Rat Peripheral Blood Lymphocytes Research Articles

Chinese medicine Di-long (Pheretima vulgaris) and its active fraction exhibit anti-rheumatoid arthritis effects by inhibiting CXCL10/CXCR3 chemotaxis in synovium

Ethnopharmacological relevanceDi-Long (Pheretima vulgaris) is a classic animal sourced traditional Chinese medicine. It has been used for the treatment of joint inflammation and arthralgia for over two thousand years due to its effects of Tong-Luo-Zhi-Tong (dredging collaterals and alleviating pain). Our previous study showed that Chinese medicine Di-Long has significant anti-rheumatoid arthritis (RA) effects. Aim of the studyConsidering Di-Long as a potential source of active compounds with specific anti-RA therapeutic effects, this research was to obtain the anti-RA target-specific active fraction from Di-Long extracts (DL), and to further explore the chemical basis and verify the anti-RA mechanism of this active fraction. Materials and methodsTranscriptomic was applied to obtain the main anti-RA targets of DL on human RA fibroblast-like synoviocytes (FLS) and validated by qPCR. The target-corresponding active fraction was isolated from DL by ethanol precipitation and gel chromatography, and analyzed by nanoliter chromatography-mass spectrometry. Anti-RA effects of this active fraction was investigated by collagen-induced arthritis (CIA) in mice, and anti-RA mechanisms were verified in cocultured model of rat FLS and peripheral blood lymphocytes. ResultsWe confirmed that CXCL10/CXCR3 was the main anti-RA target of DL. The active fraction - A (2182 - 890 Da) was isolated from DL based on its CXCL10 inhibiting effects in RA-FLS. Fraction A contains 195 peptides (192 were newly discovered), 26 of which might be bioactive and were considered to be the chemical basis of its anti-RA effects. Fraction A significantly ameliorated the joint destruction and overall inflammation in CIA mice, and downregulated CXCR3 expression in mice joint. Fraction A inhibited the chemotaxis of Th-cells in rat peripheral blood lymphocytes towards the TNF-α-induced rat FLS through CXCL10/CXCR3 pathway. ConclusionsOur work indicated that active fraction from DL containing small peptides exhibits promising therapeutic effects for RA through inhibiting CXCL10/CXCR3 chemotaxis.

Chinese herbal formula, modified Xianfang Huoming Yin, alleviates the inflammatory proliferation of rat synoviocytes induced by IL-1β through regulating the migration and differentiation of T lymphocytes.

Xianfang Huoming Yin (XFH) is a traditional Chinese herbal formula, which has the effect of clearing heat and detoxifying toxins, dispersing swellings, activating blood circulation, and relieving pain. It is usually applied to treat various autoimmune diseases, including Rheumatoid arthritis (RA). The migration of T lymphocytes plays an indispensable role in the pathogenesis of RA. Our previous studies demonstrated that modified Xianfang Huoming Yin (XFHM) could modulate the differentiation of T, B, and NK cells, and contribute to the restoration of immunologic balance. It also could downregulate the production of pro-inflammatory cytokines by regulating the activation of NF-κ B and JAK/STAT signaling pathways in the collagen-induced arthritis mouse model. In this study, we want to investigate whether XFHM has therapeutic effects on the inflammatory proliferation of rat fibroblast-like synovial cells (FLSs) by interfering with the migration of T lymphocytes in vitro experiments. High performance liquid chromatography-electrospray ionization/mass spectrometer system was used to identify the constituents of the XFHM formula. A co-culture system of rat fibroblast-like synovial cells (RSC-364cells) and peripheral blood lymphocytes stimulated by interleukin-1 beta (IL-1β) was used as the cell model. IL-1β inhibitor (IL-1βRA) was used as a positive control medicine, and two concentrations (100μg/mL and 250μg/mL) of freeze-dried XFHM powder were used as intervention measure. The lymphocyte migration levels were analyzed by the Real-time xCELLigence analysis system after 24h and 48h of treatment. The percentage of CD3+CD4+ T cells and CD3+CD8+ T cells, and the apoptosis rate of FLSs were detected by flow cytometry. The morphology of RSC-364cells was observed by hematoxylin-eosin staining. The protein expression of key factors for T cell differentiation and NF-κ B signaling pathway-related proteins in RSC-364cells were examined by western-blot analysis. The migration-related cytokines levels of P-selectin, VCAM-1, and ICAM-1 in the supernatant were measured by enzyme-linked immunosorbent assay. Twenty-one different components in XFHM were identified. The migration CI index of T cells was significantly decreased in treatment with XFHM. XFHM also could significantly downregulate the levels r of CD3+CD4+T cells and CD3+CD8+T cells that migrated to the FLSs layer. Further study found that XFHM suppresses the production of P-selectin, VCAM-1, and ICAM-1. Meanwhile, it downregulated the protein levels of T-bet, ROR γ t, IKKα/β, TRAF2, and NF-κ B p50, upregulated the expression of GATA-3 and alleviated synovial cells inflammation proliferation, contributing to the FLSs apoptosis. XFHM could attenuate the inflammation of synovium by inhibiting T lymphocyte cell migration, regulating differentiation of T cells through modulating the activation of the NF-κ B signaling pathway.

