Protective mechanisms of Leontopodium leontopodioides extracts on lipopolysaccharide-induced acute kidney injury viathe NF-κB/NLRP3 pathway / 中国天然药物

Sepsis-induced uncontrolled systemic inflammatory response syndrome (SIRS) is a critical cause of multiple organ failure. Acute kidney injury (AKI) is one of the most serious complications associated with an extremely high mortality rate in SIRS, and it lacked simple, safe, and effective treatment strategies. Leontopodium leontopodioides (Willd.) Beauv (LLB) is commonly used in traditional Chinese medicine for the treatment of acute and chronic nephritis. However, it remains unclear whether lipopolysaccharide (LPS) affects LPS-induced AKI. To identify the molecular mechanisms of LLB in LPS-induced HK-2 cells and mice, LLB was prepared by extraction with 70% methanol, while a lipopolysaccharide (LPS)-induced HK-2 cell model and an AKI model were established in this study. Renal histopathology staining was performed to observe the morphology changes. The cell supernatant and kidney tissues were collected for determining the levels of inflammatory factors and protein expression by ELISA, immunofluorescence, and Western blot. The results indicated that LLB significantly reduced the expression of IL-6 and TNF-α in LPS-induced HK-2 cells, as well as the secretion of IL-6, TNF-α, and IL-1β in the supernatant. The same results were observed in LPS-induced AKI serum. Further studies revealed that LLB remarkably improved oxidative stress and apoptosis based on the content of MDA, SOD, and CAT in serum and TUNEL staining results. Notably, LLB significantly reduced the mortality due to LPS infection. Renal histopathology staining results supported these results. Furthermore, immunofluorescence and Western blot results confirmed that LLB significantly reduced the expression of the protein related to the NF-κB signaling pathway and NLRP3, ASC, and Caspase-1 which were significantly increased through LPS stimulation. These findings clearly demonstrated the potential use of LLB in the treatment of AKI and the crucial role of the NF-κB/NLRP3 pathway in the process through which LLB attenuates AKI induced by LPS.

Effects of in vivo targeted carboxylesterase 1f gene knockdown on the Kupffer cells polarization activity in mice with acute liver failure / 中华肝脏病杂志

