Isolation, culture and identification of goat alveolar macrophages / 中国比较医学杂志

Objective In order to study the biological characteristics of macrophages and provide the materials to study the survival mechanism of intracellular parasites, we conducted this study to establish a high-purity alveolar macrophage isolation and culture method.Methods Goat lungs were lavaged with normal saline in sterile environment several times, and cells were collected and then goat alveolar macrophages were purified by density gradient centrifugation using peripheral blood mononuclear cells (PBMC) solution.The isolated goat alveolar macrophages were cultured in cell culture medium containing 10% fetal bovine serum and cell morphology was observed under an inverted microscope every day,and the phagocytic activity of the cells was detected by chicken red blood cell phagocytosis test.Flow cytometry was used to detect CD14, a characteristic monocyte-macrophage surface marker.Results The adherent cells were characterized by typical macrophage morphology, pseudopodia and protrusions, showing round and irregular shape, rich cytoplasm, and large cell body.Of the cultured macrophages, 54.5% could phagocytize chicken erythrocytes and showed good phagocytic activity.After one month of in vitro culture, 93.7% of the cells were able to express CD14 antigen, which had a macrophage-specific immunophenotype.Conclusions The alveolar macrophages obtained in this study have high purity and good bioactivity, thus provide a cell model for studying the immune mechanism of intracellular parasites.

Gap junction blockage promotes cadmium-induced apoptosis in BRL 3A derived from Buffalo rat liver cells

Gap junctions mediate direct communication between cells; however, toxicological cascade triggered by nonessential metals can abrogate cellular signaling mediated by gap junctions. Although cadmium (Cd) is known to induce apoptosis in organs and tissues, the mechanisms that underlie gap junction activity in Cd-induced apoptosis in BRL 3A rat liver cells has yet to be established. In this study, we showed that Cd treatment decreased the cell index (a measure of cellular electrical impedance) in BRL 3A cells. Mechanistically, we found that Cd exposure decreased expression of connexin 43 (Cx43), increased expression of p-Cx43 and elevated intracellular free Ca2+ concentration, corresponding to a decrease in gap junctional intercellular communication. Gap junction blockage pretreatment with 18β-glycyrrhizic acid (GA) promoted Cd-induced apoptosis, involving changes in expression of Bax, Bcl-2, caspase-3 and the mitochondrial transmembrane electrical potential (Δψm). Additionally, GA was found to enhance ERK and p38 activation during Cd-induced activation of mitogen-activated protein kinases, but had no significant effect on JNK activation. Our results indicated the apoptosis-related proteins and the ERK and p38 signaling pathways may participate in gap junction blockage promoting Cd-induced apoptosis in BRL 3A cells.

N-acetylcysteine protects against cadmium-induced oxidative stress in rat hepatocytes

Cadmium (Cd) is a well-known hepatotoxic environmental pollutant. We used rat hepatocytes as a model to study oxidative damage induced by Cd, effects on the antioxidant systems, and the role of N-acetylcysteine (NAC) in protecting cells against Cd toxicity. Hepatocytes were incubated for 12 and 24 h with Cd (2.5, 5, 10 microM). Results showed that Cd can induce cytotoxicity: 10 microM resulted in 36.2% mortality after 12 h and 47.8% after 24 h. Lactate dehydrogenase, aspartate aminotransferase, and alanine aminotransferase activities increased. Additionally, reactive oxygen species (ROS) generation increased in Cd-treated hepatocytes along with malondialdehyde levels. Glutathione concentrations significantly decreased after treatment with Cd for 12 h but increased after 24 h of Cd exposure. In contrast, glutathione peroxidase activity significantly increased after treatment with Cd for 12 h but decreased after 24 h. superoxide dismutase and catalase activities increased at 12 h and 24 h. glutathione S-transferase and glutathione reductase activities decreased, but not significantly. Rat hepatocytes incubated with NAC and Cd simultaneously had significantly increased viability and decreased Cd-induced ROS generation. Our results suggested that Cd induces ROS generation that leads to oxidative stress. Moreover, NAC protects rat hepatocytes from cytotoxicity associated with Cd.

