Deoxynucleotidyl Transferase-mediated dUTP-biotin Nick End Research Articles

Recombinant hirudin attenuates pulmonary hypertension and thrombosis in acute pulmonary embolism rat model.

Acute pulmonary embolism (APE) is classified as a subset of diseases that are characterized by lung obstruction due to various types of emboli. Current clinical APE treatment using anticoagulants is frequently accompanied by high risk of bleeding complications. Recombinant hirudin (R-hirudin) has been found to have antithrombotic properties. However, the specific impact of R-hirudin on APE remains unknown. Sprague-Dawley (SD) rats were randomly assigned to five groups, with thrombi injections to establish APE models. Control and APE group rats were subcutaneously injected with equal amounts of dimethyl sulfoxide (DMSO). The APE+R-hirudin low-dose, middle-dose, and high-dose groups received subcutaneous injections of hirudin at doses of 0.25 mg/kg, 0.5 mg/kg, and 1.0 mg/kg, respectively. Each group was subdivided into time points of 2 h, 6 h, 1 d, and 4 d, with five animals per point. Subsequently, all rats were euthanized, and serum and lung tissues were collected. Following the assessment of right ventricular pressure (RVP) and mean pulmonary artery pressure (mPAP), blood gas analysis, enzyme-linked immunosorbnent assay (ELISA), pulmonary artery vascular testing, hematoxylin-eosin (HE) staining, Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining, immunohistochemistry, and Western blot experiments were conducted. R-hirudin treatment caused a significant reduction of mPAP, RVP, and Malondialdehyde (MDA) content, as well as H2O2 and myeloperoxidase (MPO) activity, while increasing pressure of oxygen (PaO2) and Superoxide Dismutase (SOD) activity. R-hirudin also decreased wall area ratio and wall thickness to diameter ratio in APE rat pulmonary arteries. Serum levels of endothelin-1 (ET-1) and thromboxaneB2 (TXB2) decreased, while prostaglandin (6-K-PGF1α) and NO levels increased. Moreover, R-hirudin ameliorated histopathological injuries and reduced apoptotic cells and Matrix metalloproteinase-9 (MMP9), vascular cell adhesion molecule-1 (VCAM-1), p-Extracellular signal-regulated kinase (ERK)1/2/ERK1/2, and p-P65/P65 expression in lung tissues. R-hirudin attenuated pulmonary hypertension and thrombosis in APE rats, suggesting its potential as a novel treatment strategy for APE.

Adelmidrol ameliorates liver ischemia-reperfusion injury through activating Nrf2 signaling pathway

Liver ischemia/reperfusion (I/R) injury commonly occurs after various liver surgeries. Adelmidrol, an N- palmitoylethanolamide analog, has anti-inflammatory, anti-oxidant, and anti-injury properties. To investigate whether adelmidrol could reduce liver I/R injury, we established a mouse of liver I/R injury and an AML12 cell hypoxia-reoxygenation model to perform experiments using multiple indicators. Serum ALT and AST levels, and H&E staining were used to measure liver damage; MDA content, superoxide dismutase and glutathione activities, and dihydroethidium staining were used to measure oxidative stress; mRNA expression levels of tumor necrosis factor-α, interleukin (IL)-1β, IL-6, MCP-1, and Ly6G staining were used to measure inflammatory response; and protein expression of Bax, Bcl-2, C-caspase3, and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling staining were used to measure apoptosis. The experimental results showed that adelmidrol reduced liver I/R injury. In addition, adelmidrol pretreatment elevated AML12 cell activity and reduced I/R-and H/R-induced apoptosis, inflammatory injury, and oxidative stress. ML385, an inhibitor of nuclear factor erythroid2-related factor 2 (Nrf2), reverses liver I/R injury attenuated by adelmidrol. These results suggest that adelmidrol ameliorates liver I/R injury by activating the Nrf2 signaling pathway.

MiR-2113 overexpression attenuates sepsis-induced acute pulmonary dysfunction, inflammation and fibrosis by inhibition of HMGB1

PurposeSepsis-induced acute lung injury is related to high mortality. MiR-2113 possesses important functions in human diseases. This research aimed to clarify the role and mechanism of miR-2113 in sepsis-induced acute lung injury. MethodsThe expression of miR-2113 in lipopolysaccharide (LPS)-induced MLE-12 cells, serum of sepsis patients, and cecal ligation and puncture mouse models was examined using quantitative real-time PCR. The functions of miR-2113 in LPS-treated MLE-12 cells were estimated by Cell Counting Kit-8 assay, flow cytometry, enzyme-linked immunosorbent assay, Western blot, and immunofluorescence. The influences of miR-2113 in cecal ligation and puncture-induced acute lung injury in mice were assessed by hematoxylin-eosin staining, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay, acute pulmonary dysfunction analysis, lactate dehydrogenase levels and total protein concentrations in bronchoalveolar lavage fluid, and Masson staining. Also, the mechanism of miR-2113 was examined using a dual-luciferase reporter assay. ResultsMiR-2113 expression was decreased in LPS-induced MLE-12 cells, serum of sepsis patients, and cecal ligation and puncture mouse models. miR-2113 overexpression restored LPS-reduced MLE-12 cell proliferation, but alleviated LPS-induced apoptosis and markers of inflammation and fibrosis in MLE-12 cells. Moreover, we found that miR-2113 mimic reduced LPS-induced MLE-12 cell injury by negatively regulating high-mobility group box 1. In vivo data further confirmed that miR-2113 overexpression alleviated acute pulmonary dysfunction, inflammation and fibrosis in cecal ligation and puncture-induced sepsis mice. ConclusionMiR-2113 relieved sepsis-induced acute pulmonary dysfunction, inflammation and fibrosis through decreasing high-mobility group box 1.

