Selenium deficiency causes immune damage by activating the DUSP1/NF-κB pathway and endoplasmic reticulum stress in chicken spleen.

Selenium (Se) is an essential trace element and its deficiency can lead to immune dysfunction. Many studies have investigated the immune damage caused by Se deficiency in chickens, but its mechanism still needs to be explored. In this study, we fed 1-day-old Hyline male chickens with Se deficient diets (the Se content was 0.008 mg kg-1 of diet) and a basal diet (the Se content was 0.15 mg kg-1 of diet). The spleen was collected at the sixth week and used for subsequent experiments. The pathological analysis showed that Se deficiency leads to the destruction of the normal nuclear structure of the spleen cell, and we can observe obvious chromatin condensation and nuclear debris. We constructed a transcriptome database and analyzed the abundance of various genes in the spleen by transcriptome sequence. The analysis of differentially expressed genes (DEGS) showed significant changes in 337 genes, including 210 up-regulations and 127 down-regulations after feeding Se deficient diets. Se deficiency can significantly change oxidative stress and inflammatory response genes in chicken spleen. This study confirmed that Se deficiency increased the IL-2 levels, whereas it down-regulated IL-17, IFN-γ and Foxp3, which indicates that the immune dysfunction of the spleen and Th1/Th2 is imbalanced. We also found that Se deficiency down-regulated some related genes for endoplasmic reticulum Ca2+ transport, leading to endoplasmic reticulum stress (ERS). Moreover, we determined that Se deficiency triggered the low expression of DUSP1/NF-κB. In summary, our results indicate that Se deficiency can inhibit the spleen immune function of chickens by regulating the DUSP1/NF-κB pathway and ERS, leading to spleen damage in chickens. Based on transcriptomics research, our results will help further study the harmful effects of Se deficiency.

Nutritional support contributes to recuperation in a rat model of aplastic anemia by enhancing mitochondrial function.

OBJECTIVES: Acquired aplastic anemia (AA) is a hematopoietic stem cell disease that leads to hematopoietic disorder and peripheral blood pancytopenia. We investigated whether nutritional support is helpful to AA recovery. METHODS: We established a rat model with AA. A nutrient mixture was administered to rats with AA through different dose gavage once per day for 55 d. Animals in this study were assigned to one of five groups: normal control (NC; group includes normal rats); AA (rats with AA); high dose (AA + nutritional mixture, 2266.95 mg/kg/d); medium dose (1511.3 mg/kg/d); and low dose (1057.91 mg/kg/d). The effects of nutrition administration on general status and mitochondrial function of rats with AA were evaluated. RESULTS: The nutrient mixture with which the rats were supplemented significantly improved weight, peripheral blood parameters, and histologic parameters of rats with AA in a dose-dependent manner. Furthermore, we observed that the number of mitochondria in the liver, spleen, kidney, and brain was increased after supplementation by transmission electron microscopy analysis. Nutrient administration also improved mitochondrial DNA content, adenosine triphosphate content, and membrane potential but inhibited oxidative stress, thus, repairing the mitochondrial dysfunction of the rats with AA. CONCLUSIONS: Taken together, nutrition supplements may contribute to the improvement of mitochondrial function and play an important role in the recuperation of rats with AA.

Protective effect of α-lipoic acid against spleen toxicity of dimethylnitrosamine in male mice: Antioxidant and ultrastructure approaches.

The α-lipoic acid is a soft material and useful for the potential biomedical applications. The study was aimed to assess the potency of α-lipoic acid (ALA) against the toxicity of dimethylnitrosamine (DMN) on spleen of mice by assessing the antioxidants, histopathological and ultrastructure changes. The experiment was achieved on six groups of male mice as following; groups 1, 2, 3, and 4 were served as a control, ALA groups, low dose of DMN (DMN-LD; 2mgkg-1) and high dose of DMN (DMN-HD; 4mgkg-1). Group 5 was received DMN-LD plus ALA and group 6 was given DMN-HD plus ALA. The results indicated that DMN elevated lipid peroxidation, xanthine oxidase, nitric oxide, and decline the antioxidant enzymes as well as raise the C-reactive protein and tumor necrosis factor. A critical obstruction was harmonized with a reduced in lymphocyte number in the white pulp were observed. All the lymphatic nodules appeared smaller in DMN-HD group. In spleen tissues, marked changes of rough endoplasmic reticulum and appearance of three large lymphocytes were noticed. ALA/DMN treatments were improved all the oxidative damage and the ultrastructure changes. The data evince that ALA was eliminated the adverse effects of DMN on spleen of mice.

