Caffeic acid modulates activation of neutrophils and attenuates sepsis-induced organ injury by inhibiting 5-LOX/LTB4 pathway.

BACKGROUND: Sepsis is a critical systemic inflammatory syndrome which usually leads to multiple organ dysfunction. Caffeic acid (CA), a phenolic compound derived from various plants, has been proved to be essential in neuroprotection, but its role in septic organ damage is unclear. This research aimed to investigate whether CA protects against organ injury in a mouse model of cecal ligation and puncture (CLP). METHODS: CA (30 mg/kg) or vehicle was administered by intraperitoneal injection immediately after CLP. The samples of blood, lungs, and livers were collected 24 h later. Organ injury was assessed by histopathological examination (HE staining), neutrophil infiltration (myeloperoxidase fluorescence), oxidative stress levels (MDA, SOD, HO-1), and inflammatory cytokines (TNF-α, IL-1ß, and IL-6) release in lung and liver tissues. Neutrophil extracellular trap (NET) formation was analyzed by immunofluorescence. In vitro experiments were performed to investigate the potential mechanisms of CA using small interfering RNA (siRNA) techniques in neutrophils, and the effect of CA on neutrophil apoptosis was analyzed by flow cytometry. RESULTS: Results showed that CA treatment improved the 7-day survival rate and attenuated the histopathological injury in the lung and liver of CLP mice. CA significantly reduced neutrophil infiltration in the lungs and livers of CLP mice. TNF-α, IL-1ß, IL-6 and LTB4 were reduced in serum, lung, and liver of CA-treated CLP mice, and phosphorylation of MAPK (p38, ERK, JNK) and p65 NF-κB was inhibited in lungs and livers. CA treatment further increased HO-1 levels and enhanced superoxide dismutase (SOD) activity, but reduced malondialdehyde (MDA) levels and NET formation. Similarly, in vitro experiments showed that CA treatment and 5-LOX siRNA interference inhibited inflammatory activation and NET release in neutrophils, suppressed MAPK and NF-κB phosphorylation in LPS-treated neutrophils, and decreased LTB4 and cfDNA levels. Flow cytometric analysis revealed that CA treatment reversed LPS-mediated delayed apoptosis in human neutrophils, and Western blot also indicated that CA treatment inhibited Bcl-2 expression but increased Bax expression. CA treatment did not induce further changes in neutrophil apoptosis, inflammatory activation, and NET release when 5-LOX was knocked down by siRNA interference. CONCLUSIONS: CA has a protective effect on lung and liver injury in a murine model of sepsis, which may be related to inhibition of the 5-LOX/LTB4 pathway.

ß-Nicotinamide mononucleotide activates NAD+/SIRT1 pathway and attenuates inflammatory and oxidative responses in the hippocampus regions of septic mice.

Sepsis-associated encephalopathy (SAE) is one of the common serious complications in sepsis, and the pathogenesis of SAE remains unclear. Sirtuin 1 (SIRT1) has been reported to be downregulated in the hippocampus and SIRT1 agonists can attenuated the cognitive dysfunction in septic mice. Nicotinamide adenine dinucleotide (NAD+) is a key substrate to maintain the deacetylation activity of SIRT1. As an intermediate of NAD+, ß-Nicotinamide Mononucleotide (NMN) has been reported to be promising in treating neurodegenerative diseases and cerebral ischemic injury. Thus we sought to investigate the potential role of NMN in SAE treatment. The SAE model was established by cecal ligation and puncture (CLP) in vivo, and neuroinflammation model was established with LPS-treated BV-2 cells in vitro. Memory impairment was assessed by Morris water maze and fear conditioning tests. As a result, the levels of NAD+, SIRT1 and PGC-1α were significantly reduced in the hippocampus of septic mice, while the acetylation of total lysine, phosphorylation of P38 and P65 were enhanced. All these changes induced by sepsis were inverted by NMN. Treating with NMN resulted in improved behavior performance in the fear conditioning tests and Morris water maze. Apoptosis, inflammatory and oxidative responses in the hippocampus of septic mice were attenuated significantly after NMN administration. These protective effect of NMN against memory dysfunction, inflammatory and oxidative injuries were reversed by the SIRT1 inhibitor, EX-527. Similarly, LPS-induced activation of BV-2 cells were attenuated by NMN, EX-527 or SIRT1 knockdown could reverse such effect of NMN in vitro. In conclusion, NMN is protective against sepsis-induced memory dysfunction, and the inflammatory and oxidative injuries in the hippocampus region of septic mice. The NAD+/SIRT1 pathway might be involved in one of the mechanisms of the protective effect.