Oryza sativa L. Indica Seed Coat Ameliorated Concanavalin A-Induced Acute Hepatitis in Mice via MDM2/p53 and PKCα/MAPK1 Signaling Pathways.

Acute hepatitis (AH) is a common liver disease with an increasing number of patients each year, requiring the development of new treatments. Hence, our work aimed to evaluate the therapeutic effect of Oryza sativa L. indica (purple rice) seed coat on concanavalin A (ConA)-induced AH and further reveal its potential mechanisms. Purple rice seed coat extract (PRE) was extracted with hydrochloric acid ethanol and analyzed through a widely targeted components method. We evaluated the effects of PRE on AH through histopathological examination, liver function, gut microbiota composition, and the intestinal barrier. The potential targets of PRE on AH were predicted by bioinformatics. Western blotting, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay (TUNEL) staining, and corresponding kits were used to investigate PRE effects on predicting targets and associated signaling pathways in AH mice. In AH model mice, PRE treatment increased transformed mouse 3T3 cell double minute 2 (MDM2) expression to inhibit apoptosis; it also markedly downregulated protein kinase C alpha (PKCα), prostaglandin-endoperoxide synthase 1 (PTGS1), and mitogen-activated protein kinase 1 (MAPK1) activity to alleviate inflammation. Thus, PRE treatment also recovered the intestinal barrier, decreased the lipopolysaccharide (LPS) levels of plasma and the liver, enhanced liver function, and improved the composition of intestinal microbiota. In general, PRE targeting MDM2, PKCα, MAPK1, and PTGS1 ameliorated ConA-induced AH by attenuating inflammation and apoptosis, restoring the intestinal barrier, enhancing the liver function, and improving the gut microbiota, which revealed that the purple rice seed coat might hold possibilities as a therapeutic option for AH.

JNK/MAPK pathway regulation by BEX2 gene silencing in alcoholic hepatitis mice: Effects on oxidative stress.

Alcoholic hepatitis (AH) is a severe alcoholic-related liver disease that is a leading cause of morbidity and mortality, for which effective treatments are lacking. Brain-expressed X-linked gene 2 (BEX2) has been implicated in various diseases, but its association with AH has received limited attention. Thus, this study investigated BEX2's impact on the progression of AH by affecting the c-Jun NH2-terminal kinase/mitogen-activated protein kinase (JNK/MAPK) pathway. Microarray dataset GSE28619 from the Gene Expression Omnibus database was used to identify differentially expressed genes in AH. Immunohistochemistry, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL), Western blot analysis, and flow cytometry were used to measure various factors in the liver tissue of AH mice. BEX2 expression was significantly upregulated in the model. BEX2 gene silencing increased the levels of glutathione peroxidase and superoxide dismutase while decreasing malondialdehyde content; phosphorylation of JNK, c-JUN, and p38MAPK; apoptosis rate; and the extent of JNK/MAPK pathway activation. These findings provide valuable insights into the mechanisms underlying AH development and highlight the potential role of BEX2 gene expression as a promising therapeutic target for AH.

MiR-148b caused liver injury in rats with traumatic hemorrhagic shock by inhibiting SIRT6 expression.

The purpose of this study was to investigate the role of miR-148b in liver injury in rats with traumatic hemorrhagic shock (THS) and to elucidate its potential mechanism. The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum of rats were detected by enzyme-linked immune sorbent assay (ELISA), and the injury of rat liver was analyzed by hematoxylin-eosin (H&E) staining. Apoptosis of rat hepatocytes and normal rat liver cell line (BRL3A) was identified by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay and flow cytometry, respectively. MiR-148b and sirtuin 6 (SIRT6) expression was measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot. Lactate dehydrogenase (LDH) content and cell viability were measured by commercial kits and cell counting kit-8 (CCK-8) assay, respectively. The binding sites of miR-148b and SIRT6 were predicted by the Starbase database and verified by dual luciferase reporter assay. MiR-148b expression in THS rats or ischemia-reperfusion (I/R)-treated cells was higher than in the control group. Overexpression of miR-148b further promoted the effects of I/R, which enhanced the levels of ALT, AST and LDH, cell apoptosis of liver tissue or BRL3A cells and decreased the expression of SITR6. Besides, miR-148b negatively correlated with SIRT6, and upregulated the expression of SIRT6 could partly reverse the effect of miR-148b. Hepatocyte injury induced by I/R was achieved by regulating miR-148b /SIRT6 axis.

Inhibition of checkpoint kinase prevents human oocyte apoptosis induced by chemotherapy and allows enhanced tumour chemotherapeutic efficacy.

