Percentage Of PCNA-positive Cells Research Articles

Mechanisms of electroacupuncture at "Zusanli"(ST36) in delaying colon "inflammation-cancer transformation"

To investigate the mechanism of electroacupuncture (EA) "Zusanli" (ST36) in delaying colon "inflammation-cancer transformation" in mice by anti-inflammatory. C57BL/6J mice were randomly divided into normal, model and EA groups, with 12 mice in each group. The mouse model of colorectal cancer (CRC) was established by intrape-ritoneal injection of azomethane (AOM) and feeding dextran sodium sulfate (DSS). At the beginning of the 2nd cycle, EA was applied to bilateral ST36 for 30 min once every other day for 12 times. The number of colon tumors in each group was observed, and the weight and length of colon were recorded. The contents of interleukin (IL)-1β, IL-6, IL-17A, IL-23, tumor necrosis factor (TNF)-α and CXC chemokine ligand 1 (CXCL1) of serum and colon tissue were detected by MSD multifactorial assay.The apoptosis of local cells in colon tumor was observed by TUNEL staining. Cell proliferation in colon tumor was observed by immunohistochemistry. Compared with the normal group, the colon length was significantly shortened (P<0.05) and the colon mass was significantly increased (P<0.001) in the model group, the contents of IL-1β, IL-6, TNF-α, IL-17A and CXCL1 of serum and colon tissue were significantly increased (P<0.05, P<0.01, P<0.001), and the content of IL-23 was increased in colon tissue (P<0.05) in the model group. Compared with the model group, the colon mass was decreased (P<0.05) and the contents of IL-1β, IL-6, TNF-α and IL-17A in serum were decreased (P<0.05), while the contents of IL-17A, CXCL1 and IL-23 in colon tissue were decreased (P<0.05) in the EA group, the percentage of local apoptotic cells in the EA group was increased (P<0.001), the percentage of PCNA positive cells was decreased (P<0.001), the number of tumors and the tumor volume were significantly decreased (P<0.01, P<0.05). The contents of IL-6, TNF-α, IL-17A and IL-23 in serum of CRC mice were positively correlated with tumor burden (P<0.05).The contents of IL-1β, TNF-α, CXCL1 and IL-23 in colon tissue of CRC mice were positively correlated with tumor burden (P<0.05). Electroacupuncture at ST36 can inhibit the inflammatory response of AOM/DSS inflammatory associated CRC mice and delay the "inflammation-cancer transformation" of colon.

Hepatocyte Growth Factor Overexpression Slows the Progression of 4NQO-Induced Oral Tumorigenesis.

ObjectivesTo investigate the role of hepatocyte growth factor (HGF)/c-Met signaling in oral malignant transformation.MethodsWe used immunohistochemistry to investigate HGF and c-Met expression in 53 oral squamous cell carcinoma (OSCC) specimens and 21 adjacent nontumor specimens and evaluated the associations between HGF and c-Met expression and clinicopathological parameters. Additionally, HGF-overexpression transgenic (HGF-Tg) and wild-type (Wt) mice were treated with 4-nitroquinoline-1-oxide (4NQO) to induce oral carcinogenesis for 16 weeks. At 16, 20, and 24 weeks, tongue lesions were collected for clinical observation; estimation of HGF, c-Met, and PCNA expression; apoptosis (TUNEL) assays; and RNA sequencing (RNA-seq).ResultsHGF and c-Met were positively expressed in 92.5% and 64% of OSCC samples, respectively. High HGF expression was significantly associated with smaller tumor size (p = 0.006) and inferior TNM stage (p = 0.032). No correlation between HGF and c-Met levels and other clinical parameters or prognosis was noted. In addition, HGF and c-Met expression was elevated in 4NQO-induced lesions of Wt mice. Compared with Wt mice, HGF-Tg mice have lower tumor incidence, number, volume, and lesion grade. In addition, the percentage of PCNA-positive cells in Wt mice was significantly higher than that in HGF-Tg mice at different time points. At 16 weeks, HGF-Tg mice exhibited less apoptotic cells compared with Wt mice (p < 0.000), and these levels gradually increased until the levels were greater than that of Wt mice at 24 weeks (p < 0.000). RNA-seq data revealed that 140 genes were upregulated and 137 genes were downregulated in HGF-Tg mice. KEGG enrichment analysis showed that upregulated differentially expressed genes (DEGs) are highly correlated with oxidative and metabolic signaling and that downregulated DEGs are related to MAPK and PI3K-AKT signaling.ConclusionsHGF and c-Met expression is upregulated in OSCC tissues and is associated with the occurrence and development of OSCC. HGF overexpression in normal oral epithelial tissue can inhibit 4NQO-induced tumorigenesis potentially through inhibiting proliferation and accelerating apoptosis via MAPK and PI3K-AKT signaling.