Protective effect of Achyranthes bidentata polysaccharides on diabetic mice induced by streptozocin / 中国药理学与毒理学杂志

OBJECTIVE To investigate the potential protective effect of Achyranthes bidentata polysaccharides (ABP) on diabetes mice induced by streptozocin. METHODS Male ICR mice were divided into normal control, diabetes model and ABP 50 and 100 mg·kg~(-1) (ip, once daily for 15 d) treatment groups. On the day before ABP administration and after ABP administration for 8 and 15 d, the blood glucose content was detected with a glucometer and intraperitoneal glucose tolerance test was also conducted. After ABP administration for 15 d, the mice were sacrificed and body weight, heart, liver, spleen and kidneys weights were measured. The serum insulin concentration was determined by radioimmunoassay kit. The serum activities of glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT) and alkaline phosphatase (ALP), and serum concentrations of triglyceride (TG), total cholesterol (TC), calcium and phosphorus were measured by colorimetric method. Enzyme linked immunosorbent assay was employed to detect the concentrations of leptin, adiponectin, tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in serum. RESULTS Compared with mice in normal control group, the body weight and serum insulin concentration decreased and blood glucose increased in diabetic model mice. ABP 50 and 100 mg·kg~(-1) treated mice were able to normalize glucose concentrations better following a glucose tolerance test, and the blood glucose level decreased by 27.4% and 16.3%, respectively, compared with that of diabetic model mice. The relative weights of spleen and kidneys, blood glucose level, serum TG and TC concentrations, and GOT, GPT and ALP activities in mice treated with ABP 50 mg·kg~(-1) were obviously lower than those of diabetes model mice. Serum leptin concentration was also markedly decreased near to normal level. However, serum concentrations of adiponectin, TNF-α and IL-6 were significantly increased comparing with diabetes model mice. ABP 100 mg·kg~(-1) had no obvious effect on serum TG and TC levels, and GPT and ALP activities. Its effects on the other parameters indicated above were similar to those in ABP 50 mg·kg~(-1) group. For the serum concentrations of insulin, calcium and phosphorus, no statistical difference could be observed among the different groups. CONCLUSION ABP possesses protective effect against streptozocin-induced diabetes in mice.

STUDY ON THE INFLUENCE OF CALCIUM, PHOSPHORUS ON PROLIFERATION, DIFFERENTIATION AND MINERALIZATION OF OSTEOBLASTS IN VITRO / 营养学报

Objective To clarify the influence of Ca and P on bone metabolism by investigating theeir effects on proliferation, differentiation and mineralization of osteoblasts (OBs) in vitro. Method OBs were cultured in media with Ca(1, 2,4 mmol/L),P(1, 2,4 mmol/L),Ca:P 1:2 (1 mmol/L Ca+2 mmol/L P), and Ca:P 2:1 (2 mmol/L Ca+1 mmol/L P). Cell proliferation, activity of alkaline phosphatase (ALP), content of osteopontin (OPN) and collagen type I (Col-I) of OBs, and their corresponding mRNA expression were measured. Results Compared with control group,Ca at 4 mmol/L promoted OBs proliferation significanty (P

Anti-?_2-glycoprotein Ⅰ antibodies in patients with systemic lupus erythematosus / 中华风湿病学杂志

Objective To investigate the prevalence and significance of anti-?2-glycoprotein Ⅰ (anti-?2-GPⅠ) in patients with systemic lupus erythematosus (SLE). Methods Anti-?2-GPⅠ level was determined in 112 SLE patients, 40 patients with rheumatoid arthritis (RA), 30 patients with Sj?gren′s syndrome (SS) and 40 healthy individuals by enzyme-linked immunosorbent assay (ELISA). The laboratory and clinical features (thrombosis, fetal loss, etc) were analyzed retrospectively from the clinical database. Results Sensitivities of anti-?2-GPⅠ were 21.4%, 15.0%, 6.7% in SLE, RA and SS, respectively. The specificity of anti-?2-GPⅠ was 88.6% in SLE. Correlations between thrombosis and titers of ACL-IgG/IgM and anti-?2-GPⅠ were statistically significant. No association was found between anti-?2-GPⅠ and other clinical and laboratory features. Conclusion The results indicate that anti-?2-glycoprotein Ⅰ antibodies are common and may play a role in SLE with thrombosis. Combination of ACL and anti-?2-GPⅠ can facilitate the diagnosis of SLE with thrombosis.

