Evaluación in vivo de la pirogenicidad de bioproductos fúngicos con potencial prebiótico / In vivo pyrogenicity evaluation of fungal bioproducts with prebiotic potential / Avaliação in vivo da pirogenicidade de bioprodutos fúngicos com potencial prebiótico

RESUMEN Introducción: Los productos naturales con actividad farmacológica requieren de evaluaciones preclínicas que justifiquen su empleo sobre una base científica. El ensayo de pirógenos es una prueba dentro de la Farmacología de Seguridad que se realiza para determinar la presencia de endotoxinas y constituye un método valioso, para demostrar la seguridad de bioderivados con potencial prebiótico en el campo de la inmunonutrición. Objetivo: Evaluar la pirogenicidad de bioproductos fúngicos de Pleurotus ostreatus (extractos acuosos del micelio y cuerpos fructíferos) y un biopreparado de levadura Kluyveromyces marxianus, empleando el ensayo de pirógenos en conejos Nueva Zelanda. Método: Se ensayaron concentraciones de 1,0 y 10,0 mg/mL de cada muestra por vía endovenosa en dosis de 0,5 y 5,0 mg/kg de peso. El diseño experimental cumplió las buenas prácticas de laboratorio según lo establecido por el International Council for Laboratory Animals Science y se realizó de acuerdo a los procedimientos normalizados de trabajo del Centro de Toxicología y Biomedicina, Santiago de Cuba. Resultados: Los extractos de Pleurotus ostreatus y el biopreparado de levadura (0,5 mg/kg) no mostraron signos de pirogenicidad. En los resultados del biopreparado (5,0 mg/kg), los valores de temperatura caen en un rango de incertidumbre, según la Farmacopea de Estados Unidos (USP) y se sugirió repetir el estudio. Conclusiones: Los extractos de Pleurotus ostreatus y el biopreparado de Kluyveromyces marxianus (0,5 mg/kg) no indujeron un aumento de temperatura significativo en los animales, lo cual sugiere que en estos bioproductos no existen niveles de endotoxinas que puedan provocar pirogenicidad.

ABSTRACT Introduction: Natural products with pharmacological activity require preclinical evaluations to justify their uses scientifically. The pyrogen assay is a safety pharmacology test performed to determine the presence of endotoxins and it is a valuable method to demonstrate the bio-derivative products safety and their prebiotic potential in the field of immunonutrition. Objective: To evaluate the pyrogenicity of fungal bioproducts from Pleurotus ostreatus (aqueous extracts from mycelium and fruiting bodies) and a biopreparation from Kluyveromyces marxianus yeast, using a pyrogen assay in New Zealand rabbits. Method: Concentrations of 1.0 and 10.0 mg/mL of each sample were tested intravenously at doses of 0.5 and 5.0 mg/kg body weight. The experimental design complied with good laboratory practices as established by the International Council for Laboratory Animal Science and was carried out according to the standard work procedures of the Centro de Toxicología y Biomedicina, Santiago de Cuba. Results: Pleurotus ostreatus extracts and the yeast biopreparation (0.5 mg/kg) showed no signs of pyrogenicity. In the biopreparation results (5.0 mg/kg), temperature values fall in the uncertainty range according to the United States Pharmacopoeia (USP), and therefore it was suggested to repeat the study. Conclusions: Pleurotus ostreatus extracts and Kluyveromyces marxianus biopreparation (0.5 mg/kg) did not induce a significant temperature increase in the animals, thereby suggesting that there are no endotoxin levels in such bioproducts that could cause pyrogenicity.

RESUMO Introdução: Produtos naturais com atividade farmacológica requerem avaliações pré-clínicas que justifiquem seu uso em bases científicas. O ensaio de pirogênio é um teste dentro da Farmacologia de Segurança que é realizado para determinar a presença de endotoxinas e é um método valioso para demonstrar a segurança de bioderivados com potencial prebiótico no campo da imunonutrição. Objetivo: Avaliar a pirogenicidade de bioprodutos fúngicos de Pleurotus ostreatus (extratos aquosos do micélio e corpos de frutificação) e de uma biopreparação da levedura Kluyveromyces marxianus, utilizando o ensaio pirogênico de coelho da Nova Zelândia. Método: Concentrações de 1,0 e 10,0 mg/mL de cada amostra foram testadas por via intravenosa nas doses de 0,5 e 5,0 mg/kg de peso. O desenho experimental obedeceu às boas práticas laboratoriais estabelecidas pelo Conselho Internacional para a Ciência dos Animais de Laboratório e foi realizado de acordo com os procedimentos de trabalho padrão do Centro de Toxicologia e Biomedicina, Santiago de Cuba. Resultados: Os extratos de Pleurotus ostreatus e a biopreparação de leveduras (0,5 mg/kg) não apresentaram sinais de pirogenicidade. Nos resultados da biopreparação (5,0 mg/kg), os valores de temperatura estão dentro de uma faixa de incerteza, segundo a Farmacopeia dos Estados Unidos (USP) e foi sugerido repetir o estudo. Conclusões: Os extratos de Pleurotus ostreatus e a biopreparação de Kluyveromyces marxianus (0,5 mg/kg) não induziram um aumento significativo da temperatura nos animais, o que sugere que não há níveis de endotoxinas nesses bioprodutos que possam causar pirogenicidade.

The role and mechanism of intestinal microbiota metabolites in Alzheimer′s disease / 中华神经科杂志

The intestinal microbiota refers to the microbial group that exists in the intestine, and its composition disorder may affect human health. Many studies have found that intestinal microbiota and their metabolites may be closely related to the pathologies of Alzheimer′s disease (AD) through the gut-brain axis. This article will review the roles and possible mechanisms of lipopolysaccharide, functional bacterial amyloid proteins and bile acids, which are common metabolites of intestinal microbiota, in the pathogenesis of AD, and provide valuable information for exploring the pathogenesis of AD.