Bioactive compounds anthocyanins as a factor in the nutritional recovery of the body's adaptive potential after intense physical activity in the experiment: assessment of immunological and hematological indicators of adaptation

Restoring the adaptive potential of an athlete is of paramount importance not only for the implementation of his training and competitive activities, but also for maintaining health. One of the leading place in complex recovery programs in sports is given to full-fledged optimal nutrition, which provides for meeting the body's requirements not only in energy, macro- and micronutrients, but also in minor bioactive compounds. The use of anthocyanin-containing products is a promising strategy for the normalization of metabolic and immune disorders that develop as a result of intense physical and neuro-emotional stress not only in athletes, but also in other groups of people exposed to these factors, including military personnel undergoing training in conditions close to combat. This determines the relevance of this study. The aim of the research was to study the effect of an anthocyanin-enriched diet on hematological profile and cellular immunity in rats after intense physical activity. Material and methods. The experiment was carried out for 4 weeks on 4 groups of male Wistar rats with an initial body weight of ~300 g. The motor activity of the animals of the 1st (control) and 2nd groups was limited by the standard keeping animals in the vivarium, while physically active rats of the 3rd and 4th groups received additional physical activity - training on a treadmill. Before the end of the experiment, the animals of 3rd and 4th groups were given debilitating physical activity on a treadmill (until the rats refused to continue the exercise). Rats of all 4 groups received a standard semi-synthetic diet, water ad libitum. Animals in 2nd and 4th groups were additionally fed blueberry and blackcurrant extract (30% anthocyanins) as part of the diet at a daily dose of 15 mg anthocyanins/kg body weight. Hematological parameters were determined on a Coulter ACT TM 5 diff OV hematological analyzer. Expression of CD45R, CD3, CD4, CD8a, CD161 receptors on rat peripheral blood lymphocytes was determined by direct immunofluorescent staining of whole blood cells using a panel of monoclonal antibodies conjugated with fluorescent dyes: APC, FITC, PE. The measurements were carried out on an FC-500 flow cytometer. Results. Intense physical activity in rats of the 3rd group did not lead to a significant change in erythrocyte parameters compared with the control group. Enrichment of the diet with blueberry and black currant extract (the 2nd and the 4th groups) provided a significant (p<0.05) increase in blood content of hemoglobin (Hb) (150.7±0.9 and 154.4±2.0 vs 145.4±0.9 g/l in control), hematocrit (44.95±0.21 and 46.18±0.64 vs 43.78±0.32%) and the average content of Hb in erythrocytes (18.00±0.20 and 18.03±0.24 vs 17.35±0.24 pg). The absolute content of leukocytes and other cellular elements of the leukocyte formula, as well as leukocyte indices in rats of the experimental groups didn't significantly differ from those of the control rats, which confirms the absence of an inflammatory process. Intense physical activity and anthocyanin enrichment of the diet didn't have a significant effect on rat platelet parameters. Enrichment of the diet of rats of the 4th group with blueberry and black currant extract led to the activation of cellular immunity, as evidenced by a significant (p<0.01) increase in the percentage (from the total content of T-lymphocytes) of T-helpers (70.13 ±1.34 vs 63.75±0.99%) and a decrease in the relative content of cytotoxic T-lymphocytes (28.65±1.38 vs 34.71±0.95%) in comparison with those in rats of the 3rd group and at the level of the trend (р<0.1) - from the 1st group indexes (66.87±1.20 and 31.87±1.26%, accordingly). Intense physical activity led to a decrease in immunoregulatory index in rats of the 3rd group (1.86±0.07) compared with the control (2.13±0.12) (p<0.1), and in animals of the 4th group this indicator was significantly higher (2.50±0.14, p<0.05). In animals of the 3rd group a statistically significant (p<0.05) decrease in the relative content of NK cells in peripheral blood was found compared to the control. Enrichment of the diet of physically active rats with blueberry and black currant extract led to a significant (p<0.05) increase in the percentage of NK cells compared to this indicator in rats of the 3rd group (4.87±0.75 vs 2.08±0.18%) and had no significant difference with the indicator in rats of the control group (4.32±0.98%). Conclusion. The enrichment of the rats' diet with blueberry and blackcurrant extract containing a daily dose of 15 mg of anthocyanins per kg of body weight provides an increase in blood Hb content, hematocrit and the average content Hb in erythrocytes. It has been established that intense physical activity induces the cellular immunity suppression. The activating effect of anthocyanins on adaptive cellular immunity and NK cells, which are lymphocytes of innate immunity, was revealed. The data obtained indicate the effectiveness of the use of bioactive compounds (anthocyanins) to increase the adaptive potential of the organism.