Could inhibition of the checkpoint kinase (CHEK) pathway protect human oocytes and even enhance the anti-tumour effects, during chemotherapy? CHEK inhibitors prevented apoptosis of human oocytes induced by chemotherapy and even enhanced the anti-tumour effects. CHEK inhibitors showed ovarian protective effects in mice during chemotherapy, while their role in human oocytes is unclear. This experimental study evaluated the ovarian reserve of young patients (120 patients) with cancer, exposed or not exposed to taxane and platinum (TP)-combined chemotherapy. Single RNA-sequencing analysis of human primordial oocytes from 10 patients was performed to explore the mechanism of oocyte apoptosis induced by TP chemotherapy. The damaging effects of paclitaxel (PTX) and cisplatin on human oocytes were also evaluated by culturing human ovaries in vitro. A new mouse model that combines human ovarian xenotransplantation and patient-derived tumour xenografts was developed to explore adjuvant therapies for ovarian protection. The mice were randomly allocated to four groups (10 mice for each group): control, cisplatin, cisplatin + CK1 (CHEK1 inhibitor, SCH 900776), and cisplatin + CK2 (CHEK2 inhibitor, BML277). In the prospective cohort study, human ovarian follicles were counted and serum AMH levels were evaluated. RNA-sequencing analysis was conducted, and staining for follicular damage (phosphorylated H2AX histone; γH2AX), terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL) assays and assessments of apoptotic biomarkers (western blot and immunofluorescence) were conducted in human ovaries. After the treatments, histological analysis was performed on human ovarian samples to investigate follicular populations, and oocyte damage was measured by γH2AX staining, BAX staining, and TUNEL assays. At the same time, the tumours were evaluated for volume, weight, and apoptosis levels. Patients who received TP chemotherapy showed decreased ovarian reserves. Single RNA-sequencing analysis of human primordial oocytes indicated that TP chemotherapy induced apoptosis of human primordial oocytes by causing CHEK-mediated TAp63α phosphorylation. In vitro culture of human ovaries showed greater damaging effects on oocytes after cisplatin treatment compared with that after PTX treatment. Using the new animal model, CHEK1/2 inhibitors prevented the apoptosis of human oocytes induced by cisplatin and even enhanced its anti-tumour effects. This protective effect appeared to be mediated by inhibiting DNA damage via the CHEK-TAp63α pathway and by generation of anti-apoptotic signals in the oocytes. N/A. This was a preclinical study performed with human ovarian samples, and clinical research is required for validation. These findings highlight the therapeutic potential of CHEK1/2 inhibitors as a complementary strategy for preserving fertility in female cancer patients. This work was financially supported by the National Natural Science Foundation of China (nos. 82001514 and 81902669) and the Fundamental Research Funds for the Central Universities (2021yjsCXCY087). The authors declare no conflict of interest.

Bloodletting Acupuncture at Jing-Well Points on Hand Induced Autophagy to Alleviate Brain Injury in Acute Altitude Hypoxic Rats by Activating PINK1/Parkin Pathway.

To explore the protective effect of bloodletting acupuncture at twelve Jing-well points on hand (BAJP) on acute hypobaric hypoxia (AHH)-induced brain injury in rats and its possible mechanisms. Seventy-five Sprague Dawley rats were divided into 5 groups by a random number table (n=15), including control, model, BAJP, BAJP+3-methyladenine (3-MA), and bloodletting acupuncture at non-acupoint (BANA, tail tip blooding) groups. After 7-day pre-treatment, AHH models were established using hypobaric oxygen chambers. The levels of S100B, glial fibrillary acidic protein (GFAP), superoxide dismutase (SOD), and malondialdehyde (MDA) in serum were measured by enzyme-linked immunosorbent assay. Hematoxylin-eosin staining and the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling method were used to assess hippocampal histopathology and apoptosis. Transmission electron microscopy assay was used to observe mitochondrial damage and autophagosomes in hippocampal tissues. Flow cytometry was used to detect mitochondrial membrane potential (MMP). The mitochondrial respiratory chain complexes I, III and IV activities and ATPase in hippocampal tissue were evaluated, respectively. Western blot analysis was used to detect the protein expressions of Beclin1, autophagy protein 5 (ATG5), microtubule-associated protein 1 light chain 3 beta (LC3B), phosphatase and tensin homolog induced kinase 1 (PINK1), and Parkin in hippocampal tissues. The mRNA expressions of Beclin1, ATG5 and LC3-II were analyzed by quantitative real-time polymerase chain reaction. BAJP treatment reduced hippocampal tissue injury and inhibited hippocampal cell apoptosis in AHH rats. BAJP reduced oxidative stress by decreasing S100B, GFAP and MDA levels and increasing SOD level in the serum of AHH rats (P<0.05 or P<0.01). Then, BAJP increased MMP, the mitochondrial respiratory chain complexes I, III and IV activities, and the mitochondrial ATPase activity in AHH rats (all P<0.01). BAJP improved mitochondrial swelling and increased the autophagosome number in hippocampal tissue of AHH rats. Moreover, BAJP treatment increased the protein and mRNA expressions of Beclin1 and ATG5 and LC3-II/LC3-I ratio in AHH rats (all P<0.01) and activated the PINK1/Parkin pathway (P<0.01). Finally, 3-MA attenuated the therapeutic effect of BAJP on AHH rats (P<0.05 or P<0.01). BAJP was an effective treatment for AHH-induced brain injury, and the mechanism might be through reducing hippocampal tissue injury via increasing the PINK1/Parkin pathway and enhancement of mitochondrial autophagy.

