Terminal Dexynucleotidyl Transferase Research Articles

Long Non-Coding RNA Plasmacytomavariant Translocation1 as ceRNA Adsorbs miR-195 to Relieve Osteoarthritis Symptoms

This study aimed to investigate the role of the long non-coding RNA plasmacytomavariant translocation1 (LncRNA PVT1) in the development of osteoarthritis (OA). The study used mice and performed DMM surgery to establish an OA model. PVT1 and miR-195 agomir were inhibited in the knee joints, and cartilage tissue specimens were collected for gene expression analysis, apoptotic protein detection, histopathological observation, and Mankin’s score evaluation. Enzymelinked immunosorbent assay (ELISA) was used to measure the levels of inflammatory cytokines interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in peripheral blood and cartilage tissues. Terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) staining was used to detect cartilage cell apoptosis. The results indicated that the OA group had higher plasmacytomavariant translocation1 (PVT1) expression and lower miR-195 expression than the sham-operated group. PVT1 was found to act as a molecular sponge for miR-195 through a dual-luciferase reporter assay. Knocking down PVT1 or upregulating miR-195 significantly attenuated inflammation and apoptosis in the bone joints of OA mice. This study provides new insights into the mechanism of OA progression and suggests that regulating the PVT1-miR-195 axis may be beneficial for improving the inflammatory microenvironment of cartilage tissue and apoptosis.

Ketone Body β-Hydroxybutyric Acid Ameliorates Dopaminergic Neuron Injury Through Modulating Zinc Finger Protein 36/Acyl-CoA Synthetase Long-Chain Family Member Four Signaling Axis-Mediated Ferroptosis

β-hydroxybutyrate (BHB) is one of main component of ketone body, which plays an important protective role in various tissues and organs. Whereas, its exact regulatory roles and mechanisms in Parkinson's disease (PD) have not been full elucidated. In this study, SN4741 cells and C57BL/6 mice were treated with 1-methyl-4-phenylpyridinium ion (MPP+)/1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to establish the PD model in vitro and in vivo. Cell viability and damage to dopaminergic neurons were measured by cell counting kit 8, Calcein-AM/PI staining, terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling and hematoxylin & eosin staining. Corresponding assay kits and BODIPY 581/591 C11 probe evaluated oxidative stress and intracellular iron levels. Western blot examined the ferroptosis-related proteins. MPTP/MPP+-treatment reduced cell viability but triggered oxidative stress and ferroptosis in SNA4741 cells and brain tissues of mice. However, these effects were dramatically reversed by BHB and Fer-1 treatment. Mechanistically, Zinc finger protein 36 (ZFP36) was a target of BHB, and its depletion could reverse the anti-oxidative stress and anti-ferroptosis roles of BHB. Moreover, ZFP36 could directly bound to acyl-CoA synthetase long-chain family member 4 (ACSL4) mRNA to decay its expression, thus negatively modulating ACSL4-mediated oxidative stress and ferroptosis. Summary, BHB alleviated oxidative stress and ferroptosis of dopaminergic neurons in PD via modulating ZFP36/ACSL4 axis, which provided some new understanding for PD prevention and treatment.

Punicalagin attenuates osteoarthritis progression via regulating Foxo1/Prg4/HIF3α axis.

Punicalagin (PUN) is a common anti-inflammatory polyphenol. However, the function and mechanism of PUN in osteoarthritis remains unknown. Chondrocytes were isolated from rats, and confirmed by toluidine blue staining and immunofluorescence. Chondrocytes were challenged by lipopolysaccharide (LPS), and rat osteoarthritis model was established by Hulth method. The secretion of inflammatory factors, cell viability and apoptosis were tested via enzyme linked immunosorbent assay (ELISA), MTT and flow cytometry. The levels of forkhead box O1 (Foxo1), proteoglycan 4 (Prg4), hypoxia-inducible factor-3α (HIF3α), autophagy-related genes or extracellular matrix (ECM)-related proteins were examined via quantitative reverse transcription polymerase chain reaction (qRT-PCR), western blot or immunohistochemistry. The cartilage tissue damage was assessed via hematoxylin-eosin (HE) staining, toluidine blue staining and terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick and labeling (TUNEL) staining. LPS triggered inflammatory injury in chondrocytes. PUN promoted autophagy to mitigate LPS-induced inflammatory injury. Foxo1 silence attenuated the effect of PUN on LPS-mediated autophagy inhibition and inflammatory injury. Promotion of Prg4/HIF3α axis abolished the influence of Foxo1 knockdown on LPS-mediated chondrocytes injury. PUN mitigated the inflammatory injury in rat osteoarthritis model by promoting autophagy and inhibiting inflammation and ECM degradation via Foxo1/Prg4/HIF3α axis. PUN attenuates LPS-induced chondrocyte injury and osteoarthritis progression by regulating Foxo1/Prg4/HIF3α axis.

