Parthanatos initiated by ROS-induced DNA damage is involved in intestinal epithelial injury during necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) involves intestinal epithelial damage and inflammatory response and is associated with high morbidity and mortality in infants. To improve therapeutic prospects, elucidating underlying molecular mechanisms of intestinal epithelial damage during NEC is of the essence. Poly (ADP-ribose) polymerase 1 (PARP1)-dependent parthanatos is a programmed inflammatory cell death. In the present study, the presence of parthanatos-associated proteins PARP1 and poly (ADP-ribose) (PAR), along with high expression of DNA damage-associated biomarkers, 8-hydroxy-2'-deoxyguanosine (8-OHdG) and phosphorylation of histone H2AX (γH2AX), were discovered in the intestinal tissues of NEC infants. Additionally, the upregulated expression of PARP1 and PAR in NEC intestinal tissues correlated distinctly with clinical indices indicative of NEC incidence and severity. Furthermore, we demonstrated that inhibiting the expression of parthanatos-associated proteins, by either pharmacological blockage using 3-aminobenzamide (3-AB), an inhibitor of PARP1, or genetic knockout using Parp1-deficient mice, resulted in substantial improvements in both histopathological severity scores associated with intestinal injury and inflammatory reactions. Moreover, in an in vitro NEC model, reactive oxygen species (ROS)-induced DNA damage promoted the formation of PAR and nuclear translocation of apoptosis-inducing factor (AIF), thus activating PARP1-dependent parthanatos in Caco-2 cells and human intestinal organoids. Our work verifies a previously unexplored role for parthanatos in intestinal epithelial damage during NEC and suggests that inhibition of parthanatos may serve as a potential therapeutic strategy for intervention of NEC.

Suppression of endoplasmic reticulum stress-dependent autophagy enhances cynaropicrin-induced apoptosis via attenuation of the P62/Keap1/Nrf2 pathways in neuroblastoma.

Autophagy has dual roles in cancer, resulting in cellular adaptation to promote either cell survival or cell death. Modulating autophagy can enhance the cytotoxicity of many chemotherapeutic and targeted drugs and is increasingly considered to be a promising cancer treatment approach. Cynaropicrin (CYN) is a natural compound that was isolated from an edible plant (artichoke). Previous studies have shown that CYN exhibits antitumor effects in several cancer cell lines. However, it anticancer effects against neuroblastoma (NB) and the underlying mechanisms have not yet been investigated. More specifically, the regulation of autophagy in NB cells by CYN has never been reported before. In this study, we demonstrated that CYN induced apoptosis and protective autophagy. Further mechanistic studies suggested that ER stress-induced autophagy inhibited apoptosis by activating the p62/Keap1/Nrf2 pathways. Finally, in vivo data showed that CYN inhibited tumour growth in xenografted nude mice. Overall, our findings suggested that CYN may be a promising candidate for the treatment of NB, and the combination of pharmacological inhibitors of autophagy may hold novel therapeutic potential for the treatment of NB. Our paper will contribute to the rational utility and pharmacological studies of CYN in future anticancer research.

Induction of Trained Immunity Protects Neonatal Mice Against Microbial Sepsis by Boosting Both the Inflammatory Response and Antimicrobial Activity.

Background: Neonates are susceptible to a wide range of microbial infection and at a high risk to develop severe sepsis and septic shock. Emerged evidence has shown that induction of trained immunity triggers a much stronger inflammatory response in adult monocytes/macrophages, thereby conferring protection against microbial infection. Methods: This study was carried out to examine whether trained immunity is inducible and exerts its protection against microbial sepsis in neonates. Results: Induction of trained immunity by Bacillus Calmette-Guerin (BCG) plus bacterial lipoprotein (BLP) protected neonatal mice against cecal slurry peritonitis-induced polymicrobial sepsis, and this protection is associated with elevated circulating inflammatory cytokines, increased neutrophil recruitment, and accelerated bacterial clearance. In vitro stimulation of neonatal murine macrophages with BCG+BLP augmented both inflammatory response and antimicrobial activity. Notably, BCG+BLP stimulation resulted in epigenetic remodeling characterized by histone modifications with enhanced H3K4me3, H3K27Ac, and suppressed H3K9me3 at the promoters of the targeted inflammatory and antimicrobial genes. Critically, BCG+BLP stimulation led to a shift in cellular metabolism with increased glycolysis, which is the prerequisite for subsequent BCG+BLP-triggered epigenetic reprogramming and augmented inflammatory response and antimicrobial capacity. Conclusion: These results illustrate that BCG+BLP induces trained immunity in neonates, thereby protecting against microbial infection by boosting both inflammatory and antimicrobial responses.

