Acute exposure to polystyrene nanoparticles promotes liver injury by inducing mitochondrial ROS-dependent necroptosis and augmenting macrophage-hepatocyte crosstalk.

BACKGROUND: The global use of plastic materials has undergone rapid expansion, resulting in the substantial generation of degraded and synthetic microplastics and nanoplastics (MNPs), which have the potential to impose significant environmental burdens and cause harmful effects on living organisms. Despite this, the detrimental impacts of MNPs exposure towards host cells and tissues have not been thoroughly characterized. RESULTS: In the present study, we have elucidated a previously unidentified hepatotoxic effect of 20 nm synthetic polystyrene nanoparticles (PSNPs), rather than larger PS beads, by selectively inducing necroptosis in macrophages. Mechanistically, 20 nm PSNPs were rapidly internalized by macrophages and accumulated in the mitochondria, where they disrupted mitochondrial integrity, leading to heightened production of mitochondrial reactive oxygen species (mtROS). This elevated mtROS generation essentially triggered necroptosis in macrophages, resulting in enhanced crosstalk with hepatocytes, ultimately leading to hepatocyte damage. Additionally, it was demonstrated that PSNPs induced necroptosis and promoted acute liver injury in mice. This harmful effect was significantly mitigated by the administration of a necroptosis inhibitor or systemic depletion of macrophages prior to PSNPs injection. CONCLUSION: Collectively, our study suggests a profound toxicity of environmental PSNP exposure by triggering macrophage necroptosis, which in turn induces hepatotoxicity via intercellular crosstalk between macrophages and hepatocytes in the hepatic microenvironment.

Glutamine sustains energy metabolism and alleviates liver injury in burn sepsis by promoting the assembly of mitochondrial HSP60-HSP10 complex via SIRT4 dependent protein deacetylation.

Burns and burn sepsis, characterized by persistent and profound hypercatabolism, cause energy metabolism dysfunction that worsens organ injury and systemic disorders. Glutamine (Gln) is a key nutrient that remarkably replenishes energy metabolism in burn and sepsis patients, but its exact roles beyond substrate supply is unclear. In this study, we demonstrated that Gln alleviated liver injury by sustaining energy supply and restoring redox balance. Meanwhile, Gln also rescued the dysfunctional mitochondrial electron transport chain (ETC) complexes, improved ATP production, reduced oxidative stress, and protected hepatocytes from burn sepsis injury. Mechanistically, we revealed that Gln could activate SIRT4 by upregulating its protein synthesis and increasing the level of Nicotinamide adenine dinucleotide (NAD+), a co-enzyme that sustains the activity of SIRT4. This, in turn, reduced the acetylation of shock protein (HSP) 60 to facilitate the assembly of the HSP60-HSP10 complex, which maintains the activity of ETC complex II and III and thus sustain ATP generation and reduce reactive oxygen species release. Overall, our study uncovers a previously unknown pharmacological mechanism involving the regulation of HSP60-HSP10 assembly by which Gln recovers mitochondrial complex activity, sustains cellular energy metabolism and exerts a hepato-protective role in burn sepsis.

Synergistic Inhibitory Effect of Multiple Polyphenols from Spice on Acrolein during High-Temperature Processing.

Acrolein (ACR) is a toxic unsaturated aldehyde that is produced during food thermal processing. Here, we investigated the synergistic effect of polyphenols in binary, ternary, and quaternary combinations on ACR by the Chou-Talalay method, and then explored the synergistic effect of cardamonin (CAR), alpinetin (ALP), and pinocembrin (PIN) in fixed proportion from Alpinia katsumadai Hayata (AKH) combined with curcumin (CUR) in the model, and roasted pork using LC-MS/MS. Our results showed that their synergistic effect depended on the intensification of their individual trapping ACR activities, which resulted in the formation of more ACR adducts. In addition, by adding 1% AKH (as the carrier of CAR, ALP, and PIN) and 0.01% CUR (vs. 6% AKH single) as spices, more than 71.5% (vs. 54.0%) of ACR was eliminated in roast pork. Our results suggested that selective complex polyphenols can synergistically remove the toxic ACR that is produced in food processing.

