Role of HO-1 in endotoxin-induced acute lung injury in mice: relationship with regulation of mitochondrial quality control / 中华麻醉学杂志

Objective:To evaluate the role of heme oxygenase-1 (HO-1) in endotoxin-induced acute lung injury (ALI) and the relationship with the regulation of mitochondrial quality control in mice.Methods:Clean-grade healthy male adult C57BL/6 mice, aged 6-8 weeks, weighing 20-25 g, were selected.HO-1 inducible gene knockout mice (HO-1 -/-) were prepared based on CRISPER/Cas9-mediated EGE system, and HO-1 gene overexpression mice (HO-1 + /+ ) were prepared by transfection of HO-1 overexpressed adenovirus vector.The mice were divided into 2 groups ( n=6 each) using a random number table method: control group (group WT, group HO-1 -/-, group HO-1 + /+ ) and endotoxin-induced ALI group (group ALI, group HO-1 -/-+ ALI, group HO-1 + /+ + ALI). Lipopolysaccharide 15 mg/kg was injected through the tail vein to develop the model of endotoxin-induced ALI, and the equal volume of normal saline was given instead in each control group.The mice were sacrificed by bloodletting at 12 h after lipopolysaccharide or normal saline administration.The lung tissues were harvested for microscopic examination of the pathological changes which were scored, for determination of GSH and GSSG contents, for observation of the ultrastructure of mitochondria (with a transmission electron microscope) and survival within 12 h, for measurement of mitochondrial membrane potential (MMP) levels, and for determination of the expression of mitochondrial quality control-related proteins mitochondrial fusion protein 2 (Mfn2) and dynamin-related protein 1 (Drp1), peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α), nuclear respiratory factor 1 (NRF1), mitophagy marker protein PTEN-induced kinase 1 (PINK1) and E3 ubiquitin-protein ligase Parkin.The ratio of GSH/GSSG was calculated. Results:Compared with control group (group WT, group HO-1 + /+ and group HO-1 -/-), the 12-h survival rate and MMP were significantly decreased, the lung injury score was increased, GSH content and GSH/GSSG ratio were decreased, and the content of GSSG was increased in endotoxin-induced ALI groups (group ALI, group HO-1 + /+ + ALI and group HO-1 -/-+ ALI) ( P<0.05). Compared with group ALI, the 12-h survival rate and MMP were significantly decreased, the lung injury score was increased, the GSH content and GSH/GSSG ratio were decreased, the GSSG content was increased, and the expression of HO-1, Mfn2, PGC-1α, NRF1, PINK1 and Parkin was down-regulated, and Drp1 expression was up-regulated in group HO-1 -/-+ ALI, and 12-h survival rate and MMP were significantly increased, lung injury score was decreased, GSH content and GSH/GSSG ratio were increased, GSSG content was decreased, the expression of HO-1, Mfn2, PGC-1α, NRF1, PINK1 and Parkin was up-regulated, and the expression of Drp1 was down-regulated in group HO-1 + /+ + ALI ( P<0.05). Conclusions:HO-1 is involved in the process of endotoxin-induced ALI in mice, which is related to the regulation of mitochondrial quality control.

Effects of electroacupuncture on mitochondrial fusion and fission during intestinal injury in mice with endotoxemia and role of HO-1 / 中华麻醉学杂志

Objective:To evaluate the effects of electroacupuncture (EA) on mitochondrial fusion and fission during intestinal injury in mice with endotoxemia and the role of heme oxygenase-1 (HO-1).Methods:Fifty SPF-grade healthy male C57BL/6 mice, aged 8 weeks, weighing 18-22 g, were divided into 5 groups ( n=10 each) using a random number table method: control group (group C), endotoxemia group (group E), endotoxemia plus EA group (group E+ EA), endotoxemia plus EA plus hemin group (group E+ EA+ H) and endotoxemia plus EA plus Znpp-Ⅸ group (group E+ EA+ Znpp-Ⅸ). Lipopolysaccharide (LPS) was intraperitoneally injected to develop the model of endotoxemia.Before LPS injection, the HO-1 inducer hemin 100 mg/kg was intraperitoneally injected in group E+ EA+ H, and the HO-1 inhibitor zinc protoporphyrin Ⅸ 10 μmol/kg was intraperitoneally injected in group E+ EA+ Znpp-Ⅸ.At 4, 3, 2 and 1 days and 30 min prior to development of the model, Zusanli and Hegu acupoints were stimulated with electric stimulator (disperse-dense wave, frequency 2 Hz/15 Hz, at a voltage of 1 mA) for 30 min, retaining the needle until the end of the experiment on the day of development of the model.Mice were sacrificed at 6 h after development of the model, and the small intestinal tissue was obtained from the terminal ileum for examination of the pathological results (with a light microscope) and ultrastructure of mitochondria (with an electron microscope) and for determination of the levels of reactive oxygen species (ROS), ATP and diamine oxidase (DAO) and expression of dynamin-related protein 1 (Drp1), mitofusin 1 (Mfn1) and HO-1 (by Western blot). Results:Compared with group C, the level of ROS was significantly increased, ATP content and DAO activity were decreased, the expression of HO-1 and Drp1 was up-regulated, the expression of Mfn1 was down-regulated ( P<0.05), and pathological damage to small intestine tissues was found in group E. Compared with group E, the level of ROS was significantly decreased, ATP content and DAO activity were increased, the expression of HO-1 and Mfn1 was up-regulated, the expression of Drp1 was down-regulated ( P<0.05), and pathological damage to small intestine tissues was significantly attenuated in group E+ EA.Compared with group E+ EA, the level of ROS was significantly decreased, ATP content and DAO activity were increased, the expression of HO-1 and Mfn1 was up-regulated, the expression of Drp1 was down-regulated ( P<0.05), and pathological damage to small intestine tissues was significantly attenuated in group E+ EA+ H, and the level of ROS was significantly increased, ATP content and DAO activity were decreased, the expression of HO-1 and Mfn1 was down-regulated, the expression of Drp1 was up-regulated ( P<0.05), and pathological damage to small intestine tissues was accenuated in group E+ EA+ Znpp-Ⅸ. Conclusions:EA can promote mitochondrial fusion, inhibit mitochondrial fission, and alleviate intestinal damage in mice with endotoxemia, and the mechanism is related to the up-regulation of HO-1 expression.

