Natural Course of Common Bile Duct Microlithiasis.

BACKGROUND: Common bile duct microlithiasis (CBDM) with a diameter of ≤ 3 mm can pass spontaneously without causing any symptoms, but in some cases, it can also cause severe cholangitis and pancreatitis. The optimal strategy for managing CBDM is yet to be determined. METHODS: Data of 154 patients with CBDM were collected and divided into two groups: with endoscopic retrograde cholangiopancreatography (with ERCP, n = 82) and without ERCP (n = 72). Clinical outcomes, including the incidence of unfavorable outcomes (UOs), such as cholangitis and pancreatitis, were observed and compared between the two groups. RESULTS: The incidence of UOs was significantly lower in the ERCP group than in the without ERCP group (3.7% vs. 23.6%, respectively, p < 0.001). Moreover, the total number of readmissions was also lower in the ERCP group than in the without ERCP group (p < 0.001). A multivariate analysis adjusted for age, sex, and the American Society of Anesthesiologists (ASA) class revealed that endoscopic sphincterotomy (EST) and cholecystectomy were associated with a lower risk of UOs. CONCLUSION: The high rate of UOs in CBDM patients without ERCP suggests that its natural clinical course may not be as favorable as previously suggested. This finding implies that efforts should be made to clear the bile ducts.

Low-volume regimen without additional liquids or adjunctive agents versus standard bowel preparation in non-constipated patients: a propensity score matching analysis.

AIMS: Split-dose, 4-L polyethylene glycol (PEG, HSD) is currently the first-line choice for unselected or difficult colon preparations. Almost all low-volume bowel preparations (BPs) include a large volume of additional liquid and adjunctive agents to improve cleansing efficiency. However, neither HSD nor additional liquids or adjunctive agents of low-volume regimens may be necessary for low-risk patients. The aim of this study was to compare the cleansing efficiency between split-dose, low-volume (2-L) PEG without additional liquids or adjunctive agents (LSD) and HSD in non-constipated patients. METHODS: A retrospective study was performed from January 2013 to December 2015. Consecutive non-constipated patients who received LSD or HSD BPs were enrolled into LSD and HSD groups. Propensity score matching (PSM) was used to reduce selection bias and potential confounders. The primary outcome was bowel cleansing quality, as evaluated by the Boston Bowel Preparation Scale (BBPS). The adenoma detection rate (ADR), the most important secondary outcome, was also recorded. Follow-up was conducted in 2016. RESULTS: After excluding those participants who meet exclusion criteria or lost follow-up, 1656 non-constipated patients underwent LSD (n = 999) or HSD (n = 657) BP. Most patients had a BBPS score ≥6 (LSD vs. HSD, 93.6 vs. 92.9%, p = .166). The segmental BBPS scores were ≥2 in 92 and 91.9% in the LSD and HSD groups, respectively. The overall ADR was 16.7% in the LSD group and 17.5% in the HSD group (p = .334). CONCLUSION: For non-constipated patients, LSD is not inferior to HSD in cleansing efficiency, while more willing to repeat the same BP.

Quantitative analysis of collagen and capillaries of 3.8-µm laser-induced cutaneous thermal injury and wound healing.

The biological effects of cutaneous thermal injury and wound healing after 3.8-µm laser radiation were investigated by observing the effects of different radiation doses on in vivo cutaneous tissue. A 3.8-µm laser with a radiation dose that changes from small (5.07) to large (15.74 J/cm2) was used to irradiate mouse skin with the 2 × 4 grid array method. The healing progress of laser-injured spots, pathological morphology (H&E staining), and collagen structure changes (Sirius Red staining) were dynamically observed from one hour to 21 days after laser radiation, and the capillary count and collagen content were quantitatively and comparatively analyzed. When the radiation doses were 5.07, 6.77, 8.21, and 9.42 J/cm2, a white coagulation spot predominantly occurred, and when the radiation doses were 11.09 12.23, 14.13, 15.74 J/cm2, a small injured spot predominantly occurred. One hour after radiation, the collagen structure was obviously damaged. Three to fourteen days after radiation, the hyperplasia and morphology of the collagen in the 5.07 J/cm2 group were significantly better than those in the other dose groups. The number of capillaries in the 5.07 J/cm2 and 6.77 J/cm2 groups was significantly higher than that in the normal group (P < 0.01 or P < 0.05). Twenty-one days after radiation, only the collagen in the 5.07 J/cm2 group was densely arranged, and it was basically close to the normal group level. The collagen content in the 5.07 J/cm2 group was approximately 10.7%, but it was still lower than that in the normal group (with a collagen content of approximately 14.1%). The collagen in the other dose groups was diminished and had not returned to the normal group level. As the dose of the 3.8-µm laser increased, skin thermal injury gradually increased, the full-thickness skin increased, and the collagen content decreased, showing better dose-dependent and time-dependent effect relationships. The increase in capillaries in the early stage of laser radiation and the significant increase in collagen content in the middle and late stages of laser radiation were two important factors that promoted wound healing.

