Terminal Deoxynucleotidyl Transferase-mediated dUTP-biotin Research Articles

Effect of Aconitum diphtheria on the Proliferation, Apoptosis, and Inflammatory Response of Rheumatoid Arthritis Fibroblast-Like Synoviocytes.

Rheumatoid arthritis (RA), a highly disabling autoimmune disease, is characterized by joint damage and synovial hyperplasia. This paper was designed to explore the effect and mechanisms of Aconitum diphtheria on the proliferation and apoptosis of RA fibroblast-like synoviocytes (RA-FLS). First, RA-FLS was treated with different doses of A. diphtheria extracts. Then, an inverted biological microscope was adopted to observe the RA-FLS morphology. Subsequently, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling staining, Cell Counting Kit-8 and western blot were utilized to analyze the apoptosis, proliferation, and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway-related proteins in RA-FLS, respectively. The experimental results disclosed that, after treatment of RA-FLS with A. diphtheria extracts, the cells became round and the intercellular space was increased. Besides, the RA-FLS proliferation was effectively inhibited by A. diphtheria extracts, while the apoptosis was promoted. Additionally, A. diphtheria extracts could effectively downregulate the expression levels of p-NF-κB (p50), NF-κB (p50), p-NF-κB (p65), and NF-κB (p65). Collectively, A. diphtheria can effectively inhibit RA-FLS proliferation and promote apoptosis in a dose-dependent manner. Similarly, A. diphtheria is able to downregulate p-NF-κB and NF-κB protein expression, but there is no dose-response relationship.

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.

Effects of cortisone in zebrafish (Danio rerio): Insights into gut microbiota interactions and molecular mechanisms underlying DNA damage and apoptosis

Cortisone can enter aquatic ecosystems and pose a risk to organisms therein. However, few studies have explored the effects of cortisone on the gut microbiota of aquatic organisms. Here, we exposed zebrafish (Danio rerio) to cortisone at environmentally relevant concentrations (5.0, 50.0, or 500.0 ng L−1) for 60 days to explore its toxicological effects and their association with gut microbiota changes. The terminal deoxynucleotidyl transferase-mediated dUTP–biotin nick-end labeling assay revealed that exposure to 50 ng L−1 cortisone significantly increased the intestinal cell apoptosis rate, 8-hydroxydeoxyguanosine contents, and caspase-3 and caspase-8 activities. Moreover, the transcriptome analysis results demonstrated a notable downregulation in the expression of most differentially expressed genes associated with apoptosis pathways, as well as changes in DNA replication, oxidative stress, and drug metabolism pathways; these results indicated the occurrence of cortisone-induced stress response in zebrafish. Molecular docking analysis revealed that cortisone can bind to caspase-3 through hydrogen bonds and hydrophobic interactions but that no such interactions occur between cortisone and caspase-8. Thus, cortisone may induce oxidative DNA damage and apoptosis by activating caspase-3. Finally, the 16S rRNA sequencing results demonstrated that cortisone significantly affected microbial community structures and functions in the intestinal ecosystem. These changes may indicate gut microbiota response to cortisone-induced intestinal damage and inflammation. In conclusion, the current results clarify the mechanisms underlying intestinal response to cortisone exposure and provide a basis for evaluating the health risks of cortisone in animals.

Meldonium, as a potential neuroprotective agent, promotes neuronal survival by protecting mitochondria in cerebral ischemia–reperfusion injury

