Solasonine Causes Redox Imbalance and Mitochondrial Oxidative Stress of Ferroptosis in Lung Adenocarcinoma.

Ferroptosis, a type of iron-dependent oxidative cell death caused by excessive lipid peroxidation, is emerging as a promising cancer therapeutic strategy. Solasonine has been reported as a potential compound in tumor suppression, which is closely linked to ferroptosis. However, ferroptosis caused by solasonine is insufficiently identified and elaborated in lung adenocarcinoma, a fatal disease with high morbidity and mortality rates. First, the biochemical and morphological changes in Calu-1 and A549 cells exposed to solasonine are observed using a cell death assay and a microscope. The cell viability assay is performed after determining the executive concentration of solasonine to assess the effects of solasonine on tumor growth in Calu-1 and A549 cells. The ferroptosis is then identified by using ferroptosis-related reagents on CCK-8, lipid peroxidation assessment, Fe2+, and ROS detection. Furthermore, the antioxidant system, which includes GSH, Cys, GPx4, SLC7A11, and mitochondrial function, is measured to identify the potential pathways. According to the results, solasonine precisely exerts antitumor ability in lung adenocarcinoma cells. Ferroptosis is involved in the solasonine-induced cell death, as well as the accumulation of lipid peroxide, Fe2+, and ROS. Moreover, the failures of antioxidant defense and mitochondrial damage are considered to make a significant contribution to the occurrence of ferroptosis caused by solasonine. The study describes the potential process of ferroptosis caused by solasonine when dealing with lung adenocarcinoma. This encouraging evidence suggests that solasonine may be useful in the treatment of lung cancer.

The improvement effects of edible bird's nest on proliferation and activation of B lymphocyte and its antagonistic effects on immunosuppression induced by cyclophosphamide.

Edible bird's nest (EBN) is regarded as an immune-enhancing food in the People's Republic of China. The aim of this study is to demonstrate the efficiency of EBN in improving the immunity of mouse both in vivo and in vitro. We observed the effects of EBN on spleen lymphocytes proliferation and activation, as well as immunoglobulin isotypes as indicators. In addition, we evaluated the content of total sIgA in the intestinal juice to assess mucosal immunity. The results showed that EBN could promote the proliferation and activation of B-cells and increase IgE, IgA, IgM, and IgG3 levels. We also found that EBN extract can promote the secretion of sIgA in the small intestine. Using cyclophosphamide (CY), we established an immunosuppressed mouse model in which we identified a reversal influence on the ratio of CD3(+)/CD19(+) cells, which indicates that EBN also protects B-cells from the damage induced by CY. We also applied polymyxin B to exclude the interference of lipopolysaccharide throughout the experiment. In conclusion, we found that EBN can reduce the intestinal immune injury induced by CY by accelerating the proliferation and activation of B-cells and enhancing antibody secretion of B-cells.

[Effects of anhydroicaritin on the immunologic function of mouse macrophages].

AIM: To investigate the effects of anhydroicaritin (AHI) on the immunologic function of mouse macrophages stimulated by lipopolysaccharide (LPS) in vitro and its related immunosuppressive mechanism. METHODS: Mouse bone marrow-derived macrophages were isolated. Then, the drug toxicology of different concentrations of AHI on macrophages was measured by CCK-8 assay. The amount of NO produced in macrophages was detected by Griess kit. The phagocytosis of macrophages to E.coli BioParticles was assayed by flow cytometry (FCM). The expression of CD69, which was the marker of early activation of macrophages, was measured by FCM combined with two-color immunofluorescent staining of cell surface antigen. Cytometric bead array (CBA) kit was used to detect the production of cytokines of macrophages stimulated by LPS. RESULTS: AHI (2.5, 5, 10 µmol/L) significantly reduced the production of NO in macrophages stimulated by LPS, and inhibited the phagocytosis of activated macrophages. The results of FCM analysis showed that AHI decreased proportions of CD69 on LPS-stimulated macrophages. Furthermore, AHI downregulated the secretion of cytokines of LPS-induced macrophages. CONCLUSION: AHI, which exhibits immunosuppressive effect on the mouse macrophages stimulated by LPS, is promising to be developed as an immunosuppressive reagent.

[Effects of ginkgolide B on proliferation, phagocytosis, NO and ROS production of murine peritoneal macrophages in vitro].

