Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling Research Articles

Protective effect of ulinastatin on severe pulmonary infection under immunosuppression and its molecular mechanism.

The objective of the present study was to investigate the protective effect of ulinastatin on severe pulmonary infection under immunosuppression, and its molecular mechanism. Mice were treated with methylprednisolone and lipopolysaccharide (LPS) to establish the model of severe pulmonary infection under immunosuppression. Mice were randomly divided into group A (model group; treated with equal volumes of saline), group B (treated with 1×105 U/kg ulinastatin), and group C (normal control group). Bronchoalveolar lavage fluid (BALF) was collected, and the concentrations of cytokines in BALF were measured by enzyme-linked immunosorbent assay (ELISA). Pathological changes in lung tissues were observed by hematoxylin and eosin (H&E) staining. The mRNA levels of M1 and M2 macrophage markers in lung tissues were detected by real-time polymerase chain reaction (PCR). Specific protein levels in lung tissues were measured by western blotting. Apoptosis in lung tissues was detected by the terminal-deoxynucleoitidyl transferase mediated nick end-labeling (TUNEL) method. The concentrations of TNF-α, IL-6, and IL-1β in BALF, the mRNA levels of the three M1 macrophage markers, and the protein levels of p-Janus Kinase 2 (p-JAK2), p-signal transducer and activator of transcription-3 (p-STAT-3), cleaved caspase-9, and cleaved poly-ADP-ribose polymerase (PARP), and the number of apoptotic cells in lung tissues in group A were significantly higher than those in groups B and C (P<0.05), whereas the concentrations of IL-4, IL-10, and IL-13 and the mRNA levels of the three M2 macrophage markers were significantly lower than those in groups B and C (P<0.05). Immunofluorescence showed that the nuclei of lung epithelial macrophages in group A became smaller and moved towards the side of nuclear membranes. In conclusion, ulinastatin can improve the inflammatory response caused by severe infection under immunosuppression, which balances the inflammatory microenvironment and inhibits apoptosis at least partially through inhibiting JAK2/STAT-3 and/or caspase pathway activity, ultimately playing a role in lung protection.

Hyperoside protects against hypoxia/reoxygenation induced injury in cardiomyocytes by suppressing the Bnip3 expression

AimsRole of hyperoside in protecting cardiomyocytes from ischemia/reperfusion induced injury has been proved. However, possible protecting mechanisms remain unclear. To fix the problem, an essential pro-apoptotic protein Bnip3 was studied in our experiments. Methods and resultsNeonatal rat cardiomyocytes were used and submitted to hypoxia for 8h followed by reoxygenation for 2h to simulate the ischemia/reperfusion injury. Hypoxia/reoxygenation(H/R) induced damage to cardiomyocytes and the protective effect of hyperoside were examined by means of MTT assay. H/R-induced apoptosis was assessed by Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling(TUNEL) and DNA Ladder assay. mRNA expression of Bnip3 was determined by use of quantitative real-time reverse transcription polymerase chain reaction assay. Protein levels of Bnip3, Bax, Bcl-2 and cleaved caspase-3 were examined using western-blot assay. Our results showed that H/R caused great damage to cardiomyocytes, upregulated the protein expressions of Bnip3, Bax, cleaved caspase3, and decreased the expression of the anti-apoptotic protein of Bcl-2. Whereas, compared with the H/R group, a decrease in activities of Bnip3, Bax, cleaved caspase3, and a promoting expression of Bcl-2 were detected in the H/R goup pretreated with hyperoside. ConclusionIt was concluded in our study that H/R-induced apoptotic effect in cardiomyocytes could be attenuated by hyperoside, and the protective role of hyperoside, if not completely, could be partly through the suppression of the pro-apoptotic gene Bnip3.

SIRT1 signaling pathway mediated the protective effects on myocardium of rats after endurance training and acute exhaustive exercise

Objective: To detect the expression of SIRT1 and Ac-FOXO1 in rats after endurance training and acute exhaustive exercise, and explicit the myocardial protective effect of SIRT1. Methods: Rats were randomly divided into four groups: control group(n=20), exhaustive exercise group (E group, n=20), exhaustive exercise group + endurance training (TE group, n=18), exhaustive exercise group + endurance training + selective SIRT1 inhibitor (TSE group, n=17). The Control and E groups were fed routinely for 5 weeks. The TE and TSE groups were subjected to swimming exercise for 5 weeks for endurance exercising. The TSE group was intraperitoneally injected with selective SIRT1 inhibitor Sirtinol(2 mg/kg) at 30 minutes before endurance exercising. The E, TE and TSE groups were subjected to exhaustive exercise. The myocardial tissues of rats were collected after exhaustive exercise. Real-time polymerase chain reaction (PCR) and Western blot analysis were performed to detect the myocardial mRNA and protein expressions of SIRT1 and Ac-FOXO1. The myocardial protein expression of Bax and Bcl-2 was also detected by Western blot. Terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) assay was used to assess the apoptosis of myocardial cells. Results: Compared with Control group, the SIRT1 and Bcl-2 expression in the myocardial tissue was obviously decreased, while the Ac-FOXO1, Bax, and the myocardial cell apoptosis were significantly increased in E group (all P<0.01). Compared with E group, the expression of SIRT1 and Bcl-2 was obviously up-regulated (both P<0.01), while the Ac-FOXO, Bax and the myocardial cell apoptosis was significantly reduced in TE group (all P<0.01). Compared with TE group, the SIRT1 and Bcl-2 expression was obviously lower (both P<0.01), while Ac-FOXO1, Bax, and the cell apoptosis were significantly higher in group TSE (all P<0.01). Conclusion: Endurance training could protect myocardium by reducing the myocardial oxidative stress injury and apoptosis via activating SIRT1 signaling pathway, up-regulating the myocardial expression of SIRT1 and regulating the deacetylation of FOXO1.

