Deoxynucleotidyl Transferase-mediated dUTP-biotin Nick Research Articles

Network pharmacology-based approach to explore the underlying mechanism of sinomenine on sepsis-induced myocardial injury in rats.

Background: Sepsis, a systemic disease, usually induces myocardial injury (MI), and sepsis-induced MI has become a significant contributor to sepsis-related deaths in the intensive care unit. The objective of this study is to investigate the role of sinomenine (SIN) on sepsis-induced MI and clarify the underlying mechanism based on the techniques of network pharmacology. Methods: Cecum ligation and puncture (CLP) was adopted to induce sepsis in male Sprague-Dawley (SD) rats. Serum indicators, echocardiographic cardiac parameters, and hematoxylin and eosin (H&E) staining were conducted to gauge the severity of cardiac damage. The candidate targets and potential mechanism of SIN against sepsis-induced MI were analyzed via network pharmacology. Enzyme-linked immunosorbent assay was performed for detecting the serum concentration of inflammatory cytokines. Western blot was applied for evaluating the levels of protein expression. Terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labeling assay was applied to assess cardiomyocyte apoptosis. Results: SIN significantly improved the cardiac functions, and attenuated myocardial structural damage of rats as compared with the CLP group. In total, 178 targets of SIN and 945 sepsis-related genes were identified, and 33 overlapped targets were considered as candidate targets of SIN against sepsis. Enrichment analysis results demonstrated that these putative targets were significantly associated with the Interleukin 17 (IL-17) signal pathway, inflammatory response, cytokines-mediated signal pathway, and Janus Kinase-Signal Transducers and Activators of Transcription (JAK-STAT) pathway. Molecular docking suggested that SIN had favorable binding affinities with Mitogen-Activated Protein Kinase 8 (MAPK8), Janus Kinase 1 (JAK1), Janus Kinase 2 (JAK2), Signal Transducer and Activator of Transcription 3 (STAT3), and nuclear factor kappa-B (NF-κB). SIN significantly reduced the serum concentration of Tumor Necrosis Factor-α (TNF-α), Interleukin 1 Beta (IL-1β), Interleukin 6 (IL-6), Interferon gamma (IFN-γ), and C-X-C Motif Chemokine Ligand 8 (CXCL8), lowered the protein expression of phosphorylated c-Jun N-terminal kinase 1 (JNK1), JAK1, JAK2, STAT3, NF-κB, and decreased the proportion of cleaved-caspase3/caspase3. In addition, SIN also significantly inhibited the apoptosis of cardiomyocytes as compared with the CLP group. Conclusion: Based on network pharmacology analysis and corresponding experiments, it was concluded that SIN could mediate related targets and pathways to protect against sepsis-induced MI.

Cysteine-rich 61(CYR61) alleviates cyclophosphamide-induced proliferation inhibition in ovarian granulosa cells via suppressing NLRP3/caspase1-mediated pyroptosis.

We investigated the level of Cysteine-rich 61 (CYR61) in premature ovarian failure as well as its regulatory molecular mechanism in this study. Cyclophosphamide (CTX) was used to induce OGCs (rat ovarian granulosa cells) and rats to establish in vivo and in vitro premature ovarian failure models. H&E staining was used to detect the pathological changes of ovarian histopathology. Si-NLRP3 (NOD-like receptor thermal protein domain associated protein 3, NLRP3) and si-CYR61 were transfected into OGCs using lipofectamine 3000. RT-qPCR and western blot were used to detect the expressions of CYR61 in ovarian tissue and OGCs. It showed that the expression of CYR61 was significantly down-regulated in premature ovarian failure model. Cell viability was detected using a Cell Counting Kit-8 (CCK-8) kit. TUNEL (Terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labeling) staining was used to detect the apoptosis. 5-Ethynyl-2'-deoxyuridine (EdU) and SA-β-gal (senescence-associated β-galactosidase) staining were used to assess the proliferation and senescence. The expression of CYR61 in OGCs and ovarian tissues were detected by immunofluorescence and immunohistochemical staining. Overexpression of CYR61 significantly promoted OGCs proliferation and inhibited pyroptosis and apoptosis. Western blot was used to detect the protein expressions of p53 and p21 in OGCs. Flow cytometry was used to detect the pyroptosis. CYR61 overexpression inhibited the expression of NLRP3 and caspase-1 in CTX-induced OGCs according to western blot results. Moreover, we found that CYR61 overexpression down-regulated the protein expressions of p53 and p21 in CTX-induced OGCs. CYR61 inhibited CTX-induced OGCs senescence, and the mechanism may be related to the regulation of caspase-1/NLRP3-induced pyroptosis.