Relationship between SIRT3 and mitochondrial function in mice with endotoxin-induced lung injury / 中华麻醉学杂志

Objective:To evaluate the relationship between silent information regulator 2 homologue 3 (SIRT3) and mitochondrial function in mice with endotoxin-induced lung injury.Methods:Twenty clean-grade healthy adult male wild C57BL/6 (SIRT3 + /+ ) mice, 20 SIRT3 knockout (SIRT3 -/-) mice, weighing 20-25 g, aged 6-8 weeks, were studied.SIRT3 + /+ mice and SIRT3 -/- mice were divided into 4 groups ( n=5 each) according to the random number table method: blank control group (group C, group SIRT3 -/-C), endotoxin-induced lung injury group (group L, group SIRT3 -/-L), endotoxin-induced lung injury plus resveratrol group (group L+ R, group SIRT3 -/-L+ R), and resveratrol group (group R, group SIRT3 -/-R). Resveratrol 15 mg/kg was intraperitoneally injected once a day for 7 consecutive days in L+ R, R, SIRT3 -/-L+ R and SIRT3 -/-R groups, while the equal volume of normal saline was injected in the rest groups.Lipopolysaccharid 15 mg/kg was injected via the tail vein to develop a mouse model of endotoxin-induced lung injury at 30 min after resveratrol injection on 7th day, in L+ R and SIRT3 -/-L+ R groups and at the corresponding time points in L and SIRT3 -/-L groups, while the equal volume of normal saline was injected in the other groups.Blood samples were collected from the orbital venous plexus at 12 h after injection of normal saline or lipopolysaccharid for determination of serum total oxidation state (TOS) and total antioxidant state (TAS) levels by the xylenol orange method and ABTS colorimetric method, and the oxidative stress index (OSI) was calculated.After the mice were sacrificed, the lung tissues were taken for microscopic examination of the pathological changes which were scored and for determination of the mitochondrial membrane potential (MMP) (by JC-1 method), cellular oxygen consumption rate (OCR) (by the specific fluorescent probe method), and expression of SIRT3 (by Western blot). Results:Compared with group C or group SIRT3 -/-C, the lung injury score, serum TOS concentration and OSI were significantly increased, TAS concentration, MMP and OCR were decreased, and SIRT3 expression was down-regulated in L, L+ R, SIRT3 -/-L and SIRT3 -/-L+ R groups ( P<0.05). Compared with group L, the lung injury score, serum TOS concentration and OSI were significantly decreased, TAS concentration, MMP and OCR were increased, and SIRT3 expression was up-regulated in group L+ R, and lung injury score, serum TOS concentration and OSI were significantly increased, TAS concentration, MMP and OCR were decreased, and SIRT3 expression was down-regulated in group SIRT3 -/-L ( P<0.05). Compared with group L+ R, the lung injury score, serum TOS concentration and OSI were significantly increased, the TAS concentration, MMP and OCR were decreased, and the expression of SIRT3 was down-regulated in group SIRT3 -/- L+ R ( P<0.05). There was no significant difference in the indicators mentioned above between group SIRT3 -/-L+ R and group SIRT3 -/-L ( P>0.05). Conclusions:Down-regulation of SIRT3 expression can lead to impaired mitochondrial function, which is involved in the pathophysiological mechanism of endotoxin-induced lung injury.

Role of NAD +-mediated deacetylation activity of SIRT1 in endotoxin-induced acute lung injury in mice / 中华麻醉学杂志

Objective:To evaluate the role of nicotinamide adenine dinucleotide (NAD + )-mediated deacetylation activity of silent information regulator 1 (SIRT1) in endotoxin-induced acute lung injury (ALI) in mice. Methods:Twenty-five SPF clean-grade healthy male C57BL/6 mice including 10 wild-type (WT) and 15 NMNAT1 conditional-knockout (KO) mice, aged 6-8 weeks, weighing 20-25 g, were selected.The WT mice were divided into 2 groups ( n=5 each) using a random number table method: control group (group WT+ C) and ALI group (group WT+ ALI). The KO mice were divided into 3 groups ( n=5 each) using a random number table method: control group (group KO+ C), ALI group (group KO+ ALI) and ALI plus NAD + precursor substances nicotinamide mononucleotide (NMN) group (KO+ LPS+ NMN group). ALI was produced with lipopolysaccharide (LPS) 15 mg/kg injected intravenously.NMN 500 mg/kg was intraperitoneally injected at 1 h before injection of LPS in KO+ ALI+ NMN group, while the equal volume of normal saline was given instead in control group.Blood samples were collected from the abdominal aorta at 12 h after LPS or normal saline injection for blood gas analysis, and the animals were then sacrificed and the lung tissues were removed for microscopic examination of pathologic changes which were scored and for determination of wet/dry weight ratio (W/D ratio), and interleukin-6 (IL-6), IL-1β and tumor necrosis factor-alpha (TNF-α) contents (by enzyme-linked immunosorbent assay)and content of NAD + (using a spectrophotometer) and levels of SIRT1, acetylated nuclear factor kappaB (Ac-NF-κB), acetylated p53 (Ac-p53), acetylated FoxO1 (Ac-FoxO1) and acetylated PGC1α (Ac-PGC1α) (by Western blot). Results:Compared with group C, pH value and PaO 2 were significantly decreased, the PaCO 2, W/D ratio, lung injury score, contents of IL-6, IL-1β, TNF-α and NAD + were increased, expression of SIRT1 was up-regulated, and expression of Ac-NF-κB, Ac-p53, Ac-FoxO1 and Ac-PGC1α was down-regulated in group ALI ( P<0.05). Compared with group WT+ ALI, pH value and PaO 2 were significantly decreased, the PaCO 2, W/D ratio, lung injury score, contents of IL-6, IL-1β and TNF-α were increased, NAD + content was decreased, expression of SIRT1 was down-regulated, and expression of Ac-NF-κB, Ac-p53, Ac-FoxO1 and Ac-PGC1α was up-regulated in group KO+ ALI ( P<0.05). Compared with group KO+ ALI, pH value and PaO 2 were significantly increased, the PaCO 2, W/D ratio, lung injury score, contents of IL-6, IL-1β and TNF-α were decreased, NAD + content was increased, expression of SIRT1 was up-regulated, and expression of Ac-NF-κB, Ac-p53, Ac-FoxO1 and Ac-PGC1α was down-regulated in group KO+ ALI+ NMN ( P<0.05). Conclusion:The enhanced NAD + -mediated deacetylation activity of SIRT1 is involved in the endogenous protective mechanism in mice with endotoxin-induced ALI.

