Effects of neuro-immuno-modulation on healing of wound combined with local radiation injury in rats.

PURPOSE: To investigate effects of neuro-immuno-modulation on wound healing by observing changes of cytokines and hypothalamic-pituitary-adrenal (HPA) axis hormones in acute stress reaction in rats with wound and combined local radiation injury. METHODS: Sixty female Wistar rats (weighting 200 ± 20 g) were randomly divided into normal control group, wound group and combined wound-local radiation (CWR) group (25 Gy local radiation post wound), 20 rats in each group. Contents of IL-1ß, IL-6 and IFN-γ and IL-4 in serum were measured and changes of adrenocorticotropic hormone (ACTH) and glucocorticoid (GC) in serum were analyzed by using enzyme-linked immunosorbent assay and radioimmunologic assay, respectively at different time points post wound and radiation. RESULTS: (1) The level of IFN-γ, one of the Th1 cell cytokines increased significantly at 14 d post CWR, which was markedly higher than that in control group and wound group. However, the level of IL-4, IL-1ß and IL-6, one of the Th2 cell cytokines, did not show obvious change. (2) Ratio of Th1/Th2 (IFN-γ/IL-4) in wound group and CWR group increased significantly at 7 d after wound and radiation, which suggested that Th1/Th2 balance drifted to Th1 immune response. The ratio of Th1/Th2 in wound group returned to the normal level up to 14 d after the wound and radiation, while the Th1/Th2 ratio in CWR group increased persistently and was much higher than that in control and wound groups. (3) Level of serous ACTH and GC in CWR group increased at 3 d post wound and radiation, and among them, level of GC showed statistically significant increase, which was much higher than that in control and wound groups. CONCLUSION: Level of serous neurohormone GC in rats increased significantly immediately after wound and radiation; while the level of IFN-γ showed significant increase only up to 14 d after wound and radiation, and the Th1/Th2 imbalance sustained till 28 d post wound and radiation. In order to reduce acute damage caused by CWR, organic immune system and nerve system showed up a marked regulate effects simultaneously and mutually. Nonetheless, the excessive stress induced by CWR causes disturbance of immunoregulation, which is one of the key reasons for delayed wound healing in CWR.

Effect of TRAIL in combination with DDP on the expression of MDR1 gene in gastric cancer cells.

INTRODUCTION: Gastric cancer is one of the most common malignant tumor, and gastric cancer is the second most common cause of cancer mortality worldwide. Although chemotherapy is one of the most important treatment options for gastric cancer, and could improve the overall survival rate and quality of live, one significant reason for its failure is multidrug resistance (MDR). AIM: To study the effect of tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) combined with chemotherapeutic drug cisplatin (DDP) on the expression of multidrug resistance gene 1 (MDR1) in the gastric cancer cell line SGC-7901/VCR. MATERIAL AND METHODS: SGC-7901/VCR cells were cultured with DDP and TRAIL in various concentrations. The apoptosis rate was separately measured by a flow cytometer in DDP (sub-toxic dose) alone, TRAIL (200 µg/l) alone and in a combination of the two. Expression levels of MDR1 mRNA and P-glycoprotein (P-gp) were detected by RT-PCR and ELISA analysis, respectively. RESULTS: The apoptosis rate in the combination group was significantly higher than that in the other groups (p < 0.05). According to the results of RT-PCR and ELISA, the expressions of MDR1 mRNA and P-gp in the combination group were statistically significant different compared with other groups (p < 0.05). CONCLUSIONS: The combination of TRAIL with DDP could reverse MDR phenotype in gastric cancer cell line SGC7901/VCR. The mechanism may be involved in the down-regulation of MDR1 mRNA and P-gp, which may play an essential role in overcoming the chemotherapeutic resistance of gastric cancer cells. This study indicates that a combination of chemotherapy and TRAIL may be an effective strategy to treat MDR gastric cancer.

[Effects and mechanism of CD4+ CD25+ regulatory T cells in mouse experimental colitis treated by CLYSTER No. 1].

