Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling Research Articles

CDK5 Upregulated by ELF3 Transcription Promotes IL-1β-induced Inflammation and Extracellular Matrix Degradation in Human Chondrocytes.

Osteoarthritis (OA) is a common chronic disease with age-associated increase in both incidence and prevalence. The cyclin-dependent kinase 5 (CDK5), which is a member of the CDK family, is involved in many chronic diseases. This study was performed to explore the functional role of CDK5 in OA and to discuss the detailed molecular mechanisms. The expressions of CDK5 and ELF3 before or after transfection were detected with reverse transcription-quantitative PCR (RT-qPCR) and western blot. 5-ethynyl-2'-deoxyuridine (Edu) and terminal deoxynucleoitidyl transferase-mediated nick-end labeling (TUNEL) assays were used to detect the proliferation and apoptosis of C28/I2 cells. The levels of inflammatory cytokines were estimated using enzyme-linked immunosorbent assay (ELISA) while the expressions of proteins implicated in extracellular matrix (ECM) degradation- and apoptosis were detected using western blot. Additionally, the activity of CDK5 promoters and its binding with ELF3 were detected using luciferase activity assay and chromatin immunoprecipitation (CHIP) assay. In the present study, it was discovered that the mRNA and protein expressions of CDK5 were significantly increased in IL-1β-induced C28/I2 cells. After depleting CDK5 expression, the apoptosis, inflammation and ECM in C28/I2 cells with IL-1β induction were suppressed. It was also found that ELF3 expression was increased in IL-1β-induced C28/I2 cells and acted as a transcription factor binding to the CDK5 promoter to regulate its transcriptional expression. The further experiments evidenced that ELF3 overexpression partially reversed the inhibitory effects of CDK5 deficiency on IL-1β-induced apoptosis, inflammation and ECM in C28/I2 cells. Collectively, CDK5 that upregulated by ELF3 transcription could promote the development of OA.

Effect of Shoutai Pills on expression of key glycolytic proteins and apoptosis related factors at maternal fetal interface in mouse model of threatened abortion with syndrome of kidney deficiency

This study aims to explore the mechanism of Shoutai Pills in treating threatened abortion. According to the random number table method, ICR female mice were randomized into a normal group, a model group, a dydrogesterone group, and a Shoutai Pills group, with 15 mice in each group. Mice were administrated with normal saline(normal and model groups) or the suspension of Shoutai Pills or dydrogesterone by gavage at 9:00 am every day. At 16:00 every day, mice in the normal group were administrated with an equal volume of distilled water, while those in the model, Shoutai Pills, and dydrogesterone groups were administrated with hydrocortisone solution by gavage for 4 consecutive days. ICR female and male mice were caged in a ratio of 2∶1 during the pre-estrous or estrous period. From the first day of pregnancy, drug administration was continued for 5 consecutive days. On day 6, mice were administrated with mifepristone by gavage to establish the model of kidney deficiency-induced abortion. On day 6 of pregnancy, 10 female ICR mice were randomly selected from each group, and the uterus was collected for observation of the pathological changes of trophoblasts at the maternal-fetal interface by hematoxylin-eosin(HE) staining. The protein levels of key enzymes of glycolysis, hexokinase 2(HK2), enolase 1(ENO1), pyruvate kinase M2(PKM2), and lactate dehydrogenase A(LDHA), were determined by Western blot and immunofluorescence. The expression of apoptosis-related proteins including B cell lymphoma-2(Bcl-2), Bcl-2-associated protein X(Bax), and cysteinyl aspartate-specific proteinase-3(caspase-3) was determined by Western blot and real-time PCR. Terminal-deoxynucleoitidyl transferase-mediated nick-end labeling was employed to examine apoptosis. The embryo loss rate of the remaining five female mice was calculated by trypan blue staining method on day 14 of pregnancy. On day 14 of pregnancy, the embryo loss rate of the normal group was 5.00%, which was lower than that(27.78%) in the model group(P<0.05). Dydrogesterone and Shoutai Pills groups showed reduced embryo loss rates(10.26% and 7.50%, respectively) compared with the model group. On day 6 of pregnancy, compared with the normal group, the model group showed down-regulated expression of HK2, ENO1, PKM2, LDHA, and Bcl-2 and up-regulated expression of Bax and caspase-3(P<0.05). Compared with the model group, dydrogesterone and Shoutai Pills up-regulated the expression of HK2, ENO1, PKM2, LDHA, and Bcl-2 and down-regulated the expression of Bax and caspase-3(P<0.05). Compared with that in the normal group, the apoptosis rate in the model group increased(P<0.05). Compared with the model group, dydrogesterone and Shoutai Pills reduced the apoptosis rate(P<0.05). In conclusion, Shoutai Pills can reduce the embryo loss rate and protect embryos by promoting aerobic glycolysis at the maternal-fetal interface and inhibiting the apoptosis of trophoblasts in mice.

