ABSTRACT
In this experiment, we investigated the effects of adding chlorogenic acid (CGA) to the diet on growth performance, immune function, inflammation response, antioxidant capacity and its related mechanisms of common carp (Cyprinus carpio). A total of 600 fish were selected and randomly divided into five treatment groups and fed with CGA containing 0 mg/kg (CK), 100 mg/kg (L100), 200 mg/kg (L200), 400 mg/kg (L400) and 800 mg/kg (L800) for 56 days. The results of the experiment were as follows: addition of CGA significantly increased the WGR, SGR, FER, and PER of common carp (P < 0.05). The addition of 400-800 mg/kg of CGA significantly increased the serum levels of LZM, AKP activity, C3 and C4 concentration, and increased immune function of common carp (P < 0.05). Regarding antioxidant enzyme activities, adding CGA significantly increased SOD, CAT, and GsH-Px activities, while decreasing MDA content (P < 0.05). Compared with the CK group, the mRNA expression levels of NF-κB, TNF-α, and IL-1ß were decreased. The IL-10 and TGF-ß were increased in the liver and intestines of the CGA supplemented group. Meanwhile, the addition of CGA also significantly up-regulated the mRNA expression levels of Nrf2, HO-1, SOD, CAT, and GPX (P < 0.05). CGA also positively contributed to the development of the carp intestinal tract, as demonstrated by decreased serum levels of DAO, D-LA, and ET-1. And the mucosal fold height was increased significantly with increasing levels of CGA. In conclusion, the addition of CGA in the feed can enhance the growth performance, immune function and antioxidant capacity of common carp, and improve the health of the intestine and liver. According to the results of this experiment, the optimal addition amount in common carp diets was 400 mg/kg.
Subject(s)
Antioxidants , Carps , Animals , Antioxidants/metabolism , NF-kappa B/metabolism , Carps/metabolism , NF-E2-Related Factor 2/metabolism , Chlorogenic Acid/pharmacology , Signal Transduction , Dietary Supplements , Diet/veterinary , Intestines , Liver/metabolism , Immunity, Innate , RNA, Messenger/metabolism , Superoxide Dismutase/metabolism , Animal Feed/analysisABSTRACT
N-acetylcysteine (NAC) positively contributes to enhancing animal health, regulating inflammation and reducing stress by participating in the synthesis of cysteine, glutathione, and taurine in the body. The present study aims to investigate the effects of dietary different levels of NAC on the morphology, function and physiological state of hepatopancreas in juvenile common carp (Cyprinus carpio). 450 common carps were randomly divided into 5 groups: N1 (basal diet), N2 (1.5 g/kg NAC diet), N3 (3.0 g/kg NAC diet), N4 (4.5 g/kg NAC diet) and N5 (6.0 g/kg NAC diet), and fed for 8 weeks. The results indicated that dietary 3.0-6.0 g/kg NAC reduced hepatopancreas lipid vacuoles and nuclear translocation, and inhibited apoptosis in common carp. Simultaneously, the activities of hepatopancreas alanine aminotransferase and aspartate aminotransferase progressively increased with rising dietary NAC levels. Dietary NAC enhanced the non-specific immune function of common carp, and exerted anti-inflammatory effects by inhibiting the MAPK/NF-κB signaling pathway. Additionally, dietary 3.0-6.0 g/kg NAC significantly improved the antioxidant capacity of common carp, which was associated with enhanced glutathione metabolism, clearance of ROS and the activation of Nrf2 signaling pathway. In summary, NAC has the potential to alleviate inflammation, mitigate oxidative stress and inhibit apoptosis via the MAPK/NF-κB/Nrf2 signaling pathway, thereby improving hepatopancreas function and health of common carp. The current findings provide a theoretical basis for promoting the application of NAC in aquaculture and ecological cultivation of aquatic animals.
Subject(s)
Antioxidants , Carps , Animals , Antioxidants/metabolism , NF-kappa B/metabolism , Acetylcysteine/pharmacology , Carps/metabolism , NF-E2-Related Factor 2/metabolism , Hepatopancreas/metabolism , Signal Transduction , Diet/veterinary , Inflammation/veterinary , Glutathione , Dietary SupplementsABSTRACT
Investigated mitigating effects of sodium butyrate (SB) on the inflammatory response, oxidative stress, and growth inhibition of common carp (Cyprinus carpio) (2.94 ± 0.2 g) are caused by glycinin. Six isonitrogenous and isoenergetic diets were prepared, in which the basal diet was the control diet and the Gly group diet contained 80 g/kg glycinin, while the remaining 4 diets were supplemented with 0.75, 1.50, 2.25, and 3.00 g/kg SB, respectively. The feeding trial lasted for 8 weeks, and the results indicated that supplementing the diet with 1.50-2.25 g/kg of SB significantly improved feed efficiency and alleviated the growth inhibition induced by glycinin. Hepatopancreas and intestinal protease activities and the content of muscle crude protein were significantly decreased by dietary glycinin, but supplement 1.50-2.25 g/kg SB partially reversed this result. SB (1.50-2.25 g/kg) increased the activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the hepatopancreas and reduced the activities of AST and ALT in the serum. Glycinin significantly reduced immune and antioxidant enzyme activities, whereas 1.50-2.25 g/kg SB reversed these adverse effects. Furthermore, compared with the Gly group, supplement 1.50-2.25 g/kg SB eminently up-regulated the TGF-ß and IL-10 mRNA, and down-regulated the IL-1ß, TNF-α, and NF-κB mRNA in hepatopancreas, mid-intestine (MI), and distal intestine (DI). Meanwhile, supplement 1.50-2.25 g/kg SB activated the Keap1-Nrf2-ARE signaling pathway and upregulate CAT, SOD, and HO-1 mRNA expression in hepatopancreas, MI, and DI. Summarily, glycinin induced inflammatory response, and oxidative stress of common carp ultimately decreased the digestive function and growth performance. SB partially mitigated these adverse effects by activating the Keap1-Nrf2-ARE signaling pathway and inhibiting the NF-κB signaling pathway.