Objective: To investigate the effect of targeted carboxylesterase 1f (Ces1f) gene knockdown on the polarization activity of Kupffer cells (KC) induced by lipopolysaccharide/D-galactosamine (LPS/D-GalN) in mice with acute liver failure. Methods: The complex siRNA-EndoPorter formed by combining the small RNA (siRNA) carrying the Ces1f-targeting interference sequence and the polypeptide transport carrier (Endoporter) was wrapped in β-1, 3-D glucan shell to form complex particles (GeRPs). Thirty male C57BL/6 mice were randomly divided into a normal control group, a model group (LPS/D-GalN), a pretreatment group (GeRPs), a pretreatment model group (GeRPs+LPS/D-GalN), and an empty vector group (EndoPorter). Real-time fluorescent quantitative PCR and western blot were used to detect Ces1f mRNA and protein expression levels in the liver tissues of each mouse group. Real-time PCR was used to detect the expression levels of KC M1 polarization phenotypic differentiation cluster 86(CD86) mRNA and KC M2 polarization phenotypic differentiation cluster 163 (CD163) mRNA in each group. Immunofluorescence double staining technique was used to detect the expression of Ces1f protein and M1/M2 polarization phenotype CD86/CD163 protein in KC. Hematoxylin-eosin staining was used to observe the pathological damage to liver tissue. A one-way analysis of variance was used to compare the means among multiple groups, or an independent sample nonparametric rank sum test was used when the variances were uneven. Results: The relative expression levels of Ces1f mRNA/protein in liver tissue of the normal control group, model group, pretreatment group, and pretreatment model group were 1.00 ± 0.00, 0.80 ± 0.03/0.80 ± 0.14, 0.56 ± 0.08/0.52 ± 0.13, and 0.26 ± 0.05/0.29 ± 0.13, respectively, and the differences among the groups were statistically significant (F = 9.171/3.957, 20.740/9.315, 34.530/13.830, P < 0.01). The percentages of Ces1f-positive Kupffer cells in the normal control group, model group, pretreatment group, and pretreatment model group were 91.42%, ± 3.79%, 73.85% ± 7.03%, 48.70% ± 5.30%, and 25.68% ± 4.55%, respectively, and the differences between the groups were statistically significant (F = 6.333, 15.400, 23.700, P < 0.01). The relative expression levels of CD86 mRNA in the normal control group, model group, and pretreatment model group were 1.00 ± 0.00, 2.01 ± 0.04, and 4.17 ± 0.14, respectively, and the differences between the groups were statistically significant (F = 33.800, 106.500, P < 0.01). The relative expression levels of CD163 mRNA in the normal control group, the model group, and the pretreatment model group were 1.00 ± 0.00, 0.85 ± 0.01, and 0.65 ± 0.01, respectively, and the differences between the groups were statistically significant (F = 23.360, 55.350, P < 0.01). The percentages of (F4/80(+)CD86(+)) and (F4/80(+)CD163(+)) in the normal control group and model group and pretreatment model group were 10.67% ± 0.91% and 12.60% ± 1.67%, 20.02% ± 1.29% and 8.04% ± 0.76%, and 43.67% ± 2.71% and 5.43% ± 0.47%, respectively, and the differences among the groups were statistically significant (F = 11.130/8.379, 39.250/13.190, P < 0.01). The liver injury scores of the normal control group, the model group, and the pretreatment model group were 0.22 ± 0.08, 1.32 ± 0.36, and 2.17 ± 0.26, respectively, and the differences among the groups were statistically significant (F = 12.520 and 22.190, P < 0.01). Conclusion: Ces1f may be a hepatic inflammatory inhibitory molecule, and its inhibitory effect production may come from the molecule's maintenance of KC polarization phenotypic homeostasis.

Effects of total ginsenosides from Panax ginseng stems and leaves on gut microbiota and short-chain fatty acids metabolism in acute lung injury mice / 中国中药杂志

This study aimed to investigate the biological effects and underlying mechanisms of the total ginsenosides from Panax ginseng stems and leaves on lipopolysaccharide(LPS)-induced acute lung injury(ALI) in mice. Sixty male C57BL/6J mice were randomly divided into a control group, a model group, the total ginsenosides from P. ginseng stems and leaves normal administration group(61.65 mg·kg~(-1)), and low-, medium-, and high-dose total ginsenosides from P. ginseng stems and leaves groups(15.412 5, 30.825, and 61.65 mg·kg~(-1)). Mice were administered for seven continuous days before modeling. Twenty-four hours after modeling, mice were sacrificed to obtain lung tissues and calculate lung wet/dry ratio. The number of inflammatory cells in bronchoalveolar lavage fluid(BALF) was detected. The levels of interleukin-1β(IL-1β), interleukin-6(IL-6), and tumor necrosis factor-α(TNF-α) in BALF were detected. The mRNA expression levels of IL-1β, IL-6, and TNF-α, and the levels of myeloperoxidase(MPO), glutathione peroxidase(GSH-Px), superoxide dismutase(SOD), and malondialdehyde(MDA) in lung tissues were determined. Hematoxylin-eosin(HE) staining was used to observe the pathological changes in lung tissues. The gut microbiota was detected by 16S rRNA sequencing, and gas chromatography-mass spectrometry(GC-MS) was applied to detect the content of short-chain fatty acids(SCFAs) in se-rum. The results showed that the total ginsenosides from P. ginseng stems and leaves could reduce lung index, lung wet/dry ratio, and lung damage in LPS-induced ALI mice, decrease the number of inflammatory cells and levels of inflammatory factors in BALF, inhibit the mRNA expression levels of inflammatory factors and levels of MPO and MDA in lung tissues, and potentiate the activity of GSH-Px and SOD in lung tissues. Furthermore, they could also reverse the gut microbiota disorder, restore the diversity of gut microbiota, increase the relative abundance of Lachnospiraceae and Muribaculaceae, decrease the relative abundance of Prevotellaceae, and enhance the content of SCFAs(acetic acid, propionic acid, and butyric acid) in serum. This study suggested that the total ginsenosides from P. ginseng stems and leaves could improve lung edema, inflammatory response, and oxidative stress in ALI mice by regulating gut microbiota and SCFAs metabolism.