Tanshinone Ⅱ_A inhibits ferroptosis via Nrf2 signaling pathway to protect liver in rats of non-alcoholic fatty liver disease

This study investigated the protective effect of tanshinone Ⅱ_A(TSⅡ_A) on the liver in the rat model of non-alcoholic fatty liver disease(NAFLD) and the mechanism of TSⅡ_A in regulating ferroptosis via the nuclear factor E2-related factor 2(Nrf2) signaling pathway. The rat model of NAFLD was established with a high-fat diet for 12 weeks. The successfully modeled rats were assigned into model group, low-and high-dose TSⅡ_A groups, and inhibitor group, and normal control group was set. Enzyme-linked immunosorbent assay was employed to determine the content of superoxide dismutase(SOD) and malondialdehyde(MDA) in the serum of rats in each group. A biochemical analyzer was used to measure the content of aspartate aminotransferase(AST), alaninl aminotransferase(ALT), total cholesterol(TC), and triglycerides(TG). Hematoxylin-eosin(HE) staining was used to detect pathological damage in liver tissue. Terminal-deoxynucleoitidyl transferase-mediated nick end labeling(TUNEL) was employed to examine the apoptosis of the liver tissue. Oil red O staining, MitoSOX staining, and Prussian blue staining were conducted to reveal lipid deposition, the content of reactive oxygen species(ROS), and iron deposition in liver tissue. Western blot was employed to determine the expression of Nrf2, heme oxygenase-1(HO-1), glutathione peroxidase 4(GPX4), ferroptosis suppressor protein 1(FSP1), B cell lymphoma-2(Bcl-2), and Bcl-2 associated X protein(Bax) in the liver tissue. The result showed that TSⅡ_A significantly reduced the content of MDA, AST, ALT, TC, and TG in the serum, increased the activity of SOD, decreased the apoptosis rate, lipid deposition, ROS, and iron deposition in the liver tissue, up-regulated the expression of Nrf2, HO-1, FSP1, GPX, and Bcl-2, and inhibited the expression of Bax in the liver tissue of NAFLD rats. However, ML385 partially reversed the protective effect of TSⅡ_A on the liver tissue. In conclusion, TSⅡ_A could inhibit ferroptosis in the hepatocytes and decrease the ROS and lipid accumulation in the liver tissue of NAFLD rats by activating the Nrf2 signaling pathway.

MiR-181a-5p knockdown ameliorates sevoflurane anesthesia-induced neuron injury via regulation of the DDX3X/Wnt/β-catenin signaling axis.

Sevoflurane is one of the most widely used inhaled anesthetics. MicroRNAs (miRNAs) have been demonstrated to affect sevoflurane anesthesia-induced neuron damage. The purpose of this study was to investigate the role and mechanism of miR-181a-5p in sevoflurane-induced hippocampal neuronal injury. Primary hippocampal neurons were identified using microscopy and immunofluorescence. The viability and apoptosis of sevoflurane anesthesia-induced neurons were detected by cell counting kit-8 (CCK-8) assay and terminal-deoxynucleoitidyl transferase-mediated nick end-labeling (TUNEL) staining assay, respectively. The levels of apoptosis- and oxidative stress-related proteins as well as the markers in the Wnt/β-catenin signaling pathway were examined by immunoblotting. Enzyme-linked immuno-sorbent assays were performed to examine the levels of inflammatory cytokines. Luciferase reporter assay was conducted to validate the combination between miR-181a-5p and DEAD-box helicase 3, X-linked (DDX3X). Sevoflurane exposure led to significantly inhibited hippocampal neuron viability and elevated miR-181a-5p expression. Knockdown of miR-181a-5p alleviated sevoflurane-induced neuron injury by reducing cell apoptosis, inflammatory response, and oxidative stress. Additionally, DDX3X was targeted and negatively regulated by miR-181a-5p. Moreover, miR-181a-5p inhibitor activated the Wnt/β-catenin pathway via DDX3X in sevoflurane-treated cells. Rescue experiments revealed that DDX3X knockdown or overexpression of Wnt antagonist Dickkopf-1 (DKK1) reversed the suppressive effects of miR-181a-5p inhibitor on cell apoptosis, inflammatory response, and oxidative stress in sevoflurane-treated neuronal cells. MiR-181a-5p ameliorated sevoflurane-triggered neuron injury by regulating the DDX3X/Wnt/β-catenin axis, suggesting the potential of miR-181a-5p as a novel and promising therapeutic target for the treatment of sevoflurane-evoked neurotoxicity.

Mechanism of n-butanol fraction of Wenxia Formula combining with gefitinib in treating non-small cell lung cancer based on network pharmacology and in vitro experiment

This study combined network pharmacology, molecular docking, and in vitro experiments to explore the potential mechanism of the active components of the n-butanol fraction of Wenxia Formula(NWXF) combined with gefitinib(GEF) in treating non-small cell lung cancer(NSCLC). Ultra-performance liquid chromatography-quadrupole Orbitrap mass spectrometry(UPLC-Q-Orbitrap MS) was employed to detect the main chemical components of NWXF. The active components of NWXF were retrieved from SwissADME, and the candidate targets of these active components were retrieved from SwissTargetPrediction. Online Mendelian Inheritance in Man(OMIM) and GeneCards were searched for the targets of NSCLC. Cytoscape 3.9.0 and STRING were employed to build the protein-protein interaction(PPI) network with the common targets shared by NWXF and NSCLC. Gene Ontology(GO) annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment were performed in DAVID to predict the potential mechanisms. Finally, molecular docking between the main active ingredients and key targets was conducted in SYBYL-X 2.0. The methyl thiazolyl tetrazolium(MTT) assay was employed to evaluate the inhibitory effects of NWXF and/or GEF on the proliferation of human non-small cell lung cancer cells(A549 and PC-9). Additionally, the impact of NWXF on human embryonic lung fibroblast cells(MRC-5) was assessed. The effectiveness of the drug combination was evaluated based on the Q value. The terminal-deoxynucleoitidyl transferase mediated nick-end labeling(TUNEL) assay was employed to examine the apoptosis of A549 and PC-9 cells treated with NWXF and/or GEF. Quantitative real-time PCR(qRT-PCR) was employed to measure the mRNA levels of epidermal growth factor receptor(EGFR), c-Jun N-terminal kinase(JNK), and Bcl2-associated X protein(Bax) in the A549 and PC-9 cells treated with NWXF and/or GEF. Western blot was employed to determine the protein levels of EGFR, p-EGFR, JNK, p-JNK, and Bax in the A549 and PC-9 cells treated with NWXF and/or GEF. A total of 77 active components, 488 potential targets, and 49 key targets involved in the treatment of NSCLC with NWXF were predicted. The results of GO annotation showed that NWXF may treat NSCLC by regulating the biological processes such as cell proliferation, apoptosis, and protein phosphorylation. KEGG enrichment revealed that the key targets of NWXF in treating NSCLC were enriched in the mitogen-activated protein kinase(MAPK), phosphatidylinositol 3-kinase(PI3K)-protein kinase B(AKT), hypoxia-inducible factor-1(HIF-1), and microRNA-related signaling pathways. Molecular docking results showed that 91.9% of the docking scores were greater than 5, indicating the strong binding capability between main active components and key targets. The cell experiments demonstrated that NWXF combined with GEF synergistically inhibited the proliferation, promoted the apoptosis, decreased p-EGFR/EGFR and p-JNK/JNK values, down-regulated the mRNA levels of EGFR and JNK, and up-regulated the mRNA and protein levels of Bax in A549 and PC-9 cells. In conclusion, NWXF combined with GEF can regulate the EGFR/JNK pathway to promote the apoptosis of NSCLC cells, thus treating NSCLC.