Subject(s)
Carps , Globulins , Soybean Proteins , Animals , Carps/metabolism , Butyric Acid/pharmacology , Kelch-Like ECH-Associated Protein 1/metabolism , NF-kappa B/metabolism , NF-E2-Related Factor 2/metabolism , Dietary Supplements , Diet/veterinary , Antioxidants/pharmacology , Antioxidants/metabolism , Oxidative Stress , RNA, Messenger/metabolism , Animal Feed/analysisABSTRACT
Monosodium urate (MSU) crystals, the etiological agent of gout, are formed in joints and periarticular tissues due to long-lasting hyperuricemia. Although MSU crystal-triggered NLRP3 inflammasome activation and interleukin 1ß (IL-1ß) release are known to have key roles in gouty arthritis, recent studies revealed that MSU crystal-induced necrosis also plays a critical role in this process. However, it remains unknown what forms of necrosis have been induced and whether combined cell death inhibitors can block such necrosis. Here, we showed that MSU crystal-induced necrosis in murine macrophages was not dependent on NLRP3 inflammasome activation, as neither genetic deletion nor pharmacological blockade of the NLRP3 pathway inhibited the necrosis. Although many cell death pathways (such as ferroptosis and pyroptosis) inhibitors or reactive oxygen species inhibitors did not have any suppressive effects, necroptosis pathway inhibitors GSK'872 (RIPK3 inhibitor), and GW806742X (MLKL inhibitor) dose-dependently inhibited MSU crystal-induced necrosis. Moreover, a triple combination of GSK'872, GW806742X, and IDN-6556 (pan-caspase inhibitor) displayed enhanced inhibition of the necrosis, which was further fortified by the addition of MCC950 (NLRP3 inhibitor), suggesting that multiple cell death pathways might have been triggered by MSU crystals. Baicalin, a previously identified inhibitor of NLRP3, inhibited MSU crystal-induced inflammasome activation and suppressed the necrosis in macrophages. Besides, baicalin gavage significantly ameliorated MSU crystal-induced peritonitis in mice. Altogether, our data indicate that MSU crystals induce NLRP3-independent necrosis, which can be inhibited by combined inhibitors for multiple signaling pathways, highlighting a new avenue for the treatment of gouty arthritis.
Subject(s)
Arthritis, Gouty , Gout , Animals , Arthritis, Gouty/chemically induced , Arthritis, Gouty/drug therapy , Arthritis, Gouty/metabolism , Gout/drug therapy , Gout/metabolism , Inflammasomes/metabolism , Interleukin-1beta/metabolism , Mice , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Necrosis/chemically induced , Necrosis/drug therapy , Signal Transduction , Uric AcidABSTRACT
The realization of surface-enhanced Raman spectroscopy (SERS) to be a reliable quantitative analytical technique requires sensitive and reproducible enhancing substrates. Here, uniform three-dimensional (3D) Ag nanorod (AgNR) substrates with well-defined interlayer spacings are prepared through the air-liquid interface-assisted self-assembly of AgNR in a layer-by-layer manner. The correlation of the SERS performance with the 3D AgNR structures is performed by SERS mapping the substrates. SERS mapping reveals the excellent enhancement uniformity of the 3D substrates with the relative standard deviation (RSD) less than 10%. It finds that both of the number of layers (NL) and the length of the AgNR have effects on the SERS performance of the 3D AgNR substrates. It is demonstrated that the intergaps between layers contribute much to the SERS intensity of the 3D AgNR by creating the interlayer (out-of-plane) plasmonic coupling. The impact of the excitation wavelengths (532, 633, and 785 nm) on SERS performance is also determined. The optimal 3D AgNR structures achieved by the correlation study is further used to detect a set of related molecules (l-tryptophan (Trp), l-phenylalanine (Phe), urea, and melamine). The 3D AgNR SERS of the analytes exhibits linear responses over wide concentration ranges. The sensitivity of the 3D AgNR SERS is proved by comparing to that of the current methods. Moreover, the 3D AgNR substrates maintain the performance stability during 4 weeks of storage.