AIM: To explore the potential immunomodulatory effects and related mechanisms of ginkgolide B (GB), a known potent antagonist of platelet-activating factor receptor, we investigated the proliferation, phagocytosis, NO and ROS production of macrophage. METHODS: After murine peritoneal macrophages (PMs) preparation, PMs were treated with different concentrations of GB before culture time and then activated by LPS. Drug toxicology and PM proliferation were measured by MTT assays. Fluorescent beads ingestion and flow cytometry were used to assess phagocytosis of LPS-activated PMs. Griess reagent system was used to determine the amount of LPS-induced NO production. H2DCFDA labeling and flow cytometry were used to trace ROS level of both rest and LPS-activated PMs. RESULTS: In a dose-dependent manner, GB (5, 10, and 20 µmol/L) significantly suppressed the phagocytosis as well as NO and ROS production at 24 h and inhibited cell proliferation at 48 h after LPS stimulation. CONCLUSION: According to these interesting effects of GB on macrophage behaving and functioning, it's quite reasonable to do further studies of GB as a nature occurring immunomodulator candidate.

[Comparison of proteomics between acute myeloid leukemia and acute lymphoid leukemia].

The aim of this study was to explore the distinct protein profiles of different subtype of acute myeloid leukemia (AML), including M(1), M(2), M(3) and acute lymphoid leukemia (ALL) by differential proteomic expression analysis. The proteins of bone marrow leukemia cells from AML and ALL patients were separated by two-dimensional electrophoresis (2-DE). 2-DE patterns were analyzed by PDQuest 7.4 software and the differentially expressed proteins were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) and bioinformatics. The results indicated that 21 differentially expressed proteins were found by 2-DE and 15 were identified by MS to be significantly differentially expressed. In AML, seven proteins were highly expressed such as MPO, PRDX3, CALR and ECH1 and so on, and eight proteins were highly expressed in ALL, including ARHGDIB, PFN1 and ACTG1 and so on. It is concluded that the distinct protein profiles between AML and ALL have been proved. It may be helpful for the identification of new targets for specific treatment approaches and the molecular markers for the early diagnosis of leukemia.

[Effects of salidroside on proliferation, apoptosis, phagocytosis, ROS and NO production of murine peritoneal macrophages in vitro].

AIM: To investigate the effects of salidroside(Sal) on proliferation, apoptosis, phagocytosis, the production of ROS and NO of murine peritoneal macrophages in vitro as well as its immunoregulation. METHODS: The single cell suspension of murine peritoneal macrophages was prepared under sterile condition, then co-cultured with different concentrations of Sal(80, 160 and 320 µmol/L)for 4 hours prior to stimulation with LPS and IFN-γ, the proliferation of macrophages was measured by MTT colorimetry. The effect of Sal on the apoptosis of Sytox® Green-labelled peritoneal macrophages induced by CHX was detected by Fluorescence enzyme-labelled meter. FCM was used to detect the effect of Sal on phagocytosis of peritoneal macrophages. Fluorescence enzyme-labelled meter was used to measure the effects of Sal on ROS of H(2);DCFDA-labelled macrophages induced by LPS and IFN-γ. Griess Gragent was used to detect the role of Sal in production of NO in peritoneal macrophages activated by LPS and IFN-γ. RESULTS: MTT result demonstrated that Sal could promote the proliferation of peritoneal macrophages activated by LPS and IFN-γ at the final concentrations of 80, 160, 320 µmol/L, respectively (P<0.05). The result of Fluorescence enzyme-labelled meter detected showed that Sal at the final concentration of 160 µmol/L could inhibit apoptosis of peritoneal macrophages induced by CHX(P<0.01). FCM analysis showed that different concentrations of Sal significantly promoted the phagocytosis of peritoneal macrophages which include un-activated and activated by LPS and IFN-γ(P<0.05). Fluorescence enzyme-labelled meter showed that Sal could reduce the production of ROS in activated peritoneal macrophages induced by LPS and IFN-γ(P<0.05). Sal also increased the production of NO in activated peritoneal macrophages induced by LPS and IFN-γ(P<0.05). CONCLUSION: Sal can promote proliferation of peritoneal macrophages stimulated by LPS and IFN-γ, and it can inhibit apoptosis of peritoneal macrophages induced by CHX, Sal also can promote the phagocytosis of peritoneal macrophages which include un-activated and activated by LPS and IFN-γ, Sal can reduce the production of ROS in activated peritoneal macrophages induced by LPS and IFN-γ, while Sal can promote the production of NO in activated peritoneal macrophages induced by LPS and IFN-γ.