Dexmedetomidine facilitates the expression of nNOS in the hippocampus to alleviate surgery-induced neuroinflammation and cognitive dysfunction in aged rats.

Postoperative cognitive dysfunction (POCD) is a common complication in the postoperative nervous system of elderly patients. Surgery-induced hippocampal neuroinflammation is closely associated with POCD. Dexmedetomidine (DEX) is an effective α2-adrenergic receptor agonist, which can reduce inflammation and has neuroprotective effects, thereby improving postoperative cognitive dysfunction. However, the mechanism by which DEX improves POCD is currently unclear. The purpose of the present study was therefore to identify how DEX acted on POCD. Male Sprague Dawley rats with exposed carotid arteries were used to mimic POCD. Locomotor activity was accessed by the open field test and the Morris water maze was performed to estimate spatial learning, memory and cognitive flexibility. Following animal sacrifice, the hippocampus was collected and cell apoptosis was determined by terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling staining. Subsequently, the expression of apoptosis-related proteins Bax, Bcl-2, cleaved caspase-3 and cleaved caspase-9 was determined by western blotting and the concentrations of TNF-α, IL-6, IL-1β and IL-10 were measured in serum using ELISA. Nitric oxide synthase and neuronal nitric oxide synthase activities in the hippocampus were also measured. The T lymphocyte subsets were analyzed by flow cytometry to evaluate the immune function in each group. Compared with the surgery group, DEX ameliorated POCD by improving cognitive dysfunctions and immune function loss, and attenuated neuroinflammation and neuronal apoptosis.

Effect of Zhulian needle insertion by slow twirling technique on peroxidation and apoptosis of kidney tissue in aging rats

To observe the effect of Zhulian needle insertion by slow twirling technique on the contents of hydrogen peroxide (H2O2) and malondialdehyde (MDA) and apoptosis rate in kidney tissue of aging model rats and explore the potential mechanism of such needling technique. A total of 40 adult male SD rats were randomized into a blank control group, a model control group, a Zhulian needling technique group and a routine acupuncture group, 10 rats in each one. Except the blank control group, the sub-acute aging rat models were established by intraperitoneal injection with 10% D-galactose solution, 5 mL/kg in the rats of the other groups. In the Zhulian needling technique group and the routine acupuncture group, Zhulian needle insertion by slow twirling technique and the routine acupuncture technique were exerted at "Guanyuan" (CV 4) and "Zusanli" (ST 36) respectively. The needles were retained for 30 min, once a day, for 28 days totally. After intervention, the content of H2O2 in kidney tissue was detected by colorimetry, the content of MDA in kidney tissue was detected by thiobarbituric acid method and the apoptosis rate of kidney cells was detected by terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling. Compared with the blank control group, the contents of H2O2 and MDA in kidney tissue and kidney cell apoptosis rate were all increased in the model control group (P<0.05). Compared with the model control group, the contents of H2O2 and MDA in kidney tissue and kidney cell apoptosis rate were all reduced in the Zhulian needling technique group and the routine acupuncture group (P<0.05), and these indexes in the Zhulian needling technique group were all lower than the routine acupuncture group (P<0.05). Zhulian needle insertion by slow twirling technique delays aging probably by regulating peroxidation and apoptosis.

Overexpression of miR-431 inhibits cardiomyocyte apoptosis following myocardial infarction via targeting HIPK3.

Acute myocardial infarction (AMI) is a common acute cardiovascular crisis. Although the diagnosis and treatment of AMI are constantly improving, the mortality of AMI is still very high, and its pathogenesis is still unclear. This article focuses on the role of microRNA-431 (miR-431) in regulating myocardial apoptosis after myocardial infarction (MI) and its potential molecular mechanism. We constructed cell models and animal models of MI. Quantitative reverse-transcription polymerase chain reaction (RT-PCR) was used to detect miR-431 expression in myocardium after MI. Western blot, cell counting kit-8 (CCK-8) assay, flow cytometry and terminal dexynucleotidyl transferase(TdT)-mediated dUTP nick end labeling (TUNEL) staining were performed to detect myocardial apoptosis; pathological sections of myocardium, serum lactate dehydrogenase (LDH) levels and Caspase-3 activity in myocardium were employed to evaluate myocardial injury of MI rats; echocardiography was utilized to assess cardiac function of rats. We revealed that miR-431 expression was decreased in H2O2-treated H9c2 cells and myocardium of MI rats. The expression of Cleaved Caspase-3 (C-Caspase-3) in H9c2 cells treated with H2O2 was significantly increased, the cell viability was dramatically decreased, the apoptosis rate and the percentage of TUNEL positive cells were notably increased, but up-regulation of miR-431 could reverse these effects. At the same time, compared with the sham group, serum LDH levels were observably increased, myocardial Caspase-3 activity was also increased, and cardiac function was greatly reduced, while overexpression of miR-431 could reduce myocardial injury and improve cardiac function of MI rats. Through the Luciferase reporter gene experiment, we found that miR-431 could directly target HIPK3. In summary, overexpression of miR-431 can inhibit apoptosis after myocardial infarction via targeting HIPK3, thereby reducing myocardial injury and improving cardiac function in MI rats.