ABSTRACT
OBJECTIVE: Glucose-6-phosphate isomerase (GPI) deficiency is a rare hereditary nonspherocytic hemolytic anemia caused by GPI gene variants. This disorder exhibits wide heterogeneity in its clinical manifestations and molecular characteristics, often posing challenges for precise diagnoses using conventional methods. To this end, this study aimed to identify the novel variants responsible for GPI deficiency in a Chinese family. METHODS: The clinical manifestations of the patient were summarized and analyzed for GPI deficiency phenotype diagnosis. Novel compound heterozygous variants of the GPI gene, c.174C>A (p.Asn58Lys) and c.1538G>T (p.Trp513Leu), were identified using whole-exome and Sanger sequencing. The AlphaFold program and Chimera software were used to analyze the effects of compound heterozygous variants on GPI structure. RESULTS: By characterizing 53 GPI missense/nonsense variants from previous literature and two novel missense variants identified in this study, we found that most variants were located in exons 3, 4, 12, and 18, with a few localized in exons 8, 9, and 14. This study identified novel compound heterozygous variants associated with GPI deficiency. These pathogenic variants disrupt hydrogen bonds formed by highly conserved GPI amino acids. CONCLUSION: Early family-based sequencing analyses, especially for patients with congenital anemia, can help increase diagnostic accuracy for GPI deficiency, improve child healthcare, and enable genetic counseling.
Subject(s)
Anemia, Hemolytic, Congenital Nonspherocytic , Anemia, Hemolytic , Child , Humans , Glucose-6-Phosphate Isomerase/genetics , Glucose-6-Phosphate Isomerase/chemistry , Anemia, Hemolytic/genetics , Anemia, Hemolytic, Congenital Nonspherocytic/diagnosis , Anemia, Hemolytic, Congenital Nonspherocytic/genetics , Mutation, Missense , ExonsABSTRACT
BACKGROUND: Uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1) plays a crucial role in metabolizing and detoxifying endogenous and exogenous substances. However, its contribution to the progression of liver damage remains unclear. AIM: To determine the role and mechanism of UGT1A1 in liver damage progression. METHODS: We investigated the relationship between UGT1A1 expression and liver injury through clinical research. Additionally, the impact and mechanism of UGT1A1 on the progression of liver injury was analyzed through a mouse model study. RESULTS: Patients with UGT1A1 gene mutations showed varying degrees of liver damage, while patients with acute-on-chronic liver failure (ACLF) exhibited relatively reduced levels of UGT1A1 protein in the liver as compared to patients with chronic hepatitis. This suggests that low UGT1A1 levels may be associated with the progression of liver damage. In mouse models of liver injury induced by carbon tetrachloride (CCl4) and concanavalin A (ConA), the hepatic levels of UGT1A1 protein were found to be increased. In mice with lipopolysaccharide or liver steatosis-mediated liver-injury progression, the hepatic protein levels of UGT1A1 were decreased, which is consistent with the observations in patients with ACLF. UGT1A1 knockout exacerbated CCl4- and ConA-induced liver injury, hepatocyte apoptosis and necroptosis in mice, intensified hepatocyte endoplasmic reticulum (ER) stress and oxidative stress, and disrupted lipid metabolism. CONCLUSION: UGT1A1 is upregulated as a compensatory response during liver injury, and interference with this upregulation process may worsen liver injury. UGT1A1 reduces ER stress, oxidative stress, and lipid metabolism disorder, thereby mitigating hepatocyte apoptosis and necroptosis.
Subject(s)
Glucuronosyltransferase , Liver , Animals , Humans , Mice , Disease Models, Animal , Glucuronosyltransferase/genetics , Glucuronosyltransferase/metabolism , Liver/metabolismABSTRACT
Coronavirus infection induces a variety of cellular antiviral responses either dependent on or independent of type I interferons (IFNs). Our previous studies using Affymetrix microarray and transcriptomic analysis revealed the differential induction of three IFN-stimulated genes (ISGs), IRF1, ISG15 and ISG20, by gammacoronavirus infectious bronchitis virus (IBV) infection of IFN-deficient Vero cells and IFN-competent, p53-defcient H1299 cells, respectively. In this report, the induction kinetics and anti-IBV functions of these ISGs as well as mechanisms underlying their differential induction are characterized. The results confirmed that these three ISGs were indeed differentially induced in H1299 and Vero cells infected with IBV, significantly more upregulation of IRF1, ISG15 and ISG20 was elicited in IBV-infected Vero cells than that in H1299 cells. Induction of these ISGs was also detected in cells infected with human coronavirus-OC43 (HCoV-OC43) and porcine epidemic diarrhea virus (PEDV), respectively. Manipulation of their expression by overexpression, knockdown and/or knockout demonstrated that IRF1 played an active role in suppressing IBV replication, mainly through the activation of the IFN pathway. However, a minor, if any, role in inhibiting IBV replication was played by ISG15 and ISG20. Furthermore, p53, but not IRF1, was implicated in regulating the IBV infection-induced upregulation of ISG15 and ISG20. This study provides new information on the mechanisms underlying the induction of these ISGs and their contributions to the host cell antiviral response during IBV infection.
