Therapeutic Targeting of PKM2 Ameliorates NASH Fibrosis Progression in a Macrophage-Specific and Liver-Specific Manner

Nonalcoholic steatohepatitis(NASH)may soon become the leading cause of end-stage liver disease worldwide with limited treatment options.Liver fibrosis,which is driven by chronic inflammation and hepatic stellate cell(HSC)activation,critically determines morbidity and mortality in patients with NASH.Pyruvate kinase M2(PKM2)is involved in immune activation and inflammatory liver diseases;however,its role and therapeutic potential in NASH-related fibrosis remain largely unexplored.Bioinformatics screening and analysis of human and murine NASH livers indicated that PKM2 was upregulated in nonparenchymal cells(NPCs),especially macrophages,in the livers of patients with fibrotic NASH.Macrophage-specific PKM2 knockout(PKM2^(FL/FL)LysM-Cre)significantly ameliorated hepatic inflammation and fibrosis severity in three distinct NASH models induced by a methionine-and choline-deficient(MCD)diet,a high-fat high-cholesterol(HFHC)diet,and a western diet plus weekly carbon tetrachloride injection(WD/CCl_(4)).Single-cell transcriptomic analysis indicated that deletion of PKM2 in macrophages reduced profibrotic Ly6C^(high) macrophage infiltration.Mechanistically,PKM2-dependent glycolysis promoted NLR family pyrin domain containing 3(NLRP3)activation in proinflammatory macrophages,which induced HSC activation and fibrogenesis.A pharmacological PKM2 agonist efficiently attenuated the profibrotic crosstalk between macrophages and HSCs in vitro and in vivo.Translationally,ablation of PKM2 in NPCs by cholesterol-conjugated heteroduplex oligonucleotides,a novel oligonucleotide drug that preferentially accumulates in the liver,dose-dependently reversed NASH-related fibrosis without causing observable hepatotoxicity.The present study highlights the pivotal role of macrophage PKM2 in advancing NASH fibrogenesis.Thus,therapeutic modulation of PKM2 in a macrophage-specific or liver-specific manner may serve as a novel strategy to combat NASH-related fibrosis.

High mobility group box 1 in the central nervous system:regeneration hidden beneath inflammation

High-mobility group box 1 was first discovered in the calf thymus as a DNA-binding nuclear protein and has been widely studied in diverse fields,including neurology and neuroscience.High-mobility group box 1 in the extracellular space functions as a pro-inflammatory damage-associated molecular pattern,which has been proven to play an important role in a wide variety of central nervous system disorders such as ischemic stroke,Alzheimer’s disease,frontotemporal dementia,Parkinson’s disease,multiple sclerosis,epilepsy,and traumatic brain injury.Several drugs that inhibit high-mobility group box 1 as a damage-associated molecular pattern,such as glycyrrhizin,ethyl pyruvate,and neutralizing anti-high-mobility group box 1 antibodies,are commonly used to target high-mobility group box 1 activity in central nervous system disorders.Although it is commonly known for its detrimental inflammatory effect,high-mobility group box 1 has also been shown to have beneficial pro-regenerative roles in central nervous system disorders.In this narrative review,we provide a brief summary of the history of high-mobility group box 1 research and its characterization as a damage-associated molecular pattern,its downstream receptors,and intracellular signaling pathways,how high-mobility group box 1 exerts the repair-favoring roles in general and in the central nervous system,and clues on how to differentiate the pro-regenerative from the pro-inflammatory role.Research targeting high-mobility group box 1 in the central nervous system may benefit from differentiating between the two functions rather than overall suppression of high-mobility group box 1.