Inhibition of microRNA-802-5p inhibits myocardial apoptosis after myocardial infarction via Sonic Hedgehog signaling pathway by targeting PTCH1.

The incidence of acute myocardial infarction (AMI) has increased significantly in recent years, seriously threatening human life and health. This paper focused on the role of microRNA-802-5p (miR-802-5p) in myocardial infarction (MI) and its underlying mechanisms. Quantitative Real-Time Polymerase Chain Reaction (RT-PCR) was employed to detect miR-802-5p expression. Western blot was performed to detect protein expression. Flow cytometry and terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) staining were performed to observe myocardial apoptosis. Hematoxylin-eosin (HE) staining was used to observe the morphology of myocardial tissue. The cardiac function of rats was detected using echocardiography. The expression of miR-802-5p was increased in hypoxic-treated H9c2 cells and infarcted myocardium in MI rats. Hypoxia treatment reduced the viability of cardiomyocytes and increased the level of lactate dehydrogenase (LDH) in the cell supernatant. Hypoxia treatment increased Bax expression in myocardial cells while Bcl-2 expression decreased, and the number of apoptotic cells increased. MiR-802-5p silencing reversed these effects. Moreover, miR-802-5p silencing reduced myocardial damage in MI rats, and significantly improved cardiac function. Through the Luciferase activity assay, we proved that miR-802-5p could directly target PTCH1. The knockdown of PTCH1 reversed the protective effect of miR-802-5p silencing on hypoxic myocardium. MiR-802-5p expression was increased in hypoxia-treated H9c2 cells and infarcted myocardium in MI rats. MiR-802-5p silencing could inhibit apoptosis after MI via activating Sonic Hedgehog signaling pathway by targeting PTCH1, thereby reducing myocardial injury and improving cardiac function of MI rats.

Inhibition of microRNA-495 inhibits hypoxia-induced apoptosis in H9c2 cells via targeting NFIB.

Acute myocardial infarction (AMI) is a serious cardiovascular disease that threatens human life. MicroRNA is considered to be an important participant in the pathophysiology of AMI. This article focused on the role of microRNA-495 (miR-495) in regulating apoptosis after myocardial infarction (MI) and its underlying mechanisms. H9c2 cells were cultured in an incubator containing 1% O2 to establish a cell model of MI. Quantitative reverse-transcription polymerase chain reaction (RT-PCR) was utilized to detect miR-495 expression in H9c2 cells. The effects of miR-495 and NFIB on hypoxia-treated H9c2 cells were observed by Western blot, lactate dehydrogenase (LDH) detection, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay, flow cytometry, and terminal dexynucleotidyl transferase(TdT)-mediated dUTP nick end labeling (TUNEL) staining. Luciferase reporter gene experiment was used to prove the regulatory relationship between miR-495 and NFIB. Hypoxia induced injury to H9c2 cells, which was manifested by decreased cell viability, increased LDH release, increased pro-apoptotic proteins (Bax, Cleaved Caspase-3) expression, decreased anti-apoptotic protein (Bcl-2) expression, and increased in the rate of apoptosis and TUNEL positive cells. MiR-495 expression was remarkably increased in H9c2 cells treated with hypoxia. Inhibiting miR-495 expression markedly alleviated the hypoxia-induced injury in H9c2 cells, while silencing NFIB aggravated the hypoxia-induced damage. In addition, NFIB was confirmed to be the target of miR-495. MiR-495 expression was increased in hypoxia-treated H9c2 cells. Silencing miR-495 could significantly inhibit hypoxia-induced apoptosis of H9c2 cells by targeting NFIB.