Food additive octyl gallate eliminates acrolein and inhibits bacterial growth in oil-rich food.

Acrolein (ACR) is predominantly generated from oil-rich food during thermos- processing. Accumulation of ACR in vivo through food consumption has been associated with an increased risk of developing chronic diseases. Here, we investigated the inhibitory effect of octyl gallate (OG), a new food additive tolerant to high-temperature, alkaline and fat-soluble saturations, on the generation of ACR in OG-ACR, oil-Rancimat models, and real-world frying. Our results demonstrate that approximately 80% and 60% of ACR was eliminated by OG in the two models, respectively, and OG-ACR was detected in the deep-frying process using LC-MS/MS. The reaction pathways were clarified by synthesis and OG-ACR and OG-2ACR adduct structural elucidation. Our work reveals that the antibacterial activity of OG-ACR against Escherichia coli (gram-negative) was four times higher than that of OG. Thus, OG can be developed as a promising novel ACR scavenger for high-temperature food processing and an antibacterial agent in food storage.

Acute exposure to gold nanoparticles aggravates lipopolysaccharide-induced liver injury by amplifying apoptosis via ROS-mediated macrophage-hepatocyte crosstalk.

BACKGROUND: Gold nanoparticles (AuNPs) are increasingly utilized in industrial and biomedical fields, thereby demanding a more comprehensive knowledge about their safety. Current toxicological studies mainly focus on the unfavorable biological impact governed by the physicochemical properties of AuNPs, yet the consequences of their interplay with other bioactive compounds in biological systems are poorly understood. RESULTS: In this study, AuNPs with a size of 10 nm, the most favorable size for interaction with host cells, were given alone or in combination with bacterial lipopolysaccharide (LPS) in mice or cultured hepatic cells. The results demonstrated that co exposure to AuNPs and LPS exacerbated fatal acute liver injury (ALI) in mice, although AuNPs are apparently non-toxic when administered alone. AuNPs do not enhance systemic or hepatic inflammation but synergize with LPS to upregulate hepatic apoptosis by augmenting macrophage-hepatocyte crosstalk. Mechanistically, AuNPs and LPS coordinate to upregulate NADPH oxidase 2 (NOX2)-dependent reactive oxygen species (ROS) generation and activate the intrinsic apoptotic pathway in hepatic macrophages. Extracellular ROS generation from macrophages is then augmented, thereby inducing calcium-dependent ROS generation and promoting apoptosis in hepatocytes. Furthermore, AuNPs and LPS upregulate scavenger receptor A expression in macrophages and thus increase AuNP uptake to mediate further apoptosis induction. CONCLUSIONS: This study reveals a profound impact of AuNPs in aggravating the hepatotoxic effect of LPS by amplifying ROS-dependent crosstalk in hepatic macrophages and hepatocytes.

Affinity-Guided Isolation and Identification of Procyanidin B2 from Mangosteen (Garcinia mangostana L.) Rinds and its In Vitro LPS Binding and Neutralization Activities.

Garcinia mangostana L. (mangosteen) is a tropical fruit that has been used for medicinal purposes in Southeast Asia for centuries. With an interest in its applications to treat infection, we sought to investigate the bioactive constituents of mangosteen and identified the phenolic compound procyanidin B2 from the mangosteen pericarp by examining lipopolysaccharide (LPS) binding capacity. The LPS binding and neutralization activities of procyanidin B2 were determined by a combination of biophysical and in silico techniques. The affinity of procyanidin B2 to LPS was 1.61 × 10-5 M. Procyanidin B2 significantly neutralized LPS and selectively inhibited the LPS-induced release of tumor necrosis factor (TNF)-α from RAW264.7 cells in a dose-dependent manner. Binding thermodynamics revealed favorable hydrogen bonding and hydrophobic interactions between procyanidin B2 and LPS. Molecular simulations suggested that hydrogen bonding and hydrophobic interactions were involved in the binding process. These findings have, for the first time, shed light on the anti-inflammatory properties of procyanidin B2 through LPS binding and neutralization and provided a promising lead for the development of antiendotoxin agents.