Xuebijing alleviates LPS-induced acute lung injury by downregulating pro-inflammatory cytokine production and inhibiting gasdermin-E-mediated pyroptosis of alveolar epithelial cells / 中国天然药物

Acute lung injury/acute respiratory distress syndrome (ALI/ARDS) is characterized by diffuse alveolar injury primarily caused by an excessive inflammatory response. Regrettably, the lack of effective pharmacotherapy currently available contributes to the high mortality rate in patients with this condition. Xuebijing (XBJ), a traditional Chinese medicine recognized for its potent anti-inflammatory properties, exhibits promise as a potential therapeutic agent for ALI/ARDS. This study aimed to explore the preventive effects of XBJ on ALI and its underlying mechanism. To this end, we established an LPS-induced ALI model and treated ALI mice with XBJ. Our results demonstrated that pre-treatment with XBJ significantly alleviated lung inflammation and increased the survival rate of ALI mice by 37.5%. Moreover, XBJ substantially suppressed the production of TNF-α, IL-6, and IL-1β in the lung tissue. Subsequently, we performed a network pharmacology analysis and identified identified 109 potential target genes of XBJ that were mainly involved in multiple signaling pathways related to programmed cell death and anti-inflammatory responses. Furthermore, we found that XBJ exerted its inhibitory effect on gasdermin-E-mediated pyroptosis of lung cells by suppressing TNF-α production. Therefore, this study not only establishes the preventive efficacy of XBJ in ALI but also reveals its role in protecting alveolar epithelial cells against gasdermin-E-mediated pyroptosis by reducing TNF-α release.

Mechanism of chlorogenic acid reducing lipopolysaccharide-induced acute lung injury in mice by regulating miR-223/NLRP3 axis / 中南大学学报(医学版)