Lactobacillus acidophilus CICC 6074 inhibits growth and induces apoptosis in colorectal cancer cells in vitro and in HT-29 cells induced-mouse model

• The first study of the anti-cancer mechanism of Lactobacillus acidophilus CICC 6074 was carried out. • L. acidophilus CICC 6074 exerts anticancer effects in HT-29 cell-induced colon cancer mouse model. • L. acidophilus CICC 6074 may induce HT-29 cell apoptosis through the mitochondrial pathway. • The research may provide theoretical basis for development of probiotics functional food market. This research sought to assess the anticancer effects of Lactobacillus acidophilus CICC 6074 and its underlying mechanism of action. L. acidophilus CICC 6074 inhibited HT-29 cell activity and induced its apoptosis in a dose- and time-dependent manner. HT-29 cells exposed to L. acidophilus CICC 6074 showed mitochondrial membrane potential (ΔΨm) loss, as well as Cyt c is released from the mitochondria into the cytoplasm, causing Bax, Caspase-9 and Caspase-3 to be up-regulated and Bcl-2 to be down-regulated. To further explore the mechanism by which L. acidophilus CICC 6074 achieves its anti-cancer effect, a HT-29 cell-induced colon cancer mouse model was used. Compared with a colon cancer model control group, the high-dose L. acidophilus CICC 6074 group showed obvious colon cancer cell apoptosis as measured by histological morphology, electron microscopy, and terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay. An in vivo study showed that L. acidophilus CICC 6074 induced colon cancer apoptosis by up-regulating Bax, down-regulating Bcl-2, releasing Cyt c from the mitochondria into the cytoplasm, and activating Caspase-3 and Caspase-9. These results demonstrate that L. acidophilus CICC 6074 exerts anticancer effects via the activation of mitochondrial pathways which then induce apoptosis. The research could provide new ideas and theoretical basis for development of probiotics functional food market.

Overexpression of miR-129-5p Mitigates Sepsis-Induced Acute Lung Injury by Targeting High Mobility Group Box 1

BackgroundMicroRNAs are dysregulated in sepsis. Acute lung injury is a progressive syndrome during sepsis. However, the role of miR-129-5p in the development of acute lung injury induced by sepsis remains unclear. MethodsThe acute lung injury of sepsis model was established by cecal ligation puncture (CLP)-treated mice and lipopolysaccharide (LPS)-treated murine alveolar epithelial cell line (MLE)-12 cells. The lung injury in vivo was investigated by hematoxylin and eosin staining, terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling staining, enzyme-linked immunosorbent assay, lung wet-to-dry weight ratio, and myeloperoxidase activity. The lung injury in vitro was evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide, flow cytometry, and enzyme-linked immunosorbent assay. The expression levels of miR-129-5p and high mobility group box 1 (HMGB1) were measured by quantitative real-time polymerase chain reaction and Western blot. The association between miR-129-5p and HMGB1 was validated by luciferase assay and RNA immunoprecipitation. ResultsThe expression of miR-129-5p was decreased in CLP model and LPS-treated MLE-12 cells. Overexpression of miR-129-5p attenuated inflammatory response, apoptosis, lung wet/dry weight ratio, and myeloperoxidase activity induced by CLP surgery in vivo. Moreover, addition of miR-129-5p increased cell viability and suppressed cell apoptosis and inflammatory response in vitro. HMGB1 as a target of miR-129-5p alleviated miR-129-5p–mediated injury suppression in LPS-treated MLE-12 cells. ConclusionsmiR-129-5p protects against sepsis-induced acute lung injury by decreasing HMGB1 expression, providing new target for sepsis treatment.

MicroRNA-147 inhibits myocardial inflammation and apoptosis following myocardial infarction via targeting HIPK2.

The incidence of acute myocardial infarction (AMI) is increasing year by year, and it has become one of the diseases with the highest mortality in humans. MicroRNAs (miRNAs) are involved in the regulation of many diseases, including AMI. This study aims to investigate the function of miR-147 in myocardial infarction (MI) and its underlying mechanism of action. Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) was used to detect miR-147, Bcl-2 mRNA, and Bax mRNA expressions. Enzyme-linked immunosorbent assay (ELISA) kits were used to detect the levels of inflammatory factors (TNF-α, IL-6, IL-β) and lactate dehydrogenase (LDH). Besides, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay was performed to detect cell viability. Cell apoptosis was observed using terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL). Moreover, echocardiography was utilized to measure cardiac function of rats. HIPK2 expression was detected by Western blot. MiR-147 expression was significantly decreased in rat MI model and H2O2 treated H9c2 cells. H2O2 treatment increased the expression of inflammatory factors in H9c2 cells and induced apoptosis, while such effects were inhibited by overexpression of miR-147. Overexpression of miR-147 reversed the decrease in Bcl-2 expression and the increase in Bax expression in H9c2 cells caused by H2O2. In addition, overexpression of miR-147 could improve cardiac function and reduce serum LDH levels in myocardial infarction rats. Through TargetScan, we found that HIPK2 might have a binding site for miR-147. Moreover, overexpression of miR-147 could inhibit the expression of HIPK2. In MI, miR-147 expression is decreased in the myocardium and overexpression of miR-147 can inhibit myocardial inflammation and apoptosis and improve cardiac function in rats via targeting HIPK2.