Analysis of the potential biological value of pyruvate dehydrogenase E1 subunitβin human cancer

BACKGROUND The pyruvate dehydrogenase E1 subunitβ(PDHB)gene which regulates energy metabolism is located in mitochondria.However,few studies have elucidated the role and mechanism of PDHB in different cancers.AIM To comprehensive pan-cancer analysis of PDHB was performed based on bioinformatics approaches to explore its tumor diagnostic and prognostic value and tumor immune relevance in cancer.In vitro experiments were performed to examine the biological regulation of PDHB in liver cancer.METHODS Pan-cancer data related to PDHB were obtained from the Cancer Genome Atlas(TCGA)database.Analysis of the gene expression profiles of PDHB was based on TCGA and Genotype Tissue Expression Dataset databases.Cox regression analysis and Kaplan-Meier methods were used to assess the correlation between PDHB expression and survival prognosis in cancer patients.The correlation between PDHB and receiver operating characteristic diagnostic curve,clinicopathological staging,somatic mutation,tumor mutation burden(TMB),microsatellite instability(MSI),DNA methylation,and drug susceptibility in pan-cancer was also analyzed.Various algorithms were used to analyze the correlation between PDHB and immune cell infiltration and tumor chemotaxis environment,as well as the co-expression analysis of PDHB and immune checkpoint(ICP)genes.The expression and functional phenotype of PDHB in single tumor cells were studied by single-cell sequencing,and the functional enrichment analysis of PDHB-related genes was performed.The study also validated the level of mRNA or protein expression of PDHB in several cancers.Finally,in vitro experiments verified the regulatory effect of PDHB on the proliferation,migration,and invasion of liver cancer.RESULTS PDHB was significantly and differently expressed in most cancers.PDHB was significantly associated with prognosis in patients with a wide range of cancers,including kidney renal clear cell carcinoma,kidney renal papillary cell carcinoma,breast invasive carcinoma,and brain lower grade glioma.In some cancers,PDHB expression was clearly associated with gene mutations,clinicopathological stages,and expression of TMB,MSI,and ICP genes.The expression of PDHB was closely related to the infiltration of multiple immune cells in the immune microenvironment and the regulation of tumor chemotaxis environment.In addition,single-cell sequencing results showed that PDHB correlated with different biological phenotypes of multiple cancer single cells.This study further demonstrated that down-regulation of PDHB expression inhibited the proliferation,migration,and invasion functions of hepatoma cells.CONCLUSION As a member of pan-cancer,PDHB may be a novel cancer marker with potential value in diagnosing cancer,predicting prognosis,and in targeted therapy.

Metformin-associated lactic acidosis:A mini review of pathophysiology,diagnosis and management in critically ill patients

Metformin is a common diabetes drug that may reduce lactate clearance by inhibiting mitochondrial oxidative phosphorylation,leading to metforminassociated lactic acidosis(MALA).As diabetes mellitus is a common chronic metabolic condition found in critically ill patients,pre-existing metformin use can often be found in critically ill patients admitted to the intensive care unit or the high dependency unit.The aim of this narrative mini review is therefore to update clinicians about MALA,and to provide a practical approach to its diagnosis and treatment.MALA in critically ill patients may be suspected in a patient who has received metformin and who has a high anion gap metabolic acidosis,and confirmed when lactate exceeds 5 mmol/L.Risk factors include those that reduce renal elimination of metformin(renal impairment from any cause,histamine-2 receptor antagonists,ribociclib)and excessive alcohol consumption(as ethanol oxidation consumes nicotinamide adenine dinucleotides that are also required for lactate metabolism).Treatment of MALA involves immediate cessation of metformin,supportive management,treating other concurrent causes of lactic acidosis like sepsis,and treating any coexisting diabetic ketoacidosis.Severe MALA requires extracorporeal removal of metformin with either intermittent hemodialysis or continuous kidney replacement therapy.The optimal time to restart metformin has not been well-studied.It is nonetheless reasonable to first ensure that lactic acidosis has resolved,and then recheck the kidney function post-recovery from critical illness,ensuring that the estimated glomerular filtration rate is 30 mL/min/1.73 m^(2) or better before restarting metformin.