Effect of dexmedetomidine on pyroptosis in mice with acute renal injury induced by endotoxin and the relationship with miRNA-223-3p / 中华麻醉学杂志

Objective:To evaluate the effect of dexmedetomidine on pyroptosis in mice with acute renal injury induced by endotoxin and the relationship with miRNA-223-3p.Methods:Thirty-two clean-grade healthy male ICR mice, aged 8-12 weeks, weighing 20-25 g, were divided into 4 groups ( n=8 each) using the random number table method: control group (group C), lipopolysaccharide (LPS) group (group L), LPS plus dexmedetomidine group (group LD), and LPS plus dexmedetomidine plus atipamezole group (group LDT). The model of acute renal injury induced by endotoxin was established by intraperitoneal injection of LPS 400 μg/kg, followed by intraperitoneal injection of LPS 10 mg/kg 8 h later.Dexmedetomidine 40 μg/kg was intraperitoneally injected once every 2 h for 3 times in total starting from 30 min after establishing the model in group LD.Atipamezole 750 μg/kg was intraperitoneally injected immediately after establishing the model, and 30 min later dexmedetomidine 40 μg/kg was intraperitoneally injected once every 2 h for 3 times in total in group LDT.The equal volume of normal saline was intraperitoneally injected in group C. Blood samples were collected from the heart at 24 h after establishing the model, and serum creatinine (Cr) and blood urea nitrogen (BUN) concentrations were measured with an automatic biochemical analyzer.The animals were sacrificed and the left kidney tissues were obtained for microscopic examination of pathological changes after HE staining (with a light microscope) and for determination of the expression of caspase-1 p20, NOD-like receptor thermoprotein structural domain-related protein 3 (NLRP3) and ASC protein and mRNA (by quantitative real-time polymerase chain reaction and Western blot), contents of interleukin-1beta (IL-1β) and IL-18 (by enzyme-linked immunosorbent assay), and rate of pyroptosis in renal cortical cells (by TUNEL). Results:Compared with group C, the concentrations of serum Cr and BUN were significantly increased, the expression of NLRP3, caspase-1 p20 and ASC protein and mRNA in the renal tissues was up-regulated, the contents of IL-1β and IL-18 were increased, the rate of pyroptosis in renal cortical cells was increased ( P<0.05), no significant change was found in the expression of miRNA-223-3p ( P>0.05), and pathological changes of kidney were accentuated in group L. Compared with group L, the concentrations of serum Cr and BUN were significantly decreased, the expression of NLRP3, caspase-1 p20 and ASC protein and mRNA in the renal tissues was down-regulated, the contents of IL-1β and IL-18 were decreased, the rate of pyroptosis in renal cortical cells was decreased, the expression of miRNA-223-3p was up-regulated ( P<0.05), and pathological changes of kidney were attenuated in group LD.Compared with group LD, the concentrations of serum Cr and BUN were significantly increased, the contents of IL-1β and IL-18 were increased, the expression of NLRP3, caspase-1 p20 and ASC protein and mRNA in the renal tissues was up-regulated, the rate of pyroptosis in renal cortical cells was increased, the expression of miRNA-223-3p was down-regulated ( P<0.05), and the pathological changes of kidney were accentuated in group LDT. Conclusion:The mechanism by which dexmedetomidine reduces acute renal injury may be related to up-regulating the expression of miRNA-223-3p and inhibiting pyroptosis in mice.

Effect of electroacupuncture on pyroptosis in renal tubular epithelial cells of rats with acute kidney injury induced by endotoxin / 中华麻醉学杂志

Objective:To evaluate the effect of electroacupuncture (EA) on pyroptosis in renal tubular epithelial cells of rats with acute kidney injury (AKI) induced by endotoxin.Methods:Twenty-four healthy clean-grade Sprague-Dawley rats of either gender, aged 6-8 weeks, weighing 160-182 g, were divided into 4 groups ( n=6 each) using a random number table method: control group (group C), group AKI, EA plus AKI group (group EA), sham EA at non-acupoint plus AKI group (group SEA). The model of endotoxemia was established by intraperitoneally injecting 10 mg/kg lipopolysaccharide.Bilateral 30 min EA stimulation of Zusanli and Shenyu (according to atlas of animal acupoint) was performed starting from 5 days before establishing the model (once a day) and at 30 min before lipopolysaccharide administration on the day of establishing the model, with disperse-dense waves, frequency of 15 Hz, and the needle was kept until 6 h after injection of LPS in group EA.EA was performed at the points 0.5 cm lateral to the acupoints of Zusanli and Shenyu in group SEA.At 6 h after LPS injection, blood was taken from the heart, and the concentrations of serum blood urea nitrogen (BUN) and creatinine (Cr) were detected by an automatic biochemical analyzer, and the serum concentrations of neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) by enzyme-linked immunosorbent assay.The rats were then sacrificed, and the left renal cortex was obtained for determination of pyroptosis rate of renal tubular epithelial cells (by TUNEL). The right renal cortex was obtained to detect the expression of caspase-1 and IL-1β by Western blot, and the expression of caspase-1 mRNA and IL-1β mRNA was detected by real-time polymerase chain reaction. Results:Compared with group C, the concentrations of BUN, Cr, NGAL, KIM-1, TNF-α, and IL-6 were significantly increased, the pyroptosis rate of renal tubular epithelial cells was increased, the expression of caspase-1 and IL-1β protein and mRNA in the renal cortex was up-regulated in group AKI ( P<0.05). Compared with group AKI, the concentrations of BUN, Cr, NGAL, KIM-1, TNF-α, and IL-6 were significantly decreased, the pyroptosis rate of renal tubular epithelial cells was decreased, the expression of caspase-1 and IL-1β protein and mRNA in the renal cortex was down-regulated in group SEA ( P>0.05). Conclusion:The mechanism by which EA reduces AKI may be related to inhibiting pyroptosis in renal tubular epithelial cells of rats.

