[Regulatory effects of bio-intensity electric field on transformation of human skin fibroblasts].

Objective: To investigate the regulatory effects of bio-intensity electric field on the transformation of human skin fibroblasts (HSFs). Methods: The experimental research methods were used. HSFs were collected and divided into 200 mV/mm electric field group treated with 200 mV/mm electric field for 6 h and simulated electric field group placed in the electric field device without electricity for 6 h. Changes in morphology and arrangement of cells were observed in the living cell workstation; the number of cells at 0 and 6 h of treatment was recorded, and the rate of change in cell number was calculated; the direction of cell movement, movement velocity, and trajectory velocity within 3 h were observed and calculated (the number of samples was 34 in the simulated electric field group and 30 in 200 mV/mm electric field group in the aforementioned experiments); the protein expression of α-smooth muscle actin (α-SMA) in cells after 3 h of treatment was detected by immunofluorescence method (the number of sample was 3). HSFs were collected and divided into simulated electric field group placed in the electric field device without electricity for 3 h, and 100 mV/mm electric field group, 200 mV/mm electric field group, and 400 mV/mm electric field group which were treated with electric fields of corresponding intensities for 3 h. Besides, HSFs were divided into simulated electric field group placed in the electric field device without electricity for 6 h, and electric field treatment 1 h group, electric field treatment 3 h group, and electric field treatment 6 h group treated with 200 mV/mm electric field for corresponding time. The protein expressions of α-SMA and proliferating cell nuclear antigen (PCNA) were detected by Western blotting (the number of sample was 3). Data were statistically analyzed with Mann-Whitney U test, one-way analysis of variance, independent sample t test, and least significant difference test. Results: After 6 h of treatment, compared with that in simulated electric field group, the cells in 200 mV/mm electric field group were elongated in shape and locally adhered; the cells in simulated electric field group were randomly arranged, while the cells in 200 mV/mm electric field group were arranged in a regular longitudinal direction; the change rates in the number of cells in the two groups were similar (P>0.05). Within 3 h of treatment, the cells in 200 mV/mm electric field group had an obvious tendency to move toward the positive electrode, and the cells in simulated electric field group moved around the origin; compared with those in simulated electric field group, the movement velocity and trajectory velocity of the cells in 200 mV/mm electric field group were increased significantly (with Z values of -5.33 and -5.41, respectively, P<0.01), and the directionality was significantly enhanced (Z=-4.39, P<0.01). After 3 h of treatment, the protein expression of α-SMA of cells in 200 mV/mm electric field group was significantly higher than that in simulated electric field group (t=-9.81, P<0.01). After 3 h of treatment, the protein expressions of α-SMA of cells in 100 mV/mm electric field group, 200 mV/mm electric field group, and 400 mV/mm electric field group were 1.195±0.057, 1.606±0.041, and 1.616±0.039, respectively, which were significantly more than 0.649±0.028 in simulated electric field group (P<0.01). Compared with that in 100 mV/mm electric field group, the protein expressions of α-SMA of cells in 200 mV/mm electric field group and 400 mV/mm electric field group were significantly increased (P<0.01). The protein expressions of α-SMA of cells in electric field treatment 1 h group, electric field treatment 3 h group, and electric field treatment 6 h group were 0.730±0.032, 1.561±0.031, and 1.553±0.045, respectively, significantly more than 0.464±0.020 in simulated electric field group (P<0.01). Compared with that in electric field treatment 1 h group, the protein expressions of α-SMA in electric field treatment 3 h group and electric field treatment 6 h group were significantly increased (P<0.01). After 3 h of treatment, compared with that in simulated electric field group, the protein expressions of PCNA of cells in 100 mV/mm electric field group, 200 mV/mm electric field group, and 400 mV/mm electric field group were significantly decreased (P<0.05 or P<0.01); compared with that in 100 mV/mm electric field group, the protein expressions of PCNA of cells in 200 mV/mm electric field group and 400 mV/mm electric field group were significantly decreased (P<0.05 or P<0.01); compared with that in 200 mV/mm electric field group, the protein expression of PCNA of cells in 400 mV/mm electric field group was significantly decreased (P<0.01). Compared with that in simulated electric field group, the protein expressions of PCNA of cells in electric field treatment 1 h group, electric field treatment 3 h group, and electric field treatment 6 h group were significantly decreased (P<0.01); compared with that in electric field treatment 1 h group, the protein expressions of PCNA of cells in electric field treatment 3 h group and electric field treatment 6 h group were significantly decreased (P<0.05 or P<0.01); compared with that in electric field treatment 3 h group, the protein expression of PCNA of cells in electric field treatment 6 h group was significantly decreased (P<0.01). Conclusions: The bio-intensity electric field can induce the migration of HSFs and promote the transformation of fibroblasts to myofibroblasts, and the transformation displays certain dependence on the time and intensity of electric field.