Gene therapy for neuropathic pain induced by spared nerve injury with naked plasmid encoding hepatocyte growth factor.

BACKGROUND: Neuropathic pain (NP) is a refractory disease in the clinic with a tremendous impact on the quality of life of patients. Gene therapy is a potential strategy for the management of NP. In the present study, we examined the analgesic effect and mechanism of hepatocyte growth factor (HGF) in vitro and in vivo. METHODS: We examined the proinflammatroy gene changes in lipopolysaccharide (LPS)-induced microglia BV2 cells with a quantitative real-time polymerase chain reaction of interleukin (IL)-1ß, IL-6, tumor necrosis factor (TNF)-α and inducible nitric oxide synthase (iNOS). Mechanical stimulation tests were performed five times at 5-min intervals to assess pain thresholds using Von Frey Hair in mice following spared nerve injury (SNI). The glial cell activation of spinal cord was examined by western blotting. Statistical significance was determined by a Tukey's test and a paired t-test. RESULTS: We found that recombinant human HGF protein suppressed LPS-induced BV2 cell activation in vitro, marked by the down-regulation of IL-1ß, IL-6, TNF-α and iNOS expression, as well as decrease of nitric oxide production. Moreover, intrathecal injection of naked plasmid encoding HGF gene (pUDK-HGF) significantly attenuated SNI-induced pain behaviors in mice by direct inhibition of spinal cord microglia and astrocyte activation. CONCLUSIONS: The results of the present study indicate that pUDK-HGF can reduce cytotoxicity products released from activated glial cells, which may provide a promising therapeutic strategy for treating NP.

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.

[Molecular mechanism of cisplatin to enhance the ability of TRAIL in reversing multidrug resistance in gastric cancer cells].

OBJECTIVE: To study the molecular mechanism of cisplatin to enhance the ability of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in reversing multidrug resistance in vincristine-resistant human gastric cancer SGC7901/VCR cells. METHODS: MTT assay was used to measure the 50% inhibiting concentration (IC50) and cell survival in SGC7901 and SGC7901/VCR cells after different treatments. SGC7901/VCR cells were treated with different concentrations of DDP, different concentrations of TRAIL alone or in combination, and then the mRNA and protein levels of several genes were determined by RT-PCR, RT-qPCR and Western-blot analysis. After targeted silencing with specific siRNA and transfection of recombinant plasmid c-myc into the SGC7901/VCR cells, the mRNA and protein levels of DR4, DR5 and c-myc were determined by RT-PCR and Western-blot analysis. RESULTS: After combined treatment with TRAIL and DDP of the SGC7901/VCR cells, the IC50 of VCR, DDP, ADM, and 5-Fu treatment was significantly decreased compared with the control group or TRAIL-treated group (P < 0.05). After treatment with 0, 10, 50 ng/ml TRAIL in combination with 0.4 µg/ml DDP, the SGC7901/VCR cells showed significantly higher activation of caspase 3, down-regulation of DNA-PKcs/Akt/GSK-3ß signaling pathway, and higher inhibition of MDR1(P-gp) and MRP1 than those treated with TRAIL alone (P < 0.01 for all). The mRNA and protein levels of DR4, DR5, c-myc were significantly decreased after silencing c-myc with specific siRNA in the SGC7901/VCR cells (P < 0.01 for all), and were significantly increased after transfection of recombinant plasmid c-myc into the SGC7901/VCR cells (P < 0.01 foe all). After the treatment with 10 ng/ml TRAIL, 0.25 µg/ml DDP + 10 ng/ml TRAIL and 0.5 µg/ml DDP + 10 ng/ml TRAIL, the relative expression level of c-myc protein in the SGC7901/VCR cells was 0.314 ± 0.012, 0.735 ± 0.026, and 0.876 ± 0.028, respectively, and the relative expression of cytochrome C was 0.339 ± 0.036, 0.593 ± 0.020 and 0.735 ± 0.031, respectively, and the relative expression levels of DR4, DR5, active-caspase 3 and active-caspase 9 in the SGC7901/VCR cells were also increased along with increasing DDP concentrations. CONCLUSIONS: The activation of DNA-PKcs/Akt/GSK-3ß signaling pathway and high expression of MDR1 and MRP1 play an important role in the multi-drug resistance properties of SGC7901/VCR cells. After combining with TRAIL, DDP can enhance the expression of DR4 and DR5 through up-regulating c-myc and enhancing the activation of caspase 3 and caspase 9 by facilitating mitochondrial release of cytochrome C. It may be an important molecular mechanism of DDP-induced sensitization of TRAIL to reverse the multidrug resistancein SGC7901/VCR cells.