EFFECTS OF 1?,25(OH)_2D_3 ON THE EXPRESSION OF RECEPTOR ACTIVATOR OF NF-?B LIGAND AND OSTEOPROTEGERIN IN RAT OSTEOBLASTS IN VITRO / 营养学报

Objective To investigate the effect of 1?,25-dihy-droxyvitamin D3 (1?,25(OH)2D3) on the expression of receptor activator of NF-?B ligand (RANKL),osteoprotegerin (OPG) and RANKL mRNA,OPG mRNA,of rat osteobalsts (OB). Method The expression of RANKL and OPG was detected by the method of Immunohistochemistry and ELISA. RANKL mRNA and OPG mRNA were determined through FQ-PCR. Results Compared with the control group and the group with 1 nmol/L 1?,25(OH)2D3,10 and 100 nmol/L 1?,25(OH)2D3 can significantly induce the expression of RANKL and RANKL mRNA. 1,10 nmol/L 1?,25(OH)2D3 can stimulate the expression of OPG and OPG mRNA significantly,while 100 nmol/L 1?,25(OH)2D3 can inhibit the expression of OPG mRNA significantly. There were no difference in the rate of RANKL/OPG and RANKL mRNA/OPG mRNA between the control group and the group with 1 nmol/L 1?,25(OH)2D3,however the rate of RANKL/OPG and RANKL mRNA/OPG mRNA in the group with 10,100 nmol/L were higher than the control group and the group with 1 nmol/L 1?,25(OH)2D3. Conclusion Lower dosage of 1?,25(OH)2D3 (1 nmol/L) had no significant effect on bone turnover,but higher dosage of 1?,25(OH)2D3 (10,100 nmol/L) can enhance bone turnover through facilitating the formation and activity of osteoclasts via enhancing RANKL mRNA/OPG mRNA and RANKL/OPG..

EFFECT OF 1?,25-DIHYDROXYVITAMIN D_3 ON CYTOSKELETON,GAP JUNCTION INTERCELLULAR COMMUNICATION AND [Ca~(2+)]_i OF OSTEOBLASTS / 营养学报

Objective To study the effect of 1?,25-dihydroxyvitamin D3[1,25-(OH)2D3] on cytoskeleton,gap junction intercellular communication (GJIC) and intracellular Ca2+ ([Ca2+]i) in osteoblasts (OB) in vitro. Method OB were isolated from calvaria bone. After 20 min and after 24 h treated by 1,25-(OH)2 VD3 (0,10-9,10-8,10-7 mol/L),[Ca2+]i was evaluated. F-actin and GJIC were observed after 24 and 48 h incubation later. Results Compared with the control group,[Ca2+]i in all 1,25-(OH)2 D3 groups was increased significantly at 20 min. [Ca2+]i in 10-9 mol/L 1,25-(OH)2D3 group was the lowest at 24 h after treatment. OB in 10-8 and 10-7 mol/L 1,25-(OH)2D3 groups were flat,and stress fibers were formed. The expression of F-actin in control group and 10-9 mol/L 1,25-(OH)2D3 group was reduced at 48 h after treatment. Compared with the control group,GJIC was weakened very significantly after treated with 10-9 mol/L 1,25-(OH)2D3 at 48 h,but enhanced very significantly in the group with 10-8 and 10-7 mol/L. Conclusion Higher dosage of 1,25-(OH)2D3 can maintain the morphology of OB and stimulate the communication among OB,but lower dosage can inhibit it.