MiR-942-5p alleviates septic acute kidney injury by targeting FOXO3.

Sepsis refers to the systemic inflammatory response caused by infection. Acute kidney injury (AKI) in sepsis is very common, and there are many complicated mechanisms for the occurrence of septic AKI. This article aimed to study the role of miR-942-5p in inflammation and apoptosis of septic AKI and its potential mechanism. Real Time-quantitative Polymerase Chain Reaction (RT-qPCR) was performed to detect the expression of RNAs. The protein expression was detected using Western blot. The contents of inflammatory factors in the cell supernatant were detected using commercial enzyme-linked immunosorbent assay (ELISA) kits. Cell Counting Kit-8 (CCK-8) assay was utilized to compare the cell viability of each group. Terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) staining and flow cytometry were used to observe cell apoptosis. MiR-942-5p expression was reduced in lipopolysaccharide (LPS)-treated HK-2 cells. MiR-942-5p mimic could observably increase miR-942-5p expression. The overexpression of miR-942-5p dramatically inhibits the expression of inflammatory factors and Bax, but increase Bcl-2 expression. MiR-942-5p overexpression greatly reversed the LPS-induced decrease in viability of HK-2 cells. In addition, we observed that LPS can markedly increase the number of apoptosis, while miR-942-5p mimic can reduce it. Taken together, our results demonstrated that miR-942-5p expression was reduced in the LPS-treated HK-2 cells, and miR-942-5p overexpression can inhibit LPS-induced inflammation and apoptosis of HK-2 cells via targeting FOXO3.

Effects of thymosin β4 on neuronal apoptosis in a rat model of cerebral ischemia‑reperfusion injury.

The aim of the present study was to investigate the protective effects of thymosin β4 (Tβ4) on neuronal apoptosis in rat middle cerebral artery occlusion ischemia/reperfusion (MCAO I/R) injury, and determine the mechanisms involved in this process. Forty-eight adult male Sprague-Dawley rats were randomly divided into three groups (n=16 per group): A sham control group, an ischemia/reperfusion group (I/R group), and a Tβ4 group. The focal cerebral I/R model was established by blocking the right MCA for 2 h, followed by reperfusion for 24 h. The Zea-Longa method was used to assess neurological deficits. Cerebral infarct volume was assessed using 2,3,5-triphenyltetrazolium chloride staining, and pathological changes were observed via hematoxylin and eosin staining. The terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay was used to detect apoptosis. The expression of glucose-regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), and caspase-12 (CASP12) protein was assessed using immunohistochemistry and western blotting 24 h after reperfusion. Infarct volume and neuronal damage in the I/R and Tβ4 groups were significantly greater than those observed in the sham group. The Zea-Longa score, neuronal apoptosis, and expression of GRP78, CHOP, and CASP12 in the I/R and Tβ4 groups were significantly higher than those reported in the sham group. However, the Longa score and neuronal apoptosis were lower in the Tβ4 group compared to the I/R group. The expression of GRP78 was significantly increased, whereas that of CHOP and CASP12 was significantly decreased in the Tβ4 group compared to the I/R group. The present data revealed that Tβ4 can inhibit neuronal apoptosis by upregulating GRP78 and downregulating CHOP and CASP12, thereby reducing cerebral I/R injury.

A study on role and mechanism of TLR4/NF-κB pathway in cognitive impairment induced by cerebral small vascular disease.