[The effect of ginsenoside Rb1 on phagocytosis, the production of cytokines and NO of murine macrophage in vitro].

AIM: Investigate the effects of Rb1 on phagocytosis and the production of the pro-inflammatory cytokines and NO of murine macrophage, to elucidate the machanism of the immuno-regulating effect of Rb1. METHODS: The single cell suspension of murine peritoneal macrophages was prepared under sterile condition. Then, the drug toxicology of different concent rations of Rb1 on macrophages was measured by MTT assay. The phagocytosis of macrophages to microbeads was observed through flow cytometry(FCM). The amount of NO produced in macrophages was detected by Griess kit. Used Cytometric Bead Arry(CBA) kit to detect the production of cytokines of Macrophages stimulated by LPS. RESULTS: In a dose-dependent manner, Rb1(5, 10, 20 µmol/L) can significantly inhibit the phagocytose of macrophage stimulated by LPS, but improve this ability of unactivated macrophage (P<0.05). Rb1 can also reduce the production of NO by macrophage(P<0.01). Rb1 can regulate the production of cytokines of macrophage stimulated by LPS. CONCLUSION: Rb1 can inhibit the transduction of LPS signals and regulate the content of cytokines, thus to decrease the production of NO. However, to the unactivated macrophages, Rb1 up-regulates phagocytic functions and promotes the innate immunity to resist invasion of pathogens.

[p38 mitogen-activated protein kinase inhibitor suppresses the expression of pro-inflammatory cytokines in liver from brain dead rats].

OBJECTIVE: To observe the suppressive effect on the expression of pro-inflammatory cytokines in liver from brain dead (BD) rats through inhibition of p38 mitogen-activated protein kinase (MAPK) signaling pathway by SB203580. METHODS: A total of 30 male Wistar rats weighing from 180 to 200 g were randomly divided into 3 experimental groups: (1) BD group (n = 10): brain death was induced in rats; (2) BD+SB203580 group (n = 10): brain death was successfully induced and SB203580 (10 mg/kg) was given through dorsal vein of penis. After brain death artificial ventilation was maintained for 6 hours and only those with mean arterial blood pressure more than 80 mm Hg (1 mm Hg = 0.133 kPa) were accepted as BD donors. (3) Control group (n = 10): living healthy rats. The expressions of TNFalpha and IL-1beta mRNA in liver tissues were analyzed by RT-PCR and the protein expressions of TNFalpha, IL-1beta and phosphorylated p38MAPK were detected by Western blot. RESULTS: The phosphorylated p38MAPK detected in the liver in BD group was significantly increased compared with the control group (q = 172.53, P < 0.01), and the expressions of TNFalpha and IL-1beta mRNA and proteins in liver were also significantly increased in BD group compared with the control group (q = 123.99, 135.35, 243.09 and 192.23, respectively, P < 0.01). The phosphorylated p38MAPK was decreased in BD+SB203580 group and significantly decreased compared with the BD group (q = 63.90, P is less than 0.05), but higher than that in control group (q = 108.63, P < 0.01). The expressions of TNFalpha and IL-1beta mRNA and proteins in liver were significantly decreased in BD+SB203580 group compared with the BD group (q = 55.11, 98.13, 61.03 and 50.85, respectively, P < 0.01), but higher than that in control group (q = 68.89, 37.22, 182.06 and 141.38, respectively, P < 0.01). SB203580 can suppress the expression of pro-inflammatory cytokines in the liver of brain dead rats through the inhibition of p38MAPK signaling pathway which may reduce the immunogenicity of donor livers.

Quantitative proteomics analysis of chondrogenic differentiation of C3H10T1/2 mesenchymal stem cells by iTRAQ labeling coupled with on-line two-dimensional LC/MS/MS.

