Lipopolysaccharide “Two-hit” Induced Refractory Hypoxemia Acute Respiratory Distress Model in Rats

To establish a stable and reliable model of refractory hypoxemia acute respiratory distress syndrome （ARDS） and examine its pathological mechanisms, a total of 144 healthy male Wistar rats were randomized into 4 groups： group Ⅰ （saline control group）, group Ⅱ （LPS intravenous ＂single-hit＂ group）, group Ⅲ （LPS intratracheal ＂single-hit＂ group） and Group IV （LPS ＂two-hit＂ group）. Rats were intravenously injected or intratracheally instilled with a large dose of LPS （10 mg/kg in 0.5 mL） to simulate a single attack of ARDS, or intraperitoneally injected with a small dose of LPS （1 mg/kg） followed by tracheal instillation with median dose of LPS （5 mg/kg） to establish a ＂two-hit＂ model. Rats in each group were monitored by arterial blood gas analysis and visual inspection for three consecutive days. Arterial blood gas values, lung wet/dry weight ratio and pathological pulmonary changes were analyzed to determine the effects of each ALI/ARDS model. Concentrations of TNF-α, IL-1 and IL-10 in the bronchoalveolar lavage fluid （BALF） and blood plasma were meastired by using enzyme-linked immunosorbent assays （ELISA）. Our resulsts showed that single LPS-stimulation, whether through intravenous injection or tracheal instillation, could only induce ALl and temporary hypoxemia in rats. A two-hit LPS stimulation induces prolonged hypoxemia and specific pulmonary injury in rats, and is therefore a more ideal approximation of ARDS in the animal model. The pathogenesis of LPS two-hit-induced ARDS is associated with an uncontrolled systemic inflammatory response and inflammatory injury. It is concluded that the rat ARDS model produced by our LPS two-hit method is more stable and reliable than previous models, and closer to the diagnostic criteria of ARDS, and better mimics the pathological process of ARDS.

Effect of Radix Isatidis on the Expression of Moesin mRNA Induced by LPS in the Tissues of Mice

To investigate the effect of the anti-endotoxic part of Radix Isatidis on the expression of moesin mRNA in murine tissues induced by lipopolysaccharide （LPS）, the sample solution of F0z2 part from Radix Isatidis was intrapefitoneally administered to experimental mice, and the lipopolysaccharide （LPS） were injected into the tail vein, and then the tissues of liver, kidney and spleen were colleted and cut into slices. The mRNA was detected by moesin mRNA hybridization in situ. The staining results were observed under microscope. It was found that moesin mRNA expression was increased in the tissues of liver, kidndy and spleen in mice treated with LPS, while in the mice pre-treated with F022 part from Radix Isatidis, the LPS-induced moesin mRNA expressions in these tissues were inhibited in a dose-dependant manner. Our study showed that F022 part from Radix Isatidis can inhibit the LPS-induced expression of moesin mRNA in the tissues of liver, kidney and spleen in mice.

Lipopolysaccharides in Cyanobacteria: A Brief Overview

Cyanobacteria are an interesting group of photosynthetic prokaryotes with a great potential in drug discovery and scientific research. Due to their high degree of diversification, they have been able to adapt to almost all ecological niches. Similarly to Gram-negative bacteria, cyanobacterial cell wall contains Lipopolysaccharides (LPSs) in the outer membrane layer. LPSs are molecules that possess the ability to elicit an innate immune response via Toll-like receptor 4 (TLR-4) activation. Cyanobacterial LPSs have been studied to a minor extent compared to Gram-negative bacterial LPSs. However, available data revealed important differences between the LPSs of these two groups of organisms, both in term of structure and biological activity. This review summarizes the current knowledge about cyanobacterial LPSs, highlighting their peculiarity and their potentiality compared to more characterized bacterial LPSs.