OBJECTIVES@#Chlorogenic acid has various physiological activities such as antibacterial, anti-inflammatory, and antiviral activities. Studies have shown that chlorogenic acid can alleviate the inflammatory response of mice with acute lung injury (ALI), but the specific mechanism is still unclear. This study aims to investigate whether chlorogenic acid attenuates lipopolysaccharide (LPS)-induced ALI in mice by regulating the microRNA-223 (miR-223)/nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) axis.@*METHODS@#SPF grade BALBc male mice were randomly divided into a control group, a model group, a chlorogenic acid group, a chlorogenic acid+miR-223 negative control (miR-223 NC) group, and a chlorogenic acid+miR-223 inhibitor (miR-223 antagomir) group, 10 mice in each group. Except the control group, the other groups were instilled with 4 mg/kg LPS through the airway to establish the ALI mouse model. After the modeling, the mice in the chlorogenic acid group were continuously given chlorogenic acid (100 mg/kg) by gavage for 7 d. The chlorogenic acid+miR-223 NC group and the chlorogenic acid+miR-223 antagomir group were given 100 mg/kg chlorogenic acid by gavage every day, and then were injected with 10 μL of miR-223 NC (0.5 nmol/μL) and miR-223 antagomir (0.5 nmol/μL) respectively for 7 consecutive days.The control group and the model group were replaced with normal saline. The lung tissues of mice were taken to measure the ratios of lung wet to dry weight (W/D). The bronchoalveolar lavage fluid of mice was collected to measure the levels of TNF-α, IL-6, and IL-1β by ELISA kit and to count the number of eosinophils (EOS), lymphocytes, neutrophils under light microscope. After HE staining, the pathological changes of lung tissues were observed and lung injury was scored. qRT-PCR method were used to determine the expression levels of miR-223 in lung tissues. Western blotting was used to determine the expression levels of NLRP3 protein in mouse lung tissues. Luciferase reporter assay was used to analyze the targeting relationship of miR-223 to NLRP3.@*RESULTS@#Compared with the control group, the lung W/D value, the lung injury score and the level of inflammatory factors in the bronchoalveolar lavage fluid were significantly increased in the model group (all P<0.05); the infiltration of inflammatory cells in the lung tissue was severe; the alveolar space was significantly increased; the alveolar wall was significantly thickened; the number of EOS, lymphocytes, and neutrophils in the bronchoalveolar lavage fluid was significantly increased (all P<0.05); the expression levels of miR-223 in lung tissue were significantly decreased (P<0.05); and the protein expression levels of NLRP3 were significantly increased (P<0.05). Compared with the model group, the W/D value of lungs, lung injury score, and levels of inflammatory factors in bronchoalveolar lavage fluid were significantly decreased in the chlorogenic acid group, the chlorogenic acid+miR-223 NC group, and the chlorogenic acid+miR-223 antagomir group (all P<0.05); lung tissues damage was alleviated; the numbers of EOS, lymphocytes, and neutrophils in bronchoalveolar lavage fluid were significantly decreased (all P<0.05); the expression levels of miR-223 in lung tissues were significantly increased (P<0.05); and the expression levels of NLRP3 protein were significantly decreased (P<0.05). Compared with the chlorogenic acid group, the lung W/D value, lung injury score, and inflammatory factor levels in the bronchoalveolar lavage fluid were significantly increased in the chlorogenic acid+miR-223 antagomir group (all P<0.05); lung tissue damage was aggravated; the number of EOS, lymphocytes and neutrophils in bronchoalveolar lavage fluid significantly increased (all P<0.05); the expression levels of miR-223 in lung tissues were significantly decreased (P<0.05); and the expression levels of NLRP3 protein were significantly increased (P<0.05). The results of luciferase reporter assay showed that miR-223 had a targeting relationship with NLRP3.@*CONCLUSIONS@#Chlorogenic acid may increase the level of miR-223, target the inhibition of NLRP3 expression, reduce LPS-induced inflammatory response in ALI mice, and alleviate pathological damage of lung tissues.

Baicalin inhibits LPS/IFN-γ-induced inflammation via TREM2/TLR4/NF-κB pathway in BV2 cells / 中国中药杂志

This study investigated the mechanism of baicalin on lipopolysaccharide(LPS)/interferon γ(IFN-γ)-induced inflammatory microglia based on the triggering receptor expressed on myeloid cells 2(TREM2)/Toll-like receptor 4(TLR4)/nuclear factor kappaB(NF-κB) pathway. Specifically, LPS and IFN-γ were used to induce inflammation in mouse microglia BV2 cells. Then the normal group, model group, low-dose(5 μmol·L~(-1)) baicalin group, medium-dose(10 μmol·L~(-1)) baicalin group, high-dose(20 μmol·L~(-1)) baicalin group, and minocycline(10 μmol·L~(-1)) group were designed. Cell viability was detected by CCK-8 assay and cell morphology was observed under bright field. The expression of interleukin-1β(IL-1β), interleukin-4(IL-4), inducible nitric oxide synthase(iNOS), interleukin-6(IL-6), interleukin-10(IL-10), and arginase-1(Arg-1) mRNA was detected by real-time quantitative PCR, the protein expression of tumor necrosis factor-α(TNF-α), IL-1β, TREM2, TLR4, inhibitor kappaB-alpha(IκBα), p-IκBα, NF-κB p65 and p-NF-κB p65 by Western blot, and transfer of NF-κB p65 from cytoplasm to nucleus by cellular immunofluorescence. Compared with the normal group, most of the BV2 cells in the model group tended to demonstrate the pro-inflammatory M1 amoeba morphology, and the model group showed significant increase in the mRNA levels of IL-1β, IL-6, and iNOS, decrease in the mRNA levels of IL-4, IL-10, and Arg-1(P<0.01), rise of the protein expression of TNF-α, IL-1β, TLR4, p-IκBα, and p-NF-κB p65(P<0.01), reduction in TREM2 protein expression, and increase in the expression of NF-κB p65 in nucleus. Compared with the model group, baicalin groups and minocycline group showed the recovery of BV2 cell morphology, significant decrease in the mRNA levels of IL-1β, IL-6 and iNOS, increase in the mRNA levels of IL-4, IL-10, and Arg-1(P<0.01), reduction in the protein expression of TNF-α, IL-1β, TLR4, p-IκBα, and p-NF-κB p65(P<0.05), rise of TREM2 protein expression, and decrease in the expression of NF-κB p65 in nucleus. In summary, these results suggest that baicalin can regulate the imbalance between TREM2 and TLR4 of microglia and inhibit the activation of downstream NF-κB, thus promoting the polarization of microglia from pro-inflammatory phenotype to anti-inflammatory phenotype.