Interleukin 33 inhibits lipopolysaccharide-induced high permeability of cardiac microvascular endothelial cells / 中华内科杂志

Objective:To investigate the effect of interleukin-33 (IL-33) on lipopolysaccharide (LPS)-induced permeability of rat cardiac microvascular endothelial cells (RCMECs).Methods:RCMECs were cultured in vitro to be divided into control group, LPS group, IL-33 group and LPS+IL-33 group. The effect of IL-33 on the proliferation of RCMECs was detected by cell counting reagent (CCK8). Fluorescein isothiocyanate (FITC)-dextran assay was used to evaluate the permeability of RCMECs. The expression of vascular endothelial calmodulin, ras homologous gene family (Rho) member A (RhoA) and phosphorylated Rho-associated coiled-coil-containing protein kinase (p-ROCK2) proteins were tested by western blot. High-throughput sequencing and gene ontology (GO) were performed for gene expression in LPS and LPS+IL-33 groups.Results:No significant effect of IL-33 at 10-50 ng/ml on the proliferation of RCMECs was observed ( P>0.05). Compared with the control group, the permeability of RCMECs (permeability coefficient ratio 1.404±0.029 vs. 1.000±0.200, P<0.05) was significantly increased in LPS group and the expression of vascular endothelial calmodulin (relative gray value 0.429 5±0.012 9 vs. 0.594 9±0.014 2, P<0.05) was down-regulated, while the permeability of monolayers (permeability coefficient ratio, 0.948±0.013, P<0.01) was decreased in LPS+IL-33 group and the expression of vascular endothelial calmodulin (relative grayscale value 0.549 1±0.012 0, P<0.005) was up-regulated compared with the LPS group. High-throughput sequencing data revealed that the differential genes downregulated in the LPS and LPS+IL-33 groups were associated with cytoskeleton and Rho signaling pathway. Compared with the control group, RhoA (relative gray value 0.211 4±0.009 9 vs. 0.135 0±0.007 6, P<0.000 1) and p-ROCK (relative gray value 0.656 3±0.013 2 vs. 0.503 6±0.036 2, P<0.000 1) protein expression was upregulated in the LPS group. When compared with LPS group, RhoA (relative gray value 0.157 7±0.010 7, P=0.000 2), p-ROCK (relative gray value 0.427 7±0.003 8, P<0.000 1) protein expression was decreased in LPS+IL-33 group. Conclusion:IL-33 may improve LPS-induced hyperpermeability of RCMECs by inhibiting RhoA and p-ROCK protein expression in Rho/Rho-associated coiled-coil-containing protein kinase signaling pathway.

Effect of electrical stimulation on lipopolysaccharide-induced activation of M1 microglia / 中华麻醉学杂志

Objective:To evaluate the effect of electrical stimulation on lipopolysaccharide (LPS)-induced activation of M1 microglia.Methods:The well-growing BV2 microglia cells were divided into 3 groups ( n=18 each) using a random number table method: control group (group C), group LPS, LPS and electrical stimulation group (group LE). The cells were cultured for 24 h in normal culture atmosphere in group C. In group LPS and group LE, the LPS medium culture 100 ng/ml was added, and the cells were cultured for 24 h. In group LE, cells were stimulated with 100 mV/mm direct current for 4 h before LPS incubation.The levels of tumor necrosis factor-α (TNF-α) and leukocyte interleukin-1β (IL-1β) were determined by enzyme-linked immunosorbent assay.The expression of the M1 microglia surface markers CD32 and inducible nitric oxide synase (iNOS) was detected using immunofluorescent staining.The expression of CD32 and iNOS mRNA was detected using quantitative real-time polymerase chain reaction. Results:Compared with group C, the concentrations of TNF-α and IL-1β were significantly increased, and the expression of CD32 and iNOS protein and mRNA was up-regulated in LPS and LE groups ( P<0.05). Compared with group LPS, the concentrations of TNF-α and IL-1β were significantly decreased, and the expression of CD32 and iNOS protein and mRNA was down-regulated in group LE ( P<0.05). Conclusions:Electrical stimulation can inhibit LPS-induced activation of M1 microglia and thus alleviate the inflammatory responses.

Research on Syringin protecting C2C12 myotube viability through regulating NF- κB/PPAR γ1 pathway / 国际中医中药杂志

Objective:To discuss the protective effect of Syringin (SYR) on myotube cell atrophy induced by lipopolysaccharide (LPS) and its molecular mechanism.Methods:After C2C12 myoblasts were differentiated into myotubes, they were divided into normal control group, model group and syringin group according to the random number table method. The cultured medium of model group and syringin group were added with LPS with a concentration of 200 ng/ml; the cultured medium of the syringin group was also added with 10 μmol/L syringin for 24 h. CCK8 was used to detect cell viability. In cell supernatant, NO release was detected with Griess and TNF-α level was detected by ELISA kit. The expression of NF-κB, PPAR γ1, MyHC were detected by Western blot.Results:Compared with the model group, the viability of cells [(101.08±8.92)%, (79.53±5.19)% vs. (69.07±7.16)%] in the 10 μmol/L and 100 μmol/L syringin groups were significantly increased ( P<0.01 or P<0.01), of which 10 μmol/L syringin had better effect. Compared with the model group, the level of NO [(2.92±0.33) μmol/L vs. (3.57±0.41) μmol/L] in the syringin group was significantly decreased after 6 hours of intervention ( P<0.01), and the cells in the syringin group after 24 hours of intervention, the level of TNF-α [(2.73±0.29) pg/ml vs. (4.15±0.29) pg/ml] was significantly decreased ( P<0.01), and the protein expression of cellular NF-κB (0.95±0.24 vs. 1.16±0.28) was significantly decreased ( P<0.05), the protein expression of MyHC (0.79±0.15 vs. 0.70±0.16) was increased ( P<0.05). Conclusion:SYR could inhibit the inflammatory response induced by LPS, promote the activity of myotubes, and antagonize the damage of LPS to myotube cells.

