Transferase-mediated dUTP Nick End Labeling Research Articles

Molecular characterization and expression analysis of B-cell lymphoma-2 in Trachinotus ovatus and its role in apoptotic process.

B-cell lymphoma-2 (Bcl-2) is the first identified member of the Bcl-2 family that performs an anti-apoptotic function in mammals. However, its role in teleosts is not fully understood. In this study, Bcl-2 of Trachinotus ovatus (TroBcl2) was cloned, and its role in apoptosis was investigated. In this study, Bcl-2 of Trachinotus ovatus (TroBcl2) was cloned by PCR. Quantitative real-time PCR (qRT-PCR) was used to detect its mRNA expression level in healthy condition and after LPS stimulation. Subcellular localization was performed by transfecting the pTroBcl2-N3 plasmid into golden pompano snout (GPS) cells and observed under an inverted fluorescence microscope DMi8 and further verified by immunoblotting. In vivo overexpression and RNAi knockdown method were performed to evaluate the role of TroBcl2 in apoptosis. The anti-apoptotic activity of TroBcl2 was detected by flow cytometry. The effect of TroBcl2 on the mitochondrial membrane potential (MMP) was measured by an enhanced mitochondrial membrane potential assay kit with JC-1. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method was performed to evaluate the role of TroBcl2 in the DNA fragmentation. Immunoblotting was used to verify whether TroBcl2 inhibits the release of cytochrome c from mitochondria into the cytoplasm. The Caspase 3 and Caspase 9 Activity Assay Kits were used to investigate the effect of TroBcl2 on caspase 3 and caspase 9 activities. The effects of TroBcl2 on the expression of apoptosis-related and nuclear factor- κB (NF-κB) signaling pathway-related genes in vitro were evaluated by qRT-PCR and Enzyme linked immunosorbent assay (ELISA). Luciferase reporter assay was used to evaluate the activity in NF-κB signaling pathway. The full-length coding sequence of TroBcl2 contains 687 bp and encodes a protein containing 228 amino acids. Four conserved Bcl-2 homology (BH) domains and one invariant "NWGR" motif located in BH1 were identified in TroBcl2. In healthy T. ovatus, TroBcl2 was widely distributed in the eleven tested tissues, and higher expression levels were found in immune-related tissues, such as spleen and head kidney tissues. After stimulation with lipopolysaccharide (LPS), the expression of TroBcl2 in the head kidney, spleen, and liver was significantly upregulated. In addition, subcellular localization analysis revealed that TroBcl2 was localized in both the cytoplasm and nucleus. Functional experiments showed that TroBcl2 inhibited apoptosis, possibly by reducing mitochondrial membrane potential loss, decreasing DNA fragmentation, preventing cytochrome c release into cytoplasm, and reducing the caspase 3 and caspase 9 activations. Moreover, upon LPS stimulation, overexpression of TroBcl2 suppressed the activation of several apoptosis-related genes, such as BOK, caspase-9, caspase-7, caspase-3, cytochrome c, and p53. Furthermore, knockdown of TroBcl2 significantly increased the expression of those apoptosis-related genes. In addition, TroBcl2 overexpression or knockdown induced or inhibited, respectively, the transcription of NF-κB and regulated the expression of genes (such as NF-κB1 and c-Rel) in the NF-κB signaling pathway as well as the expression of the downstream inflammatory cytokine IL-1β. Overall, our study suggested that TroBcl2 performs its conserved anti-apoptotic function via the mitochondrial pathway and may serve as an anti-apoptotic regulator in T. ovatus.

Mechanism Repositioning Based on Integrative Pharmacology: Anti-Inflammatory Effect of Safflower in Myocardial Ischemia–Reperfusion Injury

Safflower (Carthamus tinctorius. L) possesses anti-tumor, anti-thrombotic, anti-oxidative, immunoregulatory, and cardio-cerebral protective effects. It is used clinically for the treatment of cardio-cerebrovascular disease in China. This study aimed to investigate the effects and mechanisms of action of safflower extract on myocardial ischemia-reperfusion (MIR) injury in a left anterior descending (LAD)-ligated model based on integrative pharmacology study and ultra-performance liquid chromatography-quadrupole time-of-flight-tandem mass spectrometer (UPLC-QTOF-MS/MS). Safflower (62.5, 125, 250 mg/kg) was administered immediately before reperfusion. Triphenyl tetrazolium chloride (TTC)/Evans blue, echocardiography, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, lactate dehydrogenase (LDH) ability, and superoxide dismutase (SOD) levels were determined after 24 h of reperfusion. Chemical components were obtained using UPLC-QTOF-MS/MS. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were used to analyze mRNA and protein levels, respectively. Safflower dose-dependently reduced myocardial infarct size, improved cardiac function, decreased LDH levels, and increased SOD levels in C57/BL6 mice. A total of 11 key components and 31 hub targets were filtered based on the network analysis. Comprehensive analysis indicated that safflower alleviated inflammatory effects by downregulating the expression of NFκB1, IL-6, IL-1β, IL-18, TNFα, and MCP-1 and upregulating NFκBia, and markedly increased the expression of phosphorylated PI3K, AKT, PKC, and ERK/2, HIF1α, VEGFA, and BCL2, and decreased the level of BAX and phosphorylated p65. Safflower shows a significant cardioprotective effect by activating multiple inflammation-related signaling pathways, including the NFκB, HIF-1α, MAPK, TNF, and PI3K/AKT signaling pathways. These findings provide valuable insights into the clinical applications of safflower.