Which Distant Organ is Most Affected by Lower Extremity Ischemia-Reperfusion?

It has been experimentally shown that reperfusion injury occurs in many remote organs after ischemia-reperfusion (I/R) of the lower extremity. However, which distant organ is affected more after I/R of the lower extremity has not been investigated. In this study, we investigate which remote organ is predominantly affected after lower extremity I/R. Twenty male Sprague-Dawley rats were randomly divided into 2 groups: sham (group 1) and lower extremity I/R (group 2). In group 2, 1hr of ischemia of the left lower extremity was followed by 24hr of reperfusion of the limb. After reperfusion, the lung, liver, kidney, heart, and small intestine tissues were harvested in both groups. In the I/R group, the malondialdehyde levels were significantly higher in the heart and small intestine tissues than those in other tissues (P < 0.05). In addition, in the I/R group, the glutathione and glutathione peroxidase activities were also higher in the heart tissues than those in other tissues (P < 0.05). However, these results were not significant because the malondialdehyde, glutathione, and glutathione peroxidase levels of the heart tissues in the control group were higher than those of the other tissues. Therefore, no statistically significant difference was found between the tissues in terms of the histological damage score we created and the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-positive cell numbers. There was no difference in the severity of reperfusion injury between the tissues we examined after lower extremity I/R. This suggests that every distal organ should be carefully monitored after lower extremity I/R.

Protective effects of gastrin-releasing peptide receptor antagonist RC-3095 in an animal model of hepatic ischemia/reperfusion injury.

We aimed to evaluate effects of RC-3095 on mice with hepatic ischemia followed by reperfusion (I/R) injury and further explore the possible underlying mechanism. Mice were subjected to partial hepatic ischemia for 60min followed by different durations of reperfusion. Levels of gastrin-releasing peptide (GRP) and GRP receptor (GRPR) in the blood and liver were detected by enzyme-linked immunosorbent assay (ELISA) or western blotting (WB) after 3, 6, 12, or 24h of reperfusion. RC-3095 or normal saline (control) was given i.p. at the time of reperfusion. Expressions of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and IL-10 in blood and liver samples were examined with ELISA. Neutrophil influx into the liver was assessed by flow cytometry and myeloperoxidase assay. Hematoxylin-eosin staining of the liver and terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling assay were used to determine hepatic injury and hepatocellular necrosis. Activation of nuclear factor (NF)-κB and p38/extracellular regulated protein kinase (ERK) mitogen activated protein kinase (MAPK) was investigated with WB. The expression of GRP was upregulated within 3h after reperfusion and remained elevated for up to 24h in the liver, whereas GRPR was also upregulated after 3 or 6h of reperfusion, but returned to baseline levels within 24h. RC-3095 significantly reduced the inflammatory hepatic injury, liver neutrophil accumulation, and hepatocellular apoptosis, probably by inhibiting activation of NF-κB or p38/ERK MAPK. These findings supported that GRP-GRPR played an important role in hepatic I/R injury, and RC-3095 ameliorated liver damage by suppressing the inflammatory response and hepatocellular necrosis.

Dexmedetomidine attenuates spinal cord ischemia\u2013reperfusion injury through both anti-inflammation and anti-apoptosis mechanisms in rabbits

BackgroundDexmedetomidine (Dex) can improve neuronal viability and protect the spinal cord from ischemia–reperfusion (I/R) injury, but the underlying mechanisms are not fully understood. This study investigated the effects of dexmedetomidine on the toll-like receptor 4 (TLR4)-mediated nuclear factor κB (NF-κB) inflammatory system and caspase-3 dependent apoptosis induced by spinal cord ischemia–reperfusion injury.MethodsTwenty-four rabbits were divided into three groups: I/R, Dex (10 µg/kg/h prior to ischemia until reperfusion), and Sham. Abdominal aortic occlusion was carried out for 30 min in the I/R and Dex groups. Hindlimb motor function was assessed using the Tarlov scoring system for gait evaluation. Motor neuron survival and apoptosis in the ventral grey matter were assessed by haematoxylin–eosin staining and terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labelling staining. The expression and localisation of ionised calcium-binding adaptor molecule 1, TLR4, NF-κB and caspase-3 were assessed by immunoreactivity analysis. The levels of interleukin 1β and tumour necrosis factor α were assessed using enzyme-linked immunosorbent assays.ResultsPerioperative treatment with dexmedetomidine was associated with a significant preservation of locomotor function following spinal cord ischemia–reperfusion injury with increased neuronal survival in the spinal cord compared to control. In addition, dexmedetomidine suppressed microglial activation, inhibited the TLR4-mediated NF-κB signalling pathway, and inhibited the caspase-3 dependent apoptosis.ConclusionsDexmedetomidine confers neuroprotection against spinal cord ischemia–reperfusion injury through suppression of spinal cord inflammation and neuronal apoptosis. A reduction in microglial activation and inhibition of both the TLR4-mediated NF-κB signalling pathway and caspase-3 dependent apoptosis are implicated.