Effects of exogenous hydrogen sulfide on pulmonary hypertension in rabbits with endotoxic shock / 中国应用生理学杂志

Objective: To investigate the effects of exogenous hydrogen sulfide (H2S) on pulmonary vascular reactivity induced by endotoxic shock (ES) in rabbits. Methods: In this experiment, the model of endotoxic shock (ES) was induced by injection of lipopolysaccharides (LPS) to New Zealand big eared white rabbit through jugular vein (8 mg/0.8 ml/kg), the intervention was performed by H2S donor(sodium hydrosulfide, NaHS) which was injected intraperitoneally (28 μmol/kg) 15 min in advance. New Zealand rabbits were randomly divided into 4 groups(n=8):control group, LPS group, LPS+NaHS group and NaHS group. The changes of mean arterial pressure (MAP) and mean pulmonary arterial pressure (MPAP) were detected. The tension of pulmonary artery ring (PARs) was detected byin vitro vascular ring technique. The ultrastructure of pulmonary artery wall and pulmonary artery endothelial cells were observed by light microscope and scanning electron microscope. Results: ①MAP was decreased while MPAP was increased in rabbits after LPS injection, and ES animal model was established successfully. Compared with LPS group, mPAP of rabbit in LPS+NaHS group was decreased significantly (all P＜0.05). ②Compared with normal control group, pulmonary artery of rabbits in LPS group had an increased contractile response to phenylephrine (PE) and a decreased relaxation response to acetylcholine (ACh) (both P＜0.01); Compared with LPS group, pulmonary artery of rabbits in LPS+NaHS group had a decreased contractile response to PE and an increased relaxation response to ACh (both P＜0.05). ③Under light microscope, the structure of vascular endothelial cells was continuous in the normal control group, the elastic fibers were intact in the subcutaneous layer, and the smooth muscle layer was arranged neatly. LPS can shed some of the pulmonary artery endothelial cells, break the subcutaneous elastic fibers, and disorder the smooth muscle layer structure. Compared with LPS group, the injury of pulmonary artery wall in LPS+NaHS group was ameliorated. The morphology of pulmonary artery wall was normal in NaHS group. It is showed that some endothelial cells of pulmonary artery were missing in LPS group by Scanning electron microscopy. The morphology of pulmonary artery endothelial cells in LPS+NaHS group was similar to that in the control group: slightly widened intercellular space was observed, and no cell exfoliation was observed. Conclusion: These results suggest that exogenous H2S can protect pulmonary artery endothelial cells and regulate the reactivity changes of pulmonary artery during ES, which may be one of the mechanisms reducing PAH in ES rabbits.

Cloning and tissue expression analysis of the LepROT gene of Rana dybowskii / 生物工程学报

Leptin receptor overlapping transcript (LepROT) plays multiple roles in the regulation of immune systems. However, very little information is available about the anti-infectious mechanisms of amphibians LepROT. In this study, the cDNA sequence of the Rana dybowskii LepROT gene was determined by using RT-PCR and bioinformatics analysis. Then, the Aeromonas hydrophila (Ah) and lipopolysaccharides (LPS) infected models of R. dybowskii was constructed to obtain histopathological characteristics. Constitutive expression of LepROT mRNA and NF-κB signaling pathway were detected by real-time quantitative PCR. The full-length cDNA of LepROT gene was 396 bp and encoded 131 amino acids. Amino acid sequence analysis revealed LepROT shares 93.74% and 86.39% identity with homologues from other amphibians and mammals respectively, and the LepROT gene was quite conserved among different species. After infection, the relative expression levels of LepROT, NF-κB, IKKα and IKKβ mRNA were all significantly upregulated (P < 0.01), but showed a diverse temporal pattern of up-regulation in different tissues. Therefore, it was proposed that the LepROT gene of R. dybowskii might activate the NF-κB signaling pathway to exert anti-infectious effects, thus providing evidence for further extending the biological function of LepROT.

Effect of Echinococcus multilocularis secreted antigen on the phenotype and function of mouse bone marrow - derived dendritic cells induced by lipopolysaccharide / 临床肝胆病杂志

Objective To investigate the effect of different concentrations of Echinococcus multilocularis secretion antigen (Em-sAg) on the phenotype and function of mouse bone marrow-derived dendritic cells (BMDCs) induced by lipopolysaccharide (LPS). Methods The bone marrow precursor cells isolated from the mouse bone marrow cavity were stimulated by mouse recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) to form BMDCs, and then cell morphology was observed under an inverted microscope. After the purity of BMDCs was identified by flow cytometry, BMDCs were divided into control group, positive control group (LPS 1 μg/ml), LPS+3 mg/ml Em-sAg group, LPS+1.5 mg/ml Em-sAg group, LPS+0.75 mg/ml Em-sAg group, and LPS+0.375 mg/ml Em-sAg group. Flow cytometry was used to measure the expression of BMDC surface molecules (CD80, CD86, and MHC-Ⅱ molecules) in each group, and ELISA was used to measure the expression level of the cytokine IL-12p70. A one-way analysis of variance was used for comparison of normally distributed continuous data between multiple groups, and the least significant difference t -test was used for further comparison between two groups. Results Observation under an inverted microscope showed that after 8-10 days of culture, the cells had burr-like protrusions and were in a state of complete suspension. Flow cytometry showed that the positive rate of CD11c was above 70% and most of the cultured cells were identified as BMDCs based on this. Flow cytometry further showed that compared with the control group, the LPS group had significant increases in the cell molecules CD80, CD86, and MHC-Ⅱ on surface (all P 0.05). ELISA showed that there was a significant difference in the level of IL-12 p70 between groups ( F =73.140, P < 0.05); compared with the control group, the LPS group had a significant increase in the expression level of IL-12p70 after stimulation ( P < 0.05); compared with the positive control group, the LPS+3 mg/ml Em-sAg group, the LPS+1.5 mg/ml Em-sAg group, the LPS+0.75 mg/ml Em-sAg group, and the LPS+0.375 mg/ml Em-sAg group had a significant reduction in the expression level of IL-12p70 ( P < 0.05), and the degree of reduction in the pro-inflammatory factor IL-12p70 increased with the increase in the concentration of Em-sAg. Conclusion Different concentrations of Em-sAg can inhibit LPS-induced maturity of BMDCs and the expression of the pro-inflammatory cytokine IL-12p70.