Subject(s)
Coronavirus Infections , Gammacoronavirus , Infectious bronchitis virus , Animals , Humans , Antiviral Agents/pharmacology , Chlorocebus aethiops , Coronavirus Infections/veterinary , Cytokines/genetics , Exoribonucleases , Infectious bronchitis virus/genetics , Swine , Tumor Suppressor Protein p53 , Ubiquitins , Vero CellsABSTRACT
The objective of this study was to detective the accuracy of model superimposition and automatic analysis for upper and lower dentition width in Invisalign Progress Assessment during the process of clear aligners. 19 cases were included in this study. Pre-treatment dental cast (T0) and post-treatment dental cast after staged treatment (T1) were available for three-dimensional model superimposition. Subsequently, movements of maxillary teeth in the horizontal plane (cross-section) after staged treatment and width of upper and lower dentition were measured by three-dimensional model superimposition in the real world and Invisalign Progress Assessment separately. Consequently, the data collected from these two methods were compared. In Invisalign Progress Assessment, movements of maxillary teeth in the horizontal plane after staged treatment was 2.31 (1.59,3.22) [median (upper quartile, lower quartile)] millimeter (mm), while in three-dimensional model superimposition, the result was 1.79 (1.21,3.03) mm. The difference between the two groups is significant (P < 0.05). Intercanine width upper, intermolar width upper, intercanine width lower, and intermolar width lower were 36.55 ± 2.76 mm, 56.98 ± 2.62 mm, 28.16 ± 1.85 mm, 53.21 ± 2.72 mm separately in Invisalign Progress Assessment and were 36.48 ± 2.78 mm, 56.89 ± 2.58 mm, 28.05 ± 1.85 mm, 53.16 ± 2.64 mm separately in three-dimensional model analysis, which was no significant difference among these groups (P > 0.05). The data from Invisalign Progress Assessment was not in parallel with what was achieved from model superimposition with palate as a reference completely. The accuracy of model superimposition in Invisalign Progress Assessment needs further investigation, whereas the accuracy of model analysis in Invisalign Progress Assessment was accurate. Thereby, results from Invisalign Progress Assessment should be interpreted with caution by the orthodontist in the clinic.
Subject(s)
Orthodontic Appliances, Removable , Tooth Movement Techniques , Retrospective Studies , PalateABSTRACT
In this study, the alleviative effects of poly-ß-hydroxybutyrate (PHB) in bioflocs on oxidative stress, inflammation and apoptosis of common carp (Cyprinus carpio) induced by lipopolysaccharide (LPS) were evaluated. Common carp were irregularity divided into 5 groups and fed five diets with 0 % (CK), 2 %, 4 %, 6 % and 8 % PHB. After 8-week feeding trial, LPS challenge was executed. Results showed that appropriate level of PHB enhanced serum immune function by reversing LPS-induced the decrease of C3, C4, IgM, AKP, ACP and LZM in serum, alleviated LPS-induced intestinal barrier dysfunction by decreasing the levels of 5-HT, D-LA, ET-1 and DAO in serum, increasing ZO-1, Occludin, Claudin-3 and Claudin-7 mRNA, improving intestinal morphology. Moreover, dietary PHB reversed LPS-induced the decrease of AST and ALT in hepatopancreas, while in serum exhibited the opposite trend. Suitable level of PHB reversed LPS-induced the reduction of GSH-PX, CAT, T-SOD and T-AOC in intestines and hepatopancreas, whereas MDA showed the opposite result. PHB alleviated LPS-induced the decrease of Nrf2, HO-1, CAT, SOD and GSH-PX mRNA, the increase of Keap1 mRNA. Appropriate level of PHB alleviated LPS-induced inflammation and apoptosis by up-regulating TGF-ß, IL-10 and Bcl-2 mRNA, down-regulating NF-κB, TNF-α, IL-6, Bax, Caspase-3, Caspase-8 and Caspase-9 mRNA. Furthermore, PHB inhibited activation of NLRP3 inflammasomes by reducing the levels of NLRP3, Caspase-1, ASC, IL-1ß and IL-18 mRNA and protein. In addition, the increases of dietary PHB linearly and quadratically affected LPS-induced adverse effects on common carp. Summary, this study suggested that appropriate level of dietary PHB alleviated LPS-induced oxidative stress, inflammation, apoptosis and the activation of NLRP3 inflammasome in common carp. And the appropriate level of PHB in common carp diets was 4 %.