The chondrogenic potential of multipotent mesenchymal stem cells (MSCs) makes them a promising source for cell-based therapy of cartilage defects; however, the exact intracellular molecular mechanisms of chondrogenesis as well as self-renewal of MSCs remain largely unknown. To gain more insight into the underlying molecular mechanisms, we applied isobaric tag for relative and absolute quantitation (iTRAQ) labeling coupled with on-line two-dimensional LC/MS/MS technology to identify proteins differentially expressed in an in vitro model for chondrogenesis: chondrogenic differentiation of C3H10T1/2 cells, a murine embryonic mesenchymal cell line, was induced by micromass culture and 100 ng/ml bone morphogenetic protein 2 treatment for 6 days. A total of 1756 proteins were identified with an average false discovery rate <0.21%. Linear regression analysis of the quantitative data gave strong correlation coefficients: 0.948 and 0.923 for two replicate two-dimensional LC/MS/MS analyses and 0.881, 0.869, and 0.927 for three independent iTRAQ experiments, respectively (p < 0.0001). Among 1753 quantified proteins, 100 were significantly altered (95% confidence interval), and six of them were further validated by Western blotting. Functional categorization revealed that the 17 up-regulated proteins mainly comprised hallmarks of mature chondrocytes and enzymes participating in cartilage extracellular matrix synthesis, whereas the 83 down-regulated were predominantly involved in energy metabolism, chromatin organization, transcription, mRNA processing, signaling transduction, and cytoskeleton; except for a number of well documented proteins, the majority of these altered proteins were novel for chondrogenesis. Finally, the biological roles of BTF3l4 and fibulin-5, two novel chondrogenesis-related proteins identified in the present study, were verified in the context of chondrogenic differentiation. These data will provide valuable clues for our better understanding of the underlying mechanisms that modulate these complex biological processes and assist in the application of MSCs in cell-based therapy for cartilage regeneration.

[Proteomic study of the effect of recombinant hFGF-10 adenovirus on HaCat cells].

This study is to investigate the effect of recombinant hFGF-10 adenovirus on the proteome of HaCat cells, and to speculate further the possible mechanism of the effect of hFGF-10 on HaCat cells via differentially expressed proteins identified. Two-dimensional gel electrophoresis (2-DE) combined with tandem time-of-flight mass spectrometry was applied to identify the differentially expressed protein spots on the 2-DE maps of the whole-cell proteins from Ad infected and rAd-hFGF-10 infected HaCat cells. The mRNA and protein levels of the differentially expressed proteins were confirmed with semi-quantitative RT-PCR and Western blotting. The results showed that the 2-DE maps with high resolution were obtained, and four selected differentially expressed proteins involved in cell apoptosis, cytoskeleton regulation and protein degradation were identified with MALDI-TOF/TOF. The mRNA and protein levels of one of the differentially expressed proteins, VDAC2, were up-regulated in HaCat cells infected with the recombinant hFGF-10 adenovirus. The differentially expressed protein, VDAC2, may be related to the bioactivities of hFGF-10.

[Effects of Kaempferol on activation, proliferation and cell cycle of mouse T lymphocytes in vitro.].

AIM: To study the effects of Kaempferol on activation, proliferation and cell cycle of murine T lymphocytes, and to elucidate the mechanism of the immunosuppressive effect of Kaempferol. METHODS: Lymphocytes were prepared from lymphoid nodes of mouse, and were stimulated with polyclonal activators ConA, and then were co-cultured with Kaempferol of different final concentration. The effect on the expression of CD69 (the early marker of the activated T cells) on T lymphocytes were measured by flow cytometry combined with two colored monoclonal antibodies flow cytometry. While, the effect of Kaempferol on the proliferation of T lymphocytes in response to ConA stimulation was determined by MTT, cell cycle was detected by PI staining. RESULTS: Kaempferol (final concentration is 10, 20 and 40 mumol/L) can inhibit the expression of CD69 on activated T lymphocytes in a dose-dependent manner (P<0.05). The expression rate of CD69 on T cells in response to ConA was (39.11+/-1.17)%. After treatment with Kaempferol, the expression rate of CD69 reduced to (30.64+/-0.23)%, (27.95+/-0.04)% and (5.63+/-0.37)%, respectively (P< 0.05); The result of MTT showed that Kaempferol can inhibit the proliferation of T lymphocytes stimulated by ConA in a dose-dependent manner (P<0.05); The result of PI staining showed that Kaempferol can arrest cell cycle at S phage and G2/M at all experimental concentration (P<0.05). CONCLUSION: Kaempferol can effectively inhibit the activation, proliferation of mouse T lymphocytes in respond to ConA, arrest cell cycle at S phage and G2/M in vitro.