BackgroundStroke is a globally dangerous disease capable of causing irreversible neuronal damage with limited therapeutic options. Meldonium, an inhibitor of carnitine-dependent metabolism, is considered an anti-ischemic drug. However, the mechanisms through which meldonium improves ischemic injury and its potential to protect neurons remain largely unknown.MethodsA rat model with middle cerebral artery occlusion (MCAO) was used to investigate meldonium’s neuroprotective efficacy in vivo. Infarct volume, neurological deficit score, histopathology, neuronal apoptosis, motor function, morphological alteration and antioxidant capacity were explored via 2,3,5-Triphenyltetrazolium chloride staining, Longa scoring method, hematoxylin and eosin staining, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay, rotarod test, transmission electron microscopy and Oxidative stress index related kit. A primary rat hippocampal neuron model subjected to oxygen–glucose deprivation reperfusion was used to study meldonium’s protective ability in vitro. Neuronal viability, mitochondrial membrane potential, mitochondrial morphology, respiratory function, ATP production, and its potential mechanism were assayed by MTT cell proliferation and cytotoxicity assay kit, cell-permeant MitoTracker® probes, mitochondrial stress, real-time ATP rate and western blotting.ResultsMeldonium markedly reduced the infarct size, improved neurological function and motor ability, and inhibited neuronal apoptosis in vivo. Meldonium enhanced the morphology, antioxidant capacity, and ATP production of mitochondria and inhibited the opening of the mitochondrial permeability transition pore in the cerebral cortex and hippocampus during cerebral ischemia–reperfusion injury (CIRI) in rats. Additionally, meldonium improved the damaged fusion process and respiratory function of neuronal mitochondria in vitro. Further investigation revealed that meldonium activated the Akt/GSK-3β signaling pathway to inhibit mitochondria-dependent neuronal apoptosis.ConclusionOur study demonstrated that meldonium shows a neuroprotective function during CIRI by preserving the mitochondrial function, thus prevented neurons from apoptosis.

External pressure induces the dysfunction of spermatogonia via triggering the intrinsic pathway of apoptosis.

Cryptorchidism, the failure of testes to descend into the scrotum, exposes the testes to higher temperature and external pressure. Scholars from Razi University found through research conducted at different pressure gradients (0, 25, 50, and 100 mmHg) and time gradients (2 and 4 h) that high hydrostatic pressure may lead to sperm apoptosis. In this work, we investigated the effect of external pressure on spermatogonia, exploring a new mechanism of male infertility caused by cryptorchidism. Various pressure gradients (0, 25, 50, and 100 mmHg) were applied to spermatogonia for different durations (0, 2, and 4 h) in the Cell Counting Kit-8 (CCK8) experiment. Morphological changes, cell ultrastructure, apoptosis rates, and the expression of apoptosis-related proteins (bax, bcl-2, caspase-3, and caspase-9) were assessed through immunofluorescence, electron microscopy, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay, flow cytometry, immunohistochemistry, real-time quantitative polymerase chain reaction (qPCR), and western blot. The cell viability assay showed that higher external pressure had a greater negative time-dependent impact on cell viability. Immunofluorescence results indicated that external pressure stimuli altered the morphology of spermatogonia. The results of TUNEL assay and flow cytometry demonstrated that external pressure stimuli induced apoptosis in spermatogonia. Transmission electron microscopy (TEM) observations showed the generation of apoptotic bodies, mitochondrial swelling, vacuolization, and mitochondrial cristae fusion. The results of immunohistochemistry indicated that pressure induced the expression of caspase-3 and caspase-9 proteins. qPCR and western blot analyses revealed an increased ratio of bax/bcl-2 and expression of caspase-3 and caspase-9. Methazolamide (cytochrome C inhibitor) blocked the pressure-induced cell apoptosis and inhibited the activation of caspase-3 while Z-IETD-FMK (caspase-8 inhibitor) did not. External pressure promotes spermatogonia apoptosis through the intrinsic apoptosis pathway, which may be one of the mechanisms of male infertility induced by cryptorchidism.

Effects of Maternal Smoke Exposure on Endoplasmic Reticulum Stress and Autophagy in the Lungs of Offspring Mice.