MiR-125b blocks Bax/Cytochrome C/Caspase-3 apoptotic signaling pathway in rat models of cerebral ischemia-reperfusion injury by targeting p53

ABSTRACTObjective: To explore the potential effect of miR-125b on p53-mediated regulation of Bax/Cytochrome C/Caspase-3 apoptotic signaling pathway in rats with cerebral ischemia-reperfusion (CIR) injury.Methods: Sprague-Dawley (SD) rats were used to conduct CIR injury and injected with miR-125b mimic/inhibitor or p53 inhibitor (Pifithrin-α, PFT-α). Dual-luciferase reporter gene assay was used to analyze the targeting relationship between miR-125b and p53. Longa scoring and Triphenyl tetrazolinm chloride (TTC) staining were used to test the neurologic function and determine infarct size, respectively. Hematoxylin-eosin (HE) and Nissl’s stainings were conducted to observe the morphology of cortical neurons. Neuronal nuclei (NeuN) expression was detected by immunohistochemical staining. QRT-PCR was performed to detect the expressions of miR-125b and p53. TUNEL staining and Western blotting was used to determine neuronal apoptosis and expressions of Bax/Cytochrome C/Caspase-3 signaling pathway-related proteins, respectively.Results: Our results showed that miR-125b could directly target p53. As observed, overexpression of miR-125b could obviously reduce the neurological score, infarct size, and brain water content after CIR in rats, which also improved the morphology of cortical neurons, increased the number of neurons, reduced neuronal apoptosis, and inhibited the expressions of Bax/Cytochrome C/Caspase-3 pathway. Moreover,the similar results were observed in rats with CIR after injected with PFT-α. But no significant differences in each index were found in CIR group and CIR + anti-miR-125b + PFT-α group.Conclusion: MiR-125b exerts protective effects on CIR injury through inhibition of Bax/Cytochrome C/Caspase-3signaling pathway via targeting p53, which is likely to be a promising treatment for CIR.Abbreviations: 3’–UTR: 3–untranslated region; CIR: cerebral ischemia–reperfusion; CIS: cerebral ischemic stroke; PFT–α: Pifithrin–α; PVDF: polyvinylidene fluoride; SD: Sprague–Dawley; TBST: tris buffered saline with tween. TTC staining: Triphenyl tetrazolinm chloride staining; TUNEL: Terminal deoxynucleotidyl transferase–mediated dUTP–biotin nick end labeling.

Loss of expression rather than cytoplasmic mislocalization of RUNX3 predicts worse outcome in non-small cell lung cancer

Functional inactivation of human runt-related transcription factor 3 (RUNX3) through mutation or epigenetic silencing has been well-documented in many cancerous entities. In addition to gene mutation and promoter hypermethylation, cytoplasmic mislocalization has emerged as another major manifestation of RUNX3 dysfunction in malignancies including breast, colorectal and gastric cancers. The aim of the present study was to investigate whether patients with non-small cell lung cancer (NSCLC) and different RUNX3 expression patterns would have different overall survival (OS), and the associations between different patterns of clinicopathological parameters and clinical outcome. Expressions of RUNX3 and Ki-67 were immunohistochemically detected in normal lung tissue (n=5) and surgically resected tissues from NSCLC patients (n=188). The optimal cutoff of RUNX3 was determined by X-tile software associated with their survival. Apoptotic index in cancerous tissue was evaluated using the terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labelling method. The prognostic significance of different expression patterns of RUNX3 was determined by means of Kaplan-Meier survival estimates and log-rank tests. It was revealed that loss of RUNX3 expression in NSCLC was correlated with a low cancerous apoptotic index (P<0.001), shorter OS and worse prognosis (P=0.0142), while no statistical difference of apoptotic index (P=0.73) or survival (P=0.3781) was determined between patient subgroups with different localization of RUNX3 expression, which was quite different from the situation demonstrated in other malignancies. In conclusion, loss of expression rather than cytoplasmic mislocalization of RUNX3 predicted worse outcome in NSCLC, which was quite different from what manifested in other cancer types, and thus, the underlying mechanism may deserve further investigation.