Gastric electrical stimulation improves enteric neuronal survival.

Research and clinical experience with vagotomy have confirmed that damage to the central nervous system severely affects physiological movement in the gastrointestinal system. The aim of this study was to investigate the effects of synchronized dual-pulse gastric electrical stimulation (SGES) on the apoptosis of enteric neurons and the possible pathways involved in these effects in vagotomized rats. For this purpose, Male Sprague-Dawley (SD) rats were randomized into a control group, an early subdiaphragmatic vagotomized group (ESDV group), an early subdiaphragmatic vagotomized group with short-term SGES (ESDV + SSGES group), a terminal subdiaphragmatic vagotomized group (TSDV group) and a terminal subdiaphragmatic vagotomized group with long-term SGES (TSDV + LSGES group). The expression levels of connexin 43 (Cx43), glial cell line-derived neurotrophic factor (GDNF), p-Akt, pan-Akt and PGP9.5 were assessed by RT-qPCR, western blot analysis and immunofluorescence staining. Apoptosis was determined by terminal-deoxynucleoitidyl transferase-mediated nick-end labeling (TUNEL) assay. We found that Cx43 expression was decreased in the ESDV and TSDV groups, but was significantly upregulated in the SSGES and LSGES groups. In addition, the GDNF and PGP9.5 expression levels were significantly decreased in the ESDV group compared with the control and TSDV groups and were upregulated in both the SSGES and LSGES groups. The LSGES group exhibited a clear increase in p-Akt expression compared with the TSDV group. Fewer TUNEL-positive cells were observed in the SSGES and LSGES groups than in the ESDV and TSDV groups. More TUNEL-positive cells were found in the stomach of rats subjected to subdiaphragmatic vagotomy. On the whole, our data indicate that SGES improved enteric neuronal survival, possibly through GDNF and the phosphatidylinositol 3-kinase (PI3K)/Akt pathways.

Construction of oncolytic adenovirus carrying kringle 1 domain of hepatocyte growth factor gene and its lethal effect to gastric cancer cells

Objective To construct an double-regulated oncolytic adenovirus carrying kringle 1 domain of hepatocyte growth factor (HGFK1) gene which exerts anti-tumor and anti-angiogenesis effect and observe its lethal effect to gastric cancer cells. Methods Adenoviral E1a and E1b genes were controlled by Human telomerase reverse transcriptase promoter (hTERT) and hypoxia regulatory element (HRE). TH-Ad-HGFK1-EGFP or TH-Ad-EGFP was constructed by inserting HGFK1 gene into the viral genome or not. The adenovirus was divided into three groups: the experimental group A (TH-Ad-HGFK1-EGFP), the experimental group B (TH-Ad-EGFP) and the control group C (Ad-EGFP). Gastric cancer cells SGC7901 and the normal human fibroblasts cell HF were infected by adenovirus, then the proliferation of adenovirus was counted by the half-cell culture infection dose (TCID50) method. Methyl thiazolyl tetrazolium (MTT) method was used to detect the inhibitory effect. 4, 6-Diamidino-2 -Phenylindole, Dihydrochloride (DAPI) method and Terminal -deoxynucleoitidyl Transferase Mediated Nick End Labeling (TUNEL) method was used to observe the cell apoptosis. Results TH-Ad-HGFK1-EGFP and TH-Ad-EGFP was confirmed constructed successfully by DNA sequencing and PCR analyses. All the titer of the adenovirus was 2×1010 pfu/ml. The TCID50 results showed the proliferation of group A and group B in SGC7901 cells was 1.47×104 and 1.60×104 fold, which were significantly higher than that in group C. However in HF cells, the fold change of replication of group A and group B was only 110 and 124, which was significantly lower than that in group C. Group A showed potent inhibitory effect on the growth of SC7901 cells, but Group B and group C showed no obviously inhibitory effect. The inhibition ratio of group A (MOI=100, 72 h and 96 h) on SGC7901 cells was (71.34±8.27)%, (74.56±1.78)%, which had significant difference compared with those in group B (23.40±2.29)%, (31.94±6.62) % (P=0.002, P=0.003) or group C (21.18±2.32%, 21.18±2.32%)(P=0.003, P=0.000). The inhibition ratio of group A and group B (MOI=100, 96 h) on HF cells was 18.94±0.88% and 22.84±3.34%, which were significant lower than that in group C [(53.25±3.50)%, P=0.003, P=0.001]. Group A also showed cell apoptosis on SGC7901 cells (18.66±4.04%), which had significant difference in other groups. Conclusion The oncolytic adenovirus TH-Ad-HGFK1-EGFP showed remarkably replication capability, promoted apoptosis and inhibited proliferation of SGC7901. Key words: Gastric cancer; Kringle 1 domain of hepatocyte growth factor; Oncolytic adenovirus; SGC7901