Aqueous extract of Epimedium sagittatum mitigates pulmonary fibrosis in mice

This study aims to investigate the intervention effect of the aqueous extract of Epimedium sagittatum Maxim on the mouse model of bleomycin(BLM)-induced pulmonary fibrosis, so as to provide data support for the clinical treatment of pulmonary fibrosis. Ninety male C57BL/6N mice were randomized into normal(n=10), model(BLM, n=20), pirfenidone(PFD, 270 mg·kg~(-1), n=15), and low-, medium-, and high-dose E. sagittatum extract(1.67 g·kg~(-1), n=15; 3.33 g·kg~(-1), n=15; 6.67 g·kg~(-1), n=15) groups. The model of pulmonary fibrosis was established by intratracheal instillation of BLM(5 mg·kg~(-1)) in the other five groups except the normal group, which was treated with an equal amount of normal saline. On the day following the modeling, each group was treated with the corresponding drug by gavage for 21 days. During this period, the survival rate of the mice was counted. After gavage, the lung index was calculated, and the morphology and collagen deposition of the lung tissue were observed by hematoxylin-eosin(HE) and Masson staining, respectively. The levels of reactive oxygen species(ROS) in lung cell suspensions were measured by flow cytometry. The levels of glutathione peroxidase(GSH-Px), total superoxide dismutase(T-SOD), and malondialdehyde(MDA) the in lung tissue were measured. Terminal-deoxynucleoitidyl transferase-mediated nick-end labeling(TUNEL) was employed to examine the apoptosis of lung tissue cells. The content of interleukin-6(IL-6), chemokine C-C motif ligand 2(CCL-2), matrix metalloproteinase-8(MMP-8), transforming growth factor-beta 1(TGF-β1), alpha-smooth muscle actin(α-SMA), E-cadherin, collagen Ⅰ, and fibronectin in the lung tissue was measured by enzyme-linked immunosorbent assay(ELISA). The expression levels of F4/80, Ly-6G, TGF-β1, and collagen Ⅰ in the lung tissue were determined by immunohistochemistry. The mRNA levels of CCL-2, IL-6, and MMP-7 in the lung tissue were determined by qRT-PCR. The content of hydroxyproline(HYP) in the lung tissue was determined by alkaline hydrolysation. The expression of α-SMA and E-cadherin was detected by immunofluorescence, and the protein levels of α-SMA, vimentin, E-cadherin in the lung tissue were determined by Western blot. The results showed the aqueous extract of E. sagittatum increased the survival rate, decreased the lung index, alleviated the pathological injury, collagen deposition, and oxidative stress in the lung tissue, and reduced the apoptotic cells. Furthermore, the aqueous extract of E. sagittatum down-regulated the protein levels of F4/80 and Ly-6G and the mRNA levels of CCL-2, IL-6, and MMP-7 in the lung tissue, reduced the content of IL-6, CCL-2, and MMP-8 in the alveolar lavage fluid. In addition, it lowered the levels of HYP, TGF-β1, α-SMA, collagen Ⅰ, fibronectin, and vimentin, and elevated the levels of E-cadherin in the lung tissue. The aqueous extract of E. sagittatum can inhibit collagen deposition, alleviate oxidative stress, and reduce inflammatory response by regulating the expression of the molecules associated with epithelial-mesenchymal transition, thus alleviating the symptoms of bleomycin-induced pulmonary fibrosis in mice.