An optimal combination of five main monomer components in Wuzi Yanzong Pill that prevents neural tube defects and reduces apoptosis and oxidative stress

Ethnopharmacological relevanceWuzi Yanzong Pill (WYP) is a classic traditional Chinese medicine (TCM) formula that is used for reproductive system diseases. Previous studies showed that WYP had a preventive effect on the development of neural tube defects (NTDs) induced by all-trans retinoic acid (atRA) in mice. Aim of the studyThis study aimed to determine the optimal combination of main monomer components in WYP on preventing NTDs and to understand the underlying mechanism. Materials and methodsAn optimal combination was made from five representative components in WYP including hyperoside, acteoside, schizandrol A, kaempferide and ellagic acid by orthogonal design method. In a mouse model of NTDs induced by intraperitoneal injection of atRA, pathological changes of neural tube tissues were observed by Hematoxylin & Eosin (HE) staining, neural tube epithelial cells apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL), protein changes related to apoptosis, anti-apoptosis, and antioxidant factors were detected with Western blot. Potential targets and mechanisms of monomer compatibility group (MCG) acting on NTDs were analyzed by bioinformatics. ResultsTreatment with different combinations of WYP bioactive ingredients resulted in varying decreases in the incidence of NTDs in mice embryos. The combination of MCG15 (200 mg/kg of hyperoside, 100 mg/kg of acteoside, 10 mg/kg of schizandrol A, 100 mg/kg of kaempferide and 1 mg/kg of ellagic acid) showed the most significant reduction in NTD incidence. Mechanistically, MCG15 inhibited apoptosis and oxidative stress, as evidenced by reduced TUNEL-positive cells, downregulation of caspase-9, cleaved caspase-3, Bad, and Bax, and upregulation of Bcl-2, as well as decreased MDA and increased SOD, CAT, GSH, HO-1, and GPX1 levels. Bioinformatics analysis showed that MCG15 acted on the PI3K/Akt signaling pathway, which was confirmed by Western blot analysis showing increased expression of p-PI3K, p-Akt/Akt, and Nrf2 related indicators. ConclusionWe have identified an optimal combination of five bioactive components in WYP (MCG15) that prevented NTDs in mice embryos induced by atRA by activating the PI3K/Akt signaling pathway and inhibiting apoptosis and oxidative stress.

The emerging role of lysine succinylation in ovarian aging

BackgroundOvarian aging is a process of decline in its reserve leading to ovary dysfunction and even reduced health quality in offspring. However, aging-related molecular pathways in the ovary remain obscure. Lysine succinylation (Ksuc), a newly post-translational modification (PTM), has been found to be broadly conserved in both eukaryotic and prokaryotic cells, and associated with multiple pathophysiological processes. There are no relevant reports revealing a link between the molecular mechanisms of ovarian aging and Ksuc.MethodsThe level of Ksuc in ovaries of aged and premature ovarian insufficiency (POI) mice were detected by immunoblotting and immunohistochemical. To further explore the role of Ksuc in ovarian aging, using in vitro mouse ovary tissue culture and an in vivo mouse model with changed Ksuc level.ResultsIncreased Ksuc in ovaries of aged and POI mice and distribution of Ksuc in various types of mice ovarian cells and the high level of Ksuc in granulosa cells (GCs) were revealed. Histological assessments and hormone levels analyses showed that the high Ksuc level down-regulated the ovarian index and the anti-Müllerian hormone (AMH) and estrogen levels, and increased follicular atresia. Moreover, in the high Ksuc groups, the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) intensities and the expression of Cleaved-caspase-3 increased and the expression of B-cell lymphoma-2 (Bcl-2) decreased together with positively-expressed P21, an aging-related marker. These results suggest that ovarian aging is likely associated with alteration in Ksuc.ConclusionThe present study has identified Ksuc in mouse ovary and found that high Ksuc level most likely contributes to ovarian aging which is expected further investigation to provide new information for delaying physiological ovarian aging and treating pathological ovarian aging.

Protective effects of histone deacetylase 6 specific inhibitor tubastatin A on subarachnoid hemorrhage in rats and the underlying mechanisms.

