• The Journal of Korean Medicine Ophthalmology and Otolaryngology and Dermatology
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• v.29 no.3
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• pp.14-26
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• 2016

Objectives : This study was performed to investigate the protective effects and mechanisms of Salvia miltiorrhizae Radix extract (SME) on endotoxin shock.Methods : We used two models; LPS-induced sepsis model for in vivo model, and murine peritoneal macrophages responses for in vitro. SME was administrated orally to mice. After 1 hr, LPS was injected intraperitoneally. Survival rate was checked each time per 12 hr for 5 days. Mice were sacrificed 3 hr after LPS injection, then blood samples and organs were harvested. Cytokines secretion was measured by ELISA. Organs tissues were observed with microscope. Murine peritoneal macrophages were cultured for 1 hr either in a medium alone or in a medium that contained SME, as indicated. Then, the cells were treated with LPS for 24 hr. mRNA levels of cytokines were measured by real-time RT-PCR. Cytokine levels in the supernatants were measured by ELISA. The amount of nitrite was measured by using the Griess method to evaluate NO production. The cell lysates were analysed by Western blotting using antibodies for iNOS and β-actin was used as an internal control to monitor equal protein loading.Results : SME improverd the survival rate of mice model. SME inhibited the secretion of inflammatory cytokines and organs damages on Endotoxin Shock model. SME suppressed cytokine expression, cytokine secretion,NO production, iNOS expression in LPS-induced murine peritoneal macrophages.Conclusions : The results suggest that SME has protective effects on endotoxin shock through suppression of inflammatory cytokines, organ damages, NO production and so on.

• The Korean Journal of Food And Nutrition
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• v.28 no.3
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• pp.343-350
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• 2015

The aim of this study was to evaluate the potential anti-inflammorty properties of herbs via MAPKs such as ERK, p38, JNK. Fifty-one kinds of each herbal medicine, that were extracted with ethanol, were used in the inhibitory assay of cytokine (TNF-${\alpha}$, IL-$1{\beta}$ and IL-6) and NO. of these, 10 species of herbal medicines, Angelica dahurica, Atractylodes lancea, Cnidium officinale, Duchesnea chrysantha, Oldenlandia diffusa, Lonicera japonica, Paeonia lactiflora, Pinus thunbergii, Rehmannia glutinosa and Rubus coreanus, were screened as potential inhibitors (< $300{\mu}g/mL$) of NO, TNF-${\alpha}$, IL-$1{\beta}$ and IL-6. Among the 10 species, Lonicera japonica showed potential anti-inflammatory effects Lonicera japonica extract of $200{\mu}g/mL$ inhibited the phosphorylation of ERK, p38 and JNK. In addition, Lonicera japonica extract at 20 mg/kg increased survival rate from LPS-induced endotoxin shock by 3 fold.

• Asian-Australasian Journal of Animal Sciences
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• v.18 no.2
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• pp.192-196
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• 2005

A study was conducted to monitor the changes in serum protein profile, cholesterol and blood glucose during endotoxic shock in buffalo calves and also to assess the role of prophylactic supplementation of vitamin E and selenium in alleviating the endotoxic effects. Fifteen male buffalo calves (6-8 months of age) were divided into three groups: Group I (control)-infused with 0.9% saline solution; Group II-infused with E. coli endotoxin at 5${\mu}g/kg$ body weight in normal saline solution; Group III- supplemented prophylactically with 250 mg vitamin E and 7.5 mg selenium by i/m injections at weekly intervals for one month prior to the induction of endotoxic shock. The blood samples were collected at 0, 1, 3, 6, 9, 12, 24, 48 and 72 h after the induction of shock. Endotoxin caused a significant (p<0.05) hypoproteinemia from 3-12 h post infusion in group II but this hypoproteinemia was less pronounced and only from 3-9 h post infusion in vitamin E and selenium supplemented calves. Hypoglycemia was observed in group II from 3-24 h and blood glucose level returned to normal at 72 h. However hypoglycemia was mild in group III and blood glucose returned to normal at 48h. Hypocholesterolaemia and hypoalbuminemia were found in both groups II and III but these changes were less pronounced in group III i.e. vitamin E and Se supplemented calves. Serum electrophoretic protein patterns of group III were quite similar to those of control group but animals of group II had different electrophoretic pattern. It was concluded that the antioxidant effects of vitamin E and Se prevent the liver against oxidative stress during endotoxic shock.

