions, such as nutrient metabolism, programmed cell death, and tumor suppression. In the central nervous system, FOXOs are reported to be pivotal regulators of learning and memory, neurite outgrowth, and axonal degeneration. However, the role of FOXOs in TBI is still unknown. Here, we investigate changes in the expression of FOXOs in the acute stage following TBI. First, we evaluated the expression of FOXO proteins in the brains of humans after TBI. A TBI model was then established in mice, and the ipsilateral cerebral cortex was collected at 3 h, 6 h, 9 h, 12 h, 24 h, and 72 h post-TBI. The dynamic expression of Foxo proteins was observed. Neuron-specific localization of Foxos was detected by double immunofluorescence staining. Following TBI, FOXO proteins in the brains of humans were significantly increased. In mice, Foxo protein levels generally peaked at 24 h. By examining co-localization with neurons, the proportion of Foxo(+) neurons was found to increase following TBI and peak at 24 h. This study reveals the time-dependent and neuron-specific expression of Foxos following TBI in mice, providing insight to enhance understanding of the role of Foxos in TBI.

Deferoxamine reduces amyloid-beta peptides genesis and alleviates neural apoptosis after traumatic brain injury.

Traumatic brain injury (TBI) is recognized as the most influential risk factor for neurodegenerative diseases later in life, including Alzheimer's disease. The aberrant genesis of amyloid-ß peptides, which is triggered by TBI, is associated with the development of Alzheimer's disease. Evidence suggests that iron plays a role in both the production of amyloid-ß and its neurotoxicity, and iron overload has been noted in the brain after TBI. We therefore investigated the effects of an iron-chelating treatment on amyloid-ß genesis in a weight-drop model of TBI in mice. Human brain samples were obtained from patients undergoing surgery for severe brain trauma. The Institute of Cancer Research mice were treated with deferoxamine by intraperitoneal injection after TBI induction. Changes in amyloid-ß(1-42) were assessed using western blot and immunohistochemical staining. Ferritin was also detected using western blot to investigate iron deposition in the mice brain. Immunofluorescent terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling was also performed to evaluate neural apoptosis. The amyloid-ß(1-42) was markedly elevated after TBI in both humans and mice. Deferoxamine treatment in mice significantly decreased the levels of both amyloid-ß(1-42) and ferritin in the brain, and reduced TBI-induced neural cell apoptosis. The iron chelator deferoxamine can alleviate the increase of amyloid-ß(1-42) in the brain after TBI, and may therefore be a potential therapeutic strategy to prevent TBI patients from undergoing neurodegenerative processes.

Lactic acid promotes metastatic niche formation in bone metastasis of colorectal cancer.

BACKGROUND: To investigate the effect of lactic acid (LA) on the progression of bone metastasis from colorectal cancer (CRC) and its regulatory effects on primary CD115 (+) osteoclast (OC) precursors. METHODS: The BrdU assay, Annexin-V/PI assay, TRAP staining and immunofluorescence were performed to explore the effect of LA on the proliferation, apoptosis and differentiation of OC precursors in vitro and in vivo. Flow cytometry was performed to sort primary osteoclast precursors and CD4(+) T cells and to analyze the change in the expression of target proteins in osteoclast precursors. A recruitment assay was used to test how LA and Cadhein-11 regulate the recruitment of OC precursors. RT-PCR and Western blotting were performed to analyze the changes in the mRNA and protein expression of genes related to the PI3K-AKT pathway and profibrotic genes. Safranin O-fast green staining, H&E staining and TRAP staining were performed to analyze the severity of bone resorption and accumulation of osteoclasts. RESULTS: LA promoted the expression of CXCL10 and Cadherin-11 in CD115(+) precursors through the PI3K-AKT pathway. We found that CXCL10 and Cadherin-11 were regulated by the activation of CREB and mTOR, respectively. LA-induced overexpression of CXCL10 in CD115(+) precursors indirectly promoted the differentiation of osteoclast precursors through the recruitment of CD4(+) T cells, and the crosstalk between these two cells promoted bone resorption in bone metastasis from CRC. On the other hand, Cadherin-11 mediated the adhesion between osteoclast precursors and upregulated the production of specific collagens, especially Collagen 5, which facilitated fibrotic changes in the tumor microenvironment. Blockade of the PI3K-AKT pathway efficiently prevented the progression of bone metastasis caused by lactate. CONCLUSION: LA promoted metastatic niche formation in the tumor microenvironment through the PI3K-AKT pathway. Our study provides new insight into the role of LA in the progression of bone metastasis from CRC. Video Abstract.