Research on the mechanism of antidepressive effect of Suanzaoren Decoction through TLR4/MyD88/NF-κB pathway and Wnt/β-catenin pathway

Ethnopharmacological relevanceIncreased inflammatory response and disruption of neuroplasticity are important mechanisms in the hypothesis of the pathogenesis of depression. Thus, these two aspects are conducive to the development of treatments for depression. Suanzaoren Decoction (SZRD) is a classic traditional Chinese medicine compound for the treatment of insomnia, which can clinically relieve depression symptoms, but its antidepressant pharmacological mechanism remains to be elucidated. Aim of this studyBased on the hypothesis of inflammation and neuroplasticity in depression, this study aimed to investigate the antidepressant effect of SZRD and its specific molecular mechanism through chronic unpredictable mild stress (CUMS) induced SD rat model and lipopolysaccharide (LPS) induced BV2 cell neuroinflammation model. Materials and methodsThe body weight and behavioral indexes of CUMS model rats treated with orally or without oral SZRD for 4 weeks were detected. Hematoxylin and eosin staining was used to observe brain pathological damage. Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling (TUNEL) staining was used to observe neuronal apoptosis. Immunofluorescence, ELISA kit and Western blotting were used to detect the inflammatory index Iba-1 and inflammatory factors, as well as the important inflammatory pathway TLR4/MyD88/NF-κB. Enzyme linked immunosorbent assay (ELISA) and western blotting were used to detect neuroplasticity indexes proteins-brain-derived neurotrophic factor (BDNF), presynaptic membrane protein-synaptophysin (SYP), and postsynaptic protein- 95(PSD95), and the key pathway Wnt/β-catenin. The possible mechanism of SZRD antidepressant was further explored in LPS-induced BV2 cells. ResultsIn vivo and in vitro experiments showed that SZRD treatment significantly reversed the depression-like behaviors in rats, decreased the levels of inflammatory factors and increased the expression levels of BDNF, SYP, PSD95 in depression model rats. Furthermore, SZRD treatment inhibited the activation of TLR4/MyD88/NF-κB and Wnt/β-catenin pathways and reduced the massive nuclear translocation of NF-κB and β-catenin. The addition of NF-κB pathway agonists could partially offset the inhibitory effect of SZRD on the Wnt pathway, and the addition of Wnt pathway agonists could also partially offset the inhibitory effect of SZRD on the TLR4 pathway. ConclusionThis study suggestted that SZRD may exert its antidepressant effect by regulating TLR4/MyD88/NF-κB pathway and Wnt/β-catenin pathway in combination.

Multilevel chitosan-gelatin particles loaded with P4HA1 siRNA suppress glioma development.

It has been reported that prolyl 4-hydroxylase subunit alpha 1 (P4HA1) promoted tumor growth and metastasis of glioma; thus, targeting P4HA1 may be a promising therapeutic strategy against glioma. In consideration of the instability of siRNA in vivo, the chitosan-gelatin microspheres loaded with P4HA1 siRNA (P4HA1 siRNA@CGM) were employed. Firstly, the gel electrophoresis and hemolytic test were performed to assess the stability and blood compatibility of P4HA1 siRNA@CGM. Then, methyl thiazolyl tetrazolium (MTT), cell colony formation, Transwell assay, wound healing assay, gliosphere formation, tube formation, and Western blot were performed to assess the effects of P4HA1 siRNA@CGM on the biological functions of glioma. Finally, 125I-labeled P4HA1 siRNA@CGM was injected into the xenograft mice, radionuclide imaging was recorded, Ki67 and terminal deoxynucleoitidyl transferase-mediated nick end labeling (TUNEL) staining was performed to assess the effects of P4HA1 siRNA@CGM on tumor growth and apoptosis of glioma in vivo. The results showed that P4HA1 siRNA and P4HA1 siRNA@CGM not only markedly inhibited the proliferation, metastasis, gliosphere formation, and the protein levels of interstitial markers (N-cadherin and vimentin) and the transcription factors of epithelial-mesenchymal transition (EMT) (Snail, Slug, and Twist1) in glioma cells, but also inhibited the tube formation in human brain microvascular endothelial cells (HBMECs), and P4HA1 siRNA@CGM exhibited the better inhibitory effects than P4HA1 siRNA. Above results suggested the feasibility of P4HA1 siRNA@CGM in the clinical treatment of glioma.

Echinacoside hampers malignant progression of breast cancer MCF-7 cells by modulating AKR1B10/ERK signal transduction

This study analyzes the impact of echinacoside(ECH) in the proliferation, metastasis and adriamycin(ADR) resistance of breast cancer(BC) MCF-7 cells via the modulation of aldo-keto reductase family 1 member 10(AKR1B10)/extracellular signal-regulated kinase(ERK) pathway. The chemical structure of ECH was firstly confirmed. MCF-7 cells were treated with different concentration(0, 10, 20, 40 μg·mL~(-1)) of ECH for 48 h. Western blot was used to analyze expression of AKR1B10/ERK pathway-associated proteins and cell counting kit-8(CCK-8) assay to determine cell viability. MCF-7 cells were collected and classified into control group, ECH group, ECH + Ov-NC group, and ECH + Ov-AKR1B10 group. Then Western blot was employed to analyze the expression of AKR1B10/ERK pathway-associated proteins. CCK-8 and 5-ethynyl-2'-deoxyuridine(EdU) assay were used to examine cell proliferation. Cell migration was appraised with scratch assay, Transwell assay, and Western blot. Eventually, MCF-7 cells were treated with ADR for 48 h to induce ADR resistance. Cell viability was tested by CCK-8 assay and cell apoptosis was estimated based on terminal-deoxynucleoitidyl transferase mediated nick end labeling(TUNEL) assay and Western blot. Based on Protein Data Bank(PDB) and molecular docking, the binding affinity of ECH to AKR1B10 was assessed. Various doses of ECH decreased the expression of AKR1B10/ERK pathway-associated proteins in a dose-dependent manner and declined cell viability compared with the control group. Compared with the control group, 40 μg·mL~(-1) ECH blocked the AKR1B10/ERK pathway in MCF-7 cells and inhibited the proliferation, metastasis and ADR resistance of the cells. Compared with the ECH + Ov-NC group, ECH + Ov-AKR1B10 group showed the recovery of some biological behaviors of MCF-7 cells. ECH also targeted AKR1B10. ECH can inhibit the proliferation, metastasis, and ADR resistance of BC cells by blocking AKR1B10/ERK pathway.