Role of Sirt3 in lipopolysaccharide-induced abnormal mitochondrial dynamics in renal tubular epithelial cells / 中华肾脏病杂志

Objective:To observe the expression of sirtuin 3 (Sirt3) and mitochondrial damage-associated proteins in lipopolysaccharide (LPS)-induced acute kidney injury mouse model and renal tubular epithelial cells, and to explore the role of Sirt3 in LPS-induced abnormal mitochondrial dynamics in renal tubular epithelial cells.Methods:Eighteen specific pathogen free (SPF) male C57BL/6 mice were randomly assigned to control group, LPS 24 h group and LPS 48 h group. The control group was intraperitoneally injected with physiological saline (0.1 ml/10 g), and LPS 24 h group and LPS 48 h group were intraperitoneally injected with LPS (10 mg/kg) solution. Renal functional indexes of mice were analyzed by automatic biochemical analyzer. The pathological change of the kidney was observed by HE staining, and the expressions of dynamin-related protein-1 (Drp1), optic atrophy type 1 (Opa1) and Sirt3 were evaluated by Western blotting. Expression and distribution of Sirt3 in kidney was assessed by immunohistochemistry. Human renal tubular epithelial cells (HK-2) were exposed to 10 μg/ml LPS for 24 h, and the expression of Drp1, Opa1 and Sirt3 were detected by Western blotting. Cell apoptosis was assessed by Hoechst-33342 staining. After transfection to HK-2 cells with pcDNA3.1-Sirt3 recombinant plasmid, the expressions of Sirt3, Drp1, Opa1 and cell apoptosis were detected by the same methods as above.Results:(1) The levels of blood urea nitrogen and serum creatinine in LPS group were significantly higher than those in control group (both P<0.05), and the pathological changes of kidney were obvious. (2) Compared with the control group, the expression of mitochondrial fission-associated protein Drp1 in renal tissue of LPS group was significantly higher ( P<0.05), and the expression of mitochondrial fusion associated protein Opa1 was significantly lower ( P<0.05). (3) Compared with the control group, the expression of Sirt3 in LPS group was significantly lower ( P<0.05), and immunohistochemistry results showed that Sirt3 was mainly expressed in glomerular vascular endothelial cells and renal tubular epithelial cells. (4) In vitro, LPS stimulation induced increased Drp1 expression in HK-2 cells ( P<0.05), decreased Opa1 and Sirt3 expression (both P<0.05), and increased apoptosis ( P<0.05). (5) LPS-induced mitochondrial dynamics disturbance and apoptosis were alleviated by pcDNA3.1-Sirt3 recombinant plasmid transfection. Conclusions:LPS can induce down-regulation of Sirt3 expression and disturbance of mitochondrial dynamics, and Sirt3 may play a protective role in LPS-induced acute kidney injury by regulating mitochondrial dynamics.

Role of TIPE2 in endotoxin-induced acute lung injury in mice / 中华麻醉学杂志

Objective:To evaluate the role of tumour necrosis factor-α-induced protein 8-like 2 (TIPE2) in the acute lung injury (ALI) induced by endotoxin in mice.Methods:Forty SPF healthy adult male BALB/c mice, aged 6-8 weeks, weighing 20-25 g, were divided into 4 groups ( n=10 each) using a random number table method: vehicle plasmid group (VP group), vehicle plasmid plus ALI group (VP+ ALI group), TIPE2 adeno-associated virus overexpression group (T group) and TIPE2 adeno-associated virus overexpression plus ALI group (T+ ALI group). The mice in VP and VP+ ALI groups were injected with empty adeno-associated virus, while the mice in T and T+ ALI groups were intratracheally given adeno-associated virus carrying TIPE interference sequence.Three weeks later, the model of endotoxin-induced ALI was established.Lipopolysaccharide (LPS) 5 mg/kg was intratracheally given in VP+ ALI and T+ ALI groups, and the equal volume of phosphate buffered saline (PBS) was given in VP and T groups.Blood samples were obtained from the abdominal aorta at 24 h after injection of LPS for blood gas analysis, oxygenation index (OI) was calculated, and tumor necrosis factor-alpha (TNF-α) in serum were detected by enzyme-linked immunosorbent assay.The animals were then sacrificed, and lung tissues were removed for examination of pathological changes which were scored after haematoxylin and eosin staining, for calculation of the wet/dry weight ratio (W/D ratio) and for determination of myeloperoxidase (MPO) activity and the expression of TIPE2, phosphorylated c-Jun N-terminal kinase (p-JNK) and nuclear factor kappa B(NF-κB) (by Western blot). Results:Compared with VP group, the lung injury score, W/D ratio, MPO activity and concentration of serum TNF-α were significantly increased, PaO 2 and OI were decreased, expression of TIPE2 was down-regulated and expression of p-JNK and NF-κB was up-regulated in VP+ ALI group ( P<0.05). Compared with VP+ ALI group, the lung injury score, W/D ratio, MPO activity and concentration of serum TNF-α were significantly decreased, PaO 2 and OI were increased, expression of TIPE2 was up-regulated and expression of p-JNK and NF-κB was down-regulated in T+ ALI group ( P<0.05). Conclusion:The down-regulation of TIPE2 expression is involved in the process of ALI induced by endotoxin in mice.