PSA controls hepatic lipid metabolism by regulating the NRF2 signaling pathway.

The activity of proteinase is reported to correlate with the development and progression of nonalcoholic fatty liver disease (NAFLD). Puromycin-sensitive aminopeptidase (PSA/NPEPPS) is an integral nontransmembrane enzyme that functions to catalyze the cleavage of amino acids near the N-terminus of polypeptides. A previous study suggested that this enzyme acts as a regulator of neuropeptide activity; however, the metabolic function of this enzyme in the liver has not been explored. Here, we identified the novel role of PSA in hepatic lipid metabolism. Specifically, PSA expression was lower in fatty livers from NAFLD patients and mice (HFD, ob/ob, and db/db). PSA knockdown in cultured hepatocytes exacerbated diet-induced triglyceride accumulation through enhanced lipogenesis and attenuated fatty acid ß-oxidation. Moreover, PSA mediated activation of the master regulator of antioxidant response, nuclear factor erythroid 2-related factor 2 (NRF2), by stabilizing NRF2 protein expression, which further induced downstream antioxidant enzymes to protect the liver from oxidative stress and lipid overload. Accordingly, liver-specific PSA overexpression attenuated hepatic lipid accumulation and steatosis in ob/ob mice. Furthermore, in human liver tissue samples, decreased PSA expression correlated with the progression of NAFLD. Overall, our findings suggest that PSA is a pivotal regulator of hepatic lipid metabolism and its antioxidant function occurs by suppressing NRF2 ubiquitination. Moreover, PSA may be a potential biomarker and therapeutic target for treating NAFLD.

GASC1 promotes hepatocellular carcinoma progression by inhibiting the degradation of ROCK2.

Hepatocellular carcinoma (HCC) is a devastating malignancy without targeted therapeutic options. Our results indicated that the histone demethylase GASC1 signature is associated with later tumor stage and poorer survival in HCC patients. GASC1 depletion led to diminished HCC proliferation and tumor growth. A distinct heterogeneity in GASC1 levels was observed among HCC cell populations, predicting their inherent high or low tumor-initiating capacity. Mechanistically, GASC1 is involved in the regulation of several components of the Rho-GTPase signaling pathway including its downstream target ROCK2. GASC1 demethylase activity ensured the transcriptional repression of FBXO42, a ROCK2 protein-ubiquitin ligase, thereby inhibiting ROCK2 degradation via K63-linked poly-ubiquitination. Treatment with the GASC1 inhibitor SD70 impaired the growth of both HCC cell lines and xenografts in mice, sensitizing them to standard-of-care chemotherapy. This work identifies GASC1 as a malignant-cell-selective target in HCC, and GASC1-specific therapeutics represent promising candidates for new treatment options to control this malignancy.

Diphenyleneiodonium enhances P2X7 dependent non-opsonized phagocytosis and suppresses inflammasome activation via blocking CX43-mediated ATP leakage.

The beneficial effects of antioxidants against oxidative stress have been well described. However, the pharmacological impacts of antioxidants other than inhibiting the production of reactive oxygen species (ROS) remain less understood. This study demonstrated that diphenyleneiodonium (DPI), a canonical NADPH oxidase 2 (NOX2) inhibitor, effectively promoted non-opsonized bacterial phagocytosis. Indeed, DPI abrogated the elevation in the extracellular ATP level of Escherichia coli (E. coli) -infected murine peritoneal macrophages, thereby restoring the association of the purinergic receptor P2X7 with non-muscle myosin heavy chain 9 (MYH9) to upregulate the P2X7 -dependent phagocytosis of E. coli. DPI also suppressed inflammasome activation and reduced necroptosis in E. coli-infected macrophages by decreasing extracellular ATP levels. Mechanistically, DPI upregulated p38 MAPK phosphorylation to suppress the expression and activity of the hemichannel protein connexin 43 (CX43), leading to the inhibition of CX43-mediated ATP efflux in E. coli-infected macrophages. In a murine E. coli infection model, DPI effectively reduced ATP release, decreased bacterial load and inhibited inflammasome activation, thereby improving survival and ameliorating organ injuries in model mice. In summary, our study demonstrates a previously unknown function of DPI in conferring protection against bacterial infection and suggests a putative antimicrobial strategy of modulating CX43 -dependent ATP leakage.