Effect of dexmedetomidine on pyroptosis in rats with endotoxin-induced acute lung injury / 中华麻醉学杂志

Objective:To evaluate the effect of dexmedetomidine on pyroptosis in rats with endotoxin-induced acute lung injury (ALI).Methods:Sixty SPF male Sprague-Dawley rats, aged 6 weeks, weighing 200-220 g, were divided into 5 groups ( n=10 each) by a random number table method: control group (group C), ALI group and different doses of dexmedetomidine groups (D 1-3 groups). In ALI group and D 1-3 groups, LPS 5 mg/kg was intraperitoneally injected to establish endotoxin-induced ALI model.Immediately after establishing the model, dexmedetomidine 12.5, 25.0 and 50.0 μg/kg were intraperitoneally injected in D 1-3 groups, and the equal volume of normal saline was intraperitoneally injected in group C, once a day for 14 consecutive days.After the end of administration, the rats were sacrificed, the left bronchus was lavaged, and the left bronchoalveolar lavage fluid (BALF) was collected for determination of the concentrations of interleukin-1beta (IL-1β), IL-6 and tumor necrosis factor-alpha (TNF-α) (by enzyme-linked immunosorbent assay), and the lung tissue was taken for determination of the wet/dry weight ratio (W/D ratio) and expression of cleaved-caspase-1, N-terminal of the spliceosome (GSDMD-N), IL-18 and IL-1β (by Western blot) and for microscopic examination of the pathological changes (with a light microscope). Results:Compared with group C, the W/D ratio of lung tissues was significantly increased, the concentrations of IL-1β, IL-6 and TNF-α in BALF were increased, the expression of cleaved-caspase-1, GSDMD-N, IL-1β and IL-18 in lung tissues was up-regulated ( P<0.05), and the pathological damage was aggravated in ALI group and D 1-3 groups.Compared with group ALI, the W/D ratio of lung tissues was significantly decreased, and the concentrations of IL-1β, IL-6 and TNF-α in BALF were decreased, the expression of cleaved-caspase-1, GSDMD-N, IL-1β and IL-18 in lung tissues was down-regulated in a dose-dependent manner ( P<0.05), and the pathological damage was significantly reduced in D 1-3 groups. Conclusion:The mechanism by which dexmedetomidine attenuates endotoxin-induced ALI may be related to inhibition of pyroptosis and reduction of inflammatory responses in rats.

Study on synergistic effect of Qingkailing Injection and Shengmai Injection on organ injury in endotoxemia rats / 中国中药杂志

As a dangerous disease with rapid progression, endotoxemia is easy to induce the damage to multiple organs. However, its specific and efficient treatment methods are still lacking at present. Both Qingkailing Injection(QKLI) and Shengmai Injection(SMI) have been proved effective in anti-inflammation, anti-endotoxin and organ protection. In this study, carrageenan and endotoxin were injected successively into rats to establish an endotoxemia model. Different doses of QKLI and SMI were administered to the endotoxemia rats by intraperitoneal injection separately or in combination. Then the count of white blood cells, the number of platelets, the content of cytokines, biochemical indexes, organ coefficient and pathological changes of main organs in the rats were detected. The results showed that the rats in the model group had obvious symptoms of endotoxemia, i.e., leucopenia, thrombocytopenia, increase in cytokines(IL-6 and TNF-α) and biochemical indexes of liver and kidney function as well as pathological damage to liver, kidney and lung. QKLI alone can alleviate the above symptoms of endotoxemia and the organ injury. SMI alone is less effective in improving disseminated intravascular coagulation(DIC) and cytokine secretion complicated with endotoxemia, but capable of reducing the inflammation degree of the lung, liver and kidney. The combination of QKLI and SMI remarkably increased the number of platelets in the peripheral blood, improved the liver and kidney function and reduced inflammatory factors, with lung, liver, kidney and other organ structures protected well. Moreover, the improvement effect of the combination of QKLI and SMI was stronger than those of the two injections alone at fixed doses, indicative of a synergistic effect.

Study of thermo-regulation as a worsening marker of experimental sepsis in an animal model / Estudo da termorregulação como marcador de agravamento do quadro de sepse experimental em modelo animal / Estudio de la termorregulación como marcador asociado al empeoramiento del cuadro de la sepsis experimental en un modelo con animales

Objective: to analyze variations in body temperature and in plasma nitrate and lactate concentrations in rats submitted to the experimental sepsis model. Method: a total of 40 rats divided equally into five groups. The induction of endotoxemia was performed with intravenous administration of lipopolysaccharide, 0.5 mg/Kg, 1.5 mg/Kg, 3.0 mg/Kg, and 10 mg/Kg, respectively. The control group received 0.5 mL of saline solution. The experiment lasted six hours, with evaluations performed at 0 (baseline data), 2nd, 4th, and 6thhours. Results: The animals that received doses up to 3.0 mg/kg showed a significant increase in body temperature compared to the group with 10 mg/kg, which showed a decrease in these values. The increase in plasma nitrate and lactate concentrations in the groups with lipopolysaccharide was significantly higher than in the group that received the saline solution and was correlated with the increase in body temperature. Conclusion: the variations in body temperature observed in this study showed the dose-dependent effect of lipopolysaccharide and were correlated with the increase in the concentrations of nitrate and plasma lactate biomarkers. The implications of this study are the importance of monitoring body temperature, together with the assessment of these pathophysiological markers, which suggest worsening in the prognosis of sepsis.