Subarachnoid hemorrhage (SAH) is a serious cerebrovascular disease. Early brain injury (EBI) and cerebral vasospasm are the main reasons for poor prognosis of SAH patients. The specific inhibitor of histone deacetylase 6 (HDAC6), tubastatin A (TubA), has been proved to have a definite neuroprotective effect on a variety of animal models of acute and chronic central nervous system diseases. However, the neuroprotective effect of TubA on SAH remains unclear. This study aims to investigate the expression and localization of HDAC6 in the early stage of SAH, and to evaluate the protective effects of TubA on EBI and cerebral vasospasm after SAH and the underlying mechanisms. Adult male SD rats were treated with modified internal carotid artery puncture to establish SAH model. In the first part of the experiment, rats were randomly divided into 6 groups: a sham group, a SAH-3 h group, a SAH-6 h group, a SAH-12 h group, a SAH-24 h group, and a SAH-48 h group. At 3, 6, 12, and 24 h after SAH modeling, the injured cerebral cortex of rats in each group was taken for Western blotting to detect the expression of HDAC6. In addition, the distribution of HDAC6 in the cerebral cortex of the injured side was measured by immunofluorescence double staining in SAH-24 h group rats. In the second part, rats were randomly divided into 4 groups: a sham group, a SAH group, a SAH+TubAL group (giving 25 mg/kg TubA), and a SAH+TubAH group (giving 40 mg/kg TubA). At 24 h after modeling, the injured cerebral cortex tissue was taken for Western blotting to detect the expression levels of HDAC6, endothelial nitric oxide synthase (eNOS), and inducible nitric oxide synthase (iNOS), terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining to detect apoptosis, and hematoxylin and eosin (HE) staining to detect the diameter of middle cerebral artery. The protein expression of HDAC6 began to increase at 6 h after SAH (P<0.05), peaked at 24 h (P<0.001), and decreased at 48 h, but there was still a difference compared with the sham group (P<0.05). HDAC6 is mainly expressed in the cytoplasm of the neurons. Compared with the sham group, the neurological score was decreased significantly and brain water content was increased significantly in the SAH group (both P<0.01). Compared with the SAH group, the neurological score was increased significantly and brain water content was decreased significantly in the SAH+TubAH group (both P<0.05), while the improvement of the above indexes was not significant in the SAH+TubAL group (both P>0.05). Compared with the sham group, the expression of eNOS was significantly decreased (P<0.01) and the expressions of iNOS and HDAC6 were significantly increased (P<0.05 and P<0.01, respectively) in the SAH group. Compared with the SAH group, the expression of eNOS was significantly increased, and iNOS and HDAC6 were significantly decreased in the SAH+TubA group (all P<0.05). Compared with the SAH group, the number of TUNEL positive cells was significantly decreased and the diameter of middle cerebral artery was significantly increased in the SAH+TubA group (both P<0.05) . HDAC6 is mainly expressed in neurons and is up-regulated in the cerebral cortex at the early stage of SAH. TubA has protective effects on EBI and cerebral vasospasm in SAH rats by reducing brain edema and cell apoptosis in the early stage of SAH. In addition, its effect of reducing cerebral vasospasm may be related to regulating the expression of eNOS and iNOS.

Paeoniflorin protects 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinson's disease mice by inhibiting oxidative stress and neuronal apoptosis through activating the Nrf2/HO-1 signaling pathway.

This study aimed to explore the neuroprotective effects of paeoniflorin on oxidative stress and apoptosis in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson's disease (PD) mice. The effects of paeoniflorin on motor function in mice were evaluated by behavioral test. Then substantia nigra of mice were collected and neuronal damage was assessed using Nissl staining. Positive expression of tyrosine hydroxylase (TH) was detected by immunohistochemistry. Levels of malondialdehyde, superoxide dismutase (SOD) and glutathione were measured by biochemical method. terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay was used to detect apoptosis of dopaminergic neurons. Western blotting and real-time fluorescence quantitative PCR were used to detect the protein and mRNA expressions of Nrf2, heme oxygenase-1 (HO-1), B-cell lymphoma-2(Bcl-2), Bax and cleaved caspase-3. Paeoniflorin treatment significantly ameliorated the motor performance impairment in MPTP-induced PD mice. Moreover, it notably increased the positive expression rate of TH and reduced the damage and apoptosis of dopaminergic neurons in the substantia nigra. Furthermore, paeoniflorin increased the levels of SOD and glutathione and decreased the malondialdehyde content. It also promoted Nrf2 nuclear translocation, increased the protein and mRNA expressions of HO-1 and Bcl-2 and reduced the protein and mRNA expressions of BCL2-Associated X2 (Bax) and cleaved caspase-3. Treatment with the Nrf2 inhibitor, ML385, notably reduced the effects of paeoniflorin in MPTP-induced PD mice. Neuroprotective effects of paeoniflorin in MPTP-induced PD mice may be mediated via inhibition of oxidative stress and apoptosis of dopaminergic neurons in substantia nigra through activation of the Nrf2/HO-1 signaling pathway.

AEBP1 Contributes to Breast Cancer Progression by Facilitating Cell Proliferation, Migration, Invasion, and Blocking Apoptosis.

The aberrant expression of adipocyte enhancer binding protein 1 (AEBP1) has been observed in many cancers and it seems to be involved in the tumorigenesis, progression, and metastasis in numerous tumor types. However, the contribution of AEBP1 in breast cancer (BCa) remains inexplicable. Information related to the diagnostic significance and expression of AEBP1 in BCa was obtained from the public dataset Kaplan-Meier Plotter (http://kmplot.com/analysis/) and the dataset UALCAN (https://ualcan.path.uab.edu/index.html). The MTT (methyl thiazolyl tetrazolium) assay, colony formation assay, Transwell® assay, and FACS (fluorescence-activated cell sorting) assay were used to detect the proliferation, invasive and apoptotic ability of cells before and after treatment. In addition, we constructed an AEBP1 overexpression vector and silenced AEBP1, combined with Real-Time Quantitative Reverse Transcription PCR (qRT-PCR), western blot, immunohistochemistry and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling) assay to investigate the prognostic significance, biological functions and potential mechanisms of AEBP1 in BCa. Higher expression of AEBP1 mRNA (message RNA) was observed in BCa patients with later-stage, who obtained poorer overall survival. Meanwhile, compared with adjacent noncancerous tissues, AEBP1 protein expression was dramatically upregulated in the BCa ones. Furthermore, overexpressed AEBP1 enhanced cell proliferation, migration, invasion, and blocked cell apoptosis in BCa cells. Moreover, the research certificated that AEBP1 upregulated the expression of MMP (matrix metalloproteinase)-2, 9, vimentin, N-cadherin (neural-cadherin), phosphorylation of ERK (extracellular signal-regulated kinase), Smad2/3 (Abbreviated from Sma for nematode and Mad for Drosophila) and AKT (V-akt murine thymoma viral oncogene homolog), while down-regulated the expression of E-cadherin (epithelial cadherin) and PTEN (phosphatase and tensin homolog deleted on chromosome 10). To inhibit cell apoptosis, enforced expression of AEBP1 effectively blocked the cleavage of caspase 9 and p53 (protein 53) and promoted the expression of anti-apoptotic protein Bcl-2 (B-cell lymphoma-2). Finally, AEBP1 accelerated subcutaneously transplanted tumor growth in nude mice by increasing the expression of the cell proliferation biomarker ki67, the phosphorylation of AKT, and blocked apoptosis in vivo. In summary, these data suggested the important role of AEBP1 in the BCa progression, which could be used as a potential biomarker for prognostic hallmark and a novel therapeutic strategy.

