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.

The roles of serine protease, intracellular and extracellular phenoloxidase in activation of prophenoloxidase system, and characterization of phenoloxidase from shrimp haemocytes induced by lipopolysaccharide or dopamine

We investigated the effects of lipopolysaccharide （LPS） and dopamine （DA） on the activation of the prophenoloxidase （proPO） system of Litopenaeus vannamei. LPS and DA were shown with a negative dose-dependent effect on hyalne cells （HC）, semi-granular cells （SGC）, large granular cells （LGC）, and total haemocyte count （THC）. When haemocytes were treated with LPS or DA, serine proteinase activity and intracellular phenoloxidase （PO） activity were significantly reduced, but extracellular PO activity increased significantly. These findings indicated that the reduction in haemocyte counts was mainly because of the degranulation and activation of the proPO system from semi-granule and large granule cells. The PKC inhibitor, chelerythrine, and the TPK inhibitor, genistein, had an inhibitory effect on extracellular PO activity, while serine proteinase and intracellular PO activity increased. This suggests that the LPS and DA induce the activation of proPO in haemocytes via PKC and TPK-related signaling pathways, but serine proteinase may be activated only by PKC, as the genistein effects were not statistically significant. Electrophoresis analysis revealed that POs induced by LPS or DA have the same molecular mass and high diphenolase activity. Two PO bands at 526 kDa and 272 kDa were observed in PAGE, while in the haemocyte lysate supematant （HLS）, only a 272-kDa band was observed. This band was resolved after SDS-PAGE under non-reducing and reducing conditions into two groups of POs, 166 kDa and 126 kDa, and 78.1 kDa and 73.6 kDa, respectively, suggesting that PO in L. vannamei is an oligomer, which may have different compositions intra- and extracellularly.

The therapeutic mechanism of Shenyuan Gan in lipopolysaccharide-induced neuroinflammation in BV2 microglial cells

Objective To study the therapeutic effects of Shenyuan Gan(参远苷,SYG)on the inflammat-ory response in BV2 microglial cells induced by lipopolysaccharide(LPS).Methods The cytotoxicity of SYG to BV2 microglial cells was evaluated using a Cell Counting Kit-8(CCK-8)assay,and the effect of SYG concentrations on LPS-induced BV2 microglial cells was studied.The morphological changes were observed using an optical microscope.The nitric oxide(NO)concentration in cell culture supernatant was determined using Griess re-agent.The expression of cytokines and inflammatory mediators were also measured by an en-zyme-linked immunosorbent assay(ELISA).Western blot analysis was used to determine the levels of inducible NO synthase(iNOS),nuclear factor-kappa B(NF-κB)p65,alpha inhibitor of NF-κB(IκB-α),phosphorylation-IκB-α(p-IκB-α),NOD-like receptor 3(NLRP3),and cas-pase-1 expression.Moreover,the expression of iNOS,NLRP3,and ionized calcium binding adapter molecule 1(Iba1)was also observed using immunofluorescent staining.Results SYG had a low cytotoxic effect on BV2 microglial cells and could significantly decr-ease LPS-induced morphological changes of BV2 microglial cells(P<0.05).ELISA results showed that SYG significantly inhibited the LPS-induced increase in interleukin(IL)-1βand IL-6 in BV2 microglia cells(P<0.05),and Western blot analysis showed that the phosphoryla-tion levels of iNOS,NF-κB p65,and IκB-αas well as NLRP3 and caspase-1 expression were also significantly decreased,and IκB-αexpression was increased after SYG treatment(P<0.05,compared with the LPS-treated group).The immunofluorescence results were consist-ent with the Western blot results,and Iba1 staining indicated that the cell morphology tended to be resting.These results indicate that SYG has a certain inhibitory effect on LPS-induced inflammation in BV2 microglial cells.Conclusion SYG can inhibit LPS-induced release of inflammatory factors in BV2 microglial cells by affecting the phosphorylation levels of NF-κB p65 and IκB-α.SYG is a valuable candid-ate for treating neuroinflammation-related diseases.

LIPOPOLYSACCHARIDE－INDUCED CYTOTOXICITY AGAINST CULTURED MOUSE HEPATOCYTES AND THE ROLE OF NONPARENCHYMAL LIVER CELLS

Lipopolysaccharide (LPS) was found to induce significant hepatocytotoxicity against cultured mouse hepatocytes. Degeneration and necrosis of cultured hepatocytes and decrease of hepatocyte viability were prominent. The aspartate transferase level and 3H-TdR release in the medium were significantly increased after treatment, and the degree of these changes paralleled with LPS concentration. Various other parameters showed no significant difference between the hepatocytes cultured alone and those cocultured with nonparenchymal liver cells. However, if the nonparenchymal liver cells were isolated from mice which had been pretreated with D-galactosamine (GalN) not only was the hepatocyotoxicity induced by LPS enhanced, but the cells also showed certain cytotoxicity against cultured hepatocytes even without LPS, These results suggest that nonparenchymal liver cells might promote LPL-induced hepatocyte injury.

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.