Ca^(2+)-亚氨二醋酸金属离子亲和色谱法吸附内毒素(英文)

Endotoxins(also known as lipopolysaccharides(LPS)) are undesirable by-products of recombinant proteins,purified from Escherichia coli.LPS can be considered stable under a wide range of temperature and pH,making their removal one of the most difficult tasks in downstream processes during protein purification.The inherent toxicity of LPS makes their removal an important step for the application of these proteins in several biological assays and for a safe parenteral administration.Immobilized metal affinity chromatography(IMAC) enables the affinity interactions between the metal ions(immobilized on the support through the chelating compound) and the target molecules,thus enabling high-efficiency separation of the target molecules from other components present in a mixture.Affinity chromatography is applied with Ca2+-iminodiacetic acid(IDA) to remove most of the LPS contaminants from the end product(more than90%).In this study,the adsorption of LPS on an IDA-Ca2+ was investigated.The adsorption Freundlich isotherm of LPS-IDA-Ca2+ provides a theoretical basis for LPS removal.It was found that LPS is bound mainly by interactions between the phosphate group in LPS and Ca2+ ligands on the beads.The factors such as pH(4.0 or 5.5) and ionic strength(1.0 mol/L) are essential to obtain effective removal of LPS for contaminant levels between endotoxin' concentration values less than100 EU/mL and 100 000 EU/mL.This new protocol represents a substantial advantage in time,effort,and production costs.

Nuclear Factor kappa B p65 Expression in Mouse Cochlea

Nuclear factor kappa B(NF-κB) is one of the best-characterized transcription factors playing important roles in many cellular responses to a large variety of stimuli,including inflammatory cytokines, phorbol esters, growth factors, and bacterial and viral products. The aim of this study is to demonstrate NF-κB expression in the mouse cochlea and its enhancement in response to lipopolysaccharides(LPS) and kanamycin(KA) treatment. Methods KA treatment consisted of subcutaneous KA injections at 700 mg/kg twice a day with an eight-hour interval between the two injections for 3 or 7 days. For animals in the LPS treatment group, a single dose of 0.3 mg LPS dissolved in 0.2 ml sterile saline were injected into both bullae through the tympanic membrane and kept there for 3 hours. Animals in the control group received subcutaneous saline injection for 7 days. Following immmunohistochemichal processing with rabbit polyclonal anti-NF-κB p65 antibodies, cryosections of the cochlea were examined for expression of NF-κB p65 in various structures in the cochlea. Results NF-κB p65 expression, identified by presence of brown reaction products characteristic of DAB immunohistochemistry, was visible in the spiral ligament, spiral prominence, tectorial membrane(TM), spiral ganglion and nerve fibers. Relatively weak NF-κB p65 expression was also visualized in the organ of Corti. Within the organ of Corti, the inner hair cells(IHC), outer hair cells(OHC), inner pillar cells(IP), outer pillar cells (OP), Deiter’s cells(DC), and Boettcher’s cells exhibited stronger staining than the inner sulcus cells, Hensen’s cells(HC) and Claudius’cells. No NF-κB p65 expression was seen in the nucleus of the IHC and OHC. NF-κB p65 expression was increased in animals exposed to LPS or KA, demonstrating significant differences in the staining between control animals and LPS/KA-treated animals. NF-κB p65 expression was not significantly different between LPS treated and KA treated animals or between 3 and 7 days in KA-treated animals. Conclusion LPS and KA exposure increases expression of NF-κB p65 in the mouse cochlea.

Study on the protective effect of icariin in different concentrations on the inflammatory response of osteoblasts in fetal rats

