Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling Research Articles

Xiayuxue Decoction ([symbols; see text]) attenuates hepatic stellate cell activation and sinusoidal endothelium defenestration in CCl4-induced fibrotic liver of mice.

To investigate the effects of ancient Chinese medical formula Xiayuxue Decoction ([symbols; see text], XYXD) on activation of hepatic stellate cells (HSCs) and defenestration of sinusoidal endothelial cells (SECs) in CCl4-induced fibrotic liver of mice. High performance liquid chromatography was used to identify the main components of XYXD and control the quality of extraction. C57BL/6 mice were induced liver fibrosis by CCl4 exposure and administered with XYXD for 6 weeks simultaneously. Liver tissue was investigated by hematoxylin-eosin and Sirius-red staining. Sinusoidal fenestrations were observed by scanning electronic microscopy and fluorescent immunohistochemistry of PECAM-1 (CD31). Whole liver lysates were detected of α-smooth muscle actin (α-SMA) and type-I collagen by Western blot. Primary rat HSCs-T6 cells were analyzed by detecting α-SMA, F-actin, DNA fragmentation through confocal microscopy, Western blot, terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) assay and cellomics arrayscan, respectively. Amygdalin and emodin in XYXD were identified. XYXD (993 mg/kg) inhibited Sirius red positive area up to 70.1% (P<0.01), as well as protein levels of α-SMA and type-I collagen by 42.0% and 18.5% (P<0.05) respectively. In vitro, XYXD (12.5 μg/mL, 50 μg/mL) suppressed the activation of HSCs and reversed the myofibroblastic HSCs into quiescent, demonstrated as inhibition of fluorescent F-actin by 32.3% and 46.6% (P<0.05). Besides, XYXD induced the apoptosis of HSC-T6 cells by 20.0% (P<0.05) and 49.5% (P<0.01), evidenced by enhanced TUNEL positivity. Moreover, ultrastructural observation suggested XYXD inhibited defenestration of SECs, which was confirmed by 31.1% reduction of protein level of CD31 (P<0.05). XYXD inhibited both HSCs activation and SECs defenestration which accompany chronic liver injuries. These data may help to understand the underlying mechanisms of XYXD for prevetion of chronic liver diseases.

Signal transducer and activator of transcription-3 inhibitor WP1066 affects human tongue squamous cell carcinoma proliferation and apoptosis in vitro and in vivo

To investigate the antitumour molecular mechanisms of WP1066 (STAT-3 inhibitor ) to human tongue squamous cell carcinoma in vitro and in vivo. WP1066 was used to inhibit the p-STAT-3 expression in Tscca human tongue squamous cell carcinoma cell line .Real-time PCR was used to detect the microRNA-21 expression after treatment with DMSO and WP1066. Methyl thiazolyl tatrozolium (MTT) assay was employed to determine cell survival. Flow cytometry (FCM) was used to measure apoptosis. The expression level of STAT-3/p-STAT-3, programmed cell death protein 4 (PDCD-4), antigen 67 (Ki-67), B cell lymphoma 2 (Bcl-2) and cleaved cysteinyl aspartate specific proteinase-3 (CCASP-3) was examined by Western blotting. Luciferase reporter gene assay was conducted to verify the regulation of STAT-3 to microRNA-21. Tscca xenograft tumor model was established in BALB/c nude mice and the tumors were divided into control, DMSO and WP1066 treated groups. The tumor tissues were measured by immunohistochemistry stain and terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) assay. STAT-3/p-STAT-3 protein was suppressed after treatment with WP1066 (STAT-3:F = 15.336, P = 0.004, p-STAT-3: F = 52.837, P = 0.000). MicroRNA-21 relative expression level was down-regulated (F = 8.197, P = 0.019). Cell survival rate was significantly reduced after treatment with WP1066 than control groups (F = 94.388, P = 0.000). Early apoptosis rate increased after treatment with WP1066 (F = 217.080, P = 0.000) . PDCD-4 and cleaved cysteinyl aspartate specific proteinase-3 (CCASP-3) protein expression was increased after treatment with WP1066 (PDCD-4:F = 8.771, P = 0.017; CCASP-3: F = 26.611, P = 0.001) .Ki-67 and Bcl-2 protein was down regulated (Ki-67:F = 5.854, P = 0.039; Bcl-2:F = 125.502, P = 0.000). Luciferase reporter gene assay proved that STAT-3 combined with specific promoter region of microRNA-21.In vivo, the tumor volume after treatment with WP1066 was significantly smaller than control groups by the end of observation (F = 15.390, P = 0.000) .Immunological histological chemistry indicated that PDCD-4 and CCASP-3 protein expression was up-regulated simultaneously while Ki-67 and Bcl-2 protein of tumor tissue was down-regulated after treatment with WP1066 than control groups. TUNEL assay suggested that apoptosis index rose after treatment with WP1066 than control groups (F = 133.368, P = 0.000) . WP1066 affected Tscca cancer cell proliferation and apoptosis via inhibiting STAT-3/microRNA-21.WP1066 provided new direction and possibility to human tongue squamous cell carcinoma therapy.