Effects of non-muscle myosin Ⅱ silenced bone marrow-derived mesenchymal stem cells transplantation on lung extracellular matrix in rats after endotoxin/lipopolysaccharide-induced acute lung injury / 中华烧伤杂志

Objective: To investigate the effects of non-muscle myosin Ⅱ (NMⅡ) gene silenced bone marrow-derived mesenchymal stem cells (BMMSCs) on pulmonary extracellular matrix (ECM) and fibrosis in rats with acute lung injury (ALI) induced by endotoxin/lipopolysaccharide (LPS). Methods: The experimental research methods were adopted. Cells from femur and tibial bone marrow cavity of four one-week-old male Sprague-Dawley rats were identified as BMMSCs by flow cytometry, and the third passage of BMMSCs were used in the following experiments. The cells were divided into NMⅡ silenced group transfected with pHBLV-U6-ZsGreen-Puro plasmid containing small interference RNA sequence of NMⅡ gene, vector group transfected with empty plasmid, and blank control group without any treatment, and the protein expression of NMⅡ at 72 h after intervention was detected by Western blotting (n=3). The morphology of cells was observed by an inverted phase contrast microscope and cells labeled with chloromethylbenzoine (CM-DiⅠ) in vitro were observed by an inverted fluorescence microscope. Twenty 4-week-old male Sprague-Dawley rats were divided into blank control group, ALI alone group, ALI+BMMSC group, and ALI+NMⅡ silenced BMMSC group according to the random number table, with 5 rats in each group. Rats in blank control group were not treated, and rats in the other 3 groups were given LPS to induce ALI. Immediately after modeling, rats in ALI alone group were injected with 1 mL normal saline via tail vein, rats in ALI+BMMSC group and ALI+NMⅡ silenced BMMSC group were injected with 1×107/mL BMMSCs and NMⅡ gene silenced BMMSCs of 1 mL labelled with CM-DiⅠ via tail vein, and rats in blank control group were injected with 1 mL normal saline via tail vein at the same time point, respectively. At 24 h after intervention, the lung tissue was collected to observe intrapulmonary homing of the BMMSCs by an inverted fluorescence microscope. Lung tissue was collected at 24 h, in 1 week, and in 2 weeks after intervention to observe pulmonary inflammation by hematoxylin eosin staining and to observe pulmonary fibrosis by Masson staining, and the pulmonary fibrosis in 2 weeks after intervention was scored by modified Ashcroft score (n=5). The content of α-smooth muscle actin (α-SMA), matrix metalloproteinase 2 (MMP-2), and MMP-9 was detected by immunohistochemistry in 2 weeks after intervention (n=3), the activity of superoxide dismutase (SOD), malondialdehyde, myeloperoxidase (MPO) was detected by enzyme-linked immunosorbent assay at 24 h after intervention (n=3), and the protein expressions of CD11b and epidermal growth factor like module containing mucin like hormone receptor 1 (EMR1) in 1 week after intervention were detected by immunofluorescence staining (n=3). Data were statistically analyzed with one-way analysis of variance, Bonferroni method, and Kruskal-Wallis H test. Results: At 72 h after intervention, the NMⅡprotein expression of cells in NMⅡ silenced group was significantly lower than those in blank control group and vector group (with P values <0.01). BMMSCs were in long spindle shape and grew in cluster shaped like vortexes, which were labelled with CM-DiⅠ successfully in vitro. At 24 h after intervention, cell homing in lung of rats in ALI+NMⅡ silenced BMMSC group was more pronounced than that in ALI+BMMSC group, while no CM-DiⅠ-labelled BMMSCs were observed in lung of rats in blank control group and ALI alone group. There was no