Cigarette smoke (CS) induces autophagy and endoplasmic reticulum (ER) stress in the lungs. Research suggests that maternal exposure to CS during pregnancy leads to decreased lung function in offspring. However, the effects of maternal CS exposure on lung autophagy and ER stress in offspring during pregnancy remain unclear. C57BL/6J female mice were divided into the AA (air treatment during both pre-pregnancy and pregnancy), AS (air treatment during pre-pregnancy and CS treatment during pregnancy), SA (CS treatment during pre-pregnancy and air treatment during pregnancy), and SS (CS treatment during both pre-pregnancy and pregnancy) groups. The male offspring mice were selected to the study and euthanized 49 days after birth for the study. Hematoxylin and eosin (HE) staining was employed to observe pathological alterations, while transmission electron microscopy (TEM) was utilized to examine ultrastructure and autophagic vesicles. Additionally, the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method was applied to identify apoptosis in lung tissues. Immunofluorescence, Real-Time PCR, and Western Blot analyses were conducted to assess the expression of ER stress and autophagy-related markers in lung tissues. The findings revealed that exposure to CS heightened the extent of pathological damage and the abundance of autophagosomes in the lungs of offspring mice. TUNEL results indicated an increased fluorescence intensity in the AS, SA and SS groups, with the most significant in AS and SS groups. Furthermore, CS exposure augmented the fluorescence intensity and expression of ER stress and autophagy-related proteins. The expression of C/EBP-homologous protein (CHOP) exhibited no discernible difference between the SA and SS groups but showed a significant increase in the AS group. Conversely, the expression levels of glucose-regulated protein 78 (GRP78), Caspase-12, Beclin-1, and microtubule-associated protein 1 light chain 3 (LC3) exhibited no significant difference between the AS and SA groups, whereas they were significantly upregulated in the SS group. Preconceptional and gestational exposure to CS heightened ER stress and autophagy in the lungs of mouse offspring. However, in mothers who smoked, withdrawal from CS during pregnancy led to a reduction in ER stress and autophagy in the lungs of their offspring.

Lipid-induced lipotoxic damage in liver and hepatocytes of black seabream Acanthopagrus schlegelii is mediated by endoplasmic reticulum stress

Liver is considered the critical tissue for metabolic processing of dietary nutrients including lipids. High-fat diets (HFD) can induce hepatic lipotoxic injury, but the mechanism is unclear. This study aimed to elucidate the underlying role of endoplasmic reticulum stress (ERS) in mediating HFD-induced hepatic lipotoxic injury in black seabream (Acanthopagrus schlegelii) via in vitro and in vivo experiments. For the in vitro experiment, isolated hepatocytes from black seabream were incubated for 24 h in medium (Control), or medium supplemented with 200 μM oleic acid (OA) alone or in combination with 20 μM 4-phenyl butyric acid (OA + 4-PBA). In the in vivo experiment, black seabream juveniles (3.30 ± 0.10 g) were fed diets containing lipid at 12.4% (Control) or 18.6% (HFD) for 8 weeks, after which the HFD-fed fish were divided into three groups and, on alternate days over a period of 8 days, subjected to intraperitoneal injections of 50 mg/kg 4-PBA in hydroxypropyl-β-cyclodextrin (HBC) (treatment 4-PBA) or carrier alone (treatment HFD + HBC) versus no injection (HFD). The contents of triglyceride and non-esterified fatty acid, as well as expression levels of genes and proteins involved in lipid metabolism, ERS, apoptosis and inflammation were determined in liver and isolated hepatocytes, and oil-red O staining, ultrastructural observation, flow cytometry assay of apoptosis rate, and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay (TUNEL) staining were performed on hepatocytes. The in vitro experiment showed that 4-PBA treatment significantly decreased lipid accumulation, and the OA-induced expression levels of genes/proteins linked to ERS, lipogenesis, inflammation and apoptosis in hepatocytes. Transmission Electron Microscope (TEM) imaging showed that 4-PBA treatment mitigated the ER swelling induced by OA. These results were confirmed in the in vivo experiment, where 4-PBA injection reduced lipid deposition and expression levels of genes/proteins related to ERS, lipogenesis, inflammation and apoptosis in liver compared to the HFD treatment. In conclusion, these results confirmed that ERS plays a contributing regulatory role in HFD- and OA-induced lipotoxic damage including lipid accumulation, apoptosis and inflammation in liver and isolated hepatocytes of black seabream, which supply a new perspective in the cross-talk of ERS and hepatic lipotoxic damage in hepatocytes/liver of marine fish and provide theoretical guidance for the prevention and treatment of hepatic lipotoxic damage.