The implication of targeting PD-1:PD-L1 pathway in treating sepsis through immunostimulatory and anti-inflammatory pathways.

Sepsis currently remains a major contributor to mortality in the intensive care unit (ICU), with 48.9 million cases reported globally and a mortality rate of 22.5% in 2017, accounting for almost 20% of all-cause mortality worldwide. This highlights the urgent need to improve the understanding and treatment of this condition. Sepsis is now recognized as a dysregulation of the host immune response to infection, characterized by an excessive inflammatory response and immune paralysis. This dysregulation leads to secondary infections, multiple organ dysfunction syndrome (MODS), and ultimately death. PD-L1, a co-inhibitory molecule expressed in immune cells, has emerged as a critical factor in sepsis. Numerous studies have found a significant association between the expression of PD-1/PD-L1 and sepsis, with a particular focus on PD-L1 expressed on neutrophils recently. This review explores the role of PD-1/PD-L1 in immunostimulatory and anti-inflammatory pathways, illustrates the intricate link between PD-1/PD-L1 and sepsis, and summarizes current therapeutic approaches against PD-1/PD-L1 in the treatment and prognosis of sepsis in preclinical and clinical studies.

Calcitonin gene-related peptide induces the histone H3 lysine 9 acetylation in astrocytes associated with neuroinflammation in rats with neuropathic pain.

AIMS: Calcitonin gene-related peptide (CGRP) as a regulator of astrocyte activation may facilitate spinal nociceptive processing. Histone H3 lysine 9 acetylation (H3K9ac) is considered an important regulator of cytokine and chemokine gene expression after peripheral nerve injury. In this study, we explored the relationship between CGRP and H3K9ac in the activation of astrocytes, and elucidated the underlying mechanisms in the pathogenesis of chronic neuropathic pain. METHODS: Astroglial cells (C6) were treated with CGRP and differentially enrichments of H3K9ac on gene promoters were examined using ChIP-seq. A chronic constriction injury (CCI) rat model was used to evaluate the role of CGRP on astrocyte activation and H3K9ac signaling in CCI-induced neuropathic pain. Specific inhibitors were employed to delineate the involved signaling. RESULTS: Intrathecal injection of CGRP and CCI increased the number of astrocytes displaying H3K9ac in the spinal dorsal horn of rats. Treatment of CGRP was able to up-regulate H3K9ac and glial fibrillary acidic protein (GFAP) expression in astroglial cells. ChIP-seq data indicated that CGRP significantly altered H3K9ac enrichments on gene promoters in astroglial cells following CGRP treatment, including 151 gaining H3K9ac and 111 losing this mark, which mostly enriched in proliferation, autophagy, and macrophage chemotaxis processes. qRT-PCR verified expressions of representative candidate genes (ATG12, ATG4C, CX3CR1, MMP28, MTMR14, HMOX1, RET) and RTCA verified astrocyte proliferation. Additionally, CGRP treatment increased the expression of H3K9ac, CX3CR1, and IL-1ß in the spinal dorsal horn. CGRP antagonist and HAT inhibitor attenuated mechanical and thermal hyperalgesia in CCI rats. Such analgesic effects were concurrently associated with the reduced levels of H3K9ac, CX3CR1, and IL-1ß in the spinal dorsal horn of CCI rats. CONCLUSION: Our findings highly indicate that CGRP is associated with the development of neuropathic pain through astrocytes-mediated neuroinflammatory responses via H3K9ac in spinal dorsa horn following nerve injury. This study found that CGRP act on their astrocytic receptors and lead to H3K9 acetylation (H3K9ac), which are mainly associated with proliferation-, autophagy-, and inflammation-related gene expression. The number of astrocytes with H3K9ac expression is increased after nerve injury. Inhibition of CGRP attenuates the development of neuropathic pain, which was accompanied by the suppression of H3K9ac, CX3CR1, and IL-1ß expression in CCI rats.