obvious inflammatory cell infiltration in lung tissue of rats in blank control group at all time points, while inflammatory cell infiltration in lung tissue of rats in ALI+BMMSC group and ALI+NMⅡ silenced BMMSC group was significantly less than that in ALI alone group at 24 h after intervention, and alveolar wall turned to be thinner and a small amount of congestion in local lung tissue appeared in rats of the two groups in 1 week and 2 weeks after intervention. In 1 week and 2 weeks after intervention, collagen fiber deposition in lung tissue of rats in ALI alone group, ALI+BMMSC group, and ALI+NMⅡ silenced BMMSC group was significantly aggravated compared with that in blank control group, while collagen fiber deposition in lung tissue of rats in ALI+BMMSC group and ALI+NMⅡ silenced BMMSC group was significantly improved compared with that in ALI alone group. In 2 weeks after intervention, modified Ashcroft scores for pulmonary fibrosis of rats in ALI alone group, ALI+BMMSC group, and ALI+NMⅡ silenced BMMSC group were 2.36±0.22, 1.62±0.16, 1.06±0.26, respectively, significantly higher than 0.30±0.21 in blank control group (P<0.01). Modified Ashcroft scores for pulmonary fibrosis of rats in ALI+BMMSC group and ALI+NMⅡ silenced BMMSC group were significantly lower than that in ALI alone group (P<0.01), and modified Ashcroft score for pulmonary fibrosis of rats in ALI+NMⅡ silenced BMMSC group was significantly lower than that in ALI+BMMSC group (P<0.01). In 2 weeks after intervention, the content of α-SMA in lung tissue of rats in ALI+BMMSC group and ALI+NMⅡ silenced BMMSC group were significantly decreased compared with that in ALI alone group (P<0.05 or P<0.01). The content of MMP-2 in lung tissue of rats in the 4 groups was similar (P>0.05). The content of MMP-9 in lung tissue of rats in ALI alone group was significantly increased compared with that in blank control group (P<0.01), and the content of MMP-9 in lung tissue of rats in ALI+BMMSC group and ALI+NMⅡ silenced BMMSC group was significantly decreased compared with that in ALI alone group (P<0.01). At 24 h after intervention, the activity of malondialdehyde, SOD, and MPO in lung tissue of rats in ALI alone group, ALI+BMMSC group, and ALI+NMⅡ silenced BMMSC group were significantly increased compared with that in blank control group (P<0.01), the activity of malondialdehyde in lung tissue of rats in ALI+NMⅡ silenced BMMSC group and the activity of SOD in lung tissue of rats in ALI+BMMSC group and ALI+NMⅡ silenced BMMSC group were significantly increased compared with that in ALI alone group (P<0.05 or P<0.01), and the activity of SOD in lung tissue of rats in ALI+NMⅡ silenced BMMSC group was significantly decreased compared with that in ALI+BMMSC group (P<0.01). The activity of MPO in lung tissue of rats in ALI+BMMSC group and ALI+NMⅡ silenced BMMSC group was significantly decreased compared with that in ALI alone group (P<0.01), and the activity of MPO in lung tissue of rats in ALI+NMⅡ silenced BMMSC group was significantly decreased compared with that in ALI+BMMSC group (P<0.01). In 1 week after intervention, the protein expression of CD11b in lung tissue of rats in ALI+NMⅡ silenced BMMSC group was significantly increased compared with those in the other three groups (P<0.05 or P<0.01), while the protein expressions of EMR1 in lung tissue of rats in the four groups were similar (P>0.05). Conclusions: Transplantation of NMⅡ gene silenced BMMSCs can significantly improve the activity of ECM components in the lung tissue in LPS-induced ALI rats, remodel its integrity, and enhance its antioxidant capacity, and alleviate lung injury and pulmonary fibrosis.

