Rat Peripheral Blood Research Articles

The mechanism of acupuncture on hyperplasia of mammary glands by the pathway of vagus nerve

Objective The experiment was designed to study the therapeutic mechanism of Tong Tiao acupuncture in the treatment of mammary gland hyperplasia (MGH) on the rat model. Methods A total of 75 healthy unpregnant female SD rats were randomly divided into six groups: blank, model, control group, vagus cutting therapy, sham operation, and vagus cutting with non-treatment group. All the groups except the blank group were prepared by MGH rat model. The vagus cutting therapy and vagus cutting with non-treatment group were treated by cutting the right cervical vagus nerve, while the sham operation group was treated by exposing the vagus nerve without any operation. All the treatment groups were treated with Tong Tiao. The rat papillary height was measured. The breast tissue morphology and breast ultrastructure were observated. And the flow cytometry in rat peripheral blood T lymphocyte subsets CD3+, CD4+, CD8+, CD4+/CD8+ were calculated. Results Compared with the model group, the papillary height (1.22 mm ± 0.21 mm, 1.24 mm ± 0.20 mm vs. 1.53 mm ± 0.16 mm) in the normal treatment and the sham treatment group decreased significantly the vagus cutting therapy and vagus cutting non-treatment group showed no difference (P>0.05). Compared with the model group, the Mammary gland morphology and ultrastructure in the normal treatment group, the sham operation group and the vagus cutting therapy groups were improved, but the vagus cutting non-treatment group showed on improvement. Compared with model group, the serum CD3+(61.5% ± 10.5%, 61.1% ± 12.4%, 59.8% ± 11.8% vs. 57.7% ± 9.4%), CD4+(37.2% ± 8.9%, 37.9% ± 10.9%, 36.1% ± 12.2% vs. 34.6% ± 6.9%), CD4+/CD8+(1.59 ± 0.8, 1.58 ± 0.8, 1.38 ± 0.6 vs. 1.23 ± 0.7) in the normal treatment group, the sham treatment group and vagus cutting therapy group, elevated significantly (P 0.05). Compared with model group, the serum CD8+ (24.2% ± 11.3%, 24.5% ± 10.6%, 25.5% ± 8.8% vs. 27.7% ± 9.9%) in the normal treatment group, sham treatment group and vagus cutting therapy group decreased significantly lower (P 0.05). Conclusions the mechanism of acupuncture treatment for MGH may be related to the vagus nerve by regulating the CD3+, CD4+, CD8+ and CD4+/CD8+ levels of T lymphocytes. Key words: Electroacupuncture; Vagus nerve; Mammary gland hyperplasia; T lymphocyte subsets; Rats

Radioprotective effect of melatonin on expression of Cdkn1a and Rad50 genes in rat peripheral blood.

Ionizing radiation is a critical threat to biomolecules, especially DNA. Various combinatorial compounds have been studied to protect this biomolecule. Melatonin has been reported as a direct and indirect free radical scavenger, but in this study, we explored the effect of melatonin on assisting in DNA repair by expression of Cdkn1a and Rad50; both of these genes are involved in DNA repair signaling, induced by radiation in rat peripheral blood. Rats were irradiated with single whole-body linear accelerator X-ray radiation doses of 2 and 8 Gy with or without melatonin (100 mg/kg body weight) pretreatments. The rats were randomly divided into nine groups and given an intraperitoneal injection of melatonin or the same volume of vehicle alone 1 h before radiation. Blood samples were taken 8, 24, and 48 h postradiation to measure gene expression of Cdkn1a and Rad50 using quantitative reverse transcription polymerase chain reaction technique. Melatonin pretreatment increased the expression of Cdkn1a and Rad50 in 8 and 24 h postradiations (2 and 8 Gy) (P < 0.05), and there was no significant difference in 48 h postradiation compared to the radiation-only and vehicle plus radiation (2 and 8 Gy) groups. Based on our results, pretreatment with melatonin (100 mg/kg) may ameliorates injurious effects of 2 and 8 Gy ionization radiation by increasing the expression level of Cdkn1a and Rad50 in rat peripheral blood and assist in DNA double-strand breaks repair, especially during the early postradiation.

The impact of bone marrow-derived mesenchymal stem cells on neovascularisation in rats with brain injury

The impact of bone marrow-derived mesenchymal stem cells (BM-MSCs) on neovascularisation in rats with craniocerebral injury (CI) was investigated. The rate CI model was established by the free-fall method, and then rats were randomly divided into atransplanted group (EG) and acontrol group (CG). Rats in the transplanted group were injected with BM-MSCs in the lateral ventricle, while those in the control group were injected with normal saline. Modified neuro-logical severity scores (mNSSs) were compared. Furthermore, CD34+CD133 double-labelled cells (CD34+CD133+ cells) in the peripheral blood and expression of CD31 and neuron-specific enolase in injured tissues were detected. There were significant intra- and intergroup differences in modified neurological severity scores at different time points. The number of CD34+CD133+ cells in the peripheral blood of CG initially decreased, but it increased later and peaked 6 h after injury, then gradually decreased and returned to normal 24 h after injury. Cells in the periphe-ral blood of EG continued to increase until 24 h after injury, reaching anumber higher than that in CG (p < 0.05). The postoperative expression of neuron-specific enolase in EG was higher than that in CG (p < 0.05). The positive expression of CD31 was lower in the two groups before surgery, but the expression in EG was higher than that in CG after surgery (p < 0.05). Bone marrow-derived mesenchymal stem cells transplantation can increase the number of endothelial progenitor cells in the peripheral blood of rats with traumatic brain injury and increase the expression of peripheral angiogenetic markers and neuronal markers. The neurological function in EG improved significantly compared to that of CG.