Inhibitory Activity on the Formation of Reactive Carbonyl Species in Edible Oil by Synthetic Polyphenol Antioxidants.

Food lipids play an important role in food quality, and their attributes contribute to texture, flavor, and nutrition. However, high-temperature processing leads to lipid peroxidation, degradation, and the formation of reactive carbonyl species (RCS), such as acrolein (ACR), glyoxal (GO), and methylglyoxal (MGO). We investigated the changes in the peroxidation value (POV), Rancimat induction time, formation and total amount of RCS, and inhibitory effects of synthetic polyphenol antioxidants on ACR/GO/MGO in plant oils during heating processing through an accelerated oxidation test using Rancimat. With increasing temperature and heating time, the amounts of ACR, GO, and MGO in oil increased and the level of ACR was about several times higher than that of GO and MGO. We also found that some amounts of ACR, GO, and MGO were produced at the initial stage before reaching the peak value of POV, even before oil oxidative rancidity, and the common antioxidant butyl hydroxyanisole (BHA)/butylated hydroxytoluene (BHT) could not remove them once they were generated. This is first time to purify PG-ACR-MGO and elucidate the structure based on analysis of their high resolution mass spectrometry and 1H, 13C, and two-dimensional nuclear magnetic resonance. We further found that PG rather than BHT and BHA efficiently trapped ACR, OG, and MGO to form adducts in oil and roasted beef burgers with corn oil. Additionally, after incubation at 80 °C, the trapping order of PG was as follows: ACR, MGO, and GO, and the adduct of PG-ACR was formed within 1 min; after 10 min, PG-MGO was generated; and three adducts formed at 15 min. However, PG could not trap ACR, GO, or MGO to form adducts at room temperature. This study provided novel knowledge to advance our understanding of the ability of synthetic polyphenol antioxidants to scavenge RCS simultaneously, such as ACR, MGO, and GO. Our findings demonstrated that PG, as an inhibitor of RCS, is suitable for medium- and high-temperature food processing but not for normal-temperature storage.

Gold nanoparticles synergize with bacterial lipopolysaccharide to enhance class A scavenger receptor dependent particle uptake in neutrophils and augment neutrophil extracellular traps formation.

Gold nanoparticles (AuNPs) are extensively utilized in biomedical fields. However, their potential interaction with host cells has not been comprehensively elucidated. In this study, we demonstrated a size-dependent effect of AuNPs to synergize with bacterial lipopolysaccharide (LPS) in promoting neutrophil extracellular traps (NETs) release in human peripheral neutrophils. Mechanistically, LPS was more efficient to contact with 10 nm AuNPs and promote their uptake in neutrophils compared to 40 and 100 nm AuNPs, leading to a synergistic upregulation of class A scavenger receptor (SRA) which mediated AuNPs uptake and triggered activation of extracellular regulated protein kinase (ERK) and p38. Blocking SRA or inhibiting ERK and p38 activation remarkably abrogated the effect of AuNPs and LPS to induce NETs formation. Further experiments demonstrated that AuNPs and LPS augmented the production of cytosolic reactive oxygen species (ROS) in p38 and ERK dependent manner, through upregulating and activating NADPH oxidase 2 (NOX2). Accordingly, scavenging of ROS or inhibiting the NOX2 dampened NETs release induced by combined AuNPs and LPS treatment. AuNPs and LPS also synergized to upregulate reactive oxygen species modulator 1 (ROMO1) via activating ERK, thereby increasing mitochondrial ROS generation and promoting the release of NETs. In summary, we provide new evidences about the synergy of AuNPs and LPS to augment cellular responses in neutrophils, which implicates the need to consider the amplifying effect by pathogenic stimuli when utilizing nanomaterials in infectious or inflammatory conditions.