Regulatory effects of bio-intensity electric field on transformation of human skin fibroblasts / 中华烧伤杂志

Objective: To investigate the regulatory effects of bio-intensity electric field on the transformation of human skin fibroblasts (HSFs). Methods: The experimental research methods were used. HSFs were collected and divided into 200 mV/mm electric field group treated with 200 mV/mm electric field for 6 h and simulated electric field group placed in the electric field device without electricity for 6 h. Changes in morphology and arrangement of cells were observed in the living cell workstation; the number of cells at 0 and 6 h of treatment was recorded, and the rate of change in cell number was calculated; the direction of cell movement, movement velocity, and trajectory velocity within 3 h were observed and calculated (the number of samples was 34 in the simulated electric field group and 30 in 200 mV/mm electric field group in the aforementioned experiments); the protein expression of α-smooth muscle actin (α-SMA) in cells after 3 h of treatment was detected by immunofluorescence method (the number of sample was 3). HSFs were collected and divided into simulated electric field group placed in the electric field device without electricity for 3 h, and 100 mV/mm electric field group, 200 mV/mm electric field group, and 400 mV/mm electric field group which were treated with electric fields of corresponding intensities for 3 h. Besides, HSFs were divided into simulated electric field group placed in the electric field device without electricity for 6 h, and electric field treatment 1 h group, electric field treatment 3 h group, and electric field treatment 6 h group treated with 200 mV/mm electric field for corresponding time. The protein expressions of α-SMA and proliferating cell nuclear antigen (PCNA) were detected by Western blotting (the number of sample was 3). Data were statistically analyzed with Mann-Whitney U test, one-way analysis of variance, independent sample t test, and least significant difference test. Results: After 6 h of treatment, compared with that in simulated electric field group, the cells in 200 mV/mm electric field group were elongated in shape and locally adhered; the cells in simulated electric field group were randomly arranged, while the cells in 200 mV/mm electric field group were arranged in a regular longitudinal direction; the change rates in the number of cells in the two groups were similar (P>0.05). Within 3 h of treatment, the cells in 200 mV/mm electric field group had an obvious tendency to move toward the positive electrode, and the cells in simulated electric field group moved around the origin; compared with those in simulated electric field group, the movement velocity and trajectory velocity of the cells in 200 mV/mm electric field group were increased significantly (with Z values of -5.33 and -5.41, respectively, P<0.01), and the directionality was significantly enhanced (Z=-4.39, P<0.01). After 3 h of treatment, the protein expression of α-SMA of cells in 200 mV/mm electric field group was significantly higher than that in simulated electric field group (t=-9.81, P<0.01). After 3 h of treatment, the protein expressions of α-SMA of cells in 100 mV/mm electric field group, 200 mV/mm electric field group, and 400 mV/mm electric field group were 1.195±0.057, 1.606±0.041, and 1.616±0.039, respectively, which were significantly more than 0.649±0.028 in simulated electric field group (P<0.01). Compared with that in 100 mV/mm electric field group, the protein expressions of α-SMA of cells in 200 mV/mm electric field group and 400 mV/mm electric field group were significantly increased (P<0.01). The protein expressions of α-SMA of cells in electric field treatment 1 h group, electric field treatment 3 h group, and electric field treatment 6 h group were 0.730±0.032, 1.561±0.031, and 1.553±0.045, respectively, significantly more than 0.464±0.020 in simulated electric field group (P<0.01). Compared with that in electric field treatment 1 h group, the protein expressions of α-SMA in electric field treatment 3 h group and electric field treatment 6 h group were significantly increased (P<0.01). After 3 h of treatment, compared with that in simulated electric field group, the protein expressions of PCNA of cells in 100 mV/mm electric field group, 200 mV/mm electric field group, and 400 mV/mm electric field group were significantly decreased (P<0.05 or P<0.01); compared with that in 100 mV/mm electric field group, the protein expressions of PCNA of cells in 200 mV/mm electric field group and 400 mV/mm electric field group were significantly decreased (P<0.05 or P<0.01); compared with that in 200 mV/mm electric field group, the protein expression of PCNA of cells in 400 mV/mm electric field group was significantly decreased (P<0.01). Compared with that in simulated electric field group, the protein expressions of PCNA of cells in electric field treatment 1 h group, electric field treatment 3 h group, and electric field treatment 6 h group were significantly decreased (P<0.01); compared with that in electric field treatment 1 h group, the protein expressions of PCNA of cells in electric field treatment 3 h group and electric field treatment 6 h group were significantly decreased (P<0.05 or P<0.01); compared with that in electric field treatment 3 h group, the protein expression of PCNA of cells in electric field treatment 6 h group was significantly decreased (P<0.01). Conclusions: The bio-intensity electric field can induce the migration of HSFs and promote the transformation of fibroblasts to myofibroblasts, and the transformation displays certain dependence on the time and intensity of electric field.

Effects of electroacupuncture on luteal regression and steroidogenesis in ovarian hyperstimulation syndrome model rat.