Involvement of brain-gut axis in treatment of cerebral infarction by β-asaron and paeonol

Cerebral infarction (CI) causes severe brain damage with high incidence. This study aimed to investigate the involvement of brain-gut axis in the treatment of CI by combined administration of β-asaron and paeonol. Rat middle cerebral artery occlusion (MCAO) model was established, the interleukin-1beta (IL-1β) and tumor necrosis factor α (TNF-α) in the rat peripheral blood were determined by ELISA assay, and brain tissue damage was evaluated by TUNNEL assay. The correlation of cholecystokinin (CCK) and nuclear factor-kappaB (NF-κB) signaling components between intestinal mucosa and prefrontal cortex of MCAO rats treated with β-asaron and paeonol were analyzed by quantitative RT-PCR and western blotting. In vitro transwell co-culture was performed to confirm the correlated expression. The expression of CCK and NF-κB signaling components were closely correlated between the intestinal mucosa and prefrontal cortex of MCAO rats treated with β-asaron and paeonol. The combined administration also regulates the IL-1β and TNF-α in the MCAO rat peripheral blood and ameliorate the brain damage in MCAO rats. Elevated expression of related genes was observed in the cortical neurons co-cultured with intestinal mucosal epithelial cells treated by β-asaron and paeonol. The brain-gut axis mediates the therapeutic effect of β-asaron and paeonol for cerebral infarction through CCK and NF-κB signaling.

Cholinergic and cytoprotective signaling cascades mediate the mitigative effect of erythropoietin on acute radiation syndrome.

The present investigation aimed to evaluate the radiomitigative efficacy of the recombinant human erythropoietin (EPO) against acute radiation syndrome (ARS) in a rat model. Rats were irradiated with a single sublethal dose of γ-radiation (7 Gy; total body irradiation; TBI) on the 1st day of experimental course, then received EPO (5000 IU/kg; i.p.) 24 h after irradiation, and rats were observed for 30 days of survival analysis. Administration of EPO improved 30-day survival, alleviated TBI-induced myelosuppression and pancytopenia, by augmenting lymphocytes and other white blood cells in the peripheral blood of rats, while bone marrow and spleen cellularity were restored. EPO post-exposure treatment alleviated hepatotoxicity biomarkers and restored splenic function. EPO abrogated radiation-induced oxidative stress through the upregulation of the cholinergic anti-inflammatory nicotinic acetylcholine receptor (α-7-nAChR) and the pro-survival Janus kinase-2 and signal transducers and activators of transcription JAK-2/STAT-3 signaling mediated via enhancing nuclear factor erythroid-2 related factor-2 (Nrf-2) cytoprotective machinery in liver and spleen of irradiated rats. Moreover, EPO treatment prevented hepatic and splenic apoptosis. The present study establishes the implication of α-7-nAChR-JAK-2/STAT-3-Nrf-2 signaling cascade in the radiomitigative potential of EPO against ARS.

Myocardial infarction does not affect circulating haematopoietic stem and progenitor cell self-renewal ability in a rat model.

What is the central question of this study? Although peripheral blood haematopoietic stem and progenitor cells are potentially important in regeneration after acute myocardial infarction, their self-renewal ability in the post-acute phase has not yet been addressed. What is the main finding and its importance? In rat peripheral blood, we show that myocardial infarction does not negatively affect circulating haematopoietic stem and progenitor cell self-renewal ability 2 weeks after acute infarction, which suggests a constant regenerative potential in the myocardial infarction post-acute phase. Given the importance of peripheral blood haematopoietic stem and progenitor cells (HPCs) in post-acute regeneration after acute myocardial infarction (MI), the aim of the present study was to investigate the number and secondary replating capacity/self-renewal ability of HPCs in peripheral blood before and 2weeks after MI. In female Lewis inbred rats (n=9), MI was induced by ligation of the left coronary artery, and another nine underwent sham surgery, without ligation, for control purposes. Myocardial infarction was confirmed by troponinI concentrations 24h after surgery. Peripheral blood was withdrawn and fractional shortening and ejection fraction of the left ventricle were assessed before (day0) and 14days after MI or sham surgery (day14). After mononuclear cell isolation, primary and secondary functional colony-forming unit granulocyte-macrophage (CFU-GM) assays were performed in order to detect the kinetics of functional HPC colony counts and cell self-renewal ability in vitro. The CFU-GM counts and cell self-renewal ability remained unchanged (P>0.05) in both groups at day14, without interaction between groups. In the intervention group, higher day0 CFU-GM counts showed a relationship to lower fractional shortening on day14 (ρ=-0.82; P<0.01). Myocardial infarction did not negatively affect circulating HPC self-renewal ability, which suggests a constant regenerative potential in the post-acute phase. A relationship of cardiac contractile function 14days after MI with circulating CFU-GM counts on day0 might imply functional colony count as a predictive factor for outcome after infarction.