Systemic Amyloidosis Model on Young Mice.

Subcutaneous daily injection (with neglect of aseptics) of 0.5 ml solution of soybean cream substitute (10% volume in distilled water) during 30 days caused systemic amyloidosis in 30-day-old mice. All the known methods for induction of systemic amyloidosis are based on the use of old animals, as senile tissue bradytrophy allows effective simulation of amyloidosis.

Effects of dietary n-6/n-3 polyunsaturated fatty acid ratios on the mass, and histological and ultrastructures of liver, spleen and thymus of 70-day-old Yangzhou goslings.

The objective of this study was to determine the effects of dietary n-6/n-3 polyunsaturated fatty acid (PUFA) ratios on the organ indexes, and histological and ultrastructures of organs including liver, spleen and thymus in 70-day-old Yangzhou goslings. One-hundred and sixty 21-day-old Yangzhou goslings were randomly divided into 4 groups and fed 4 diets varying in the n-6/n-3 PUFA ratio from 3:1 up to 12:1. After 1-week acclimation, the feeding experiment lasted for 6 weeks. At the end of the experimental period, goslings were slaughtered and the liver, spleen and thymus were weighed, and their histological and ultrastructures were examined. The results showed that the organ indices in the 3:1 group were remarkably higher than in the other three groups, whereas the mitochondrial square did not differ among four groups. The histological and ultrastructures of the liver, spleen and thymus were not affected by the diets with the lower n-6/n-3 PUFA ratios (3:1 and 6:1). However, feeding diets with the higher n-6/n-3 PUFA ratios (9:1 and 12:1), the nuclear chromatin was concentrated and marginalized; the cell membrane was contracted inwardly and disrupted; the mitochondrial membrane was damaged to some degree. In conclusion, the diet containing higher content of n-3 PUFA might improve immune capacity of goslings the animal by accelerating the growth and maintaining cellular structures of organs like liver, spleen and thymus.

[Ginkgo biloba extract enhances the immune function of spleen and thymus in SD rats].

OBJECTIVE: To study the effect of Ginkgo biloba extract (GBE) on the immune function of spleen and thymus in SD rats. METHODS: Forty SD rats were randomly divided into four groups (10 rats each group). Three experimental groups were given GBE daily by gavage in doses of 40, 120, 360 mg/(kg.d), respectively. Animals in the control group were fed the same amount of PBS. After 28 days, the rats were sacrificed by chloral hydrate anesthesia. The spleen and thymus were harvested to determine the organ index first. MTT assay was used to detect the concanavalin A (ConA)-induced splenic lymphocyte proliferation and transformation. Neutral red assay was performed to measure the rat peritoneal macrophage phagocytosis. The ultrastructural changes of spleen and thymus were observed under scanning electron microscope. RESULTS: Administration of GBE in the rats increased the mass indexes of rat thymus and spleen, dose-dependently elevated the lymphocyte proliferative responses and enhanced the peritoneal macrophage phagocytosis. In experimental groups, the numbers of mature spleen and thymus lymphocytes were significantly raised in comparison with the control rats. CONCLUSION: GBE plays a regulatory role in immune function of the rat by increasing the mass of immune organs, increasing the number of mature T lymphocytes as well as their proliferative responses, and enhancing the phagocytic capacity of peritoneal macrophages.

Antitumor activity of Pulsatilla chinensis (Bunge) Regel saponins in human liver tumor 7402 cells in vitro and in vivo.