ADAMTS13 inhibits H2O2-induced human venous endothelial cell injury to attenuate deep-vein thrombosis by blocking the p38/ERK signaling pathway.

Deep vein thrombosis (DVT) is a common complication in hematologic malignancies and immunologic disorders. Endothelial cell injury and dysfunction comprise the critical contributor for the development of DVT. A disintegrin and metalloproteinase with thrombospondin motifs 13 (ADAMTS13), a plasma metalloprotease that cleaves von Willebrand factor, acts as a critical regulator in normal hemostasis. This study was aimed to explore the role of ADAMTS13 in endothelial cell injury during DVT and the possible mechanism. First, human umbilical vein endothelial cells (HUVECs) were exposed to hydrogen peroxide (H2O2). Then, the mRNA and protein expressions of ADAMTS13 were evaluated with the reverse transcription-quantitative polymerase chain reaction and western blot. After treatment with recombinant ADAMTS13 (rADAMTS13; rA13), the viability and apoptosis of H2O2-induced HUVECs were assessed by cell counting kit-8 assay and terminal-deoxynucleoitidyl transferase-mediated nick end labeling staining. In addition, the levels of prostaglandin F1-alpha, endothelin-1, and reactive oxygen species were detected using the enzyme-linked immunosorbent assay and dichloro-dihydro-fluorescein diacetate assay. The expressions of proteins related to p38/extracellular signal-regulated kinase (ERK) signaling pathway were estimated with the western blot. Then, p79350 (p38 agonist) was used to pretreat cells to analyze the regulatory effects of rA13 on p38/ERK signaling in H2O2-induced HUVEC injury. The results revealed that ADAMTS13 expression was significantly downregulated in H2O2-induced HUVECs. The reduced viability and increased apoptosis of HUVECs induced by H2O2 were revived by ADAMTS13. ADAMTS13 also suppressed the oxidative stress in HUVECs after H2O2 treatment. Besides, ADAMTS13 was found to block p38/ERK signaling pathway, and p79350 reversed the impacts of ADAMTS13 on the damage of HUVECs induced by H2O2. To sum up, ADAMTS13 could alleviate H2O2-induced HUVEC injury through the inhibition of p38/ERK signaling pathway.

Catalpol induces apoptosis in breast cancer in vitro and in vivo: Involvement of mitochondria apoptosis pathway and post-translational modifications

Breast cancer is a fatal cancer with the highest mortality in female. New strategies for anti-breast cancer are still urgently needed. Catalpol, an iridoid glycoside extracted from the traditional Chinese medicinal plant Rehmannia glutinosa, has shown anticancer efficacy in various cancer cells. However, its effect on breast cancer remains unclear. In this study, we aim to investigate the anti-breast cancer activity of catalpol and elucidate its underlying mechanism. Cell counting kit-8 (CCK-8) and morphology change showed that catalpol could inhibit the proliferation and viability of MCF-7 cells. Catalpol administration reduced the tumor volume in xenograft model. Catalpol induced apoptosis in MCF-7 cells confirmed by Hoechst 33342 staining and Annexin V-FITC/PI double staining. In vivo, catalpol also induced apoptosis as seen from the increased level of terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) in tumor. According to JC-1 and Dichlorodi-hydrofluorescein Diacetate (DCFH-DA) staining, loss of mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) generation was found in MCF-7 cells treated with catalpol. Furthermore, catalpol also increased the level of cytoplasmic cytochrome c and activity of caspase-3 in MCF-7 cells. Likewise, histopathological and immunohistochemical (IHC) assay also found that catalpol enhanced the levels of cytochrome c and caspase-3 in breast cancer tissues. Ultimately, acetylation, 2-hydroxyisobutyrylation and lactylation were dramatically increased, whereas succinylation, malonylation and phosphorylation were markedly decreased in the breast cancer tumor treated with catalpol. Taken together, catalpol inhibited breast cancer in vitro and in vivo through induction of apoptosis via mitochondria apoptosis pathway and regulation of protein post-translational modifications (PTMs). Thus, it can be considered as an excellent candidate compound for treatment of breast cancer.

Effect of acupuncture on expression of endoplasmic reticulum Ca2+ and Caspase-12 protein in hippocampal neurons of rats with convulsive brain injury

To observe the effect of acupuncture on endoplasmic reticulum calcium, apoptosis number and Caspase-12 protein expression in hippocampal neurons of convulsive rats, so as to explore its mechanisms underlying improvement of convulsion. SD rats were randomly divided into normal control, model and acupuncture groups, with 36 rats in each group. Rats in the normal control group received intraperitoneal injection (i.p.) of normal saline (2 mL), and those of the other 2 groups received i.p. of pentylenetetrazole (50 mg/kg) for establishing convulsion model. Manual acupuncture stimulation was applied to "Baihui"(GV20) and "Dazhui"(GV14) for 30 min after modeling. The hippocampal tissues were taken at 2, 12 and 48 h after modeling. The endoplasmic reticulum Ca2+ concentration (optical density, OD) was detected by using fluorescence probe technique and laser confocal microscopy, and the number of apoptosis of hippocampal neurons at the 3 time points detected by using terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) stain. The expression of Caspase-12 protein in hippocampus at 3 time points was observed by immunohistochemistry. In comparison with the normal group, the number of apoptotic cells of hippocampal neurons and the expression levels of Caspase-12 protein in hippocampus at 2, 12 and 48 h after seizures were obviously increased (P<0.01), and the OD value of Ca2+ at 3 time points significantly decreased (P<0.01) in the model group.Following acupuncture intervention, the increased levels of the number of apoptotic cells of hippocampal neurons and the expression of Caspase-12 protein in hippocampus at 3 time points and the decreased levels of OD value of Ca2+ at 3 time points were reversed in the acupuncture group (P<0.05, P<0.01). Acupuncture intervention is effective in reducing the apoptosis of hippocampal neurons in convulsion rats, which may be related to its functions in down-regulating Caspase-12 expression and promoting influx of Ca2+ in the hippocampal neurons.