• IMMUNE NETWORK
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• v.10 no.3
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• pp.99-103
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• 2010

Background: Glycogen synthase kinase $3{\beta}$ ($GSK3{\beta}$) is a ubiquitous serine/threonine kinase that is regulated by serine phosphorylation at 9. Recent studies have reported the beneficial effects of a number of the pharmacological $GSK3{\beta}$ inhibitors in rodent models of septic shock. Since most of the $GSK3{\beta}$ inhibitors are targeted at the ATP-binding site, which is highly conserved among diverse protein kinases, the development of novel non-ATP competitive $GSK3{\beta}$ inhibitors is needed. Methods: Based on the unique phosphorylation motif of $GSK3{\beta}$, we designed and generated a novel class of $GSK3{\beta}$ inhibitor (GSK3i) peptides. In addition, we investigated the effects of a GSK3i peptide on lipopolysaccharide (LPS)-stimulated cytokine production and septic shock. Mice were intraperitoneally injected with GSK3i peptide and monitored over a 7-day period for survival. Results: We first demonstrate its effects on LPS-stimulated pro-inflammatory cytokine production including interleukin (IL)-6 and IL-12p40. LPS-induced IL-6 and IL-12p40 production in macrophages was suppressed when macrophages were treated with the GSKi peptide. Administration of the GSK3i peptide potently suppressed LPS-mediated endotoxin shock. Conclusion: Collectively, we present a rational strategy for the development of a therapeutic GSK3i peptide. This peptide may serve as a novel template for the design of non-ATP competitive GSK3 inhibitors.

• Asian-Australasian Journal of Animal Sciences
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• v.16 no.11
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• pp.1577-1582
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• 2003

This study was conducted to determine the effects of prophylactic supplementation of vitamin E and Se on oxidative damage and antioxidant status. Fifteen healthy male buffalo (Bubalus bubalis) calves between the age of 6 to12 months were divided into three groups of five animals each: Group I-control, group II-endotoxic shock group infused with lyophilized E coli endotoxin @ 5 ${\mu}g$/kg body wt, and group III-supplemented with vitamin E @ 250 mg and Se @ 7.5 mg, one month prior to induction of endotoxic shock. All the animals in group II and group III exhibited signs of endotoxic shock. When the endotoxic shock was induced, there was significant (p<0.05) increase in the circulating levels of malonyl dialdehyde MDA (an indicator of lipid peroxidation). In the supplemented group III the magnitude of formation of MDA was also less as compared to group II at every stage of study. There was significant (p<0.05) decrease in circulating levels of SOD, GSH-Px, Catalase and G-6-PD activity from the normal (0 h) value with passage of time. As a result of endotoxic shock, these values reached a lowest value, and then showed a tendency towards the 0 h value. Prophylactic supplementation with vitamin E and Se was successful in reducing the quantum of oxidative damage due to formation of free radicals because of endotoxic shock.

• The Journal of Korean Obstetrics and Gynecology
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• v.21 no.2
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• pp.152-165
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• 2008

Purpose: It is the purpose of this study to investigate the anti-inflammatory effects and mechanism of cheukbaekjurpihwan(CBJPH) extract on LPS (lipopolysaccharide)-induced inflammatory mediators in murine peritoneal macrophages. Methods: To evaluate anti-inflammatory effects of CBJPH extract, the production of cytokines(TNF-${\alpha}$(tumor necrosis factor-alpha), IL(interleukin)-6, IL-12) and NO(nitric oxide) was measured in vitro and in vivo. And western blot analysis has been done to look into the mechanism. Results: CBJPH extract reduced LPS-induced NO, TNF-${\alpha}$ and IL-6, IL-12 productions in peritoneal macrophages. CBJPH extract inhibited the activation of JNK(c-Jun N-terminal kinase), but didn't inhibit the activation of MAPKs (mitogen-activated protein kinases) such as p38, ERK1/2(extracelluar signal-regulated kinase1/2) and the degradation of $I_{\kappa}B-{\alpha}$(inhibitory kappa B-alpha) in the LPS-stimulated peritoneal macrophages. CBJPH extract suppressed LPS-induced endotoxin shock and the productions of TNF-${\alpha}$, but not of IL-6, after an oral administration of CBJPH extract Conclusion: CBJPH extract suppressed the productions of LPS-induced NO and cytokines by preventing JNK from phosphorylation, which may provide a clinical basis for anti-inflammatory properties of CBJPH.