TWEAK Knockdown Alleviates Post-Cardiac Arrest Brain Injury via the p38 MAPK/NF-κB Pathway.

Cardiac arrest (CA) and subsequent cardiopulmonary resuscitation (CPR) can cause brain injury, which is one of the factors affecting the recovery of brain function in CA patients. There is increasing evidence that tumor necrosis factor-like weak apoptosis-inducing factor (TWEAK) is associated with the brain injury diseases. This study was aimed to investigate the modulation mechanism of TWEAK involved in brain injury after cardiac arrest/subsequent cardiopulmonary resuscitation (CA/CPR). For in vivo experiments, healthy male Sprague-Dawley (SD) rats were applied to establish CA/CPR model, and oxygen-glucose deprivation/reoxygenation (OGD/R)-stimulated neurons model was established in vitro. TWEAK short hairpin RNAs (shRNAs) were injected into the lateral ventricle of CA/CPR rats or transfected into OGD/R cell culture to analyze the consequent alteration in neurological scores, behavioral tests, cell proliferation, cell apoptosis, and neuroinflammation through immunofluorescence staining, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining and enzyme linked immunosorbent assay (ELISA). There were high expressions of TWEAK and fibroblast growth factor-inducible 14 (Fn14) in the cerebral cortex of CA/CPR rats and OGD/R-stimulated neuronal cells. TWEAK knockdown attenuated cell apoptosis, inflammation and showed better behavioral tests in CA/CPR rats. Furthermore, TWEAK shRNAs obviously facilitated cell proliferation, suppressed apoptosis and inflammation after OGD/R injury. Western blotting results stated that TWEAK silencing promoted phosphorylated p38 (p-p38) and phosphorylated p65 (p-p65) expressions. TWEAK might be involved in the pathogenesis of CA/CPR through inhibiting p38 MAPK/NF-κB pathway.

Circular RNA protein tyrosine kinase 2 aggravates pyroptosis and inflammation in septic lung tissue by promoting microRNA-766/eukaryotic initiation factor 5A axis-mediated ATP efflux

Sepsis is characterized by an acute inflammatory response to infection, often with multiple organ failures, especially severe lung injury. This study was implemented to probe circular RNA (circRNA) protein tyrosine kinase 2 (circPTK2)-associated regulatory mechanisms in septic acute lung injury (ALI). A cecal ligation and puncture-based mouse model and an lipopolysaccharides (LPS)-based alveolar type II cell (RLE-6TN) model were generated to mimic sepsis. In the two models, inflammation- and pyroptosis-related genes were measured. The degree of lung injury in mice was analyzed by hematoxylin and eosin (H&E) staining and the apoptosis was by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling staining. In addition, pyroptosis and toxicity were detected in cells. Finally, the binding relationship between circPTK2, miR-766, and eukaryotic initiation factor 5A (eIF5A) was detected. Data indicated that circPTK2 and eIF5A were up-regulated and miR-766 was down-regulated in LPS-treated RLE-6TN cells and lung tissue of septic mice. Lung injury in septic mice was ameliorated after inhibition of circPTK2. It was confirmed in the cell model that knockdown of circPTK2 effectively ameliorated LPS-induced ATP efflux, pyroptosis, and inflammation. Mechanistically, circPTK2 mediated eIF5A expression by competitively adsorbing miR-766. Taken together, circPTK2/miR-766/eIF5A axis ameliorates septic ALI, developing a novel therapeutic target for the disease.

LncRNA MIAT Upregulates NEGR1 by Competing for miR-150-5p as a Competitive Endogenous RNA in SCIRI Rats.