Resveratrol Alleviates Inflammatory Responses and Oxidative Stress in Rat Kidney Ischemia-Reperfusion Injury and H2O2-Induced NRK-52E Cells via the Nrf2/TLR4/NF-κB Pathway

Background: Ischemia-reperfusion injury (IRI) is one of the major causes of postoperative renal allograft dysfunction, which is mainly the result of proinflammatory reactions including inflammatory responses, oxidative stress, and metabolic disorders. Resveratrol (RSV) plays an important role in protecting various organs in IRI because it reduces oxidative stress, lessens the inflammatory response, and exerts anti-apoptotic effects. The aim of this study was to demonstrate the renoprotective effect of RSV in inhibiting inflammatory responses, reducing oxidative stress, and decreasing cell apoptosis in vivo and in vitro. Methods: RSV was administered before renal ischemia and H₂O₂ induction. Serum and kidneys were harvested 24 h after reperfusion and NRK-52E cells were collected 4 h after H₂O₂ stimulation. Serum creatinine and blood urea nitrogen were used to assess renal function. Hematoxylin and eosin staining was performed to assess histological injury. Quantitative real-time PCR and enzyme-linked immunosorbent assay were used to assess proinflammatory cytokine expression. Oxidative stress–related proteins, such as Nrf2 and TLR4, were evaluated by western blot. Terminal deoxynucleotidyl transferase–mediated dUTP-biotin nick end labeling assay was used to detect apoptotic cells in tissues, and western blot was used to evaluate the expression of caspase-3, -8, and -9 in this study. Results: RSV inhibited inflammatory responses and improved renal function after renal IRI. Additionally, RSV decreased oxidative stress and reduced cell apoptosis by upregulating Nrf2 expression, downregulating the TLR4/NF-κB signaling pathway, and by decreasing caspase-3 activity and caspase cascades. Conclusion: Our study demonstrated the mechanisms underlying RSV renoprotection. We found that RSV exerts its greatest effects by blocking inflammatory responses, lowering oxidative stress, and reducing apoptosis via the Nrf2/TLR4/NF-κB pathway.

Neuroprotective Effects of Luteolin Against Spinal Cord Ischemia-Reperfusion Injury by Attenuation of Oxidative Stress, Inflammation, and Apoptosis.

Luteolin (LU) is a widely distributed flavonoid with multitarget effects. The objective of this study was to determine whether LU could reduce the ischemia-reperfusion injury of the spinal cord (SCII) in a rat model. Forty-eight rats were divided into four groups: sham, SCII, SCII+L-LU (50 mg/kg), and SCII+H-LU (100 mg/kg). Abdominal aortic occlusion was carried out for 40 min in all groups. Hindlimb motor functions were evaluated using the Tarlov scoring system. Nissl and terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labeling (TUNEL) staining were used to detect cell survival and apoptosis in the spinal cord. Spinal cord samples were taken for determination of malondialdehyde, xanthine oxidase, superoxide dismutase, and glutathione peroxidase activities. The levels of tumor necrosis factor-α, interleukin (IL)-1β, and IL-18 were assessed using ELISA kits to examine the inflammatory responses in the spinal cord. Western blot analysis was used to examine the expression of nuclear factor erythroid 2-related factor (Nrf2) and nod-like receptor pyrin domain-containing 3 protein (NLRP3) levels. We found that LU pretreatment significantly improved the locomotor function of rats after SCII, increased neuron survival, and inhibited apoptosis in the spinal cord. Furthermore, the oxidative stress and inflammatory response were significantly suppressed upon treatment with LU. Finally, LU upregulated Nrf2 levels and downregulated NLRP3 protein expression in SCII tissues. Thus, LU exhibited a neuroprotective effect following SCII by alleviating oxidative stress and inhibiting inflammatory responses and cell apoptosis. The possible mechanism may be related to the activation of Nrf2 and inhibition of NLRP3 inflammasome pathway.

Cellular interactions of the phosphorylated form of AKT in prostate cancer

Phospho-Akt (P-Akt1) promotes proliferation and increased survival in vitro and plays an important role in prostate cancer (PCa) progression as well as the prediction of the probability of recurrence. In this study, the goal was to demonstrate the involvement and impact of P-Akt1 on cellular interactions, biomechanisms, and pathways in PCa. Tissue microarrays from 640 PCa patients were immunostained with various antibodies. Ki-67 was used to measure proliferation index, and terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labeling was used for apoptotic index. Increased expression of P-Akt1 was associated with an increased proliferation but inversely correlated with apoptotic index. Higher levels of P-Akt1 are associated with both higher levels of cytoplasmic p27 and higher levels of nuclear p27, suggesting an involvement in both cytoplasmic entrapment and phosphorylation of p27. P-Akt1 expression significantly correlated with nuclear and cytoplasmic staining of FHKR and GSK. The strongest correlations were found with the P- forms of both, suggesting enzyme kinetics in the latter. Here, phosphorylation is the principal method of FHKR and GSK inactivation. P-Akt1 correlated with nuclear transcription factor kappa B, suggesting a role in the inhibition through phosphorylation of nuclear transcription factor kappa B. The results of the current study are unique because of the scope of the markers and the size of the population used. In vitro- and in vivo-derived information of P-Akt1 and its downstream effectors demonstrates significant involvement in PCa. Our data suggest that PCa uses multiple mechanisms to regulate this pathway and substantiate the concept of redundancy in cancer pathway regulation. Consequently, new hypothesis-driven studies can be derived from this information.