Retinal hypoxia after experimental subarachnoid hemorrhage.

BACKGROUND AND PURPOSE: The patients who survive subarachnoid hemorrhage (SAH) often have long-term neurological complications. There are no reports about the pathological change of retina after SAH. METHODS: An experimental model of SAH was established by injecting autologous blood into the prechiasmatic cistern of Sprague-Dawley rats. Hematoxylin and eosin (HE) staining was performed to show the alternation of morphology in retina after SAH. To detect the retinal new vessels (NVs), CD31 was labelled by immunofluorescence and immunohistochemistry. The time-course expressions of vascular endothelial growth factor (VEGF)-A and hypoxia-inducible factor-1α (HIF-1 α) was also revealed by Western blot analysis. RESULTS: A clear reduction of retinal ganglion cells (RGCs) was noticed after SAH. The immunofluorescence and immunohistochemical staining of CD31 reveals a large number of NVs in RGC layer after SAH compared with the normal controls. The level of VEGF-A in the retina after SAH was increased and peaked at 12h and 14 d. The expression of HIF-1α in the retina increased as early as 3 h after SAH, reached a peak at 12 h after SAH. CONCLUSIONS: The results showed that SAH induced the retina hypoxia resulting in the reduction of RGCs, increase of NVs and activation of NVs related HIF-1α/VEGF-A pathway.

Neuroprotective role of glutathione peroxidase 4 in experimental subarachnoid hemorrhage models.

BACKGROUND AND PURPOSE: Early brain injury is an essential pathological process after subarachnoid hemorrhage (SAH), with many cell death modalities. Ferroptosis is a newly discovered regulated cell death caused by the iron-dependent accumulation of lipid peroxidation, which can be prevented by glutathione peroxidase 4 (GPX4). Our study aimed to investigate the role of GPX4 in neuronal cell death after experimental SAH. METHODS: In vivo experimental SAH was induced by injecting autologous arterial blood into the prechiasmatic cistern in male Sprague-Dawley rats. Meanwhile, the in vitro SAH model was performed with primary rat cortical neurons cultured in medium containing hemoglobin (Hb). Adenovirus was used to overexpress GPX4 before experimental SAH. GPX4 expression was detected by western blot and immunofluorescence experiments. Malondialdehyde (MDA) was measured to evaluate the level of lipid peroxidation. Nissl staining was employed to assess cell death in vivo, whereas lactate dehydrogenase (LDH) release was used to evaluate cell damage in vitro. The brain water content and neurological deficits were evaluated to determine brain injury. RESULTS: Endogenous GPX4 was mainly expressed in neurons, and its expression decreased at 24 h after experimental SAH. Overexpression of GPX4 significantly reduced lipid peroxidation and cell death in the experimental SAH models both in vivo and in vitro. Moreover, overexpression of GPX4 ameliorated brain edema and neurological deficits at 24 h after SAH. CONCLUSIONS: The decrease of GPX4 expression potentially plays an important role in ferroptosis during early brain injury after SAH. Overexpression of GPX4 has a neuroprotective effect after SAH.

Cholecystokinin type 2 receptor in colorectal cancer: diagnostic and therapeutic target.