Effects of Treadmill Exercise on Liver Apoptosis in Fluoride-Exposed Mice.

Hepatotoxicity induced by excessive fluoride (F) exposure has been extensively studied in both humans and animals. Chronic fluorosis can result in liver apoptosis. Meanwhile, moderate exercise alleviates apoptosis caused by pathological factors. However, the effect of moderate exercise on F-induced liver apoptosis remains unclear. In this research, sixty-four three-week-old Institute of Cancer Research (ICR) mice, half male and half female, were randomly divided into four groups: control group (distilled water); exercise group (distilled water and treadmill exercise); F group [100mg/L sodium fluoride (NaF)]; and exercise plus F group (100mg/L NaF and treadmill exercise). The liver tissues of mice were taken at 3months and 6months, respectively. Hematoxylin-eosin (HE) staining and situ terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) results showed that nuclear condensation and apoptotic hepatocytes occurred in the F group. However, this phenomenon could be reversed with the intervention of treadmill exercise. The results of QRT-PCR and western blot displayed NaF- induced apoptosis via tumor necrosis factor recpter1 (TNFR1) signaling pathway, while treadmill exercise could restore the molecular changes caused by excessive NaF exposure.

Effect of Isothiocyanate Therapy on Apoptosis of Trophoblast Cells in Pregnant Rats Hyperglycemia Atmosphere

Abstract: One of the factors that influence the complications of pregnancy and childbirth is diabetes mellitus. In this study, it was intended to create experimental diabetic conditions in experimental white rats with the administration of aloksan for 5 days. A total of 30 mice were checked for sugar levels on day 7 after inducing alloxane to see that the mice were already in a hyperglycemic state. Then divided into 6 groups, each group there are 5 white pregnant rats. Group 1 was the negative group (without being given aloksan), group 2 was positive group (given aloksan as much as 150 mg / day / kg BB), groups 3, 4, 5 and 6 were given Aloksan 150mg / day / kg and each was given simultaneously isothiocyanates with a dose of 0.1; 0,2; 0.4 and 0.8 mg/day/kg BB. DNA fragmentation is one of the characteristics of apoptosis. In this study DNA fragmentation was examined by the TUNEL method (Terminal deoxynucleotidyl Transferase-mediated dUTP Nick End Labeling). The average number of trophoblast cells in white pregnant rats hyperglycemia that experienced apoptosis decreased as the dose of isotiocyanate therapy increased in rats that had been induced by alloxane. And the best dose is at a dose of 0.8 mg / day / kg BB.

Tumor suppressor circPDE4D inhibits the progression of colorectal cancer and regulates oxaliplatin chemoresistance

Colorectal cancer (CRC) is one of the most common cancers worldwide, and it frequently develops resistance to chemotherapy. It was discovered that circular RNAs, which function as microRNA sponges, are involved in the pathogenesis of many cancers. This study aimed to investigate the biological functions of a circRNA derived from phosphodiesterase 4D (circPDE4D, hsa_circ_0072568) and its potential mechanism in oxaliplatin-resistant CRC. CircPDE4D expression were validated in human CRC cell lines and tissues. CircPDE4D siRNAs (si-circPDE4D) and LV003-circPDE4D plasmid were applied to investigate the function of circPDE4D. A quantitative real-time polymerase chain reaction was used to detect the levels of circPDE4D, its predicted sponge miRNAs, and their target genes. Cell proliferation was assessed by MTS（3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)–2H-tetrazolium） assay. Cell migration and invasion capacity were evaluated by transwell assay. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling was used to stain apoptotic cells. The results showed that circPDE4D expression was downregulated in CRC cells and tissues. Transfection with si-circPDE4D promoted cell proliferation, migration, and invasion, and inhibited apoptosis in DLD1 cells. Transfection with LV003-circPDE4D showed the opposite effect. Besides, circPDE4D presented higher expression in HCT116/L cells than that in HCT116 cells. Si-circPDE4D or lv003-circPDE4D transfection increased or decreased cell proliferationin in both two cells. Moreover, si-circPDE4D transfection inhibited cell apoptosis, while LV003-circPDE4D induced apoptosis in HCT116/L cells. LV003-CircPDE4D reduced hsa-miR-569 expression while increasing SPI1 expression in HCT116/L. CircPDE4D could inhibit tumorigenesis and progression of both CRC and oxaliplatin-resistant CRC, providing insight for the development of therapeutic strategies.