Pulsatilla chinensis (Bunge) Regel is a Chinese medicinal herb for "blood-cooling" and detoxification. Now it is used for the treatment of malignant tumor, but the antitumor mechanisms and toxic side effects of P. chinensis are unclear. The present study was undertaken to investigate if P. chinensis saponins (PRS) possesses anticancer effects and toxic side effects in human liver tumor 7402 cells in vitro and vivo. 7402 cells were treated with different concentrations of PRS for 24h. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cell apoptosis was assessed by flow cytometry. The in vivo effect of PRS on 7402 tumor cells transplanted in athymic nude mice was investigated. 15 saponins were isolated and identified from PRS. PRS inhibited the proliferation of human liver tumor 7402 cells in vitro by apoptosis. 19 days after administration of PRS (100, 200mg/kg), the weight of tumor mass was markly decreased in nude mice. The anti-tumor effect of PRS in vivo was associated with a significant increase in the 7402 apoptosis rate. Although PRS inhibited the weight of mice, it showed almost no effect on leukocyte number, liver and spleen weight index. Light microscopic histopathological examination showed that PRS had no specific lesion in organ. These results suggested that P. chinensis saponins exert potential anticancer activity in treating tumors in nude mice and no toxic side effects.

Investigation of toxicological effects of Shuanghuanglian injection in Beagle dogs by metabonomic and traditional approaches.

In this study, clinical biochemistry, hematology, histopathology and (1)H nuclear magnetic resonance spectroscopy-based metabonomic approaches were applied to investigate the toxicological effects of Shuanghuanglian (SHL) injection after intravenous administration (dosed at 4, 12 and 36 mL stock/kg) in Beagle dogs for 30 d. Decreases in red blood cells, hemoglobin, mean cell volume, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration were observed in the high-dose group. Elevated reticulocytes, total bilirubin and direct bilirubin were also observed in this group. Moreover, significant hemosiderosis and Prussian blue positivity were detected in the liver, spleen and kidney from high-dose group animals, and transmission electron microscopy examination revealed an appreciable number of acanthrocytes in the liver. These results collectively indicate that SHL injection has the potential to cause hemolytic anemia. Metabonomic analysis showed increases in serum lactate, choline and phosphocholine but a decrease in taurine in treated groups and these findings may underlie the toxicological mechanism of SHL injection. In summary, SHL injection shows hemolytic effects in Beagle dogs; moreover, serum choline and phosphocholine as well as lactate and taurine may be the biomarkers for hemolytic anemia induced by SHL injection.

Characterization of gold nanorods in vivo by integrated analytical techniques: their uptake, retention, and chemical forms.

Integrated analytical techniques were used to study the tissue distribution and structural information of gold nanorods (Au NRs) in Sprague-Dawley rats through tail intravenous injection. Before in vivo experiments were conducted, careful characterization of Au NRs was performed. The zeta potential proved that adsorption of bovine serum albumin on Au NRs turned the surface charges from positive to negative as in an in vitro simulation. The biodistribution of Au NRs was investigated quantitatively by inductively coupled plasma mass spectrometry at different time points after injection. As target tissues, both liver and spleen were chosen to further demonstrate the intracellular localization of Au NRs by the combination of transmission electron microscopy and energy-dispersive X-ray spectroscopy. Moreover, synchrotron-radiation-based X-ray absorption spectroscopy was employed and it was observed that long-term retention of Au NRs in liver and spleen did not induce obvious changes in the oxidation states of gold. Therefore, the present systematic method can provide important information about the fates of Au NRs in vivo and can also be extended to study the biological effects of other metallic nanomaterials in the future.

Expression of osteogenic proteins during the intrasplenic transplantation of Meckel's chondrocytes: A histochemical and immunohistochemical study.