OBJECTIVE: To explore the effects and mechanism of CD4+ CD25+ regulatory T cells (Tregs) in mouse experimental colitis treated by CLYSTER No. 1. METHOD: The mouse model of experimental colitis was established by dinitrochlorobenzene (DNCB)-acetic acid (AA) in mice DNCB and AA. Adult KM mouse were randomly divided into four groups: normal control group, experimental colitis model group, SASP and Chinese medicine therapeutic groups. Proportion of CD4 CD25+ Tregs in peripheral blood (PB) and mesenteric lymph node (MLN) was estimated by flow cytometry at the end of one or two week after treating with SASP and CLYSTER No. 1. RESULT: The model of experimental colitis in mouse was successfully established. Compared with normal control group, the proportion of CD4 CD25 Tregs was markedly decreased in PB and MLN of model control group of experimental colitis. But it was significantly increased in therapeutic groups of SASP and CLYSTER No. 1, and their CD4+ CD25+ Tregs in PB and MLN were much more than the model control group at the end of one or two weeks after treating with SASP and CLYSTER No. 1. CONCLUSION: CD4+ CD25+ Tregs with strong immune suppression could play a central role in the initiation and development of mouse experiment colitis, and the CLYSTER No. 1 might exert its therapeutic effects on UC by the regulation of number and function of CD4+ CD25+ Tregs.

Reactive oxygen species, but not mitochondrial membrane potential, is associated with radiation-induced apoptosis of AHH-1 human lymphoblastoid cells.

The aim of the study was to investigate the sensitivity of AHH-1 human lymphoblastoid cells to radiation and its relevance to intracellular events, specifically alteration in cellular energy-producing systems. AHH-1 human lymphoblastoid cells were irradiated with 6 Gy of gamma radiation, and then were collected at the indicated time points. Parallel studies were conducted to assess the effects of radiation on the cell proliferation and apoptotic index. Mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) production were monitored. A marked decrease of cell viability was observed as early as 12 h postirradiation and fraction of apoptotic cells was highest at 24 h. Intracellular ROS generation measured with 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) appeared to be highest as early as 30 min postirradiation and resumed to normal level at 6 h. Unexpectedly, the fluorescence intensity of Rhodamine 123 for measuring MMP did not change during the first 3h after radiation and exhibited an aberrant increase at 6 h. The results suggest that AHH-1 cells are sensitive to radiation-induced apoptosis and ROS generation is an early phase in the apoptosis process. Moreover, the results might cast doubts on those studies using Rhodamine 123 which hypothesized that the fall in MMP is one of the early events of apoptosis.

Increase of CD4(+)CD25(+) T cells in Smad3(-/-) mice.

AIM: To investigate the changes of lymphocyte subpopulations, especially CD4(+)CD25(+) T regulatory cells in Smad3(-/-) mice. METHODS: Hematological changes and changes of lymphocyte subpopulations were detected in Smad3(-/-) mice using cell counter and flow cytometry, respectively, and compared to their littermate controls. RESULTS: The numbers of neutrophils and lymphocytes in peripheral blood were significantly increased in Smad3(-/-) mice compared to littermate controls. CD19(+) expressing cells in blood and spleen, and CD8(+) T cells in thymus were all markedly decreased in Smad3(-/-) mice. More important, Smad3(-/-) mice had an increased population of CD4(+)CD25(+) T cells in peripheral lymphoid tissues, including thymus, spleen, and lymph nodes. CONCLUSION: These observations suggest that the changes of lymphocyte subpopulations might play a role in susceptibility to inflammation of Smad3(-/-) mice.

Bcl-xL overexpression restricts gamma-radiation-induced apoptosis.

Bcl-xL belongs to a family of proteins which inhibit apoptosis in a number of stimuli including ionizing radiation. To better understand the effects and mechanisms of Bcl-xL on the apoptosis of lymphocytes and provide experimental basis to treat immune injury induced by radiation, we used normal human lymphoblastoid AHH-1 cells that were engineered to overexpress Bcl-xL proteins. Our results showed that overexpressed Bcl-xL reduced time-dependent increase of apoptosis induced by ionizing radiation. Reactive oxygen species (ROS) generation and Bax protein expression in the transfected AHH1-Bcl-xL cells were also lower compared to parental AHH-1 cells. Unexpectedly, the fluorescence intensity of Rhodomine 123 (Rh 123) for measuring mitochondrial membrane potential (MMP) did not change at all detected time points. These results possess a vital significance for insights into a new strategy for gene therapy of radiation-induced immune injury.