AIMS: Electroacupuncture (EA) is an effective and safe therapeutic method widely used for treating clinical diseases. Previously, we found that EA could decrease serum hormones and reduce ovarian size in ovarian hyperstimulation syndrome (OHSS) rat model. Nevertheless, the mechanisms that contribute to these improvements remain unclear. MATERIALS AND METHODS: HE staining was used to count the number of corpora lutea (CL) and follicles. Immunohistochemical and ELISA were applied to examine luteal functional and structural regression. Immunoprecipitation was used for analyzing the interaction between NPY (neuropeptide Y) and COX-2; western blotting and qRT-PCR were used to evaluate the expressions of steroidogenic enzymes and PKA/CREB pathway. KEY FINDINGS: EA treatment significantly reduced the ovarian weight and the number of CL, also decreased ovarian and serum levels of PGE2 and COX-2 expression; increased ovarian PGF2α levels and PGF2α/PGE2 ratio; decreased PCNA expression and distribution; and increased cyclin regulatory inhibitor p27 expression to have further effect on the luteal formation, and promote luteal functional and structural regression. Moreover, expression of COX-2 in ovaries was possessed interactivity increased expression of NPY. Furthermore, EA treatment lowered the serum hormone levels, inhibited PKA/CREB pathway and decreased the expressions of steroidogenic enzymes. Hence, interaction with COX-2, NPY may affect the levels of PGF2α and PGE2 as well as impact the proliferation of granulosa cells in ovaries, thus further reducing the luteal formation, and promoting luteal structural and functional regression, as well as the ovarian steroidogenesis following EA treatment. SIGNIFICANCE: EA treatment could be an option for preventing OHSS in ART.

Effect of luteolin on gene expression in mouse H22 hepatoma cells.

The purpose of our study was to observe the effects of luteolin on the expression of the genes ICAM-1, LFA-3, and PCNA in H22 hepatoma tissue. Sixty ICR (Institute of Cancer Research) mice with H22 hepatoma were randomly divided into five groups: a normal saline control group, low-, medium-, and high-dose luteolin groups, and a cyclophosphamide group. The mice were euthanized the day after administration withdrawal and subcutaneous tumor tissue was extracted. Quantitative fluorescence RT-PCR was used to detect the expression of ICAM-1, LFA-3, and PCNA in H22 hepatoma tissue in the mice. Luteolin was found to up-regulate the expression of ICAM-1 in H22 hepatoma tissue, of which the middle-dose group had the most obvious effect, showing a significant difference (P < 0.01) as compared to the normal saline group. Each dose group of luteolin significantly down-regulated the expression of LFA-3 in H22 hepatoma tissue, showing significant differences as compared to the saline control group (P < 0.01). The medium- and high-dose luteolin groups significantly reduced the expression of PCNA in H22 hepatoma tissue of ICR mice, where the effect of the high-dose group was the most obvious, and the difference between the two luteolin groups and the normal saline group was statistically significant (P < 0.01). Luteolin may inhibit tumor angiogenesis and tumor cell proliferation by down-regulation of LFA- 3 and PCNA and up-regulation of ICAM-1 in tumor tissue of tumor-bearing mice, thereby achieving its anti-tumor effect.

Effect of Selenium Supplementation on Apoptosis and Cell Cycle Blockage of Renal Cells in Broilers Fed a Diet Containing Aflatoxin B1.

The aim of the study was to investigate the competency of selenium (Se) in counteracting the adverse effects of aflatoxin B1 (AFB1) on apoptosis, cell cycle, and proliferation of nephritic cells. Two hundred forty 1-day-old healthy male avian broilers were randomly divided into four groups and fed basal diet (control group), 0.3 mg/kg AFB1 diet (AFB1 group), 0.4 mg/kg Se diet (+Se group), and 0.3 mg/kg AFB1 + 0.4 mg/kg Se diet (AFB1 + Se group), respectively. Compared to the control group, the number of apoptotic renal cells and expressions of Bax and caspase-3 messenger RNA (mRNA) were significantly increased, while the expression of Bcl-2 was significantly decreased in the AFB1 and the +Se groups (p < 0.01). A significantly decreased proliferating cell nuclear antigen (PCNA) expression and arrested G0/G1 phases of the cell cycle were also seen in the AFB1 and the +Se groups when compared with those of the control group. Moreover, these parameters were restored to the control group levels in the AFB1 + Se group. These results suggested that sodium selenite supplied in the diet could effectively inhibit AFB1-induced apoptosis and cell cycle blockage in renal cells of broiler.

Antenatal taurine improves neuronal regeneration in fetal rats with intrauterine growth restriction by inhibiting the Rho-ROCK signal pathway.