Spinal cord ischemia-reperfusion injury (SCIRI) can cause a pathological state of irreversible delayed death of neurons in the spinal cord tissue and a range of complications, such as spinal cord dysfunction and motor function impairment. This study aimed to determine whether the long-stranded non-coding ribonucleic acid (lncRNA), myocardial infarction-associated transcript (MIAT), could upregulate neuronal growth regulator 1 (NEGR1) by competing for miR-150-5p as a competitive endogenous RNA in a rat SCIRI model. The MIAT knockdown vector or the corresponding blank vector was injected into the spinal cord of healthy sprague Dawley (SD) rats. Administration of the MIAT knockdown vector led to the establishment of the SCIRI rat model. Basso, Beattie & Bresnahan locomotor rating scale (BBB) assessment of hind limb motion. Pathological changes in the spinal cord were observed via hematoxylin and eosin staining and eosin staining. Quantitative polymerase chain reaction was performed to determine the expression levels of the candidate microRNAs and predicted candidate genes, and the relationship between them. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay (TUNEL) staining was used to detect apoptosis in the spinal cord tissue of rats in each group. Western blotting was performed to determine the expression of the apoptosis-related proteins, caspase-9, caspase-3, and BCL2-Associated X (Bax)/B-cell lymphoma-2 (Bcl-2). The luciferase reporter gene was used to assess the interaction among the lncRNA, MIAT, and miR-150-5, and the interaction between miR-150-5 and NEGR1. The sh-lncRNA, MIAT, improved exercise status, and pathological changes in the spinal cord of SCIRI rats, inhibited apoptosis, increased the expression of miR-150-5p, and reduced the expression of NEGR1. Compared with mimics-NC, the transfection of miR-150-5p significantly decreased the relative fluorescence activity ratio of MIAT 3'-untranslated region (3'-UTR) wild-type Human embryonic kidney cells 293 (HEK-293 cells). Compared with mimics-negative control (NC), the transfection of miR-150-5p significantly decreased the relative fluorescence activity ratio of NEGR1 3'-UTR wild-type HEK-293 cells. MIAT can affect the symptoms of SCIRI in rats. Furthermore, as a competitive endogenous RNA, MIAT upregulates NEGR1 by competing with miR-150-5p in SCIRI rats.

Neuroprotective effects of Chrysanthemum morifolium on cerebral ischemia- reperfusion injury contributes to the oxidative stress suppression and related Keap1/Nrf2 pathway

ABSTRACT Background Ischemic stroke, the cause of death and disability worldwide, is closely related to oxidative stress damage. Chrysanthemum has profound antiantioxidant activity. We aimed to verify whether Chrysanthemum morifolium extract (CME) influences brain injury in cerebral ischemia-reperfusion injury (CR/RI) model. Methods In vitro, rat hippocampal H19-7 neurons were pretreated with CME, CR/RI was simulated with oxygen glucose deprivation/reoxygenation (OGD/R). The cell viability, apoptosis, lactate dehydrogenase release, reactive oxygen species (ROS) generation, malonaldehyde (MDA) content and superoxide dismutase（SOD） activity were detected. In vivo, middle cerebral artery occlusion (MCAO) model rats were pre-administered with CME, and then behavioral test, triphenyltetrazolium chloride (TTC), hematoxylin-eosin staining (HE), terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL), ROS immunofluorescence, MDA and SOD activity were tested. Furthermore, Keap1/Nrf2 signaling of CME in CI/RI was investigated. Results In OGD/R induced in H19-7 cells, CME increased OGD/R-induced cell viability and reduced cell apoptosis, which was reversed by siNrf2 transfection . In MCAO rats, CME improved the neurological deficits and alleviated brain injury. However, co-treatment with MLK385 counteracted these neuroprotective effects of CME on MCAO rats. Conclusion CME could significantly reduce oxidative stress and nerve injury in vitro and in vivo models of CI/RI by regulating the Keap1/Nrf2 pathway.

Bloodletting Acupuncture at Jing-Well Points Alleviates Myocardial Injury in Acute Altitude Hypoxic Rats by Activating HIF-1α/BNIP3 Signaling-Mediated Mitochondrial Autophagy and Decreasing Oxidative Stress.

To explore the protective effect and possible mechanisms of bloodletting acupuncture at Jing-well points (BAJP) pre-treatment on acute hypobaric hypoxia (AHH)-induced myocardium injury rat. Seventy-five rats were randomly divided into 5 groups by a random number table: a control group (n=15), a model group (n=15), a BAJP group (n=15), a BAJP+3-methyladenine (3-MA) group (n=15), and a BANA (bloodletting at nonacupoint; tail bleeding, n=15) group. Except for the control group, the AHH rat model was established in the other groups, and the corresponding treatment methods were adopted. Enzyme-linked immunosorbent assay (ELISA) was used to detect creatine kinase isoenzyme MB (CK-MB) and cardiac troponins I (CTnI) levels in serum and superoxide dismutase (SOD) and malondialdehyde (MDA) levels in myocardial tissue. Hematoxylin-eosin (HE) staining was used to observe myocardial injury, and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining was used to observe cell apoptosis. Transmission electron microscopy detection was used to observe mitochondrial damage and autophagosomes in the myocardium. The mitochondrial membrane potential of the myocardium was analyzed with the fluorescent dye JC-1. Mitochondrial respiratory chain complex (complex I, III, and IV) activities and ATPase in the myocardium were detected by mitochondrial respiratory chain complex assay kits. Western blot analysis was used to detect the autophagy index and hypoxia inducible factor-1α (HIF-1α)/Bcl-2 and adenovirus E1B 19k Da-interacting protein 3 (BNIP3) signaling. BAJP reduced myocardial injury and inhibited myocardial cell apoptosis in AHH rats. BAJP pretreatment decreased MDA levels and increased SOD levels in AHH rats (all P<0.01). Moreover, BAJP pretreatment increased the mitochondrial membrane potential (P<0.01), mitochondrial respiratory chain complex (complexes I, III, and IV) activities (P<0.01), and mitochondrial ATPase activity in AHH rats (P<0.05). The results from electron microscopy demonstrated that BAJP pretreatment improved mitochondrial swelling and increased the autophagosome number in the myocardium of AHH rats. In addition, BAJP pretreatment activated the HIF-1α/BNIP3 pathway and autophagy. Finally, the results of using 3-MA to inhibit autophagy in BAJP-treated AHH rats showed that suppression of autophagy attenuated the treatment effects of BAJP in AHH rats, further proving that autophagy constitutes a potential target for BAJP treatment of AHH. BAJP is an effective treatment for AHH-induced myocardial injury, and the mechanism might involve increasing HIF-1α/BNIP3 signaling-mediated autophagy and decreasing oxidative stress.