To investigate the role and potential mechanism of Toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-κB) signaling pathway in cognitive impairment induced by cerebral small vascular disease (CSVD), so as to provide a reference for the clinical treatment of CSVD-induced cognitive impairment. Mice with TLR4 gene knockout (n = 20) and those with wild-type TLR4 gene (n = 40) aged 8-10 weeks old were divided into blank control group (Control group, n = 20), wild-type + CSVD group (WT + CSVD group, n = 20) and TLR4 gene knockout + CSVD group (TLR4 KO + CSVD group, n = 20). Allogeneic thrombosis (particle diameter: 50-70 mm) was injected to the mouse's external carotid artery to create a model of learning and memory dysfunction. Step-down test and Y-type maze test were utilized to examine the learning and memory abilities of the mice. Reverse transcription-polymerase chain reaction (RT-PCR) and immunoblotting techniques were adopted to measure the levels of apoptosis-related genes [B-cell lymphoma/leukemia-2 (Bcl-2), Bcl-2-associated X protein (Bax), C-caspase-3 and T-caspase-3] in the brain tissues of mice. Terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) method was applied to detect the apoptosis of neuronal cells in the brain tissues. Meanwhile, the levels of oxidative stress markers, including superoxide dismutase (SOD), gp91 and malondialdehyde (MDA), were measured. Finally, the expression level of TLR4/NF-κB pathway was detected. The latency in the step-down test in the WT + CSVD group was remarkably longer than that in the Control group, and the number of errors was evidently larger than that in the Control group (p < 0.05). At the same time, in the WT + CSVD group, the expression levels of pro-apoptotic genes Bax and C-caspase-3 were up-regulated markedly, while the expression level of anti-apoptotic gene Bcl-2 declined notably (p < 0.05). TUNEL results showed that the number of apoptotic cells in the brain tissues in the WT + CSVD group was about 12 times that in the Control group (p < 0.05). Meanwhile, the SOD expression level was lowered, and the MDA expression level was elevated in the brain tissues in the WT + CSVD group. In addition, the TLR4/NF-κB pathway was prominently activated in the mice in the WT + CSVD group (p < 0.05). After TLR4 gene knockout, the cognitive functions of the mice were improved markedly, and the apoptosis of neuronal cells and oxidative stress in the brain tissues were suppressed significantly in the meantime. Moreover, the activation of the TLR4/NF-κB signaling pathway was also inhibited. The TLR4/NF-κB pathway is involved in the occurrence and development of CSVD-induced cognitive impairment through regulating oxidative stress and cell apoptosis.

Nobiletin Inhibits Cell Viability via the SRC/AKT/STAT3/YY1AP1 Pathway in Human Renal Carcinoma Cells.

Nobiletin is a polymethoxy flavonoid isolated from Citrus depressa and Citrus reticulata. It has been reported that nobiletin can suppress tumors. We primarily explored the antitumor effects of nobiletin and the associated potential mechanisms in ACHN and Caki-2 renal carcinoma cells. A CCK-8 assay and cloning experiments were used to assess cell viability, and a transwell assay and scratch test were used to assess metastatic ability. The cell cycle was analyzed by flow cytometry, whereas apoptosis was analyzed using flow cytometry and a terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay. Protein expression was examined by Western blot and immunofluorescence. Renal cancer cells were subcutaneously transplanted into nude mice for in vivo studies. The data showed that nobiletin administration significantly dose- and time-dependently suppressed renal cancer cell proliferation; moreover, nobiletin treatment induced cell cycle arrest in the G0/G1 phase and promoted apoptosis. Immunofluorescence analysis indicated that nobiletin decreased the nuclear localization of signal transducer and activator of transcription 3 (STAT3) and YY1-associated protein 1 (YY1AP1). Western blot showed that the levels of phosphorylated SRC, phosphorylated AKT serine/threonine kinase (AKT), and phosphorylated STAT3 were decreased, whereas that of phosphorylated YY1AP1 was increased. The results further showed that application of insulin-like growth factor 1 (IGF1) was able to reverse the nobiletin-induced changes in the levels of phosphorylated AKT, phosphorylated STAT3, and phosphorylated YY1AP1, and could also reverse the antitumor effects of nobiletin. The results of in vivo experiments showed that, compared to the control, tumor volume and weight were both reduced following nobiletin treatment. In conclusion, our study demonstrated that nobiletin can inhibit renal carcinoma cell viability and provides a novel therapeutic approach for the treatment of kidney cancer.

Resveratrol protects myocardial apoptosis induced by ischemia-reperfusion in rats with acute myocardial infarction via blocking P13K/Akt/e-NOS pathway.