The Rho-ROCK signal pathway is an important mediator of inhibitory signals that blocks central nervous cell regeneration. Here, we investigated whether antenatal taurine improved neuronal regeneration in fetal rats with intrauterine growth restriction (IUGR) by inhibiting this pathway. Thirty pregnant rats were randomly divided into three groups: control, IUGR, and IUGR + antenatal taurine supplementation (taurine group). The mRNA levels of Ras homolog gene A (Rho A), Rho-associated coiled-coil forming protein kinase 2 (ROCK2), and proliferating cell nuclear antigen (PCNA) were detected using real-time quantitative PCR. RhoA, ROCK2 and PCNA-positive cells were counted using immunohistochemistry. Antenatal taurine supplementation decreased RhoA and Rock2 mRNA expression, increased PCNA mRNA expression, and significantly decreased RhoA, ROCK2-positive and increased PCNA-positive cell counts in IUGR fetal rat brain tissues (p < 0.05). Thus, antenatal taurine supplementation inhibited the expression of key Rho-ROCK signal molecules and improved IUGR fetal brain development.

The effect of hyperbaric oxygen on intracephalic angiogenesis in rats with intracerebral hemorrhage.

Healthy SD rats were randomly divided into 3 groups as sham operation (group A), ICH (group B), and HBO2 (group C). The behavioral change and angiogenesis in brain tissue of rats in each group were observed. The protein expression of PCNA, vWF, HIF1-α, and VEGF in rat brain was measured by immunohistochemistry, while the mRNA expression level of HIF1-α and VEGF was determined using quantitative real-time PCR. This study has investigated the effect of HBO2 on intracephalic angiogenesis in rats with intracerebral hemorrhage (ICH). There were significant differences in behavior score between HBO2 and ICH groups at 14, 21, and 28 days. A large number of vessel-like structures and microvessels were observed in perihematomal brain tissues in HBO2 group. There were significant differences in HIF1-α and VEGF protein and HIF1-α mRNA level between HBO2 and ICH groups at 14, 21, and 28 days; at 7, 14, 21, and 28 days, the differences in PCNA and vWF protein expression between the 2 groups were statistically significant. At 21 and 28 days, the expression levels of VEGF mRNA in the 2 groups differed significantly from each other. Our results indicate that HBO2 can significantly promote the expression of HIF1-α and VEGF at both mRNA and protein levels in rats with ICH, increase the protein expression of both PCNA and vWF, promote the formation of new blood vessels, and promote the recovery of behavioral ability, hence resulting in a rapid rehabilitation.

Protective effect of sildenafil on liver injury induced by intestinal ischemia/reperfusion.

BACKGROUND: This study evaluated the protective effect of sildenafil on liver injury induced by intestinal ischemia-reperfusion. METHODS: Forty female Sprague Dawley rats were divided into 4 groups: sham-control (SC), ischemia (I), ischemia-reperfusion (IR), and ischemia-reperfusion+sildenafil (SIL; sildenafil gavaged at 50mg/kg before operating). A 2-h ischemia-reperfusion was performed by clamping the superior mesenteric artery. Liver function, plasma alanine (ALT) and aspartate (AST) aminotransferase, and intestinal and liver malondialdehyde (MDA) were measured at the end of the experiment. Intestinal and liver tissue damage was examined by histology. Liver samples were immunologically stained for endothelial nitric oxide synthase (eNOS) and proliferating cell nuclear antigen (PCNA). RESULTS: The ALT and AST levels were highest in the IR group and were lower in the SIL group (p<0.05). Intestinal MDA levels were statistically higher in the IR group than in the SC, I and SIL groups. Liver MDA levels were significantly higher in the IR group than in the I and SC groups (p<0.05) and higher than in the SIL group (p>0.05). Intestinal damage based on Chiu scoring was more severe in the IR than in the SIL group (p<0.05). Sildenafil reduced damage and also increased eNOS and PCNA immunoreactivity in liver tissue. CONCLUSIONS: Sildenafil shows a protective effect on intestinal ischemia-reperfusion-induced liver injury, possibly by decreasing vascular resistance through increased nitric oxide levels.

Role of the phytoestrogenic, pro-apoptotic and anti-oxidative properties of silymarin in inhibiting experimental benign prostatic hyperplasia in rats.

Androgen and estrogen play an important role in the pathogenesis of benign prostatic hyperplasia (BPH). Estrogen exerts its action through two distinct estrogen receptors (ERs) either ER-α or ER-ß. The phytoestrogenic property of silymarin (SIL) has been previously characterized. Thus, this study examined the protective effect of SIL against testosterone-induced BPH in rats. In an initial dose-response study, SIL in a dose of 50mg/kg was the most effective in preventing the rise in prostate weight, prostate weight/body weight ratio and histopathologic changes induced by testosterone. Testosterone significantly decreased ER-ß and increased ER-α and AR expressions as compared to the control group and these effects were significantly ameliorated by SIL. Furthermore, SIL significantly protected against testosterone-provoked decline in mRNA expression of P21(WAF1/Cip1) and Bax/Bcl-xl ratio as well as caspase-3 activity. SIL minimized the number of proliferating cell nuclear antigen (PCNA) positive cells as compared to testosterone-treated group. Moreover, SIL significantly blunted the inducible NF-κB expression and restored the oxidative status to within normal values in the prostatic tissues. Collectively these findings elucidate the effectiveness of SIL in preventing testosterone-induced BPH in rats. This could be attributed, at least partly, to its phytoestrogenic, pro-apoptotic and anti-oxidative properties.