The effects of YHTJF on lung ischemia/reperfusion injury in mice

To explore whether yiqi huoxue tongluo jiedu fang (YHTJF, Traditional Chinese Medicine) alleviates the injury during lung ischemia/reperfusion (I/R) in mice through inhibiting oxidative stress or not. C57BL/6J male mice (n=70) were randomly divided into 7 groups:control (C), carboxyl methyl cellulose-Na(CMC·Na) + normal control (CC), carboxyl methyl cellulose-Na + sham (CS), carboxyl methyl cellulose-Na + I/R (CIR), carboxyl methyl cellulose-Na + YHTJF-Low, CMC-Na + YHTJF-Middle, CMC-Na + YHTJF-High (CYL, CYM, CYH). The mice in CYL, CYM and CYH group were treated with YHTJF by intraperitoneal injection every day, while the carboxyl methyl cellulose-Na was administered with the same volume of CYL in CC, CS and CIR group. After 3 h-reperfusion, the left lung tissues were harvested to determine the lung wet/dry weight (W/D), the total lung water content (TLW), and the index of quantita-tive evaluation for alveolar damage (IQA). Morphological observation and terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) were applied to evaluate the structural changes and the apoptosis index (AI) of the lung tissues. The expressions superoxide of dis-mutase(SOD), malondialdehyde(MDA) and myeloperoxidase(MPO) in the lung tissues were detected by kits. Compared with group C, the W/D, TLW, IQA, AI, lung tissue structural changes, and the expressions of MDA and MPO in group I/R were increased obviously (P < 0.01), and the expression of SOD was decreased, while there was no significant difference between group CC and CS. Compared with group I/R, the parameters of these experiments in group CYL, CYM, CYH were all decreased, and the expression of SOD was increased, while the reduction in group CYM was the most remarkable among them (P < 0.01). YHTJF may attenuate the I/R injury of the lung by the inhibition of apoptosis via ROS pathway.

Hepatoprotective effect of exosomes from human-induced pluripotent stem cell–derived mesenchymal stromal cells against hepatic ischemia-reperfusion injury in rats

BackgroundThis study aimed to evaluate the effect of exosomes produced by human-induced pluripotent stem cell–derived mesenchymal stromal cells (hiPSC-MSCs-Exo) on hepatic ischemia-reperfusion (I/R) injury. MethodsExosomes were isolated and concentrated from conditioned medium using ultracentrifugation and ultrafiltration. hiPSC-MSCs-Exo were injected systemically via the inferior vena cava in a rat model of 70% warm hepatic I/R injury, and the therapeutic effect was evaluated. The serum levels of transaminases (aspartate aminotransferase [AST] and alanine aminotransferase [ALT]) were measured using an automatic analyzer. The expression of inflammatory factors was measured using enzyme-linked immunosorbent assay (ELISA). Histological changes indicated changes in pathology and inflammatory infiltration in liver tissue. Apoptosis of hepatic cells in liver tissue was measured using terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) staining along with apoptotic markers. ResultshiPSCs were efficiently induced into hiPSC-MSCs with typical MSC characteristics. hiPSC-MSCs-Exo had diameters ranging from 50 to 60 nm and expressed exosomal markers (CD9, CD63 and CD81). Hepatocyte necrosis and sinusoidal congestion were markedly suppressed with a lower Suzuki score after hiPSC-MSCs-Exo administration. The levels of the hepatocyte injury markers AST and ALT were significantly lower in the treated group than in the control group. Inflammatory markers, such as tumor necrosis factor (TNF)-α, interleukin (IL)-6 and high mobility group box 1 (HMGB1), were significantly reduced after administration of hiPSC-MSCs-Exo, which suggests that the exosomes have a role in suppressing the inflammatory response. Additionally, in liver tissues from the experimental group, the levels of apoptotic markers, such as caspase-3 and bax, were significantly lower and the levels of oxidative markers, such as glutathione (GSH), glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD), were significantly higher than in the control group. These data point to an anti-apoptotic, anti-oxidative stress response role for hiPSC-MSCs-Exo. ConclusionsOur results demonstrated that hiPSC-MSCs-Exo alleviate hepatic I/R injury, possibly via suppression of inflammatory responses, attenuation of the oxidative stress response and inhibition of apoptosis.