Ginsenoside Rd Attenuates DNA Damage by Increasing Expression of DNA Glycosylase Endonuclease VIII-like Proteins after Focal Cerebral Ischemia.

Background:Ginsenoside Rd (GSRd), one of the main active ingredients in traditional Chinese herbal Panax ginseng, has been found to have therapeutic effects on ischemic stroke. However, the molecular mechanisms of GSRd's neuroprotective function remain unclear. Ischemic stroke-induced oxidative stress results in DNA damage, which triggers cell death and contributes to poor prognosis. Oxidative DNA damage is primarily processed by the base excision repair (BER) pathway. Three of the five major DNA glycosylases that initiate the BER pathway in the event of DNA damage from oxidation are the endonuclease VIII-like (NEIL) proteins. This study aimed to investigate the effect of GSRd on the expression of DNA glycosylases NEILs in a rat model of focal cerebral ischemia.Methods:NEIL expression patterns were evaluated by quantitative real-time polymerase chain reaction in both normal and middle cerebral artery occlusion (MCAO) rat models. Survival rate and Zea-Longa neurological scores were used to assess the effect of GSRd administration on MCAO rats. Mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) damages were evaluated by the way of real-time analysis of mutation frequency. NEIL expressions were measured in both messenger RNA (mRNA) and protein levels by quantitative polymerase chain reaction and Western blotting analysis. Apoptosis level was quantitated by the expression of cleaved caspase-3 and terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labeling assay.Results:We found that GSRd administration reduced mtDNA and nDNA damages, which contributed to an improvement in survival rate and neurological function; significantly up-regulated NEIL1 and NEIL3 expressions in both mRNA and protein levels of MCAO rats; and reduced cell apoptosis and the expression of cleaved caspase-3 in rats at 7 days after MCAO.Conclusions:Our results indicated that the neuroprotective function of GSRd for acute ischemic stroke might be partially explained by the up-regulation of NEIL1 and NEIL3 expressions.

Rhizome extracts of Curcuma zedoaria Rosc induce caspase dependant apoptosis via generation of reactive oxygen species in filarial parasite Setaria digitata in vitro

Human lymphatic filariasis (LF) is mainly caused by filarial parasite Wuchereria bancrofti and is the second leading cause of long term and permanent disability in tropical countries. To date, incapability to eliminate long lived adult parasites by current drugs remains the major challenge in the elimination of LF. Hence, in the current study, the efficacy of rhizome extracts of Curcuma zedoaria (a plant traditionally used in Sri Lanka in the management of LF) was evaluated as an effective filaricide in vitro. Sequential solvent extracts of C. zedoaria rhizomes were screened for in vitro antifilarial activity at 0.01–1 mg/mL concentrations by motility inhibition assay and 3-(4, 5 dimethylthiazol-2-yl)-2, 5 diphenyl tetrazolium bromide (MTT) reduction assay using cattle parasite Setaria digitata as a model organism. Exposure of parasites to hexane and chloroform extracts of C. zedoaria caused a dose dependant reduction in motility and viability of microfilariae (IC50 = 72.42 μg/mL for hexane extract, 191.14 μg/mL for chloroform extract) and adult parasites (IC50 = 77.07 μg/mL for hexane extract, 259.87 μg/mL for chloroform extract). Both extracts were less toxic to human peripheral blood mononuclear cells when compared to filariae. A dose dependant increase in caspase 3/CED 3 and a decrease in total protein content, cyclooxygenase (COX) and protein tyrosine phosphatase (PTP) activities were observed in adult parasites treated with hexane or chloroform extract. A significant degree of chromatin condensation and apoptotic body formation were also observed in these worms by Hoechst 33342 and terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labeling (TUNEL) staining respectively. Dose dependant chromosomal DNA laddering was observed in treated adult worms but not in microfilariae in response to both extracts. Oxidative stress parameters such as reduction in reduced glutathione (GSH) levels and increase in glutathione s transferase (GST), superoxide dismutase (SOD) and catalase activities, increased reactive oxygen levels (ROS) and lipid peroxidation were also observed indicating that an apoptotic event is induced by reactive oxygen species.