Induction of Heat-Shock Protein 70 Expression by Geranylgeranylacetone Shows Cytoprotective Effects in Cardiomyocytes of Mice under Humid Heat Stress

BackgroundIncreasing evidence has revealed that humid heat stress (HHS) causes considerable damage to human health. The cardiovascular system has been suggested to be the primary target of heat stress, which results in serious cardiovascular diseases. However, there is still a lack of effective approaches for the prevention and treatment of cardiovascular diseases induced by HHS.ObjectiveHeat-shock proteins (Hsps), especially Hsp70, are reported to provide effective cytoprotection under various stress stimuli. In the present study, we evaluated the cytoprotective effect of geranylgeranylacetone (GGA), which was previously been reported to induce Hsp70 expression in cardiomyocytes under HHS.Methods and Principal FindingsUsing a mouse model of HHS, we showed that the pretreatment of GGA enhanced Hsp70 expression under HHS, as examined by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. We then examined the effect of GGA pretreatment on the cardiomyocyte apoptosis induced by HHS using terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) staining, and found that GGA pretreatment inhibited mitochondria-mediated apoptosis. GGA pretreatment could reverse the effect of HHS on cell apoptosis by increasing expression of Bcl-2, decreasing cytochrome c in cytosol, and increasing cytochrome c in mitochondria. However, GGA pretreatment had no effect on the oxidative stress induced by HHS as determined by levels of superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione (GSH).ConclusionWe have demonstrated that GGA pretreatment suppressed HHS-induced apoptosis of cardiomyocytes through the induction of Hsp70 overexpression.

Role of autophagy and apoptosis in wound tissue of deep second-degree burn in rats.

The pathogenesis of burn wound progression is poorly understood. Contributing factors include continuous loss of blood perfusion, excessive inflammation, and elevated apoptosis levels in wound tissue. Macroautophagy (here referred to simply as "autophagy") is associated with many chronic diseases. The authors hypothesized that autophagy is involved in burn wound progression in a rat model of deep second-degree burn. Deep second-degree burns were modeled using a brass rod heated to 100°C applied for 6 seconds to the back skin of Wistar rats. Full-thickness biopsies were obtained from burned and nonburned controls at several times postburn. Western blotting and immunohistochemical (IHC) staining determined expression of the autophagy markers Light Chain 3 (LC3) and beclin-1. Apoptosis was determined by terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) assay and laser Doppler flowmetry (LDF)-measured tissue perfusion. Myeloperoxidase (MPO) activity assay measured inflammation. Hematoxylin and eosin (H&E) and Masson's trichrome staining-determined pathology and wound depth. The LC3 and beclin-1 protein level in burn wounds decreased to one-fourth of normal levels (p<0.01) over 24 hours and then began to increase but still did not reach their normal level. TUNEL-positive cells in burn wounds were 3.7-fold (p<0.01) elevated over 48 hours and then decreased slightly, yet still remained higher than in normal skin. The burn wound progressed in depth over 72 hours. In addition, significant decrease in LDF values and upregulation of MPO activity were observed. Enhanced LC3-positive cells were observed in the deep dermal layer of burn wounds as shown by IHC staining. A reduction in autophagy and blood flow and an increase in apoptosis and inflammation were observed in burn wounds early during the course of burn injury progression. This suggests that autophagy, complemented by apoptosis, play important roles in burn progression. Enhanced autophagy in the deep dermis may be a prosurvival mechanism against ischemia and inflammation after burn injury.