Anti-inflammatory and immunosuppressive effect of phloretin.

This study investigated the effect of phloretin (Ph) on the proliferation, activation, and cell-cycle distribution of mouse T lymphocytes and NO production and phagocytosis of macrophages. Carboxyfluorescein diacetatesuccinimidyl ester (CFDA-SE) staining plus flow cytometry assay was employed to obtain the proliferation-related index (PI) of lymphocytes. The expression levels of CD69 and CD25 on T lymphocytes stimulated with Con A were evaluated with flow cytometry after staining with fluorescent monoclonal antibody. Cell-cycle distribution of T lymphocytes was analyzed by propidium iodide staining. Griess kit was used to evaluate the NO production and fluorescent microbeads were used to analyze the phagocytosis ability of macrophages. Our results showed that phloretin (40, 60, and 80 micromol x L(-7)) significantly inhibited the proliferation of T lymphocytes and the PI reduced from 1.41 +/- 0.13 to 1.34 +/- 0.16, 1.19 +/- 0.12 and 1.07 +/- 0.06, respectively. Phloretin significantly inhibited the expression of CD69 and CD25 (P < 0.01). The cell cycle distribution analysis showed that phloretin could induce a cell cycle arrest at G0/G1 phase. NO production of LPS +IFN-gamma group of macrophages was (26.72 +/- 3.57) micromol x L(-1), and was significantly reduced by phloretin (P < 0.01). And phagocytosis rate of macrophages was significantly reduced by phloretin (P < 0.01). The results demonstrate that phloretin might be developed into a new immuosuppressive drug.

[Effects of Hesperetin on cell activation and proliferation of murine T lymphocytes in vitro].

AIM: The effect of Hesperetin (Hes) on activation and proliferation of murine T lymphocytes in vitro as well as its mechanism of action is investigated to provide a theory for developing an immunosuppressive agent. METHODS: The lymphocytes were cocultured with Concanavalin A (ConA) and Hes together. Fluorescence conjugated monoclonal antibodies and flow cytometry were used to detect the expression of CD69 of activated T lymphcytes in vitro in response to ConA. MTT test was used to estimate the effect of Hes on proliferation of lymphocytes stimulated by ConA and the toxic effect of Hes on lymphocytes. Proliferation of lymphocytes stimulated by ConA was detected by carboxyl fluorescein diacetate-succinimide ester (CFDA-SE) staining combined with flow cytometry, and the proliferation index (PI) was analyzed by means of ModFit software. RESULTS: The survival rate of lymphocytes shows that DMSO(0.2 mL/L)and Hes (25-75 micromol/L) had little side effect on murine lymphocytes in vitro; Hes can inhibit the activation and proliferation of T lymphocytes in response to ConA with noticed dosage-effect relation. CONCLUSION: Hes may has broad prospects to be developed as immunosuppressive drug through further studies.

[Inhibitory effects of pretreatment with Buyanghuanwu decoction on inflammatory cytokine expressions in the kidneys of rats after induction of brain death].

OBJECTIVE: To observe the inhibitiory effects of pretreatment with Buyanghuanwu decoction (BYHWT) on inflammatory cytokine expressions in the kidneys and the level of reactive oxygen species (ROS) by peripheral blood neutrophils of rats after induction of brain death (BD), and to investigate the effect of BYHWT on the improvement of kidney quality from BD donor. METHODS: 30 male Wistar rats were randomly divided into 3 groups: control group, BD model group and BYHWT group. 6 hours after successful onset of brain death,only the BD rats whose mean arterial blood pressure were higher than 80 mmHg were accepted as donors. Kidneys were harvested and peripheral blood was taken from BD rats. RT-PCR was used to detect the expressions of TNF-a and IL-lpfl mRNA. Western blot was adopted to analyze the expressions of both TNF-alpha and IL-lp protein,and the expression of phosphorylated p38 mitogen-activated protein kinase (p-p38MAPK). Reactive oxygen species( ROS) in the peripheral blood neutrophils were labeled with CM-H2DCFDA and then detected with Flow Cytometry. RESULTS: The expressions of both TNF-alpha and IL-1beta mRNA and protein, and the p-p38MAPK proteins all significantly increased in BD group compared with control group (P < 0.01). However, those in BYHWT group statistically decreased compared with BD group (P < 0.05), but they significantly increased in comparison with control group (P < 0.01). There was a close relation between the expression of p-p38MAPK protein and the expressions of both TNF-a and IL-1beta mRNA and protein. ROS level significantly increased in BD group (P < 0.05 ), whereas it significantly decreased in BYHWT group (P < 0.05). There was no statistically significant difference between BYHWT group and control group (P > 0.05). CONCLUSION: Pretreatment of the rats with BYHWT prior to the induction of rat brain death, can significantly suppress the expressions of inflammatory cytokines and ROS level in the kidneys of rats from BD. It might be related to the blockage of key target points in p38MAPK signaling pathway. Therefore pretreatment with BYHWT could hopefully be an ideal way to improve the quality of kidneys from brain dead donors prior to transplantation.