Adjunctive antimicrobial photodynamic treatment of experimentally induced periodontitis in rats with ovariectomy.

BACKGROUND: The aim of this study is to compare antimicrobial photodynamic therapy (aPDT) as an adjunctive therapy to scaling and root planing (SRP) for the treatment of experimentally induced periodontitis in rats with ovariectomy (OVX) that are or are not treated with estrogen replacement. METHODS: A total of 270 female rats were divided into three groups: 1) normal rats; 2) rats with OVX; and 3) rats with OVX with estrogen replacement. Periodontal disease was induced through the introduction of a cotton thread around the mandibular left first molar. After 7 days, the ligature was removed, and the rats were randomly divided into the following treatment groups: 1) SRP plus saline solution; 2) SRP plus low-level laser therapy (LLLT); and 3) SRP plus toluidine blue O irrigation followed by LLLT. Ten rats from each group were euthanized at days 7, 15, and 30 after dental treatment. Bone loss (BL) in the furcation region was evaluated using histometric and immunohistochemical analyses. RESULTS: aPDT treatment resulted in reduced BL compared with SRP treatment at all time points. Additionally, rats treated with aPDT exhibited reduced numbers of tartrate-resistant acid-phosphatase-positive cells and more proliferating cell nuclear antigen-positive cells in all treatment groups regardless of estrogen status. Whereas rats treated with aPDT showed weak immunoreactivity to the receptor activator of nuclear factor-κ B ligand at day 7 post-treatment, strong osteoprotegerin immunoreactivity was observed at day 15 post-treatment. CONCLUSION: aPDT is an effective adjunctive therapy for the treatment of periodontitis in rats with OVX that are or are not given estrogen replacement therapy.

Effects of growth differentiation factor-9 and FSH on in vitro development, viability and mRNA expression in bovine preantral follicles.

The present study investigated the role of growth differentiation factor (GDF)-9 and FSH, alone or in combination, on the growth, viability and mRNA expression of FSH receptor, proliferating cell nuclear antigen (PCNA) and proteoglycan-related factors (i.e., hyaluronan synthase (HAS) 1, HAS2, versican, perlecan) in bovine secondary follicles before and after in vitro culture. After 12 days culture, sequential FSH (100 ng mL⁻¹) from Days 0 to 6 and 500 ng mL⁻¹ from Days 7 to 12) increased follicular diameter and resulted in increased antrum formation (P<0.05). Alone, 200 ng mL⁻¹ GDF-9 significantly reduced HAS1 mRNA levels, but increased versican and perlecan mRNA levels in whole follicles, which included the oocyte, theca and granulosa cells. Together, FSH and GDF-9 increased HAS2 and versican (VCAN) mRNA levels, but decreased PCNA mRNA expression, compared with levels in follicles cultured in α-minimum essential medium supplemented with 3.0 mg mL⁻¹ bovine serum albumin, 10 µg mL⁻¹ insulin, 5.5 µg mL⁻¹ transferrin, 5 ng mL⁻¹ selenium, 2 mM glutamine, 2mM hypoxanthine and 50 µg mL⁻¹ ascorbic acid (α-MEM⁺). Comparisons of uncultured (0.2 mm) and α-MEM⁺ cultured follicles revealed that HAS1 mRNA expression was higher, whereas VCAN expression was lower, in cultured follicles (P<0.05). Expression of HAS1, VCAN and perlecan (HSPG2) was higher in cultured than in vivo-grown (0.3 mm) follicles. In conclusion, FSH and/or GDF-9 promote follicular growth and antrum formation. Moreover, GDF-9 stimulates expression of versican and perlecan and interacts positively with FSH to increase HAS2 expression.

Regulation of glioblastoma stem cells by retinoic acid: role for Notch pathway inhibition.

It is necessary to understand mechanisms by which differentiating agents influence tumor-initiating cancer stem cells. Toward this end, we investigated the cellular and molecular responses of glioblastoma stem-like cells (GBM-SCs) to all-trans retinoic acid (RA). GBM-SCs were grown as non-adherent neurospheres in growth factor supplemented serum-free medium. RA treatment rapidly induced morphology changes, induced growth arrest at G1/G0 to S transition, decreased cyclin D1 expression and increased p27 expression. Immunofluorescence and western blot analysis indicated that RA induced the expression of lineage-specific differentiation markers Tuj1 and GFAP and reduced the expression of neural stem cell markers such as CD133, Msi-1, nestin and Sox-2. RA treatment dramatically decreased neurosphere-forming capacity, inhibited the ability of neurospheres to form colonies in soft agar and inhibited their capacity to propagate subcutaneous and intracranial xenografts. Expression microarray analysis identified ∼350 genes that were altered within 48 h of RA treatment. Affected pathways included retinoid signaling and metabolism, cell-cycle regulation, lineage determination, cell adhesion, cell-matrix interaction and cytoskeleton remodeling. Notch signaling was the most prominent of these RA-responsive pathways. Notch pathway downregulation was confirmed based on the downregulation of HES and HEY family members. Constitutive activation of Notch signaling with the Notch intracellular domain rescued GBM neurospheres from the RA-induced differentiation and stem cell depletion. Our findings identify mechanisms by which RA targets GBM-derived stem-like tumor-initiating cells and novel targets applicable to differentiation therapies for glioblastoma.