The inhibitory influence of toluene on mare ovarian granulosa cells can be prevented by fennel

The influence of environmental contaminant toluene and of plant fennel (Foeniculum vulgare Mill.) on reproduction are reported, but the mechanisms of their action and the protective effect of fennel on contaminant influence remain to be elucidated. In this study, we hypothesized that toluene and fennel directly affects basic ovarian cell functions, and that fennel can be used as an appropriate natural protective agent against the potential adverse effects of toluene. This study aimed to examine the action of toluene (20 μg/mL) and fennel extract (0, 1, 10, 100 μg/mL), and assess their combination on viability, proliferation, apoptosis, and hormone release by cultured healthy mare ovarian granulosa cells. Viability, proliferation (percentage of PCNA-positive cells), apoptosis and release of progesterone, oxytocin and prostaglandin F were evaluated by using Trypan blue exclusion tests, immunocytochemistry and enzyme immunoassays, respectively. Toluene, when given alone, inhibited viability, proliferation, apoptosis, progesterone, prostaglandin F and IGF-I. However, it did not affect oxytocin release. Moreover, Fennel, when given alone, inhibited viability, progesterone, and prostaglandin F release, as well as stimulating proliferation and oxytocin release. In addition, Fennel did not affect apoptosis. When given in combination with toluene, fennel was able to suppress, and even invert, the effects of toluene on viability, proliferation, apoptosis, prostaglandin F, and IGF-I. However, it did not alter its effect on progesterone release. Moreover, fennel induced the inhibitory effect of toluene on oxytocin output. The findings of our study suggest direct adverse effects of toluene on the basic ovarian functions of mares. Lastly, we also observed the direct influence of fennel on these functions, as well as its ability to be a natural protector against the action of toluene on the ovarian functions of mares.

Resveratrol prevented experimental pulmonary vascular remodeling via miR-638 regulating NR4A3/cyclin D1 pathway

ObjectiveResveratrol has shown benefit for pulmonary hypertension improvement. Our previous reports showed NR4A3/cyclin D1 pathway promoted pulmonary arterial smooth muscle cells (PASMCs) proliferation. This study tried to explore the mechanism underlying this process, focusing on the role of resveratrol in regulation of miRNA and NR4A3. MethodsRats were injected with monocrotaline (MCT) to establish pulmonary hypertension (PH) models. Resveratrol was used to prevent pulmonary vascular remodeling. Primary rat PASMCs were cultured in vitro and stimulated by platelet-derived growth factor (PDGF) with or without resveratrol. Cells proliferation and expression of miR-638 as well as NR4A3 were evaluated. ResultsMCT resulted in significant pulmonary vascular remodeling and down-regulation of miR-638, which could be suppressed by resveratrol. Moreover, PDGF-induced PASMC proliferation and miR-638 down-regulation were both significantly prevented by resveratrol treatment in vitro. MiR-638 mimics markedly inhibited PASMC proliferation and percentage of PCNA-positive cells in vitro. But anti-miR-638 could markedly promote cells proliferation and percentage of PCNA-positive cells. The luciferase reporter assay showed that NR4A3 was a direct target of miR-638. The loss-of-function and gain-of-function experiments indicated that NR4A3 promoted proliferation via cyclin D1 pathway. ConclusionOur data indicated that resveratrol prevented MCT-induced pulmonary vascular remodeling via miR-638 regulating NR4A3/cyclin D1 pathway.

ZHX2 inhibits proliferation and promotes apoptosis of human lung cancer cells through targeting p38MAPK pathway.

To investigate the effect of ZHX2 on lung cancer cells proliferation and apoptosis. The mRNA and protein expression of ZHX2 were detected by qRT-PCR and western blot, respectively. The human lung cancer cells were divided into Control, NC, ZHX2, SB, and ZHX2 + Ani groups. The cell proliferation was detected by CCK-8 assay and the cell migration and invasion were detected by Transwell assay. Cell apoptosis was detected by flow cytometry. Apoptosis and p38MAPK signaling pathway related proteins were detected by western blot. The nude mice model of lung cancer xenograft was constructed. The tumor volume and tumor weight were measured. The expression of PCNA protein in tumor tissues was detected by immunohistochemistry. The apoptosis of tumor cells was detected by TUNEL staining. The ZHX2 and p38MAPK signaling pathway related proteins in tumor tissues were detected by western blot. The expression of ZHX2 gene and protein in the cancer cell lines were significantly decreased. Compared with control and NC groups, the cells proliferation, migration and invasion were inhibited in ZHX2 and SB groups, while the apoptosis and apoptosis related proteins were increased (p< 0.05). Meanwhile, compared with ZHX2 group, the tumor growth rate, volume, weight, the percentage of PCNA-positive cells, and p-P38 MAPK/P38 MAPK were increased significantly in ZHX2 + Ani group, while the apoptotic index and the expression of MMP-9 protein were significantly decreased (p< 0.05). ZHX2 could inhibit proliferation and promote apoptosis of lung cancer cells by inhibiting p38MAPK signaling pathway.