Subject(s)
Carps , Lipopolysaccharides , Animals , Lipopolysaccharides/toxicity , Kelch-Like ECH-Associated Protein 1 , NLR Family, Pyrin Domain-Containing 3 Protein , NF-E2-Related Factor 2 , Inflammation/chemically induced , Inflammation/drug therapy , Oxidative Stress , Inflammasomes , Apoptosis , RNA, Messenger , Superoxide Dismutase/pharmacologyABSTRACT
Type H vessels couple angiogenesis with osteogenesis, while sympathetic cues regulate vascular and skeletal function. The crosstalk between sympathetic nerves and type H vessels in bone remains unclear. Here, we first identify close spatial connections between sympathetic nerves and type H vessels in bone, particularly in metaphysis. Sympathoexcitation, mimicked by isoproterenol (ISO) injection, reduces type H vessels and bone mass. Conversely, beta-2-adrenergic receptor (ADRB2) deficiency maintains type H vessels and bone mass in the physiological condition. In vitro experiments reveal indirect sympathetic modulation of angiogenesis via paracrine effects of mesenchymal stem cells (MSCs), which alter the transcription of multiple angiogenic genes in endothelial cells (ECs). Furthermore, Notch signaling in ECs underlies sympathoexcitation-regulated type H vessel formation, impacting osteogenesis and bone mass. Finally, propranolol (PRO) inhibits beta-adrenergic activity and protects type H vessels and bone mass against estrogen deficiency. These findings unravel the specialized neurovascular coupling in bone homeostasis and regeneration.
ABSTRACT
BACKGROUND: Preterm infants with long-term parenteral nutrition (PN) therapy are at risk for cholestasis associated with total parenteral nutrition (PNAC). This study examined the safety and efficacy of ursodeoxycholic acid (UDCA) in preventing PNAC in preterm infants. Our research aimed to investigate the prophylactic effect of preventive oral UDCA on PNAC in preterm infants. METHODS: We compared oral administration of UDCA prophylaxis with no prophylaxis in a randomized, open-label, proof-of-concept trial in preterm neonates with PN therapy. The low-birth-weight preterm infants (< 1800 g) who were registered to the neonatal intensive care unit (NICU) within 24 hours after birth were randomized. The main endpoint was the weekly values of direct bilirubin (DB) of neonates during the NICU stay. RESULTS: Eventually, a total of 102 preterm neonates from January 2021 to July 2021 were enrolled in this prospective study (42 in the UDCA group and 60 in the control group). Notably, the peak serum level of DB [13.0 (12-16) vs. 15.2 (12.5-19.6) µmol/L, P < 0.05)] was significantly lower in the UDCA group than that in the control group without prevention. The peak serum level of total bilirubin (101.1 ± 34 vs. 116.5 ± 28.7 µmol/L, P < 0.05) was also significantly lower in the UDCA group than in the control group. Furthermore, the proportion of patients who suffered from neonatal cholestasis (0.0% vs. 11.7%, P < 0.05) in the UDCA group was significantly lower. CONCLUSION: UDCA prophylaxis is beneficial in preventing PNAC in NICU infants receiving prolonged PN.
Subject(s)
Cholestasis , Ursodeoxycholic Acid , Cholestasis/etiology , Cholestasis/prevention & control , Humans , Infant , Infant, Newborn , Infant, Premature , Parenteral Nutrition, Total , Prospective Studies , Retrospective Studies , Ursodeoxycholic Acid/therapeutic useABSTRACT
Discovery of anti-inflammatory drugs that can suppress T lymphocyte activation and proliferation by inhibiting TCR/CD3 and IL-2/IL-2R signaling is still needed in clinic, though rapamycin and other related reagents have made great success. Taraxasterol (TAS) is an active ingredient of dandelion, an anti-inflammatory medicinal herb with low in vivo toxicity that has long been used in China. Yet the action mechanism of TAS on lymphocytes remains elusive. The anti-inflammatory effects of TAS were evaluated in C57BL/6 mouse primary lymphocytes stimulated with concanavalin A (Con A) in vitro and in mouse model of Con A-induced acute hepatitis in vivo. Our results showed that TAS significantly suppressed Con A-induced acute hepatitis in a mouse model, reducing the hepatic necrosis areas, the release of aminotransferases, and the production of IL-2 and other inflammatory cytokines. Supporting this, in vitro study also showed that TAS reduced the production of IL-2 and the expression of IL-2 receptor subunit α (CD25) upon the stimulation of Con A, which was likely mediated by suppressing NF-κB activation. The downstream pathways of IL-2/IL-2R signaling, including the activation of PI3K/PDK1/mTOR, STAT3 and STAT5, were also suppressed by TAS. Consistently, Con A-induced T cell proliferation was also inhibited by TAS in vitro. Our data indicate that TAS can suppress both T lymphocyte activation and cell proliferation by down-regulating IL-2 expression and its signaling pathway thereby ameliorating Con A-induced acute hepatitis, highlighting TAS as a potential drug candidate for treating inflammatory diseases including autoimmune hepatitis.