Vitamin D3 improved erectile function recovery by regulating autophagy and apoptosis in a rat model of cavernous nerve injury.

Vitamin D3 is an important element in improving erectile function. However, the mechanisms of vitamin D3 remain unknown. Thus, we explored the effect of vitamin D3 on erectile function recovery after nerve injury in a rat model and investigated its possible molecular mechanisms. Eighteen male Sprague-Dawley rats were used in this study. The rats were randomly divided into three groups: the control, bilateral cavernous nerve crush (BCNC), and BCNC + vitamin D3 groups. BCNC model was established in rats by surgery. The intracavernosal pressure and the ratio of intracavernosal pressure to mean arterial pressure were utilized to evaluate erectile function. Masson trichrome staining, immunohistochemistry, terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling and western blot analysis were performed on penile tissues to elucidate the molecular mechanism. The results indicated that vitamin D3 alleviated hypoxia and suppressed the fibrosis signalling pathway by upregulating the expression of eNOS (p = 0.001), nNOS (p = 0.018) and α-SMA (p = 0.025) and downregulating the expression of HIF-1α (p = 0.048) and TGF-β1 (p = 0.034) in BCNC rats. Vitamin D3 promoted erectile function restoration by enhancing the autophagy process through decreases in the p-mTOR/mTOR ratio (p = 0.02) and p62 (p = 0.001) expression and increases in Beclin1 expression (p = 0.001) and the LC3B/LC3A ratio (p = 0.041). Vitamin D3 application improved erectile function rehabilitation by suppressing the apoptotic process through decreases in the expression of Bax (p = 0.002) and caspase-3 (p = 0.046) and an increase in the expression of Bcl2 (p = 0.004). Therefore, We concluded that vitamin D3 improved the erectile function recovery in BCNC rats by alleviating hypoxia and fibrosis, enhancing autophagy and inhibiting apoptosis in the corpus cavernosum.

Bavachinin protects the liver in NAFLD by promoting regeneration via targeting PCNA

IntroductionNonalcoholic fatty liver disease (NAFLD) is the most common liver disease all over the world, and no drug is approved for the treatment of NAFLD. Bavachinin (BVC) is proven to possess liver-protecting effect against NAFLD, but its mechanism is still blurry. ObjectivesWith the use of Click Chemistry-Activity‐Based Protein Profiling (CC-ABPP) technology, this study aims to identify the target of BVC, and investigate the mechanism by which BVC exerts its liver-protecting effect. MethodsThe high fat diet induced hamster NAFLD model is introduced to investigate BVC’s lipid-lowering and liver-protecting effects. Then, a small molecular probe ofBVC is designed and synthesized based on theCC-ABPP technology, and BVC’s target is fished out. A series of experiments are performed to identify the target, including competitive inhibition assay, surface-plasmon resonance (SPR), cellular thermal shift assay (CETSA), drug affinity responsive target stability (DARTS) assay, and co-immunoprecipitation (Co-IP). Afterward, the pro-regeneration effects of BVC are validated in vitro and in vivo through flow cytometry, immunofluorescence, and the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). ResultIn the hamster NAFLD model, BVC shows lipid-lowing effect and improvement on the histology. PCNA is identified as the target of BVC with the method mentioned above, and BVC facilitates the interaction between PCNA and DNA polymerase delta. BVC promotes HepG2 cells proliferation which is inhibited by T2AA, an inhibitor suppresses the interaction between PCNA and DNA polymerase delta. In NAFLD hamsters, BVC enhances PCNA expression and liver regeneration, reduces hepatocyte apoptosis. ConclusionThis study suggests that, besides the anti-lipemic effect, BVC binds to the pocket of PCNA facilitating its interaction with DNA polymerase delta and pro-regeneration effect, thereby exerts the protective effect against HFD induced liver injury.

