Effect and mechanism of Qingre Huashi decoction on drug-resistant Helicobacter pylori.

BACKGROUND: Helicobacter pylori (HP), the most common pathogenic microorganism in the stomach, can induce inflammatory reactions in the gastric mucosa, causing chronic gastritis and even gastric cancer. HP infection affects over 4.4 billion people globally, with a worldwide infection rate of up to 50%. The multidrug resistance of HP poses a serious challenge to eradication. It has been de-monstrated that compared to bismuth quadruple therapy, Qingre Huashi decoction (QHD) combined with triple therapy exhibits comparable eradication rates but with a lower incidence of adverse reactions; in addition, QHD can directly inhibit and kill HP in vitro. AIM: To explore the effect and mechanism of QHD on clinically multidrug-resistant and strong biofilm-forming HP. METHODS: In this study, 12 HP strains were isolated in vitro after biopsy during gastroscopy of HP-infected patients. In vitro, the minimum inhibitory concentration (MIC) values for clinical HP strains and biofilm quantification were determined through the E-test method and crystal violet staining, respectively. The most robust biofilm-forming strain of HP was selected, and QHD was evaluated for its inhibitory and bactericidal effects on the strain with strong biofilm formation. This assessment was performed using agar dilution, E-test, killing dynamics, and transmission electron microscopy (TEM). The study also explored the impact of QHD on antibiotic resistance in these HP strains with strong biofilm formation. Crystalline violet method, scanning electron microscopy, laser confocal scanning microscopy, and (p)ppGpp chromatographic identification were employed to evaluate the effect of QHD on biofilm in strong biofilm-forming HP strains. The effect of QHD on biofilm and efflux pump-related gene expression was evaluated by quantitative polymerase chain reaction. Non-targeted metabolomics with UHPLC-MS/MS was used to identify potential metabolic pathways and biomarkers which were different between the NC and QHD groups. RESULTS: HP could form biofilms of different degrees in vitro, and the intensity of formation was associated with the drug resistance of the strain. QHD had strong bacteriostatic and bactericidal effects on HP, with MICs of 32-64 mg/mL. QHD could inhibit the biofilm formation of the strong biofilm-forming HP strains, disrupt the biofilm structure, lower the accumulation of (p)ppGpp, decrease the expression of biofilm-related genes including LuxS, Spot, glup (HP1174), NapA, and CagE, and reduce the expression of efflux pump-related genes such as HP0605, HP0971, HP1327, and HP1489. Based on metabolomic analysis, QHD induced oxidative stress in HP, enhanced metabolism, and potentially inhibited relevant signaling pathways by upregulating adenosine monophosphate (AMP), thereby affecting HP growth, metabolism, and protein synthesis. CONCLUSION: QHD exerts bacteriostatic and bactericidal effects on HP, and reduces HP drug resistance by inhibiting HP biofilm formation, destroying its biofilm structure, inhibiting the expression of biofilm-related genes and efflux pump-related genes, enhancing HP metabolism, and activating AMP in HP.

GFRAL Is Widely Distributed in the Brain and Peripheral Tissues of Mice.

In 2017, four independent publications described the glial cell-derived neurotrophic factor (GDNF) receptor alpha-like (GFRAL) as receptor for the growth differentiation factor 15 (GDF15, also MIC-1, NAG-1) with an expression exclusively in the mice brainstem area postrema (AP) and nucleus tractus solitarii (NTS) where it mediates effects of GDF15 on reduction of food intake and body weight. GDF15 is a cell stress cytokine with a widespread expression and pleiotropic effects, which both seem to be in contrast to the reported highly specialized localization of its receptor. This discrepancy prompts us to re-evaluate the expression pattern of GFRAL in the brain and peripheral tissues of mice. In this detailed immunohistochemical study, we provide evidence for a more widespread distribution of this receptor. Apart from the AP/NTS region, GFRAL-immunoreactivity was found in the prefrontal cortex, hippocampus, nucleus arcuatus and peripheral tissues including liver, small intestine, fat, kidney and muscle tissues. This widespread receptor expression, not taken into consideration so far, may explain the multiple effects of GDF-15 that are not yet assigned to GFRAL. Furthermore, our results could be relevant for the development of novel pharmacological therapies for physical and mental disorders related to body image and food intake, such as eating disorders, cachexia and obesity.