Critical role of myeloid differentiation factor 88 in necrotizing enterocolitis

The importance of toll-like receptor 4 in necrotizing enterocolitis (NEC) has been intensively studied, but its downstream signaling and the potential regulatory mechanisms remain unidentified. Our study focused on the role of myeloid differentiation factor 88 (MyD88), the first downstream adaptor of toll-like receptor 4 inflammatory and apoptotic signaling in the pathogenesis of NEC. MyD88 knockout (MyD88(-/-)-Ko) mice and lentivirus-mediated stable MyD88-knockdown cell line (IEC-6) were used. NEC was induced by formula gavage, cold, hypoxia, combined with lipopolysaccharide (LPS) in vivo, or LPS stimulation in vitro. NEC was evaluated by histology and multiple inflammatory cytokines. Enterocyte apoptosis was evaluated by terminal-deoxynucleoitidyl transferase-mediated nick end labeling (TUNEL) or Annexin analysis. Inflammatory or apoptotic molecules including NF-κB, Toll/IL-1R domain-containing adaptor-inducing IFN-β, interferon regulatory factor 3, Bax, Bcl-2, and caspases were examined by quantitative real-time PCR (qRT-PCR). In the MyD88-Ko group, NEC severity and intestinal enterocyte apoptosis rate were reduced, the expression of NF-κB, caspases, and Bax, were all downregulated, while Toll/IL-1R domain-containing adaptor-inducing IFN-β and were upregulated, and antiapoptotic gene Bcl-2 remained stable. Cytokine levels of interleukin (IL)-6, IL-1β, and tumor necrosis factor-α (TNF-α) were also all decreased. MyD88-dependent signaling is the prevailing inflammatory and apoptotic signaling in toll-like receptor 4 downstream signaling; MyD88-Ko resulted in reduced inflammatory severity and apoptosis, though MyD88-independent signaling can also be activated, but is of less dominant for the development of NEC.

The Regulation of Cellular Adhesion Geometry on Apoptosis of Mesenchymal Stem Cell

The mechanical stimulation from extracellular matrix could regulate physiological behavior of cells through the mechanism of mechanotransduction. Previous researches had shown that apoptosis could be regulated by the size of the cell adhesion area.However, the regulation of cell apoptosis by different adhesion shape with the same area is still unclear. This workfocused on the regulation of apoptosis for bone marrow mesenchymal stem cells (MSCs) by different circularity and area of adhesion geometry. We manufactured micro-pattern surface which was suitable for adhesion of MSCs by the technique of micro-contact printing. Three typesof geometry for individual is land of micro-pattern were designed. We adopted terminal-deoxynucleoitidyl transfer as emediated nick end labeling (TUNEL) method to detectcell apoptosis. This research shows that the adhesion geometry which has smaller area and greater circularity will promote apoptosis of MSCs. This indicates that MSCsmay prefer to live on the surface without any restrict. Ourstudies focused on the significantly important problem about interaction between extracellular matrix and physiological behavior of mesenchymal stem cells.

Effects of aminoguanidine on retinal apoptosis in mice with oxygen-induced retinopathy.

To explore the protective effects of aminoguanidine (AG) on retinal apoptosis in mice with oxygen-induced retinopathy (OIR). A total of 80 C57BL/6J mice, aged 7 days, were randomly divided into four groups: normal, high oxygen, high oxygen saline and high oxygen treated with AG. In the normal group, mice were housed in normoxic conditions from postnatal day P7 to P17. Mice in the other 3 groups were placed under hyperoxic conditions (75±2%O2) in an oxygen-regulated chamber for 5 days and subsequently placed in normoxic conditions for 5 days. Mice in the AG group were treated once daily, from P12 to P17, with AG hemisulfate (100mg/kg body weight, intraperitoneally) dissolved in physiological saline. An equivalent amount of 0.9% physiological saline was administered, as above, to mice in the high oxygen saline group. Ten mice were randomly selected from each group on P14 and on P17, euthanized and the retinas examined. Apoptotic cells in the retina were detected using the terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) method. The expression of nitric oxide synthase (iNOS) in the retina was detected by immunohistochemistry and changes in rod cells were observed using electron microscopy. TUNEL-positive cells and iNOS immunoreactive neurons were present in the inner nuclear and ganglion cell retinal layers of mice in the high oxygen group. The number of TUNEL-positive cells was significantly greater in the high oxygen group compared with the normal group (t=-20.81, P 14d <0.05; t=-15.05, P 17d <0.05). However, the number of TUNEL-positive cells in the AG treatment group was significantly lower (t=-13.21, P 14d<0.05; t=-6.61, P 17d <0.05) compared with the high oxygen group. The expression of iNOS was significantly higher in the high oxygen group compared with the normal group (t=-21.95, P 14d<0.05; t=-17.30, P 17d<0.05). However, the expression of iNOS in the AG treatment group was significantly lower (t=-12.17, P 14d<0.05; t=-10.30, P 17d<0.05) compared with the high oxygen group. The outer segments of the rods were disorganized and short in the high oxygen group. Rod morphology appeared to be slightly improved in the AG group. AG may protect retinal neurons in OIR by inhibiting apoptosis. The mechanism may be related to iNOS.