Trapping of Acrolein by Curcumin and the Synergistic Inhibition Effect of Curcumin Combined with Quercetin.

Acrolein (ACR) is a toxic unsaturated aldehyde that is formed during different steps of thermal food processing. Here, we explored the kinetics of curcumin and ACR and elucidated the pathway of curcumin trapping ACR by preparing a mono-adduct of ACR (CMA-1) conjugated with curcumin. The synergistic scavenging effect and mechanism of curcumin combined with quercetin on ACR was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Comparing the uses of curcumin and quercetin both individually and in combination, we found that quercetin in combination resulted in more curcumin being transformed into CMA-2, while curcumin in combination made the amount of di-ACR conjugated to quercetin (QDA) increase. We also added combined curcumin and quercetin into grilled chicken wings to demonstrate that curcumin and quercetin could scavenge ACR by forming their own ACR adducts and antioxidant activity during the process. Our results have noted a new strategy, in which some combinations of dietary polyphenols might contribute to the removal of toxic ACR produced during thermal food processing.

Artesunate promotes the proliferation of neural stem/progenitor cells and alleviates Ischemia-reperfusion Injury through PI3K/Akt/FOXO-3a/p27kip1 signaling pathway.

Stroke is one of the leading causes of death worldwide that also result in long-term disability. Endogenous neural stem/progenitor cells (NSPCs) within subventricular (SVZ) and dentate gyrus (DG) zone, stimulated by cerebral infarction, can promote neural function recovery. However, the proliferation of eNSPCs triggered by ischemia is not enough to induce neural repair, which may contribute to the permanent disability in stroke patients. In this study, our results showed that following the treatment with artesunate (ART, 150 mg/kg), the functional recovery was significantly improved, the infarct volume was notably reduced, and the expression of Nestin, a proliferation marker of NSPCs in the infarcted cortex, was also increased. Additionally, the proliferative activity of NSPCs with or without oxygen-glucose deprivation/reperfusion was significantly promoted by ART treatment, and the therapeutic concentration was 0.8 µmol/L (without OGD/R) or 0.4 µmol/L (with OGD/R) in the in vitro model. Furthermore, the effects of ART can be abolished by the treatment of PI3K inhibitor wortmannin. The expression levels of related molecules in PI3K/Akt/FOXO-3a/p27kip1 signaling pathway (p-AKT, p-FOXO-3a, p27kip1) were examined using western blotting. The results suggested ART could inhibit the transcriptional function of FOXO-3a by inducing its phosphorylation, subsequently downregulating p27kip1 and enhancing neural stem cell proliferation in the infarcted cortex via PI3K/AKT signaling, further alleviating ischemia-reperfusion injury after ischemic stroke.

Quantitative Iron Neuroimaging Can Be Used to Assess the Effects of Minocycline in an Intracerebral Hemorrhage Minipig Model.

Iron-mediated toxicity is a key factor causing brain injury after intracerebral hemorrhage (ICH). This study was performed to investigate the noninvasive neuroimaging method for quantifying brain iron content using a minipig ICH model and assess the effects of minocycline treatment on ICH-induced iron overload and brain injury. The minipig ICH model was established by injecting 2 ml of autologous blood into the right basal ganglia, which were then subjected to the treatments of minocycline and vehicle. Furthermore, the quantitative susceptibility mapping (QSM) was used to quantify iron content, and diffusion tensor imaging (DTI) was performed to evaluate white matter tract. Additionally, we also performed immunohistochemistry, Western blot, iron assay, Perl's staining, brain water content, and neurological score to evaluate the iron overload and brain injury. Interestingly, we found that the ICH-induced iron overload could be accurately quantified by the QSM. Moreover, the minocycline was quite beneficial for protecting brain injury by reducing the lesion volume and brain edema, preventing brain iron accumulation, downsizing ventricle enlargement, and alleviating white matter injury and neurological deficits. In summary, we suggest that the QSM be an accurate and noninvasive method for quantifying brain iron level, and the minocycline may be a promising therapeutic agent for patients with ICH.