Objetivo: analisar as variações na temperatura corporal e nas concentrações de nitrato e lactato plasmáticos em ratos submetidos ao modelo de sepse experimental. Método: foram utilizados 40 ratos divididos igualmente em cinco grupos. A indução da endotoxemia foi realizada com administração endovenosa de lipopolissacarídeo, respectivamente 0,5 mg/Kg, 1,5 mg/Kg, 3,0 mg/Kg e 10 mg/Kg. O grupo controle recebeu 0,5 mL de solução salina. O experimento teve duração de seis horas, com avaliações realizadas na 0 (dados basais), 2a, 4a e 6a hora. Resultados: os animais que receberam doses de até 3,0 mg/Kg apresentaram aumento significativo na temperatura corporal em relação ao grupo com 10 mg/Kg, que apresentou diminuição nesses valores. O aumento nas concentrações de nitrato e lactato plasmáticos nos grupos com lipopolissacarídeo foi significativamente superior ao grupo que recebeu salina e esteve correlacionado com o aumento na temperatura corporal. Conclusão: as variações na temperatura corporal observadas neste estudo mostraram efeito dose dependentes de lipopolissacarídeo e estiveram correlacionadas com o aumento nas concentrações dos biomarcadores nitrato e lactato plasmáticos. O estudo traz como implicações, a importância no monitoramento da temperatura corporal, em conjunto com a avaliação destes marcadores fisiopatológicos, os quais sugerem agravamento no prognóstico da sepse.

Objetivo: analizar las variaciones de la temperatura corporal y de las concentraciones de nitrato y lactato en plasma en ratones sometidos a un modelo de sepsis experimental. Método: se utilizaron 40 ratones divididos en cinco grupos iguales. La inducción de la endotoxemia se realizó mediante administración intravenosa de 0,5 mg/Kg, 1,5 mg/Kg, 3,0 mg/Kg y 10 mg/Kg de lipopolisacárido, respectivamente. El grupo de control recibió 0,5 mL de solución salina. El experimento duró seis horas, con evaluaciones realizadas a la hora 0 (datos de referencia) y a la 2a, 4a y 6ahora. Resultados: los animales que recibieron dosis de hasta 3,0 mg/kg presentaron un aumento significativo de la temperatura corporal, en comparación con el grupo al que se le administró 10 mg/kg, que presentó una disminución de dichos valores. En los grupos a los que se les administró lipopolisacárido, el aumento en las concentraciones de nitrato y lactato en plasma fue significativamente mayor que en el grupo al que se le administró la solución salina y estuvo correlacionado con el aumento de la temperatura corporal. Conclusión: las variaciones de la temperatura corporal observadas en este estudio mostraron que los efectos dependieron de la dosis de lipopolisacárido, y estuvieron correlacionadas con el aumento en la concentración de biomarcadores, como el nitrato y lactato en plasma. El estudio reveló la importancia del control de la temperatura corporal, junto con la evaluación de estos marcadores fisiopatológicos, que sugieren un empeoramiento en el pronóstico de la sepsis.

Sepsis and cirrhosis in growing animals: description of a new experimental model and its pathological and immunological reliability

OBJECTIVES: In cirrhotic children, infection events and sepsis are more frequent and more severe due to immune dysfunction. The objectives of the current study were therefore to develop an experimental model of infection and sepsis in cirrhotic weaning growing rats, by the use of bile duct ligation (BDL) and cecal ligation and puncture (CLP). Additionally, the correlation of the clinico-histopathological data and serial cytokine levels in septic cirrhotic and non-cirrhotic animals was studied. METHODS: Young Wistar rats of age 21 days and of weight between 70-90 g were divided into 12 groups according to the surgical procedure performed: sham (sacrificed after 2 or 4 weeks), BDL (sacrificed after 2 or 4 weeks), CLP (2- or 4-week old animals sacrificed after 12 or 24 hours), BDL+CLP (2- or 4-week old animals sacrificed after 12 hours). Histopathological studies and determination of serum levels of cytokines IL-1 beta, IL-10, and TNF-alpha, for studies of systemic infection, were performed. Murine sepsis scores (MSS) based on the clinical aspects just before euthanasia were also included. RESULTS: A transitory increase in IL-1, IL-10, and TNF-alpha levels was observed, with different patterns according to the groups. Two-hit groups tended to present with higher values of serum cytokines and histopathological scores than their septic non-cirrhotic counterparts. There was a correlation between mortality rate and MSS (p<0.0001). CONCLUSION: The model is feasible and may be utilized in studies on liver cirrhosis and infection in growing animals.

Molecular mechanism underlying the inhibitory effect of propofol on lipopolysaccharide-induced pyroptosis of mouse bone marrow-derived macrophages / 南方医科大学学报

OBJECTIVE@#To investigate the molecular mechanism underlying the inhibitory effect of propofol on pyroptosis of macrophages.@*METHODS@#Macrophages derived from bone marrow were extracted and divided into three groups: control group, LPS+ATP group and propofol+LPS+ATP group. The control group was not given any treatment; LPS+ATP group was given LPS 1 μg/mL stimulation for 4 h, then ATP 4 mM stimulation for 1 h; Propofol+LPS+ATP group was given propofol+LPS 1 μg/mL stimulation for 4 h, then ATP stimulation for 1 h. After treatment, the supernatant and cells of cell culture were collected. the cell activity was detected by CCK8 and flow cytometry. The inflammatory cytokines IL-1βand IL-18 were detected by Elisa. Western blot was used to detect the expression of caspase-1 protein and TLR4 on cell membran Immunohistochemical fluorescence was used to detect apoptosis of cells.@*RESULTS@#LPS+ATP significantly decreased the viability of the macrophages and increased the cellular production of IL-1β and IL-18, activation of caspase-1 protein and the expression of TLR-4 on the cell membrane ( < 0.05). Treatment with propofol obviously reversed the changes induced by LPS+ATP.@*CONCLUSIONS@#LPS+ATP can induce pyroptosis of mouse bone marrow-derived macrophages, and propofol effectively inhibits such cell death, suggesting that propofol anesthesia is beneficial during operation and helps to regulate the immune function of in patients with sepsis.

