Intraperitoneal Injection Of LPS Research Articles

Saiga antelope horn suppresses febrile seizures in rats by regulating neurotransmitters and the arachidonic acid pathway

BackgroundSaiga antelope horn (SAH) is a traditional Chinese medicine for treating febrile seizure (FS) with precise efficacy, but its mechanism of action and functional substances are still unclear. Given the need for further research on SAH, our group conducted studies to elucidate its mechanisms and active substances.MethodsAn FS rat pup model was constructed through intraperitoneal injection of LPS and hyperthermia induction. Behavioural indicators of seizures, hippocampal histopathological alterations, serum levels of inflammatory cytokines and hippocampal levels of neurotransmitters were observed and measured to investigate the effects of SAH on FS model rats. Hippocampal metabolomics and network pharmacology analyses were conducted to reveal the differential metabolites, key peptides and pathways involved in the suppression of FS by SAH.ResultsSAH suppressed FS, decreased the inflammatory response and regulated the Glu-GABA balance. Metabolomic analysis revealed 13 biomarkers of FS, of which SAH improved the levels of 8 differential metabolites. Combined with network pharmacology, a “biomarker-core target-key peptide” network was constructed. The peptides of SAH, such as YGQL and LTGGF, could exert therapeutic effects via the arachidonic acid pathway. Molecular docking and ELISA results indicated that functional peptides of SAH could bind to PTGS2 target, inhibiting the generation of AA and its metabolites in hippocampal samples.ConclusionIn summary, the functional peptides contained in SAH are the main material basis for the treatment of FS, potentially acting through neurotransmitter regulation and the arachidonic acid pathway.Graphical abstract

#1524 SKLB023 alleviates kidney inflammation and apoptosis via inhibiting TLR4-mediated NF-κB p65 and MAPK signaling pathways in septic AKI mice

Abstract Background and Aims Sepsis is a severe and complex clinical syndrome resulting from the host's inflammatory response to infection. Acute kidney injury (AKI) is one of the major complications of sepsis with high morbidity and mortality, namely sepsis-associated acute kidney injury (S-AKI). Despite current advances, effective drugs for treatment of S-AKI are scarce. SKLB023 is a small molecule compound designed based on 5-benzylidene-thiazolidine-2,4-dione, which has a potent anti-inflammatory effect with favorable efficacy in rheumatoid arthritis. This study aims to evaluate the efficacy of SKLB023 in treating S-AKI and to explore the underlying mechanisms. Method S-AKI was induced by cecal ligation puncture (CLP) and intraperitoneal injection of LPS (10 mg/kg) in male C57BL/6 mice. SKLB023 (25 mg/kg, 50 mg/kg) was administrated by gavage three days in advance and 30 minutes earlier on the day of modeling. Mice death per group was recorded for 100 hours after CLP to evaluate the effect of SKLB023 on the survival rate of S-AKI mice. Other mice were sacrificed 16 hours after model establishment, blood and kidney specimens were collected. Renal function was measured by the detection of serum creatinine (SCr) and blood urea nitrogen (BUN). Kidney pathological injury was evaluated by the expression of kidney injury markers and the assessment of PAS/HE stainings. In vitro experiments were performed with LPS stimulation of TCMK-1 cells and SKLB023 intervention, and the doses were determined by CCK-8 assay. Possible molecular mechanisms involved in SKLB023 against S-AKI were screened by bioinformatics analysis and further validated in vivo and vitro. RT-PCR, western blot, immunofluorescence, and immunohistochemistry were applied to detect renal inflammation and apoptosis. To determine the role of TLR4, TLR4 siRNA and TLR4-KO mice were applied in S-AKI model, and TLR4 expression and the activities of NF-κB p65 and MAPK signaling pathway were assessed. One-way ANOVA was performed for comparison between groups, and logrank analysis was used for comparison of survival curve. P < 0.05 was considered as statistically significant. Results The S-AKI model was successfully established by CLP and LPS injection with obvious renal dysfunction and kidney pathological damage. The mice administrated with SKLB023 had a higher survival rate, lower levels of Scr and BUN, and improved kidney pathological injury than the mice in the CLP group. Transcriptomic analysis identified TLR4-mediated downstream signaling pathways as the potential mechanism of SKLB023 and further experiments in vivo and in vitro proved that SKLB023 regulated the expression of TLR4 and the activities of NF-κB p65 and MAPK signaling pathway in S-AKI mice and LPS-stimulated TCMK-1 cells. Moreover, TLR4 inhibited the expression of inflammatory cytokines including TNF-α, IL-1β, IL-6, iNOS, COX-2, HMGB-1 and alleviated kidney apoptosis in S-AKI mice and LPS-treated TCMK-1 cells. Additionally, knockout of TLR4 remarkably improved inflammation, apotosis, and kidney injury in LPS-treated mice and TCMK-1 cells. Conclusion SKLB023 confers renoprotective effects in S-AKI by modulating inflammation and apoptosis. TLR4 is a key target in S-AKI, and SKLB023 inhibits inflammation and apoptosis by inhibiting the activities of TLR4 mediated NF-κB p65 and MAPK pathways. SKLB023 may be proposed as one of the potential preventive and therapeutic agents for S-AKI.