Biodistribution and Immunogenicity of Allogeneic Mesenchymal Stem Cells in a Rat Model of Intraarticular Chondrocyte Xenotransplantation.

Xenogeneic chondrocytes and allogeneic mesenchymal stem cells (MSC) are considered a potential source of cells for articular cartilage repair. We here assessed the immune response triggered by xenogeneic chondrocytes when injected intraarticularly, as well as the immunoregulatory effect of allogeneic bone marrow-derived MSC after systemic administration. To this end, a discordant xenotransplantation model was established by injecting three million porcine articular chondrocytes (PAC) into the femorotibial joint of Lewis rats and monitoring the immune response. First, the fate of MSC injected using various routes was monitored in an in vivo imaging system. The biodistribution revealed a dependency on the injection route with MSC injected intravenously (i.v.) succumbing early after 24 h and MSC injected intraperitoneally (i.p.) lasting locally for at least 5 days. Importantly, no migration of MSC to the joint was detected in rats previously injected with PAC. MSC were then administered either i.v. 1 week before PAC injection or i.p. 3 weeks after to assess their immunomodulatory function on humoral and adaptive immune parameters. Anti-PAC IgM and IgG responses were detected in all PAC-injected rats with a peak at week 2 postinjection and reactivity remaining above baseline levels by week 18. IgG2a and IgG2b were the predominant and long-lasting IgG subtypes. By contrast, no anti-MSC antibody response was detected in the cohort injected with MSC only, but infusion of MSC before PAC injection temporarily augmented the anti-PAC antibody response. Consistent with a cellular immune response to PAC in PAC-injected rats, cytokine/chemokine profiling in serum by antibody array revealed a distinct pattern relative to controls characterized by elevation of multiple markers at week 2, as well as increases in proliferation in draining lymph nodes. Notably, systemic administration of allogeneic MSC under the described conditions did not diminish the immune response. IL-2 measurements in cocultures of rat peripheral blood lymphocytes with PAC indicated that PAC injection induced some T-cell hyporesponsiveness that was not enhanced in the cohorts additionally receiving MSC. Thus, PAC injected intraarticularly in Lewis rats induced a cellular and humoral immune response that was not counteracted by the systemic administration of allogeneic MSC under the described conditions.

Combined Impact of Gamma and Laser Radiation on Peripheral Blood of Rats in vivo

The impact of γ radiation of 137Сs (doses of 1 and 3 Gy), low-intensity laser radiation (λ = 670 nm, 5.3 or 10.6 J/cm2) as well as the influence of consecutive laser and γ radiation on peripheral blood and blood cells (erythrocytes, leukocytes, lymphocytes, granulocytes) were studied by analyzing the number of blood cells, blood absorption spectra, and activity of antioxidant defense enzymes. Two series of experiments were performed on four groups of rats. The rats of the control group (group 1) were not exposed to γ or laser radiation. In the experimental groups, single irradiation of the whole body of rats with γ radiation (group 2), three- or four-day over-vein irradiation of blood in the tail vein by low-intensity laser radiation (group 3), and successive three- or four-day irradiation of blood by laser and then a single irradiation of the whole body with γ radiation (group 4) were performed. It was shown that changes of the blood cell content in the experimental groups are accompanied by changes in the spectral characteristics of the blood and the activity of antioxidant defense enzymes. The radioprotective effect of low-intensity laser radiation is manifested as an increase in the average number of leukocytes and lymphocytes in the group as compared with the postradiation, as well as an increase in the activity of antioxidant protection enzymes. The possibility of using low-intensity optical radiation for correction of hematological disorders caused by ionizing radiation is discussed.

Abstract P334: Persistent Adrenomedullin Derivative Inhibits Development of Hypertension