Effect of Wenyang Jiedu Huayu Granule on TLR4 mRNA, TNF-α and Hepatocyte Apoptosis in IETM Rats with Hepatic Failure / 中国实验方剂学杂志

Objective::To investigate the relationship between Toll-like receptor 4 (TLR4) mRNA and its downstream inflammatory factor-tumor necrosis factor-α (TNF-α) and hepatocyte apoptosis in mice with intestinal endotoxemia (IETM) of hepatic failure, and explore the regulatory mechanism of Wenyang Jiedu Huayu granule on endotoxin-induced hepatocyte apoptosis. Method::The 85 SPF male SD rats were randomly divided into normal group, model group, TLR4 monoclonal antibody group and Wenyang Jiedu Huayu granule group. D-galactosamine (D-Gal) intraperitoneal injection was performed to establish the IETM model of hepatic failure. The TLR4 monoclonal antibody group and the Wenyang Jiedu Huayu granule group were given Wenyang Jiedu Huayu granule solution by gavage 5 days before the modeling. The normal group and the model group were given isovolumetric distilled water. Each group was given by gavage until sacrifice. Rats in each group were randomly sacrificed at 24, 48, 72 h, respectively, and samples were collected. The levels of serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were detected at 24, 48, 72 h. The pathological changes of liver tissue were observed by hematoxylin and eosin(HE) staining. The expression of TLR4 in liver tissue was detected by real-time fluorescence quantitative PCR (Real-time PCR). The expression of TNF-α in liver tissue was detected by enzyme-linked immunosorbent assay (ELISA). The apoptotic rate of hepatocytes was detected by flow cytometry. Result::Compared with the normal group, ALT and AST were increased in model group, while the pathological injury degree of liver tissue was significantly increased. The expressions of TLR4 mRNA and TNF-α were increased (P<0.05, P<0.01), whereas the apoptosis rate of liver cells was significantly increased (P<0.05, P<0.01). Compared with the model group, ALT and AST were decreased in Wenyang Jiedu Huayu granule group (P<0.01), and the degree of pathological injury of liver tissue was significantly reduced (P<0.05). The expressions of TLR4 mRNA and TNF-α were significantly decreased (P<0.01), and the apoptosis rate of liver cells was also reduced (P<0.01). Conclusion::TLR4 mRNA and TNF-α are positively correlated with hepatocyte apoptosis in liver failure. Wenyang Jiedu Huayu granule can improve liver function, alleviate liver cell injury and reduce liver cell apoptosis in IETM mice with hepatic failure. The mechanism may be related to its ability to down-regulate the expression of liver TLR4 mRNA, inhibit the release of TNF-α, and reduce the rate of hepatocyte apoptosis.

Effect of Warming and Heat-clearing Method on Expressions of FoxP3 and ROR-γt in Colon Tissue of Rats with Intestinal Endotoxemia Caused by Acute-on-chronic Liver Failure / 中国实验方剂学杂志

Objective::To study the effect of warming and heat-clearing method (Wenyang Jiedu Huayu decoction) on the expressions of Forkhead box P3 (FoxP3), Retinoic acid-related orphan receptor gamma t (ROR-γt) in colon tissue of mice with acute-on-chronic liver failure (ACLF), in order to explore the possible regulatory mechanism on intestinal endotoxemia (IETM) in liver failure mice. Method::The 130 SD rats were randomly divided into normal group (10 rats) and model group (120 rats). The ACLF mice model was established through the subcutaneous injection with bovine serum albumin and the intraperitoneal injection with D-galactosamine(D-Gal) and lipopolysaccharide (LPS). The model mice were randomly divided into model group, heat-clearing group (Yinchenhao decoction, 6.68 g·kg-1), warming group (Yinchen Zhufu decoction, 7.09 g·kg-1) and warming and heat-clearing group (Wenyang Jiedu Huayu decoction, 19.53 g·kg-1). The normal group and the model group were given distilled water by gastric lavage, while the other groups were given equal volume of corresponding Chinese herbal medicines for a week. The value of each index at 1, 12 and 24 h was measured. The ratio of Treg/Th17 cell in peripheral blood were detected and calculated by flow cytometry. Real-time fluorescence quantitative PCR (Real-time PCR) was used to detect the expressions of FoxP3 and ROR-γt in colon tissues of mice at different time points. In situ hybridization and immunohistochemistry were used to observe the expressions of FoxP3 and ROR-γt genes and proteins. Result::Compared with normal group, the ratio of Treg/Th17 in the model group decreased significantly at each time point (P<0.01). Compared with the model group, the Treg/Th17 ratio increased only in the warming and heat-clearing method group (P<0.05). Compared with normal group, the expression of ROR-γt in the model group was significantly higher (P<0.01), and the expression of ROR-γt in the model group was higher than FoxP3.Compared with the model group, the expressions of FoxP3 and ROR-γt mRNA in the heat-clearing group and the warming group decreased at each time point (P<0.05), and the expressions of FoxP3 and ROR-γt in the warming and heat-clearing method group decreased significantly (P<0.01). The expressions of FoxP3 and ROR-γt mRNA in warming and heat-clearing group decreased compared with those in the warming group and heat-clearing group (P<0.05). Conclusion::The mechanism of the warming and heat-clearing method on IETM in liver failure may be related to the regulation of FoxP3 and ROR-γt expressions.

Prevention and Treatment of Postoperative Complications in Equines / Prevención y tratamiento de complicaciones postoperatorias en equinos / Prevenção e tratamento de complicações pós-operatórias em equinos

Abstract Colic surgery is one of the most expensive procedures in equine clinics throughout the world. Although costs of surgery and anesthesia can be standardized to some extent, the cost of aftercare is less predictable. It can be altered considerably by the development of complications, such as surgical site infection, postoperative ileus, and formation of post-operative hernias, among others. Colic surgery places enormous demands on personnel, time, and resources. Nevertheless, it is a life-saving procedure. Complications increase cost through prolonged nursing care, technician salaries, extended treatment with antibiotics and other drugs, repeated laboratory analysis, and high-volume fluid therapy. Additional surgery, such as repeat celiotomy or repair of incisional complications, increases the costs considerably. Because none of these can be anticipated in most cases, a critical part of case management is a complete evaluation of expected and probable unexpected costs for the owner.