Electroacupuncture Ameliorates Cerebral Ischemia-reperfusion Injury by Inhibiting Pyroptosis through the Sirtuin-1 Pathway

Abstract Objective: NOD-like receptor protein 3 (NLRP3)-mediated pyroptosis is pivotal in the pathological development of cerebral ischemia/reperfusion injury (CIRI). Although previous research has shown that electroacupuncture (EA) can alleviate CIRI through sirtuin-1 (SIRT1), the mechanism has not been well elucidated. Our study aimed to clarify whether the neuroprotective functions of EA are related to the reduction in NLRP3-mediated pyroptosis through the SIRT1 pathway. Materials and Methods: Rats received daily pretreatment with EA for 5 consecutive days before undergoing middle cerebral artery occlusion surgery. The Longa score was used to assess neurologic function. Infarct volume and morphological alterations were analyzed using 2,3,5-triphenyltetrazolium chloride and hematoxylin and eosin staining. In addition, neuronal pyroptosis was identified by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling/caspase-1 and neuronal nuclear antigen/caspase-1 immunofluorescence double staining. Levels of expression of pyroptosis markers were assessed by Western blotting and enzyme-linked immunosorbent assay. Results: EA improved deficits in neurologic function and minimized cerebral infarct volume. Mechanistically, a number of neuronal pyroptotic cells and protein levels of NLRP3, apoptosis-associated speck-like protein containing a CARD, and gasdermin D in the cerebral cortex were markedly reduced by EA treatment, and conversely, SIRT1 levels were increased. Notably, the specific SIRT1 inhibitor, EX527, reversed the effects of EA. Conclusions: EA potentially exerts a neuroprotective effect against CIRI through the SIRT1 pathway in NLRP3-mediated pyroptosis.

The Long Noncoding RNA Gall Bladder Cancer-Associated Suppressor of Pyruvate Carboxylase Inhibits the Proliferation, Migration, and Invasion of Colorectal Cancer Cells and Induces Their Apoptosis.

This study aimed to determine the role of the long noncoding RNA (lncRNA) gall bladder cancer-associated suppressor of pyruvate carboxylase (SOD2-1) in the progression of colorectal cancer (CRC). A total of 23 pairs of specimens, including CRC tissues and adjacent normal tissues, were collected, and the expression of lncRNA SOD2-1 (lnc-SOD2-1) was measured. lnc-SOD2-1 function was examined using HCT15 and HCT116 cells. A lnc-SOD2-1 overexpression vector was designed and transfected into both cell lines. MTS and colony formation assays were used to determine cell viability. Flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling assays were performed to measure apoptosis. Cell migration and invasion were evaluated using the Transwell assay. Migration and invasion markers were validated using quantitative reverse transcription-polymerase chain reaction and western blot analysis. The results indicated that the expression of lnc-SOD2-1 was downregulated in CRC tissues. lnc-SOD2-1 overexpression evidently decreased cell viability and led to the formation of fewer cell colonies. lnc-SOD2-1 overexpression induced ~ twofold higher apoptosis than the control group. lnc-SOD2-1 overexpression reduced the proportion of migratory and invasive cells to 50% and 75% of the control group, respectively. lnc-SOD2-1 overexpression significantly decreased the expression of matrix metalloproteinase-2 and -9. In conclusion, lnc-SOD2-1 may act as a tumor suppressor that inhibits the proliferation, migration, and invasion of CRC cells and induces their apoptosis.

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.