Objective:To verify whether icariin has drug toxicity to osteoblasts and its pre protective effect on inflammatory osteoblasts induced by lipopolysaccharide.Methods:(1)The osteoblasts of newborn SD rats were extracted,cultured and passaged.By observing the morphology of osteoblasts and ALP staining,the activity and proliferation of osteoblasts were determined.(2)CCK-8 method was used to observe the effect of Icariin on osteoblast activity.(3)Different concentrations of lipopolysaccharide(LPS)were used to induce inflammatory osteoblasts in fetal rats.CCK-8 method was used to select the best concentration for induction.(4)This experiment was divided into control group,low concentration group,middle concentration group and high concentration group.CCK-8 method was used to observe whether icariin could protect osteoblasts from inflammatory reaction.Results:(1)The number of osteoblasts in the third generation increased,the shape of osteoblasts overlapped like tiles,most of osteoblasts grew in the center,the center was dense and the specific shape was difficult to see.After ALP staining,the positive cells showed gray black granules in cytoplasm and irregular cell body shape under inverted microscope;(2)DWhen the concentration of icariin was lower than 1μg/ml,it had no significant effect on osteoblasts(P<0.05);when it was higher than 10μg/ml,icariin had no significant effect on osteoblasts(P<0.05);(3)When the concentration of lipopolysaccharide was higher than 80μg/ml,there was a significant trend of inflammatory damage to osteoblasts,which was statistically significant(P<0.01).Therefore,80μg/ml was selected as the best injury concentration in this experiment;(4)When the concentration of icariin was lower than 1μg/ml,there was no significant pre protective effect on the inflammatory response of osteoblasts(P<0.05);when it was higher than 10μg/ml,there was significant pre protective effect on the inflammatory response of osteoblasts(P<0.05).Conclusion:When the concentration of icariin reaches a certain level,it can promote the proliferation of osteoblasts and has a certain pre protective effect on inflammatory osteoblasts.

Antipyretic Effect and Action Mechanism of Jinzhen Oral Liquid

[Objectives]To investigate the antipyretic effect and action mechanism of Jinzhen Oral Liquid.[Methods]The yeast-induced fever model was used to determine the rectal temperature of rats at 6,8,and 10 h after yeast injection.The contents of IL-6 and TNF-αin the rat serum and the contents of 5-HT and NE in the hypothalamus were detected by radioimmunoassay;the model of yeast-induced fever in rat was established by intraperitoneal injection of lipopolysaccharide(LPS),rectal temperature of the rats was determined,the contents of TNF-αand IL-1βin serum,cyclic adenosine monophosphate(cAMP)and prostaglandin E2(PGE2)in hypothalamus were detected by radioimmunoassay.[Results]After the intervention of Jinzhen Oral Liquid,the body temperature of rats in the yeast-induced fever model was significantly reduced,the IL-6 and TNF-αcontents in serum were significantly reduced,the 5-HT content in the hypothalamus was significantly reduced,while the NE content was significantly increased;after 2 h of intraperitoneal injection of LPS,the body temperature of the model rats increased significantly;after the intervention of Jinzhen Oral Liquid,the increase in rectal temperature of LPS-induced fever model rats decreased,and the TNF-αand IL-1βcontents in serum and cAMP and PGE2 contents in hypothalamus of model rats were significantly decreased.[Conclusions]Jinzhen Oral Liquid has a significant antipyretic effect,and its action mechanism may be related to reducing the inflammatory factors in the serum,thereby inhibiting the generation of pyrogenic media in the hypothalamus.

Regulation Mechanism of TFP on TGF-β1/STAT3 Signaling Pathway in Immune-mediated Liver Injury in Mice

[Objectives]To study the effect of total flavonoids extracted from Polygonum perfoliatum L.(TFP)on immune-mediated liver injury induced by bacillus Calmette-Guerin plus lipopolysaccharide(BCG+LPS)in mice,and to explore its action mechanism.[Methods]60 Kunming mice were divided into normal group,model group,control group(bifendate)and TFP low,medium and high dose groups according to random number table method,with 10 mice in each group.On the first day of modeling,mice were injected with 0.2 mL of BCG solution(12.5 mg/mL)through the tail vein,and on the eleventh day,0.2 mL of LPS(37.5μg/mL)were injected into the tail vein to prepare a mouse model of immune-mediated liver injury;from the first day of modeling,the normal group and the model group were administered intragastrically with the corresponding volume of distilled water,and the bifendate group and the TFP high,medium,and low dose groups were administered intragastrically with the corresponding doses once a day for 11 d.After the last time administration,fasting but giving water for 16 h,took blood from eyes,then collected the liver tissue.The levels of alanine transaminase(ALT)and aspartate transaminase(AST)in serum were detected by biochemical method;transforming growth factor-β1(TGF-β1),intercellular adhesion molecule-1(ICAM-1),interleukin-6(IL-6)and interleukin-1β(IL-1β)expression levels in liver tissue were detected by enzyme-linked immunosorbent assay(ELISA);phosphorylated protein tyrosine kinase JAK-2(p-JAK2),phosphorylated signal transducer and activator of transcription 3(p-STAT3)protein expression levels were detected by Western Blot method;the degree of liver tissue lesions was detected by HE staining.[Results]Compared with the model group,the levels of ALT and AST in the serum of mice in each dose group of TFP(high dose 600 mg/kg,medium dose 400 mg/kg,and low dose 200 mg/kg)were reduced,and the activities of T-SOD and GSH-Px were increased;the content or expression ofβ1,ICAM-1,IL-6,IL-1βdecreased,and the expression of p-JAK2 and p-STAT3 protein decreased;pathological sections showed that the degree of inflammatory necrosis and the degree of lesions in the liver tissues of each dose group of TFP were reduced by varying degrees.[Conclusions]TFP has a protective effect on BCG+LPS-induced immune-mediated liver injury in mice.The mechanism may be related to regulating the phosphorylation level of JAK2 and inhibiting the inflammatory reaction,thereby regulating the TGF-β1/STAT3 signaling pathway and improving the immune-mediated liver injury.