Effects of Low-dose Gamma Radiation on Expression of Apoptotic Genes in Rat Peripheral Blood Lymphocyte.

Background: Exposure to high-dose ionizing radiation is known as a human carcinogen factor, but our information about the effects of low-dose ionizing radiation such as occupational exposures is limited. The main concern of scientific community is biological consequences due to low-dose radiations.Objective: This study aims to evaluate the effects of low-dose γ-radiation on expression changes of apoptotic genes (bax and bcl-2) in the rat peripheral blood lymphocytes.Material and Methods: In this experimental study, 42 adult male rats were classified into 6 groups, which was exposed to various doses values ranged from 20 mGy to 1000 mGy by γ-rays from a Co-60 source. Blood samples were provided for analysis of gene expression 24 h after gamma radiation by relative quantitative Reverse Transcription - Polymerase Chain Reaction (RT-PCR). Radiation sensitivity of rat lymphocytes was measured by the bax/bcl-2 ratio as a predictive marker for radio-sensitivity. Results: The results of this study showed that low dose of gamma radiation can induce down-regulation of bax in rat peripheral blood lymphocytes. Despite other mechanisms of cellular radio-protection, changes in expression of these apoptotic genes can be the primary pathway in responses of the lymphocytes radio-protection to the exposure. Our study revealed a significant decrease in the bax/bcl-2 ratio at 50 mGy dose compare to control and the other irradiated groups (p < 0.05).Conclusion: These results suggest that changes in the bax/bcl-2 ratio especially in radiation workers, as a key factor in apoptosis, can be considered as a biological marker in low-dose gamma radiation.

Oxidized Cell-Free DNA Role in the Antioxidant Defense Mechanisms under Stress.

The present study focuses on the investigation of the oxidized cell-free DNA (cfDNA) properties in several experimental models, including cultured cerebellum cells, peripheral blood lymphocytes (PBL), plasma, and hippocampus under an acute and chronic unpredictable stress model in rats. Firstly, our study shows that Spectrum Green fluorescence-labeled oxidized cfDNA fragments were transferred into the cytoplasm of 80% of the cerebellum culture cells; meanwhile, the nonoxidized cfDNA fragments do not pass into the cells. Oxidized cfDNA stimulates the antioxidant mechanisms and induction of transcription factor NRF2 expression, followed by an activation of NRF2 signaling pathway genes—rise of Nrf2 and Hmox1 gene expression and consequently NRF2 protein synthesis. Secondly, we showed that stress increases plasma cfDNA concentration in rats corresponding with the duration of the stress exposure. At the same time, our study did not reveal any significant changes of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) level in PBL of rats under acute or chronic stress, probably due to the significantly increased Nrf2 expression, that we found in such conditions. 8-oxodG is one of the most reliable markers of DNA oxidation. We also found an increased level of 8-oxodG in the hippocampal homogenates and hippocampal dentate gyrus in rats subjected to acute and chronic stress. Taken together, our data shows that oxidized cfDNA may play a significant role in systemic and neuronal physiological mechanisms of stress and adaptation.