To elucidate the protective role of resveratrol (RSV) in myocardial apoptosis induced by ischemia-reperfusion injury in rats with acute myocardial infarction (AMI), and to explore its underlying mechanism. The AMI rat model was successfully established by ligation of the left anterior descending coronary artery. Rat cardiomyocytes were isolated and cultured. Cells were divided into four groups, including: control group (no specific treatment), AMI group (acute ischemia-reperfusion treatment), AMI+RSV group (RSV pretreatment for 24 h before acute ischemia-reperfusion) and AMI+ RSV+LY group (RSV pretreatment combined with 40 μmol/L phosphatidylinositide 3-kinases (PI3K) pathway inhibitor LY294002 for 24 h before acute ischemia-reperfusion). Morphology of apoptotic cardiomyocytes in each group was observed by Hoechst staining. The proliferation, apoptosis and cell cycle progression of cardiomyocytes were determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay, terminal dexynucleotidyl transferase(TdT)-mediated dUTP nick end labeling (TUNEL) assay and flow cytometry, respectively. Finally, the protein levels of genes relative to PI3K/Akt/eNOS pathway were detected by Western blot. Hoechst staining showed a large number of necrotic cells, cell retraction, enhanced refractive index and enlarged cell gap in AMI group. A small number of necrotic cells were found in AMI+RSV group, which was significantly fewer than that of AMI group. Meanwhile, remaining cells presented normal morphology. However, a great number of necrotic cells were observed in AMI+RSV+LY group, which was obviously more than that of AMI+RSV group. Compared with control group, cells in AMI group showed significantly decreased proliferative rate, increased early phase, late phase and total one of apoptosis. In AMI group, the ratio of G0/G1 phase was remarkably increased, whereas those of S and G2/M phases were decreased. Moreover, the expression levels of phosphorylated Akt (p-Akt) and phosphorylated e-NOS (p-eNOS) were significantly downregulated in AMI group. In AMI+RSV group, cell apoptosis, cell cycle progression and levels of p-Akt and p-eNOS showed the opposite trends as those of AMI group. However, LY294002 pretreatment reversed the protective role of RSV in cellular behaviors of cardiomyocytes. RSV protects cardiomyocyte apoptosis from ischemia-reperfusion injury through regulating phosphorylation levels of proteins relative to PI3K/Akt/e-NOS pathway.

Grape seed proanthocyanidin extract improves myocardial function in rats with myocardial infarction by inhibiting PARP-1 activity

Objective To observe the improvement effect of grape seed proanthocyanidin extract (GSPE) on myocardial function in rats with myocardial infarction and to explore its mechanism. Methods Myocardial infarction model rats were randomly divided into five groups: model group, GSPE low, middle and high group, poly ADP-ribose polymerase-1 (PARP-1) inhibitor BSI-201 group, with 15 rats in each group, and another 15 rats were as the sham operation control group. Rats in GSPE low, medium and high dose groups were administrated with 100 mg/ml GSPE, 200 mg/ml GSPE and 400 mg/ml GSPE respectively; BSI-201 group was given 120 μmol/L BSI-201 of gavage, and the control group and model group were treated with equal volume of normal saline of gavage, 1 times a day for 4 weeks. Doppler echocardiography was used to evaluate cardiac function in rats; myocardial infarction volume was detected by three-phenyl tetrazolium chloride (TTC) staining; hematoxylin and eosin (HE) staining was used to detect histopathological changes of myocardial tissue; terminal dexynucleotidyl transferase (TdT) mediated dUTP nick end labeling (TUNEL) assay was used to detect the apoptosis of myocardial cells; Western blot was used to analyze the expressions of Bax, cleaved-caspase3, Bcl-2 and PAR in myocardial tissue. Results Compared with the control group, the heart rate, left ventricular end diastolic pressure, myocardial infarction area and cell apoptosis rate were significantly increased in the model group, and the mean arterial pressure and left ventricular systolic pressure were decreased significantly (P<0.05); Myocardial cells were broken and necrotic, irregular fibers arranged in the myocardium, and a large number of inflammatory cells were infiltrated; the protein expressions of Bax, cleaved-caspase3 and PAR in myocardial tissues were increased significantly (P<0.05), and the protein expression of Bcl-2 was significantly decreased (P<0.05). Compared with the model group, the heart rate, the left ventricular end diastolic pressure, myocardial infarction area and cell apoptosis rate in the GSPE low, middle and high dose groups and the BSI-201 group were significantly decreased, and the mean arterial pressure and the left ventricular systolic pressure were increased significantly (P<0.05); the degree of histopathological damage was alleviated; the expressions protein of Bax, cleaved-caspase3 and PAR in myocardial tissues were decreased significantly (P<0.05), and the protein expression of Bcl-2 was increased significantly (P<0.05). Conclusions Grape seed proanthocyanidin extract may inhibit cardiomyocyte apoptosis and improve myocardial function of rats with myocardial infarction by inhibiting PARP-1 activation. Key words: Proanthocyanidins/PD; Poly(ADP-ribose) polymerases/DE; Myocardial infarction/DT; Apoptosis; Rats

Vitamin D receptor regulates high-level glucose induced retinal ganglion cell damage through STAT3 pathway.