Efficacy of Compound Therapy by Ginseng and Ciprofloxacin on Bacterial Prostatitis.

Genitourinary tract infections play a significant role in male infertility. Infections of reproductive sex glands, such as the prostate, impair function and indirectly affect male fertility. The general aim of this study is to investigate the protective effect of Korean red ginseng (KRG) on prostatitis in male rats treated with ciprofloxacin (CIPX). In this experimental study, we randomly divided 72 two male Wistar rats into 9 groups. The groups were treated as follows for 10 days: i. Control (no medication), ii. Sham [(normal saline injection into the vas deferens and oral administration of phosphate-buffered saline (PBS)], iii. Ginseng, iv. CPIX, v. CIPX+ginseng, vi. Uropathogenic Escherichia coli (E. coli) (UPEC), vii. UPEC+ginseng, viii. UPEC+CIPX, and ix. UPEC+ginseng+CIPX. The rats were killed 14 days after the last injection and the prostate glands were removed. After sample preparation, routine histology was performed using hematoxylin and eosin staining. The terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling (TUNEL) method was used to determine the presence of apoptotic cells. The severity score for acinar changes and inflammatory cell infiltration in the UPEC+CIPX group did not significantly different from the UPEC group. However this score significantly decreased in the UPEC+CIPX+ginseng group compared to the UPEC group. Apoptotic index of all ginseng treated groups significantly decreased compared to the UPEC and CPIX groups. These results suggested that ginseng might be an effective adjunct in CIPX treatment of prostatitis. The combined use ginseng and CIPX was more effective than ginseng or CIPX alone.

Myofibrillogenesis regulator-1 attenuated hypoxia/reoxygenation-induced apoptosis by inhibiting the PERK/Nrf2 pathway in neonatal rat cardiomyocytes.

The purpose of this study was to investigate the role of myofibrillogenesis regulator-1 (MR-1) in cardiomyocyte apoptosis induced by hypoxia/reoxygenation (H/R), through protein kinase R-like ER kinase (PERK)/nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. To address this aim, an H/R model of neonatal rat cardiomyocytes was used. MR-1 was overexpressed using an adenoviral vector system and knocked down using MR-1 specific siRNA. Apoptosis was assessed by using Annexin V/PI double staining, terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling assay, and the Bcl-2/Bax ratio. Western blotting was used to detect the protein levels of MR-1, glucose-regulated protein 78 (GRP78), total and phosphorylated PERK, Nrf2, activating transcription factor 4 (ATF4), C/EBP homologous protein (CHOP), Bcl-2 and Bax. Immunofluorescence staining was used to assess the subcellular location of Nrf2. We found that H/R induced significant apoptosis in neonatal rat cardiomyocytes. MR-1 overexpression attenuated H/R-induced apoptosis, decreased GRP78 (P < 0.01) and CHOP expression (P < 0.05), and increased the Bcl-2/Bax ratio (P < 0.01). MR-1 overexpression suppressed H/R-induced PERK phosphorylation, Nrf2 nuclear translocation, and ATF4 expression (P < 0.01). While MR-1 knockdown aggravated H/R-induced apoptosis, increased expression of GRP78 and CHOP (P < 0.05), and decreased the Bcl-2/Bax ratio (P < 0.01). MR-1 knockdown significantly increased H/R-induced PERK phosphorylation (P < 0.05), Nrf2 nuclear translocation, and ATF4 expression (P < 0.01). These findings suggest that MR-1 alleviates H/R-induced cardiomyocyte apoptosis through inhibition of the PERK/Nrf2 pathway.

Acetylcholine inhibits tumor necrosis factor α activated endoplasmic reticulum apoptotic pathway via EGFR-PI3K signaling in cardiomyocytes.

Previous findings have shown that acetylcholine (ACh) decreased hypoxia-induced tumor necrosis factor alpha (TNF α) production, thus protected against cardiomyocyte injury. However, whether and how ACh affects TNF α-induced endoplasmic reticulum (ER) stress and cell apoptosis remain poorly defined. This study was aimed at determining the effect of ACh in H9c2 cells after TNF α stimulation. Presence of ER stress was verified using the ER stress protein markers glucose regulatory protein 78 (GRP78) and C/EBP homologous protein (CHOP). Cell apoptosis was shown by caspase-3 activation and terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling. Exogenously administered ACh significantly decreased these TNF α-induced changes. Moreover, when the cells were exposed to nonspecific muscarinic receptor (M AChR) inhibitor atropine, methoctramine (M2 AChR inhibitor) or the epidermal growth factor receptor (EGFR) inhibitor AG1478, the cardioprotection elicited by ACh was diminished. Furthermore, the above effects were also blocked by M2 AChR or EGFR siRNA, indicating that EGFR transactivation by M2 AChR may be the major pathway responsible for the benefits of ACh. In addition, LY294002, a phosphatidylinositol-3-kinase (PI3K) inhibitor, displayed the similar trends as AG1478, suggesting that PI3K/Akt signaling may be the downstream of EGFR in ACh-elicited anti-apoptotic property. Together, these data indicate that EGFR-PI3K/Akt signaling is involved in M2 AChR-mediated ER apoptotic pathway suppression and the subsequent survival of H9c2 cardiomyocytes. We have identified a novel pathway underlying the cardioprotection afforded by ACh.