Relationship between over-expression of endocannabinoid receptor 2 and macrophage pyroptosis in mice / 中华麻醉学杂志

Objective:To evaluate the relationship between the over-expression of endocannabinoid receptor 2 (CB2R) and macrophage pyroptosis in mice.Methods:Bone marrow-derived macrophages of mice were transfected by lentivirus vector and successfully screened out two stable cell lines: lentivirus LV5 negative control cells (LV5-NC) and lentivirus LV5CB2R overexpressing cells (OE). Two cell lines were respectively divided into 3 groups ( n=18 each) using a random number table method: control group (LV5-NC-C group, OE-C group), LPS/ATP group (LV5-NC-LPS/ATP group, OE-LPS/ATP group) and CB2R specific agonist HU308 group (LV5-NC-HU308 group, OE-HU308 group). Cells in group C were commonly cultured.In LPS/ATP group, cells were incubated with LPS at a final concentration of 0.5 μg/ml for 5 h, and then incubated with ATP at the final concentration of 5 mmol/L for 1 h. In group LPS/ATP+ HU308, cells were incubated for 5 h with LPS at the final concentration of 0.5 μg/ml and HU308 at the final concentration of 10 μmol/L and then with ATP at the final concentration of 5 mmol/L for 1 h. The expression of CB2R, nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3), caspase-1, and gasdermin D (GSDMD) mRNA was detected by real-time polymerase chain reaction, the expression of caspase-1 was detected by Western blot, and the concentrations of interleukin-18 (IL-18) and IL-1β in the culture medium were determined by enzyme-linked immunosorbent assay. Results:In each cell line, compared with group C, the expression of NLRP3, caspase-1 and GSDMD was significantly up-regulated, and the concentrations of IL-18 and IL-1β were increased in group LPS/ATP ( P<0.05). Compared with group LPS/ATP, the expression of NLRP3, caspase-1 and GSDMD was significantly down-regulated, the concentrations of IL-18 and IL-lβ were decreased in group HU308 ( P<0.05). There was no significant differences in the indicators mentioned above between group V5-NC-C and group OE-C, between group LV5-NC-LPS/ATP and group OE-LPS/ATP, and between group LV5-NC-HU308 and OE-HU308 ( P>0.05). Conclusion:Over-expression of CB2R gene cannot effectively inhibit the occurrence of macrophage pyroptosis, and only activation of CB2R can inhibit it in mice.

The impact of Distinctive Root Canal Instrumentation Systems on Endotoxin Lessening from the Root Canal: A Systematic Review and Meta-Analysis

ABSTRACT Objective: To determine the impact of distinctive instrumentation systems of the root canals on the endotoxin lessening through the root canals. Material and Methods: From the electronic databases, MEDLINE, PubMed, Cochrane Library, Embase, ISI, Google Scholar have been used to perform a systematic literature review between 2015 and 2020. Therefore, a software program (Endnote X9) has been utilized for managing electronic titles. Searches were performed with keywords, "root canal," "instrumentation," "endotoxin," "root canal preparation," "biofilm" "endodontics," and "lipopolysaccharide." This systematic review has been conducted on the basis of the key consideration of the PRISMA Statement-Preferred Reporting Items for the Systematic Review and Meta-analysis. Results: Hence, 163 potentially important abstracts and research topics have been discovered by electronic searches and three studies (3 RCTs) have been included. According to the outputs, any statistically significant differences have been not found between the rotary files and reciprocation (SMD 0.51, 95% CI [0.11, 0.90], p=0.011) (I2 = 49.5%; p=0.138). Conclusion: Analyses indicated that instrumentation methods decreased the content of endotoxin from the root canals.

Antigenic stimulation during pregnancy modifies specific iga1 and iga2 subclasses in human colostrum according to the chemical composition of the antigen

ABSTRACT Background: Several studies have evaluated the effect of infectious diseases and vaccine protocols during pregnancy on maternal milk immunoglobulin (Ig) levels, to understand the protection conferred by lactation on newborns. Colostrum is the primary source of maternal IgA for the newborn. IgA participates in protection mechanisms in the neonate's mucosa. In humans, IgA has two subclasses with differential anatomical distribution among mucosal compartments. Total IgA levels in maternal milk vary after antigen stimulation and have differential affinities in function of the chemical composition of the antigens. We studied the effect of antigenic stimulation during pregnancy on the concentrations of specific IgA1 and IgA2 subclasses in human colostrum. Methods: We analyzed data from 113 women in Mexico City and compared the amount of IgA subclasses in colostrum against three antigens: two from vaccine protocols (tetanus toxoid and pneumococcal polysaccharides) and lipopolysaccharide, a ubiquitous antigen in the gastrointestinal tract. Results: In agreement with the previous reports, we showed that IgA1 from colostrum mainly recognized protein antigens; in sharp contrast, IgA2 was mostly directed against polysaccharide antigens. These levels increased in women who had previous contacts through vaccination or infections during pregnancy. Conclusions: Antigen interaction during pregnancy increased the amount of specific IgA subclasses, depending on the chemical composition of the antigen.