Traumatic Brain Injury-Mediated Neuroinflammation and Neurological Deficits are Improved by 8-Methoxypsoralen Through Modulating PPARγ/NF-κB Pathway.

8-Methoxypsoralen (8-MOP) has anti-inflammatory, antioxidant and tissue-repairing abilities. Here, we probed the function and mechanism of 8-MOP in traumatic brain injury (TBI). The in-vivo TBI model was constructed in Sprague-Dawley (SD) rats using controlled cortical impact (CCI) surgery. In parallel, BV2 microglia and HT22 neurons were activated by lipopolysaccharide (LPS) to establish an in-vitro model. The modified neurological score (mNSS) and the Morris water maze experiment were employed to evaluate the rats' neurological functions. The rats' brain edema was assessed by the dry and wet method, and neuronal apoptosis in damaged brain tissues was monitored by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) and Nissl's staining. Immunohistochemistry (IHC) was applied to verify Iba1-microglial activation in brain lesions of rats. The expression of inflammatory cytokines in BV2 microglia and HT22 neurons in the injured lesion of TBI rats was examined by the enzyme-linked immunosorbent assay (ELISA). The levels of iNOS, COX2, TLR4, PPARγ, STAT3, and NF-κB in brain lesions, BV2 microglia and HT22 neurons were compared by Western blot. As a result, 8-MOP administration reduced inflammation and LPS-induced neuronal damage in BV2 microglia. In vivo, 8-MOP treatment relieved neurological deficits in TBI rats, improved cognitive, learning and motor functions and mitigated brain edema and neuroinflammation induced by TBI. Furthermore, LPS or TBI activated the NF-κB and STAT3 pathways and repressed the PPARγ expression. However, 8-MOP treatment attenuated NF-κB and STAT3 phosphorylation and elevated PPARγ levels. Hence, 8-MOP exerts neuroprotective and anti-inflammatory effects in TBI rats by modulating the PPARγ/NF-κB pathway.

The Role of the GRP78/PERK/ATF4 Pathway in the Ability of Gua Lou Gui Zhi Decoction to Attenuate Apoptosis by Inhibiting Endoplasmic Reticulum Stress after Ischemia-Reperfusion Injury.

Endoplasmic reticulum stress (ERS) is a key part of the apoptotic cascade that is initiated after cerebral ischemia-reperfusion injury and is very important for research on poststroke rehabilitation. In addition, the unfolded protein response (UPR) plays an important role in ERS because it activates downstream apoptotic signal transduction and induces apoptosis through the glucose-regulated protein 78 (GRP78)/protein kinase R (PKR)-like ER kinase (PERK)/activating transcription factor 4 (ATF4) pathway. The Gua Lou Gui Zhi Decoction (GLGZD) ameliorated neuronal apoptosis of ischemia-reperfusion injury caused by middle cerebral artery occlusion (MCAO) had been proved in our previous study. The present study aims to underly the regulatory ability of GLGZD in ERS-induced apoptosis mediated by the GRP78/PERK/ATF4 pathway. GLGZD was analyzed by HPLC. The effects of GLGZD were obversed on MCAO-induced ischemic rats. The cerebral infarct volume was detected by 2,3,5-Triphenyl-2H-Tetrazolium Chloride (TTC) Staining. Terminal Deoxynucleotidyl Transferase-Mediated dUTP-Biotin Nick End Labeling (TUNEL) were used to detect apoptosis. Transmission Electron Microscopy (TEM), Ca2+ levels and reactive oxygen species (ROS) detection were used to determine the function of endoplasmic reticulum. The GRP78/PERK/ATF4 signaling pathway was assessed by western blotting and immunohistochemistry. Our results showed that GLGZD exerted its effects on ischemia-reperfusion injury by significantly promoting the restoration of the quantity and morphology of the rough ER and reducing the neuronal apoptosis rate in the ischemic cortex. Moreover, both of the intracellular ROS and Ca2+ levels in ischemic cortical cells were found significantly reduced by GLGZD. The GLGZD-treated group showed increased levels of phosphorylation in both of PERK and eukaryotic translation initiation factor 2α (eIF2α), activation of cysteinyl aspartate-specific proteinase-3 (Caspase-3), upregulation of the total protein levels of sarcoplasmic/endoplasmic Ca2+ ATPase 2α (SERCA 2α) and B-cell leukemia/lymphoma gene 2 (Bcl-2). These findings suggest that GLGZD reduces oxidative stress-induced injury and promotes a dynamic calcium balance, thereby inhibiting ERS and exerting an antiapoptotic effect on neuronal ischemic injury, which are closely related to the activation of GRP78/PERK/ATF4 signaling pathway.