Effect of electroacupuncture of "Zusanli"(ST36) and "Guanyuan" (CV4) on apoptosis and expression of apoptosis-related proteins of synoviocytes in adjuvant-induced arthritis rats

To observe the effect of electroacupuncture (EA) of "Zusanli"(ST36) and "Guanyuan"(CV4) on the apoptosis rate of synoviocytes and protein expression of Fas, FasL and Caspase-3 in synovial tissue of adjuvant-induced arthritis (AIA) rats, so as to explore its mechanisms underlying improvement of rheumatoid arthritis （RA）. A total of 24 rats were randomly divided into normal, model, medication and EA groups, with 6 rats in each group. The AIA model was established by injection of complete Freund's adjuvant (CFA, 0.1 mL) into the left hindlimb paw. The rats in the medication group received intraperitoneal injection of 0.35 mg/kg of methotrexate, twice a week for 4 weeks. The rats in the EA group received EA stimulation of ST36 and CV4 (20 Hz/50 Hz, 1 mA) for 20 min, 6 times a week for 4 weeks. The left hind paw volume was measured using a paw volume meter, and histopathological changes of synovial tissue were observed by light microscope after H.E. staining. The serum contents of tumor necrosis factor-α（TNF-α） and interleukin-1 （IL-1） were measured by ELISA. The apoptosis of synoviocytes was detected by terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL), and the expression of apoptosis-associated proteins Fas, FasL and Caspase-3 in synovium were detected by Western blot. Compared with the normal group, the left hind paw volume from day 3 to 24 after administration of CFA, serum IL-1 and TNF-α contents were significantly increased (P<0.01), while the expressions of Fas, FasL and Caspase-3 proteins and apoptotic rate of synoviocytes were significantly decreased in the model group (P<0.01). In comparison with the model group, the paw volume from day 17 to 24 after modeling, the serum IL-1 and TNF-α contents were significantly reduced (P<0.01), while the apoptotic rate of synoviocytes, expressions of Fas protein in both medication and EA groups, Caspase-3 protein in the acupuncture group and FasL protein in the medication group were increased (P<0.01, P<0.05). Compared with the medication group, the expression of FasL protein was decreased in EA group (P<0.05). H.E. stain showed obvious hyperplasia of the synovial lining layer, and disordered arrangement of synovial cells, with edema and enlargement in some cells in the model group, which was relatively milder in both medication and EA groups. EA of ST36 and CV4 can promote the apoptosis of synoviocytes and the expressions of Fas and FasL proteins in AIA rats, which may contribute to its role in relieving synovitis through activating Fas/FasL signaling.

Buyang Huanwu Decoction Ameliorates Damage of Erectile Tissue and Function Following Bilateral Cavernous Nerve Injury.

To verify the effect of Buyang Huanwu Decoction (BHD) in ameliorating erectile dysfunction (ED) after radical prostatectomy (RP). The composition of BHD was verified by ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS/MS) analysis. Bilateral cavernous nerve crush injury (BCNI) in rats was used to mimic the neurovascular injury occurring after RP. By the envelope method, forty rats were randomly divided into 4 groups as follows: sham (cavernous nerves exposed only), model (BCNI), low-dosage BHD [LBHD, 12.8 g/(kg·d)], and high-dosage BHD [HBHD, 51.2 g/(kg·d)] groups, 10 rats in each group, feeding for 3 weeks respectively. Erectile function was evaluated by measuring intracavernosal pressure (ICP). Changes in the histopathology of corpus cavernosum (CC) were examined by hematoxylin-eosin staining. Meanwhile, the fibrosis of CC was measured by Masson's trichrome staining and Western blot was used to detect the expressions of collagen I, transforming growth factor beta 1 (TGF- β 1) and α-smooth muscle actin (α-SMA). Apoptosis index was detected by terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) and Western blot for determining the expressions of B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X (Bax). The oxidative stress in the CC were assessed by the superoxide dismutase (SOD), malondialdehyde (MDA) and reactive oxygen species (ROS) levels. The proteins expression of c-Jun N-terminal kinase (JNK) and c-Jun were detected by Western blot. In addition, the expression of α-SMA and p-c-Jun in the CC was observed by double immunofluorescence staining. The UPLC-QTOF-MS/MS analysis showed that BHD contained calycosin-7-O- β -D-glucoside, ononin, calycosin and formononetin. Compared with the model group, LBHD and HBHD treatment improved the ICP and the circumference, area, and weight of CC (P<0.05 or P<0.01). Furthermore, LBHD and HBHD treatments increased CC smooth muscle content and decreased apoptosis index (P<0.05 or P<0.01). LBHD and HBHD also elevated SOD and expression level of α -SMA and Bcl-2, and reduced MDA and ROS levels, as well as expression of TGF- β 1, collagen I, Bax, p-c-JNK, p-JNK in the CC compared with the model group (P<0.05 or P<0.01). The double immunofluorescence staining showed that the fluorescence degree of p-c-Jun in both LBHD and HBHD treatment groups was significantly reduced, whereas the α -SMA expression increased (P<0.05 or P<0.01). BHD can improve ED of rats with BCNI, which is related to inhibiting fibrosis, apoptosis, and oxidative stress of CC. The ROS/JNK/c-Jun signaling pathway may play an important role in the process.