TNFRSF6 induces mitochondrial dysfunction and microglia activation in the in vivo and in vitro models of sepsis-associated encephalopathy.

Sepsis-associated encephalopathy (SAE) is a serious complication of sepsis. The tumour necrosis factor receptor superfamily member 6 (TNFRSF6) gene encodes the Fas protein, and it participates in apoptosis induced in different cell types. This study aimed to explore TNFRSF6 function in SAE. The SAE mouse model was established by intraperitoneal injection of LPS in TNFRSF6-/- mice and C57BL/6J mice. Microglia were treated with LPS to establish the cell model. The learning, memory and cognitive functions in mice were tested by behavioral tests. Nissl staining was utilized for determining neuronal injury. Microglial activation was tested by immunofluorescence assay. ELISA was utilized for determining TNF-α, IL-1β, IL-6, and IL-10 contents. Mitochondrial dysfunction was measured by mitochondrial oxygen consumption, ATP content, ROS production, and JC-1 assay. TNFRSF6 was upregulated in the LPS-induced mouse model and cell model. TNFRSF6 deficiency notably alleviated the impaired learning, memory and cognitive functions in SAE mice. Furthermore, we found that TNFRSF6 deficiency could alleviate neuronal injury, microglial activation, and inflammation in SAE mice. Additionally, mitochondrial dysfunction in the SAE mice was improved by TNFRSF6 depletion. In the LPS-induced microglia, we also proved that TNFRSF6 knockdown reduced inflammatory response inhibited ROS production, and alleviated mitochondrial dysfunction. TNFRSF6 induced mitochondrial dysfunction and microglia activation in the in vivo and in vitro models of SAE.

Induction of lipopolysaccharide during pregnancy will suppress IL-6 production but not activin B

During inflammation, proinflammatory cytokines are released by various immune cells including IL-6 and activin B. Production of proinflammatory cytokines can be suppressed by estradiol and an increase in estradiol can be found in pregnancy. The aim of this study was to determine the response of IL-6 and activin b in pregnant mice through LPS induction. There were two groups of mice consisting of 8 mice which were not pregnant and 13 pregnant mice in the 2nd week. Inflammation was induced by intraperitoneal injection of LPS Escherichia coli serotype O111:B4. After 4 hours of treatment, the mice serum was collected from their intracardiac blood to measure estradiol, IL-6 and activin B using ELISA. Pregnancy significantly reduced IL-6 levels (P=0.015) but there was no significant difference in estradiol (P=0.169) and activin B (P=0.583) levels. We also found that there was no association of E2 with IL-6 (P=0.637) and activin B (P=0.306). Pregnancy influences the inflammatory response, especially IL-6. This condition can occur because during pregnancy physiological changes occur in the body which involve various complex mechanisms in regulating the immune system.

Dietary taurine effect on intestinal barrier function, colonic microbiota and metabolites in weanling piglets induced by LPS.

Diarrhea in piglets is one of the most important diseases and a significant cause of death in piglets. Preliminary studies have confirmed that taurine reduces the rate and index of diarrhea in piglets induced by LPS. However, there is still a lack of relevant information on the specific target and mechanism of action of taurine. Therefore, we investigated the effects of taurine on the growth and barrier functions of the intestine, microbiota composition, and metabolite composition of piglets induced by LPS. Eighteen male weaned piglets were randomly divided into the CON group (basal diet + standard saline injection), LPS group (basal diet + LPS-intraperitoneal injection), and TAU + LPS group (basal diet + 0.3% taurine + LPS-intraperitoneal injection). The results show that taurine significantly increased the ADG and decreased the F/G (p < 0.05) compared with the group of CON. The group of TAU + LPS significantly improved colonic villous damage (p < 0.05). The expression of ZO-1, Occludin and Claudin-1 genes and proteins were markedly up-regulated (p < 0.05). Based on 16s rRNA sequencing analysis, the relative abundance of Lactobacilluscae and Firmicutes in the colon was significantly higher in the LPS + TAU group compared to the LPS group (p < 0.05). Four metabolites were significantly higher and one metabolite was significantly lower in the TAU + LPS group compared to the LPS group (p < 0.01). The above results show that LPS disrupts intestinal microorganisms and metabolites in weaned piglets and affects intestinal barrier function. Preventive addition of taurine enhances beneficial microbiota, modulates intestinal metabolites, and strengthens the intestinal mechanical barrier. Therefore, taurine can be used as a feed additive to prevent intestinal damage by regulating intestinal microorganisms and metabolites.