Zhao’s Weitiao No 3 inhibits tumor growth in gastric cancer-bearing nude mice

Purpose: To investigate the effect of Zhao’s Weitiao No. 3 (ZW3) on the progression of gastric cancer (GC) in mice, and to determine its effects on the proliferation and apoptosis of gastric cancer tissues.
 Methods: Tumor growth in vivo was used to determine the effect of ZW3 (15, 30, and 60 mg/kg) on GC growth in mice. Immunohistochemical and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assays were conducted to evaluate the effects of ZW3 on the proliferation and apoptosis of mouse GC tissues, while quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and immunoblot assays were conducted to determine its effects on GC cell apoptosis.
 Results: ZW3 inhibited tumor growth in tumor-bearing mice, suppressed the expression of proliferation-related proteins (p < 0.001), increased the apoptosis of tumor cells in tumor-bearing mice (p < 0.001), and suppressed the expression of apoptosis-related proteins in mouse tumor tissues (p < 0.001).
 Conclusion: Thus, ZW3 is a potential drug for the management of GC. However, further investigations are required to determine its full potential

Neutrophil Membrane-Coated Mesoporous Silica Nanoparticles Loaded with Hydrocortisone Alleviate DSS-Induced Colitis in Mice

The incidence and prevalence of ulcerative colitis (UC) are increasing worldwide. Hydrocortisone enema (HC) is widely used in UC treatment; however, this approach is limited by the easy degradation of HC in the intestinal environment and low intestinal uptake efficiency. Accordingly, the development of methods to improve the targeting efficiency of corticosteroids is an important issue. Mesoporous silica nanoparticles (MSN) and biomimetic nanovesicles (derived from the natural cell membrane structure) provide a basis for improved drug delivery and uptake. In this research, we constructed neutrophil membrane-coated MSN loaded with HC for the treatment of UC. MSN-HC and neutrophil-derived membrane nanoparticles (MSN-HC@NM NPs) were obtained by mechanical extrusion and characterized. The effects of free HC, MSN, MSN-HC, and MSN-HC@NM NPs were compared using dextran sodium sulfate (DSS)-induced colitis model, including terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assays and inflammatory factors in colon tissues. The MSN-HC@NM NPs alleviated DSS-induced colitis by reducing colon apoptosis and ameliorating the destruction of the colonic barrier and inflammation in mice. This research provides a hydrocortisone delivery system with good biosafety and the potential for clinical translation.

Perfluorocarbon restrains inflammation and cell apoptosis in rats with lung ischemia reperfusion injury via down regulation of TLR4/NF κB signaling pathway

Purpose: To exaluate the effect of perfluorocarbon on lung ischemia-reperfusion injury in rats, and to unravel the potential underlying mechanism.Methods: A total of 36 Sprague-Dawley (SD) rats were randomly assigned to sham group, model group, and perfluorocarbon group (12 rats per group). The levels of inflammatory factors (TNF-α and IL1β) were determined using enzyme-linked immunosorbent assay (ELISA). Cell apoptosis was evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay, while Western blotting was conducted to determine the protein expressions of TLR4 and NF-κB.Results: The levels of inflammatory factors in the model and perfluorocarbon groups were significantly higher than those in operation group (p < 0.05), while their levels in perfluorocarbon group were significantly lower than in model group (p < 0.05). The mRNA expression levels of TNF-α and IL-1β in lung tissues rose significantly in both model and perfluorocarbon groups when compared with those in sham group (p < 0.05), but declined significantly in the perfluorocarbon group in comparison with those in model group (p < 0.05). Furthermore, the perfluorocarbon group exhibited a significantly lower cell apoptosis than model group (p < 0.05). The relative protein expression levels of TLR4 and NF-κBdeclined significantly in perfluorocarbon group than in model group.Conclusions: Perfluorocarbon down-regulates the TLR4/NF-κB signaling pathway, and inhibits inflammation an

Effect of Chitosan Drug-Loaded Nanoparticles on Proliferation of Gastric Cancer Cells and Expression of Prdx6 Protein

Since there are many problems in the clinical application of traditional chemotherapy drugs for gastric cancer (GC), including poor water solubility and insufficient targeting, the preparation of docetaxel nanoparticles (NPs) with chitosan (CS) as the carrier under nanotechnology can improve its water solubility and targeting and thus improve the bioavailability and antitumor effect. GX1 was used as a GC target molecule, N-deoxycholic acid (DCA) hydroxyethyl CS was used as a nanodrug carrier, and docetaxel was used as an antitumor angiogenesis drug to fabricate GC target NPs. The physical characterization was analyzed, the sustained release performance was further analyzed, and the anticancer performance was tested from in vitro and in vivo perspectives. After the NPs were transfected into GC cells, Prdx6 protein level in the cancer cells was determined by western blotting, and the survival rate of GC cells was determined by cholecystokinin octapeptide (CCK-8). Experiments showed that the encapsulation efficiency and drug loading rate of GC-targeted NPs reached 51.8% and 24.3%, respectively. The average particle size was 148.7 nm, and the surface showed smooth and homogeneous aggregation. docetaxel in the NPs belongs to pH-responsive sustained release, and the drug diffusion type belongs to non-Fickian diffusion. in vitro tests showed that GX1 can improve the uptake rate of the NPs by gastric vascular endothelial cells, and in vivo tests showed that the NPs can greatly limit the growth rate of GC tumors in nude mice (P < 0.01). Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) results of tumor sections showed that NPs could induce a large amount of tumor cell apoptosis. The expression of Prdx6 protein in GC cells was decreased after transfection with NPs (P < 0.05), and NPs notably inhibited the proliferation of GC cells (P < 0.01).