Novel pharmacological inhibition of JMJD3 improves necrotizing enterocolitis by attenuating the inflammatory response and ameliorating intestinal injury.

Necrotizing enterocolitis (NEC), an acute intestinal inflammatory disease of premature infants, is one of the leading causes of death in neonates. Effective measures for clinical treatment are limited and there is a pressing need in searching for new therapeutic strategies. Jumonji domain-containing protein D3 (JMJD3), a histone H3 lysine 27 (H3K27) demethylase plays a proinflammatory role in sepsis and neuroinflammation. However, whether JMJD3 is involved in the pathogenesis of NEC has not been elucidated. Here we report that overexpressed JMJD3 was revealed in the intestine of NEC patients by bioinformatic analysis. Moreover, upregulated JMJD3 and suppressed H3K27me3 were detected in both NEC patients and neonatal mice subjected to experimental NEC. Importantly, administration of GSK-J4, a specific JMJD3 inhibitor, rescued neonatal mice from NEC-associated lethality by suppressing proinflammatory response with attenuated IL-6, TNF-α, and MCP-1 levels and ameliorating intestinal injury with reversed claudin-1, occludin, and E-cadherin expression. Remarkably, administration of GSK-J4 attenuated intestinal injury by inhibiting activation of intestinal necroptosis in NEC mice. Administration of GSK-J4 regulated intestinal inflammation via NF-κB and JAK2/STAT3 pathway. These results indicate that JMJD3 is involved in the development of NEC and inhibition of JMJD3 overexpression by mean of GSK-J4 could be a potential therapeutic approach in the prevention and treatment of NEC.

Reduced Incidence of Necrotizing Enterocolitis due to the Anti-Inflammatory Effects of CXCL14 in Intestinal Tissue.

Bioinformatic analysis indicated that downregulated CXCL14 will occur in the intestinal tissue of patients with necrotizing enterocolitis (NEC). To reveal the relationship between CXCL14 and mucosal immune regulation, we designed and implemented the experiments to explore the potential function of CXCL14 in the pathogenesis of NEC. Firstly, this study confirmed that the expression of CXCL14 decreased in the intestinal tract of NEC children. Secondly, the experiments results showed that CXCL14 could ameliorate the inflammatory injury of intestinal tissue through the suppressive effect on the expression of TNF-α and INF-γ in vivo. Finally, we explained that activation of the TLR4 can reduce the expression level of CXCL14 in the intestinal tissue of mouse pups. Collectively, our study suggested that CXCL14 may negatively regulate the inflammatory response in intestinal tissue and play an essential role in NEC development and progression.

Shionone Attenuates Sepsis-Induced Acute Kidney Injury by Regulating Macrophage Polarization via the ECM1/STAT5 Pathway.