Morphological Characteristics of the Thymus and Spleen and the Subpopulation Composition of Lymphocytes in Peripheral Blood during Systemic Inflammatory Response in Male Rats with Different Resistance to Hypoxia.

On the model of the systemic inflammatory response (SIRS), induced by lipopolysaccharide (LPS), the morphological and functional changes in the thymus and spleen and the subpopulation composition of peripheral blood lymphocytes of rats differing in resistance to hypoxia were studied. It was demonstrated that the level of endotoxin in blood serum after 3 hours of LPS administration in susceptible-to-hypoxia rats was 64 times higher than in the control group, while in tolerant-to-hypoxia animals it was only 8 times higher in 6 hours. After 24 hours of LPS injection, only in susceptible-to-hypoxia rats did the level of C-reactive protein in blood serum increase. There is a difference in the dynamics of morphological changes of lymphoid organs after LPS injection in tolerant- and susceptible-to-hypoxia animals. After 3 hours of LPS administration, the tolerant-to-hypoxia rats showed no changes in the thymus, spleen, and subpopulation composition of lymphocytes in peripheral blood. After 6 hours there was only a decrease in B-lymphocytes and increase in cytotoxic T-lymphocytes and NK cells. After 1 day of LPS injection, the tolerant-to-hypoxia rats had devastation in PALS of the spleen. After 3 hours of LPS injection the susceptible-to-hypoxia animals had reactive changes in the lymphoid organs: decrease of the thymus cortex, narrowing of the marginal zones of spleen lymphoid follicles, widening of their germinal centers, and a decrease in the absolute number of cytotoxic T-lymphocytes, NK cells, and B-lymphocytes. After 24 hours of LPS injection the tolerant-to-hypoxia animals had a greater absolute number of T-lymphocytes and NK cells in comparison with the susceptible rats. Thus, in animals with different resistance to hypoxia the LPS-induced SIRS is characterized by different dynamics of morphological and functional changes of the thymus and spleen. The obtained data will serve as a basis for the development of new individual approaches to the prevention and treatment of infectious and inflammatory diseases.

Effects of long term oral acrylamide administration on alpha naphthyl acetate esterase and acid phosphatase activities in the peripheral blood lymphocytes of rats

ABSTRACTAcrylamide is an important industrial chemical; it also is formed in starch-rich foodstuffs during baking, frying and roasting. Most acrylamide exposure occurs by ingestion of processed foods. We investigated possible immunotoxic effects of extended administration of low doses of acrylamide in rats. To do this, we measured alpha-naphthyl acetate esterase (ANAE) and acid phosphatase (ACP-ase) activities in peripheral blood lymphocytes. Male and female weanling Wistar rats were administered 2 or 5 mg acrylamide/kg/day in drinking water for 90 days. Peripheral blood was sampled at the end of the administration period. We found ANAE staining in eosinophils and T-lymphocytes, but not in monocytes, platelets, B-lymphocytes and neutrophils. ACP-ase was found in B-lymphocytes. We found a significant reduction of the ratio of ANAE:ACP-ase in lymphocytes of the experimental animals compared to controls. We found no statistically significant differences between the doses or sexes. We found that acrylamide ingested in processed foods might affect the immune system adversely by decreasing the population of mature T- and B-lymphocytes.