Immunomodulatory effect of pachymaran on cyclosporine A(CsA)-induced lung injury in mice

Objective To investigate the immunomodulatory effect of pachymaran on cyclosporine A(CsA)-induced lung injury in mice.Methods(i) Fifty male BALB/c mice were randomly divided into five groups(10 mice in each group): normal control(NC) group, 30, 45, and 60 mg/kg CsA groups, and lipopolysaccharide(LPS) group. Except for the NC group, other groups underwent CsA modeling. The NC group was treated with phosphate-buffered saline(PBS), the LPS group with 10 mg/kg LPS eight hours before mice euthanized, and the 30, 45, and 60 mg/kg CsA groups with corresponding doses of CsA for seven consecutive days. After treatment, the body and organ mass of each group were weighed, and the lung, thymus, and spleen indexes were calculated. Hematoxylin-Eosin(HE) staining was performed to observe histopathological changes in the lungs of the mice. The protein expression levels of interleukin(IL)-2 and IL-1β in the blood were detected using enzyme-linked immunosorbent assay(ELISA), and those of surfactant protein D(SP-D), IL-2, and IL-6 in lung tissues were detected by immunohistochemistry(IHC). The mRNA expression levels of SP-D, IL-1β, IL-6, and myeloperoxidase(MPO) in the lung tissues were detected by quantitative reverse transcriptase-polymerase chain reaction(q RT-PCR).(ii) Another 60 BALB/c mice were divided into six groups(10 mice in each group) : NC group,model control(MC) group, 50, 100, and 200 mg/kg pachymaran groups, and polyinosinicpolycytidylic acid [poly(I:C)] group. Except for the NC group, other groups underwent45 mg/kg CsA modeling. The NC and MC groups were treated with distilled water, the pachymaran groups with corresponding doses pachymaran, and the poly(I:C) group with 0.1 mg/kg poly(I:C) for seven days.The mice were euthanized to obtain tissues and serum for detection.Detection methods were identical to those described in(i) above.Results(i) CsA(30 mg/kg) increased the lung index of mice(P < 0.001), and decreased the spleen index(P < 0.01), thymus index(P < 0.05), and the serum level of IL-2(P < 0.05). CsA(45 mg/kg) decreased the spleen, thymus indexes, and the serum level of IL-2(P < 0.01) in mice, and increased the serum level of IL-1β(P < 0.05) and the protein level of lung SP-D(P <0.001). CsA(60 mg/kg) increased the lung index of mice(P < 0.01), the serum level of IL-1β(P < 0.05), the protein level of lung SP-D(P < 0.01), and the mRNA levels of lung MPO and SP-D( P < 0.05), and decreased the thymus index of mice(P < 0.01). HE staining showed that 30, 45, and60 mg/kg CsA, and LPS caused pathological changes in the lung tissue of mice.(ii) After pachymaran intervention in MC mice, the spleen and thymus indexes(P < 0.05) were increased in the 100 and 200 mg/kg pachymaran groups, and the lung index was decreased(P < 0.05).Moreover, 50 mg/kg pachymaran increased the thymus index(P < 0.05) and decreased the lung index(P < 0.01) in MC group. Pachymaran(50, 100, and 200 mg/kg) improved lung tissue injury, reduced the serum level of IL-1β(P < 0.001), and the mRNA levels of MPO and SPD in lung tissues(P < 0.05) of mice. Pachymaran(100 mg/kg) increased the protein level of lung IL-2(P < 0.01), decreased the protein level of lung SP-D(P < 0.01), and the mRNA level of IL-1β(P < 0.001) in the lung tissues of mice. Pachymaran(200 mg/kg) increased the serum level of IL-2(P < 0.01) and lung IL-6 of mice(P < 0.05). Pachymaran(50 and 200 mg/kg) increased the mRNA level of IL-6 in the lung tissues of mice(P < 0.05).Conclusion While the immune function of mice was suppressed by CsA, the lung tissue was also damaged. Pachymaran can improve the immunosuppression induced by CsA and improve the lung tissue injury in immunosuppressed mice.

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.

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.

Antepenultimate residue at the C-terminus of NADPH oxidase RBOHD is critical for its function in the production of reactive oxygen species in Arabidopsis

Production of reactive oxygen species(ROS)is a conserved immune response primarily mediated by NADPH oxidases(NOXs),also known in plants as respiratory burst oxidase homologs(RBOHs).Most microbe-associated molecular patterns(MAMPs)trigger a very fast and transient ROS burst in plants.However,recently,we found that lipopolysaccharides(LPS),a typical bacterial MAMP,triggered a biphasic ROS burst.In this study,we isolated mutants defective in LPS-triggered biphasic ROS burst(delt)in Arabidopsis,and cloned the DELT1 gene that was shown to encode RBOHD.In the delt1-2 allele,the antepenultimate residue,glutamic acid(E919),at the C-terminus of RBOHD was mutated to lysine(K).E919 is a highly conserved residue in NADPH oxidases,and a mutation of the corresponding residue E568 in human NOX2 has been reported to be one of the causes of chronic granulomatous disease.Consistently,we found that residue E919 was indispensable for RBOHD function in the MAMP-induced ROS burst and stomatal closure.It has been suggested that the mutation of this residue in other NADPH oxidases impairs the protein’s stability and complex assembly.However,we found that the E919K mutation did not affect RBOHD protein abundance or the ability of protein association,suggesting that the residue E919 in RBOHD might have a regulatory mechanism different from that of other NOXs.Taken together,our results confirm that the antepenultimate residue E is critical for NADPH oxidases and provide a new insight into the regulatory mechanisms of RBOHD.

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.

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.