Evaluation of apoptosis using TUNEL assay and caspase-3 immunostaining in NMU induced rat mammary tumours

Avoidance of apoptosis is an important event of tumourigenesis. Tumour progression is dependent on balance between cell proliferation and apoptosis. The present study was carried out to evaluate apoptosis in N-methyl nitrosourea (NMU) induced rat mammary tumours by terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labelling (TUNEL) assay in correlation with caspase-3 and PCNA immunostaining. A total of 22 female virgin Wistar rats of 28–30 days of age were selected for the experiment. NMU was injected at 50th, 80th and 110th days of age in 16 rats and 6 control group rats received the vehicle only. Six numbers of mammary tumours were successfully induced in 4 (25%) rats with average latency period of 87±2.63 days. The mean counts of PCNA and caspase-3 immunostaining were 57.67±9.45 and 1.82±0.21, respectively. The mean count of the TUNEL positive cells were 2.44±0.69. It was observed that apoptosis was evident by TUNEL assay and caspase-3 immunostaining in highly proliferative and more aggressive NMU induced rat mammary tumours, similar to that observed in human breast tumours.

Panax Quinquefolium Saponin Attenuates Ventricular Remodeling After Acute Myocardial Infarction by Inhibiting Chop-Mediated Apoptosis

Panax quinquefolium saponin (PQS) alleviates hypoxia-reoxygenation injury of cardiomyocytes in vitro by inhibiting excessive endoplasmic reticulum stress (ERS)-related apoptosis. We hypothesized that inhibition of excessive ERS-related apoptosis contributes to cardioprotection in ventricular remodeling after acute myocardial infarction (AMI). Sprague-Dawley rats subjected to AMI were randomly treated with water, PQS (50 mg/kg per day, 100 mg/kg per day, or 200 mg/kg per day), or taurine (300 mg/kg per day), an ERS inhibitor, for 4 weeks. Left ventricular (LV) fractional shortening, ejection fraction, and structure were then evaluated using echocardiography. Myocardial infarct size was measured by Evans blue and 2,3,5-triphenyhetrazolium chloride staining. The hydroxyproline level was assayed using the colorimetric method. Cardiomyocyte apoptosis was detected using terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labeling. In addition, expression of ERS molecules in the noninfarcted myocardium was detected using Western blotting. We found that PQS treatment significantly reduced infarct size and LV dilation and improved LV ejection fraction and fractional shortening in rat hearts. Panax quinquefolium saponin treatment also decreased hydroxyproline level in noninfarcted myocardium. Panax quinquefolium saponin treatment significantly decreased expression of glucose regulating protein 78, calreticulin, C/EBP homologous protein (CHOP), and Bax protein, as well as increased Bcl-2 protein expression in noninfarcted myocardium. Panax quinquefolium saponin treatment (200 mg/kg per day) mimicked the results achieved from the taurine-treated rats. Expression of CHOP positively correlated with the apoptosis index of cardiomyocytes in the noninfarcted myocardium (r = 0.797, P < 0.01). Taken together, PQS treatment significantly improves AMI-induced LV remodeling, and this may be attributed to inhibiting CHOP-mediated ERS-related apoptosis.

Grape seed proanthocyanidins ameliorate pancreatic beta-cell dysfunction and death in low-dose streptozotocin- and high-carbohydrate/high-fat diet-induced diabetic rats partially by regulating endoplasmic reticulum stress