Inhibition of CaMKK2 Decreases Progression of Post-traumatic Osteoarthritis in a Rabbit ACL Transection Model

Background and Hypothesis:Post-traumatic osteoarthritis (PTOA) is a multifactorial degenerative disease of the joint affecting 20-50% of all joint injuries with a total annual cost of $15 billion. There are no current disease-modifying therapies for PTOA. Mechanical stress due to ligament tear or impact injury triggers the release of inflammatory mediators in the joint. Resulting collagen damage, loss of proteoglycans, and cell death triggers further release of inflammatory mediators and reactive oxygen species. This cycle of inflammation leads to PTOA. We hypothesize that inhibition of Ca2+/CaM dependent protein kinase kinase 2 (CaMKK2), a kinase associated with the inflammatory effects in PTOA, will mitigate the disease-propagating mechanisms.
 Methods:We utilized a rabbit model of PTOA which involved surgical transection of the anterior cruciate ligament (ACL) to generate joint instability. Rabbits were then treated tri-weekly with either STO-609 (CaMKK2 inhibitor, 0.033 mg/kg) or saline (control) for 16 weeks. Rabbits were sacrificed at 16 weeks post-surgery. Tibiofemoral joints were harvested for staining with safranin O fast green (SO) and PTOA grading via Osteoarthritis Research Society International (OARSI) guidelines. Apoptosis was assessed with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). RNA isolation of cartilage and subchondral bone tissue was conducted for qRT-PCR. Gene expression of MMP-13, IL-6, IL-1B, ACAN, COL2A1 was quantified and normalized to GAPDH.
 Results:Histology and gross morphology showed increased PTOA severity in saline controls compared to STO-609 treated rabbits. There was no significant difference in chondrocyte apoptosis in STO-609 treated rabbits compared to saline controls based on TUNEL staining. Gene expression analyses are in progress.
 Potential Impact:This study addresses the unmet clinical need for novel disease-modifying therapeutics for PTOA. Preliminary results show that inhibition of CaMKK2 has the potential to decrease cartilage degradation after joint injuries.

Comprehensive Analysis of RNA-Binding Protein-Related lncRNA in Breast Invasive Carcinoma.

RNA-binding proteins (RBPs), which form complexes or single/multiple RNA-binding domains, have a functional role in regulating and determining the function or stability of the bound RNAs in various cancers, including breast invasive carcinoma (BRCA). However, the biological functions and clinical implications of RBP-related long noncoding RNAs (lncRNAs) in BRCA remain largely unknown. Herein, we first identified and characterized RBP-related lncRNAs in BRCA. Then we built an RBP-related lncRNA signature (RBPLSig) and explored the clinical evaluation and prediction performance of the RBPLSig by bioinformatic analysis. In addition, to optimize treatment plans, prediction online tools was developed to predict the patient survival rate. Lastly, to verify the function of lncRNA WAC antisense RNA 1 (WAC-AS1), the experiments such as Quantitative real-time PCR (qRT-PCR), lncRNA knockdown, CCK-8, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining were performed. We also gained the potential mechanisms of the druggable compounds of the WAC-AS1 related RBP gene, putative NSUN6, using molecular docking. The results showed that RBPLSig, as an independent prognostic factor for BRCA patients, was involved in numerous malignancy-associated immunoregulatory pathways. We found different immune statuses and responses to immunotherapy, chemotherapy, and targeted therapy between the high- and low-risk groups stratified by RBPLSig. Our data broaden the comprehensive understanding of the biological functions of RBP-related lncRNAs, and demonstrate a novel and independent RBPLSig to assess prognosis and the immune microenvironment, thus helping to guide treatment decisions for BRCA.

Electroacupuncture Improves Blood-Brain Barrier and Hippocampal Neuroinflammation in SAMP8 Mice by Inhibiting HMGB1/TLR4 and RAGE/NADPH Signaling Pathways.