INTRODUCTION: Cholecystokinin type 2 receptor (CCK2R), which mediates the action of gastrin and cholecystokinin (CCK), has been demonstrated to promote the proliferation of colorectal cancer (CRC). A number of studies showed that CCK2R overexpressed in gastric cancer and pancreatic cancer but few in CRC. The correlation between CCK2R expression and clinicopathological characteristics is also not clear. METHODS: This study investigated CCK2R expression in a wide range of cell lines and clinical CRC samples, and explored expression pattern and prognostic value of CCK2R in relation to clinicopathological parameters. The location and expression levels of CCK2R were measured by immunocytochemical (ICC), qRT-PCR and Western blot. The druggability and antineoplastic effects of CCK2R as a therapeutic target were investigated using an anti-CCK2R targeting recombinant toxin named rCCK8PE38 by CCK-8 assay. RESULTS: Compared with paracarcinoma tissues, tumor samples showed overexpression of CCK2R (p = 0.028) including both CRC tissue and plasma samples, with plasma detection showing a significant indication for CCK2R evaluation. Aberrant expression correlated significantly with histological type (p = 0.032) and p53 status (p < 0.01), and patients with CCK2R overexpression had significantly lower disease-free survival. Application of rCCK8PE38 demonstrated the specificity and druggability of CCK2R as a therapeutic target, providing a strategy for clinical case screening of drugs targeting CCK2R. CONCLUSION: This study highlighted the aberrant expression and clinical correlation of CCK2R and reveals its diagnostic, prognostic and treatment value in CRC. We hypothesize that CCK2R serve as a target for the diagnosis and treatment of this cancer.

Oxytocin in the periaqueductal gray mainly comes form the hypothalamic supraoptic nucleus to participate in pain modulation.

Oxytocin (OXT) that effects the nociception process is mainly synthesized and secreted in the hypothalamic supraoptic nucleus (SON). Although the periaqueductal gray (PAG) hardly synthesizes OXT, OXT in PAG also plays a role in pain regulation. The communication investigates whether OXT in the PAG comes from SON to influence pain modulation. RT-PCR was used to analyze OXT mRNA expression and radioimmunoassay to measure OXT concentration. The results showed that (1) pain stimulation enhanced OXT mRNA expression in the SON at 10 min (268.1 ±â€¯39.2%, p < 0.001) and 20 min (135.4±37.9%, p < 0.05) treatment and did not change in the PAG; (2) OXT level increase in SON perfusion liquid during pain stimulation [236.7±22.1% at 10 min (p < 0.001), 223.1±12.4% at 20 min (p<0.001), 56.1 ±â€¯15.7% at 30 min (p < 0.01) and 11.2±14.2% at 40 min] was earlier than that in PAG perfusion liquid [17.8±9.7% at 10 min, 375.6±35.1% at 20 min (p <  0.001), 123.2±17.7% at 30 min (p <  0.001) and 52.7±22.4% at 40 min (p < 0.05)]; (3) SON excitation (L-glutamate sodium microinjection) induced OXT level increase in PAG perfusion liquid in a dose-dependent manner; (4) the bilateral SON cauterization completely controlled and the right SON cauterization partly reversed the pain stimulation induced-OXT concentration increase in PAG perfusion liquid. The data suggested that OXT in PAG came from SON, which might influence the pain process.

Molecular characterization and differential expression analysis of interleukin 1ß from Ovis aries.

The interleukin-1 family is an important component of the innate immune system and plays an important role in regulating immune responses on the invasion of intracellular parasites in the acquired immune system. Interleukin 1ß (IL-1ß) is one of the members of the IL-1 family that predominantly activates downstream signaling pathways to play immunological functions of stimulating T and B lymphocyte activation and promoting the various syntheses of inflammatory substances in conjunction with other cytokines. Here, a full-length IL-1ß cDNA (OaIL-1ß) of sheep (Ovis aries) was cloned using rapid amplification of cDNA ends (RACE), which consists of 1494 bp and contains a 5'-UTR region with a length of 83 bp, a complete ORF of 801 bp in length, and a 3'-UTR region with a length of 642 bp. Recombinant protein OaIL-1ß was expressed and purified, and the monoclonal antibody against IL-1ß of sheep is prepared. Western blotting results showed that the sheep IL-1ß protein was detected in the heart, liver, lung, kidney, stomach, intestine, muscle, lymph nodes and leukocytes with the highest expression in the muscle and the lowest expression in the lung. Different bacteria treating sheep white blood cells induced differential expression of OaIL-1ß. Compared with the normal sheep, OaIL-1ß in the buffy coat was differentially expressed in the Brucella melitensis-challenged group and the B. suis S2 strain-inoculated group. However, whether IL-1ß may be considered as a molecular biomarker for differing Brucella-infected animals from brucellosis-vaccinated animals or not need to be further studied.