[Effect of icariin on intermediate and advanced activation of murine T lymphocytes in vitro].

AIM: To study the effect of icariin (ICA) on the intermediate and advanced activation of murine T lymphocytes stimulated by concanvalin A (ConA) in vitro. METHODS: Single cell suspensions were prepared from murine lymphoid nodes under germ free condition. The drug toxicity of ICA on T lymphocytes was measured by MTT after 24 h. Before they were stimulated by ConA (the final concentration was 5 mg/L), T cells were treated with different concentrations of icariin for 4 h. The expression of CD25 and CD71, the marker of intermediate and advanced activation of T lymphocytes, were measured by flow cytometry (FCM) combined with two-colour immunofluorescent staining of cell surface antigen after 24 h and 30 h. The change of cytokine contents such as IL-2, IL-4, IL-10, TNF-alpha and IFN-gamma in the supernatant of intermediate activation was measured by Luminex 100. RESULTS: ICA at the final concentration of 0.3, 1.5, 3.0 micromol/L inhibited the expression of CD25 with dose-dependent effect but it had no effect on that of CD71. ICA decreased the secretion of IL-2, IL-4 and IL-10 but increased that of TNF-alpha and IFN-gamma. CONCLUSION: ICA can not only decrease CD25, IL-2, IL-4 and IL-10 but also enhance the cell-mediated immunity reaction. So it may have a function of two-way regulation of immune balance.

[Effects of Forskolin on proliferation cell-cycle distribution and activation of the murine T lymphocytes in vitro].

OBJECTIVE: To investigate the effects of Forskolin on activation, proliferation, and cell-cycle distribution of murine CD3+ T lymphocytes, and study the mechanisms of its immunosuppressive effect. METHODS: Singel cell suspensions were prepared from murine lymph nodes. Fluorescence conjugated monoclonal antibodies and flow cytometry were used to detect the expression of CD69 activated by Con A, the proliferation index of activated mouse T lymphocytes was analyzed by CFDA-SE staining, the distribution of the cell cycle was analyzed by PI staining. RESULTS: Forskolin (10(-7), 10(-6), 10(-5) M) could inhibit both the expression of CD69 on CD3+ T lymphocytes and T lymphocyte proliferation index stimulated by Con A in a dose-dependent manner. The C0/G1 of T lymphocytes increased but the S, G2/M phase decreased. CONCLUSION: Forskolin can inhibit the activation and proliferation of murine T lymphocytes in vitro, and arrest activated T lymphocytes from G0/G1 to S or G2/M. Forskolin is a potential immunosupressive agent.

[Effect of shuanghuanglian injection on the activation and proliferation of T lymphocytes in mice in vitro].

AIM: To study the effect of shuanghuanglian injection on the activation and proliferation of T lymphocytes in mice in vitro. METHODS: The toxic effect of SHL on lymphocytes in mice was estimated by MTT test; Double-fluorescent plus flow cytometry to analyze the effect of SHL on the activation of T lymphocytes in mice stimulated by ConA in vitro; MTT test and CFDA-SE plus flow cytometry were used to detect the effect of SHL on the proliferation of T lymphocytes in mice induced by ConA in vitro. RESULTS: SHL had little side effect on lymphocytes in mice in vitro; At the mass concentration of 60, 80, 100, 120 mg/L, SHL inhibited the activation of T lymphocytes in mice stimulated by ConA in vitro (P<0.01); Both MTT test and CFDA-SE stain sign that SHL inhibited the activation of T lymphocytes in mice induced by ConA in vitro at the mass concentration of 60, 80, 100, 120 mg/L (P<0.01). CONCLUSION: SHL can inhibit the activation and proliferation of T lymphocytes in mice in vitro.