Determination of the changes on the small intestine of pregnant mice by histological, enzyme histochemical, and immunohistochemical methods.

The aim of the present study was to determine the changes on the small intestine in mice during pregnancy using histological, enzyme histochemical, and immunohistochemical methods. A total of 24 Swiss albino female mice were divided as non-pregnant/control, first week, second week, and third week of pregnancy (n=6). Tissue samples obtained from the duodenum, jejunum, and ileum were processed by means of routine histological techniques and stained with Crossmon's triple staining. Alkaline phosphatase (ALP) was demonstrated with the simultaneous azo-coupling method. Proliferating cell nuclear antigen (PCNA) was demonstrated with the streptavidin-biotin-peroxidase complex method. The numerical data of the parameters were obtained and analyzed statistically. Villus height, villus width, and the rate of villus height/crypt depth were decreased in the duodenum, jejunum, and ileum in the last week of pregnancy compared with the control group. Changes in the crypt depth of the duodenum, jejunum, and ileum in pregnancy were found. The muscle width increased in pregnancy. It was identified that the ALP reactivity statistically significantly increased in the duodenum, jejunum, and ileum in pregnancy. The percentage of PCNA-positive cells in the duodenum, jejunum, and ileum increased in the first and second weeks of pregnancy, whereas it decreased in the third week of pregnancy compared with non-pregnant control animals. In conclusion, villus parameters, ALP reactivity, and percentage of PCNA-positive cells in the small intestine were affected during pregnancy.

Effect of decreased activity of 5’-adenosine monophosphate activated protein kinase on breast development in adolescent mice

Objective To investigate the effects of decreased 5’-activated protein kinase (AMPK) activity in mouse mammary epithelium on the proliferation of ductal epithelial cells and the development of teminal end bud (TEB) in adolescent mice. Methods AMPK DN transgenic mice were successfully constructed. The fourth pair of mammary gland tissues from the right abdomen of 10 WT and DN mice were stained by whole tissue staining and PCNA immunohistochemical staining. The duct coverage area, initial duct length, number of ductal branches per millimeter, percentage of ductal invasion length, terminal tip of mammary gland tissues of WT and DN mice were analyzed. TEB average quantity, average maximum area of TEB. The ratio of PCNA positive cells to the total number of glandular epithelial cells was calculated. Results At 5th week, the number of ductal branches per millimeter in DN mice and WT mice was (1.39±0.18) mm vs. (1.21±0.11) mm, the average number of TEB was (13.99±3.14) vs. (10.65±2.09) and the average maximum area of TEB was (0.104±0.014) mm2 vs. (0.082±0.013) mm2, respectively, with significant difference (t=2.698, 2.800, 3.642, P<0.05); at 6th week, the number of ductal branches per millimeter in DN mice and WT mice was (1.59±0.013) mm2, respectively. 31) vs. (1.41±0.34) mm, the average number of TEB (15.86±3.26) vs. (12.47±2.34), the average maximum area of TEB (0.118±0.017) mm2vs. (0.087±0.014) mm2, the difference was statistically significant (t=2.406, 2.671, 4.451, P<0.05). The percentage of PCNA positive cells in mammary epithelial cells of 5 w DN mice (86.47±4.67) was significantly higher than that of WT mice (65.32±3.87)%, and 6 w DN mice (65.32±3.87)%. The percentage of cells in WT group was significantly higher than that in WT group (85.37±4.35) (63.47±3.45)%. The difference was statistically significant (t=4.614, 12.474, P< 0.05). Conclusion The decrease of AMPK activity in mouse mammary epithelium can promote the proliferation of ductal epithelial cells and the development of TEBs in adolescent mice. Key words: Mouse mammary epithelial cells; Adolescence; 5’-activated protein kinase; Proliferation; Teminal end bud

Influence of green tea constituents on cultured porcine luteinized granulosa cell functions