TRIM45 aggravates microglia pyroptosis via Atg5/NLRP3 axis in septic encephalopathy

BackgroundNeuroinflammation mediated by microglial pyroptosis is an important pathogenic mechanism of septic encephalopathy (SAE). It has been reported that TRIM45 is associated with tumours and inflammatory diseases. However, the role of TRIM45 in SAE and the relationship between TRIM45 and microglial pyroptosis are unknown. In this study, we found that TRIM45 played an important role in regulating microglial pyroptosis and the molecular mechanism.MethodsSAE was induced by intraperitoneal injection of LPS in WT and AAV-shTRIM45 mice. BV2 cells were treated with LPS/ATP in vitro. Cognitive function was assessed by the Morris water maze. Nissl staining was used to evaluate histological and structural lesions. ELISA was used to dectect neuroinflammation. qPCR was used to detect the mRNA levels of inflammatory cytokines, NLRP3, and autophagy genes. Western blotting and immunofluorescence analysis were used to analyse the expression of the proteins. Changes in reactive oxygen species (ROS) in cells were observed by flow cytometry. Changes in mitochondrial membrane potential in BV2 cells were detected by JC-1 staining. Peripheral blood mononuclear cells were extracted from blood by density gradient centrifugation and then used for qPCR, western blotting and flow detection. To further explore the mechanism, we used the overexpression plasmids TRIM45 and Atg5 as well as siRNA-TRIM45 and siRNA-Atg5 to analyse the downstream pathway of NLRP3. The protein and mRNA levels of TRIM45 in peripheral blood mononuclear cells from sepsis patients were examined.ResultsKnocking down TRIM45 protected against neuronal damage and cognitive impairment in septic mice. TRIM45 knockdown inhibited microglial pyroptosis and the secretion of inflammatory cytokines in vivo and in vitro, which was mediated by NLRP3/Gsdmd-N activation. Overexpression of TRIM45 could activate NLRP3 and downstream proteins. Further examination showed that TRIM45 regulated the activation of NLRP3 by altering Atg5 and regulating autophagic flux. It was also found that overexpression and knockdown of TRIM45 affected the changes in ROS and mitochondrial membrane potential. Thus, knocking down TRIM45 could reduce microglial pyroptosis, the secretion of proinflammatory cytokines, and neuronal damage and improve cognitive function. In addition, the level of TRIM45 protein in septic patients was increased. There was a positive linear correlation between APACHE II score and TRIM45, between SOFA score and TRIM45. Compared to group GCS > 9, level of TRIM45 were increased in group GCS ≤ 8.ConclusionTRIM45 plays a key role in neuroinflammation caused by LPS, and the mechanism may involve TRIM45-mediated exacerbation of microglial pyroptosis via the Atg5/NLRP3 axis.Graphical

An integrated RNA-Seq and network pharmacology approach for exploring the preventive effect of Corydalis bungeana Turcz. Extract and Acetylcorynoline on LPS-induced acute lung injury

Ethnopharmacological relevanceCorydalis bungeana Turcz. (KDD) is a Chinese herbal medicine with anti-inflammatory, lung cleansing, detoxification and other functions. Clinically, it is commonly used to treat respiratory infections. This study uses ALI as the research model, which is consistent with the clinical use of KDD. Acetylcorynoline (AC) is the main alkaloid component of the KDD extracts, and network pharmacology studies suggest that it may be the main active ingredient in the prevention of ALI. Aim of the studyThe aim of this study is to explore the underlying mechanisms and to study the efficacy material basis of KDD in anti-ALI effect by LPS-induced mice and using a combination of RNA sequencing (RNA-Seq) technology and network pharmacology. Materials and methodsEstablish a mouse model of ALI by intraperitoneal injection of LPS (5 mg/kg). The main active ingredients of KDD were identified and analyzed by high performance liquid chromatography with quadrupole time-of-flight mass spectrometry (HPLC-QTOF-MS) and network pharmacology. IL-18, IL-1β, and IL-6 levels in serum and bronchoalveolar lavage fluid (BALF), lung histopathological changes, and lung myeloperoxidase (MPO) activity were assessed. We investigated the possible molecular mechanisms of KDD and AC in an LPS-induced mouse ALI models with RNA-Seq technology. In addition, the anti-inflammatory effect of AC was verified in vitro by establishing an LPS-stimulated RAW264.7 inflammation model. Molecular docking further validated AC as the efficacy material basis of KDD in anti-ALI. ResultsBased on HPLC-QTOF-MS technology and network pharmacology, KDD is more strongly associated with lung tissue, and that AC may be the main active ingredient of KDD. Subsequently, in vivo experiments results showed that KDD and AC reduced the levels of pro-inflammatory cytokines in serum and BALF, reduced MPO levels and reduced inflammatory damage in the lungs. To elucidate its underlying mechanism, based on RNA-Seq analysis techniques performed in lung tissue, enrichment analysis showed that KDD and AC intervened through the NLR signaling pathway, thereby mitigating LPS-induced ALI. Then, RT-qPCR, IF, WB and other technologies were used to verify the anti-ALI core difference genes of KDD and AC from the gene transcription and protein expression levels of the NLR signaling pathway, and confirmed the anti-ALI. In vitro experimental results also showed that AC has anti-inflammatory effects in RAW264.7. Finally, the biotransformation and molecular docking results also further indicated that AC is the active ingredient of KDD in anti-ALI. ConclusionsStudies have shown that KDD has a good therapeutic effect on ALI, and AC is the main pharmacodynamic material basis for its therapeutic effect in ALI.

A novel co-culture model for investigation of the effects of LPS-induced macrophage-derived cytokines on brain endothelial cells.