The PTEN/Akt Signaling Pathway Mediates Myocardial Apoptosis in Swine After Coronary Microembolization

Phosphatase and the tensin homolog deleted on chromosome ten (PTEN) has been recognized as a promoter of apoptosis in various tissues and has been shown to be upregulated in circumstances of coronary microembolization (CME). We hypothesized that the upregulation of PTEN correlates with CME-induced myocardial apoptosis. Swine CME was induced by an intracoronary injection of inert plastic microspheres (diameter of 42 μm) into the left anterior descending coronary, with or without pretreatment of the PTEN small-interfering RNA (siRNA). Echocardiological measurements, a pathological examination, Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling (TUNEL) staining, and Western blotting, were performed to assess their functional, morphological, and molecular effects in CME. PTEN was aberrantly upregulated in cardiomyocytes following CME. Downregulation of PTEN in vivo via siRNA was associated with improved cardiac function and attenuated myocardial apoptosis; concomitantly inhibited the expression of key proapoptotic proteins, such as phosphorylated Bad (p-Bad); cleaved caspase-3; and enhanced the expression of key antiapoptotic proteins, such as phosphorylated protein kinase B (p-Akt). However, there was no difference in the Akt-regulated downstream protein IκB kinases (IKKα, IKKβ, and IKKγ) among the sham, CME, and control siRNA groups. This study demonstrates, for the first time, that the PTEN/Akt signaling pathway contributes to cardiomyocyte apoptosis. The data generated from this study provide a rationale for the development of PTEN-based therapeutic strategies for CME-induced myocardial injury.

Follistatin-like 1-engineered mesenchymal stem cells prevent myocardial ischemic reperfusion injury

To investigate the role of follistatin-like protein 1 (FSTL1) on bone marrow mesenchymal stem cells (BM-MSCs)-mediated cardioprotection during myocardial ischemia/reperfusion injury. Rat bone marrow mesenchymal stem cells were isolated by whole bone marrow adherence method in vitro. A total of 60 Wistar rats were randomly divided into 4 groups: control group, ischemic /reperfusion injury (IRI) group, ischemia/reperfusion injury group treated with natural BM-MSCs (IRI+ MSC group), ischemia/reperfusion injury group treated with BM-MSCs which did not contain FSTL1 (IRI+ MSC FSTL1 siRNA group). Survival analysis was used to analyze survival time of rats, besides, expression of FSTL1 was detected by Western blotting. Myocardial pathological changes were detected by Hematoxylin and Eosin (HE) staining. Terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) and enzyme-linked immunosorbent assay (ELISA) were used to determine the associated biomarkers and apoptosis 7 days after operation. Compared with IRI group, rats in IRI+ MSC group had a higher survival rate and lived longer. Meanwhile, IRI+ MSC group had higher FSTL1 expression in blood and myocardial tissues than IRI group. Control group showed significantly lower apoptosis rate of myocardial cells[(1.4±0.1)% vs (29.8±4.5)%, P<0.05], less histological changes and infarction areas (0 vs 24.48±4.27, P<0.05) than IRI group. Compared with IRI group, IRI+ MSC group had an improvement of apoptosis rate[(4.2±0.3)% vs (29.8±4.5)%, P<0.05], less histological injury and infarction areas (15.12±3.82 vs 24.48±4.27, P<0.01). IRI+ MSC group had lower expression of LDH, MDA, CK and higher expression of SOD than IRI group (P<0.05). However, IRI+ MSC FSTL1 siRNA group showed weaker protection of myocardial cells than IRI+ MSC group after knockdown of FSTL1 (P<0.05). FSTL1, which was secreted by BM-MSCs, plays a protective role in myocardial IRI.

The neuroprotective effect of apelin-13 in a mouse model of intracerebral hemorrhage

Adipocytokine apelin-13 is a peptide which could reportedly protect the brain against ischemic reperfusion injury and traumatic brain injury (TBI). Whether apelin-13 has any roles to play in intracerebral hemorrhage (ICH) has not been clarified. We aimed to investigate the roles of apelin-13 in ICH and effects on ICH-induced apoptosis. Firstly, CD-1 mice were subjected to infusion of Type IV collagenase (to induce ICH) or saline (for shams) into the left striatum. ICH animals received intracerebroventricular administration of vehicle, apelin-13 (50μg dissolved in 5μl saline) immediately after ICH. The motor function and the cerebral water content (CWC) as well as blood brain barrier (BBB) disruption were measured, coupled with determination of ICH-induced neural cell death by Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling (TUNEL). The apoptosis-associated proteins caspase-3 and Bcl-2 as well as the brain edema-associated proteins aquaporin-4 (AQP4) and MMP-9 were all assessed with western blotting. The results showed that apelin-13 decreased CWC and reduced Evans blue leakage into injured hemispheres, with the motor function significantly improved. Additionally, apelin-13 also acutely decreased the number of ICH-induced TUNEL-positive (TUNEL+) cells at 48h after ICH. The expressions of AQP4, MMP-9, caspse-3 and Bcl-2 were all downregulated by apelin-13 at 24h and 48h after ICH. All these results revealed that apelin-13 attenuated brain edema and reduced cellular death by suppressing apoptosis after ICH in mice.

Inhibition of p21-Activated Kinase 1 by IPA-3 Promotes Locomotor Recovery After Spinal Cord Injury in Mice.