Meckel's chondrocytes, derived from the ectomesenchyme, have the potential to transform into other phenotypes. In this study, we transplanted cell pellets of Meckel's chondrocytes into isogenic mouse spleens and analyzed their phenotypic transformation into osteogenic cells using histological and immunohistochemical methods. With the increasing duration of transplantation, chondrocytes were incorporated into splenic tissues and formed a von Kossa-positive calcified matrix containing calcium and phosphoric acid, similar to that of intact bone. Type I, II, and X collagens, and the bone-marker proteins osteocalcin, osteopontin, osteonectin, and bone morphogenetic protein-2 (BMP-2) were immunolocalized in the matrix formed by the transplanted chondrocytes. Osteopontin and osteonectin were detected in the calcified matrix at earlier stages than osteocalcin and BMP-2. Type II collagen was expressed during the first week of transplantation, and type X collagen-positive cells appeared scattered during the initial stage of calcification, these collagens being later replaced by type I collagen formed by osteocyte-like cells. Electron microscopic observations revealed that chondrocytes surrounded by the calcified matrix transformed into spindle-shaped osteocytic cells accompanying the formation of bone-type thick-banded collagen fibrils. These results suggest that phenotypic switching of Meckel's chondrocytes can occur under in vivo conditions at a cellular morphological level.

[Regulating effects of Paecilomyces cicadae polysaccharides on immunity of aged rats].

OBJECTIVE: To investigate the effects of Paecilomyces cicadae polysaccharide (PCPS) on the immunological function of aged rats in vivo. METHOD: The young and old rats were administered with normal saline as control groups, and the rats from test group were sc given 50, 100, 200 mg x kg(-1) x d(-1) dosage of PCPS for 3 weeks. The phagocytizing rate and index of PMphi, AMphi to S. aureas were observed, and the colorimetric MTI was used to analyze the proliferative activity of spleenocytes which had been stimulated with ConA or LPS. We also inspected the ability varing of ACP, LDH, ARG of spleen, and observed the ultramicro structure of spleen under the SEM. RESULT: The phagocytosis of Mphi was lower in aged group than that in young' s group, and the proliferative activity of spleenocytes was lower too. The activities of ACP, LDH, ARG of spleen were extremely decreased (P < 0.01) in aged rats as well. The proliferative activity and phagocytotic rate were both extremely increased in PCPS groups (P < 0.01), and the mitochondrion and endoplasmic reticulum of spleen were accrementition as well (P < 0.01). CONCLUSION: PCPS could enhance the phagocytizing function of PMphi, AMphi of aged rats in vivo, and strengthen the immune function of spleen and its proliferative activity as well. Then the immunity of aged rats could be improved. The PCPS may be an anti-aging agent.

Achyranthes aspera stimulates the immunity and enhances the antigen clearance in Catla catla.

Achyranthes aspera, an Indian medicinal plant (family: Amaranthaceae) was incorporated in artificial fish diet, and fed to catla Catla catla. After 4 weeks of feeding, fish were immunized with bovine serum albumin (BSA), spleen and blood were sampled on weekly intervals for four times after immunization. Antigen-specific antibody level in serum was determined by ELISA. Antigen clearance was determined in spleen by immuno-electron microscopy. Achyranthes has significantly (P<0.05) enhanced the BSA-specific antibody titers than the untreated control group throughout the study period. The efficiency of antigen clearance was also enhanced in C. catla treated with Achyranthes.

Alcoholic turmeric extract simultaneously activating murine lymphocytes and inducing apoptosis of Ehlrich ascitic carcinoma cells.

In the present investigation ethanolic turmeric extract has been found to play diabolically opposite role on murine lymphocytes and on Ehlrich ascitic carcinoma cells. Turmeric stimulates the lymphocytes into the effector pathway as studied through in vitro viability, blastogenesis and (3)H-TdR incorporation and also seems to be healthy under scanning electron microscopy (SEM). SEM revealed the formation of cytoplasmic blebs and plasma membrane disintegration of tumor cells with ethanolic turmeric extract treatment, suggesting turmeric to be initiating apoptosis of tumor cells. Thus, in the present work viability of the cells, blastogenesis, DNA synthesis and SEM study establish the fact that turmeric is a conducive agent for lymphocytes and inhibitory as well as apoptotic for tumor cells.

Photofrin II induces cytokine secretion by mouse spleen cells and human peripheral mononuclear cells.