Protective mechanism of glutamine on the expression of proliferating cell nuclear antigen after cisplatin-induced intestinal mucosal injury.

BACKGROUND: Glutamine prevents the intestinal mucosal injury induced by chemotherapy. However, the mechanism has not yet been elucidated. Proliferating cell nuclear antigen (PCNA) is expressed in the nuclei of cells during the DNA synthesis phase of the cell cycle, and PCNA is also involved in the DNA damage tolerance pathway known as post-replication repair. We hypothesized that glutamine supplementation might stimulate the intestinal epithelial cell cycle interruption induced by chemotherapy. The effect of supplemental glutamine after cisplatin-induced intestinal mucosal injury on the expression of PCNA was investigated. MATERIALS AND METHODS: The male Wister rats were divided into three groups; a control group (control n = 5), which received standard rat diet; the Cis group (cisplatin 6 mg/kg i.p., n = 5), and the Cis + Gln group [cisplatin + Ala-Glutamine (0.5 g/day × 3 days p.o., n = 5)]. After 1, 3, and 7 days of chemotherapy, PCNA, and glutamine transporter (ASCT2) expression in the small intestine (jejunum and ileum) was investigated. RESULTS: The expression of PCNA in the crypt of the small intestine (jejunum and ileum) decreased after chemotherapy, while the expression strongly increased by glutamine administration, even if it was after chemotherapy. On day 1, both the mRNA expression of the glutamine transporter (ASCT2) and PCNA expression in crypt cells were significantly increased by administration of glutamine (Cis + Gln group). The increased expression of ACST2 appeared earlier than in the Cis group. In the Cis + Gln group, the PCNA expression was normalized on day 3, and the expression was same as that in the control group on day 3. CONCLUSION: Glutamine supplementation rapidly improved the expression of PCNA after cisplatin-induced intestinal mucosal injury. The effects of glutamine may be due to an anti-oxidant effect, but the amino acid might also attenuate the initial mucosal injury and improve intestinal cell turnover.

Regulation of p21, MMP-1, and MDR-1 expression in human colon carcinoma HT29 cells by Tian Xian liquid, a chinese medicinal formula, in vitro and in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: Tian-Xian liquid (TXL), a commercially available Chinese medicine decoction, has been used as an anticancer dietary agent for more than 10 years without reported side effects. AIM OF THE STUDY: The safety and quality consistency of TXL and its mechanisms of action on antiproliferation, antimetastasis, and reversion of multidrug resistance (MDR) regimens were explored. MATERIALS AND METHODS: In this study, an atomic absorption spectrophotometer and reversed phase high performance liquid chromatography with photodiode array detection (HPLC-DAD) were used to evaluate the main toxic elements and the quality consistency among different batches of TXL extracts, respectively. HT29 human colon cancer cell line and tumor-bearing nude mice were used. TXL was provided by China-Japan Feida Union Company Limited. The effect of TXL on in vitro proliferation of HT29 human colon cancer cell line was examined. The percentages of treated cells distributed in different phases of the cell cycles were analyzed by flow cytometry. Antiproliferative effect after treatment with TXL was assessed by determination of the protein levels of p21, cyclinD1, PCNA, and cdk-2, which are the key regulators for cell cycle progression. Meanwhile, the protein levels of MMP-1 and MDR-1 (multidrug resistance protein-1) were also determined to assess the effect of TXL on antimetastasis and reversion of MDR regimen, respectively. RESULTS: The contents of main toxic elements were lower in TXL extract compared with the standard set by the Department of Health of the Government of Hong Kong Special Administrative Region (SAR). Our HPLC results showed that the relative standard deviations of the amount of the 5 standards were less than 5% in different batches of TXL. Immunoblotting analysis revealed a dramatic induction of cyclin kinase inhibitor p21 as well as an inhibition of cyclinD1, PCNA, and cdk-2 in the TXL-treated in vitro models, thereby, impeding cell progression from G1/S phase. Results obtained from the in vivo study also demonstrated that TXL upregulated the protein level of p21 and downregulated the protein levels of MMP-1 and MDR-1. CONCLUSIONS: Results obtained from the present investigation not only demonstrate the safety and quality of TXL extract but also demonstrate that TXL possesses antiproliferative and antimetastatic activities and brings about reversion of MDR on HT29 cell and on xenografted tissue in tumor-implanted nude mice.