Subject(s)
Anti-Inflammatory Agents/therapeutic use , Chemical and Drug Induced Liver Injury/drug therapy , Interleukin-2/immunology , Sterols/therapeutic use , T-Lymphocytes/drug effects , Triterpenes/therapeutic use , Animals , Anti-Inflammatory Agents/pharmacology , Cell Proliferation/drug effects , Chemical and Drug Induced Liver Injury/blood , Chemical and Drug Induced Liver Injury/immunology , Chemical and Drug Induced Liver Injury/pathology , Concanavalin A , Cytokines/blood , Female , Liver/drug effects , Liver/immunology , Liver/pathology , Mice, Inbred C57BL , Signal Transduction/drug effects , Sterols/pharmacology , T-Lymphocytes/immunology , Triterpenes/pharmacologyABSTRACT
BACKGROUND: Endoplasmic reticulum (ER) stress contributes to the pathogenesis of chronic liver diseases, but how hepatocytes respond to ER stress has not been clarified. Alpha-fetoprotein (AFP) is secreted by hepatoma cells and elevated levels of serum AFP are associated with development of liver malignancies. AIM: To investigate whether and how AFP could regulate ER stress and hepatocyte injury. METHODS: The distribution of AFP and the degrees of ER stress in liver tissues and liver injury were characterized by histology, immunohistochemistry, and Western blot in biopsied human liver specimens, two mouse models of liver injury and a cellular model. The levels of AFP in sera and the supernatants of cultured cells were quantified by chemiluminescence. RESULTS: High levels of intracellular AFP were detected in liver tissues, particularly in the necrotic areas, from patients with chronic liver diseases and mice after carbon tetrachloride (CCl4) administration or induction of ER stress, but not from the controls. The induced intracellular AFP was accompanied by elevated activating transcription factor-6 (ATF6) expression and protein kinase R-like ER kinase (PERK) phosphorylation in mouse livers. ER stress induced AFP expression in LO2 cells and decreased their viability. ATF6, but not PERK, silencing mitigated the ER-stress-induced AFP expression in LO2 cells. Conversely, AFP silencing deteriorated the ER stress-mediated LO2 cell injury and CCl4 administration-induced liver damages by increasing levels of cleaved caspase-3, the C/enhancer binding protein homologous protein expression, mixed lineage kinase domain-like pseudokinase and PERK phosphorylation, but decreasing ATF6 expression. CONCLUSION: ER stress upregulated intra-hepatocyte AFP expression by activating ATF6 during the process of liver injury and intracellular AFP attenuated hepatocyte apoptosis and necroptosis by alleviating ER stress.
Subject(s)
Endoplasmic Reticulum Stress , Liver Diseases , Animals , Apoptosis , Hepatocytes/pathology , Humans , Liver Diseases/pathology , Mice , Necroptosis , alpha-Fetoproteins/metabolismABSTRACT
Hepatocyte nuclear factor alpha (HNF1α), endoplasmic reticulum (ER) stress, and hepatocyte apoptosis contribute to severe acute exacerbation (SAE) of liver injury. Here, we explore HNF1α-ER stress-hepatocyte apoptosis interaction in liver injury. LO2, HepG2 and SK-Hep1 cells were treated with thapsigargin (TG) or tunicamycin (TM) to induce ER stress. Carbon tetrachloride (CCl4) was used to induce acute liver injury in mice. Low-dose lipopolysaccharide (LPS) exacerbated liver injury in CCl4-induced mice. Significant apoptosis, HNF1α upregulation, and nuclear factor kappa B (NF-κB) activation were observed in human-derived hepatocytes during ER stress. Knockdown of Rela, NF-κB p65, inhibited the HNF1α upregulation. Following CCl4 treatment ER stress, apoptosis, HNF1α expression and RelA phosphorylation were significantly increased in mice. HNF1α knockdown reduced activating transcription factor 4 (ATF4) expression, and aggravated ER stress as well as hepatocyte apoptosis in vivo and in vitro. The double fluorescent reporter gene assay confirmed that HNF1α regulated the transcription of ATF4 promoter. LPS aggravated CCl4-induced liver injury and reduced HNF1α, and ATF4 expression. Therefore, in combination, HNF1α and ER stress could be mutually regulated forming a feedback loop, which helps in protecting the injured liver by down-regulating hepatocyte apoptosis. Low-dose LPS aggravates hepatocyte apoptosis and promotes the SAE of liver injury by interfering with the feedback regulation of HNF1α and ER stress in acute liver injury.