Experimental substantiation of the possibility of using the cytochemical parameters of peripheral blood to assess the functional state of the organism of the pilot

The activity of succinate (mitochondrial), lactate, α-glycerophosphate, glucose-6-phosphate peripheral blood lymphocytes in rats under the conditions of modeling acute and chronic gravitational stress was studied by quantitative spectrophotometry of cytochemical reaction products to reveal the localization of oxidative enzymes. The dynamics of morphofunctional transformations is determined, the severity of which depends on the duration of the effect (the rotation time of the animals on the centrifuge). For acute gravitational stress, changes in the activity of mitochondrial succinate dehydrogenase are characteristic, and in the various stages of chronic gravitational stress, glycolysis (increase in lactate dehydrogenase and α-glycerophosphate dehydrogenase activity) and plastic metabolism (increase in glucose-6-fosfata dehydrogenase activity) are characteristic. These changes are explained by the inclusion of neurohumoral regulatory mechanisms that ensure synchronization of oxidative metabolic processes in the cells of organs of various body systems under unfavorable conditions of their functioning. Significant correlation links between the activity of lymphocyte enzymes with the same indices of neurons of the celiac sympathetic node, adenocytes of the cortical and medulla of the adrenal glands have been established. This suggests that the activity of lymphocyte enzymes can be used as indirect indicators of the processes occurring in the organs of the sipato-adrenomedular and pituitary-adrenocortical systems under conditions of acute and chronic gravitational stress.

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.

Study of commonly used organophosphate pesticides that induced oxidative stress and apoptosis in peripheral blood lymphocytes of rats.

In a previous study, we have found that organophosphate (OP) pesticides such as chlorpyrifos (CPF), methyl parathion (MPT), and malathion (MLT) significantly induced genotoxicity in peripheral blood lymphocytes of rats. To explore the mechanism of OP-induced genotoxicity, we measured the formation of DNA interstrand cross-links (DICs) and apoptosis in peripheral blood lymphocytes of rats. Peripheral blood lymphocytes of rats were treated with CPF, MPT, and MLT individually and in combination at concentrations of 0.1 and 0.25 LC50 for 2, 4, 8, and 12 h at 37°C. Lipid peroxidation (LPO) was measured as a biomarker of oxidative stress. Apoptosis induced by CPF, MPT, and MLT individually and in combination was determined by measuring the intracellular level of active caspase-3 and caspase-9 by spectrofluorimetry. We found significant dose- and time-dependent increases in LPO, DICs formation and increase of intracellular active caspase-3 and caspase-9 in exposed peripheral blood lymphocytes of rats. These findings suggest that the studied pesticides have potential to induce oxidative stress, cause DNA adduct formation, and cause failure of adduct repair, which leads to apoptosis that is partially mediated by activation of intracellular caspase-3 and caspase-9.

Human fetal mesenchymal stem cell secretome enhances bone consolidation in distraction osteogenesis.

BackgroundDistraction osteogenesis (DO) is one of the most dramatic reconstructive techniques for inducing bone regeneration, but it involves an undesirably long period for bone consolidation. Developing innovative approaches to enhance bone consolidation is a burning need. Human fetal mesenchymal stem cells (hFMSCs) have been shown to express more primitive developmental genes than those of human adult mesenchymal stem cells (hAMSCs), which is a preferable source for cell therapy and tissue regeneration. In the present study, we investigated the immunogenicity of using the human mesenchymal stem cell (MSC) secretome on rat cells, the effects of secretome on osteogenic differentiation of rat bone marrow-derived MSCs (rBMSCs), and the potential application of hFMSC secretome in promoting bone consolidation in a rat DO model.MethodsSecretome was collected from MSC culture and was used to treat rBMSCs. Following secretome treatment, cell proliferation, alkaline phosphatase staining, Alizarin Red S staining, and mRNA expression of osteogenic differentiation-related genes (including ALP, Runx2, OCN, OPN, and Osx) in the rBMSCs were checked, as well as mixed rat peripheral blood lymphocyte reaction. hFMSC secretome was injected locally into the regenerates from the end of lengthening every 3 days in the rat DO model, until termination. The regenerates were subject to weekly x-rays, micro-computed tomography (μCT) and mechanical testing examination. The bone quality was assessed by histology and immunohistochemistry examinations.ResultsCompared to the secretome from rBMSCs and hAMSCs, hFMSC secretome had the best osteogenic induction ability and low immunogenicity. hFMSC secretome with different doses showed no effect on cell viability. hFMSC secretome at the dose of 100 μg/μl could significantly increase the expression of alkaline phosphatase and all the osteogenic marker genes, as well as the amount of calcium deposits in the rBMSCs. Finally, the local application of hFMSC secretome in distraction regenerates in a rat DO model significantly improved bone consolidation according to the results of μCT, mechanical test, and histological and immunohistochemistry analysis.ConclusionsThe current study demonstrated that hFMSC secretome promotes osteogenesis of rBMSCs and bone consolidation during DO. hFMSC secretome may be a new therapeutic strategy to enhance bone consolidation in patients undergoing DO treatment.