Human adrenomedullin (hAM) consists of a 52 amino acid peptide that is amidated and has a disulfide bond. The bioactive peptide hAM has a variety of physiological functions, such as vasodilatation, hormone secretion, neurotransmission, embryogenesis, wound healing and immunoregulation. hAM has shown several therapeutic effects in experimental models of various diseases, including ischemic heart disease, inflammatory bowel disease, stroke and retinochoroidal disease. However, these therapies required continuous administration of hAM as the half-life of native hAM is quite short in blood. To resolve these issues, the aim of this study was to synthesize novel hAM derivative, and to examine its effect in spontaneously hypertensive rats (SHR). First, we conjugated the hAM N-terminal with 60kDa polyethylene glycol (PEG) (60 kDa PEG-hAM). To demonstrate that PEG binds covalently to α-amino residue of the N-terminal tyrosine of hAM, 60 kDa PEG-hAM was digested with cyanogen bromide and the AM(6-52) thus obtained was confirmed by ion exchange chromatography and MALDI-MS. Next, to determine plasma hAM concentrations, either 60 kDa PEG-hAM (10 nmol/kg) or native hAM was administrated subcutaneously with Wistar rat. We compared hAM concentrations in the peripheral blood of rats after injection with either 60 kDa PEG-hAM or native hAM. The hAM concentrations in the 60 kDa PEG-hAM group after 1, 7 and 10 days were 2600, 740 and 280 pM, respectively. In contrast, the hAM concentrations in the native hAM group after 1 day was 6.7pM, not detect 7 and 10 days. In addition, 60kDa PEG-hAM stimulated cAMP production in cultured human embryonic kidney cells expressing a specific AM receptor. Finally, we compared blood pressor after administrated subcutaneously with either 60 kDa PEG-hAM (20 nmol/kg) or saline in SHR with high salt diet. After 9 days, a single subcutaneous administration of 60 kDa PEG-hAM inhibited development of hypertension with SHR. In summary, the plasma half-life of 60 kDa PEG-hAM was much longer than native hAM, and subcutaneous injection of 60 kDa PEG-hAM in a single dose inhibited development of hypertension with SHR. Therefore, these results suggest that 60 kDa PEG-hAM is a possible therapeutic agent for the treatment of hypertension.

Emodin alleviates severe acute pancreatitis-associated acute lung injury by decreasing pre-B-cell colony-enhancing factor expression and promoting polymorphonuclear neutrophil apoptosis.

The present study aimed to evaluate the protective effects of emodin on severe acute pancreatitis (SAP)-associated acute lung injury (ALI), and investigated the possible mechanism involved. SAP was induced in Sprague-Dawley rats by retrograde infusion of 5% sodium taurocholate (1 ml/kg), after which, rats were divided into various groups and were administered emodin, FK866 [a competitive inhibitor of pre-B-cell colony-enhancing factor (PBEF)] or dexamethasone (DEX). DEX was used as a positive control. Subsequently, PBEF expression was detected in polymorphonuclear neutrophils (PMNs) isolated from rat peripheral blood by reverse transcription-quantitative polymerase chain reaction and western blotting. In addition, histological alterations, apoptosis in lung/pancreatic tissues, apoptosis of peripheral blood PMNs and alterations in the expression of apoptosis-associated proteins were examined by hematoxylin and eosin staining, terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling assay, Annexin V/propidium iodide (PI) assay and western blotting, respectively. Serum amylase activity and wet/dry (W/D) weight ratios were also measured. An in vitro study was also conducted, in which PMNs were obtained from normal Sprague-Dawley rats and were incubated with emodin, FK866 or DEX in the presence of lipopolysaccharide (LPS). Apoptosis of PMNs and the expression levels of apoptosis-associated proteins were examined in cultured PMNs in vitro by Annexin V/PI assay and western blotting, respectively. The results demonstrated that emodin, FK866 and DEX significantly downregulated PBEF expression in peripheral blood PMNs. In addition, emodin, FK866 and DEX reduced serum amylase activity, decreased lung and pancreas W/D weight ratios, alleviated lung and pancreatic injuries, and promoted PMN apoptosis by regulating the expression of apoptosis-associated proteins: Fas, Fas ligand, B-cell lymphoma (Bcl)-2-associated X protein, cleaved caspase-3 and Bcl-extra-large. In addition, the in vitro study demonstrated that emodin, FK866 and DEX significantly reversed the LPS-induced decrease of apoptosis in PMNs by regulating the expression of apoptosis-associated proteins. In conclusion, the present study demonstrated that emodin may protect against SAP-associated ALI by decreasing PBEF expression, and promoting PMN apoptosis via the mitochondrial and death receptor apoptotic pathways.

Neuroprotection by IFN-γ via astrocyte-secreted IL-6 in acute neuroinflammation.

Inflammation eliminates pathogenic infections while also threatening the integrity of the central nervous system. In this study, using in vivo and in vitro models of acute neuroinflammation, we investigated the mechanisms by which inflammation and astrocytes affect neuronal apoptosis. The in vitro model mimicked acute neuroinflammation by incubation in IFN-γ-containing media with primary cultured cerebellar granule neurons, with or without cultured astrocytes. This quickly induced neuronal apoptosis characterized by cleaved caspase-3 expression, Hoechst 33342 staining, and intercellular Ca2+ influx, whereas the presence of astrocytes significantly protected neurons from these effects. IFN-γ in the inflammation media also promoted astrocyte secretion of IL-6, essential for protection. The supernatants of rat peripheral blood mononuclear cells stimulated by lymphocyte mitogen lipopolysaccharide or concanavalin A were used as inflammation media to verify the results. The in vivo model involved a peripheral challenge with lipopolysaccharide, with or without recombinant IFN-γ, in C57BL/6 mice. This confirmed the in vitro results: anti-IFN-γ antibodies exacerbated the acute course of neuroinflammation and led to neurocyte apoptosis in vivo. The pro-inflammatory cytokine IFN-γ provided neuroprotection during acute neuroinflammation via induction of astrocyte-secreted IL-6. The findings provide novel insights into the mechanisms of neuroprotection by IFN-γ during acute neuroinflammation, and may impact therapies for inflammation-related central nervous system injury and disease.