The aim of the study was to examine the action of green tea polyphenols (GTPP) and their main constituent, epigallocatechin-3-gallate (EGCG), on porcine ovarian granulosa cells. For this purpose, the effect of GTPP and EGCG on cultured porcine ovarian granulosa cell functions, including proliferation, apoptosis, steroidogenesis and response to insulin-like growth factor I (IGF-I) was examined. Proliferation (the accumulation of proliferating cell nuclear antigen (PCNA) and cyclin B1), apoptosis (the accumulation of bax and caspase 3) and the release of steroid hormones (progesterone and testosterone) were evaluated by using immunocytochemistry and enzyme immunoassay. The addition of both GTPP and EGCG reduced the percentage of both PCNA- and cyclin B1-positive cells, increased the proportion of cells containing bax and caspase 3 and stimulated progesterone release. GTPP had a biphasic effect on testosterone output – stimulating at dose 1 μg/ml and inhibiting at doses 10 and 100 μg/ml, whilst EGCG did not affect testosterone secretion. IGF-I, when administered alone, promoted % of cells containing PCNA, suppressed bax accumulation, and stimulated progesterone release (only at dose 100 ng/ml). Testosterone release increased after the addition of IGF-I at 1 ng/ml, but decreased after IGF-I addition at 10 or 100 ng/ml. Both GTPP and EGCG suppressed or even reversed the effects of IGF-I on percentage of PCNA-positive cells, bax, testosterone output, and promoted IGF-I action on progesterone release. These observations suggested the inhibitory actions of green tea constituents on porcine ovarian granulosa cell functions were mediated through various regulatory mechanisms: suppression of the ovarian cell cycle in the S and G2 phases; promotion of cytoplasmic apoptosis; alteration of steroid hormone release; and, predominantly, prevention of the action of the hormonal stimulator IGF-I on ovarian cells. It can be also suggested that the major GTPP effects may result from the presence of EGCG.

Comparative evaluation of hesperetin loaded nanoparticles for anticancer activity against C6 glioma cancer cells

The aim of this study was to evaluate anti-cancer properties of hesperetin (Hsp) and hesperetin-loaded poly(lactic-co-glycolic acid) nanoparticles (HspNPs) for glioblastoma treatment. Nanoparticles prepared by single emulsion method had a size of less than 300 nm with 70.7 ± 3.9% reaction yield and 26.4 ± 1.1% Hsp loading capacity. Treatment of C6 glioma cells with HspNPs for 24 and 48 h resulted in dose- and time-dependent decrease in cell viability, with approximate IC50 of 28 and 21 μg/mL, respectively (p = .036 for 24 h, p = .025 for 48 h). The percentage of PCNA positive cells decreased to 20% and 10%, respectively, for Hsp- and HspNP-treated cells at concentration of 100 µg/mL. Treatment with increasing concentrations of HspNPs (25, 50, 75 and 100 µg/mL) resulted in 9.1-, 7-, 12.5- and 12.7-fold in increase in apoptotic cell number. Optimum doses of Hsp and HspNPs were found to increase oxidative damage in C6 glioma cells. MDA levels, an indicator of lipid peroxidation, were found to be significantly elevated at 75 and 100 µg/mL exposure concentration of HspNPs with (p = .002) and (p = .018), respectively for 48-h treatment. The results obtained with this study showed biocompatible polymeric nanoparticle systems has great advantages to enhance anti-cancer activity and poor solubility of therapeutic agents. Overall our findings suggest that Hsp-loaded PLGA nanoparticle systems showed significant anti-cancer activity and HspNPs could be used as promising novel anti-cancer agent.

The Effects and Mechanism of Atorvastatin on Pulmonary Hypertension Due to Left Heart Disease.

BackgroundPulmonary hypertension due to left heart disease (PH-LHD) is one of the most common forms of PH, termed group 2 PH. Atorvastatin exerts beneficial effects on the structural remodeling of the lung in ischemic heart failure. However, few studies have investigated the effects of atorvastatin on PH due to left heart failure induced by overload.MethodsGroup 2 PH was induced in animals by aortic banding. Rats (n = 20) were randomly divided into four groups: a control group (C), an aortic banding group (AOB63), an atorvastatin prevention group (AOB63/ATOR63) and an atorvastatin reversal group (AOB63/ATOR50-63). Atorvastatin was administered for 63 days after banding to the rats in the AOB63/ATOR63 group and from days 50 to 63 to the rats in the AOB63/ATOR50-63 group.ResultsCompared with the controls, significant increases in the mean pulmonary arterial pressure, pulmonary arteriolar medial thickening, biventricular cardiac hypertrophy, wet and dry weights of the right middle lung, percentage of PCNA-positive vascular smooth muscle cells, inflammatory infiltration and expression of RhoA and Rho-kinase II were observed in the AOB63 group, and these changes concomitant with significant decreases in the percentage of TUNEL-positive vascular smooth muscle cells. Treatment of the rats in the AOB63/ATOR63 group with atorvastatin at a dose of 10 mg/kg/day significantly decreased the mean pulmonary arterial pressure, right ventricular hypertrophy, pulmonary arteriolar medial thickness, inflammatory infiltration, percentage of PCNA-positive cells and pulmonary expression of RhoA and Rho-kinase II and significantly augmented the percentage of TUNEL-positive cells compared with the AOB63 group. However, only a trend of improvement in pulmonary vascular remodeling was detected in the AOB63/ATOR50-63 group.ConclusionsAtorvastatin prevents pulmonary vascular remodeling in the PH-LHD model by down-regulating the expression of RhoA/Rho kinase, by inhibiting the proliferation and increasing the apoptosis of pulmonary arterial smooth muscle cells, and by attenuating the inflammation of pulmonary arteries.