To investigate the molecular mechanisms underlying the beneficial effect of electroacupuncture (EA) in experimental models of Alzheimer's disease (AD) in vivo. Senescence-accelerated mouse prone 8 (SAMP8) mice were used as AD models and received EA at Yingxiang (LI 20, bilateral) and Yintang (GV 29) points for 20 days. For certain experiments, SAMP8 mice were injected intravenously with human fibrin (2 mg). The Morris water maze test was used to assess cognitive and memory abilities. The changes of tight junctions of blood-brain barrier (BBB) in mice were observed by transmission electron microscope. The expressions of fibrin, amyloid- β (Aβ), and ionized calcium-binding adapter molecule 1 (IBa-1) in mouse hippocampus (CA1/CA3) were detected by reverse transcription-quantitative polymerase chain reaction (qRT-PCR), Western blot or immunohistochemical staining. The expression of fibrin in mouse plasma was detected by enzyme-linked immunosorbent assay. The expressions of tight junction proteins zonula occludens-1 and claudin-5 in hippocampus were detected by qRT-PCR and immunofluorescence staining. Apoptosis of hippocampal neurons was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. Fibrin was time-dependently deposited in the hippocampus of SAMP8 mice and this was inhibited by EA treatment (P<0.05 or P<0.01). Furthermore, EA treatment suppressed the accumulation of Aβ in the hippocampus of SAMP8 mice (P<0.01), which was reversed by fibrin injection (P<0.05 or P<0.01). EA improved SAMP8 mice cognitive impairment and BBB permeability (P<0.05 or P<0.01). Moreover, EA decreased reactive oxygen species levels and neuroinflammation in the hippocampus of SAMP8 mice, which was reversed by fibrin injection (P<0.05 or P<0.01). Mechanistically, EA inhibited the promoting effect of fibrin on the high mobility group box protein 1 (HMGB1)/toll-like receptor 4 (TLR4) and receptor for advanced glycation end products (RAGE)/nicotinamide adenine dinucleotide phosphate (NADPH) signaling pathways (P<0.01). EA may potentially improve cognitive impairment in AD via inhibition of fibrin/A β deposition and deactivation of the HMGB1/TLR4 and RAGE/NADPH signaling pathways.

Silencing of long noncoding RNA X-inactive specific transcript alleviates Aβ1-42-induced microglia-mediated neurotoxicity by shifting microglial M1/M2 polarization.

This experimental study aims to investigate the role of long noncoding RNA X-inactive specific transcript (lncRNA XIST) in the microglial polarization and microglia-mediated neurotoxicity in Alzheimer's disease (AD). The levels of XIST and microRNA-107 (miR-107) were detected by quantitative real-time polymerase chain reaction. The spatial learning and memory capability of APPswe/PS1dE9 (APP/PS1) mice were evaluated by the Morris water maze test. The morphology of mouse hippocampus cells was evaluated by hematoxylin and eosin staining. The Iba1-positive microglia were labeled by immunohistochemistry staining. The protein levels were determined by western blot and enzyme-linked immunosorbent assay. Neurotoxicity was evaluated by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, caspase-3 activity, and Cell Counting Kit-8 assay. The XIST, miR-107, and AD targets were predicted by bioinformatics analysis. The level of XIST was increased in APP/PS1 mice, and XIST silencing ameliorated AD progression. XIST silencing suppressed microglia activation, microglial M1 polarization, and proinflammatory factor levels, but promoted microglial M2 polarization in APP/PS1 mice and Aβ1-42-treated BV-2 cells. XIST knockdown reduced Aβ1-42-induced microglia-mediated apoptosis and enhanced cell viability in HT22 cells. XIST silencing down-regulated miR-107 level and attenuated Aβ1-42-caused suppression of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling. Those effects of XIST silencing were attenuated by miR-107 inhibitor or LY294002. Downregulation of XIST lessened Aβ1-42-induced microglia-mediated neurotoxicity by modulating microglial M1/M2 polarization, which may be mediated by the miR-107/PI3K/Akt pathway.

Genkwanin suppresses mitochondrial dysfunction to alleviate IL-1β-elicited inflammation, apoptosis, and degradation of extracellular matrix in chondrocytes through upregulating DUSP1.

Osteoarthritis (OA) is a form of chronic degenerative disease contributing to elevated disability rate among the elderly. Genkwanin is an active component extracted from Daphne genkwa possessing pharmacologic effects. Here, this study is designed to expound the specific role of genkwanin in OA and elaborate the probable downstream mechanism. First, the viability of chondrocytes in the presence or absence of interleukin-1 beta (IL-1β) treatment was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay was used to assess cell apoptosis. Inflammatory response was estimated through enzyme-linked immunosorbent assay and Western blot. In addition, immunofluorescence staining and Western blot were utilized to measure the expression of extracellular matrix (ECM)-associated proteins. Dual-specificity protein phosphatase-1 (DUSP1) expression was tested by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot. Following DUSP1 elevation in genkwanin-treated chondrocytes exposed to IL-1β, inflammatory response and ECM-associated factors were evaluated as forementioned. In addition, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine iodide staining was to assess the mitochondrial membrane potential. Adenosine triphosphate (ATP) level was examined with ATP assay kit, and RT-qPCR was used to test mitochondrial DNA expression. Results indicated that genkwanin administration enhanced the viability while ameliorated the apoptosis, inflammatory response, and ECM degradation in IL-1β-induced chondrocytes. Besides, genkwanin treatment fortified DUSP1 expression in IL-1β-exposed chondrocytes. DUSP1 interference further offsets the impacts of genkwanin on the inflammation, ECM degradation, and mitochondrial dysfunction in IL-1β-challenged chondrocytes. In short, genkwanin enhanced DUSP1 expression to mitigate mitochondrial dysfunction, thus ameliorating IL-1β-elicited inflammation, apoptosis, and degradation of ECM in chondrocytes.