[Establishment and optimization of two-dimensional gel electrophoresis technique of proteome from bone marrow leukemia cells].

The aim of this study was to establish and optimize two-dimensional electrophoresis method for human bone marrow leukemia cells in order to obtain the profiles with high resolution and reproducibility. The total protein was extracted and separated by isoelectric focusing (IEF) and SDS polyacrylamide gel electrophoresis (SDS-PAGE). The gels were stained with silver nitrate or Coomassie brilliant blue, and then scanned and analyzed with PDQuest 7.4 analysis software. The effects of different protein preparation methods and electrophoresis conditions on the profiles were compared. The results indicated that by optimizing preparation of protein sample and electrophoresis protocols, clear profiles with 780 +/- 73 well separated protein spots on an average were obtained and the match rate was 82 +/- 5% between reproducible gels from leukemia cells of different sub-type. It is concluded that the two-dimensional electrophoresis method of proteome from human bone marrow leukemia cells is established successfully and is suitable for the further comparative proteomic research between leukemia of different types.

[Effects of natural killer cells on engraftment and reconstitution of hematopoiesis and immunity in mice undergoing allogeneic bone marrow transplantation].

The aim of this study was explore the effect of natural killer (NK) cells on engraftment and reconstitution of hematopoiesis and immunity in mice undergoing allogeneic bone marrow transplantation (allo-BMT). Lethally and nonlethally irradiated BALB/c (H-2(d)) mice were transplanted with C57BL/6 (H-2(b)) bone marrow plus donor peripheral T cells and/or NK cells. Recipient CD34(+), H-2K(b+), CD3(+) and CD19(+) cells were detected by flow cytometry; peripheral blood leukocytes were counted by auto-cytometry; survival rates, engraftment, hematopoietic and immune recovery of recipients in different transplant groups were then observed. The results indicated that as compared with lethally irradiated and allo-BMT group without infusion of NK cells, the survival rate in lethally irradiated and allo-BMT group with infusion of NK cells significantly enhanced (survival rates at 60 days were 70% and 0.0% respectively); leukocyte count, expression level of CD19(+) cell and CD34(+) cell count recovered rapidly; expression level of H-2K(b+) cells obviously increased [(86.68 +/- 4.45)% vs (4.68 +/- 0.32)%]; expression level of CD3(+) cell at day 28 after transplantation obviously decreased [(33.69 +/- 3.36)% vs (50.40 +/- 5.06)%, p < 0.01], at day 60 there was not significant difference between the 2 groups (p > 0.05). In nonlethally irradiated and allo-BMT group without NK cell infusion, the expression level of H-2K(b+) at day 30 after transplantation significantly decreased, and reduced to level before transplantation at day 60; while expression of H-2K(b+) yet could be detected with > 80% at day 60 after transplantation in group infused with high and low concentration of NK cells. It is concluded that in allo-BMT mice, alloreactive NK cell inhibits graft rejection, enhances engraftment, promotes the reconstitution of hematopoiesis and immunity, and increases survival rates.

[Effect of forsythia suspense extract on phagocytosis of peritoneal macrophages and NO production in vitro].

AIM: To investigate the effect of forsythia suspensa (FS) extract on phagocytosis of peritoneal macrophages and NO production in vitro. METHODS: The peritoneal macrophagess were isolated from BALB/c mice. After stained with CFDA-SE, the DH5alpha were co-cultured with peritoneal macrophagess for 3 h. The effect of FS extract on cyto-phagocytesis in vitro was analyzed by flow cytometry. The peritoneal macrophages were stimulated and activated by LPS in vitro. The effect of FS extract on NO production of the peritoneal macrophages in vitro was measured by NO assay kit. RESULTS: FCM analysis showed that FS extract significantly promoted the phagocytosis of peritoneal macrophages at the final concentration of 40, 80, 160 mg/L, respectively (P<0.05). It also decreased the production of NO at different concentration induced by LPS (P<0.05). CONCLUSION: FS extract can promote phagocytosis of peritoneal macrophages and inhibit NO production in vitro.