• Microbiology and Biotechnology Letters
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• v.41 no.2
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• pp.242-248
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• 2013

Endotoxins are part of the outer membrane of the cell wall of gram-negative bacteria and are continuously released during bacterial growth. Endotoxins typically induce severe sepsis and septic shock, which cause more than 50% of mortalities. Endotoxins are easily measured in the serum by the limulus amebocyte lysate (LAL) test. However, a nonspecific result is obtained, because the high concentration of serum proteins disturbs the enzyme reaction of the LAL test. In order to solve this problem, the LAL test was performed in this study after the centrifugation of the boiled serum samples to remove the impurities. As a result, among the various conditions examined, endotoxin measurement with the LAL test was the most accurate and repeatable after centrifugation of the boiled serum at $100^{\circ}C$. Moreover, the endotoxin was accurately and repeatedly measured from the prepared sera of mice that had been administered an intraperitoneal injection of purified lipopolysaccharides (LPS) or E. coli. Therefore, the application of centrifugation to remove impurities from boiled serum gives an accurate measurement of endotoxins in the sera of normal subjects or patients, and this will lead to the improved diagnosis and prevention of diseases caused by endotoxins. In addition, the centrifugation of boiled serum samples should be considered and included in the development of endotoxin test kits.

• Journal of Microbiology and Biotechnology
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• v.19 no.7
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• pp.727-733
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• 2009

Heat shock protein 70 kDa (hsp70), a molecular chaperone involved in folding of nascent proteins, has been studied for its ability to activate innate and specific immunity. High purity hsp70 preparation is generally required for immunization experiments, because endotoxins and other immunologically active contaminants may affect immune responses independently of hsp70. We have developed a novel modification of E. coli-expression medium that enabled a simple two-step production and purification method for endotoxin-free recombinant hsp70. During Ni-NTA-based affinity purification of hsp70, a contaminating protein from host E. coli cells, L-glutamine-n-fructose-6-phosphate aminotransferase (GFAT), was identified. By testing various compounds, supplementation of growth medium with a GFAT metabolite,N-acetylglucosamine, was found to reduce GFAT expression and increase the total hsp70 yield five times. The new protocol is based on column purification of His-tagged hsp70 protein produced by E. coli with the modified medium, followed by endotoxin removal by Triton X-114 extraction. This approach yielded hsp70 with high purity and minimal endotoxin contamination, making the final product acceptable for immunization experiments. In summary, a simple modification of growth medium allowed production of recombinant mouse hsp70 in high yield and purity, thus compatible with immunological studies. This protocol may be useful for production of other Histagged proteins expressed in E. coli.

• Korean journal of applied entomology
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• v.33 no.3
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• pp.178-183
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• 1994

The prolein components of each crystal toxin of Bacillus thuringiensis kurstaki and alzawai were separated by SDS-PAGE. The major clqistal proteins from both shins were composed of paiypeptides having molecular weights of 130 kd and 64 kd. Digestive mixture of both. toxins with t~ypsin and gut juices shared 62 kd polypeptide which may be major actwe toxh. However, aizawal produced much less amount of 62 kd polypeptide than kurstaki did. On ingestion with Bt &-endotoxin, larvae of Hyphantria cunea developed 45 kd stress protein in the several tissues including fat body, which was induced by heat and cold shock.

• Journal of Microbiology and Biotechnology
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• v.31 no.1
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• pp.25-32
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• 2021

Inflammatory reactions activated by lipopolysaccharide (LPS) of gram-negative bacteria can lead to severe septic shock. With the recent emergence of multidrug-resistant gram-negative bacteria and a lack of efficient ways to treat resulting infections, there is a need to develop novel anti-endotoxin agents. Antimicrobial peptides have been noticed as potential therapeutic molecules for bacterial infection and as candidates for new antibiotic drugs. We previously designed the 9-meric antimicrobial peptide Pro9-3 and it showed high antimicrobial activity against gram-negative bacteria. Here, to further examine its potency as an anti-endotoxin agent, we examined the anti-endotoxin activities of Pro9-3 and elucidated its mechanism of action. We performed a dye-leakage experiment and BODIPY-TR cadaverine and limulus amebocyte lysate assays for Pro9-3 as well as its lysine-substituted analogue and their enantiomers. The results confirmed that Pro9-3 targets the bacterial membrane and the arginine residues play key roles in its antimicrobial activity. Pro9-3 showed excellent LPS-neutralizing activity and LPS-binding properties, which were superior to those of other peptides. Saturation transfer difference-nuclear magnetic resonance experiments to explore the interaction between LPS and Pro9-3 revealed that Trp3 and Tlr7 in Pro9-3 are critical for attracting Pro9-3 to the LPS in the gram-negative bacterial membrane. Moreover, the anti-septic effect of Pro9-3 in vivo was investigated using an LPS-induced endotoxemia mouse model, demonstrating its dual activities: antibacterial activity against gram-negative bacteria and immunosuppressive effect preventing LPS-induced endotoxemia. Collectively, these results confirmed the therapeutic potential of Pro9-3 against infection of gram-negative bacteria.