The aim of our study was to find out if Photofrin II, a cytotoxic drug used routinely in photodynamic therapy (PDT), can induce immune responses in vitro, and to compare its effects with those of the protoporphyrin 9, hemin, which also has antitumor properties. We tested the effect of these porphyrins on lymphocyte proliferation and secretion of interleukin-2, interleukin-3, tumor necrosis factor alpha (TNFalpha) and interferon gamma (IFNgamma), by human or murine mononuclear cells (MNC) without an activating light. Both the Photofrin II- and hemin-treated cells showed a significant increase in cytokine secretion in the presence of suboptimal concentrations of mitogen. Moreover, Photofrin II and hemin significantly increased production of TNFalpha and IFNgamma even in the absence of mitogen. The cellular binding sites of Photofrin II and hemin to MNC were localized by electromicroscopy or fluorescence. Combined stimulation of cells by mitogens and porphyrins maintained optimal vital ionic balance of potassium, sodium and chlorine in the lymphocytes. In the cells thus treated there was a significant increase in intracellular calcium, a vital second messenger for lymphokine secretion. We demonstrate that the effect of Photofrin II on the immune system involves enhanced cytokine secretion which may account for the subsequent tumor eradication by PDT.

[Effect of HE-NE laser acupuncture on the spleen in rats].

The splenic sinuses, lymphocytes and antigen presenting cells in the spleen of rat stimulated by He-Ne laser acupuncture were observed by using TEM to investigate the ultrastructural changes of them. The width of endothelial interstice was increased, hence a lot of blood cells oozed out of the splenic sinuses. There were numerous activated T cells which contained abundant mitochondria and ribosome in the periarterial lymphatic sheaths. The B cells were gradually differentiated into large lymphocytes, immature and mature plasmatic cells with a lot of endoplasmic reticula. They were prominently increased in the splenic nodules. The macrophages had short processes with numerous folds and microvilli and tended to neighboring lymphocytes. The nucleus pores were increased. There were a lot of vacuoles, phagosome and mitochondria were closed to macrophages and lymphocytes. In conclusion the activities of the cellular immunity and humoral immunity were enhanced by laser.

RZRs, a new family of retinoid-related orphan receptors that function as both monomers and homodimers.

Members of the superfamily of nuclear receptors share the greatest homology in their DNA-binding domains. We have used reverse transcription-polymerase chain reaction and highly degenerate primers based on the amino acid sequence of the zinc finger motif of known nuclear receptors to identify novel members of the family. Starting with rat brain RNA, we have isolated an orphan receptor that we call RZR beta. The sequence of its nearly full-length complementary DNA shows great similarity to RZR alpha, a receptor we recently identified from human umbilical vein endothelial cells. These RZR subtypes represent members of a new family of orphan nuclear receptors that most likely regulate specific gene expression. Sequence comparison with other known nuclear receptors reveals great similarity for both RZR subtypes to retinoic acid and retinoid-X receptors. By Northern blot analyses, we found RZR beta messenger RNA only in brain, whereas RZR alpha is expressed in many tissues. We show here that the RZRs bind as monomers to natural retinoid response elements formed by (A/G)GGTCA half-sites. However, a T-residue in the -1 position of this motif greatly enhances the DNA binding affinity of RZRs, whereas the -2 position has no influence. We show that RZRs can bind as homodimers on response elements formed by palindromes, inverted palindromes, or direct repeats of two TAGGTCA half-sites. Interestingly, these response elements display dramatically reduced affinity for retinoic acid receptor-retinoid-X receptor heterodimers. Thus, the 5'-flanking sequence of hexameric half-sites appears to be crucial to direct the activity of several nuclear receptors. On monomeric as well as dimeric binding sites, RZRs show constitutive transactivational activity that can be enhanced by unidentified components of fetal calf serum.

Prechaperonin 60 and preornithine transcarbamylase share components of the import apparatus but have distinct maturation pathways in rat liver mitochondria.