Anti-inflammatory effects of Abelmoschus manihot(L.)Medik.on LPS-induced cystitis in mice:potential candidate for cystitis treatment based on classic use

Abelmoschus manihot(L.)Medik.(A.manihot)is a traditional Chinese herbal medicine with a variety of pharmacological properties.It was first recorded in Jiayou Materia Medica dating back to the Song dynasty to eliminate urinary tract irritation by clearing away heat and diuretic effect.However,its pharmacological action on urinary tract infections has not been investigated.The present study aims to evaluate the anti-inflammatory activity of A.manihot on a mouse model of lipopolysaccharide(LPS)-induced cystitis.The results showed that A.manihot decreased white blood cell(WBC)count in urine sediments of the cystitis mice,alleviated bladder congestion,edema,as well as histopathological damage,reduced the expression levels of tumor necrosis factor-α,interleukin-6,and interleukin-1βsimultaneously.Moreover,A.manihot administration significantly downregulated the expression levels of TLR4,MYD88,IκBα,p-IκBα,NF-κB p65,and p-NF-κB p65 in LPS-induced cystitis mice.These findings demonstrated the protective effect of A.manihot against LPS-induced cystitis,which is attributed to its anti-inflammatory profile by suppressing TLR4/MYD88/NF-κB pathways.Our results suggest that A.manihot could be a potential candidate for cystitis treatment.

Effect of Palrnatine on lipopolysaccharide-induced acute lung injury by inhibiting activation of the Akt/NF-κB pathway

Inflammation plays an important role in the development of acute lung injury(ALI).Severe pulmonary inflammation can cause acute respiratory distress syndrome(ARDS)or even death.Expression of proinflammatory interleukin-1β(IL-1β)and inducible nitric oxide synthase(iNOS)in the process of pulmonary inflammation will further exacerbate the severity of ALI.The purpose of this study was to explore the effect of Palrnatine(Pa)on lipopolysaccharide(LPS)-induced mouse ALI and its underlying mechanism.Pa,a natural product,has a wide range of pharmacological activities with the potential to protect against lung injury.Western blotting and quantitative real-time polymerase chain reaction(qRT-PCR)assays were performed to detect the expression and translation of inflammatory genes and proteins in vitro and in vivo.Immunoprecipitation was used to detect the degree of P65 translocation into the nucleus.We also used molecular modeling to further clarify the mechanism of action.The results showed that Pa pretreatment could significantly inhibit the expression and secretion of the inflammatory cytokine IL-1β,and significantly reduce the protein level of the proinflammatory protease iNOS,in both in vivo and in vitro models induced by LPS.Further mechanism studies showed that Pa could significantly inhibit the activation of the protein kinase B(Akt)/nuclear factor-κB(NF-κB)signaling pathway in the LPS-induced ALI mode and in LPS-induced RAW264.7 cells.Through molecular dynamics simulation,we observed that Pa was bound to the catalytic pocket of Akt and effectively inhibited the biological activity of Akt.These results indicated that Pa significantly relieves LPS-induced ALI by activating the Akt/NF-κB signaling pathway.