Chryseriol attenuates the progression of OVA-induced asthma in mice through NF-κκB/HIF-1αα and MAPK/STAT1 pathways

Asthma is a hackneyed chronic inflammatory disease of the airway. Chryseriol (CSR) is a kind of flavonoid, and has the effect of bronchiectasis, indicating its potential application for treating respiratory diseases. However, the functions of CSR in asthma have not been reported till now. The histopathologic changes of the lung tissues were assessed by hematoxylin and eosin staining. The cell apoptosis was identified through terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling assay. Total numbers of eosinophils, neutrophils, and macrophages were assessed under microscope. The levels of interleukin (IL)-1β, IL-4, IL-5, and IL-13 were detected by enzyme-linked-immunosorbent serologic assay. The airway hyper-responsiveness (AHR) was evaluated by the whole body plethysmography. The levels of methane dicarboxylic aldehyde, superoxide dismutase, glutathione S-transferase, and glutathione in lung homogenates were confirmed by using corresponding commercial kits. The protein expressions were examined by Western blot analysis. The ovalbumin (OVA) was utilized to establish asthma mouse model. At first, it was revealed that CSR treatment reduced lung injury in OVA-stimulated mice. Moreover, cell apoptosis was enhanced after OVA stimulation but was attenuated by CSR treatment. In addition, CSR treatment decreased the infiltration of inflammatory cells and the production of inflammatory factors in OVA-treated mice. Further investigations demonstrated that CSR treatment relieved AHR in OVA-stimulated mice. The oxidative stress was strengthened in OVA-treated mice, but these effects were relieved by CSR treatment. Lastly, it was discovered that CSR treatment retarded nuclear factor kappa B (NF-κB)/hypoxia-inducible factor 1 alpha (HIF-1α) and p38 mitogen-activated protein kinase (MAPK)/signal transducer and activator of transcription 1 (STAT1) pathways in OVA-triggered asthma mice. Our findings proved that CSR attenuated the progression of OVA-induced asthma in mice through inhibiting NF-κB/HIF-1α and MAPK/STAT1 pathways. This work might highlight the functions of CSR in the treatment of asthma.

Isosteviol plays a protective role on hepatic ischemia and reperfusion injury in mice through MAPK/NF-κB signaling pathway.

Hepatic ischemia and reperfusion (I/R) injury is of common occurrence during liver surgery and transplantation, isosteviol (ISV) is an acid hydrolysate of stevioside, the major component of Stevia rebaudiana. Stevioside and its metabolites have been shown to have varieties of pharmacological activities, However, the effect of ISV on hepatic I/R injury has not determined. The purpose of this paper is to study the effect of ISV on mice with hepatic I/R injury and further investigate its underlying mechanism. The blood vessels supplying the left/middle lobe of the liver in mice were clamped to cause liver ischemia for 1h, and then removed the clamp to conduct reperfusion for 6 h. ISV or saline was injected intraperitoneally after reperfusion. The expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6 and IL-10 in serum and tissues were evaluated by enzyme linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction (qRT-PCR). The infiltration of neutrophils and macrophages into the liver tissue was determined by flow cytometry and myeloperoxidase. Liver hematoxylin-eosin (HE) staining, terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL) and Annexin V probe were used to determine liver injury and hepatocyte apoptosis. western blots (WB) was used to investigate the activation of nuclear factor kappa-B (NF-κB) and c-JunNH2 terminal kinase (JNK), p38 and extracellular regulated protein kinase (ERK), while the expression of apoptosis-related proteins B-cell lymphoma-2 (BCL-2), BCL2-associated X protein (BAX), caspase-3 was detected. ISV reduced aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels to alleviate liver injury. ISV significantly reduced the release of inflammatory cytokines and the accumulation of liver neutrophils and macrophages. Meanwhile, ISV can promote the expression of anti-apoptosis-related protein BCL-2 and inhibit the expression of pro-apoptotic protein BAX and the activation of the protease caspase-3, and reduce the occurrence of hepatocyte apoptosis. Finally, ISV can reduce the phosphorylation level and activation of NF-κB, JNK, p38 and ERK. ISV inhibits the occurrence of inflammation and hepatocyte apoptosis through mitogen-activated protein kinase (MAPK)/NF-κB signaling pathway to relieve liver injury.