The Emerging Roles of E3 Ligases and DUBs in Neurodegenerative Diseases.

Despite annual increases in the incidence and prevalence of neurodegenerative diseases, there is a lack of effective treatment strategies. An increasing number of E3 ubiquitin ligases (E3s) and deubiquitinating enzymes (DUBs) have been observed to participate in the pathogenesis mechanisms of neurodegenerative diseases, on the basis of which we conducted a systematic literature review of the studies. This review will help to explore promising therapeutic targets from highly dynamic ubiquitination modification processes.

P2X7Rs: new therapeutic targets for osteoporosis.

Increasing evidence suggests that both the occurrence and progression of osteoporosis are associated with inflammation, especially in primary osteoporosis. The maintenance of skeletal homeostasis is dependent on the complex regulation of bone metabolism. Numerous evidence suggested that purinoceptor networks are essential for bone homeostasis. In this review, the relationship between inflammation and the development of osteoporosis and the role of P2X7 receptor (P2X7R) in regulating the dynamic regulation of bone reconstruction were covered. We also discussed how P2X7R regulates the balance between resorption and bone formation by osteoblasts and reviewed the relevance of P2X7R polymorphisms in skeletal physiology. Finally, we analyzed potential targets of P2X7R for osteoporosis.

Mitochondrial-derived damage-associated molecular patterns amplify neuroinflammation in neurodegenerative diseases.

Both mitochondrial dysfunction and neuroinflammation are implicated in neurodegeneration and neurodegenerative diseases. Accumulating evidence shows multiple links between mitochondrial dysfunction and neuroinflammation. Mitochondrial-derived damage-associated molecular patterns (DAMPs) are recognized by immune receptors of microglia and aggravate neuroinflammation. On the other hand, inflammatory factors released by activated glial cells trigger an intracellular cascade, which regulates mitochondrial metabolism and function. The crosstalk between mitochondrial dysfunction and neuroinflammatory activation is a complex and dynamic process. There is strong evidence that mitochondrial dysfunction precedes neuroinflammation during the progression of diseases. Thus, an in-depth understanding of the specific molecular mechanisms associated with mitochondrial dysfunction and the progression of neuroinflammation in neurodegenerative diseases may contribute to the identification of new targets for the treatment of diseases. In this review, we describe in detail the DAMPs that induce or aggravate neuroinflammation in neurodegenerative diseases including mtDNA, mitochondrial unfolded protein response (mtUPR), mitochondrial reactive oxygen species (mtROS), adenosine triphosphate (ATP), transcription factor A mitochondria (TFAM), cardiolipin, cytochrome c, mitochondrial Ca2+ and iron.

TIGAR plays neuroprotective roles in KA-induced excitotoxicity through reducing neuroinflammation and improving mitochondrial function.

Excitotoxicity refers to the ability of excessive extracellular excitatory amino acids to damage neurons via receptor activation. It is a crucial pathogenetic process in neurodegenerative diseases. TP53 is confirmed to be involved in excitotoxicity. It is demonstrated that TP53 induced glycolysis and apoptotic regulator (TIGAR)-regulated metabolic pathway can protect against neuronal injury. However, the role of TIGAR in excitotoxicity and specific mechanisms is still unknown. In this study, an in vivo excitotoxicity model was constructed via stereotypical kainic acid (KA) injection into the striatum of mice. KA reduced TIGAR expression levels, neuroinflammatory responses and mitochondrial dysfunction. TIGAR overexpression could reverse KA-induced neuronal injury by reducing neuroinflammation and improving mitochondrial function, thereby exerting neuroprotective effects. Therefore, this study could provide a potential therapeutic target for neurodegenerative diseases.

NADPH and Mito-Apocynin Treatment Protects Against KA-Induced Excitotoxic Injury Through Autophagy Pathway.