Clinical targeting recombinant immunotoxins for cancer therapy.

Recombinant immunotoxins (RITs) are proteins that contain a toxin fused to an antibody or small molecules and are constructed by the genetic engineering technique. RITs can bind to and be internalized by cells and kill cancerous or non-cancerous cells by inhibiting protein synthesis. A wide variety of RITs have been tested against different cancers in cell culture, xenograft models, and human patients during the past several decades. RITs have shown activity in therapy of several kinds of cancers, but different levels of side effects, mainly related to vascular leak syndrome, were also observed in the treated patients. High immunogenicity of RITs limited their long-term or repeat applications in clinical cases. Recent advances in the design of immunotoxins, such as humanization of antibody fragment, PEGylation, and modification of human B- and T-cell epitopes, are overcoming the above mentioned problems, which predict the use of these immunotoxins as a potential therapeutic method to treat cancer patients.

Exploration of the Main Sites for the Transformation of Normal Prion Protein (PrPC) into Pathogenic Prion Protein (PrPsc).

INTRODUCTION: The functions and mechanisms of prion proteins (PrPC) are currently unknown, but most experts believe that deformed or pathogenic prion proteins (PrPSc) originate from PrPC, and that there may be plural main sites for the conversion of normal PrPC into PrPSc. In order to better understand the mechanism of PrPC transformation to PrPSc, the most important step is to determine the replacement or substitution site. MATERIAL AND METHODS: BALB/c mice were challenged with prion RML strain and from 90 days post-challenge (dpc) mice were sacrificed weekly until all of them had been at 160 dpc. The ultra-structure and pathological changes of the brain of experimental mice were observed and recorded by transmission electron microscopy. RESULTS: There were a large number of pathogen-like particles aggregated in the myelin sheath of the brain nerves, followed by delamination, hyperplasia, swelling, disintegration, phagocytic vacuolation, and other pathological lesions in the myelin sheath. The aggregated particles did not overflow from the myelin in unstained samples. The phenomenon of particle aggregation persisted all through the disease course, and was the earliest observed pathological change. CONCLUSION: It was deduced that the myelin sheath and lipid rafts in brain nerves, including axons and dendrites, were the main sites for the conversion of PrPC to PrPSc, and the PrPSc should be formed directly by the conversion of protein conformation without the involvement of nucleic acids.

Establishment of a three-step purification scheme for a recombinant protein rG17PE38 and its characteristics identification.

A protein with high purity has become an essential pre-requisite for investigating its bioactivity, molecular structure and characteristics. Therefore, the development of technologies for efficient purification of protein is urgently necessary. The objective of this study was to establish a purification protocol for a recombinant protein rG17PE38. Different forms of chromatography such as hydrophobic interaction and ion exchange chromatography were chosen as the core purification steps. The performance of each technique was optimized to meet the requirements and the purification steps were arranged in a logical way of facilitating to operate in next step. In addition, some characteristics of the protein such as stability, bioactivity and cellular location were determined. Finally, whether the protein could induce cell apoptosis was also explored. The results showed the protein purified via the suggested three-step purification scheme could obtain a purity of 95%, and its bioactivity in the form of IC50 was 17.6 ng/mL, furthermore it could keep stable at 4 °C for at least 10 days. The protein could bind on its target cell membrane specifically, and inducing cell apoptosis was demonstrated to be one of the cytotoxicity mechanisms of the protein. Results obtained in our study may provide useful information on strategies of protein purification and lay a substantial foundation for the followed animal or clinical experiments on rG17PE38.