Ninety-six male adult CD-1 mice were randomly divided into sham, spinal cord injury (SCI) + vehicle, and SCI + IPA-3 groups. Expression of matrix metalloproteinase (MMP)-2 and MMP-9, production of tumor necrosis factors (TNF)-α and interleukin (IL)-1β, tissue edema, blood-spinal cord barrier penetrability, neural cell apoptosis, and neurological function recovery were measured. The aim of the study was to evaluate the effect of specific inhibition of p21-activated kinase 1 (PAK1) by IPA-3 on SCI and the underlying mechanisms thereof. SCI is a devastating clinical condition that may result in long-lasting and deteriorating functional deficits. The major goal of SCI treatment is to limit the development of secondary injury. IPA-3, a PAK1 inhibitor, exhibited neuroprotection against secondary damage after traumatic brain injury and subarachnoid hemorrhage (SAH). MMP-2, MMP-9, and cleaved caspase-3 expression were assessed by Western blot. Inflammatory cytokines TNF-α and IL-1β were detected by enzyme-linked immunosorbent assay (ELISA). The blood-spinal cord barrier disruption was measured by water content and Evans blue extravasation of the spinal cord. Neuronal apoptosis was evaluated by Nissl staining and Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling (TUNEL) assay. The locomotor behavior of hind limb was evaluated by Basso Mouse Scale (BMS) at 1, 3, 7, 14, and 28 days post-injury. Compared with SCI + vehicle mice, IPA-3 treatment showed decreased p-PAK1, MMP-2, MMP-9, cleaved caspase-3, TNF-α, and IL-1β expression. Moreover, inhibition of PAK1 by IPA-3 reduced spinal cord water content and Evans blue extravasation, increased neuronal survival, and reduced TUNEL-positive cells at 24 hours after SCI. Furthermore, IPA-3 improved spinal cord functional recovery 7 days after SCI. Inhibition of PAK1 by IPA-3 promoted recovery of neurological function, possibly by downregulating the expression of MMP-2, MMP-9, TNF-α, and IL-1β. Our data suggest that PAK1 may be a potential therapeutic target in patients with SCI. 1.

Effect of mitochondrial apoptosis on pulmonary fibrosis in rats with severe scald injury

To explore the role of mitochondrial apoptosis on pulmonary fibrosis in rats with severe scald injury. According to the random digital table, a total number of 32 Wistar rats were divided into 4 groups: sham burn (group A), burn group (group B), 12-week post burn recovery group (group C), and 12-week post burn recovery plus a second burn injury group (group D). In group A and group B, lung tissues were harvested on post burn day 4. After received first burn injury 12 weeks, the group C and group D received separately a second sham burn injury and burn injury. Lung tissues were harvested on post burn day 4 after the second burn injury. All tissues were examined for cells apoptosis by Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling (TUNEL). Pulmonary fibrosis was assessed by Masson trichrome staining and Sirius red staining. The protein expression levels of cleaved Caspase-3, Bax and Bcl-2 were assessed by Western blot. Both Masson trichrome staining and Sirius red staining showed obvious pulmonary fibrosis in group C and group D. The apoptosis rates of group B, C and D were significantly higher than that in group A ((15.50±3.30)%, (7.88±3.10)%, (15.88±3.23)% vs (2.10±1.07)%, all P<0.05). Compared to group A, cleaved Caspase-3 levels were significantly higher in group B, C and D ((0.59±0.11), (0.33±0.08), (0.73±0.13) vs (0.16±0.05), all P<0.05). The ratio of Bax/Bcl-2 in group B, C and D also increased significantly ((2.08±0.30), (0.83±0.09), (1.54±0.12) vs (0.64±0.05), all P<0.05). Severe burn injury can induce pulmonary fibrosis and mitochondrial apoptosis may play an important role in the development of pulmonary fibrosis.

Effects of ulinastatin on long-term learning and memory function after partial hepatectomy in mice

Objective To investigate the effect of ulinastatin (UTI) on long-term learning and memory function after partial hepatectomy (PH) in mice. Methods Forty-five adult male specific pathogen free (SPF) C57BL/6J mice were randomly divided into control group (C group), PH group and UTI group (n=15 each). After the mice regained consciousness after operation, animals received intraperitoneally UTI at a dose of 50 000 U/(kg·day) for 30 consecutive days in UTI group. Animals were given intraperitoneally with the same volume of normal saline for 30 consecutive days in C group and PH group. The mice of the three groups underwent Morris water maze test at 24 h after the last pretreatment. The mice were euthanized after the test of learning and memory function, and hippocampi tissue was immediately excised for determination of wet weight to dry weight (W/D) and total water content (TWC). The expression levels of CCAAT enhancer-binding proteins homologous protein (CHOP) mRNA and protein of hippocampi tissue were detected respectively by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. Apoptosis index (AI) of hippocampi tissue was determined by terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) assay. Results Compared to C group [(17.39±7.72) s, (12.59±6.69) s; (2 122.57±543.48) mm, (1 123.69±369.32) mm], the escape latency [(54.48±9.69) s, (56.63±7.74) s] and swimming distance [(4 789.48±677.67) mm, (4 987.72±884.53) mm] were significantly prolonged (both P<0.05), W/D (5.47±0.98 vs. 2.79±0.78), TWC (4.43±0.97 vs. 1.79±0.72) and AI [(36.32±2.62)% vs. (2.69±0.75)%] were significantly increased (all P<0.05), and the expression levels of CHOP mRNA (0.92±0.36 vs. 0.38±0.08) and protein (2.79±0.79 vs. 1.06±0.23) of hippocampi tissue were significantly increased (both P<0.05) in PH group. Compared to PH group, the escape latency [(23.56±7.47) s, (13.62±6.36) s] and swimming distance [(2 234.64±890.58) mm, (1 120.67±389.74) mm] were significantly shortened (both P<0.05), W/D (2.92±0.64 vs. 5.47±0.98), TWC (1.86±0.84 vs. 4.43±0.97) and AI [(13.65±1.74)% vs. (36.32±2.62)%] were significantly reduced (all P<0.05), and the expression levels of CHOP mRNA (0.54±0.11 vs. 0.92±0.36) and protein (1.41±0.47 vs. 2.79±0.79) of hippocampi tissue were significantly reduced (both P<0.05) in UTI group. Conclusion The pretreatment of UTI could improve the long-term learning and memory function after PH in mice, which might be contributed to the inhibition of apoptosis mediated by CHOP in hippocampi tissue. Key words: Ulinastatin; CCAAT enhancer-binding proteins homologous protein; Partial hepatectomy; Cognition disorders; Postoperative complications