[Screening and cloning of hepatitis C virus non-structural protein 4A interacting protein gene in hepatocytes].

OBJECTIVE: To investigate biological functions of hepatitis C virus (HCV) non-structural protein 4A (NS4A). METHODS: Yeast-two hybrid technique was performed to seek proteins in hepatocytes interacting with HCV NS4A. HCV NS4A bait plasmid was constructed by ligating the NS4A gene with carrier plasmid pGBKT7, then it was transformed into yeast AH109 (alpha type). The transformed yeast cells were amplified and mated with yeast cells Y187 (alpha type) containing liver cDNA library plasmid pACT2 in 2 x YPDA medium. Diploid yeast cells were plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) and synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing X-alpha-gal for selection two times. After extracting plasmid from blue colonies, plasmid DNA was transformed into competent E.coli and analyzed by DNA sequencing and bioinformatics methods. RESULTS: Among twenty-two positive colonies there were eleven positive for metallothionein 2A, three for eukaryotic translation elongation factor 1 alpha 1, two for albumin, two for RNA binding motif protein 21, two for myomesin, one for cytochrome C oxidase II, and one for ATPase. CONCLUSIONS: Genes of HCV NS4A interacting proteins in hepatocytes were successfully cloned and the results pave the way for studying the biological functions of NS4A and associated proteins.

Pathways to caspase activation.

Apoptosis or programmed cell death is an active form of cell death which is essential for tissue homeostasis. Many proteins are involved in the molecular signal transduction of apoptosis. The caspase enzymes, a family of specific cysteine proteases, play a central role in cell death machinery. In this review, we mainly discuss the current understanding of several pathways to activate caspases and some key proteins related to these pathways.

[Apoptotic characteristics of spleen lymphocyte in mice irradiated by lethal dose and its relationship to the expression of Bax and Bcl-XL proteins].

OBJECTIVE: To observe the apoptotic characteristics of mouse spleen lymphocyte after lethal dose gamma-irradiation and its relationship to the expression of Bax and Bcl-XL proteins. METHODS: Two hundred and twenty-five second-grade C57 mice were randomly divided into six groups of 0, 6, 9, 12, 15 and 20 Gy. They were sacrificed by dislocation and samples were taken on 1-28 days after whole body single gamma-irradiation. Lymphocyte apoptosis and necrosis were analyzed by TdT-mediated dUTP nick end labeling (TUNEL) and flow cytometry (FCM) techniques. The expression of Bax and Bcl-XL proteins were estimated by immunohistochemical method. RESULTS: (1) The number of peripheral white blood cells of mice increased temporarily at 6 hours after radiation, thereafter, began to decrease rapidly, which reached the minimum on day 7 and recovered normal level basically one month after 6 Gy gamma-irradiation. (2) Apoptotic rate of spleen lymphocytes increased significantly, peaking at 6 hours after radiation, which was found to have a dose-response relationship during 6-24 hours after < or =12 Gy irradiation, but decreased after > or =15 Gy irradiation. (3) It was confirmed by FCM that the apoptotic rate of spleen lymphocytes increased along with the elevation of radiation dose. However, the apoptotic rate began to decrease and the necrotic rate rose distinctively after > or =15 Gy irradiation. The analysis of DNA gel electrophoresis supported above-mentioned results. (4) The expression of Bax protein in spleen lymphocyte enhanced at 6 hours after 6 Gy gamma-irradiation and peaked by 12 Gy-irradiation, showing a dose-dependent pattern, but which was not be found after > or =15 Gy gamma-irradiation. On the other hand, the expression of Bcl-XL protein reduced persistently with the increase of radiation dose, and also presented a better dose-dependent effect after < or =12 Gy irradiation. CONCLUSION: After 6-12 Gy gamma-irradiation, the apoptosis is the major death way of spleen lymphocyte, while both necrosis and apoptosis are important death pathways after > or =15 Gy irradiation. Pro-apoptotic Bax and anti-apoptotic Bcl-XL play an important role in the apoptotic regulation of spleen lymphocytes induced by lethal dose radiation.

[The effects of acute large dose of gamma-irradiation on immune function of mice].