Evaluation of caspase dependent apoptosis induced by organophosphate pesticide in peripheral blood lymphocytes of rats

Evaluation of caspase dependent apoptosis induced by organophosphate pesticide in peripheral blood lymphocytes of rats

Establishment of a γ-H2AX foci-based assay to determine biological dose of radon to red bone marrow in rats.

The biodosimetric information is critical for assessment of cancer risk in populations exposed to high radon. However, no tools are available for biological dose estimation following radon exposure. Here, we established a γ-H2AX foci-based assay to determine biological dose to red bone marrow (RBM) in radon-inhaled rats. After 1–3 h of in vitro radon exposure, a specific pattern of γ-H2AX foci, linear tracks with individual p-ATM and p-DNA-PKcs foci, was observed, and the yield of γ-H2AX foci and its linear tracks displayed a linear dose-response manner in both rat peripheral blood lymphocytes (PBLs) and bone-marrow lymphocytes (BMLs). When the cumulative doses of radon inhaled by rats reached 14, 30 and 60 working level months (WLM), the yields of three types of foci markedly increased in both PBLs and BMLs, and γ-H2AX foci-based dose estimates to RBM were 0.97, 2.06 and 3.94 mGy, respectively. Notably, BMLs displayed a more profound increase of three types of foci than PBLs, and the absorbed dose ratio between BMLs and PBLs was similar between rats exposed to 30 and 60 WLM of radon. Taken together, γ-H2AX foci quantitation in PBLs is able to estimate RBM-absorbed doses with the dose-response curve of γ-H2AX foci after in vitro radon exposure and the ratio of RBM- to PBL-absorbed doses in rats following radon exposure.

The Effect of Alpha-Lipoic Acid on Preventing Amikacin-Induced DNA Damage in Rats

Amikacin (AK) is a preferred antibacterial drug, but it is associated with significant dose dependent toxic side effects. Alpha-lipoic acid (ALA) is an effective antioxidant which protects cellular macromolecules against various drug induced toxicities. This study was designed to determine the effects of AK on the DNA of rat lymphocytes, and the possible protective effects of ALA on genotoxicity induced by AK. Three different groups of rats (n = 8) were formed: (1) Control: administered saline, (2) AK: administered 1.2 g/kg, single dose, intraperitoneally, and (3) ALA: administered 100 mg/kg, p.o. and AK combination (ALA 1 day before the AK for 5 days). DNA damage through comet assay was analyzed in peripheral blood lymphocytes of rats. Total oxidant system (TOS), total antioxidant system (TAS), and ischemia-modified albumin (IMA) as serum oxidative stress markers were measured in blood serum. AK administration to rats increased DNA damage, IMA and TOS levels (p = 0.0002, p = 0.013, p = 0.048, respectively), but decreased TAS levels (p = 0.011). ALA administration before AK injection caused a significant increase in TAS levels (p = 0.000), and caused a significant decrease in TOS, IMA, and DNA damage levels (p = 0.000, p = 0.003, 0.044, respectively). The present findings showed that AK caused DNA damage in lymphocytes of rats and ALA provided a protective effect against AK induced DNA damage by balancing the oxidant-antioxidant status, and it may therefore improve the therapeutic effects of AK.

Characterization of microRNA expression profiling in peripheral blood lymphocytes in rats with experimental autoimmune uveitis.