Celastrol mitigates inflammation in sepsis by inhibiting the PKM2-dependent Warburg effect

Background: Sepsis involves life-threatening organ dysfunction and is caused by a dysregulated host response to infection. No specific therapies against sepsis have been reported. Celastrol(Cel) is a natural anti-inflammatory compound that shows potential against systemic inflammatory diseases. This study aimed to investigate the pharmacological activity and molecular mechanism of Cel in models of endotoxemia and sepsis.Methods: We evaluated the anti-inflammatory efficacy of Cel against endotoxemia and sepsis in mice and macrophage cultures treated with lipopolysaccharide(LPS). We screened for potential protein targets of Cel using activity-based protein profiling(ABPP). Potential targets were validated using biophysical methods such as cellular thermal shift assays(CETSA) and surface plasmon resonance(SPR). Residues involved in Cel binding to target proteins were identified through point mutagenesis, and the functional effects of such binding were explored through gene knockdown.Results: Cel protected mice from lethal endotoxemia and improved their survival with sepsis, and it significantly decreased the levels of pro-inflammatory cytokines in mice and macrophages treated with LPS(P <0.05). Cel bound to Cys424 of pyruvate kinase M2(PKM2), inhibiting the enzyme and thereby suppressing aerobic glycolysis(Warburg effect). Cel also bound to Cys106 in high mobility group box 1(HMGB1) protein, reducing the secretion of inflammatory cytokine interleukin(IL)-1β. Cel bound to the Cys residues in lactate dehydrogenase A(LDHA).Conclusions: Cel inhibits inflammation and the Warburg effect in sepsis via targeting PKM2 and HMGB1 protein.

Overexpressed PKM2 promotes macrophage phagocytosis and atherosclerosis

Background:The expression of pyruvate kinase muscle 2(PKM2)is augmented in macrophages of patients with atherosclerotic coronary artery disease.The role of PKM2 in atherosclerosis is to be determined.Methods:Global and myeloid cell-specific PKM2 knock-in mice with ApoE^(-/-)background(ApoE^(-/-),PKM2^(KI/KI)and Lyz2-cre,ApoE^(-/-),and PKM2^(flox/flox))were produced to evaluate the clinical significance of PKM2 in atherosclerosis development.Wild-type and PKM2 knock-in macrophages were isolated to assess the function of PKM2 in macrophage phagocytosis.Atherosclerotic mice were treated with PKM2 inhibitor shikonin(SKN)to evaluate the therapeutic potential of PKM2 suppression in atherosclerosis.Results:Oxidized low-density lipoprotein(oxLDL)upregulated PKM2 in macrophages.PKM2 in return promoted the uptake of oxLDL by macrophages.Overexpressed PKM2 accelerated atherosclerosis in mice.SKN blocked the progress of mouse atherosclerosis.Conclusions:PKM2 accelerates macrophage phagocytosis and atherosclerosis.Targeting PKM2 is a potential therapy for atherosclerosis.

New insights into the pathogenesis of primary biliary cholangitis asymptomatic stage

Primary biliary cholangitis(PBC)is a chronic cholestatic progressive liver disease and one of the most important progressive cholangiopathies in adults.Damage to cholangiocytes triggers the development of intrahepatic cholestasis,which progresses to cirrhosis in the terminal stage of the disease.Accumulating data indicate that damage to biliary epithelial cells[(BECs),cholangiocytes]is most likely associated with the intracellular accumulation of bile acids,which have potent detergent properties and damaging effects on cell membranes.The mechanisms underlying uncontrolled bile acid intake into BECs in PBC are associated with pH change in the bile duct lumen,which is controlled by the bicarbonate(HCO3-)buffer system“biliary HCO3-umbrella”.The impaired production and entry of HCO3-from BECs into the bile duct lumen is due to epigenetic changes in expression of the X-linked microRNA 506.Based on the growing body of knowledge on the molecular mechanisms of cholangiocyte damage in patients with PBC,we propose a hypothesis explaining the pathogenesis of the first morphologic(ductulopenia),immunologic(antimitochondrial autoantibodies)and clinical(weakness,malaise,rapid fatigue)signs of the disease in the asymptomatic stage.This review focuses on the consideration of these mechanisms.