Retinal apoptosis of laser-induced retinal injury in mice after bone marrow mesenchymal stem cells transplantation

Objective To observe the retinal apoptosis of laser-induced retinal injury in mice after bone marrow mesenchymal stem cells transplantation.Methods Green fluorescent protein (GFP) labeled MSCs from C57BL/6 mice were cultured in vitro.A total of 135 C57BL/6 mice were divided into three groups including normal control group (15 mice),injured control group (60 mice) and MSCs treatment group (60 mice).Laser retinal injuries were induced by laser photocoagulation.One day after photocoagulation,0.2 ml cell suspension,which contained 1 × 106 GFP-MSCs,were injected into the mice in treatment group via tail vein,and the mice in injured control group were given equal volume of phosphate buffer solution.Animal were execute on three,seven,14 and 21 days following laser damage.Hematoxylin and eosin (HE) staining was performed to assess the changes of injured retinas.The diameters of laser spots and areas with total loss of cells in outer nuclear layer (ONL) were analyzed by image processing software.The apoptosis of retinal cells was examined by terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) staining.The migration of GFP-MSCs into the retina was observed by fluorescence microscope.Results HE staining showed that the retinal structures were integrated in normal control group.Retinal damages were observed both in injured control group and MSCs treatment group,but milder in the latter.Though the average diameter of area with total loss of cells in ONL of MSCs treatment group was less than the injured control group (t =5.769,P ＜ 0.05),the diameters of laser spots show no difference (t=0.964,P＞0.05) on day three.Both the average diameter of laser spots (t=5.180,5.417,2.381) and area with total loss of cells in ONL (t=3.530,3.224,3.162) were less in the MSCs treatment group on day seven,14 and 21 (P＜0.05).TUNEL staining shows that the apoptosis were decreased after MSCs transplantation on day three,seven,14 and 21 (t=11.142,7.479,6.678,3.953,P＜0.05).No apoptosis was observed in normal control group.Very few GFP-MSCs were observed in the retina at all time-points.They were only seen in the subretinal and choroidal neovascularization occasionally on day seven and 14.Conclusion MSCs transplantation can effectively limit the range of retinal laser damage and inhibit cell apoptosis. Key words: Mesenchymal stem cell transplantation; Retina/pathophysiology; Apoptosis

Effects of apocynin on oxidative stress and expression of apoptosis-related genes in testes of diabetic rats

Reactive oxygen species (ROS) are important in the development of diabetic testicular dysfunction. Overproduction of ROS promotes the process of apoptosis, which shows that there is a crosstalk between oxidative stress and apoptosis. Recent research has suggested that NADPH oxidase is the main source of ROS. In this study, we investigated whether the NADPH oxidase inhibitor, apocynin, can improve diabetes‑induced testicular dysfunction by suppressing oxidative stress. The streptozocin (STZ)-induced diabetic rats were administered apocynin, and the mRNA and protein expression of Bax, Bcl-2, p47phox and p67phox was examined by real-time PCR (RT-PCR) and western blot analysis. Production of ROS was measured by thiobarbituric acid reactive substances (TBARS) assay. Terminal-deoxynucleoitidyl transferase mediated nick end-labeling (TUNEL) assay was used to detect apoptosis and ELISA was used to detect total testosterone levels. The mRNA and protein expression of Bcl-2 was significantly reduced, and that of Bax, p47phox and p67phox was significantly increased in the diabetic rats compared to the control group. Apocynin significantly increased the expression of Bcl-2 and decreased the expression of Bax, p47phox and p67phox at both the mRNA and protein levels. The production of ROS and apoptotic cells significantly increased in the diabetic group compared to the control group. Apocynin significantly reduced the production of ROS and apoptotic cells and increased the total testosterone level. In conclusion, apocynin is capable of ameliorating testicular dysfunction.