BACKGROUNDS: To date, there are no specific drugs approved for the treatment of sepsis associated acute kidney injury (AKI). Shionone is a natural component with anti-inflammatory activity. In this study, we sought to determine the functional role of Shionone in sepsis-induced AKI. METHODS: Animal models of AKI were constructed by cecum ligation and puncture (CLP) surgery. C57BL/6 mice were randomly assigned to the Sham, CLP, 10 mg/kg DXM, 50 mg/kg Shionone and 100 mg/kg Shionone groups. RAW264.7 treated with lipopolysaccharides (LPS) was used as an in vitro sepsis model and cells were divided into control, LPS, 1 µg/mL Shionone and 2 µg/mL Shionone groups. The pathological status was assessed by Hematoxylin-Eosin (HE) staining assay, protein expressions were detected by immunofluorescence staining and Western blot, macrophage typing was detected by flow, and the levels of pro-inflammatory factors (IL-6, IL-12, IL-1ß, TNF-α) and anti-inflammatory factors (IL-10 and TGF-ß) were measured using the corresponding kits. RESULTS: ECM1 is highly expressed in tissue-infiltrating macrophages under inflammatory conditions. It has been observed that Shionone inhibits the expression of ECM1 and attenuates sepsis-induced injury in kidney and inflammatory factor levels in serum. In addition, Shionone may reduce inflammatory factor levels through the promotion of M2 macrophages by GM-CSF/STAT5/Arg1 pathway to alleviate sepsis induced inflammation in vitro. CONCLUSION: These findings demonstrate that Shionone can alleviate sepsis-induced AKI by promoting M2 macrophage polarization through regulating the ECM1/STAT5 pathway.

The Effect of Shionone on Sepsis-Induced Acute Lung Injury by the ECM1/STAT5/NF-κB Pathway.

Purpose: The purpose of the present study was to estimate the effect of shionone (SHI) on sepsis-induced acute lung injury (ALI). Methods: The cecal ligation and puncture (CLP) surgery was performed to induce sepsis in mice. Pulmonary hematoxylin and eosin staining, the wet/dry ratio, myeloperoxidase (MPO) activity, and the survival rate were detected. The RAW264.7 cells were treated with SHI and stimulated with lipopolysaccharide (LPS). The cells were also overexpressed by extracellular mechanism protein 1 (ECM1) adenovirus. The relative levels of granulocyte-macrophage colony-stimulating factor, IL-6, IL-1ß, TNF-α, IL-10, and TGF-ß in the serum and supernatant were measured by ELISA. The protein expressions of ECM1, p-STAT5, signal transducer and activator of transcription 5 (STAT5), p-NF-κB, nuclear factor kappa-B (NF-κB), Arg1, CD206, CD16/32, and iNOS in the CLP-induced lung tissues and LPS-induced cells were detected by western blot. The cell counts of Ly6G, F4/80, CD16/32, and CD206 were evaluated by flow cytometry. The ECM1 expression was also observed by immunohistochemistry and immunofluorescence staining. Results: As a result, the histopathological change, pulmonary edema, and the MPO activity were relieved by SHI. SHI treatment increased the percentage of neutrophil and macrophage in the bronchoalveolar lavage fluid. Besides, SHI administration inhibited pro-inflammatory cytokines and M1 phenotype indices, as well as augmented the anti-inflammatory cytokines and M2 phenotype indices. SHI also attenuated the ECM1/STAT5/NF-κB pathway both in vivo and in vitro. The overexpression of ECM1 confirmed that the regulated effect of SHI was due to ECM1 signaling. Conclusion: In conclusion, the present study suggests that SHI ameliorated sepsis-induced ALI by screwing M1 phenotype to M2 phenotype macrophage via the ECM1/STAT5/NF-κB pathway.

Effect of gut microbiota on LPS-induced acute lung injury by regulating the TLR4/NF-kB signaling pathway.

Acute lung injury (ALI) is a common acute respiratory disease treated in the clinic. Intestinal microflora disorder affect lung diseases through the gut-lung axis. In this study, we explored the regulatory mechanism of the gut flora in the host defense against lipopolysaccharide (LPS)-induced ALI through the TLR4/NF-kB pathway by constructing a gut microflora dysbiosis-model with antibiotic administration and reconstruction of the intestinal microecology. Then, high-throughput sequencing was performed, and the levels of secreted IgA (sIgA), ß-defensins, and Muc2 were measured to assess the gut flora and mucosal barrier. The expression of TLR4, NF-kB, TNF-α, IL-1ß, oxidative stress and the lung wet/dry (W/D) ratio were evaluated to assess lung damage. Hematoxylin and eosin (HE) staining was performed to evaluate the damage to the gut and lung tissues. Accordingly, gut microbiota imbalance may regulate the TLR4/NF-kB signaling pathway in the lung immune system, activating oxidative stress in the lung and mediating lung injury through the regulation of the gut barrier. However, fecal microbiota transplantation (FMT) impairs the activity of the TLR4/NF-kB signaling pathway in the lung and decreases oxidative stress in animals with ALI by restoring the gut microecology. CONCLUSIONS: Our results indicated the protective effect of gut flora in regulating immunity of LPS-induced ALI by modulating the TLR4/NF-kB signaling pathway which may induce inflammation and oxidative stress.