Inhibitory effect of carvacrol on lipopolysaccharide-induced memory impairment in rats

Neuroinflammation is an important process underlying a wide variety of neurodegenerative diseases. Carvacrol (CAR) is a phenolic monoterpene commonly used as a food additive due to its antibacterial properties, but it has also been shown to exhibit strong antioxidative, anti-inflammatory, and neuroprotective effects. Here, we sought to investigate the effects of CAR on inflammation in the hippocampus and prefrontal cortex, as well as the molecular mechanisms underlying these effects. In our study, lipopolysaccharide was injected into the lateral ventricle of rats to induce memory impairment and neuroinflammation. Daily administration of CAR (25, 50, and 100 mg/kg) for 21 days improved recognition, discrimination, and memory impairments relative to untreated controls. CAR administration significantly attenuated expression of several inflammatory factors in the brain, including interleukin-1β, tumor necrosis factor-α, and cyclooxygenase-2. In addition, CAR significantly increased expression of brain-derived neurotrophic factor (BDNF) mRNA, and decreased expression of Toll-like receptor 4 (TLR4) mRNA. Taken together, these results show that CAR can improve memory impairment caused by neuroinflammation. This cognitive enhancement is due to the anti-inflammatory effects of CAR medicated by its regulation of BDNF and TLR4. Thus, CAR has significant potential as an inhibitor of memory degeneration in neurodegenerative diseases.

Biological characteristics of dental pulp stem cells induced by lipopolysaccharide / 中国组织工程研究

BACKGROUND: During the decay process, the bacteria and toxins invade dental pulp tissue along the dentin tubule. Dental pulp stem cells proliferate and migrate to the damaged point, forming reparative dentin to avoid stimulations from bacteria outside. Lipopolysaccharide is the main component of gram-negative bacteria. Whether lipopolysaccharide affects the proliferation, migration, and odontoblastic ability of dental pulp stem cells remains to be studied. OBJECTIVE: To investigate the proliferation, migration and odontoblastic abilities in dental pulp stem cells induced by lipopolysacchari de. METHODS: Primary cultured dental pulp stem cells were stimulated by 0, 0.1,1 and 10 mg/L lipopolysaccharide. The proliferation of dental pulp stem cells was detected by MTT assay. Scratch test and Transwell assay were performed to test the migration of dental pulp stem cells. Dental pulp stem cells were cultured in mineralized solution and 1 mg/L lipopolysaccharide for 21 days. The mineralized nodule formation was detected by alizarin red staining. RT-PCR was performed to detect the expression of dentin related genes. RESULTS AND CONCLUSION: The absorbance value in the 0.1, 1 and 10 mg/L lipopolysaccharide groups at 1, 3, 5 and 7 days was significant lower than that in the 0 mg/L lipopolysaccharide group (P < 0.01), and the migration ability was higher than that in the 0 mg/L lipopolysaccharide group. After 12 days of mineralized induction, the number and area of mineralized nodule formation in the 1 mg/L lipopolysaccharide group were significantly lower than those in the 0 mg/L lipopolysaccharide group (P < 0.01). The mRNA expression levels of osteocalcin, bone sialoprotein, and alkaline phosphatase in the 1 mg/L lipopolysaccharide group were significantly lower than those in the 0 mg/L lipopolysaccharide group (P < 0.01). These results indicate that lipopolysaccharide treatment inhibits the proliferation and odontoblastic ability of dental pulp stem cells but promotes the cell migration ability.

TNF-alpha inhibitor adalimumab attenuates endotoxin induced cardiac damage in rats

Abstract Purpose To investigate the effects of adalimumab pretreatment on the lipopolysaccharide-mediated myocardial injury. Methods Twenty-eight Wistar rats were randomized into four groups (n=7). Control (C) group animals were injected once a day with intraperitoneal (i.p) 0.9 % saline for two days. In the Adalimumab (Ada) group, adalimumab was injected at a dose of 10 mg/kg/ day (i.p) for two days. Lipopolysaccharide (Lps) group rats were injected with a dose of 5 mg/kg (i.p) lipopolysaccharide. Lipopolysaccharide + Adalimumab (Lps+Ada) group rats received adalimumab before the administration of lipopolysaccharide. The animals were sacrificed 24 h after the last injection and blood samples were obtained for determination of biochemical cardiac injury markers and circulating levels of TNF-α and interleukin-6 (IL-6). Hearts were harvested for histological examination. Results Endotoxin exposure resulted in significant increases in serum cardiac injury markers, serum cytokines and histological myocardial injury scores in the Lps group. The levels of circulating cytokines, cardiac injury markers and histological injury scores for myocardial necrosis, perivascular cell infiltration, and inflammation were significantly reduced in Lps+Ada as compared to Lps group (p<0.05). Conclusions Adalimumab pretreatment reduces endotoxin-induced myocardial damage in rats. This beneficial effect is thought to be related to the reduction of cytokine release.

Microbial profile and endotoxin levels in primary periodontal lesions with secondary endodontic involvement

Abstract This study was carried out to investigate the microbial profile and endotoxin levels of endodontic-periodontal lesions of periodontal origin. Periodontal and endodontic samples were taken from periodontal pockets and necrotic root canals of 10 teeth with endodontic-periodontal lesions. Evidencing of 40 different bacterial species were determined in each endodontic and periodontal sample using the checkerboard DNA-DNA hybridization method and Kinetic chromogenic LAL assay was used for quantification of endotoxins. Fisher's exact test correlated the bacterial species with the endodontic or periodontal microbiota. The endotoxin levels (EU/mL) found in samples of the root canal and periodontal pocket were compared by the Wilcoxon test (p<0.05). Bacteria and LPS units were found in 100% of the endodontic and periodontal samples. The species E. faecium, P. acnes, G. morbillorum, C. sputigena and L. buccalis were strongly correlated with the endodontic microbiota and P. nigrescens with the periodontal microbiota. P. intermedia, P. endodontalis and V. parvula were more prevalent in both endodontic and periodontal microbiots. The endotoxin levels in the periodontal pocket (89600 EU/mL) were significantly higher than in the root canal (2310 EU/mL). It was concluded that the microbiota present in the periodontal and endodontic tissues is similar, with a higher prevalence of species of the orange complex and a higher level of endotoxin in the periodontal pockets.