In order to study effects of macrophage-derived inflammatory mediators associated with systemic inflammation on brain endothelial cells, we have established a co-culture system consisting of bEnd.3 cells and LPS-activated Raw 264.7 cells and performed its cytokine profiling. The cytokine profile of the co-culture model was compared to that of mice treated with intraperitoneal LPS injection. We found that, among cytokines profiled, eight cytokines/chemokines were similarly upregulated in both in vivo mouse and in vitro co-culture model. In contrast to the co-culture model, the cytokine profile of a common mono-culture system consisting of only LPS-activated bEnd.3 cells had little similarity to that of the in vivo mouse model. These results indicate that the co-culture of bEnd.3 cells with LPS-activated Raw 264.7 cells is a better model than the common mono-culture of LPS-activated bEnd.3 cells to investigate the molecular mechanism in endothelial cells, by which systemic inflammation induces neuroinflammation. Moreover, fibrinogen adherence both to bEnd.3 cells in the co-culture and to brain blood vessels in a LPS-treated animal model of Alzheimer's disease increased. To the best of our knowledge, this is the first to utilize bEnd.3 cells co-cultured with LPS-activated Raw 264.7 cells as an in vitro model to investigate the consequence of macrophage-derived inflammatory mediators on brain endothelial cells.

Dietary diosgenin transcriptionally down-regulated intestinal NPC1L1 expression to prevent cholesterol gallstone formation in mice

BackgroundCholesterol gallstone disease is a common disease. Reducing cholesterol burden is important to prevent/treat gallstone. In this study, we investigated the application of diosgenin (DG) to prevent the formation of gallstone in mice.MethodsAdult male C57BL/6J mice were fed with the lithogenic diet (LD) only or LD supplemented with DG or ezetimibe for 8 weeks. Incidences of gallstone formation were documented. Intestine and liver tissues were collected to measure the lipid contents and expression of genes in cholesterol metabolism. Caco2 cells were treated with DG to monitor the regulation on cholesterol absorption and the transcriptional regulation of Npc1l1 gene. Changes of gut microbiota by DG was analyzed. Intraperitoneal injection of LPS on mice was performed to verify its effects on STAT3 activation and Npc1l1 expression in the small intestine.ResultsLD led to 100% formation of gallstones in mice. In comparison, dietary DG or ezetimibe supplementary completely prevents gallstones formation. DG inhibited intestinal cholesterol absorption in mice as well as in Caco2 cells by down-regulation of Npc1l1 expression. DG could directly inhibit phosphorylation of STAT3 and its transcriptional regulation of Npc1l1 expression. Furthermore, DG could modulate gut microbiota profiles and LPS mediated STAT3 activation and Npc1l1 expression.ConclusionOur results demonstrated that dietary DG could inhibit intestinal cholesterol absorption through decreasing NPC1L1 expression to prevent cholesterol gallstone formation.

#5648 RIPK3 MEDIATES KIDNEY INJURY INDUCED BY A CYTOKINE STORM

Abstract Background and Aims Receptor-interacting protein kinase 3 (RIPK3) is traditionally involved in necroptosis, a regulated necrosis pathway which has been observed in diseases associated to cell death and inflammation. However, RIPK3 has been also associated to inflammatory responses independent of necroptosis. In previous studies we demonstrated that RIPK3 deficiency does not prevent renal injury but protects from inflammation in folic acid-induced AKI (FA-AKI), suggesting a proinflammatory role independently of cell death. In the present work, we aim to explore the role of RIPK3 in kidney injury and inflammation induced by a cytokine storm. Method Lipopolysaccharide (LPS) was used to induce cytokine storm-AKI in mice. For this, female 12- to 14-week-old wild type (WT), RIPK3-KO, MLKL-KO or NRLP3-KO C57BL/6J mice received a single intraperitoneal (i.p) injection of LPS 5 mg/kg or vehicle and were sacrificed 1h, 4h and 24 hours later. To generate chimera mice, WT and Ripk3-KO receptor mice were irradiated to deplete the autologous bone marrow (BM). BM was extracted from the femur and tibia of donor WT or Ripk3-KO mice, and 107 cells were transferred to irradiated receptor mice by intravenous injection. After 1 month, LPS-AKI was induced. Plasma was collected to assess kidney function. Kidneys were collected for RNA, protein studies, and histologic studies. Additionally, a WT group received 1.65 mg/kg of the necroptosis inhibitor Necrostatin-1 (Nec1) i.p. prior to LPS to evaluate the impact of necroptosis pathway. Liver, lungs and heart were also collected to assess the systemic inflammatory response. Additionally, cultured murine immortalized tubular MCT cells, and primary tubular cells, bone marrow dendritic cells (BMDC) and bone marrow derived macrophages (BMDM) isolated from WT and Ripk3-KO mice were studied. Cells were stimulated with 100 ng/ml LPS for 6h and RNA was studied. To analyze the impact of the inflammatory response on tubular cells, supernatants from LPS-stimulated BMDM and BMDC were collected and used to stimulate MCT cells. RNA expression was studied by RT-PCR and protein expression by Western Blotting. Results The kidney expression of RIPK3 mRNA and protein was upregulated in cytokine storm-AKI in mice, while Ripk3 deficiency improved survival and renal function, with less expression of proinflammatory cytokines and inflammatory infiltrate. Necroptosis did not seem to be implicated in cytokine storm-AKI, since neither Nec1 nor genetic MLKL ablation offered protection on renal function. Systemic inflammation in non-kidney organs was also milder with Ripk3 deficiency. In addition, inflammasome-related proteins were upregulated in cytokine storm-AKI, and this was reduced in Ripk3-KO mice. However, Nlrp3-deficient mice developed kidney injury and inflammation after LPS injection. Next, we explored the role of RIPK3 in BM-derived cells in cytokine storm-AKI by generating chimera mice. In this context, WT mice with Ripk3 deficient BM exhibited less inflammation and better renal function. This supports that RIPK3 from BM cells mediates kidney inflammation and injury during cytokine storm-AKI. In cultured cells, LPS induced RIPK3 expression in BMDMs but not in tubular cells, and RIPK3 mediated IL-6 expression in BMDMs but not in tubular cells. Moreover, IL-6 and conditioned media from LPS-exposed WT macrophages promoted proinflammatory responses in cultured tubular cells, that was partially ameliorated for RIPK3-KO LPS-macrophage conditioned medium. Conclusion In conclusion, RIPK3 mediates kidney injury and systemic inflammation induced by a cytokine storm independently of the necroptosis pathway. These results identify RIPK3 as a therapeutic target for renal inflammatory diseases.