Expression, purification and characterization of recombinant toxins consisting of truncated gastrin 17 and pseudomonas exotoxin.

Gastric cancer is a major cause of mortality and morbidity around world. However the effectiveness of the current approaches to the diagnosis and treatment of gastric cancer is limited. Recombinant targeted toxins may represent a novel direction of cancer therapy. In this study, we aimed to explore whether recombinant toxins fused with the truncated forms of G17 could target to kill cancer cells by recognizing CCK2R. Four recombinant Pseudomonas toxins PE38 fused with the forward or reverse truncated forms of G17 (G14 and G13) were successfully constructed, expressed, and purified. Their characteristics were further analyzed by SDS-PAGE, western blot and indirect immunofluorescence assay. The cytotoxicity assay demonstrated that only reversely fused recombinant toxins rG14PE38 and rG13PE38 exhibited certain toxicity on several cancer cell lines, and a competition assay indicated that the binding of the reverse gastrin-endotoxin to CCK2R (+) cells may be mediated by interaction between gastrin/gastrin-like and CCK2R.

Simple, specific, sensitive and rapid loop-mediated method for detecting Yersinia enterocolitica.

Yersinia enterocolitica (YE) is a main pathogenic bacterium causing diarrhea and yersiniosis occurs in both developed and developing countries with high incidence. YE in contaminated food is able to survive for a long duration even under cold storage, thereby enhancing the risk of food infection. In this study, a new loop-mediated isothermal amplification (LAMP) method showing the characteristics of simplicity, rapidity, high specificity and sensitivity was established by targeting outL of pathogenic YE. Two inner-primers and outer-primers were designed and LAMP reaction was optimized for Mg2+, betaine, dNTPs and inner primers concentrations, reaction temperature and time. Sensitivity and specificity of the LAMP assay was evaluated using YE genomic DNA and those of 44 different bacteria strains, respectively. Validation of LAMP detection method was by employing meat samples spiked with varying CFU of YE. The optimized LAMP assay was specific, capable of detecting 97 fg of genomic DNA (equivalent to 37 genome copies) of YE (100-fold more sensitive than PCR) and 80 CFU/ml of YE-spiked meat samples based on ethidium bromide stained amplicon bands on agarose gel-electrophoresis and on GelRed fluorescence of the LAMP reaction solution, respectively. This rapid, sensitive and specific LAMP technique should enable application in field inspection of Y. enterocolitica in food.

[Osteogenetic capacity of cross-linked adjustable anti-tuberculosis drug sustained-release artificial composite].

OBJECTIVE: To explore the osteogenetic capacity of cross-linked adjustable anti-tuberculosis drug sustained-release artificial composite (TPB/SA-RFP/PLA). METHODS: The model of femur bone defect was established in rabbits.TPB/SA-RFP/PLA complex was implanted into defect parts in the experimental group while TPB/SA/PLA in the blank control group. At Weeks 4, 8 and 12, gross specimens received radiographic, histological and immunohistochemical examinations to determine the osteogenetic performance of TPB/SA-RFP/PLA. RESULTS: As compared with the control group, TPB/SA-RFP/PLA complex had excellent osteogenic capacities while the TPB/SA/PLA group had no obvious osteogenic difference. Lane-sandhu histological and radiographic ratings demonstrated significant difference between TPB/SA-RFP/PLA (8.3 ± 0.3) and blank groups (2.2 ± 0.4) (P < 0.05). And TPB/SA/PLA showed no significant intragroup significance (P > 0.05). Two groups immunohistochemical Alkaline phosphatase was strongly positive in two test groups and weakly positive in the control group. CONCLUSION: TPB/ SA-RFP/PLA has excellent profiles of bone conductivity and regeneration.And the incorporation of rifampin does not affect its osteogenetic capacity.