Resumo Este estudo foi realizado para investigar o perfil microbiano e os níveis de endotoxina de lesões endoperiodontais de origem periodontal. Amostras periodontais e endodônticas foram obtidas de bolsas periodontais e canais radiculares necróticos de 10 dentes com lesões endoperiodontais. A investigação de 40 espécies bacterianas diferentes foram determinadas em cada amostra endodôntica e periodontal usando o método de hibridização de DNA-DNA (checkerboard) e o ensaio cinético cromogênico LAL foi usado para quantificação de endotoxinas. O teste exato de Fisher correlacionou as espécies bacterianas com a microbiota endodôntica ou periodontal. Os níveis de endotoxina (EU/mL) encontrados nas amostras do canal radicular e na bolsa periodontal foram comparados pelo teste de Wilcoxon (p<0,05). Bactérias e unidades de LPS foram encontradas em 100% das amostras endodônticas e periodontais. As espécies E. faecium, P. acnes, G. morbillorum, C. sputigena e L. buccalis foram fortemente correlacionadas com a microbiota endodôntica e P. nigrescens com a microbiota periodontal. P. intermedia, P. endodontalis e V. parvula foram mais prevalentes em ambas microbiotas endodôntica e periodontal. Os níveis de endotoxina na bolsa periodontal (89600 EU/mL) foram significativamente maiores do que no canal radicular (2310 EU/mL). Concluiu-se que a microbiota presente nos tecidos periodontal e endodôntico é semelhante, com maior prevalência de espécies do complexo laranja e maior nível de endotoxina nas bolsas periodontais.

Patho-Physiological Response of LPS Defective Brucella abortus S19Δper in Experimentally Infected Mice

Brucellosis is an anthropozoonotic infectious disease in which infected animals pose a threat to humans. This disease has considerable social, economic and international trade importance. Despite past and current efforts to eradicate brucellosis, a large number of new human cases are reported annually worldwide. In this study, patho-physiological response to Brucella abortus S19Δper infection in BALB/c mice was assessed in comparison to its parent strain, B. abortus S19 and virulent strain, B. abortus 544. Immunohistochemical analysis confirmed the presence of bacteria in liver and spleen. Comparatively lower serum Aspartate aminotranferase (AST) level and observation of less number of microgranulomas in liver indicated that B. abortus S19 Δper was less infectious and failed to cause active infection unlike S19 and 544 strain. S19Δper, thus could be a safer vaccine candidate as an alternate to the S19 vaccine strain. Hematological studies indicated clinical manifestation of thrombocytopenia in different Brucella infected mice including S19Δper. Therefore, measurement of platelets count and serum AST level may offer as reliable indicators of brucellosis in clinical cases

Role of SIRT1 signaling pathway in endotoxin-induced acute lung injury in rats / 中华麻醉学杂志

Objective To evaluate the role of silent information regulator 1 (SIRT1) signaling pathway in endotoxin-induced acute lung injury (ALI) in rats.Methods Ninety-six clean-grade healthy male Sprague-Dawley rats,aged 4 weeks,weighing 160-180 g,were divided into 4 groups (n=24 each) using a random number table method:control group (group C),ALI group,ALI + SIRT1 agonist SRT1720 group (group SRT),and ALI + SIRT1 inhibitor EX527 group (group EX).ALI was induced by injecting lipopolysaccharide (LPS) 5 mg/kg (diluted to 0.5 ml with 0.9％ normal saline) in ALI,SRT and EX groups.SRT1720 10 mg/kg was injected via the tail vein,and 30 min later ALI model was established in group SRT.EX527 1 μg/kg was injected via the tail vein,and 30 min later ALI model was established in group EX.Blood samples were collected from the abdominal aorta at 6,24 and 48 h after LPS injection for blood gas analysis,rats were then sacrificed and lungs were removed for determination of wet to dry weight ratio (W/D ratio) and expression of SIRT1,nuclear factor kappa B p65 (NF-κB p65),interleukin-6 (IL-6) and IL-1β protein and mRNA (by Western blot or real-time polymerase chain reaction) and for examination of pathological changes of lung tissues (with a light microscope).Results Compared with groups C,PaO2 was significantly decreased,and PaCO2 and W/D ratio were increased at each time point after LPS injection in ALI,SRT and EX groups,the expression of SIRT1 protein and mRNA was significantly down-regulated,and the expression of NF-κB p65,IL-6 and IL-1β protein and mRNA was upregulated at each time point after LPS injection in ALI and EX groups,and the expression of SIRT1,NFκB p65,IL-6 and IL-1β was significantly up-regulated at each time point after LPS injection in group SRT (P＜0.05).Compared with group ALI,PaO2 was significantly increased,PaCO2 and W/D ratio were decreased,the expression of SIRT1 protein and mRNA was up-regulated,the expression of NF-κB p65,IL-6 and IL-1β protein and mRNA was down-regulated at each time point after LPS injection (P＜0.05),and the pathological changes were significantly attenuated in group SRT,and no significant change was found in the parameters mentioned above at each time point after LPS injection in group EX (P＞0.05).Conclusion Inhibited SIRT1/NF-κB signaling pathway is involved in endotoxin-induced ALI in rats.