Sepsis-Induced Changes in Spectral Segregation and Kinetics of Hippocampal Oscillatory States in Rats.

Sepsis-associated encephalopathy (SAE) is a frequent severe complication of sepsis and the systemic inflammatory response syndrome, associated with high mortality and long-term neurologic consequences in surviving patients. One of the main clinical signs of SAE are discontinuous sleep periods that are fragmented by frequent awakenings. Although this brain state fragmentation strongly impacts the functionality of the nervous and other systems, its underlying network mechanisms are still poorly understood. In this work, we therefore aim to characterize the properties and dynamics of brain oscillatory states in response to SAE in an acute rat model of sepsis induced by high-dose lipopolysaccharide (LPS; 10 mg/kg). To focus on intrinsically generated brain state dynamics, we used a urethane model that spares oscillatory activity in rapid eye movement (REM)-like and nonrapid eye movement (NREM)-like sleep states. Intraperitoneal LPS injection led to a robust instability of both oscillatory states resulting in several folds more state transitions. We identified opposing shifts in low-frequency oscillations (1-9 Hz) in REM and NREM-like states under influence of LPS. This resulted in increased similarity between both states. Moreover, the state-space jitter in both states increased as well, pointing to higher within-state instability. The reduction of interstate spectral distances in 2-D state space, combined with increased within-state jitter might represent a key factor in changing the energy landscape of brain oscillatory state attractors, and hence lead to altered sleep architecture. Their emergence during sepsis might point to a mechanism underlying severe sleep fragmentation as described both in sepsis patients and SAE animal models.

Estrogen receptor subtype mediated anti-inflammation and vasorelaxation via genomic and nongenomic actions in septic mice.

Sepsis is a life-threatening disease with high mortality worldwide. Septic females have lower severity and mortality than the males, suggesting estrogen exerts a protective action, but nothing is known about the role of vascular endothelial estrogen receptor subtypes in this process. In the present study, we aimed to study the estrogen receptors on mesenteric arterioles in normal and sepsis mice and to elucidate the underlying mechanisms. Sepsis was induced in mice by intraperitoneal injection of LPS. The changes in the expression and release of the serum and cell supernatant proinflammatory cytokines, including TNF-α, IL-1β and IL-6, were measured by qPCR and ELISA, and the functions of multiple organs were analyzed. The functional activities of mouse mesenteric arterioles were determined by a Mulvany-style wire myograph. The expression of phospholipase C (PLC) and inositol 1,4,5-trisphosphate receptor (IP3R) in endothelial cells were examined by Western blot and their functions were characterized by cell Ca2+ imaging. Septic female mice had higher survival rate than the male mice, and pretreatment with E2 for 5 days significantly improved the survival rate and inhibited proinflammatory cytokines in septic male mice. E2 ameliorated pulmonary, intestinal, hepatic and renal multiple organ injuries in septic male mice; and ER subtypes inhibited proinflammatory cytokines in endothelial cells via PLC/IP3R/Ca2+ pathway. E2/ER subtypes immediately induced endothelial-derived hyperpolarization (EDH)-mediated vasorelaxation via PLC/IP3R/Ca2+ pathway, which was more impaired in septic male mice. E2/ER subtypes could rescue the impaired acetylcholine (ACh)-induced EDH-mediated vasorelaxation in septic male mice. E2 through ER subtypes mediates anti-inflammation and vasorelaxation via genomic and nongenomic actions in sepsis. Mechanistically, activation of endothelial ER subtypes reduces proinflammatory cytokines and induces EDH-mediated vasorelaxation via PLC/IP3R/Ca2+ pathway, leading to amelioration of sepsis-induced organ injury and survival rate.

Uncovering the active constituents and mechanisms of Rujin Jiedu powder for ameliorating LPS-induced acute lung injury using network pharmacology and experimental investigations.