Effect of icariin on the proteomic expression profile of bone microvascular endothelial cells of human femoral head against steroids-induced lesion

To explore the effect of icariin on the proteomic expression profile of bone microvascular endothelial cells of human femoral head against steroids-induced lesion using protein chip system in vitro. Bone microvascular endothelial cells (BMECs) in cancellous bone of femoral head were isolated and harvested in vitro.The model of BMECs injury induced by glucocorticoid and icariin preconditioning was established. The apoptosis of BMECs were detected with terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL)methods. The proteomic expression profile of BMECs in each group were detected by protein chip system. The apoptosis rates of hydrocortisone groups (46.9%±5.8%, 78.0%±8.5%) were significantly higher than that of normal group(4.8%±1.3%), meanwhile the apoptosis rate of icariin+ hydrocortisone groups(15.2%±7.7%, 44.6%±5.3%)significantly decreased compared with hydrocortisone group (P<0.001). The results of protein chip showed that the expression of granulocyte colony-stimulating factor (G-CSF) in the hydrocortisone group decreased compared with the control group, meanwhile the expression of IL-4 increased. The expression of G-CSF in the icariin+ hydrocortisone groups increased compared with the hydrocortisone group, meanwhile the expression of IL-4 decreased.No appreciable change was found in the expression of apoptotic factors. The therapy effect of icariin for steroid-induced necrosis of femoral head might be related with the protective action to BMECs and the moderating effect of promoting the proteins expression of pro-angiogenesis factor and inhibiting expression of inflammatory factor.

Antagonistic effect and mechanism of Rosuvastatin on myocardial apoptosis in rats with acute myocardial infarction

To investigate the protective effect of Rosuvastatin on myocardial cells in rats with acute myocardial infarction and its possible mechanism. Rats were randomly assigned to four groups: Control group, Sham group, AMI and Rosuvastatin group. The levels of lactate dehydrogenase (LDH) and creatine jubase (CK), the vitality of superoxide dismutase (SOD) and the content of malondialdehyde (MDA) were detected by assay kits and the levels of C-reactive protein (CRP), tumor necrosis factor (TNF) alpha and interleukin (IL)-6 expression were detected by enzyme linked immunosorbent assay (ELISA). TTC/Evans blue staining was used to determine the relative myocardial infarction area, HE staining was used to detect pathologic changes and myocardial apoptosis was detected by terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL). What's more, Western blot was used to detect the protein expression of B-cell lymphoma-2 (Bcl-2), Bax, cleaved-Caspase-3, Rock1, Rock2, I-κB and NF-κBp65. The model of acute myocardial infarction rats was established. Compared with Sham group, the myocardial pathological changes were more severe, and the apoptosis number, the production of inflammatory factors and oxidative damage were significantly increased in AMI group. Compared with AMI group, the relative area of infarction myocardium (43% ± 4% vs 31% ± 8%, P=0.004 3) was dramatically reduced, the levels of LDH (2 545.45 ± 613.67 U/L vs 1 573.43 ± 373.72 U/L, P=0.02) and CK (7.49 ± 1.75 U/ml vs 4.42 ± 1.28 U/ml, P=0.04) in serum were significantly lower (P<0.01), the myocardial pathological damage degree was relieved, the apoptosis number (41% ± 8% vs 23% ± 6%, P=0.014 7) was significantly decreased, the expression of Bax (1.17 ± 0.10 vs 0.57 ± 0.08, P=0.003) and cleaved-Caspase-3 (1.31 ± 0.07 vs 0.70 ± 0.01, P=0.004) were dramatically reduced, and the expression of Bcl-2 (0.19 ± 0.01 vs 0.32 ± 0.01, P=0.003) was enhanced in Rosuvastatin group. Furthermore, the production of inflammatory factors and oxidative damage were effectively alleviated (P<0.05), the protein expression of Rock1, Rock2 and NF-κBp65 were declined in Rosuvastatin group (P<0.01). Rosuvastatin can effectively alleviate the cardiomyocyte apoptosis induced by acute ischemia, and its main mechanism may be the inhibition on RhoA/ROCK activation and the oxidative stress and inflammation reaction mediated by it.