Role and mechanism of nonreceptor tyrosine kinase Tec in endotoxin/lipopolysaccharide-induced interleukin-8 production in human alveolar epithelial cells A549 / 中华烧伤杂志

Objective@#To investigate the role and mechanism of nonreceptor tyrosine kinase Tec in the production of pro-inflammatory cytokine interleukin-8 (IL-8) induced by endotoxin/lipopolysaccharide (LPS) in human alveolar epithelial cells A549.@*Methods@#Human alveolar epithelial cells A549 were routinely cultured and passaged in Roswell Park Memorial Institute-1640 medium containing 10% fetal bovine serum. The second or third passage of cells were collected for subsequent experiments. (1) Cells were collected and divided into 6 groups with 4 wells in each group according to the random number table. Cells in blank control group were routinely cultured for 2 h. Cells in simple LPS group were routinely cultured for 1 h and then stimulated by 1 μg/mL LPS for 1 h. Cells in simple LFM-A13 group were cultured with conventional culture medium adding 75 μmol/L LFM-A13 for 1 h and then cultured with replaced conventional culture medium for 1 h. Cells in 25 μmol/L LFM-A13+ LPS group, 75 μmol/L LFM-A13+ LPS group, and 100 μmol/L LFM-A13+ LPS group were cultured with conventional culture medium adding 25, 75, and 100 μmol/L LFM-A13 respectively for 1 h and then all stimulated by 1 μg/mL LPS added into the replaced conventional culture medium for 1 h. The protein expression of Tec in cells of each group was detected by Western blotting, and the content of IL-8 in cell culture supernatant of each group was determined by enzyme-linked immunosorbent assay. (2) Cells were collected and divided into 5 groups with 4 wells in each group according to the random number table. Cells in blank control group were routinely cultured for 2 h. Cells in small interfering RNA (siRNA) control+ LPS group were transfected with empty lentivirus for 10 h and then stimulated by 1 μg/mL LPS added into the conventional culture medium for 2 h. Cells in Tec mus-298 RNA interference (RNAi)+ LPS group, Tec mus-299 RNAi+ LPS group, and Tec mus-300 RNAi+ LPS group were transfected with lentivirus loaded with Tec mus-298 RNAi, Tec mus-299 RNAi, and Tec mus-300 RNAi respectively for 10 h and then stimulated by 1 μg/mL LPS added into the conventional culture medium for 2 h. The protein expression of Tec in cells of each group was detected by Western blotting to screen Tec-siRNA with the best silencing effect on Tec gene. (3) Cells were collected and divided into 4 groups with 4 wells in each group according to the random number table. Cells in blank control group were routinely cultured for 2 h. Cells in virus control group were transfected with empty lentivirus for 10 h and then routinely cultured for 2 h. Cells in simple LPS group were stimulated by 1 μg/mL LPS added into the conventional culture medium for 2 h. Cells in Tec-siRNA+ LPS group were transfected with lentivirus loaded with Tec-siRNA with the best silencing effect on Tec gene for 10 h and then stimulated by 1 μg/mL LPS added into the conventional culture medium for 2 h. The protein expressions of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) MAPK of cells in each group were detected by Western blotting. Data were processed with one-way analysis of variance and the least significant difference-t test.@*Results@#(1) Compared with that of blank control group, the protein expression of Tec of cells in simple LPS group was obviously increased (t=9.72, P<0.05), but the protein expression of Tec of cells in simple LFM-A13 group was not obviously changed (t=4.31, P=0.05). Compared with that of simple LPS group, the protein expression of Tec of cells in 25 μmol/L LFM-A13+ LPS group, 75 μmol/L LFM-A13+ LPS group, or 100 μmol/L LFM-A13+ LPS group was obviously decreased (t=9.72, 9.07, 16.33, P<0.05 or P<0.01). Compared with (189±22) pg/mL of blank control group, the content of IL-8 in culture supernatant of cells in simple LPS group was obviously increased [(214±10) pg/mL, t=2.18, P<0.05], but the content of IL-8 in culture supernatant of cells in simple LFM-A13 group was not obviously changed [(173±43) pg/mL, t=0.64, P>0.05]. Compared with that of simple LPS group, the content of IL-8 in culture supernatant of cells in 25 μmol/L LFM-A13+ LPS group was not obviously changed [(204±38) pg/mL, t=0.54, P>0.05], but the content of IL-8 in culture supernatant of cells in 75 μmol/L LFM-A13+ LPS group and 100 μmol/L LFM-A13+ LPS group was obviously decreased [(144±44), (137±51) pg/mL, t=3.63, 2.55, P<0.05 or P<0.01]. (2) Compared with that of blank control group, the protein expression of Tec of cells in siRNA control+ LPS group was obviously increased (t=14.24, P<0.01). Compared with that of siRNA control+ LPS group, the protein expression of Tec of cells in Tec mus-298 RNAi+ LPS group or Tec mus-299 RNAi+ LPS group was obviously decreased (t=36.03, 18.23, P<0.01), but the protein expression of Tec of cells in Tec mus-300 RNAi+ LPS group was not obviously changed (t=4.08, P>0.05). The protein expression of Tec was the lowest in cells of Tec mus-298 RNAi+ LPS group, so Tec mus-298 RNAi was used in subsequent experiment. (3) Compared with 1.16±0.16 and 0.78±0.11 of blank control group, the protein expressions of p38 MAPK and ERK MAPK of cells in virus control group were not obviously changed (1.66±0.13, 0.89±0.11, t=11.09, 3.60, P>0.05), but the protein expressions of p38 MAPK and ERK MAPK of cells in simple LPS group were obviously increased (2.83±0.29, 1.86±0.37, t=9.70, 7.23, P<0.05). Compared with those of simple LPS group, the protein expression of p38 MAPK and protein expression of ERK MAPK of cells in Tec-siRNA+ LPS group were obviously decreased (0.69±0.16, 1.03±0.24, t=13.78, 4.12, P<0.05 or P<0.01).@*Conclusions@#Tec may mediate the production and release of pro-inflammatory cytokine IL-8 from human alveolar epithelial cells A549 induced by LPS via the p38 MAPK and ERK MAPK signal pathways.