Subject(s)
Endoplasmic Reticulum Stress , Hepatocyte Nuclear Factor 1-alpha , Activating Transcription Factor 4/metabolism , Animals , Apoptosis , Endoplasmic Reticulum Stress/physiology , Feedback , Hepatocyte Nuclear Factor 1-alpha/metabolism , Hepatocytes/metabolism , Humans , Lipopolysaccharides/metabolism , Liver/metabolism , Mice , NF-kappa B/metabolismABSTRACT
Dimethyl fumarate (DMF) is a fumaric acid derivative clinically approved for the treatment of some inflammatory diseases, but the underlying mechanism for its therapeutic effects remains incompletely understood. NLR family pyrin domain containing 3 (NLRP3) inflammasome activation has critical roles in innate immune responses to various infections and sterile inflammations. In this study, we aimed to explore whether DMF affects auto-immune hepatitis (AIH) in mice induced by concanavalin A (Con A) by modulating NLRP3 inflammasome activation. The results showed that DMF suppressed the activation of NLRP3 inflammasome activation in lipopolysaccharide-primed murine bone marrow-derived macrophages upon ATP or nigericin treatment, as evidenced by reduced cleavage of pro-caspase-1, release of mature interleukin-1ß (IL-1ß) and generation of gasdermin D N-terminal fragment (GSDMD-NT). DMF also greatly reduced ASC speck formation upon the stimulation of nigericin or ATP, indicating its inhibitory effect on NLRP3 inflammasome assembly. Consistent with reduced generation of GSDMD-NT, ATP or nigericin-induced pyroptosis was markedly suppressed by DMF. Moreover, DMF treatment alleviated mitochondrial damage induced by ATP or nigericin. Interestingly, all these effects were reversed by the protein kinase A (PKA) pathway inhibitors (H89 and MDL-12330A). Mechanistically, DMF enhanced PKA signaling and thus increased NLRP3 phosphorylation at PKA-specific sites to attenuate its activation. Importantly, DMF decreased serum levels of inflammatory cytokines and ameliorated liver injury in Con A-induced AIH of mice, concomitant with reduced the generation of caspase-1p10 and GSDMD-NT and alleviating mitochondrial aggregation in the liver. Collectively, DMF displayed anti-inflammatory effects by inhibiting NLRP3 inflammasome activation likely through regulating PKA signaling, highlighting its potential application in treating AIH.
Subject(s)
Hepatitis, Autoimmune , Inflammasomes , Adenosine Triphosphate/pharmacology , Animals , Caspase 1/metabolism , Cyclic AMP-Dependent Protein Kinases , Dimethyl Fumarate/pharmacology , Dimethyl Fumarate/therapeutic use , Hepatitis, Autoimmune/drug therapy , Inflammasomes/metabolism , Interleukin-1beta/metabolism , Mice , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Nigericin/pharmacology , Nigericin/therapeutic useABSTRACT
OBJECTIVE: To develop a novel chondrocyte condensation culture strategy recapitulating developmental condensation and construct self-organised cartilaginous tissue for cartilage regeneration. METHODS: Cell-condensation aggregate (CCA) was generated using the condensation culture method by sequential cell seeding. The chondrification capacities and biocompatibilities of CCA were assessed by comparison with the cell-scaffold complex (CSC), which was constructed by cell-scaffold coculture. Preclinical studies including implantation into nude mice subcutaneously and cartilage defect repair in rabbits were performed. RESULTS: CCA constructed by condensation culture exhibited a morphology of self-organised cartilaginous tissue. Meanwhile, the condensation culture inhibited or abolished expression of HOX genes including HOXC4 and HOXD8, which was partially consistent with developmental HOX gene expression patterns and associated with enhanced regeneration capacities. Compared with CSC, CCA showed a higher capacity for chondrification and regeneration of rabbit cartilage defects. CONCLUSION: The therapeutic assessments indicate that CCA is an efficient therapeutic tool for cartilage regeneration, providing a new strategy for tissue engineering by mimicking developmental events.
Subject(s)
Cartilage, Articular , Animals , Chondrocytes , Mice , Mice, Nude , Rabbits , Regeneration , Tissue Engineering , Tissue ScaffoldsABSTRACT
Coronaviruses (CoVs) are enveloped (+) ssRNA viruses of veterinary and medical importance. Because recombinant CoVs with reporter proteins fused with viral proteins are usually non-viable or unstable, a small and quantifiable epitope tag would be beneficial to CoV research. In this study, we integrated the NanoLuc Binary Technology to the reverse genetics of infectious bronchitis virus (IBV), a prototypic gammacoronavirus. The 11-amino-acid HiBiT tag was inserted to the spike (S) or membrane (M) protein, and the recombinant IBVs (rS-HiBiT and rM-HiBiT) were characterized. Compared with the rIBV-p65 control, rS-HiBiT exhibited comparable growth kinetics, whereas rM-HiBiT replicated slightly slower. The levels of HiBiT-tagged S and M proteins in the infected cells or the culture supernatant could be both rapidly (~15 min) and efficiently (30 µL sample volume) determined using the HiBiT luminescence assay. Notably, replication of the HiBiT-tagged IBV could be monitored continuously in an infected chicken embryo, and rS-HiBiT was genetically stable for at least 20 passages. By integrating the HiBiT tagging system with CoV reverse genetics, this new reporter system may facilitate future study of CoV replication and pathogenesis.