Detection of Th17/Treg cells and related factors in gingival tissues and peripheral blood of rats with experimental periodontitis.

Objective(s):This study aimed to investigate the role and the possible mechanisms involved in the immunoregulation of experimental periodontitis by Th17/Treg.Materials and Methods:Experimental periodontitis was established by silk thread ligation with Porphyromonas gingivalis daubing in the bilateral maxillary second molar of Male Sprague-Dawley (SD) rats. Alveolar bones were scanned by Micro-CT. Histological examination was stained with H&E. The proportions of Th17 and Treg cells in peripheral blood were detected by flow cytometry. RT-PCR was used to measure the expression of RORγt, Foxp3 mRNA in the gingival tissues. The concentrations of IL-17, IL-10, and TGF-β in peripheral blood and gingival crevicular fluid were measured by ELISA.Results:Experimental rats showed profound bone resorption and inflammatory cell infiltration. The percentages of Th17 significantly increased in the peripheral blood, which was consistent with gingival tissues study that Th17 cells related transcription factor RORγt mRNA and IL-17 increased in the course of periodontitis. The percentages of CD25+Foxp3+ Treg significantly increased in the peripheral blood, which was consistent with gingival tissues study that Treg cells related transcription factor Foxp3 mRNA and cytokines IL-10 and TGF-β increased in the course of periodontitis. The ratio of Th17/Treg cells was significantly increased in the peripheral circulation, however, the Th17/Treg balance is in wave motion in inflamed gingival tissues in the different stages of periodontitis.Conclusion:Th17/Treg balance may be associated with the progression of periodontitis and pathological tissue destruction. Moreover, local inflammation would result in the up-regulation ratio of Th17/Treg in peripheral blood, which may influence some periodontally involved systemic diseases.

In vitro and in vivo assessment of inhibitory effect of stevioside on pro-inflammatory cytokines.

Stevioside is a natural non-caloric sweetener which has been reported to have anti-inflammatory activity. The aim of the present study was to examine in vitro and in vivo effects of stevioside on rats plasma levels of tumor necrosis factor- α (TNF-α), interleukin-1β (IL-1β), TNF-α and IL-1β release from lipopolysaccharide(LPS)-stimulated rat peripheral blood mononuclear cells (PBMCs). Male wistar rats weighing between 170-220 g were given stevioside (0, 500 and 1000 mg/kg BW/day) for 6 weeks. Mononuclear cells were separated from peripheral blood samples. TNF-α and IL-1β levels in plasma and the release of TNF-α and IL-1β from PBMCs were determined using rat enzyme-linked immunosorbent assay (ELISA) kits. Plasma levels of TNF-α and IL-1β were found to be non-detectable in control and groups treated with 500 and 1000 mg/kg of stevioside. Regarding TNF-α release from LPS-stimulated PBMCs, rats that were orally fed with 500 and 1000 mg/kg of stevioside were significantly different (p<0.05) from those in LPS-treated control group (186.8+18.6 and 151.4 + 15.4 vs 248.6+21.4 pg/ml). Additionally, IL-1β levels in rats treated with 500 and 1000 mg/kg of stevioside were significantly different (p<0.05) from those in LPS-treated control group (220.0+12.1 and 158.1 + 22.6 vs 294.4+16.1 pg/ml). Consumption of stevioside has an inhibitory effect on the release of TNF-α and IL-1β from LPS-stimulated PBMCs in rats.

Expression and Role of the Calcium-Sensing Receptor in Rat Peripheral Blood Polymorphonuclear Neutrophils.

The calcium-sensing receptors (CaSRs) play an important role in many tissues and organs that are involved in inflammatory reactions. Peripheral blood polymorphonuclear neutrophils (PMNs) are important inflammatory cells. However, the expression and functions of CaSR in peripheral blood PMNs are still not reported. In this study, we collected rat peripheral blood PMNs to observe the relationship between CaSR and PMNs. From the results, we found first that the CaSR protein was expressed in PMNs, and it increased after PMNs were activated with fMLP. In addition, CaSR activator cincalcet promoted the expression of CaSR and P-p65 (NF-κB signaling pathway protein) and Bcl-xl (antiapoptosis protein), and it increased the secretion of interleukin-6 (IL-6) and myeloperoxidase (MPO); meanwhile, it decreased proapoptosis protein Bax expression and the production of IL-10 and reactive oxygen species (ROS). At the same time, cincalcet also decreased the PMN apoptosis rate analyzed by flow cytometry. However, CaSR inhibitor NPS-2143 and NF-κB signaling pathway inhibitor PDTC reverse the results cited earlier. All of these results indicated that CaSR can regulate PMN functions and status to play a role in inflammation, which is probably through the NF-κB signaling pathway.