Resumen La cirugía de cólicos es uno de los procedimientos más costosos en la clínica de equinos en todo el mundo. Aunque los costos de la cirugía y la anestesia se pueden estandarizar hasta cierta medida, el costo de cuidado posterior es menos predecible y se puede alterar considerablemente mediante el desarrollo de complicaciones, tales como infección del sitio quirúrgico, ileus postoperatorio y formación de hernias postoperatorias, entre otros. La cirugía de cólicos le trae exigencias enormes al personal y requiere tiempo y recursos. Sin embargo, es un procedimiento que salva vidas. Las complicaciones aumentan el costo debido a que el cuidado de enfermería se prolonga, hay que pagar salarios a los técnicos, se amplía el tratamiento con antibióticos y otros medicamentos, hay que repetir análisis de laboratorio y se requiere terapia de fluidos con altos volúmenes. La cirugía adicional, como una celiotomía repetida o reparación de las complicaciones incisionales, aumenta los costos considerablemente. Como ninguno en la mayoría de los casos no se puede prever ninguna de estas cosas, una parte crítica del manejo de casos es la evaluación completa de los costos esperados y los probables no-esperados que cubrirá el propietario.

Resumo A cirurgia de cólica é um dos mais custosos procedimentos clínicas equinas ao redor do mundo. No entanto, os custos de cirurgia e anestesia possam ser padronizados até certo ponto, o custo dos cuidados posteriores é menos previsível e pode se alterar consideravelmente a causa de complicações, tais como infecção do local cirúrgico, íleo pós-operatório e formação de hérnias pós-operatórias, entre outras. A cirurgia de cólica impõe uma enorme demanda de pessoal, tempo e recursos, toda vez que é um procedimento que salva vidas. As complicações aumentam o custo por meio de cuidados prolongados de enfermagem, salários dos técnicos, tratamento prolongado com antibióticos e outros medicamentos, análises laboratoriais repetidas e fluidoterapia de alto volume. Cirurgias adicionais, como repetição de celiotomia ou reparo de complicações incisionais, aumentam consideravelmente os custos. Como nada disso pode ser previsto na maioria dos casos, uma parte crítica do gerenciamento de casos é a avaliação completa dos custos esperados e prováveis inesperados para o proprietário.

Comparación de Algoritmos Lineales y no Lineales para la Detección del Desacoplamiento Cardiorrespiratorio en Ratas Endotoxémicas / Comparison of Linear and Nonlinear Algorithms for Detection of Cardiorespiratory Uncoupling in Endotoxemic Rats

Resumen En estudios previos se ha demostrado que la endotoxemia inducida por lipopolisacárido (LPS) produce un desacoplamiento cardiorrespiratorio (CRP) debido a los efectos fisiológicos de la inflamación sistémica. Adicionalmente se sabe que la oxitocina tiene efectos antiinflamatorios y propiedades cardioprotectoras; sin embargo, se desconoce si ésta modifica el acoplamiento CRP. El objetivo del presente estudio fue comparar diferentes métodos matemáticos lineales y no lineales para la detección del desacoplamiento cardiorrespiratorio entre series de tiempo cardiacas y respiratorias. Se estudiaron series de tiempo R-R obtenidas de electrocardiogramas de grupos de roedores macho a los cuales se les administró solución salina o vehículo (V); lipopolisacárido (LPS); oxitocina (O) y LPS + oxitocina (LPS+Ox). Las series R-R y respiratorias derivadas del electrocardiograma (EDR) se analizaron en conjunto para cuantificar su grado de acoplamiento a través de las técnicas de correlación cruzada; entropía muestral cruzada; entropía condicional; información mutua; e información mutua de Rényi para los cuatro grupos. Se observó que la oxitocina no parece favorecer el acoplamiento CRP durante la endotoxemia inducida por LPS. Finalmente, se encontró que la entropía muestral cruzada y la entropía condicional presentaron las mayores diferencias estadísticas para identificar el desacoplamiento CRP producido por el LPS.

Abstract Previous studies have shown that LPS-induced endotoxemia causes a cardiorespiratory (CRP) uncoupling owing to the physiological effects of systemic inflammation. Also, it is known that oxytocin has anti-inflammatory effects and cardioprotective properties; however, it is unknown whether it can modify the CRP coupling. This study aimed to compare different linear and nonlinear mathematics methods for the detection of cardiorespiratory uncoupling between cardiac and respiratory time series. The R-R time series of electrocardiograms of male rodents that were administered with saline solution (V); lipopolysaccharide (LPS); oxytocin (O) and LPS + oxytocin (LPS + Ox) were studied. We tested the R-R and respiratory series derived from the electrocardiogram (EDR) for the four groups to quantify the degree of coupling with cross-correlation; cross sample entropy; conditional entropy; mutual information; and Rényi's mutual information. We found that oxytocin does not seem to favor the CRP coupling during endotoxemia induced by LPS. Finally, we observed that the cross-sample entropy and the conditional entropy presented the highest statistical differences to identify the CRP uncoupling produced by LPS.

Linderae radix ethanol extract attenuates alcoholic liver injury via attenuating inflammation and regulating gut microbiota in rats

This study aimed to explore the influence of gut microbiota alterations induced by Linderae radix ethanol extract (LREE) on alcoholic liver disease (ALD) in rats and to study the anti-inflammatory effect of LREE on ALD through the lipopolysaccharide (LPS) toll-like receptor 4 (TLR4)-nuclear factor kappa B (NF-κB) pathway. ALD rat models were established by intragastric liquor [50% (v/v) ethanol] administration at 10 mL/kg body weight for 20 days. Rats were divided into six groups: normal group (no treatment), model group (ALD rats), Essentiale group (ALD rats fed with Essentiale, 137 mg/kg), and LREE high/moderate/low dose groups (ALD rats fed with 4, 2, or 1 g LREE/kg). NF-κB and LPS levels were evaluated. Liver pathological changes and intestinal ultrastructure were examined by hematoxylin and eosin staining and transmission electron microscopy. The gut microbiota composition was evaluated by 16S rDNA sequencing. Expression levels of TLR4 and CD68 in liver tissue, and occludin and claudin-1 in intestinal tissue were measured. LREE treatment significantly reduced NF-κB and LPS levels, improved liver pathological changes, and ameliorated intestinal ultrastructure injury. Meanwhile, LREE-fed groups showed a higher abundance of Firmicutes and a lower abundance of Bacteroidetes than the rats in the model group. Administration of LREE suppressed TLR4 overexpression and promoted the expression of occludin and claudin-1 in intestine tissue. Thus, LREE could partly ameliorate microflora dysbiosis, suppress the inflammatory response, and attenuate liver injury in ALD rats. The protective effect of LREE might be related to the LPS-TLR4-NF-κB pathway.