Comprehensive semen examination in patients with pancreatic-sufficient and pancreatic-insufficient cystic fibrosis.

We examined a cohort of 93 cystic fibrosis (CF) male patients who were pancreatic-sufficient (PS-CF; n=40) or pancreatic-insufficient (PI-CF; n = 53). Complex semen examination was performed, including standard semen analysis, quantitative karyological analysis (QKA) of immature germ cells (IGCs), transmission electronic microscopy (TEM), biochemical analysis, and sperm DNA fragmentation by terminal deoxynucleotidyl transferase-mediated dUTP nickend labeling (TUNEL) assay. Azoospermia was diagnosed in 83 (89.2%) patients. The other 10 (10.8%) patients were found to be nonazoospermic and showed various spermatological diagnoses (asthenozoospermia, n = 2; asthenoteratozoospermia, n = 3; oligoasthenozoospermia, n = 1; oligoasthenoteratozoospermia, n = 3; and normozoospermia, n = 1) with no specific morphological abnormalities. Oligospermia was detected in 89.2% azoospermic and 30.0% nonazoospermic patients. Low seminal pH (<7.0) was found in 74 (89.2%) of 83 azoospermic patients. Moderate leukocytospermia (2.0 × 10 6 -2.2 × 10 6 ml -1 ) was revealed in 2.4% azoospermic and 40.0% nonazoospermic semen samples. The signs of partial meiotic arrest at prophase I were found in 4 of 6 nonazoospermic patients examined by QKA of IGCs. The content of fructose and citrate was low in oligospermic and normal in nonoligospermic semen samples. An increased percentage (>30%) of spermatozoa with noncondensed ("immature") chromatin was revealed in 2 of 6 nonazoospermic semen samples analyzed by TEM.

Gentiopicroside Ameliorates Cerebrovascular Angiogenesis, Neuronal Injury and Immune Disorder in Rats with Cerebral Ischemia/Reperfusion Injury via VEGF and Phosphorylated Nrf2 Elevation.

Cerebral ischemia-reperfusion (CI/R) injury is induction of blood flow restoration after an ischemic stroke. Gentiopicroside (GPC) is the principal active secoiridoid glycoside of Gentiana Manshurica Kitagawa. This research aimed to illuminate the function of GPC and its mechanism in CI/R injury. After CI/R injury models were constructed, GPC (25, 50 or 100 mg/kg) was then administered by gavage to rats. Rats were grouped into Sham, CI/R, CI/R+25 mg/kg GPC, CI/R+50 mg/kg GPC, and CI/R+100 mg/kg GPC. Neuronal cells were exposed to oxygen-glucose deprivation and reperfusion (OGD/R) injury to establish ischemic-like conditions in vitro, and cells were further treated with 25, 50, or 100 μM GPC. Cells were grouped into control, OGD/R, OGD/R+25 μM GPC, OGD/R+50 μM GPC, and OGD/R+100 μM GPC. GPC's function on rat cerebral injury, angiogenesis, oxidative stress, neuronal injury and immune dysfunction in vivo was estimated using hematoxylin-eosin staining, Western blot, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining, commercial kits and enzyme linked-immunosorbent assay. Meanwhile, GPC's mechanism in CI/R injury was examined via Western blot. GPC's function in vitro was estimated via Cell Counting Kit-8 assay, 5-ethynyl-2'-deoxyuridine (EdU) staining, flow cytometry. GPC alleviated cerebral injury through decreasing cerebral infarction volume, cerebral indexes, brain water contents (p < 0.05). GPC reduced oxidative stress and boosted cerebral angiogenesis in CI/R rats (p < 0.05). Meanwhile, GPC weakened neuronal cell apoptosis, and decreased neuron-specific enolase and S100beta protein levels in CI/R rats. GPC reduced inflammatory cytokines contents in serum and brain tissues of CI/R rats (p < 0.05). Moreover, GPC increased the viability and proliferation in OGD/R-treated neuronal cells, but decreased cell apoptosis (p < 0.05). Mechanistically, GPC upregulated vascular endothelial growth factor (VEGF) and phosphorylated nuclear factor E2-related factor 2 (p-Nrf2) levels in CI/R rat brain tissues (p < 0.05). GPC reduced cerebrovascular angiogenesis, neuronal injury and immune disorder in CI/R injury through elevating VEGF and p-Nrf2.