Developmental changes in intercellular junctions and Kv channels in the intestine of piglets during the suckling and post-weaning periods.

BackgroundThe intestinal epithelium is an important barrier that depends on a complex mixture of proteins and these proteins comprise different intercellular junctions. The purpose of this study was to investigate the postnatal and developmental changes in morphology, intercellular junctions and voltage-gated potassium (Kv) channels in the intestine of piglets during the suckling and post-weaning periods.ResultsSamples of the small intestine were obtained from 1-, 7-, 14-, and 21-d-old suckling piglets and piglets on d 1, 3, 5, and 7 after weaning at 14 d of age. The results showed that the percentage of proliferating cell nuclear antigen (PCNA)-positive cells and alkaline phosphatase (AKP) activity, as well as the abundances of E-cadherin, occludin, and Kv1.5 mRNA and claudin-1, claudin-3, and occludin protein in the jejunum were increased from d 1 to d 21 during the suckling period (P < 0.05). Weaning induced decreases in the percentage of PCNA-positive cells, AKP activity and the abundances of E-cadherin, occludin and zonula occludens (ZO)-1 mRNA or protein in the jejunum on d 1, 3 and 5 post-weaning (P < 0.05). There were lower abundances of E-cadherin, occludin and ZO-1 mRNA as well as claudin-1, claudin-3 and ZO-1 protein in the jejunum of weanling piglets than in 21-d-old suckling piglets (P < 0.05). The abundances of E-cadherin, occludin, ZO-1 and integrin mRNA were positively related to the percentage of PCNA-positive cells.ConclusionWeaning at 14 d of age induced damage to the intestinal morphology and barrier. While there was an adaptive restoration on d 7 post-weaning, the measured values did not return to the pre-weaning levels, which reflected the impairment of intercellular junctions and Kv channels.

In vivo inhibition followed by exogenous supplementation demonstrates galactopoietic effects of prolactin on mammary tissue and milk production in dairy cows

It has been previously shown that the long-term inhibition of milking-induced prolactin (PRL) release by quinagolide (QN), a dopamine agonist, reduces milk yield in dairy cows. To further demonstrate that PRL is galactopoietic in cows, we performed a short-term experiment that used PRL injections to restore the release of PRL at milking in QN-treated cows. Nine Holstein cows were assigned to treatments during three 5-d periods in a 3×3 Latin square design: 1) QN: twice-daily i.m. injections of 1mg of QN; 2) QN-PRL: twice-daily i.m. injections of 1mg of QN and twice-daily (at milking time) i.v. injections of PRL (2µg/kg body weight); and 3) control: twice-daily injections of the vehicles. Mammary epithelial cells (MEC) were purified from milk so that their viability could be assessed, and mammary biopsies were harvested for immunohistological analyses of cell proliferation using PCNA and STAT5 staining. In both milk-purified MEC and mammary tissue, the mRNA levels of milk proteins and BAX were determined using real-time reverse-transcription PCR. Daily QN injections reduced milking-induced PRL release. The area under the PRL curve was similar in the control and PRL injection treatments, but the shape was different. The QN treatment decreased milk, lactose, protein, and casein production. Injections of PRL did not restore milk yield but tended to increase milk protein yield. In mammary tissue, the percentage of STAT5-positive cells was reduced during QN but not during QN-PRL in comparison with the control treatment. The percentage of PCNA-positive cells was greater during QN-PRL injections than during the control or QN treatment and tended to be lower during QN than during the control treatment. In milk-purified MEC, κ-casein and α-lactalbumin mRNA levels were lower during QN than during the control treatment, but during QN-PRL, they were not different from the control treatment. In mammary tissue, the BAX mRNA level was lower during QN-PRL than during QN. The number of MEC exfoliated into milk was increased by QN injections but tended to be decreased by PRL injections. Injections of PRL also increased the viability of MEC harvested from milk. Although PRL injections at milking could not reverse the effect of QN treatment on milk production, their effects on cell survival and exfoliation and on gene expression suggest that the effect of QN treatment on the mammary gland is due to QN’s inhibition of PRL secretion.