ABSTRACT
OBJECTIVE: To investigate high risk human papillomavirus (HR-HPV) prevalence among married women in Beijing and to study the high risk factors. METHODS: During March 2007 to September 2008, a total of 6185 married women sampled from 137 communities in 12 districts were screened by HR-HPV DNA test and cytological test. The interview was carried out with unified questionnaires. The database was set up and twice entered in EpiData 3.0. After checked up, the data were analyzed in SPSS 15.0. RESULTS: (1) The HR-HPV infection rate was 9.89%. The HR-HPV infection rate of the city zone, the suburb and the exurb were 9.34%, 10.51% and 9.51% (P > 0.05). The HR-HPV infection rate of the native and the outlander were 9.53%, 11.30% (P < 0.05). (2) The age distribution of HR-HPV infection was that the rate was around 10% among 25 to 44 age groups, which was the highest (11.21%) in 30 to 34 age group; then the rate was descended as the age raising, the rate of 50 to 54 age group was the lowest (7.78%). (3) Multiple logistic regression showed that the related risk factors of HR-HPV infection mainly included 1000 RMB and above of family income per person per month, possessing more than 1 sexual partner of her husband, outlander and high levels of education. (4) The prevalence of cervical intraepithelial neoplasia (CIN) in HR-HPV positive group was significantly higher than that in HR-HPV negative group (29.76% vs 3.32%, P < 0.01). CONCLUSIONS: (1) The HR-HPV infection rate among aged 25 to 54 years was 9.9% and there was no significant difference in area distribution. (2) The high risk population which should strengthen screening was the married bearing-age women with high level of family income, outlander, high levels of education and her husband possessing more than 1 sexual partner. (3) HR-HPV infection is the main risk factor for CIN and cervical cancer, while does not provide a causal relationship with them. The high risk population should be checked regularly to understand the development of HR-HPV infection and CIN incidence.
Subject(s)
Papillomavirus Infections , Uterine Cervical Dysplasia , Beijing , Epidemiologic Studies , Female , Humans , Papillomavirus Infections/virology , Uterine Cervical Neoplasms/diagnosis , Uterine Cervical Dysplasia/virologyABSTRACT
OBJECTIVE: To evaluate the efficacy of corneal confocal microscopy (CCM) as a non-invasive test to assess diabetic peripheral neuropathy in Chinese patients diagnosed with type 2 diabetes. RESEARCH DESIGN AND METHODS: Diabetic distal symmetric polyneuropathy (DSPN) and its severity degrees were assessed based on the modified Toronto diagnostic criteria in 128 patients with type 2 diabetes (No DSPN [nâ¯=â¯49], mild DSPN [nâ¯=â¯43], moderate-to-severe DSPN [nâ¯=â¯36]) and 24 age-matched controls. CCM was also examined in all enrolled subjects. Corneal nerve fiber length (CNFL), corneal nerve branch density (CNBD) and corneal nerve fiber density (CNFD) were analyzed by Fiji imaging analysis software. The efficacy of CCM as a non-invasive test to assess diabetic peripheral neuropathy was determined. RESULTS: CNFL was 17.99⯱â¯0.66, 15.82⯱â¯0.64, 14.98⯱â¯0.63, and 12.49⯱â¯0.93 in healthy controls, T2DM patients with no, mild, and moderate-to-severe DPN, respectively. CNFL in type 2 diabetes patients with no, mild, and moderate-to-severe DSPN demonstrated a significant reduction than in healthy controls (Pâ¯=â¯.012, .003 and <.001, respectively). CNFL in patients with moderate-to-severe DSPN was significantly shorter than in patients with no or mild DSPN (Pâ¯<â¯.001 and .004, respectively). CNBD was 41.48⯱â¯3.35, 33.02⯱â¯2.50, 30.91⯱â¯2.33, and 18.00⯱â¯2.33 in healthy controls, T2DM patients with no, mild, and moderate-to-severe DPN, respectively. CNBD in healthy control was significantly higher than in type 2 diabetes patients with no, mild, and moderate-to-severe DSPN (Pâ¯=â¯.036, 0.016 and <â¯.001, respectively). CNBD in patients with moderate-to-severe DSPN was significantly lower than in patients with no or mild DSPN (Pâ¯<â¯.001 for both). CNFD was 35.32⯱â¯1.18, 35.68⯱â¯1.10, 34.54⯱â¯1.12, and 32.28⯱â¯1.76 in healthy controls, T2DM patients with no, mild, and moderate-to-severe DPN, respectively. CNFD did not differ among the four groups. In an analysis that divided CNFL, CNFD and CNBD into quartiles, there were no significant differences in electromyography findings and vibration perception threshold among the 4 groups; however, significant differences were seen in the positive distribution of temperature perception measurements following CNFL and CNBD stratification (Pâ¯=â¯.001 and <â¯.001, respectively). CONCLUSION: CCM might be a non-invasive method for detecting DSPN and its severity degree in Chinese patients diagnosed with type 2 diabetes.