Protective effect of dexmedetomidine against organ dysfunction in a two-hit model of hemorrhage/resuscitation and endotoxemia in rats

Dexmedetomidine (DEX), a selective agonist of α2-adrenergic receptors, has anti-inflammation properties and potential beneficial effects against trauma, shock, or infection. Therefore, this study aimed to investigate whether DEX might protect against multiple-organ dysfunction in a two-hit model of hemorrhage/resuscitation (HS) and subsequent endotoxemia. Eighty Wistar rats were randomized into four groups: NS (normal saline), HS/L (HS plus lipopolysaccharide), HS/L+D (HS/L plus dexmedetomidine), and HS/L+D+Y (HS/L+D plus yohimbine). Six hours after resuscitation, blood gas (PaO2) and serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urine nitrogen (BUN), creatinine (Cr), TNF-α, IL-β, IL-6, IL-8, IL-10, and nitric oxide (NO) were measured. The histopathology was assayed by staining. Malondialdehyde (MDA) and superoxide dismutase (SOD) levels and heme oxygenase-1 (HO-1) were assayed. The PaO2 levels in HS/L rats were lower whereas the ALT, AST, BUN, Cr, TNF-α, IL-β, IL-6, IL-8, IL-10, and NO levels were higher compared to the control group. The HS/L+D increased PaO2 and further increased IL-10 and decreased ALT, AST, BUN, Cr, TNF-α, IL-β, IL-6, IL-8, and NO levels of the HS/L groups. In addition, the MDA in the HS/L groups increased whereas SOD activity decreased compared to the control group. Moreover, the HO-1 expression levels were increased by DEX administration in lung, liver, and kidney tissues. Lungs, livers, and kidneys of the HS/L group displayed significant damage, but such damage was attenuated in the HS/L+D group. All of the above-mentioned effects of DEX were partly reversed by yohimbine. DEX reduced multiple organ injury caused by HS/L in rats, which may be mediated, at least in part, by α2-adrenergic receptors.

Role of NLRP3 inflammasome in the pathogenesis of alcoholic liver disease / 中华肝脏病杂志

Chronic excess alcohol intake triggers the formation of enterogenic endotoxemia. TLR4 ligand localization activates nuclear transcription factor NF-κB by inducing the up-regulation of NLRP3 inflammasome and the biologically inactive IL-1β and IL-18 precursors to form initiation of pro-inflammatory signals. Under the influence of ethanol, the damaged hepatocyte release uric acid, and adenosine triphosphate and induces NLRP3 inflammasome assembly and functional activation in Kupffer cells to promote the release of inflammatory mediators, such as interleukin-1β and interleukin-18, that cascade mediates inflammation and drive alcoholic liver disease from steatosis to inflammation and fibrosis. The NLRP3 inflammasome acts as a ligand-sensing element and plays an important role in mediating the immune and inflammatory response in the course of alcoholic liver disease. Thus, exploring the activation mechanism of NLRP3 inflammasome and its pathogenic role may provide a new idea in the clinical treatment of alcoholic liver disease.

Mechanism and research status of hemoperfusion in endotoxemia treatment / 国际生物医学工程杂志

Endotoxemia is a pathophysiological manifestation caused by the release of large amounts of endotoxin from bacteria in the blood or in the lesion. It can cause multiple organ failure, irreversible shock, and even death. The mortality rate of endotoxemiaIt is high. Bacterial endotoxin is the main cause of endotoxemia. At present, there is no safe and effective drug to treat endotoxemia in clinic. Research shows that blood purification can effectively reduce endotoxin level in the blood, then achieve the goal of treatment of endotoxemia. In this paper, the pathogenesis of endotoxemia, the development of hemoperfusion therapy technology, the mechanism and research status of endotoxin adsorption by different hemoperfusion resin were discussed, and the performance and safety requirements of hemoperfusion adsorbent materials for endotoxemia treatment were studied, so as to provide theoretical support for the synthesis of new hemoperfusion adsorption materials for the treatment of endotoxemia.

Effect of exercise training on heat shock protein 70 expression during endotoxin-induced acute lung / 中华麻醉学杂志

Objective To evaluate the effect of exercise training on heat shock protein 70 (HSP70) expression during endotoxin-induced acute lung injury (ALI) in rats.Methods Thirty-two SPF healthy male Sprague-Dawley rats,aged 8 weeks,weighing 175-220 g,were divided into 4 groups (n=8 each) using a random number table method:control group (group C),group ALI,low-intensity exercise training group (group ET1) and high-intensity exercise training group (group ET2).The rats in ET1 and ET2 groups received 2-and 4-week treadmill exercise training before establishing the ALI model,while the rats in C and ALI groups received no training.ALI was induced by intravenously injecting 5 mg/kg lipopolysaccharide via the tail vein in ALI,ET1 and ET2 groups,and the equal volume of normal saline was given instead in group C.The animals were sacrificed,and the lungs were harvested for microscopic examination of the pathological changes of lung tissues which were also scored and for determination of wet to dry weight ratio (W/D ratio),concentrations of total protein,interleukin-1beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) in bronchoalveolar lavage fluid (BALF),and expression of HSP70 and nuclear factor kappa B (NF-κB) in lung tissues by Western blot.Results Compared with group C,the W/D ratio and pathological changes of lung tissues were significantly increased,the concentrations of total protein,IL-1 β and TNF-α in BALF were increased,the expression of NF-κB was up-regulated (P＜0.05),and no significant change was found in HSP70 expression in group ALI(P＞0.05).Compared with group ALI,the W/D ratio and pathological changes of lung tissues were significantly decreased,the concentrations of total protein,IL-1β and TNF-α in BALF were decreased,the expression of HSP70 was up-regulated,and the expression of NF-κB was down-regulated in ET1 and ET2 groups (P＜0.05).Conclusion Exercise training can attenuate the endotoxin-induced ALI through relieving the inflammatory responses,which may be related to up-regulating HSP70 expression in the lung of rats.