Calcitonin gene-related peptide regulates spinal microglial activation through the histone H3 lysine 27 trimethylation via enhancer of zeste homolog-2 in rats with neuropathic pain.

BACKGROUND: Calcitonin gene-related peptide (CGRP) as a mediator of microglial activation at the transcriptional level may facilitate nociceptive signaling. Trimethylation of H3 lysine 27 (H3K27me3) by enhancer of zeste homolog 2 (EZH2) is an epigenetic mark that regulates inflammatory-related gene expression after peripheral nerve injury. In this study, we explored the relationship between CGRP and H3K27me3 in microglial activation after nerve injury, and elucidated the underlying mechanisms in the pathogenesis of chronic neuropathic pain. METHODS: Microglial cells (BV2) were treated with CGRP and differentially enrichments of H3K27me3 on gene promoters were examined using ChIP-seq. A chronic constriction injury (CCI) rat model was used to evaluate the role of CGRP on microglial activation and EZH2/H3K27me3 signaling in CCI-induced neuropathic pain. RESULTS: Overexpressions of EZH2 and H3K27me3 were confirmed in spinal microglia of CCI rats by immunofluorescence. CGRP treatment induced the increased of H3K27me3 expression in the spinal dorsal horn and cultured microglial cells (BV2) through EZH2. ChIP-seq data indicated that CGRP significantly altered H3K27me3 enrichments on gene promoters in microglia following CGRP treatment, including 173 gaining H3K27me3 and 75 losing this mark, which mostly enriched in regulation of cell growth, phagosome, and inflammation. qRT-PCR verified expressions of representative candidate genes (TRAF3IP2, BCL2L11, ITGAM, DAB2, NLRP12, WNT3, ADAM10) and real-time cell analysis (RTCA) verified microglial proliferation. Additionally, CGRP treatment and CCI increased expressions of ITGAM, ADAM10, MCP-1, and CX3CR1, key mediators of microglial activation in spinal dorsal horn and cultured microglial cells. Such increased effects induced by CCI were suppressed by CGRP antagonist and EZH2 inhibitor, which were concurrently associated with the attenuated mechanical and thermal hyperalgesia in CCI rats. CONCLUSION: Our findings highly indicate that CGRP is implicated in the genesis of neuropathic pain through regulating microglial activation via EZH2-mediated H3K27me3 in the spinal dorsal horn.

[Molecular analysis of hantavirus isolated from Shandong province].

OBJECTIVE: To compare the molecular characteristics of hantaviruses isolated from Shandong province by using PCR typing and nucleotide sequencing. METHODS: Total cellular RNA was extracted from hantaviruses infected Vero E6 cells, viral cDNA was amplified by polymerase chain reaction. PCR products of partial M Segments of 4 strains of hantaviruses were sequenced. Cross neutralization tests were performed. RESULTS: Four strains of hantavirus isolated from Rottus in Shandong province were SEO like viruses. The homology between Shandong isolates and other SEO like viruses was high at both amino acid and nucleotide levels. The homology among those 4 strains of hantaviruses was 98%. CONCLUSIONS: The SEO like hantaviruses were more conserved than other hantaviruses. The homology of SEO like hantaviruses isolated from Shandong province was as high as 98% at nucleotides level, though they were isolated at more than 10-year intervals.