Effect of Purified Paper Wasp Ropalidia marginata Venom Toxin Enzyme Activity in Blood Serum, Liver, and Gastrocnemius Muscle of Albino Mice

In the present, investigation effects of sub-lethal dose of purified paper wasp Ropalidia marginata venom toxins were evaluated on important metabolic enzymes i.e. ALP ACP, GPT, GOT, LDH, and AchE enzyme activity in serum, liver, and gastrocnemius muscles of albino mice. Alkaline phosphatase was found to be increased up to 119.9% at the 6th hr of the toxin injection in comparison to control. This elevation may be due to cytolysis. Maximum increase i.e., 153.33% level of glutamate pyruvate transaminase (GPT) was found at 6 hrs of 40% of 24-h LD50 treatment while it was found to be 151.1% at 6 hrs of 24 hr 80% of LD50, venom injection. A significant elevation was observed in LDH activity in serum, liver, and muscles, while the activity of AchE was decreased in serum, liver, and gastrocnemius muscles of albino mice after injecting the sub-lethal dose of Ropalidia marginata venom. This increase in the activity of LDH produces liver damage, massive disintegration and necrosis of hepatic cells. This elevation in LDH level led to a significant increase in the glucose catabolism and elevated oxidative stress in muscle and liver cells. It also displays insufficient oxygen supply and consequently leads to cell death. In experimental animals, venom toxin treatment decreased AchE level, and animals showed muscular paralysis. When mice were treated with 40% and 80% of 24-h LD50 of purified venom caused a significant (p < 0.05) elevation in the level of ACP, GOT, GPT, and LDH while the reduction in ALP and AChE level. Present study will be useful in the development of prototypes for study of pharmacological and therapeutic effects of various venom toxins. For this purpose structure activity relationship of enzyme and venom toxin, its due interaction to various metabolic enzymes and receptors must be explored.

Effects of Purified Indian Cattle Tick Rhipicephalus microplus Saliva Toxins on Various Enzymes in Blood Serum, Liver and Muscles of Albino Mice

In the present investigation, in vivo effects of purified ticks’ saliva toxin were evaluated on the level of certain important cellular metabolic enzymes i.e. acid phosphatase (ACP), alkaline phosphatase (ALP), glutamate pyruvate transaminase, glutamate oxaloacetate transaminase and lactic dehydrogenase. For this purpose, sub-lethal doses, 40% and 80% of 24 h LD50 purified saliva toxins of Rhipicephalus microplus (Canestrini, 1888) were injected subcutaneously in the albino mice. In treated mice saliva toxins targeted membrane-bound enzymes i.e. serum acid phosphatase and alkaline phosphatase, its level was increased from 118.30% to 163.63% at the 6th hr in comparison to the control. Besides this, the levels of serum glutamate pyruvate transaminase (GPT) and glutamate oxaloacetate transaminase (GOT) and lactic dehydrogenase (LDH) also increased up to 161.11% (at 6th hr), 148.27 (at 8th hr) and 125.45% (at 6th hr) respectively in comparison to control. An increase in the level of LDH showed insufficient oxygen supply, massive disintegration of cells and leakage of the enzyme into the circulation. It clearly indicated the toxic effects of saliva toxins on the membrane of blood cells, hepatocytes and myocardial muscle cell functions in albino mice. On the other hand activity of acetyl cholinesterase was reduced by 65.51% at the 6th hr of the saliva toxin injection in comparison to the control. This inhibition of acetyl cholinesterase activity caused the accumulation of acetylcholine molecules at the synaptic junctions and led to prolonged activation of acetylcholine receptors. It caused permanent stimulation of nerves and muscle cells that may result in muscular paralysis and finally death of the animal.