AIM: To observe the effects of large dose of gamma-irradiation on immune function of mice. METHODS: 225 cleaning-grad C57 mice, weighing(20+/-2.0) g, were randomly divided into 6 groups, and treated with 0, 6, 9, 12, 15 and 20 Gy gamma-irradiation. At different times after irradiation, lymphocytes were collected and lymphocytic apoptosis and T cell subsets were analyzed by TUNEL, May-Grunwald Giemsa (MGG) staining and flow cytometry. RESULTS: (1)At early stage(1-14) d after radiation, the apoptotic rate of peripheral blood lymphocytes increased significantly and 12 Gy radiation resulted in the highest apoptotic rate. The number of T lymphocytes and T cell subsets decreased continuously in a dose-dependent manner. CD8(+) T cells were the most sensitive in T cell subsets to irradiation. These results suggested that early severe injury might be one of the important features of immune injury caused by acute radiation. (2) One month after radiation, the apoptotic rate of lymphocytes began to decrease and T lymphocytes and their subsets recovered gradually. However, neither the lymphocytic apoptotic rate nor the number of CD3(+) T cells and CD8(+) T cells, recovered to normal level, indicating that large dose of radiation had severe remote effects on immune function. CONCLUSION: Apoptosis of a large number of peripheral blood lymphocytes in early stage after radiation may result in sharp reduction of T cell number and late immune function depression.

[Relationship between apoptosis of mouse thymic lymphocytes and expressions of bax, bcl-2 and bcl-XL after gamma-ray radiation with lethal dose].

AIM: To observe the relationship between apoptosis of mouse thymic lymphocytes and the expressions of bax, bcl-2 and bcl-X(L) after gamma-ray radiation with lethal dose and provide the basis for treatment of acute severe radiation sickness. METHODS: 250 cleaning-grade C57 mice were randomly divided into 6 groups and treated with 0, 6, 9, 12, 15 and 20 Gy of whole body single gamma-irradiation, respectivety. The mice were killed by dislocation and then the thymus and peripheral blood samples were taken at 1-28 days and 3-12 months after irradiation. The lymphocytic apoptosis was analyzed by TUNEL. The expressions of bax, bcl-2 and bcl-X(L) were detected by in situ hybridization and immunohistochemical staining. RESULTS: (1)Mouse peripheral leucocytes showed a transient elevation at 6 h after radiation and then decreased rapidly. The leucocyte's number reduced to minimal level at day 7 after 6 Gy gamma-irradiation and returned to basic normal value until one month after radiation. (2)24 h after irradiation the apoptotic rate of thymic lymphocytes increased swiftly, and apoptotic rate was positively correlated with radiation dose in the range of 6-12 Gy. There was no such a correlation after >/=15 Gy irradiation. (3)24 h after 6 Gy irradiation the apoptotic rate of thymic lymphocytes reached the maximal level. Afterwards it began to decrease, but still higher than that of control group even after 12 months. (4)3 h after 6 Gy radiation the expression of Bax protein in thymic lymphocytes increased immediately, and reached the highest value at 24 h. On the other hand, the expressions of Bcl-2 and Bcl-X(L) proteins reduced evidently at 3 h after radiation, reaching the lowest level at 24 h. Analysis of bax and bcl-2 mRNAs was concordant with the protein expression results. CONCLUSION: After 6-12 Gy gamma-ray irradiation, the apoptotic rate of thymic lymphocytes is positively correlated with the radiation doses, suggesting that apoptosis is a major way of thymic lymphocyte death after

[Apoptosis and modulatory mechanism of human AHH-1 T lymphocytes induced by gamma-irradiation].

AIM: To explore the apoptotic characteristics and modulatory mechanism of human AHH-1 T lymphoblast induced by ionizing radiation and provide an experimental basis for preventing and treating immune damage in acute radiation sickness. METHODS: TdT-mediated dUTP nick end labeling (TUNEL) and May-Grunwald Giemsa (MGG) staining, MTT colorimetry and immunohistochemical staining were used to detect the T lymphocyte apoptosis, cell survival and the expressions of Bax, Bcl-XL and caspase-3 proteins. RESULTS: (1) Over a definite dose range, apoptotic rate of AHH-1T lymphocytes increased with the elevation of radiation doses, while the lymphocytic survival rate decreased sharply, both showing a good dose-effect relationship. (2) The expression of apoptosis-accelerating protein Bax enhanced obviously with the increase of radiation doses, while apoptosis-inhibiting protein Bcl-XL trended to persistent decline. (3)The activity of activated apoptosis-executing protein caspase-3 heightened significantly with increased radiation doses, which revealed a better corresponding relationship to apoptotic rate. CONCLUSION: A great number of apoptotic AHH-1 T cells may be one of major accounts for the lymphocyte rapid reduction and depressed organism immune function induced by irradiation. Bax, Bcl-XL and caspase-3 proteins play an important role in the regulation of radiation-induced T cell apoptosis.