Background: Acute lung injury (ALI) is a common clinical disease with high mortality. Rujin Jiedu powder (RJJD) has been clinically utilized for the treatment of ALI in China, but the active constituents in RJJD and its protective mechanisms against ALI are still unclear. Methodology: ALI mice were established by intraperitoneal injection of LPS to test the effectiveness of RJJD in treating ALI. Histopathologic analysis was used to assess the extent of lung injury. An MPO (myeloperoxidase) activity assay was used to evaluate neutrophil infiltration. Network pharmacology was used to explore the potential targets of RJJD against ALI. Immunohistochemistry and TUNEL staining were performed to detect apoptotic cells in lung tissues. RAW264.7 and BEAS-2B cells were used to explore the protective mechanisms of RJJD and its components on ALI in vitro. The inflammatory factors (TNF-α, IL-6, IL-1β and IL-18) in serum, BALF and cell supernatant were assayed using ELISA. Western blotting was performed to detect apoptosis-related markers in lung tissues and BEAS-2B cells. Results: RJJD ameliorated pathological injury and neutrophil infiltration in the lungs of ALI mice and decreased the levels of inflammatory factors in serum and BALF. Network pharmacology investigations suggested that RJJD treated ALI via regulating apoptotic signaling pathways, with AKT1 and CASP3 as crucial targets and PI3K-AKT signaling as the main pathway. Meanwhile, baicalein, daidzein, quercetin and luteolin were identified as key constituents in RJJD targeting on the above crucial targets. Experimental investigations showed that RJJD significantly upregulated the expression of p-PI3K, p-Akt and Bcl-2, downregulated the expression of Bax, caspase-3 and caspase-9 in ALI mice, and attenuated lung tissue apoptosis. Four active constituents in RJJD (baicalein, daidzein, quercetin and luteolin) inhibited the secretion of TNF-α and IL-6 in LPS-induced RAW264.7 cells. Among these components, daidzein and luteolin activated the PI3K-AKT pathway and downregulated the expression of apoptosis-related markers induced by LPS in BEAS-2B cells. Conclusion: RJJD alleviates the inflammatory storm and prevents apoptosis in the lungs of ALI mice. The mechanism of RJJD in treating ALI is related to the activation of PI3K-AKT signaling pathway. This study provides a scientific basis for the clinical application of RJJD.

Activation of neuronal NADPH oxidase NOX2 promotes inflammatory neurodegeneration.

Strong evidence indicates critical roles of NADPH oxidase (a key superoxide-producing enzyme complex during inflammation) in activated microglia for mediating neuroinflammation and neurodegeneration. However, little is known about roles of neuronal NADPH oxidase in neurodegenerative diseases. This study aimed to investigate expression patterns, regulatory mechanisms and pathological roles of neuronal NADPH oxidase in inflammation-associated neurodegeneration. The results showed persistent upregulation of NOX2 (gp91phox; the catalytic subunit of NADPH oxidase) in both microglia and neurons in a chronic mouse model of Parkinson's disease (PD) with intraperitoneal LPS injection and LPS-treated midbrain neuron-glia cultures (a cellular model of PD). Notably, NOX2 was found for the first time to exhibit a progressive and persistent upregulation in neurons during chronic neuroinflammation. While primary neurons and N27 neuronal cells displayed basal expression of NOX1, NOX2 and NOX4, significant upregulation only occurred in NOX2 but not NOX1 or NOX4 under inflammatory conditions. Persistent NOX2 upregulation was associated with functional outcomes of oxidative stress including increased ROS production and lipid peroxidation. Neuronal NOX2 activation displayed membrane translocation of cytosolic p47phox subunit and was inhibited by apocynin and diphenyleneiodonium chloride (two widely-used NADPH oxidase inhibitors). Importantly, neuronal ROS production, mitochondrial dysfunction and degeneration induced by inflammatory mediators in microglia-derived conditional medium were blocked by pharmacological inhibition of neuronal NOX2. Furthermore, specific deletion of neuronal NOX2 prevented LPS-elicited dopaminergic neurodegeneration in neuron-microglia co-cultures separately grown in the transwell system. The attenuation of inflammation-elicited upregulation of NOX2 in neuron-enriched and neuron-glia cultures by ROS scavenger N-acetylcysteine indicated a positive feedback mechanism between excessive ROS production and NOX2 upregulation. Collectively, our findings uncovered crucial contribution of neuronal NOX2 upregulation and activation to chronic neuroinflammation and inflammation-related neurodegeneration. This study reinforced the importance of developing NADPH oxidase-targeting therapeutics for neurodegenerative diseases.

Renoprotective Effect Of Sambiloto (Andrographis Paniculata) Leaf Extract On Lipopolysaccharide – Induced Septic Rats