We aimed to investigate the alterations of microRNA (miRNA) genomics in peripheral blood lymphocytes in experimental autoimmune uveitis (EAU) rats versus control samples. Six Lewis rats received interphotoreceptor retinoid-binding protein (IRBP) emulsion to induce EAU. On day 12, peripheral blood lymphocytes were isolated, and total RNAs were extracted. Using microarray analysis, we analyzed the aberrant miRNAs, validated the relevant expression of differentially expressed miRNAs, and predicted the possible miRNA targets and signaling pathways. The results indicated that 36 miRNAs were upregulated and 31 miRNAs were downregulated in EAU rats versus normal samples. Real-time quantitative PCR substantiated a high degree of confidence for the differentially expressed miRNAs, and miRNA analyses showed the differentially expressed miRNA targets were involved not only in the multicellular organismal process and developmental process, but also in T cell receptor signaling pathway, B cell receptor signaling pathway and so on. Our findings show that the differentially expressed miRNAs in EAU rats were closely associated with immune signaling pathways and may be applied in early prevention, prognosis and possible therapy in uveitis, indicating that miRNAs play an important role in the development of uveitis.

The effects of Jieduquyuzishen prescription-treated rat serum on the BAFF/BAFF-R signal pathway.

Systemic lupus erythematosus (SLE) is a chronic inflammatory disease mainly characterized by B cell hyperactivity. Glucocorticoid (GC) is widely used in SLE for its potent anti-inflammatory and immunosuppressive effects. Despite its important clinical efficacy, high-dose or long-term use of GC can cause severe side effects, such as osteoporosis, osteonecrosis, cataracts, hyperglycemia, coronary heart disease and cognitive impairment. Our early clinical studies have shown that Jieduquyuzishen prescription (JP) can effectively reduce the adverse effects and improve the curative effect of GC in the treatment of SLE. The BAFF/BAFF-R signaling pathway plays an important role in the development of SLE and has been regarded as a potential target for the therapy of SLE. In this study, we attempt to investigate the effect of JP on the BAFF/BAFF-R signaling pathway to explore the mechanism of JP in reducing the toxicity and enhancing the efficacy of GC. YAC-1 cells, isolated rat peripheral blood lymphocytes, polymorphonuclear neutrophils and spleen lymphocytes were treated with drug-containing serum. The results of RT-PCR, Western blot and dual-luciferase reporter gene assays indicate that either JP or GC can inhibit the mBAFF-induced up-regulation of BAFF, BAFF-R, Bcl-2, IL-10 and NF-κB in YAC-1 cells and WEHI-231 cells. Furthermore, MTS, flow cytometry and CFSE results reveal that the proliferation and survival of lymphocytes activated by mBAFF are suppressed by JP, GC and their combination. Contrary to GC, JP can reduce the apoptosis and raise the survival of polymorphonuclear neutrophils and can’t increase the apoptosis of the peripheral blood lymphocytes and spleen lymphocytes. Therefore, it is possible that JP can down-regulate the BAFF/BAFF-R signaling pathway as effectively as GC, which may result in the dosage reduction of GC, thus decreasing the toxicity and improving the efficacy of GC-based treatment of SLE.

Evaluation of genotoxic potential of commonly used organophosphate pesticides in peripheral blood lymphocytes of rats.

Chlorpyrifos (CPF), methyl parathion (MPT), and malathion (MLT) are among the most extensively used organophosphate (OP) pesticides in India. DNA protein cross-links (DPC) and DNA strand breaks are toxic lesions associated with the mechanism(s) of toxicity of carcinogenic compounds. In the present study, we examined the hypothesis that individual and interactive genotoxic effects of CPF, MPT, and MLT are involved in the formation of DPC and DNA strand break. The DNA strand break was measured by comet assay and expressed as DNA damage index, while DPC estimation was carried out by fluorescence emission assay. The results showed that exposure of rat lymphocytes with CPF, MPT, and MLT caused significantly marked increase in DNA damage and DPC formation in time-dependent manner. MPT caused the highest damage, and these pesticides do not potentiate the toxicity of each other.