AIM: Previous research recognizes that NADPH can produce reduced glutathione (GSH) as a coenzyme and produce ROS as a substrate of NADPH oxidase (NOX). Besides, excessive activation of glutamate receptors results in mitochondrial impairment. The study aims at spelling out the effects of NADPH and Mito-apocynin, a NOX inhibitor which specifically targets the mitochondria, on the excitotoxicity induced by Kainic acid (KA) and its mechanism. METHODS: The in vivo neuronal excitotoxicity model was constructed by stereotypically injecting KA into the unilateral striatum of mice. Administrated NADPH (i.v, intravenous) 30 min prior and Mito-apocynin (i.g, intragastric) 1 day prior, respectively, then kept administrating daily until mice were sacrificed 14 days later. Nissl staining measured the lesion of striatum and survival status of neurons. Cylinder test of forelimb asymmetry and the adhesive removal test reflected the behavioral deficit caused by neural dysfunction. Determined Total superoxide dismutase (T-SOD), malondialdehyde (MDA), and GSH indicated oxidative stress. Western blot presented the expression levels of LC3-II/LC3-I, SQSTM1/p62, TIGAR, and NOX4. Assessed oxygen consumption rate using High-Resolution Respirometry. In vitro, the MitoSOX Indicator reflected superoxide released by neuron mitochondria. JC-1 and ATP assay Kit were used to detect mitochondrial membrane potential (MMP) and energy metabolism, respectively. RESULTS: In this study, we have successfully established excitotoxic model by KA in vivo and in vitro. KA induced decreased SOD activity and increased MDA concentration. KA cause the change of LC3-II/LC3-I, SQSTM1/p62, and TIGAR expression, indicating the autophagy activation. NADPH plays a protective role in vivo and in vitro. It reversed the KA-mediated changes in LC3, SQSTM1/p62, TIGAR, and NOX4 protein expression. Mito-apocynin inhibited KA-induced increases in mitochondrial NOX4 expression and activity. Compared with NADPH, the combination showed more significant neuroprotective effects, presenting more neurons survive and better motor function recovery. The combination also better inhibited the over-activated autophagy. In vitro, combination of NADPH and Mito-apocynin performed better in restoring mitochondria membrane potential. CONCLUSION: In summary, combined administration of NADPH and NOX inhibitors offers better neuroprotection by reducing NADPH as a NOX substrate to generate ROS. The combined use of NADPH and Mito-apocynin can better restore neurons and mitochondrial function through autophagy pathway.

Molecular Cloning and Functional Analysis of the NPR1 Homolog in Kiwifruit (Actinidia eriantha).

Kiwifruit bacterial canker, caused by the bacterial pathogen Pseudomonas syringae pv. actinidiae (Psa), is a destructive disease in the kiwifruit industry globally. Consequently, understanding the mechanism of defense against pathogens in kiwifruit could facilitate the development of effective novel protection strategies. The Non-expressor of Pathogenesis-Related genes 1 (NPR1) is a critical component of the salicylic acid (SA)-dependent signaling pathway. Here, a novel kiwifruit NPR1-like gene, designated AeNPR1a, was isolated by using PCR and rapid amplification of cDNA ends techniques. The full-length cDNA consisted of 1952 base pairs with a 1,746-bp open-reading frame encoding a 582 amino acid protein. Homology analysis showed that the AeNPR1a protein is significantly similar to the VvNPR1 of grape. A 2.0 Kb 5'-flanking region of AeNPR1a was isolated, and sequence identification revealed the presence of several putative cis-regulatory elements, including basic elements, defense and stress response elements, and binding sites for WRKY transcription factors. Real-time quantitative PCR results demonstrated that AeNPR1a had different expression patterns in various tissues, and its transcription could be induced by phytohormone treatment and Psa inoculation. The yeast two-hybrid assay revealed that AeNPR1a interacts with AeTGA2. Constitutive expression of AeNPR1a induced the expression of pathogenesis-related gene in transgenic tobacco plants and enhanced tolerance to bacterial pathogens. In addition, AeNPR1a expression could restore basal resistance to Pseudomonas syringae pv. tomato DC3000 (Pst) in Arabidopsis npr1-1 mutant. Our data suggest that AeNPR1a gene is likely to play a pivotal role in defense responses in kiwifruit.

Pyrrolidine dithiocarbamate and dexamethasone are novel treatments of Acute Exogenous Lipoid Pneumonia.