M2型丙酮酸激酶调控肿瘤细胞能量代谢的研究进展

肿瘤细胞未进行高效的氧化磷酸化途径,而是经有氧糖酵解将葡萄糖降解为乳酸并产生ATP,这一现象被称warburg effect[1]。糖酵解的关键酶丙酮酸激酶（pyruvate kinase,PK）作用于其底物磷酸烯醇式丙酮酸（phosphoenolpyruvate,PEP）生成丙酮酸,PK有4种亚型,不同亚型在组织中选择性表达及其动力学特征各不同。

Characteristics of CO_2 Exchange and Chlorophyll Fluorescence of Transgenic Rice with C_4 Genes

The responses of photosynthesis of phosphoenopyruvate carboxylase (PEPC), pyrurate dikinase (PPDK), NADP-malic enzyme (NADP-ME) and PPDK+PEPC transgenic rice (Oryza saltiva L.) plant to light, temperature, CO 2 and the characteristics of chlorophyll fluorescence under photoinhibition conditions were studied. The results were as follows: 1. The light-saturated photosynthetic rates of transgenic rice plants were higher than that of wild type, in which the light-saturated point of PEPC and PPDK+PEPC transgenic rice plants was 200 μmol·m -2·s -1 higher than that of untransformed rice and the light-saturated photosynthetic rates were 51.6% and 58.5% respectively. The carboxylation efficiency of PEPC transgenic rice plant increased by 49.3% and the CO 2 compensation point decreased by 26.2% than that of untransformed rice. Under high temperature (35 ℃), the photosynthetic rate of PEPC transgenic rice plant was higher over 17.5% than that of untransformed rice. 2. On the 8th day after photoinhibition treatment, the PSⅡ photochemical efficiency (F v/F m) and photochemical quenching (qP) of PEPC and PPDK+PEPC transgenic rice plants decreased by about 20%-30% while the non-photochemical quenching (qN) increased by approximately 30%. But F v/F m and qP of untransformed rice decreased by over 50% while qN increased by less than 10%. The result suggested that transgenic rice plants were more tolerant to photoinhibition.

Effects of Used Battery on Key Enzyme Activity during the Germination of Wheat Seeds

[Objective] This study aimed to explore the effects of used battery lixivium on wheat germination. [Method] The wheat seeds were treated with used battery lix- ivium at different concentrations to detect the change of activities of amylase, pro- tease, pyruvate dehydrogenase （PDH） and polyphenol oxidase （PPO） during the ger- mination period. [Result] The results showed that the used battery affected enzyme activity. With the increase of concentration of used battery lixivium, trends of the changes of amylase and protease activities were not different. The activities were en- hanced at low concentrations of lixivium, while were inhibited at high concentrations. The tends of changes of pyruvate dehydrogenase （PDH） and polyphenol oxidase （PPO） activities were not consistent with that of either amylase or protease, which showed continuous downward trends with the increasing concentration of used battery lixivium. [Conclusion] This study is of great practical significance for understanding the effects of used battery lixivium on the germination of wheat seeds.

Overexpression of HMGB1 A-box reduced lipopolysaccharide-induced intestinal inflammation via HMGB1/TLR4 signaling in vitro