To explore the protective role of Vitamin D Receptor (VDR) in retinal ganglion cells (RGCs) after high-level glucose induction, and to investigate its underlying mechanism. Primary RGCs were isolated from 24-hour-old Sprague Dawley (SD) rats and cultured in 50 mmol/L glucose. The expression of VDR in RGCs induced by 50 mmol/L glucose at different time points was determined by Real-time quantitative polymerase chain reaction (qRT-PCR) and Western blot, respectively. Subsequently, VDR siRNA was transfected into RGCs. Transfection efficiency was determined by qRT-PCR and Western blot, respectively. The protein expressions levels of VDR, signal transducer and activator of transcription 3 (STAT3) and p-STAT3 in RGCs after VDR knockdown were determined by Western blot. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) and cell counting kit-8 (CCK-8) assay were conducted to access the viability of RGCs after high-level glucose induction. Ki-67 staining was performed to detect the proliferation of RGCs. Meanwhile, the apoptosis of RGCs was evaluated by using Annexin-V FITC/PI and TUNEL (terminal dexynucleotidyl transferase(TdT)-mediated dUTP nick end labeling) assay, respectively. In addition, caspase-3 activity in RGCs was detected by relative commercial kit. After 4 days of high-level glucose induction, the viability of RGCs was remarkably decreased. VDR was highly expressed in RGCs during high-level glucose culture. The mRNA and protein expression levels of VDR were both significantly downregulated after the transfection of VDR siRNA in RGCs. Meanwhile, VDR knockdown in RGCs significantly increased the viability and proliferative ability of RGCs, whereas significantly decreased apoptotic rate and caspase-3 activity. In addition, the protein level of p-STAT3 in RGCs was remarkably downregulated after VDR knockdown. Inhibition of VDR exerts a protective role in high-level glucose induced RGCs damage by activating the STAT3 pathway.

Pro-apoptosis effects of protocatechuic acid in the early stage of infectious bursal disease virus infection

Infectious bursal disease virus (IBDV) is a very important small RNA virus in the family of Birnaviridae, which can cause severe immunosuppressive effects and pathological damages in young chickens. It can replicate in bursal lymphocytes and impede the growth and development of B cells, finally causing bursal lymphocytes apoptosis. Previous results have shown that protocatechuic acid (PCA) as an important phenolic compound could effectively improve the survival rate of chickens infected with IBDV. The current study aimed to explore how PCA influenced the pathogenesis of IBDV, especially lymphocyte apoptosis in the process of IBDV infection. The results showed that PCA could effectively alleviate bursal pathological changes at the early stage of IBDV invasion. Moreover, bursal lymphocyte apoptosis for tissue section samples was largely elevated by PCA by using the terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) method while the bursal lymphocyte apoptosis ratio was also increased by PCA by flow cytometry in the early stage of IBDV infection in vivo. Meanwhile, PCA could promote non-lymphocyte apoptosis in vitro. Further study displayed that the potential mechanisms mainly relied on regulation of the expressions of pro-apoptotic protein Bax and anti-apoptotic Bcl-2, thus speeding up the process of IBDV-infected cell apoptosis and preventing virus infection. Meanwhile, the results displayed that the PI3K/Akt and NF kappa B signal pathways might play an important role in promoting cell apoptosis after IBDV infection. Overall, PCA as a potent antiviral drug precursor is expected to be applied in the poultry industry as a substitute for clinical antiviral application.

Adeno-associated virus 2 mediated gene transfer of vascular endothelial growth factor Trap: a new treatment option for glioma

ABSTRACTBackground: Adeno-associated virus 2 mediated gene transfer of vascular endothelial growth factor Trap (AAV2-VEGF-Trap) has been reported to inhibit the growth of primary tumor as well as distant metastasis in 4T1 metastatic breast cancer models. The aim of this study was to investigate the inhibiting efficacy of AAV2-VEGF-Trap for glioma.Methods: The intracranial transplanted model of glioma in rats was established. They were treated with AAV2-VEGF-Trap, bevacizumab (BEV), temozolomide (TMZ), TMZ combined with AAV2-VEGF-Trap, TMZ combined with BEV and the control group, respectively. A 7.0 Tesla magnetic resonance (MR) was used to assess the tumor volumes and obtain the apparent diffusion coefficient (ADC) values. Immunohistochemical and terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) staining were used to evaluate the effects on tumor angiogenesis, proliferation and apoptosis.Results: The combination of TMZ with AAV2-VEGF-Trap or BEV showed greater tumor growth inhibition than the other groups, and the ADC values in these two groups were larger than that of the control group. The decreased microvessel density in treatment groups which contain AAV2-VEGF-Trap or BEV was observed. The reduced proliferation activity in groups containing TMZ and increased apoptotic tumor cells in TMZ combined with AAV2-VEGF-Trap group and TMZ combined with BEV group were detected. In addition, there were no differences in antitumor effect, ADC values, Ki-67 and CD31 staining and apoptosis analysis between the two combined therapy groups.Conclusion: AAV2-VEGF-Trap has an obvious anti-angiogenic effect and inhibits the growth of glioma just by a single intravenous injection, which is similar to BEV. Moreover, there is a synergistic antitumor effect between AAV2-VEGF-Trap and TMZ.