Thermoseed hyperthermia treatment of mammary orthotopic transplantation tumors in rats and impact on immune function.

To evaluate the effect of thermoseed inductive heating on mammary orthotopic transplantation tumors and immunologic function in rats. Walker-256 tumor cells were inoculated subcutaneously into the mammary glands of Wistar rats. Rats were allocated to five treatment groups as follows: i) C group (control group); ii) M group (magnetic field group); iii) T group (thermoseed control group); iv) H1 group (hyperthermia treatment, 45 degrees C for 30 min); v) H2 group (hyperthermia treatment, 50-55 degrees C for 10 min). Immediately, 12 and 24 h after hyperthermia, two rats were sacrificed in each group for pathological and immunohistochemical examination of the expression of PCNA and HSP70. Tumor volume was measured and long-term survival was observed. The T lymphocyte subgroup IL-2 and IFN-gamma levels were measured in C, H1 and H2 groups. Both types of hyperthermia induced necrosis and apoptosis in the tumor tissue, decreased tumor volume (P<0.05), and increased survival time (P<0.01). The expression of PCNA and HSP70 in hyperthermia group was significantly different compared to the C, M and T groups (P<0.05), Hyperthermia increased CD4+ T lymphocytes and the levels of IL-2 and IFN-gamma (P<0.05). Both types of hyperthermia can suppress the growth of mammary tumors and improve immunological function of rats.

Various dietary polyunsaturated fatty acids modulate acrylamide-induced preneoplatic urothelial proliferation and apoptosis in mice.

To investigate whether various dietary fats affected preneoplatic lesions of urinary tract in acrylamide (ACR)-treated mice. Eighty Kunming mice were initiated with ACR at dose 10mg/kg bw, and fed with different polyunsaturated fatty acid (PUFA): 6% fish oil (enriched in n-3 PUFA), 6% corn oil (enriched in n-6), 6% olein (enriched in n-9, an EFA deficiency inducer), 10% fish oil, 10% corn oil, 10% olein and a commercial chow. Animals were autopsied 22 weeks. The liver fatty acid profile showed a close correlation with dietary sources, exhibiting macroscopic and biochemical EFA-deficient (EFAD) characteristics in ACR mice with olein. The frequency of simple urothelial hyperplasia (H) and dysplasia/carcinoma in situ (D/CIS) was significantly higher in ACR mice with corn oil or olein compared to ACR mice with commercial chow. Proliferation and abnormal luminal localized mitosis, also expression of proliferating cell nuclear antigen (PCNA), significantly increased in ACR mice with corn oil and olein than in ACR mice with commercial chow; moreover, abnormal apoptotic/mitosis ratio, expression of caspase-3, decreased in ACR mice with both olein and corn oil. Fish oil took no significant effect on almost all the parameters in ACR mice in this study. Results suggest that dietary PUFA modulate preneoplastic proliferation in ACR mice; n-6 PUFA (corn oil) and EFAD status (n-9 PUFA) exhibits a promoting activity; whereas, fish oil, rich in n-3 fatty acids, exhibits somewhat attenuated effect, and needs further research.

Long-term effect of Spirulina platensis extract on DMBA-induced hamster buccal pouch carcinogenesis (immunohistochemical study).

In cancer research, the use of complementary and alternative medicine has increased over the past decade. In this study, 80 male golden Syrian hamsters were divided into four equal groups; the right buccal pouches of the hamster rats in group 1 were painted with 0.5% solution of 7, 12-dimethylbenz[a]anthracene (DMBA), three times a week for 32 weeks. The same pouches of group 2 were subjected to the same DMBA painting; but at the same time, the animals received 10 mg/daily Spirulina platensis extract for the same period. In group 3, the same regimen of DMBA painting was done but for 24 weeks only and the daily systemically S. platensis was received for the 32 weeks. In group 4, neither DMBA painting nor S. platensis administration was done but pouches were painted with saline and served as a control one. Five rats from each group were sacrificed at 12, 24, 28, and 32 weeks, respectively. The required pouches were excised, fixed, and embedded in paraffin to be immunostained with proliferating cell nuclear antigen (PCNA). The results showed that increased PCNA expression was directly related to the severity of pathological alterations from normal epithelium to dysplasia and from dysplasia to squamous cell carcinoma (SCC) in the study groups at the different extended periods of DMBA application and S. platensis extract administration. Analysis of variance and Duncan's multiple-range test for PCNA labeling index were proved a high significant difference (P < 0.01) between the different groups. From the previous results, it can be concluded that S. platensis extract has a beneficial role in regression of cancer progression.

Dual roles of cyp19a1a in gonadal sex differentiation and development in the protandrous black porgy, Acanthopagrus schlegeli.