BACKGROUND: Acute exogenous lipoid pneumonia (AELP) is characterized by pulmonary inflammation. This mainly occur in children who have ingested sewing machine oil or other mineral oils accidentally. Despite emerging evidences revealing that inhibiting inflammation improves acute exogenous lipoid pneumonia, the actual process of inhibiting inflammation remains unknown. This study aimed to evaluate the effects of PDTC and dexamethasone on AELP to gain insight into the mechanism of AELP. METHODS: The experimental rats were randomly divided into 10 groups: NS control group (NS3 group, NS5 group), Oil inhalation group (AE3 group, AE5 group), PDTC intervention group (PDTC3 group, PDTC5 group), DXM intervention group (DXM3 group, DXM5 group), PDTC + DXM combined intervention group (PDTC + DXM3 group, PDTC + DXM 5 group). Enzyme-linked immunosorbent assay (ELISA) was used to determine concentrations of macrophage migration inhibitory factor (MIF), interleukin-6 (IL-6) and tumor necrosis factor-α (TNFα) in bronchoalveolar lavage fluid (BALF) and serum samples. On the other hand, western blotting was used to measure the expression levels of nuclear factor-κB p65 (NF-κB p65) and b-cell leukemia 2 (Bcl-2) in the lungs. Hematoxylin and Eosin (H&E) staining was performed to evaluate changes in the lung tissue. The wet-to-dry lung weight ratio was subsequently used to determine the pulmonary edema of the lungs. RESULTS: There were increased MIF levels in both serum and BALF samples of the AE group. Pyrrolidine dithiocarbamate (PDTC) and dexamethasone (DXM) independently and in combination reduced pulmonary inflammation induced by the sewing machine oil by regulating MIF expression. TNF-α and IL-6 levels in serum and BALF samples of the AE group were higher than those of the NS control animals. However, their levels decreased after treatment with either PDTC, DXM or PDTC + DXM. Similarly, NF-κBp65 expression increased after oil inhalation but decreased after treatment with either PDTC, DXM or PDTC + DXM. PDTC, DXM and PDTC + DXM treatment significantly reduced pulmonary inflammation and pulmonary edema of the lung tissue following induction of acute exogenous lipoid pneumonia. CONCLUSIONS: Individual or combined use of PDTC and DXM can ameliorate pulmonary inflammation induced by inhalation of sewing machine oil by inhibiting the NF-κB pathway in young rats. These findings provide novel insights that will greatly contribute in treatment of AELP.

NADPH protects against kainic acid-induced excitotoxicity via autophagy-lysosome pathway in rat striatum and primary cortical neurons.

PURPOSE: To investigate the effects and mechanisms of NADPH on Kainic acid (KA)-induced excitotoxicity. METHODS: KA, a non-N-methyl-d-aspartate glutamate receptor agonist, was exposed to adult SD rats via intrastriatal injection and rat primary cortical neurons to establish excitotoxic models in vivo and in vitro, respectively. To determine the effects of NADPH on KA-induced excitotoxicity, neuronal survival, neurologically behavioral score and oxidative stress were evaluated. To explore the mechanisms of neuroprotective effects of NADPH, the autophagy-lysosome pathway related proteins were detected. RESULTS: In vivo, NADPH (1 mg/kg or 2 mg/kg) diminished KA (2.5 nmol)-induced enlargement of lesion size in striatum, improved KA-induced dyskinesia and reversed KA-induced activation of glial cells. Nevertheless, the neuroprotective effect of NADPH was not significant under the condition of autophagy activation. NADPH (2 mg/kg) inhibited KA (2.5 nmol)-induced down-regulation of TP-53 induced glycolysis and apoptosis regulator (TIGAR) and p62, and up-regulation of the protein levels of LC3-II/LC3-I, Beclin-1 and Atg5. In vitro, the excitotoxic neuronal injury was induced after KA (50 µM, 100 µM or 200 µM) treatment as demonstrated by decreased cell viability. Moreover, KA (100 µM) increased the intracellular levels of calcium and reactive oxygen species (ROS) and declined the levels of the reduced form of glutathione (GSH). Pretreatment of NADPH (10 µM) effectively reversed these changes. Meanwhile NADPH (10 µM) inhibited KA (100 µM)-induced down-regulation of TIGAR and p62, and up-regulation of the ratio of LC3-II/LC3-I, Beclin-1, Atg5, active-cathepsin B and active-cathepsin D. CONCLUSIONS: Our data provide a possible mechanism that NADPH ameliorates KA-induced excitotoxicity by blocking the autophagy-lysosome pathway and up-regulating TIGAR along with its antioxidant properties.