Mitochondrial preornithine transcarbamylase (p-OTC) and premalate dehydrogenase (p-MDH) are the only two matrix-located preproteins so far identified for which the proteolytic processing in vitro requires the formation of genuine processing intermediates, i-OTC and i-MDH, respectively. To establish the processing of other preproteins during import with respect to the two-step processing of p-OTC and p-MDH, the chelators EDTA and 1,10-phenanthroline were used to study the import and processing of rat prechaperonin 60 (p-cpn60) and p-OTC by mitochondria from four cpn60-containing organs. We found no evidence for a secondary processing step in the maturation of p-cpn60, but a clear requirement for two-step processing of p-OTC, even in three organs which do not contain ornithine transcarbamylase. The metal-ion requirement of the p-OTC processing activities in the organelle is consistent with the proposition that the mitochondrial processing protease (MPP) and mitochondrial intermediate peptidase (MIP) activities defined in vitro [Kalousek, F., Hendrick, J.P. & Rosenberg, L. E. (1988) Proc. Natl Acad. Sci. USA 85, 7536-7540] are responsible for precursor processing in vivo. The authenticity of two-step processing in vivo was, furthermore, established by demonstrating that i-OTC accumulates to high levels in Spodoptora frugiperda insect cells supplemented with MnCl2. The inability of the insect cells to process p-OTC fully is not a characteristic of cells grown in culture since cultured rat hepatoma cells process p-OTC to the fully processed m-OTC. Finally, we find that the import and processing of p-cpn60 and p-OTC is inhibited in an identical fashion by presequence-bovine-serum-albumin conjugates. The differences in proteolytic maturation between p-cpn60 and p-OTC are therefore not likely to result from different import pathways as the two precursors compete for common components of the import apparatus.

Ultrastructural observations in the carbonyl iron-fed rat, an animal model for hemochromatosis.

Rats fed a carbonyl iron-supplemented diet for 4-15 months were studied for iron content and morphologic changes in the liver, spleen, intestinal mucosa, pancreas and heart. All organs had an increased iron content measured by atomic absorption, with the highest concentrations in the liver and spleen. The periportal distribution of stored iron in the liver was similar to that in human hemochromatosis. In animals treated beyond 6 months Kupffer cells and sinusoidal lining cells also showed cytosiderosis. Electron microscopy provided information on ferritin and hemosiderin content and distribution within parenchymal and sinusoidal cells of the liver but no excessive fibrosis was found. Except for the spleen, the other organs showed less iron deposition. Iron-filled lysosomes (siderosomes) were found in macrophages in the intestinal lamina propria and pancreas, as well as in enterocytes, pancreatic acinar cells and heart muscle cells. Heavily iron-laden siderosomes had increased membrane instability which was demonstrated both morphologically and by measurements of latent lysosomal enzyme activities. Even though cirrhosis was not found, the distribution pattern of accumulated storage iron and lysosomal lability indicated that the carbonyl iron-fed rat is a suitable experimental model for human hemochromatosis.

Generalized lipidosis in newborn rats and Guinea pigs induced during prenatal development by administration of amphiphilic drugs to pregnant animals.

Pregnant rats and guinea pigs were treated throughout the second half of gestation with amphiphilic drugs (chlorphentermine, chlorcyclizine, chloroquine) known to induce generalized lipidosis. The offspring were sacrificed immediately after birth, and several tissues (lung, liver, kidney, spleen, pituitary gland, adrenal gland, spinal cord, hypothalamus) were examined by electron microscopy. Generalized lipidosis was found in the offspring of both species, albeit of lesser degree than in the mothers. The results show that fetal and adult tissues respond to lipidosis-inducing drugs in a qualitatively similar way; the quantitative differences found may be related to pharmacokinetic and cellular factors.

Quantitative x-ray microanalysis of spleen lysosomes after acid phosphatase reaction.

A quantitative X-ray microanalytical study of 80 spleen lysosomes after histochemical reaction for acid phosphatase was carried out. Lysosomal Fe concentrations showed a skewed distribution, with most lysosomes having relatively low concentrations. The concentration of Pb in the lysosomes (indicative of acid phosphatase activity) showed a close to normal distribution. Although the plot of lysosomal Pb concentrations against Fe concentrations showed a marked scattering of the points, there is a statistically highly significant correlation between accumulated metal and acid phosphatase activity as determined from the Pb concentrations. The relation between concentrations of P and those of Pb in the lysosomes points to the possibility that Pb(H2PO4)2 is the main reaction product of the histochemical acid phosphatase reaction.