Quercetin Induces Dose-Dependent Differential Morphological and Proliferative Changes in Rat Uteri in the Presence and in the Absence of Estrogen.

Quercetin could have profound effects on uterine morphology and proliferation, which are known to be influenced by estrogen. This study investigated the effect of quercetin on these uterine parameters in the presence and in the absence of estrogen. Ovariectomized adult female rats received peanut oil, quercetin (10, 50, and 100 mg/kg/day), estrogen, or estrogen+quercetin (10, 50, or 100 mg/kg/day) treatment for 7 consecutive days. At the end of the treatment, uteri were harvested for histological and molecular biological analyses. Distribution of proliferative cell nuclear antigen (PCNA) protein in the uterus was observed by immunohistochemistry. Levels of expression of PCNA protein and mRNA in uterine tissue homogenates were determined by Western blotting and real-time polymerase chain reaction, respectively. Our findings indicated that administration of 10 mg/kg/day of quercetin either alone or with estrogen resulted in decreased uterine expression of PCNA protein and mRNA with the percentage of PCNA-positive cells in uterine luminal and glandular epithelia markedly reduced compared with estrogen-only treatment. Changes in uterine morphology were the opposite of changes observed following estrogen treatment. Treatment with 100 mg/kg/day of quercetin either alone or with estrogen resulted in elevated PCNA protein and mRNA expression. In addition, the percentages of PCNA-positive cells in the epithelia, which line the lumen and glands, were increased with morphological features mimicking changes that occur following estrogen treatment. Following 50 mg/kg/day quercetin treatment, the changes observed were in between those changes that occur following 10 and 100 mg/kg/day quercetin treatment. In conclusion, changes in uterine morphology and proliferation following 10 mg/kg/day quercetin treatment could be attributed to quercetin's antiestrogenic properties, while changes that occur following 100 mg/kg/day quercetin treatment could be attributed to quercetin's estrogenic properties.

Role of Monochromatic Light on Development of Cecal Tonsil in Young Broilers

Previously, the different monochromatic lights have been demonstrated to affect splenocyte proliferation and melatonin (MEL) secretion in broilers. The present study was designed to evaluate the effects of different monochromatic lights on the development and immune function of broiler cecal tonsils, and to disclose the mechanisms underlying these phenomena. A total of 185 broilers (P0) including intact, sham-operated, and pinealectomized groups were exposed to blue light (BL), green light (GL), red light (RL), and white light (WL) by a light-emitting diode system for 14 days. Compared with RL groups, the GL in the intact and sham-operated groups showed larger follicle areas (66.70%), higher percentages of proliferating cell nuclear antigen (PCNA)-positive cells (33.33%), increased numbers of IgA(+) cells (48.60%), and increased antioxidase activity (33.33%-61.37%), whereas, the density of iNOS and MDA content in GL were lower (43.63%-54.43%) than that of RL. In contrast, after pinealectomy, the area of follicles, the percentage of PCNA-positive cells, the number of IgA(+) cells, and the antioxidase activity decreased in the different light treatments, but the density of iNOS and MDA content increased substantially. There was no significant difference in these parameters between broilers exposed to GL and other lights (P = 0.085-1.000). The results suggested that the enhanced effects of GL on the development and immune function of cecal tonsils in young broilers were mediated by elevated antioxidative status via up-regulation of MEL.

Exposure to ethanol and tobacco smoke in relation to level of PCNA antigen expression in pancreatic and hepatic rat cells

BackgroundPrevious results proved that simultaneous effect of tobacco smoke constituents and alcohol consumption may change toxicity of these substances and have a greater effect on hepatic and pancreatic disease and cancer risk. The aim of this study was to investigate hepatocyte and pancreatic cells regeneration after tobacco and/or ethanol treatment. MethodsIn the study, four groups of rats were used – alcohol non-addicted and addicted male and female rats. The animals from each group were exposed to tobacco smoke, to ethanol or tobacco smoke and ethanol. After the exposure, pancreas and liver were collected at two time-points - 5 and 24h. Biochemical methods were used to measure concentration of ethanol and cotinine in blood and plasma. Additionally, proliferating cell nuclear antigen labeling index (PCNA-LI), an S-phase marker was assessed by immunohistochemical staining and morphometric method. ResultsOur experimental results showed that the exposure of rats to tobacco smoke does not have influence on ethanol concentration in blood of non-addicted (male, female) and addicted (male and female) animals. The results also proved that alcohol addiction did not influence nicotine metabolism in all animals exposed to tobacco smoke. Morphological studies of tissues display significant damage in liver of addicted males, including fatty degradation, fibrosis and slight inflammatory infiltrate. Immunohistochemical studies revealed at first, significant increase of PCNA-LI and, thus, increased cell proliferation activity and damage in tissues were observed in hepatic and pancreatic cells of addicted males when compared with non-addicted males. Secondly, comparison between addicted males and addicted females revealed that PCNA-LI in females is significantly lower, both in hepatic and pancreatic tissues. And finally, animals exposed only to ethanol and to tobacco smoke plus ethanol were characterized by higher percentage of PCNA positive cells in relation to animals exposed only to tobacco smoke. ConclusionFrom the preliminary study one can conclude that the influence of ethanol and simultaneous influence of ethanol and tobacco smoke impairs liver and pancreatic functions to a greater degree than tobacco abuse.