Scavenging of Acrolein by Food-Grade Antioxidant Propyl Gallate in a Model Reaction System and Cakes.

Reactive carbonyl species (RCS), such as acrolein (ACR), glyoxal (GO), and methylglyoxal (MGO), have received extensive attention recently as a result of their high activity and toxicity in vitro and in vivo. In the present study, propyl gallate (PG), a common food antioxidant, was found to effectively trap more ACR than butylated hydroxytoluene and butylated hydroxyanisole through the formation of mono-ACR adducts (PG-ACR) and di-ACR adducts (PG-2ACR). The two adducts were successfully purified, and their structures were elucidated on the basis of their high-resolution mass spectrometry and 1H, 13C, and two-dimensional nuclear magnetic resonance data. We further identified that PG-ACR had the ability to continue to trap GO and MGO to form PG-ACR-GO and PG-ACR-MGO, respectively, by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Furthermore, we verified that PG could inhibit the production of ACR, GO, and MGO via trapping these RCS simultaneously to form the corresponding adducts in pound cakes using LC-MS/MS.

Stimulation of the class-A scavenger receptor induces neutrophil extracellular traps (NETs) by ERK dependent NOX2 and ROMO1 activation.

Neutrophil extracellular traps (NETs) play a critical role in host antimicrobial response whereas they are also implicated in the pathogenesis of inflammatory and autoimmunediseases. Generation of reactiveoxygen species (ROS) is key to NETs formation. A variety of stimulatory ligands have been found to enhance ROS production and thus trigger NETs. However, the mechanisms that connect receptor stimuli with ROS production and NETs formation remain unclear. In this study, we described a new mechanism of NETs generation in neutrophils triggered by stimulation of the class A scavenger receptor (SRA), a major subtype of scavenger receptors in response to various stimuli during infection and inflammatory disorders. By using polyinosinic acid (Poly I), a ribonucleotide ligand of SRA, we demonstrated that SRA stimulation lead to selective ERK phosphorylation, which upregulated cytosol ROS levels and induced canonical NETs formation by activating NADPH oxidase 2 (NOX2). Interestingly, our results showed that mitochondrial ROS (mtROS) production was also enhanced by the SRA dependent ERK activation through upregulation and activation of reactive oxygen species modulator 1(ROMO1), a mitochondrial membrane protein and a key mediator of mtROS. Moreover, inhibition of the SRA elicited ROMO1 activation dampened NETs release upon SRA stimulation. Overall, our study describes a new insight into the NETs release triggered by membrane SRA stimulation and mediated by ERK dependent NOX2 and ROMO1 activation.

Levels and formation of α-dicarbonyl compounds in beverages and the preventive effects of flavonoids.

ABSTRACT: Methylglyoxal (MGO) and glyoxal (GO), α-dicarbonyl compounds found in the Maillard reaction, progressively and irreversibly modify proteins. Beverages are an exogenous source of α-dicarbonyl compounds and may potentially increase MGO and GO levels in vivo. Using GC-FID method, we detected the MGO and GO contents of 86 beverages in Chinese supermarkets. The highest MGO and GO 587.5 µg/100 mL and 716.7 µg/100 mL respectively found in soyamilk and coffee. Herbal beverages, which contained bioactive components, had lower average levels of MGO (48.1 µg/100 mL) and GO (25.9 µg/100 mL). A box-and-whisker plot was used to display variation of the same group drinks, and comparing distributions between six different groups. It was further discovered that fat, protein and flavonoids, in addition to sweeteners, had notable effects on the formation of MGO and GO in soybean milk. The result of LC/MS indicated that quercetin could prevent the formation of MGO by trapping MGO to form the mono-MGO and di-MGO adducts during soybean milk manufacturing.

The Antimalarial Chloroquine Suppresses LPS-Induced NLRP3 Inflammasome Activation and Confers Protection against Murine Endotoxic Shock.