AIM: To investigate the inhibitory effects and mechanism of high mobility group box(HMGB)1 A-box in lipopolysaccharide(LPS)-induced intestinal inflammation.METHODS: Overexpression of HMGB1 A-box in human intestinal epithelial cell lines(SW480 cells) was achieved using the plasmid p EGFP-N1. HMGB1 A-box-overexpressing SW480 cells were stimulated with LPS and co-culturing with human monocyte-like cell lines(THP-1 cells) using a Transwell system, compared with another HMGB1 inhibitor ethyl pyruvate(EP). The m RNA and protein levels of HMGB1/toll-like receptor(TLR) 4 signaling pathways [including HMGB1, TLR4, myeloid differentiation factor88(MYD88), Phosphorylated Nuclear Factor κB(p NF-κB) p65] in the stimulated cells were determined by realtime polymerase chain reaction and Western blotting. The levels of the proinflammatory mediators [including HMGB1, interleukin(IL)-1β, IL-6 and tumor necrosis factor(TNF)-α] in the supernatants of the stimulated cells were determined by ELISA.RESULTS: EP downregulated the m RNA and protein levels of HMGB1, inhibited the TLR4 signaling pathways(TLR4, MYD88 and p NF-κB p65) and reduced the secretion of proinflammatory mediators(HMGB1, IL-1β, IL-6 and TNF-α) in the SW480 and THP-1 cells activated by LPS but not in the unstimulated cells. Activated by LPS, the overexpression of HMGB1 A-box in the SW480 cells also inhibited the HMGB1/TLR4 signaling pathways and reduced the secretion of these proinflammatory mediators in the THP-1 cells but not in the transfected and unstimulated cells. CONCLUSION: HMGB1 A-box, not only EP, can reduce LPS-induced intestinal inflammation through inhibition of the HMGB1/TLR4 signaling pathways.

Effects of Ethyl Pyruvate on Myocardial Apoptosis and Expression of Bcl-2 and Bax Proteins after Ischemia-reperfusion in Rats

In order to study the effects of ethyl pyruvate on cardiomyocyte apoptosis following ischemia/reperfusion （I/R） in vitro and the expression of Bcl-2 and Bax proteins, isolated rat hearts were perfused in a Langendorff model. Twenty-four rats were randomly divided into 3 groups （n=8 in each group）： control group was perfused for 120 min. In the I/R group, after 30 min stabilization the injury was induced by 30 min global ischemia followed by 60 min reperfusion. Ethyl pyruvate （EP） group was set up with the same protocol as I/R group except that it was supplied with 2 mmol/L EP 15 rain before ischemia and throughout reperfusion. Myocardial malonaldehyde （MDA） content was measured. Myocardial apoptotic index （AI） was tested by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling （TUNEL） method. The expression of anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bax in cardiac myocytes was detected by immunohistochemistry. As compared with control group, the content of MDA, myocardial AI and the expression of Bcl-2, Bax proteins were increased significantly in I/R group, but the content of MDA, myocardial AI and the expression of Bax protein were decreased obviously and the expression of Bcl-2 protein was up-regulated in EP group （P〈0.05）. These results demonstrate that EP could inhibit apoptosis of cardiac myocytes possibly via alleviating oxidative stress, up-regulating Bcl-2 and down-regulating Bax proteins.

Delayed ethyl pyruvate therapy attenuates experimental severe acute pancreatitis via reduced serum high mobility group box 1 levels in rats

AIM： To investigate the effect of delayed ethyl pyruvate （EP） delivery on distant organ injury, survival time and serum high mobility group box 1 （HMGB1） levels in rats with experimental severe acute pancreatitis （SAP）. METHODS： A SAP model was induced by retrograde injection of artificial bile into the pancreatic ducts of rats. Animals were divided randomly into three groups （n = 32 in each group）： sham group, SAP group and delayed EP treatment group. The rats in the delayed EP treatment group received EP （30 mg/kg） at 12 h, 18 h and 30 h after induction of SAP. Animals were sacrificed, and samples were obtained at 24 h and 48 h after induction of SAP. Serum HMGB1, aspartate arninotransferase （AST）, alanine arninotransferase （ALT）, blood urea nitrogen （BUN）, and creatinine （Cr） levels were measured. Lung wet-to-dry-weight （W/D） ratios and histological scores were calculated to evaluate lung injury. Additional experiments were performed between SAP and delayed EP treatment groups to study the influence of EP on survival times of SAP rats. RESULTS： Delayed EP treatment significantly reduced serum HMGB1 levels, and protected against liver, renal and lung injury with reduced lung W/D ratios （8.22 ±0.42 vs 9.76 ± 0.45, P 〈 0.01）, pulmonary histological scores （7.1 ± 0.7 vs 8.4 ± 1.1, P 〈 0.01）, serum AST （667 ± 103 vs 1 368 ± 271, P 〈 0.01）, ALT （446 ± 91 vs 653 ± 98, P 〈 0.01） and Cr （1.2 ± 0.3 vs 1.8 ± 0.3, P 〈 0.01） levels. SAP rats had a median survival time of 44 h. Delayed EP treatment significantly prolonged median survival time to 72 h （P 〈 0.01）. CONCLUSION： Delayed EP therapy protects against distant organ injury and prolongs survival time via reduced serum HMGBllevels in rats with experimental SAP. EP may potentially serve as an effective new therapeutic option against the inflammatory response and multiple organ dysfunction syndrome （MODS） in SAP patients.