Ethyl acetate extraction from a Chinese herbal formula, Jiedu Xiaozheng Yin, inhibits the proliferation of hepatocellular carcinoma cells via induction of G0/G1 phase arrest in vivo and in vitro

Jiedu Xiaozheng Yin (JXY), a polyherbal formula of traditional Chinese medicine (TCM), has been used to treat various kinds of cancer in China. However, the mechanism of its anticancer activity has yet to be elucidated. Air-dried herbs were extracted with reagents of different polarity. HepG2 cells were treated with different doses of ethyl acetate extract (EE-JXY) and chloroform extract (CE-JXY) for 24 h. Cell viability was detected by MTT assay. Colony formation ability was also evaluated. Cell cycle was evaluated by FACS. Tumor bearing BALB/c nude mice was treated with EE-JXY (0.06 g/kg) for 20 days. Tumor volume and weight were monitored. The percentage of PCNA-positive cells and the level of G1 phase proteins [cyclin-dependent kinase2 (CDK2), cyclin‑dependent kinase4 (CDK4), cyclin D and cyclin E and G2 phase proteins [cyclin-dependent kinase1 (CDK1), cyclin A and cyclin B] were detected by immunohistochemistry and western blotting. EE-JXY and CE-JXY dose-dependently inhibited the growth of HepG2 cells (P<0.01 for both). Furthermore, EE-JXY inhibited the formation of cell colonies and blocked the cell cycle to G1 phase in a dose-dependent manner (P<0.01 for all). EE-JXY showed an obviously antitumor effect in vivo (P<0.05). Further investigation showed that EE-JXY decreased the proliferation index of tumors (P<0.01) through increasing the expression of G1-related proteins (cyclin D and cyclin E, P<0.05 and P<0.01). These results suggested that JXY inhibits the growth of HepG2 cells at least via arresting the cell cycle at the G0/G1 phase.

Body Weight, Intestinal Morphometry and Cell Proliferation of Broiler Chickens Submitted to Cyclic Heat Stress

To investigate the effects of heat stress on body weight, intestinal length, mucous area, crypt’s depth, villus height and percentage of cells in proliferation activity in male broiler chickens, one hundred birds of Avian Farms strain were housed in cages and divided into two groups, the reared under heat stress and in thermoneutrality. The group reared under heat stress was submitted daily, from 12-13 h, to 38 C from first o to 27th day old and 40 C from 28 to 42nd day. The group reared in thermoneutrality was maintained for 24 o h in comfort temperature. The body weight of ten birds of each group was obtained weekly from the first until 42nd day old. Each week, five birds from each group were euthanased by cervical dislocation to obtain intestinal length, the mucous area, crypt’s depth, villus height and percentage of cells in proliferation activity. A completely randomized design was used in a factorial schema 6 x 2 (six ages: one, seven, 14, 21, 28 and 42 days and two ambient temperatures: thermoneutral and heat stress). Data were analyzed by analysis of variance and means compared by Tukey test at 5%. The birds stressed by heat presented less crypt depth, mucous area and villus height of duodenum and lower intestinal length at 42 days old. However, heat stress did not influence the percentage of PCNA positive cells, the area of the mucosa, crypt’s depth and villus height in jejunum and ileum.