Effects of outdoor activity time, screen time, and family socioeconomic status on physical health of preschool children.

Objective: Experienced 3 years of pandemic-induced home life, in the post-epidemic period, preschoolers in China are falling short of the World Health Organization's standards for screen time and outdoor activities. This notably impacts their physical well-being. The study aims to probe the associations between screen time, outdoor activities, and the physical health of preschoolers, offering insights to shape interventions targeting myopia and obesity prevention in children. Methods: A cross-sectional study was conducted in Guangdong Province, involving a representative sample of 23,992 preschoolers and their caregivers recruited through proportional stratified cluster sampling. Data collection utilized the Chinese Early Human Capability Index (CHeHCI, eHCi), a questionnaire on children's media use in daily family life, and Body Mass Index (BMI). Linear regression and binary logistic regression models were employed to analyze the impact of screen time and outdoor activity duration on the physical health of preschoolers. Results: In the high family socioeconomic status (SES) group, children had significantly less screen time compared to those in the medium and low SES groups. Outdoor activity time varied significantly based on SES, with higher SES linked to extended outdoor engagement. Additionally, children's eHCi health dimension score exhibited significant SES-related differences, showcasing higher scores for children in higher SES groups. In terms of gender differences, boys dedicated significantly more time to outdoor activities than girls, yet boys had a notably higher overweight rate. Furthermore, girls demonstrated better health outcomes based on eHCi health scores. A significant association emerged between overweight and screen time in children with high SES, indicating that prolonged screen time was linked to a higher likelihood of overweight based on BMI. Additionally, a substantial negative correlation was observed between children's eHCi health dimension score and screen time. Furthermore, children's outdoor activity time exhibited a significant positive correlation with eHCi health dimension score. Regression analysis revealed that screen time could significantly negatively predict children's physical health score, while outdoor activity time could significantly positively predict children's eHCi physical health score. Conclusion: The current study highlights that family SES, age, and gender play pivotal roles in influencing preschoolers' screen time and outdoor activity duration, with family SES being particularly influential. Higher family SES correlates with reduced screen time, increased outdoor activity, and elevated health levels among children. Importantly, children's screen time negatively predicts their health status, while outdoor time positively predicts their health status.

A Preliminary Study on Quantitative Analysis of Collagen and Apoptosis Related Protein on 1064 nm Laser-Induced Skin Injury.

To investigate the associated factors concerning collagen and the expression of apoptosis-related proteins in porcine skin injuries induced by laser exposure, live pig skin was irradiated at multiple spots one time, using a grid-array method with a 1064 nm laser at different power outputs. The healing process of the laser-treated areas, alterations in collagen structure, and changes in apoptosis were continuously observed and analyzed from 6 h to 28 days post-irradiation. On the 28th day following exposure, wound contraction and recovery were notably sluggish in the medium-high dose group, displaying more premature and delicate type III collagen within the newly regenerated tissues. The collagen density in these groups was roughly 37-58% of that in the normal group. Between days 14 and 28 after irradiation, there was a substantial rise in apoptotic cell count in the forming epidermis and granulation tissue of the medium-high dose group, in contrast to the normal group. Notably, the expression of proapoptotic proteins Bax, caspase-3, and caspase-9 surged significantly 14 days after irradiation in the medium-high dose group and persisted at elevated levels on the 28th day. During the later stage of wound healing, augmented apoptotic cell population and insufficient collagen generation in the newly generated skin tissue of the medium-high dose group were closely associated with delayed wound recovery.

[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.

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.

[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.

[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.