Colorectal cancer screening by non-invasive metabolic biomarker fecal tumor M2-PK

AIM: To evaluate the utility of the innovative fecal tumor M2-Pyruvate kinase (M2-PK) test in our daily clinical routine, as a marker for the pre-selection of patients who should subsequently undergo colonoscopy for the diagnosis or exclusion of colorectal cancer. METHODS: Fecal tumor M2-PK was measured in stool samples of 96 study participants (33 patients with colorectal cancer, 21 patients with rectal carcinoma and 42 controls) who all underwent total colonoscopy. RESULTS: In 39 of 42 individuals in the control group, fecal tumor M2-PK was below 4.0 kU/L (93% specificity). Colorectal tumors were accompanied by a highly significant increase (P < 0.001) in fecal tumor M2- PK levels (median: colon carcinoma, 23.1 kU/L; rectal carcinoma, 6.9 kU/L; colorectal carcinoma, 14.7 kU/L), which correlated with Duke’s staging and T-classification. The overall sensitivity was 78% for colorectal cancer, increasing from 60% for stage T1 to 100% for stage T4 and from 60% for Duke’s A to 90% for Duke’s D tumors. CONCLUSION: Fecal tumor M2-PK is an appropriately sensitive tool to pre-select those patients requiring colonoscopy for the further diagnostic confirmation or exclusion of colorectal cancer.

Stress Responses to Rapid Temperature Changes of the Juvenile Sea Cucumber(Apostichopus japonicus Selenka)

Activities of hexokinase （HK）, pyruvate kinase （PK）, superoxide dismutase （SOD） and catalase （CAT） and Hsp70 level were measured to evaluate the response of the commercially important sea cucumber （Apostichopus japonicus Selenka） to rapid temperature changes in laboratory. Animals were subjected to a higher temperature （from 10 to 20℃） （Tinc treatment） or to a lower temperature （from 20 to 10℃） （Tddec treatment） for 72 h. At 1, 3, 12, 24, 72 h of exposure, animals were removed and prepared for further analysis. Results showed that the effect of acute temperature changes on enzyme activities was significant. In Tinc treatment, activities of SOD and CAT increased immediately. The significant enhancement of SOD and CAT activities suggested that oxidative stress increases significantly when ambient temperature increasing from 10 to 20℃. The up-regulation of Flsp70 in Tinc and Tdec treatments indicated that Hsp70 was a bioindicator of thermal stress in the sea cucumber, and the expression pattern depended on the thermal treatment.

Application of response surface methodology in medium optimization for pyruvic acid production of Torulopsis glabrata TP19 in batch fermentation

Response surface methodology (RSM) was used to optimize the fermentation medium for enhancing pyruvic acid production by Torulopsis glabrata TP19. In the first step of optimization, with Plackett-Burman design, ammonium sulfate, glucose and nicotinic acid were found to be the important factors affecting pyruvic acid production significantly. In the second step, a 23 full factorial central composite design and RSM were applied to determine the optimal concentration of each significant variable. A second-order polynomial was determined by the multiple regression analysis of the experimental data. The optimum values for the critical components were obtained as follows: ammonium sulfate 0.7498 (10.75 g/L), glucose 0.9383 (109.38 g/L) and nicotinic acid 0.3633 (7.86 mg/L) with a predicted value of maximum pyruvic acid production of 42.2 g/L. Under the optimal conditions, the practical pyruvic acid production was 42.4 g/L. The determination coefficient (R2) was 0.9483, which ensures adequate credibility of the model. By scaling up fermentation from flask to jar fermentor, we obtained promising results.