Activation of the NLRP3 inflammasome, which catalyzes maturation of proinflammatory cytokines like IL-1ß and IL-18, is implicated and essentially involved in many kinds of inflammatory disorders. Chloroquine (CQ) is a traditional antimalarial drug and also possesses an anti-inflammatory property. In this study, we investigated whether CQ suppresses NLRP3 inflammasome activation and thereby confers protection against murine endotoxic shock. CQ attenuated NF-κB and MAPK activation and prohibited expression of IL-1ß, IL-18, and Nlrp3 in LPS treated murine bone marrow-derived macrophages (BMDMs), demonstrating its inhibitory effect on the priming signal of NLRP3 activation. Then, CQ was shown to inhibit caspase-1 activation and ASC specks formation in BMDMs, which indicates that CQ also suppresses inflammasome assembly, the second signal for NLRP3 inflammasome activation. In a murine endotoxic shock model, CQ effectively improved survival and markedly reduced IL-1ß and IL-18 production in serum, peritoneal fluid, and lung tissues. Moreover, CQ reduced protein levels of NLRP3 and caspases-1 p10 in lung homogenates of mice with endotoxic shock, which may possibly explain its anti-inflammatory activity and life protection efficacy in vivo. Overall, our results demonstrate a new role of CQ that facilitates negative regulation on NLRP3 inflammasome, which thereby confers protection against lethal endotoxic shock.

Kukoamine B promotes TLR4-independent lipopolysaccharide uptake in murine hepatocytes.

Free bacterial lipopolysaccharide (LPS) is generally removed from the bloodstream through hepatic uptake via TLR4, the LPS pattern recognition receptor, but mechanisms for internalization and clearance of conjugated LPS are less clear. Kukoamine B (KB) is a novel cationic alkaloid that interferes with LPS binding to TLR4. In this study, KB accelerated blood clearance of LPS. KB also enhanced LPS distribution in the hepatic tissues of C57 BL/6 mice, along with LPS uptake in primary hepatocytes and HepG2 cells. By contrast, KB inhibited LPS internalization in Kupffer and RAW 264.7 cells. Loss of TLR4 did not affect LPS uptake into KB-treated hepatocytes. We also detected selective upregulation of the asialoglycoprotein receptor (ASGPR) upon KB treatment, and ASGPR colocalized with KB in cultured hepatocytes. Molecular docking showed that KB bound to ASGPR in a manner similar to GalNAc, a known ASGPR agonist. GalNAc dose-dependently reduced KB internalization, suggesting it competes with KB for ASGPR binding, and ASGPR knockdown also impaired LPS uptake into hepatocytes. Finally, while KB enhanced LPS uptake, it was protective against LPS-induced inflammation and hepatocyte injury. Our study provides a new mechanism for conjugated LPS hepatic uptake induced by the LPS neutralizer KB and mediated by membrane ASGPR binding.

Consistency and pathophysiological characterization of a rat polymicrobial sepsis model via the improved cecal ligation and puncture surgery.

Sepsis is the leading cause of death for critical ill patients and an essential focus in immunopharmacological research. The cecal ligation and puncture (CLP) model is regarded as a golden standard model for sepsis study. However, this animal model is easily affected by variability problems and dramatically affects pharmacological evaluation of anti-sepsis therapies, which requires standardized procedures and stable outcomes. Herein, the traditional syringe needle based puncture method was used as the major unstable factor for CLP models. Syringe needles created varied mortality in parallel experimental groups of CLP rats; they were inconsistent for severity control as mortality in CLP rats was not correlated with change in punctures, ligation lengths, or needle sizes. Moreover, the use of drainage tubes or strips, which was supposed to strengthen drainage stability, also failed to improve consistency of traditional syringe needles. To solve the consistency problem, an improved design of CLP surgery by puncture with newly-developed three-edged needles was described herein. In contrast to traditional syringe needles, these three-edged needles ensured more stable outcomes in repetitive groups. Furthermore, increased severity was found to be consistent with the enlarged needle size, as shown by the elevated mortality, increased proinflammatory cytokines, abnormal coagulation, worsen acidosis and more severe acute lung injury. In conclusion, application of the newly-developed three-edged needles provides a simple and feasible method to improve stability when conducting CLP surgery, which is significant for pharmacological studies on sepsis.