Hsa_circ_0030042 Facilitates the Proliferation and Migration of Vascular Smooth Muscle Cells via the miR-514a-3p/FOXO1 Axis

Purpose: Circular RNA (circRNA) has been proved to play a vital role in atherosclerosis (AS) progression, and vascular smooth muscle cells (VSMCs) are involved in the progression of AS. However, the in-depth mechanism by which circRNA regulates VSMC proliferation and migration remains to be elusive. Methods: We used tumor necrosis factor-alpha (TNF-α) to treat VSMCs to establish a cell model of AS. We used Cell Counting Kit-8, terminal-deoxynucleoitidyl transferase–mediated nick end labeling, and transwell assays to assess the proliferation, apoptosis, and migration in TNF-α-induced VSMCs. Moreover, the interaction between molecules was measured by RNA-binding protein immunoprecipitation, RNA pull-down, and luciferase reporter assays. Results: Our study found that a novel circRNA hsa_circ_0030042, which is derived from its host gene forkhead box O1 (FOXO1), was upregulated in TNF-α-induced VSMCs. Silencing of hsa_circ_0030042 inhibited proliferation and migration while promoting apoptosis in TNF-α-induced VSMCs. Mechanically, hsa_circ_0030042 positively regulated FOXO1 expression via sponging miR-514a-3p. Conclusions: Our study stated the vital role of the hsa_circ_0030042/miR-514a-3p/FOXO1 axis and provides a profound understanding about the circRNA in AS.

Matrine Combined with Mammalian Target of Rapamycin Inhibitor Enhances Anti-Tumor Efficacy of dendritic cell Vaccines in hepatocellular carcinoma

ABSTRACT Dendritic cells (DCs), as the most important antigen-presenting cells, play a crucial role in T cell activation. The latest research showed that inhibition of the mammalian target of rapamycin (mTOR) could enhance DCs maturation, promoting antigen presentation. Matrine has been identified as one of the key alkaloids isolated from the roots of Sophora flavescens. In present study, we combined matrine and mTOR inhibitor KU0063794 to observe the DCs functions, especially the antigen presentation ability. DCs were activated by phosphate-buffered saline (PBS), lipopolysaccharide (LPS), LPS+KU0063794, LPS+Matrine, and LPS+KU0063794+Matrine. The surface markers in DCs, proliferation of T cells and cytokines were detected by flow cytometry, cell counting kit-8 (CCK-8) and enzyme-linked immunosorbent assay (ELISA), respectively. The lactate dehydrogenase (LDH) release test was used to detect the antitumor efficacy. The tumor growth curves were plotted by calculating tumor volume. The apoptosis was detected by Terminal-deoxynucleoitidyl Transferase-Mediated Nick End Labeling (TUNEL) method. Matrine combined with KU0063794 could enhance the maturity of DCs, T cells proliferation and cytokines secretion (P < 0.05). The cytotoxic T lymphocytes (CTL) killing efficacy of LPS+KU0063794+Matrine group was higher than other groups (P < 0.05). In vivo, the tumor weights and volumes in LPS+KU0063794+Matrine group were lower than other groups. The detections of tumor apoptosis were increased in LPS+KU0063794+Matrine group (P < 0.05). DC vaccine with mTOR inhibitor and matrine could significantly improve the efficacy of antitumor immunity in vitro and vivo. These findings illustrated that mTOR inhibitor and matrine, as two immunomodulators, could enhance DC activation and differentiation.

The effect and mechanisms of melatonin on the proliferation and apoptosis of lung cancer cells

ABSTRACT The aim of the present study was to observe the effects and mechianisms of melatonin on the proliferation and apoptosis of lung cancer (LC) cells. A549 cells were treated with a concentration gradient (0–100 μM) of melatonin for 24 hours, and cell viability was detected by XTT ((2,3-Bis-(2-methoxy-4-nitro-5-sulfophenyl) −2H-tetrazolium-5-carboxanilide)) colorimetry. Melatonin with a concentration of 50 μM was selected to interact with the LC cells for ten days, and then a colony formation assay was used to detect the proliferation of the LC cells. TUNEL (Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling) staining was used to evaluate the amount of apoptosis in the two groups. Finally, Western blotting was used to detect the expression levels of related proteins in the p38MAP (mitogen-activated protein) signaling pathway. Meanwhile, another experiment, CCK-8 cell proliferation test, was conducted to detect the OD540 absorbance of LC cells at 24, 48, 72, and 96 hours. Melatonin inhibited the proliferation of LC cells in a concentration-dependent (5–100 μM) manner (P < 0.05), and inhibited the proliferation of LC cells in a time-dependent (0–96 hour) manner (P < 0.05). Melatonin (50 μM) could significantly inhibit the colony formation ability of LC cells (P < 0.05). The ratio of LC cells in the G0/G1 phase in the melatonin group increased, while the ratio of cells in the G2/M and S phase was significantly reduced (P < 0.05). Melatonin significantly promoted the apoptosis of LC cells (P < 0.05) and activate the phosphorylation of p38 (P < 0.05).