Modulation of Radiation-induced Base Excision Repair Pathway Gene Expression by Melatonin.

Objective:Approximately 70% of all cancer patients receive radiotherapy. Although radiotherapy is effective in killing cancer cells, it has adverse effects on normal cells as well. Melatonin (MLT) as a potent antioxidant and anti-inflammatory agent has been proposed to stimulate DNA repair capacity. We investigated the capability of MLT in the modification of radiation-induced DNA damage in rat peripheral blood cells.Materials and Methods:In this experimental study, male rats (n = 162) were divided into 27 groups (n = 6 in each group) including: irradiation only, vehicle only, vehicle with irradiation, 100 mg/kg MLT alone, 100 mg/kg MLT plus irradiation in 3 different time points, and control. Subsequently, they were irradiated with a single whole-body X-ray radiation dose of 2 and 8 Gy at a dose rate of 200 MU/min. Rats were given an intraperitoneal injection of MLT or the same volume of vehicle alone 1 h prior to irradiation. Blood samples were also taken 8, 24, and 48 h postirradiation, in order to measure the 8-oxoguanine glycosylase1 (Ogg1), Apex1, and Xrcc1 expression using quantitative real-time-polymerase chain reaction.Results:Exposing to the ionizing radiation resulted in downregulation of Ogg1, Apex1, and Xrcc1 gene expression. The most obvious suppression was observed in 8 h after exposure. Pretreatments with MLT were able to upregulate these genes when compared to the irradiation-only and vehicle plus irradiation groups (P < 0.05) in all time points.Conclusion:Our results suggested that MLT in mentioned dose may result in modulation of Ogg1, Apex1, and Xrcc1 gene expression in peripheral blood cells to reduce X-ray irradiation-induced DNA damage. Therefore, administration of MLT may increase the normal tissue tolerance to radiation through enhancing the cell DNA repair capacity. We believed that MLT could play a radiation toxicity reduction role in patients who have undergone radiation treatment as a part of cancer radiotherapy.

Фізико-хімічні властивості лігандних форм гемоглобіну за експериментального стрептозотоцинового цукрового діабету й за алкогольної інтоксикації

The study of hemoglobin affinity to oxygen, oxygen capacity and the content of ligand forms has been conducted in hemolyzates of rat peripheral blood at chronic alcohol intoxication models and experimental diabetes mellitus. The findings revealed that at alcohol intoxication both hemoglobin affinity to oxygen and hemoglobin oxygen capacity validly decrease, whereas at experimental streptozotocin-induced diabetes hemoglobin the affinity to oxygen increases and oxygen capacity is quite high. The results of study demonstrated that sulf- and methemoglobin levels rise under alcohol intoxication and sulf-, met- and carboxyhemoglobin levels rise under experimental diabetes mellitus. We have also undertaken study of hemoglobin affinity to oxygen, oxygen capacity and the ratio of hemoglobin ligand forms in alcoholics and healthy donors. A valid increase in sulf- and methemoglobin and a decrease in hemoglobin affinity to oxygen in alcoholics (32.50±0.45 versus 28.60±0.54 mm Hg in the control group) with insignificant changes in oxygen capacity were revealed. Since minor ligand forms contribute to the affinity of total hemoglobin to oxygen, their quantitative characteristics are essential. Thus, data on absorption spectra which characterize nitrosyl-hemoglobin ligand form and transition of deoxy- and methemoglobin into nitrosyl-hemoglobin have been collected. A combined absorption spectrum of nitrosyl-hemoglobin has been built based on the statistical data set deoxyhemoglobin and its full transition into nitrosyl-hemoglobin (60 analyses) in the examined wavelength range. The article provides the results of the comparative analysis of electronic absorption spectra characteristic peaks of six hemoglobin ligand forms taken from peripheral blood in healthy donors in 450–750 nm wavelength range. The studies can be a pre-requisite for the development of methods for determining six hemoglobin ligand forms in a single blood sample.

The Activity of Immunoglobulin Y Anti-Mycobacterium tuberculosis on Proliferation and Cytokine Expression of Rat Peripheral Blood Mononuclear Cells.