Protandrous black porgy fish, Acanthopagrus schlegeli, have a striking life cycle, with male sex differentiation at the juvenile stage, a bisexual gonad during first 2 yr of life, and a male-to-female sex change (with vitellogenic oocytes) at 3 yr of age. The present study investigated the role of aromatase (cyp19a1a/Cyp19a1a) in gonadal development in this species, especially in relation to sexual differentiation and sex change. Fish of various ages were treated with estradiol (E2) or aromatase inhibitor (AI) to determine whether manipulation of the hormonal environment has an impact on these processes. We report an integrative immunohistochemical, cellular, and molecular data set describing these interesting phenomena. During male sex differentiation, high levels of cyp19a1a/Cyp19a1a expression were observed in the undifferentiated gonad (4 mo of age), in marked contrast to the low cyp19a1a/Cyp19a1a levels detected in the differentiated testis at the age of 5-6 mo. A low dose of E2 (0.25 mg/kg feed) stimulated testicular growth and function in sexually differentiated fish, whereas a high dose of E2 (6 mg/kg feed) induced female development. Furthermore, administration of AI suppressed male development and promoted female sexual differentiation. An increased number of figla transcripts (an oocyte-specific gene) were observed prior to cyp19a1a expression, concomitant with the development of oogonia and early primary oocytes in the ovaries of both E2- and AI-treated groups. Immunohistochemical Pcna staining showed that the regression of testicular tissue occurred prior to the development of ovarian tissue in both E2- and AI-induced females. The importance of cyp19a1a in female development was further demonstrated by the increase in cyp19a1a transcripts during the naturally occurring sex change. Transcripts of foxl2 increased in the gonads of 2- to 3-yr-old black porgy during the early stages of the natural sex change, followed by a gradual elevation of cyp19a1a levels. The levels of both genes peaked in the resulting ovarian tissue. Thus, cyp19a1a/Cyp19a1a plays dual roles in the gonadal development, namely, in testicular development during the initial period of sexual differentiation and later in ovarian development during the natural sex change.

[Relationship of TCM syndrome type of gastric mucosal epithelial growth factor, vascular endothelial growth factor and proliferative cell nuclear antigen in patients with chronic atrophic gastritis].

OBJECTIVE: To study the relationship of TCM syndrome type of gastric mucosal epithelial growth factor (EGF), vascular endothelial growth factor (VEGF) and proliferative cell nuclear antigen (PCNA) in patients with chronic atrophic gastritis (CAG) for exploring the essence of TCM type and providing a theoretical basis of clinical treatment. METHODS: TCM syndrome type of 200 patients with diagnosis of CAG confirmed by fibro-gastroscope and pathological examination were differentially classified, and the expressions of EGF, VEGF and PCNA in different types were determined using immunohistochemistry. RESULTS: Patients were differentiated as Pi-Wei deficiency type (Type I ) in 72; Gan-Wei disharmony type (Type II ) in 43; Pi-deficiency with qi stagnation type (Type III) in 32; Wei-yin deficiency type (Type IV) in 24; Pi-Wei damp-heat type (Type V) in 14; and Wei-collateral stasis obstruction type (Type VI) in 5. The difference of PCNA expression level between Type II with Type I , III and IV was significant (P < 0.05). No significant difference in expression levels of EGF and VEGF was found among the 6 types (P > 0.05). CONCLUSION: Type I and II were the dominant TCM syndrome types in CAG patients; the high expression of PCNA might be a diagnostic evidence for Gan-Wei disharmony syndrome.

Inhibition of mouse osteoblast proliferation and prostaglandin E2 synthesis by Ulmus davidiana Planch (Ulmaceae).

Ulmus davidiana Planch (Ulmaceae) (UD) is a widely used Korean herbal medicine that has been used historically in anti-inflammatory and anticancer therapy. Since UD has been known to have anti-inflammatory and protective effects on damaged tissue, inflammation and bone among other functions, this study was undertaken to address whether the water extract of the bark of UD could modulate proliferation of mouse osteoblasts in vitro and to investigate its effect on cyclooxygenase-2 (COX-2), which converts arachidonic acid to prostaglandin E2 (PGE2). Mouse osteoblasts were tested in vitro for growth inhibition, proliferating cell nuclear antigen (PCNA) expression, and COX-2 activity and expression after treatment with UD extract. Its effects were compared with those of indomethacin (a nonselective COX inhibitor) and celecoxib (a selective COX-2 inhibitor). UD demonstrated a strong growth inhibition in tested mouse osteoblasts. The IC50s were 10microg/ml for UD, 6microM for celecoxib and 42microM for indomethacin. UD, as well as celecoxib and indomethacin, suppressed PCNA expression and PGE2 synthesis in osteoblasts. UD inhibited COX-2 expression, whereas celecoxib inhibited COX-2 activity directly. UD selectively and effectively inhibits osteoblasts cell growth in vitro. Inhibition of PGE2 synthesis via suppression of COX-2 expression may be responsible for its anti-inflammatory activity.