Background: Associated with sepsis is the illness known as acute kidney injury (AKI). AKI may be mediated by uric acid, and blood creatinine levels can be utilized to diagnose the condition to measure kidney function. Sambiloto (Andrographis paniculata) is a traditional medicine that has flavonoid compounds that can reduce creatinine levels and Xanthine Oxidase Inhibitors which can reduce uric acid levels.Objective: Septic model rats generated by lipopolysaccharide were used to test the effects of sambiloto (Andrographis paniculata) leaf extract on serum creatinine and uric acid levels (LPS).Methods: This study was experimental employing 25 rats split into 5 groups as the post-test alone control group: healthy control with standard feed (HC), negative control with LPS injection(NC), Treatment (T)1 (A.Paniculata 200 mg/kgBW+LPS), T2(A.paniculata 400 mg/kgBW+LPS), and T3(A.paniculata 500mg/kgBW+ LPS). A.paniculata leaf extract was given via oral gavage on day 8-21. Intraperitoneal injection of LPS 5 mg/kgBW was given on day 22. On the 25th day, the blood serum was analyzed for creatinine levels using Jaffe method and uric acid was analyzed using the enzymatic photometric method. One-way analysis of variance (Kruskal-Wallis) and the Kruskal-Wallis test were used to evaluate the data.Results: The mean creatinine levels of HC,NC,T1,T2,T3 were 0.7±0.01;3.5±0.04;2.9±0.03;1.9±0.05; 1.3 ±0.04 mg/dl respectively. The mean uric acid levels of HC.NC.T1.T2.T3 were 1.7±0.05;8.2±0.11; 4.5±0.03;4.0±0.12;3.0±0.19 mg/dl respectively. There were significant differences (p&lt;0.05) in creatinine levels in groups T2(p=0.031) and T3(p=0.001) to NC group and serum uric acid levels in groups T1(p&lt;0.001), T2(p&lt;0.001), and T3(p&lt;0.001) to NC group which creatinine and uric acid levels were lower than NC group.Conclusion: Andrographis paniculata leaf extract has renoprotective effect against AKI in LPS-induced septic ratsKeywords: Andrographis paniculata, creatinine, uric acid, lipopolysaccharide, AKI, sepsis

Different effects of a perioperative single dose of dexamethasone on wound healing in mice with or without sepsis.

Sepsis delays wound healing owing to uncontrolled inflammation. A single perioperative dose of dexamethasone is widely used because of its anti-inflammatory effects. However, the effects of dexamethasone on wound healing in sepsis remain unclear. We discuss the methods to obtain dose curves and explore the safe dosage range for wound healing in mice with or without sepsis. A saline or LPS intraperitoneal injection was applied to C57BL/6 mice. After 24 hours, the mice received a saline or DEX intraperitoneal injection and full-thickness, dorsal wounding operation. Wound healing was observed by image record, immunofluorescence and histological staining. Inflammatory cytokines and M1/M2 macrophages in wounds were determined by ELISA and immunofluorescence, respectively. Dose-response curves reflected the safe dosage range of DEX in mice with or without sepsis, from 0.121 to 2.03 mg/kg and from 0 to 0.633 mg/kg, respectively. we found that a single dose of dexamethasone (1 mg/kg, i.p.) promoted wound healing in septic mice, but delayed wound healing in normal mice. In normal mice, dexamethasone delays inflammation, resulting in an insufficient number of macrophages during the healing process. In septic mice, dexamethasone alleviated excessive inflammation and maintained the balance of M1/M2 macrophages in the early and late healing process. In summary, the safe dosage range of dexamethasone in septic mice is wider than that in normal mice. A single dose of dexamethasone (1 mg/kg) increased wound healing in septic mice, but delayed it in normal mice. Our findings provide helpful suggestions for the rational use of dexamethasone.

Deletion of Gpatch2 does not alter Tnf expression in mice

The cytokine TNF has essential roles in immune defence against diverse pathogens and, when its expression is deregulated, it can drive severe inflammatory disease. The control of TNF levels is therefore critical for normal functioning of the immune system and health. We have identified GPATCH2 as a putative repressor of Tnf expression acting post-transcriptionally through the TNF 3’ UTR in a CRISPR screen for novel regulators of TNF. GPATCH2 is a proposed cancer-testis antigen with roles reported in proliferation in cell lines. However, its role in vivo has not been established. We have generated Gpatch2−/− mice on a C57BL/6 background to assess the potential of GPATCH2 as a regulator of Tnf expression. Here we provide the first insights into Gpatch2−/− animals and show that loss of GPATCH2 affects neither basal Tnf expression in mice, nor Tnf expression in intraperitoneal LPS and subcutaneous SMAC-mimetic injection models of inflammation. We detected GPATCH2 protein in mouse testis and at lower levels in several other tissues, however, the morphology of the testis and these other tissues appears normal in Gpatch2−/− animals. Gpatch2−/− mice are viable, appear grossly normal, and we did not detect notable aberrations in lymphoid tissues or blood cell composition. Collectively, our results suggest no discernible role of GPATCH2 in Tnf expression, and the absence of an overt phenotype in Gpatch2−/− mice warrants further investigation of the role of GPATCH2.

Isocorydine Ameliorates IL-6 Expression in Bone Marrow-Derived Macrophages and Acute Lung Injury Induced by Lipopolysaccharide.