Intestinal Epithelial Cell Exosome Launches IL-1β-Mediated Neuron Injury in Sepsis-Associated Encephalopathy

BackgroundGut–microbiota–brain axis links the relationship between intestinal microbiota and sepsis-associated encephalopathy (SAE). However, the key mediators between them remain unclear.MethodsMemory test was determined by Water maze. Intestinal flora was measured by 16S RNA sequencing. Neurotransmitter was detected by high-performance liquid chromatography (HPLC). Histopathology was determined by H&E, immunofluorescence (IF), and terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) staining. Flow cytometry was employed to determine the proportion of macrophages.ResultsFecal microbiota transplantation (FMT) relieved hippocampus impairment of SAE rats by inhibiting inflammation cytokine secretion, the expression of IBA-1 and neurotransmitter disturbance, and cell apoptosis and autophagy, accompanied by the reduced M1 polarization and M1 pro-inflammation factors produced by macrophages in mesenteric lymph nodes (MLNs). Actually, M1 polarization in SAE rats depended on intestinal epithelial cell (IEC)-derived exosome. GW4869-initiated inhibition of exosome secretion notably abolished M1 polarization and the secretion of IL-1β. However, GW4869-mediated improvement of hippocampus impairment was counteracted by the delivery of recombinant interleukin (IL)-1β to hippocampus. Mechanistically, IEC-derived exosome induced the excessive circulating IL-1β produced by CP-R048 macrophages, which subsequently induced damage and apoptosis of hippocampal neurons H19-7 in an autophagy-dependent manner. And reactivation of autophagy facilitates intestinal IL-1β-mediated hippocampal neuron injury.ConclusionCollectively, intestinal flora disturbance induced the exosome release of IECs, which subsequently caused M1 polarization in MLNs and the accumulation of circulating IL-1β. Circulating IL-1β promoted the damage and apoptosis of neurons in an autophagy-dependent manner. Possibly, targeting intestinal flora or IEC-derived exosome contributes to the treatment of SAE.

Human Umbilical Cord Mesenchymal Stem Cells Improve the Necrosis and Osteocyte Apoptosis in Glucocorticoid-Induced Osteonecrosis of the Femoral Head Model through Reducing the Macrophage Polarization.

Background and ObjectivesApoptosis is an outstanding determinant of glucocorticoid (GC)-induced osteonecrosis of the femoral head (ONFH). Human umbilical cord mesenchymal stem cells (hUC-MSCs) have been demonstrated to be associated with apoptosis in diseases models. However, the role of hUC-MSCs in GC-induced ONFH via regulating apoptosis still needs further study.Methods and ResultsIn the present study, a GC-induced ONFH model was built in vivo through a consecutive injection with lipopolysaccharide (LPS) and methylprednisolone. The necrosis and apoptosis of the femoral head was evaluated by histological and Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling (TUNEL) assay. The level of collagen and TRAP positive cells were determined by Masson and TRAP staining, respectively. M1 macrophage polarization was assessed using immunofluorescence assay. The level of proinflammatory cytokines including tumor necrosis factor (TNF)‐α, Interleukin (IL)‐1β and IL-6 of femoral head was determined by enzyme-linked immunosorbent assay (ELISA) kits. The protein expression of AKT, mTOR, p-AKT and p-mTOR was detected using western blot assay. The results showed that hUC-MSCs treatment prominently promoted the GC-induced the decrease of the collagen level and the increase of TRAP positive cells. Besides, hUC-MSCs treatment decreased necrosis and apoptosis, macrophage polarization, the level of TNF‐α, IL‐1β and IL-6, the protein expression of p-AKT and p-mTOR, and the radio of p-AKT to AKT and p-mTOR to mTOR of femoral head in vivo.ConclusionsTherefore, the present study revealed that hUC-MSCs improved the necrosis and osteocyte apoptosis in GC-induced ONFH model through reducing the macrophage polarization, which was associated with the inhibition of AKT/mTOR signaling pathway.

Etanercept Reduces Neuron Injury and Neuroinflammation via Inactivating c-Jun N-terminal Kinase and Nuclear Factor-κB Pathways in Alzheimer’s Disease: An In Vitro and In Vivo Investigation

Inflammation contributes to amyloid beta (Aβ) aggregation and neuron loss in Alzheimer’s disease (AD). Meanwhile, tumor necrosis factor-α (TNF-α) inhibitors present strong effect on suppressing inflammation. Thus, this study aimed to investigated the effect and molecular mechanism of etanercept (ETN) (a commonly used TNF-α inhibitor) on neuron injury and neuroinflammation in AD. AD cellular model was constructed by co-culture of primary embryonic neuron cells and microglial cells, followed by Aβ treatment. Subsequently, ETN was used to treat AD cellular model. Besides, APPswe/PS1M146V/tauP301L transgenic (AD) mice were respectively treated with saline or ETN by intravenous injection once per 3 days for 10 times. In vitro data revealed that cell viability and neurite outgrowth were increased, but apoptosis and levels of pro-inflammatory cytokines (including TNF-α, interleukin-1β, Interleukin-6 and C–C motif chemokine ligand 2 (CCL2)) were decreased by ETN treatment in AD cellular model. In vivo experiments found that ETN treatment improved spatial, long-term memory (reflected by Morrison water maze) and working memory (reflected by Y maze) in AD mice. Besides, ETN treatment reduced neuron injury (reflected by Hematoxylin–Eosin (HE) and terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) assays) and levels of pro-inflammatory cytokines (including TNF-α, interleukin-1β, Interleukin-6 and CCL2) in AD mice. Moreover, ETN repressed the activation of c-Jun N-terminal kinase (JNK) and nuclear factor-κB (NF-κB) pathways in AD both in vitro and in vivo. In conclusion, ETN exerts neuroprotective function via inactivating JNK and NF-κB pathways in AD, indicating the potential of ETN for improving AD management.