Cisplatin-mediated c-myc overexpression and cytochrome c (cyt c) release result in the up-regulation of the death receptors DR4 and DR5 and the activation of caspase 3 and caspase 9, likely responsible for the TRAIL-sensitizing effect of cisplatin.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) reverses multidrug resistance (MDR) and induces apoptosis in MDR gastric carcinoma cells. In our previous study, cisplatin proved to be a sensitizing agent for TRAIL. To study the synergistic effects of cisplatin and TRAIL, we investigated the mechanism by which TRAIL reverses multidrug resistance, the role of c-myc in modulating the death receptors DR4 and DR5 and the relationship between cisplatin and cytochrome c (cyt c) release in SGC7901/VCR and SGC7901/DDP cells. We found that after treatment with TRAIL, the DNA-PKcs/Akt/GSK-3ß pathway, which is positively correlated with the levels of MDR1 and MRP1, was significantly inhibited and that this tendency can be abolished by Z-DEVD-FMK (a specific caspase 3 inhibitor). We also found that suppression of c-myc by siRNA reduced the expression of DR4 and DR5 and that transfection with a pAVV-c-myc expression vector increased the expression of DR4 and DR5. Moreover, cisplatin increased the expression of c-myc in the presence of TRAIL, and there is a clear increase in cyt c release from mitochondria with the increasing concentrations of cisplatin. Meanwhile, the intrinsic death receptor pathway of caspase 9, as well as the common intrinsic and extrinsic downstream target, caspase 3, was potently activated by the release of cyt c. Together, we conclude that in TRAIL-treated MDR gastric carcinoma cells, cisplatin induces the death receptors DR4 and DR5 through the up-regulation of c-myc and strengthens the activation of caspases via promoting the release of cyt c. These effects would then be responsible for the TRAIL sensitization effect of cisplatin.

Expression of MMP1 in surgical and radiation-impaired wound healing and its effects on the healing process.

Radiation-impaired wound is characterized by delayed healing, nonhealing, and carcinogenesis. The mechanism remains unclear. Matrix metalloproteinases (MMPs) are one family of key regulators of the process of wound healing. Their abnormal expression plays important roles in the formation of some chronic skin ulcers. The objective of this project was to study the expression of MMP1 in surgical and radiation-impaired wound healing and its effects on the healing process and tissue remodeling. A rat model of radiation-impaired wound healing was used. Routine light microscopy, electron microscopy, immunohistochemistry, and in situ hybridization, all of which enabled the detection of MMP1 expression during the healing process, were performed. The wound healing process was impaired and delayed. In rats receiving 25Gy gamma-ray locally, the irradiated wounds healed 6 days later than the nonirradiated controls. The following changes in MMP1 expression were found: (1) In the early inflammatory phase and in the period of granulation tissue formation, MMP1 expression was only slightly if at all affected in the newly formed epidermis of irradiated wounds compared with controls. Later, the epidermal expression of MMP1 in radiation wounds was comparatively increased following the delay of the healing process. (2) MMP1 expression in irradiated wounds was markedly decreased in fibroblasts, endothelial cells, and macrophages compared with controls. The expression phase was prolonged because of the delay of the healing process. The reduced expression of MMP1 in granulation tissue retards such important processes as cell migration, angiogenesis, and tissue remodeling, thus slowing the healing process. The expression ofMMP1 in the proliferating keratinocytes may help re-epithelialization. However, in the late healing period, overexpression of MMP1 in the epidermis may hinder the establishment of basal membrane and the formation of granulation tissue, and affect the tissue remodeling process.

Molecular mechanism of damage and repair of mouse thymus lymphocytes induced by radiation.

OBJECTIVE: To investigate the role of apoptosis in radiation-induced mouse thymus lymphocyte damage and repair and provide the basis for understanding the molecular mechanism of radiation-induced lymphocyte damage and repair as well as the prevention and treatment of acute radiation sickness. METHODS: We studied the dynamic changes of apoptosis of mouse thymus lymphocytes and the expression of bax and bcl-2 gene products after 2, 4, 6 and 8 Gy of whole body gamma-irradiation using in situ terminal labeling, DNA electrophoresis and immunohistochemical techniques. RESULTS: At the early stage after irradiation, the percentage of apoptotic lymphocytes increased rapidly in accordance with the increasing of radiation doses, while the counts of the thymus and peripheral lymphocytes decreased sharply, showing an opposite change to lymphocyte apoptosis. After 6 Gy gamma-irradiation, typical morphological characteristics of thymus apoptotic lymphocytes in early, middle and late stages were found by transmission electron microscopy. The thymus lymphocytes displayed characteristic DNA ladders 4 hr and 8 hr after 2-6 Gy gamma-irradiation,using DNA gel electrophoresis techniques. Abnormal expression of bcl-2 and bax gene products were shown in irradiated lymphocytes. CONCLUSIONS: Apoptosis plays an important role in the process of radiation-induced mouse thymus lymphocyte damage and repair. Bcl-2 and Bax proteins may regulate the process of lymphocyte apoptosis.

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.

[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.

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.

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.

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.