Protective effects of ulinastatin on intestinal barrier damaged after cardiopulmonary resuscitation in rats

Objective To investigate the protective effects of Ulinastatin on intestinal barrier damaged after cardiopulmonary resuscitation (CPR) in rats in order to illustrate the possible mechanism. Methods Twenty-one male SD rats were divided into three groups randomly (random number) including control group (sham group, n =7 ) , cardiopulmonary resuscitation group (CPR group, n =7 ) and ulinastatin group (UTI group, n =7). The rats were anesthetized with pentobarbital sodium (45 -60 mg/kg) by intraperitoneal injection. The rats of sham group were only treated with endotracheal intubation. Ulinastatin (100 000 U/kg) were injected via caudal vein 2 hours prior to CPR, and cardiac arrest was made in rats and cardiopulmonary resuscitation was carried out in the UTI group, while equivalent volume of sterile saline was used instead in the CPR group. Blood and ileum samples were obtained at 48 hour after restoration of spontaneous circulation (ROSC). The levels of tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β) were assayed by ELISA (enzyme-linked immunosorbent assay) , the protein levels of caspase-3 were determined by western blot, the intestinal mucosa were stained by terminal- deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) and ileac mucosa were observed under transmission electron microscope. Data were processed with SPSS 17.0 software. Results The plasma levels of TNF-α and IL-1β were dramatically higher in CPR group than those in other two groups (CPR vs. sham, P <0.01; CPR vs. UTI, P <0.05). Moreover, the tight junctions between cells obviously broadened and loosened in the CPR group were found under electron microscope, however, this phenomenon was not obvious in the UTI group. A large number of apoptotic cells were observed by TUNEL assay in the CPR group, but a small number of apoptotic cells were observed in the UTI group. The protein levels of caspase-3 in the UTI group were higher than those in sham group, but lower than those in CPR group (both P <0.05 ). Conclusions Ulinastatin has protective effects on the intestinal barrier damaged after cardiopulmonary resuscitation in rats by decreasing the proinflammatory mediators in the blood, reducing the expression of caspase-3and then reducing the numbers of apoptotic intestinal cells. Key words: Ulinastatin; Cardiopulmonary resuscitation; TNF-α; IL-1β; Cyteine-containing aspartate-specific proteases; Apoptosis; Intestinal barrier; Protection

Decreased MicroRNA-26a expression causes cisplatin resistance in human non-small cell lung cancer

ABSTRACTBackground: Lung cancer is the most common cancer that is caused by perturbation of regulatory pathways rather than dysfunction of a single gene. Cisplatin (CDDP; cis-diamminedichloroplatinum II) is the first member of a class of platinum-containing anti-cancer medication, which binds to DNA and triggers apoptosis. CDDP-based chemotherapy is used to treat various types of cancers. However, the efficacy of CDDP in the treatment of non-small-cell lung cancer (NSCLC) is limited by acquired drug resistance. MicroRNAs have recently emerged as key regulators of cancers, and miR-26a is one of down-regulated miRNAs in A549/CDDPres cell line. This study aimed to investigate the role of miR-26a in CDDP resistance in NSCLC as well as the underlying mechanisms.Methods: In this study, we analyzed expressional profiles of CDDP resistance-related mRNA, miRNA, and transcription factors (TF) that regulate miRNA expression in NSCLC. A549 cells were treated with CDDP, miR-26a mimic, or miR-26a inhibitor, and followed by biological analysis including drug sensitivity assay, colony formation assay, terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling (TUNEL) assay, and cell cycle analysis. Luciferase assay was used to determine the target of miR-26a. The regulation of miR-26a in Akt pathway was measured by western blot.Results: High mobility group A (HMGA) 2 was identified as the target of miR-26a. Overexpression of miR-26a in A549 cells inhibited G1-S transition, increased cell death in response to CDDP treatment, and decreased the colony formation of A549 cells. MiR-26a significantly decreased the expression of E2F1, diminished Akt phosphorylation, and downregulated Bcl2 expression. Cell growth was suppressed by inhibiting HMGA2-mediated E2F1-Akt pathway.Conclusion: MiR-26a is responsible for A549 cell sensitivity in the treatment of CDDP through regulating HMGA2-mediated E2F1-Akt pathway.

Acute liver injury attenuation of a novel recombinant sTNFR through blocking hepatic apoptosis

Context: Tumor necrosis factor (TNF) α plays a key role in acute liver injury (ALI) induced by injection of d-galactosamine (D-Gal)/lipopolysaccharide (LPS). A novel recombinant trimeric sTNFRII, sTNFRII-gAD, has been tested to be effective in ameliorating ALI, when administered prior to ALI establishment. This study aims to validate the protective effect of sTNFRII-gAD when given after ALI setup and further explore its effect on hepatic apoptosis.Materials and methods: The treatments were carried out concomitantly with ALI establishment with clinically approved sTNFRII-Fc (the dimeric sTNFRII) as a positive control. Lethality, liver weight, and serum alanine transaminase were measured, and histological analysis was performed to evaluate liver injury induced by D-Gal/LPS. Additionally, Terminal-deoxynucleoitidyl transferase-mediated nick end labeling (TUNEL) and Western blot analyses of caspase-3 were used to examine hepatocellular apoptosis.Results: sTNFRII-gAD given after D-Gal/LPS injection turned out to attenuate animal mortality significantly (p < 0.01), and had better hepatic protection. In terms of apoptosis, both sTNFRII-gAD and sTNFRII-Fc displayed noticeable improvement of apoptosis evidenced by dramatic decline of active caspase-3 compared to the control group.Conclusions: The results demonstrated that sTNFRII-gAD therapeutically diminished the lethality induced by D-Gal/LPS, possibly through blocking hepatic apoptosis initiated by TNFα. Of note, sTNFRII-gAD was superior to sTNFRII-Fc in some respects, indicating a promising alternative for the therapeutic strategy against the diseases associated with excessive TNFα.