The Initiation of Swallowing Can Indicate the Prognosis of Disorders of Consciousness: A Self-Controlled Study.

Objective: To detect the initiation of swallowing in patients with disorders of consciousness (DOC) as well as the relationship between the initiation of swallowing and the prognosis of DOC patients. Methods: Nineteen DOC patients were included in this study, and a self-controlled trial compared five different stimuli. The five different stimuli were as follows: (1) one command, as recommended by the Coma Recovery Scale-Revised (CRS-R), which was "open your mouth"; (2) placing a spoon in front of the patient's mouth without a command; (3) placing a spoon filled with water in front of the patient's mouth without a command; (4) one command-"there is a spoon; open your mouth"-with a spoon in front of the patient's mouth; (5) one command, "there is a spoon with water; open your mouth," with a spoon filled with water in front of the patient's mouth. All 19 patients were given these five stimuli randomly, and any one of the commands was presented four times to a patient, one at a time, at 15-s intervals. The sensitivity and specificity of the initiation of swallowing in detecting conscious awareness were determined. Results: None of the patients responded to the first four stimuli. However, six patients showed initiated swallowing toward the fifth stimulus. Among those six, five patients showed improvement in their consciousness state 6 months later. The sensitivity and specificity of the initiation of swallowing for DOC patients was 83.33% [95% CIs (36%, 100%)] and 92.31% [95% CIs (64%, 100%)], respectively. Conclusions: The initiation of swallowing can be an early indication of conscious behavior and can likely provide evidence of conscious awareness. Clinical Trial Registration: www.ClinicalTrials.gov, identifier: NCT03508336; Date of registration: 2018/4/16.

Effect of Intranasal Instillation of Lipopolysaccharide on Lung Development and Its Related Mechanism in Newborn Mice.

Premature infants are prone to repeated lung infections after birth, which can disrupt the development of lung structure and function. However, the effects of postnatal pulmonary inflammation on lung development in newborn mice have not been reported and may play an important role in the development of bronchopulmonary dysplasia (BPD). This study aimed to establish a BPD model of postnatal pulmonary inflammation in premature infants and to explore its role and possible mechanisms in the pathogenesis of BPD. We exposed postnatal day 1 mice to lipopolysaccharide (LPS) and normal saline for 14 days. Pulmonary inflammation and alveolar microvascular development were assessed by histology. In addition, we also examined the expression of vascular endothelial growth factor (VEGF), VEGFR2, nuclear factor-kappa-B (NF-κB) and related inflammatory mediators [interleukin-1ß (IL-1ß), tumor necrosis factor-alpha (TNF-α), macrophage inflammatory protein-1α (MIP-1α), monocyte chemoattractant protein-1 (MCP-1)] in the lungs. Lung histology revealed inflammatory cell infiltration, alveolar simplification, and decreased microvascular density in LPS-exposed lungs. VEGF and VEGFR2 expression was decreased in the lungs of LPS-exposed neonatal mice. Furthermore, we detected elevated levels of the inflammatory mediators IL-1ß, TNF-α, MIP-1α, and MCP-1 in the lungs, which are associated with the activation of NF-κB. Intranasal instillation of LPS inhibits lung development in newborn mice, and postnatal pulmonary inflammation may participate in the pathogenesis of BPD. The mechanism is related to the inhibition of VEGF and VEGFR2 and the upregulation of inflammatory mediators through activation of NF-κB.