Isocorydine (ICD) is a type of isoquinoline alkaloid originating from Corydalis edulis, which has been used to relieve spasm, dilate blood vessels, and treat malaria as well as hypoxia in clinic. However, its effect on inflammation and underlying mechanisms remains unclear. The aim of our study was to determine the potential effects and mechanisms of ICD on pro-inflammatory interleukin-6 (IL-6) expression in bone marrow-derived macrophages (BMDMs) and acute lung injury mouse model. A mouse model of acute lung injury was established by intraperitoneal injection of LPS and treated with different doses of ICD. The body weight and food intake of mice were monitored to determine the toxicity of ICD. The tissue samples of lung, spleen and blood were taken to assess the pathological symptoms of acute lung injury and the expression levels of IL-6. Further, BMDMs isolated from C57BL/6 mice were cultured in vitro and treated with granulocyte-macrophage colony-stimulating factor (GM-CSF), LPS and different doses of ICD. CCK-8 assay and flow cytometry were performed to assess the viability of BMDMs. The expression of IL-6 was detected by RT-PCR and ELISA. RNA-seq was carried out to detect the differential expression genes of ICD-treated BMDMs. Western blotting was used to detect the change in MAPK and NF-κB signaling pathways. Our findings show that ICD ameliorates IL-6 expression and attenuates phosphorylation of p65 and JNK in BMDMs, and can protect mice from acute lung injury.

Selective activation of cholinergic neurotransmission from the medial septal nucleus to hippocampal pyramidal neurones improves sepsis-induced cognitive deficits in mice

BackgroundSepsis-associated encephalopathy is characterised by cognitive dysfunction, and might be mediated by deficits in neurotransmission. Reduced cholinergic neurotransmission in the hippocampus impairs memory function. We assessed real-time alterations of acetylcholine neurotransmission from the medial septal nucleus to the hippocampus, and explored whether sepsis-induced cognitive deficits can be relieved by activating upstream cholinergic projections. MethodLipopolysaccharide (LPS) injection or caecal ligation and puncture (CLP) was used to induce sepsis and associated neuroinflammation in wild-type and mutant mice. Adeno-associated viruses for calcium and acetylcholine imaging, and for optogenetic and chemogenetic modulation of cholinergic neurones were injected into the hippocampus or medial septum, and a 200-μm-diameter optical fibre was implanted to collect acetylcholine and calcium signals. Cholinergic activity of the medial septum was manipulated and combined with cognitive assessment after LPS injection or CLP. ResultsIntracerebroventricular LPS injection reduced postsynaptic acetylcholine (from 0.146 [0.001] to 0.0047 [0.0005]; p=0.004) and calcium (from 0.0236 [0.0075] to 0.0054 [0.0026]; p=0.0388) signals in hippocampal Vglut2-positive glutamatergic neurones, whereas optogenetic activation of cholinergic neurones in the medial septum reversed LPS-induced reductions in these two signals. Intraperitoneal LPS injection decreased acetylcholine concentration in the hippocampus (476 [20] pg ml−1 to 382 [14] pg ml−1; p=0.0001). Reduction in long-term potentiation (238 [23] % to 150 [12] %; p=0.0082) and enhancement of hippocampal pyramidal neurone action potential frequency (5.8 [1.5] Hz to 8.2 [1.8] Hz; p=0.0343) were relieved, and neurocognitive performance was improved by chemogenetic activation of cholinergic innervation of the hippocampus 3 days after LPS injection in septic mice. ConclusionsSystemic or local LPS reduced cholinergic neurotransmission from the medial septum to hippocampal pyramidal neurones, and their selective activation alleviated defects in hippocampal neuronal function and synaptic plasticity and ameliorated memory deficits in sepsis model mice through enhanced cholinergic neurotransmission. This provides a basis for targeting cholinergic signalling to the hippocampus in sepsis-induced encephalopathy.

RBM3 is associated with acute lung injury in septic mice and patients via the NF-κB/NLRP3 pathway.

Sepsis refers to host response disorders caused by infection, leading to life-threatening organ dysfunction. RNA-binding motif protein 3 (RBM3) is an important cold-shock protein that is upregulated in response to mild hypothermia or hypoxia. In this study, we aimed to investigate whether RBM3 is involved in sepsis-associated acute lung injury (ALI). Intraperitoneal injection of LPS (10mg/kg) was performed in wild type (WT) and RBM3 knockout (KO, RBM3-/-) mice to establish an in vivo sepsis model. An NLRP3 inflammasome inhibitor, MCC950 (50mg/kg), was injected intraperitoneally 30min before LPS treatment. Serum, lung tissues, and BALF were collected 24h later for further analysis. In addition, we also collected serum from sepsis patients and healthy volunteers to detect their RBM3 expression. The results showed that the expression of RBM3 in the lung tissues of LPS-induced sepsis mice and the serum of patients with sepsis was significantly increased and positively correlated with disease severity. In addition, RBM3 knockout (KO) mice had a low survival rate, and RBM3 KO mice had more severe lung damage, inflammation, lung cell apoptosis, and oxidative stress than WT mice. LPS treatment significantly increased the levels of nucleotide binding and oligomerization domain-like receptor family 3 (NLRP3) inflammasomes and mononuclear cell nuclear factor-κB (NF-κB) in the lung tissues of RBM3 KO mice. However, these levels were only slightly elevated in WT mice. Interestingly, MCC950 improved LPS-induced acute lung injury in WT and RBM3 KO mice but inhibited the expression of NLRP3, caspase-1, and IL-1β. In conclusion, RBM3 was overexpressed in sepsis patients and LPS-induced mice. RBM3 gene deficiency aggravated sepsis-associated ALI through the NF-κB/NLRP3 pathway.