Protective mechanism of the resveratrol in liver injury of obstructive jaundice in rats

Objective To explore the protective mechanism and effect of the resveratrol (Res) for liver injury of obstructive jaundice.Methods The rats were divided randomly into three groups:the sham group receiving laparotomy without bile duct ligation (BDL),the BDL group and the BDL + Res group with the Res given following BDL.The expression of the silent information regulator 1 (SIRT1) and nuclear factor-κBp56 (NF-κB) proteins were analyzed by western blotting but immunocytochemical assay was performed to examine peroxisome proliferator activated receptor-alpha (PPARα) protein.Real-time polymerase chain reaction (PCR) was performed to determine the mRNA expression of SIRT1 and PPARα.Cell apoptosis was examined by terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) staining.Results After the BDL,the cholestasis model was established.Compared B group to A group,the alanine transaminase (ALT) was higher and the mRNA and proteins expression of the SIRT1 and the PPARα was lower,but the NF-κB protein and the rate of cell apoptosis was higher (P ＜ 0.05).Compared B group to C group,the ALT was higher and the mRNA and proteins expression of the SIRT1 and the PPARα was lower,but the NF-κB protein and the rate of cell apoptosis was significantly higher (P ＜ 0.05).Conclusion This study demonstrates that the Res could alleviate liver damage and that the Res plays a beneficial role to resist inflammation and apoptosis by activating the SIRT1 which probably inhibits the expression of NF-κB protein in cholestatic liver injury.The Res also shows antioxidant effect by promoting the expression of PPARα. Key words: Resveratrol; Cholestasis; Silent information regulator 1 ; Peroxisome proliferator activated receptor-alpha; Nuclear factor-κBp56

Effects of hypoxia preconditioning on inflammatory responses and apoptosis in the rat model after spinal cord injury

Objective To observe the effects of hypoxia preconditioning on inflammatory cytokines and apoptosis after spinal cord injury in the rat model.Methods 154 adult female wistar rats were randomly divided into three groups,including the spinal cord injury group,the spinal cord injury ± hypoxia precondition (HP) group,the sham operation group.Immunohistochemistry was performed to determine the expressions of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β)in spinal cord injury area at five time points (2,6,12 h,1,3 d).BBB score was used to evaluate the function recovery of hind limb.The method of immunohistochemistry and terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) were adopted to detect the level of B cell lymphoma/leukemia-2 (bcl-2) protein and the apoptotic cells at five time points (6 h,1,3,7,14 d)after spinal cord injury.Results The integral absorbance (IA) of TNF-α in HP group was decreased at 6 h and 12 h,compared with that in spinal cord injury group (5.187±0.628 vs.7.962±2.185; 3.219±0.724 vs.5.361 ±1.246,P＜0.05).The IA ofIL-1β in HP group was lower than spinal cord injury group at three time points (6,12 h,1 d) (3.845 ± 1.669 vs.6.674 ± 1.537 ; 6.241 ± 1.142 vs.9.961 ± 2.814 ; 2.264 ± 1.476 vs.4.418 ± 0.973,P ＜ 0.05).In HP group,the IA of bcl-2 protein was significantly higher than spinal cord injury group at any time point (4.335 ±0.563 vs.3.256 ± 1.024,P ＜0.05; 6.873 ±2.032 vs.5.029 ± 1.537,P ＜0.01 ; 11.869 ± 2.560 vs.6.893 ± 2.164,P ＜ 0.01 ; 10.340 ± 1.469 vs.8.074 ± 2.175,P ＜ 0.05 ; 8.143 ± 2.376 vs.5.238 ± 1.467,P ＜0.05).The apoptosis index (AI) of HP group was decreased on 1 d,3 d,7 d,14 d (20.63 ±5.86 vs.26.37 ±4.76,P＜0.05; 29.85 ±8.12 vs.37.96 ±3.81,P＜0.05; 31.56 ±4.01 vs.41.74 ±6.69,P ＜0.01 ; 22.66 ±5.24 vs.32.87 ±9.90,P ＜0.05),compared with the spinal cord injury group.The BBB scores of HP group was higher than spinal cord injury group on 7 d and 14 d (5.68 ±1.83 vs.3.06 ±1.21; 10.21 ±2.64 vs.6.45 ±1.97,P＜0.05).Conclusion Hypoxiapreconditioning can inhibit the inflammatory response of local tissue damage and reduce apoptosis through the high expression of bcl-2 protein,thus promote the neural functional recovery after spinal cord injury. Key words: Hypoxia precondition ; Spinal cord injury; Inflammation ; Apoptosis