Objective:It has long been known that chickens, like mammals, are capable of producing antigen-specific immunoglobulin Y (IgY), which functions similar to IgG. The present study was performed to investigate the activity of IgY anti-Mycobacterium tuberculosis on proliferation, interleukin (IL)-2, and interferon (IFN)-γ expression of rat peripheral blood mononuclear cells (PBMCs).Materials and Methods:The activity of IgY anti-M. tuberculosis in different doses (25, 50, and 100 μg/ml) on rat PBMCs proliferation was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. The production of IL-2 and IFN-γ in the PBMC supernatant was determined using enzyme-linked immunosorbent assay. Investigation was performed on mRNA expression of IL-2 and IFN-γ by reverse transcription-polymerase chain reaction (RT-PCR).Results:IgY anti-M. tuberculosis significantly increased the proliferation of rat PBMC. Furthermore, IgY anti-M. tuberculosis dose dependently increased IL-2 and IFN-γ production in PBMC, suggesting that pharmacological activities of IgY anti-M. tuberculosis in PBMC may be mediated by regulating the production of cytokines. In the RT-PCR, expression of cytokines such as IL-2 and IFN-γ in PBMC cultures was increased by IgY anti-M. tuberculosis.Conclusions:We concluded that increasing IL-2 and IFN-γ productions in PBMC was related to IgY anti-M. tuberculosis, stimulating the mRNA transcription (gene expression) of these cytokines which can induce proliferation of PBMC.SUMMARY Lohman laying hens immunized intramuscularly with antigens of M. tuberculosis can produce specific IgY anti-Mycobacterium tuberculosis complexIgY anti-M. tuberculosis significantly increased the proliferation of rat peripheral blood mononuclear cell (PBMC)IgY anti-M. tuberculosis dose dependently increased interleukin 2 (IL-2) and interferon (IFN)-γ production in PBMCIn the reverse transcription-polymerase chain reaction, expression of cytokines such as IL-2 and IFN-γ in PBMC cultures was increased by IgY anti-M. tuberculosisThe increasing IL-2 and IFN-γ productions in PBMC were related to stimulation on mRNA transcription which can induce proliferation of PBMC. Abbreviations Used: IgY anti-M. tuberculosis: Immunoglobulin Y anti-Mycobacterium tuberculosis; IL-2: Interleukin-2; IFN-γ: Interferon-γ; PBMCs: Peripheral blood mononuclear cells.

Polysaccharides from Arnebia euchroma Ameliorated Endotoxic Fever and Acute Lung Injury in Rats Through Inhibiting Complement System.

Arnebiaeuchroma (Royle) Johnst (Ruanzicao) is a traditional Chinese herbal medicine (TCM). It is extensively used in China and other countries for treatment of inflammatory diseases. It is known that hyper-activated complement system involves in the fever and acute lung injury (ALI) in rats. In our preliminary studies, anti-complementary activity of crude Arnebiaeuchroma polysaccharides (CAEP) had been demonstrated in vitro. This study aimed to investigate the role and mechanism of crude Arnebiaeuchroma polysaccharides (CAEP) using two animal models, which relate with inappropriate activation of complement system. In lipopolysaccharide (LPS)-induced fever model, the body temperature and leukocytes of peripheral blood in rats were significantly increased, while the complement levels of serum were remarkably decreased. CAEP administration alleviated the LPS-induced fever, reduced the number of leukocytes, and improved the levels of complement. Histological assay showed that there were severe damages and complement depositions in lung of the ALI rats. Further detection displayed that the oxidant stress was enhanced, and total hemolytic activity and C3/C4 levels in serum were decreased significantly in the ALI model group. Remarkably, CAEP not only attenuated the morphological injury, edema, and permeability in the lung but also significantly weakened the oxidant stress in bronchoalveolar lavage fluid (BALF) in the ALI rats. The levels of complement and complement depositions were improved by the CAEP treatment. In conclusion, the CAEP treatment ameliorated febrile response induced by LPS and acute lung injury induced by LPS plus ischemia-reperfusion. CAEP exerted beneficial effects on inflammatory disease potentially via inhibiting the inappropriate activation of complement system.

EphB4/ephrinB2 ephrinB1 Interaction Mediated Chronic Myelogenous Leukemia Mesenchymal Stromal Cells Osteogenic Differentiation in Vitro and In Vivo