Differentially Gene Expression Profile Related to Inflammation in Endometrial Cells Induce by Lipopolysaccharide

Objective To investigate differentially expressed genes related to inflammation in endometrial cells induced by Lipopolysaccharide（LPS）. Methods Normal endometrium in the proliferative phase of specimen from 3 cases for the experiment was collected. The LPS group were treated with 50 μg/ml LPS. Total RNA was extracted using Trizol reagent from cells. RNA quality was assessed by determining the OD260/280 ratio by agarose gel electrophoresis, the chip was scanned by laser scanner. The acquired was analyzed. Results A total of differentially expressed genes were found, these genes were relative to many aspects. Among them, the expression of genes involved in inflammation were up-regulated by LPS, such as overexpression of IL-1β, 8, etc. Conclusion The results indicates that inflammation-related genes may be one of the mechanisms of abnormal uterine bleeding by LPS-induced.

New insights in the pathogenesis of alcohol-related liver disease:The metabolic,immunologic,and neurologic pathways

Alcohol-related liver disease(ALD)became an important health issue worldwide.Following chronic alcohol consumption,the development of ALD might be caused by metabolic and immunologic factors,such as reactive oxygen species(ROS)and pro-inflammatory cytokines.For example,hepatic cytochrome P4502E1 enzyme increases ROS production and stimulates de novo lipogenesis after alcohol exposure.In addition,damage-and pathogen-associated molecular patterns stimulate their specific receptors in nonparenchymal cells,including Kupffer cells,hepatic stellate cells(HSCs),and lymphocytes,which result in hepatocyte death and infiltration of pro-inflammatory cells(e.g.,neutrophils and macrophages)in the liver.Moreover,our studies have suggested the novel involvement of neurologic signaling pathways(e.g.,endocannabinoid and glutamate)through the metabolic synapse between hepatocytes and HSCs in the development of alcohol-related hepatic steatosis.Additionally,agouti-related protein and beta2-adrenergic receptors aggravate hepatic steatosis.Furthermore,organ-crosstalk has emerged as a critical issue in ALD.Chronic alcohol consumption induces dysbiosis and barrier disruption in the gut,leading to endotoxin leakage into the portal circulation,or lipolysis-mediated transport of triglycerides from the adipose tissue to the liver.In summary,this review addresses multiple pathogeneses of ALD,provides novel neurologic signaling pathways,and emphasizes the importance of organ-crosstalk in the development of ALD.

HMGB1 is a critical molecule in the pathogenesis of Gram-negative sepsis

Gram-negative sepsis is a severe clinical syndrome associated with significant morbidity and mortality.Lipopolysaccharide(LPS),expressed on Gram-negative bacteria,is a potent pro-inflammatory toxin that induces inflammation and coagulation via two separate receptor systems.One is Toll-like receptor 4(TLR4),expressed on cell surfaces and in endosomes,and the other is the cytosolic receptor caspase-11(caspases-4 and-5 in hu-mans).Extracellular LPS binds to high mobility group box 1(HMGB1)protein,a cytokine-like molecule.The HMGB1-LPS complex is transported via receptor for advanced glycated end products(RAGE)-endocytosis to the endolysosomal system to reach the cytosolic LPS receptor caspase-11 to induce HMGB1 release,inflammation,and coagulation that may cause multi-organ failure.The insight that LPS needs HMGB1 assistance to generate severe inflammation has led to successful therapeutic results in preclinical Gram-negative sepsis studies target-ing HMGB1.However,to date,no clinical studies have been performed based on this strategy.HMGB1 is also actively released by peripheral sensory nerves and this mechanism is fundamental for the initiation and prop-agation of inflammation during tissue injury.Homeostasis is achieved when other neurons actively restrict the inflammatory response via monitoring by the central nervous system and the vagus nerve through the cholinergic anti-inflammatory pathway.The neuronal control in Gram-negative sepsis needs further studies since a deeper understanding of the interplay between HMGB1 and acetylcholine may have beneficial therapeutic implications.Herein,we review the synergistic overlapping mechanisms of LPS and HMGB1 and discuss future treatment opportunities in Gram-negative sepsis.