D-galactose-induced mitochondrial oxidative damage and apoptosis in the cochlear stria vascularis of mice

BackgroundAge-related hearing loss, known as presbycusis, is the result of auditory system degeneration. Numerous studies have suggested that reactive oxygen species (ROS) and mitochondrial oxidative damage play important roles in the occurrence and progression of aging. The D-galactose (D-gal)-induced aging model is well known and widely utilized in aging research. Our previous studies demonstrate that administration of D-gal causes mitochondrial oxidative damage and causes subsequent dysfunction in the cochlear ribbon synapses, which in turn leads to hearing changes and early stage presbycusis. Stria vascularis (SV) cells are vital for hearing function. However, it is unclear to what extent D-gal induces oxidative damage and apoptosis in the cochlear SV of mice. In addition, the source of the causative ROS in the cochlear SV has not been fully investigated.MethodsIn this study, we investigated ROS generation in the cochlear SV of mice treated with D-gal. Hearing function was measured using the auditory brainstem response (ABR). Immunofluorescence was used to examine apoptosis and oxidative damage. Transmission electron microscopy was also used to investigate the mitochondrial ultrastructure. DNA fragmentation was determined using the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) assay. Mitochondrial membrane potential (MMP) and ATP were also measured.ResultsWe found that D-gal-treated mice exhibited a significant shift in the mean amplitude and latency of the ABR; a remarkable increase in the levels of NADPH oxidase (NOX-2), Uncoupling protein 2 (UCP2) and cleaved caspase-3 (c-Cas3) was observed, as well as an increase in the number of TUNEL-positive cells were observed in the SV of mice. Both the expression of the DNA oxidative damage biomarker 8-hydroxy-2-deoxyguanosine (8-OHdG) and a commonly occurring mitochondrial DNA deletion were markedly elevated in the SV of mice that had been treated with D-gal to induce aging. Conversely, the ATP level and MMP were significantly reduced in D-gal-induced aging mice. We also found alterations in the mitochondrial ultrastructure in the SV of aging mice, which include swollen and distorted mitochondrial shape, shortened and thickened microvilli, and the accumulation of lysosomes in the SV.ConclusionOur findings suggest that the impairment of cochlear SV during presbycusis may be caused by mitochondrial oxidative damage and subsequent apoptosis.

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.

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

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

Naringin protects against inflammation and apoptosis induced by intestinal ischemia-reperfusion injury through deactivation of cGAS-STING signaling pathway.

Effective amelioration of ischemia/reperfusion (I/R)-induced intestinal injury and revealing its mechanisms remain the challenges in both preclinic and clinic. Potential mechanisms of naringin in ameliorating I/R-induced intestinal injury remain unknown. Based on pre-experiments, I/R-injured rat intestine in vivo and hypoxia-reoxygenation (H/R)-injured IEC-6 cells in vitro were used to verify that naringin-alleviated I/R-induced intestinal injury was mediated via deactivating cGAS-STING signaling pathway. Naringin improved intestinal damage using hematoxylin and eosin staining and decreased alanine aminotransferase and aspartate aminotransferase contents in plasma. Naringin decreased inflammation characterized by reducing IL-6, IL-1β, TNF-α, and IFN-β contents in both plasma and IEC-6 cells. Naringin mitigated oxidative stress via recovering superoxide dismutase, glutathione, and malondialdehyde levels in the I/R-injured intestine. Naringin reduced the expression of apoptotic proteins, including Bax, caspase-3, and Bcl-2, and reduced terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling-positive cells both in vivo and in vitro, and decreased Hoechst 33342 signals in vitro. cGAS, STING, p-TBK1, p-IRF3, and NF-κB expressions were up-regulated both in vivo and in vitro respectively and the up-regulated indexes were reversed by naringin. Transfection of cGAS-siRNA and cGAS-cDNA significantly down-regulated and up-regulated cGAS-STING signaling-related protein expressions, respectively, and partially weakened naringin-induced amelioration on these indexes, suggesting that deactivation of cGAS-STING signaling is the crucial target for naringin-induced amelioration on I/R-injured intestine.

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.