[Radioprotection of recombinant human interleukin-3 and granulocyte-macrophage colony-stimulating factor on peripheral lymphocytes of rhesus monkey irradiated by 3.0 Gy gamma-rays].

OBJECTIVE: To observe the radioprotection of recombinant human interleukin-3 (rhIL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF) and rhIL-3 (rhIL-3+GM-CSF) on peripheral lymphocytes of rhesus monkey irradiated by 3.0 Gy gamma-rays, and attempt to provide evidence of cytokines used effectively in the therapy of acute radiation sickness. METHODS: Thirty rhesus monkey used in the experiment were randomly divided into six groups of rhIL-3 20 microg.kg -1.d -1, 60 microg.kg -1.d -1 GM-CSF 10 microg.kg -1.d -1 IL-3 20 microg.kg -1.d -1 +GM-CSF 10 microg.kg -1.d -1 radiation control and normal control. 21 d after whole body gamma-irradiation and subcutaneous injection of cytokines, T lymphocyte and its subsets, Bax/Bcl-2 proteins in lymphocytes were determined by immunohistochemical staining with alkaline phosphatase, and lymphocyte apoptosis was detected by TdT-mediated dUTP nick end labeling (TUNEL) technique. RESULTS: (1) After irradiation the quantities of peripheral lymphocyte, T cell and its subsets obviously decreased as compared with those of normal controls. For instance, the percentages of lymphocyte, T, T H and Ts cells in radiation control group reduced to 44 percent, 42 percent, 41 percent and 57 percent of normal controls, respectively. (2)After radiation the reduction of lymphocyte, T, T H and Ts cells were evidently improved by injection of GM-CSF and GM-CSF+IL-3, The T,T H cells in GM-CSF and GM-CSF+IL-3 groups were respectively elevated by 1.57 and 1.76 fold, as well as 1.48 and 1.72 fold of radiation controls. (3) A large amount of lymphocyte apoptosis was found after radiation, GM-CSF and GM-CSF+IL-3 treatment could distinctively inhibit abundant lymphocyte apoptosis induced by acute irradiation,the apoptotic rates of lymphocytes in GM-CSF and GM-CSF+IL-3 groups reduced to 41 percent and 48 percent respectively when compared with that of radiation controls. CONCLUSION: A definite dose of GM-CSF and GM-CSF+IL-3 could suppress the reduction of lymphocyte, T and T H cells and lymphocyte apoptosis induced by 3.0 Gy gamma-irradiation. It confirms that inhibition of GM-CSF and GM-CSF+IL-3 on lymphocyte reduction as well as apoptosis might be one of the major causes to alleviate radiation injury of lymphocytes and improve the immunological function.

Relationship between expression of Bax and Bcl-2 proteins and apoptosis in radiation compound wound healing of rats.

OBJECTIVE: To study the relationship between the expression of Bax, Bcl-2 proteins, and apoptosis in radiation compound wound healing of rats. METHODS: Apoptosis, Bax and Bcl-2 proteins were estimated by in situ terminal labeling (TUNEL) and immunohistochemical methods. RESULTS: (1) Changes of the apoptosis in wound healing showed three typical characteristics: early occurrence, high frequency and delayed disappearance after radiation to rats when compared with those of simple wound group, which might be an important reason for radiation-induced delayed wound healing. (2) The expression of Bax protein increased evidently with the increment of apoptosis and showed a good corresponding relationship with the apoptotic frequency in the process of wound healing. While the expression of Bcl-2 protein decreased obviously as the apoptosis reached a maximum and showed increasing tendency up to normal level when the apoptosis decreased distinctively. CONCLUSIONS: Bax and Bcl-2 proteins play an important role in the apoptotic regulation of radiation compound wound healing in rats.