Background and Objective: Osteoblasts, important of stromal cells in bone marrow microenvironment, maintain HSCs in resting state and protect its' functions. Osteoblasts derived from mesenchymal stem cells (MSCs), which can be differentiated into osteoblast in bone marrow under the regulation of cytokines. Recent studies have indicated that EphB4/ephrinB2 protein participates in the regulation of osteogenesis differentiation of MSCs in bone marrow microenvironment.Our previous study found that EphB4 receptor was over expressed in CML patients and cell lines, which played an important role to change characterize of Imatinib(IM)-resistant in chronic myeloid leukemia cells. Furthermore, we performed experiments to prove that osteogenic differentiation in MSCs from CML-initial patient significantly higher in contrast to normal human MSCs and the change of EphB4 molecules on leukemia cells may transform MSCs functions in vitro. However, the mechanism of these transformations of MSCs in vitro and what is change in vivo were still unclear. Therefore, we hypothesis that the change of EphB4 molecules on leukemia cells might play an important role to osteogenic differentiate in MSCs in vitro and in vivo, which support to leukemia progression and disruption of normal hematopoiesis.Methods and Results: MSCs were prepared from bone marrow mononuclear cells isolated from normal human or CML- chronic phase (CCP) patients’ BM and cultures in Cyagen Bone marrow culture medium at 37 °C, 5% CO2 incubator. In vitro, after stimulated with different concentrations of EphB4-Fc (0, 5, 8, 10 ug/ml) for 21 days, visualized by Alizarin Red staining, MSCs (CCP) produced maximum calcium nodules (P<0.05, n=3) in EphB4-Fc (8 ug/ml) group in contrast with other groups, accompanied by increased ephrinB1 and STAT3 phosphorylation. In vitro osteogenesis condition, after treatment with EphB4-Fc (0, 8 ug/ml) 14 days, MSCs (CCP) incubated with K562 cells. After 48 h, the IC50 (0.842±0.065, P<0.05, P<0.05 ANOVA, n=4) of K562 cells in MSCs+EphB4-Fc (8ug/ml) group increased, S phase cells percentage(56.6±4.01, P<0.05, P<0.01, ANOVA, n=4) increased and cells apoptosis rate(P<0.01, P<0.001, LSD, n=4) declined compared with K562 (control group) and K562+MSCs+EphB4-Fc (0 ug/ml). In vivo, K562-R, K562-R+MSCs (normal) (5:1), K562-R-EphB4-sh, K562-R-EphB4-sh+MSCs (normal) (5:1), MSCs (normal) cells were injected respectively into bone cavity of NOG rat (NOD/SCID/γ c-/-, n=12) rat and blank control group were also established. Examined peripheral blood in rats while hCD45+ cells > 1% is considered as leukemia model. K562-R+MSCs mice were earliest to establish leukemia model (31.75±1.26d) and had the shortest survival time(4.25±1.71d) than other groups. After treatment with IM, survival times of K562-R+MSCs mice were not significantly extended (4.7±3.055 d, pared-samples T test, P>0.05). In bone marrow of K562-R+MSCs mice, RUNX2 mRNA (0.654±0.0278; P < 0.001) over expressed in contrast to other groups. After treatment with IM, expression level of RUNX2 mRNA was significantly increased than non-treatment group. Among four leukemia groups of mice, expression levels VEGF mRNA in bone marrow were no significantly difference and there was no statistical difference existed in treatment group and non-treatment group. The same cells lines above were subcutaneously injected to establish subcutaneous transplantation tumor, respectively, in NOG rat (NOD/SCID/γ c-/-, n=8) rat. K562-R+MSCs tumors were earliest to appear (17.333±1.154 d) and had the biggest tumors volume (13116.27±165.502 mm3, P<0.001) compared to other groups. After mice treated by IM, compared with non-treatment group, K562-R+MSCs tumors had significantly increased in volume (14703.14±309.333mm3, pared-samples T test, P<0.01). VEGF mRNA (0.861±0.0648; P<0.01) in K562-R+MSCs tumor over express than other groups. After treatment, the expression level was no significantly declined (0.796±0.0688, P>0.05). The level expression of RUNX2 mRNA in four groups of subcutaneous transplantation tumors are low and had no statistical difference.Conclusion: Our experiments in vitro and in vivo illustrated that EphB4 molecule on leukemia cells may transform MSCs osteogenic differentiation to change characterize of Imatinib(IM)-resistant in CML through ephrinB1 and STAT3 phosphorylation. DisclosuresLin:Natural Science Foundation of China: Research Funding. Na:Natural Science Foundation of China: Research Funding.

Limb Remote Ischemic Postconditioning Reduces Ischemia-Reperfusion Injury by Inhibiting NADPH Oxidase Activation and MyD88-TRAF6-P38MAP-Kinase Pathway of Neutrophils.

Limb remote ischemic postconditioning (LRIP) has been confirmed to reduce the ischemia-reperfusion injury but its mechanisms are still not clear. This study clarified the mechanism of LRIP based on the nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase and Myeloid differentiation factor 88 (MyD88)-Tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6)-P38 pathway of neutrophils. Rat middle cerebral artery occlusion (MCAO) model was used in this study. Ischemia-reperfusion injury was carried out by MCAO 1.5 h followed by 24 h reperfusion. LRIP operation was performed to the left femoral artery at 0, 1 or 3 h after reperfusion. Behavioral testing, including postural reflex test, vibrissae-elicited forelimb placing test and tail hang test, showed that LRIP operated at 0 h of reperfusion could significantly ameliorate these behavioral scores. Pathological examinations, infarct size, Myeloperoxidase (MPO) activity showed that LRIP operated at 0 h of reperfusion could significantly ameliorate the pathological scores, reduce the infarct size and MPO activity in the brain and increase the MPO activity in the left leg. By using Neutrophil counting, immunofluorescence and real-time PCR techniques, we found that LRIP operated at 0 h of reperfusion could reduce neutrophil counts in the peripheral blood and downregulate the activation of neutrophil in the peripheral blood and rat brain. Western blots revealed that MyD88, TRAF6, p38 mitogen-activated protein kinase (p38-MAPK) in neutrophils and the phosphorylation of p47phox (Ser 304 and Ser 345) in neutrophil could be downregulated by LRIP. Our study suggests that LRIP inhibits the number and activation of neutrophils in the rat brain and peripheral blood linked to down-regulating the activation of NADPH oxidase in neutrophils by MyD88/TRAF6/p38-MAPK pathway.