In vitro studies on organophosphate pesticides induced oxidative DNA damage in rat lymphocytes

Organophosphate (OP) pesticides are widely used for agricultural and household pest control. We studied the genotoxicity of the commonly used OP pesticides chlorpyrifos (CPF), methyl parathion (MPT), and malathion (MLT), individually and in combination, in Wistar rat peripheral blood lymphocytes in vitro. DNA single-strand and double-strand breaks were measured by single cell gel electrophoresis (SCGE; comet assay). To test whether the DNA lesions were caused by oxidative stress, the DNA repair enzymes formamidoaminopyrimidineglycosylase (Fpg) and endonuclease (Endo III), which convert base damages to strand breaks, were used. Significant increases in strand breaks and in levels of the reactive oxygen species (ROS) superoxide anion and hydrogen peroxide were observed in lymphocytes treated with pesticides. MPT exposure caused the greatest DNA damage and ROS production, followed by CPF and ML. Our results demonstrate genotoxic potential of these OP pesticides.

Potential chemoprotective effects of selenium on diazinon-induced DNA damage in rat peripheral blood lymphocyte

The purpose of this study was to investigate the protective effects of selenium (Se) against genotoxicity induced by diazinon (DZN) in rat peripheral blood lymphocytes by micronucleus (MN) test. Animals were concurrently administered intraperitoneally with DZN in proper solvent (20mg/kg body weight (b.w.)) and Se at three different doses (0.5, 1, and 2mg/kg b.w.) for 30 consecutive days. The positive control group received DZN at the same dose without Se. After 24h of last injection, 0.5ml blood of each rat was received and cultured in culture medium for 44h. The lymphocyte cultures were mitogenically stimulated with cytochalasin B to allow the evaluation of number of MNs in cytokinesis-blocked binucleated cells. Incubation of lymphocytes with DZN induced additional genotoxicity and is shown by increase in MNs frequency in human lymphocytes. Se at low dose of 0.5mg/kg had a maximum effect and significantly reduced the MNs frequency in cultured lymphocytes (p<0.0001) that reduced the frequency of MN from 12.78±0.24% for DZN group to 4.40±0.36. The present study revealed that Se particularly at low doses has a potent antigenotoxic effect against DZN -induced toxicity in rats, which may be due to the scavenging of free radicals and increased antioxidant status.

Time-order effects of vitamin C on hexavalent chromium-induced mitochondrial damage and DNA-protein crosslinks in cultured rat peripheral blood lymphocytes

Hexavalent chromium [Cr(VI)] and its compounds have extensive applications in many industries and are widely known to cause occupational diseases as well as carcinogenic effects in humans. Mitochondrial damage, which is important in Cr(VI)‑induced cytotoxicity, may be characterized by the opening status of the permeability transition pore, the maintenance of the mitochondrial membrane potential and the level of malondialdehyde. The formation of DNA‑protein crosslinks (DPCs) in target tissues appears to be the direct and primary genotoxic effect of Cr(VI) exposure, and the lymphocytic DPCs may be viewed as a biomarker of internal Cr(VI) accumulation. It is well known that vitaminC (vitC) is an important biological reducing agent in humans and animals, which is capable of reducing Cr(VI). Regardless of the evidence from cell culture and invivo experiments of the protective effect of the antioxidant, vitC, following exposure to Cr(VI), no studies have been conducted to date to demonstrate the time‑order effects of vitC on Cr(VI)‑induced mitochondrial damage and DPC formation. In the present study, by using peripheral blood lymphocytes from Sprague‑Dawley rats, we demonstrated that vitC pre‑ and co‑treatment have a protective effect against Cr(VI)‑induced loss of cell viability and mitochondrial damage, while only vitC co‑treatment has a protective effect against the Cr(VI)‑induced increase in DPCs. The mechanistic investigation revealed that cellular reactive oxygen species levels are correlated with Cr(VI)‑induced mitochondrial damage, and that p53 expression is correlated with the Cr(VI)‑induced increase in DPCs. We concluded that vitC exerts different time‑order effects on Cr(VI)‑induced mitochondrial damage and DPC formation, and that biomarkers, including DPC and p53, may be used in the assessment of the development of Cr(VI)‑induced cancer. These findings facilitate more detailed follow‑up of the Cr(VI)‑exposure populations for secondary prevention.