Comparison of Effects of Protein Kinase A, Mitogen-activated Protein Kinase, and Cyclin-dependent Kinase Blockers on Rabbit Ovarian Granulosa Cell Functions

The aim of the present study was to define the role of protein kinase A (PKA)-, mitogen-activated protein kinase (MAPK)-, and cyclin-dependent kinase (CDK)-dependent pathways in the control of ovarian cell functions. The effects of PKA, MAPK, and CDK blockers (KT 5720, PD 98059, and olomoucine, respectively), given at doses of 0.001-10.0 μg/ml medium on functions of cultured rabbit granulosa cells were examined. Expression of PKA, MAPK/ERK1,2, secretory activity (IGF-I output), and proliferation (proliferating cell nuclear antigen, PCNA) in these cells were determined by RIA, immunocytochemistry and Western blotting. A PKA inhibitor, KT 5720 suppressed the expression of PKA and MAPK/ERK1,2, the IGF-I release, and the ratio of PCNA-positive cells in granulosa cells. A MAPK blocker, PD 98059 reduced the expression of MAPK/ERK1,2 (but not PKA), the IGF-I release, and percentage of PCNA-positive cells. A CDK blocker, olomoucine, increased the PKA expression, decreased the expression of MAPK/ERK1,2 and PCNA, but did not affect the IGF-I release. These observations confirm the involvement of PKs in control of basic ovarian functions and demonstrate the involvement of PKA in stimulation of ovarian cell proliferation and MAPK (but not CDK) and in promotion of ovarian IGF-I release. Different activity and specificity of the PKA, MAPK, and CDK blockers in their effects on PCNA and IGF-I suggests different biological role of these PKs in control of proliferative and secretory functions of rabbit ovarian cells.

Abstract 3462: A DNA methyltransferase inhibitor and all-trans retinoic acid reduce oral cavity carcinogenesis induced by the carcinogen 4-nitroquinoline 1-oxide

Abstract The transcriptional silencing of some cell cycle inhibitors and tumor suppressors, such as p16 and RARβ2, by DNA hypermethylation at CpG islands is commonly found in human oral squamous carcinoma cells. We examined the effects of the DNA methyltransferase (Dnmt) inhibitor 5-Aza-2′-deoxycytidine (5-Aza, 0.25 mg/kg body weight), all-trans retinoic acid (RA, given at 100 μg/kg body weight and 1 mg/kg body weight, respectively), and the combination of 5-Aza and the low dose RA on murine oral cavity carcinogenesis induced by the carcinogen 4-nitroquinoline 1-oxide (4-NQO) in a mouse model. All the drug treatments were performed for 15 weeks after a 10-week 4-NQO treatment. Mice in all drug treatment groups showed decreases in the average numbers of neoplastic tongue lesions. The combination of 5-Aza and RA effectively attenuated tongue lesion severity. While all drug treatments limited the increase in the percentage of PCNA positive cells and the decrease in the percentage of p16 positive cells caused by the 4-NQO treatment in mouse tongue epithelial regions without visible lesions and in the neoplastic tongue lesions, the combination of 5-Aza and RA was the most effective. Collectively, our results show that the combination of a DNA demethylating drug and RA has potential as a strategy to reduce oral cavity cancer in this 4-NQO model. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3462.

Changes of protein kinase Cα and cyclin D1 expressions in pulmonary arteries from smokers with and without chronic obstructive pulmonary disease

The purpose of this study was to investigate the changes of protein kinase Calpha (PKCalpha) and cyclin D1 expressions in pulmonary arteries from smokers with normal lung function and smokers with mild to moderate chronic obstructive pulmonary disease (COPD). The peripheral lung tissues were obtained from 10 non-smokers with normal lung function (non-smoker group), 14 smokers with normal lung function (smoker group), 11 smokers with mild to moderate COPD (COPD group). The morphological changes of pulmonary arteries were observed by HE-staining. The expressions of alpha-smooth muscle actin (alpha-SMA), proliferating cell nuclear antigen (PCNA), PKCalpha and cyclin D1 proteins in pulmonary artery smooth muscle cells (PASMCs) were immunohistochemically determined. The percentages of PCNA-positive cells were taken as the smooth muscle cells proliferation index (PI). The mRNA expressions of PKCalpha and cyclin D1 in PASMCs were evaluated by real-time fluorescence PCR. Morphometrical analysis showed that the ratio of pulmonary artery wall area to total area (WA%) in smoker group and COPD group was significantly greater than that in non-smoker group (P<0.01). The PASMCs proliferation index in smoker group and COPD group was significantly higher than that in nonsmoker group (P<0.01). The protein levels of PKCalpha and cyclin D1 in PASMCs were significantly increased in smoker group and COPD group as compared with non-smoker group (P<0.01). The mRNA expressions of PKCalpha and cyclin D1 in PASMCs were significantly elevated in smoker group and COPD group as compared with non-smoker group (P<0.01). Significant correlations were found between PKCalpha protein and WA% or PI (P<0.01). Correlations between cyclin D1 protein and WA% or PI also existed (P<0.01). The expression of PKCalpha was positively correlated with the expression of cyclin D1 at both protein and mRNA levels (P<0.01). In conclusion, increased expressions of PKCalpha and cyclin D1 might be involved in the pathogenesis of abnormal proliferation of PASMCs in smokers with normal lung function and smokers with mild to moderate COPD.