Catalytic domain of PDC-E2 contains epitopes recognized by antimitochondrial antibodies in primary biliary cirrhosis

AIM:To search for further immunodominant peptides of the pyruvate dehydrogenase complex E2-component (PDC-E2) recognized by antimitochondrial antibodies (AMA) in primary biliary cirrhosis (PBC). METHODS:Sera from 95 patients with PBC were tested by enzyme-linked immunosorbent assay against 33 synthetic overlapping peptides (25 amino acids; aa) covering the entire length of the E2-subunit of PDC-E2. Furthermore,the inner lipoyl peptide 167-184 was used in an unlip oylated and a lipoylated form as well as coupled to ovalbumin. Sera from 11 AMA negative/ANA posit ive PBC patients,63 patients with other liver disorders and 22 healthy blood donors served as controls.RESULTS:Of the 95 PBC-sera,74% reacted with the peptide 475-499 and 58% with the pept ide 407-431 located within the catalytic domain of PDC-E2. Patients with other disorders or healthy controls were positive in only up to 18%. Antibodies to the unlipoylatedand lip oylated pept ide 167-184 within the inner lipoyl domain were found in only 5% and 11% of the PBC sera,respectively; using ovalbumin-coupled peptides,the incidence increased up to 57% (unlipoylated form). CONCLUSION:Peptides within the catalytic site of PDC-E2 rather than the previously reported lipoyl binding peptide 167-184 may represent major immunodomin ant epitopes recognized by AMA in PBC.

Ethyl pyruvate prevents inflammatory factors release and decreases intestinal permeability in rats with D-galactosamine-induced acute liver failure

BACKGROUND: The pathogenesis and progression of acute liver failure (ALF) are closely associated with intestinal endotoxemia because of the high permeability of the intestinal wall. Treatment with ethyl pyruvate (EP) has been shown to protect liver failure effectively. The current study aimed to explore the relationship between proinflammatory cytokines and intestinal permeability, and to investigate whether EP administration might prevent the release of multiple proinflammatory cytokines and decrease intestinal permeability and therefore, protect the liver from injury. METHODS: The ALF model was induced by D-galactosamine in rats. The rats were randomly divided into control (saline i.p.), model (D-galactosamine, 1.2 g/kg, i.p.), prevention [EP injection (40 mg/kg) 2 hours ahead of D-galactosamine] and treatment groups (EP injection 2 hours after D-galactosamine) Samples were obtained at 12 and 24 hours after ALF induction respectively. The histology of liver and intestinal tissue was accessed. Serum alanine aminotransferase, endotoxin, D(-) lactate, diamine oxidase (DAO), tumor necrosis factor-alpha (TNF-α), interferon-γ (IFN-γ) and high mobility group box-1 (HMGB1) were evaluated. The survival of rats was also recorded. RESULTS: The rats in model group showed severe damage to liver tissue and intestinal mucosa 12 and 24 hours after ALF induction. EP significantly improved liver or intestinal injury In addition, serum endotoxin, D(-)-lactate, DAO, TNF-α IFN-γ and HMGB1 levels were significantly increased in the model group compared with the control group. There was a positive correlation between intestinal permeability andproinflammatory cytokines. EP significantly reduced serum endotoxin, D(-)-lactate, DAO, TNF-α, IFN-γ and HMGB1 levels. The median survival time was significantly prolonged in both prevention and treatment groups (126 and 120 hours compared with 54 hours in the model group). CONCLUSIONS: EP has protective and therapeutic effects on intestinal mucosa. EP decreases intestinal permeability, and inhibits the release of multiple proinflammatory cytokines in rats with ALF.