Intestinal barrier function and metabolic/liver diseases

Dietary,environmental or genetic changes contribute to the development of several metabolic and neurological diseases.Extensive research efforts have been directed towards examining how these fac-tors modulate gut bacterial composition.However,the mechanisms by which changes in gut bacterial diversity affect host function are not completely defined.Intestinal barrier dysfunction is being increasingly recognized as an early or initiating step in communicating bacterial diversity-dependent changes to the host.Here,we review the functional composition of the intestinal barrier and describe the consequences of the breach of this barrier.The functional layers of the intestinal barrier provide an initial defense and prevent the infiltration of bacteria or bacterial products from the gut lumen to the underlying lamina propria.Mesenteric lymph nodes subsequently act as the first firewall where dendritic cells from the lamina propria transport the infiltrated bacteria or bacterial products during the early stages of barrier dysfunction.When overwhelmed,the liver functions as the second firewall to detoxify bacterial endotoxins,such as lipopolysaccharide(LPS),and limit systemic involvement.Continuous translocation of LPS from the leaky gut to the liver results in liver damage or dysfunction that leads to the development of type 2 diabetes,atherosclerosis,heart disease,heart failure and also neurological dis-eases,such as Alzheimer’s and Parkinson’s disease.Thus,targeted restoration of intestinal barrier function represents a novel strategy for the attenuation of multiple diseases.

Liver tissue microbiota in nonalcoholic liver disease:a change in the paradigm of host-bacterial interactions

Nonalcoholic fatty liver disease(NAFLD)pathogenesis is explained by the complex relationship among diet and lifestyle-predisposing factors,the genetic variance of the nuclear and mitochondrial genome,associated phenotypic traits,and the yet not fully explored interactions with epigenetic and other environmental factors,including the microbiome.Despite the wealth of knowledge gained from molecular and genome-wide investigations in patients with NAFLD,the precise mechanisms that explain the variability of the histological phenotypes are not fully understood.Earlier studies of the gut microbiota in patients with NAFLD and nonalcoholic steatohepatitis(NASH)provided clues on the role of the fecal microbiome in the disease pathogenesis.Nevertheless,the composition of the gut microbiota does not fully explain tissue-specific mechanisms associated with the degree of disease severity,including liver inflammation,ballooning of hepatocytes,and fibrosis.The liver acts as a key filtration system of the whole body by receiving blood from the hepatic artery and the portal vein.Therefore,not only microbes would become entrapped in the complex liver anatomy but,more importantly,bacterial derived products that are likely to be potentially powerful stimuli for initiating the inflammatory response.Hence,the study of liver tissue microbiota offers the opportunity of changing the paradigm of host-NAFLD-microbial interactions from a“gut-centric”to a“liver-centric”approach.Here,we highlight the evidence on the role of liver tissue bacterial DNA in the biology of NAFLD and NASH.Besides,we provide evidence of metagenomic findings that can serve as the seed of further hypothesis-raising studies as well as can be leveraged to discover novel therapeutic targets.

Inflammation and intestinal leakiness in older HIVD individuals with fish oil treatment

Fish oil is a natural product that has shown efficacy for managing inflammatory conditions with few side effects.There is emerging evidence that crosstalks between gut epithelial cells and immune cells contribute to chronic infectious diseases.HIV-infected(HIVt)older adults show age-related co-morbidities at a younger age than their uninfected counterparts.Persistent inflammation related to the chronic viral infection and its sequelae is thought to contribute to this disparity.However,little is known about whether fish oil reduces intestinal inflammation in HIV t patients.We measure inflammation and gut barrier function in HIV t older adults(median age Z 52,N Z 33),following 12 weeks of fish oil supplementation(a total daily dose of 1.6 g of omega-3 fatty acids).We showed a reduction in inflammation and gut permeability as measured by CD14,inflammatory cytokines,lipopolysaccharide,and lipopolysaccharide binding protein.The results indicate that older HIV t adults may benefit from a diet supplemented with the omega-3 fatty acids found in fish oil.