BackgroundIt is increasingly being realized that failure of pancreatic beta cells to secrete enough insulin to adequately compensate for obesity and insulin resistance is the primary defects of type 2 diabetes mellitus (T2DM). Pancreatic beta cells possess a highly developed and active endoplasmic reticulum (ER), reflecting their role in folding, export and processing of newly synthesized insulin. ER stress-induced pancreatic beta-cell failure is a novel event in the pathogenesis of T2DM. Some studies with antioxidants indicated a beneficial impact on ER stress. Our previous study found that strong antioxidants, grape seed proanthocyanidins (GSPs), ameliorated ER stress to protect skeletal muscle from cell death in type 2 diabetic rats. The present study continued to investigate the effect of GSPs on beta-cell failure and ER stress in diabetic pancreas.MethodsMale Sprague–Dawley rats made type 2 diabetic with 2 injections of 25 mg/kg streptozotocin and 8 weeks of the high-carbohydrate/high-fat diet were fed a basal diet with or without GSPs administration for 16 weeks. Oral glucose tolerance, plasma glucose, serum insulin and the score of beta-cell function were measured. Morphological observation was performed by light and electron microscopic analyses. Islet cell apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labeling staining. Additionally, the level of insulin and the expression of ER stress markers in pancreatic islets were also studied using immunohistochemical staining.ResultsAfter 16 weeks treatment, the score of beta-cell function and the abnormal oral glucose tolerance of diabetic rats were partially reversed by GSPs treatment. The efficacious effect of GSPs was also manifested in the amelioration of pancreatic damage and ER dilatation by microscopic analyses. Moreover, GSPs treatment increased normal insulin content and decreased the number of apoptotic cells in diabetic islets. Importantly, GSPs treatment partially alleviated ER stress by decreasing some ER stress markers.ConclusionThese findings suggest that GSPs might have auxiliary therapeutic potential for pancreatic beta-cell dysfunction and death in T2DM.

Functional proteomics reveals the protective effects of saffron ethanolic extract on hepatic ischemia‐reperfusion injury

Hepatic ischemia-reperfusion (IR) injury is a common clinical problem and ROS may be a contributing factor on IR injury. The current study evaluates the potential protective effect of saffron ethanol extract (SEE) in a rat model upon hepatic IR injury. Caspases 3 and terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labeling (TUNEL) results showed increased cell death in the IR samples; reversely, minor apoptosis was detected in the SEE/IR group. Pretreatment with SEE significantly restored the content of antioxidant enzymes (SOD1 and catalase) and remarkably inhibited the intracellular ROS concentration in terms of reducing p47phox translocation. Proteome tools revealed that 20 proteins were significantly modulated in protein intensity between IR and SEE/IR groups. Particularly, SEE administration could attenuate the carbonylation level of several chaperone proteins. Network analysis suggested that saffron extract could alleviate IR-induced ER stress and protein ubiquitination, which finally lead to cell apoptosis. Taken together, SEE could reduce hepatic IR injury through modulating protein oxidation and our results might help to develop novel therapeutic strategies against ROS-caused diseases.

Antineoplastic effects of deoxyelephantopin, a sesquiterpene lactone from Elephantopus scaber, on lung adenocarcinoma (A549) cells

Deoxyelephantopin, a sesquiterpene lactone from Elephantopus scaber, showed inhibition of the growth of various tumor cells in vitro. In the present study, we investigated the cytotoxicity and apoptosis-inducing capacity of deoxyelephantopin on lung adenocarcinoma (A549) cells. The cytotoxic effect of deoxyelephantopin on A549 cells and normal lymphocytes was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and 50% inhibitory concentration (IC50) value was determined. The self-renewal and proliferating potential of A549 cells after treatment with deoxyelephantopin were examined by colony formation assay. Cellular morphology of deoxyelephantopin-treated cells was observed using phase-contrast microscopy. The induction of apoptosis was evaluated using acridine orange and ethidium bromide staining, Hoechst 33342 staining, terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end-labeling (TUNEL) assay, DNA fragmentation analysis and Annexin V-fluorescein isothiocyanate staining by flow cytometry. Activation of caspases was detected using fluorogenic substrate specific to caspases 2, 3, 8 and 9 and flow cytometric analysis. The total cellular DNA content and expression of cleaved poly (ADP-ribose) polymerase was also analyzed. Deoxyelephantopin exhibited cytotoxicity to A549 cells (IC50 = 12.287 μg/mL), however, there was no toxicity towards normal human lymphocytes. Deoxyelephantopin suppressed the colony-forming ability of A549 cells in a dose-dependent manner. Acridine orange, ethidium bromide and Hoechst 33342 staining showed cell shrinkage, chromosomal condensation and nuclear fragmentation, indicating induction of apoptosis. Deoxyelephantopin increased apoptosis of A549 cells, as evidenced by more TUNEL-positive cells. DNA fragmentation and Annexin V staining revealed late-stage apoptotic cell population. Deoxyelephantopin inhibited A549 cell growth by cell cycle arrest at G2/M phase and induced apoptosis through both extrinsic and intrinsic pathways. These results suggest that deoxyelephantopin has great potential as a new chemotherapeutic agent to be developed further for the treatment of lung cancer.