Liver fibrosis inhibits lethal injury through D-galactosamine/lipopolysaccharide-induced necroptosis / 中华肝脏病杂志

Objective: To explore the new mechanism of liver fibrosis through D-galactosamine/lipopolysaccharide (D-GalN/LPS)-induced necroptosis as an entry point to inhibit lethal injury. Methods: The carbon tetrachloride (CCl4)-induced mouse model of liver fibrosis was established. At 6 weeks of fibrosis, the mice were challenged with a lethal dose of D-GalN/LPS, and the normal mice treated with the same treatment were used as the control. The experiment was divided into four groups: control group (Control), acute injury group (D-GalN/LPS), liver fibrosis group (Fib), and liver fibrosis + acute challenge group (Fib + D-GalN/LPS). Quantitative PCR and immunofluorescence were used to analyze the expression of necroptosis key signal molecules RIPK1, RIPK3, MLKL and/or P-MLKL in each group. Normal mice were treated with inhibitors targeting key signaling molecules of necroptosis, and then given an acute challenge. The inhibitory effect of D-GalN/LPS-induced-necroptosis on acute liver injury was evaluated according to the changes in transaminase levels and liver histology. Liver fibrosis spontaneous ablation model was established, and then acute challenge was given. Necroptosis key signal molecules expression was analyzed in liver tissue of mice in each group and compared by immunohistochemistry. The differences between groups were compared with t-test or analysis of variance. Results: Quantitative PCR and immunofluorescence assays result showed that D-GalN/LPS-induced significant upregulation of RIPK1, RIPK3, MLKL and/or P-MLKL. Necroptosis key signal molecules inhibition had significantly reduced D-GalN/LPS-induced liver injury, as manifested by markedly reduced serum ALT and AST levels with improvement in liver histology. Necroptosis signaling molecules expression was significantly inhibited in fibrotic livers even under acute challenge conditions. Additionally, liver fibrosis with gradual attenuation of fibrotic ablation had inhibited D-GalN/LPS-induced necroptosis. Conclusion: Liver fibrosis may protect mice from acute lethal challenge injury by inhibiting D-GalN/LPS-induced necroptosis.

Anti-inflammation and anti-apoptosis effects of growth arrest-specific protein 6 in acute liver injury induced by LPS/D-GalN in mice

Abstract Purpose To investigate the effect of growth arrest-specific protein 6 (Gas6) on acute liver injury in mice and related mechanisms. Methods Thirty C57BL/6 (6-8 weeks old) mice were randomly divided into control, LPS/D-GalN, and LPS/D-GalN+Gas6 groups (10 mice in each group). The LPS/D-GalN group was intraperitoneally administered with LPS (0.25 mg/Kg) and D-GalN (400 mg/Kg) for 5h. The LPS/D-GalN+Gas6 group was intraperitoneally administered with rmGas6 one hour before intraperitoneal application of LPS/D-GalN. All subjects were sacrificed at 5 h for blood and tissue analysis. The expression of protein and mRNA was assessed by western blotting and RT-PCR, respectively. Results Compared with the control group, AST, ALT, IL-1β, TNF-α, IL-6 IL-10, MPO activity were increased in the LPS/D-GalN group. However, they were significantly inhibited by Gas6. Gas6 markedly suppressed the expression of apoptosis-related protein induced by LPS/D-GalN. Moreover, Gas6 attenuated the activation of the NF-κB signaling pathway in acute liver injury induced by LPS/D-GalN. Conclusions Gas6 alleviates acute liver injury in mice through regulating NF-κB signaling pathways. Gas6 can be a potential therapeutic agent in treating LPS/D-GalN-induced acute liver injury in the future.

Anti-inflammatory Effect of a Goji Berry Extract (Lycium barbarum) in Rats Subjected to Inflammation by Lipopolysaccharides (LPS)

Abstract The present study aimed to evaluate the anti-inflammatory potential of a Lycium barbarum (L. barbarum) fruit extract in Wistar rats submitted to a palatable diet presenting systemic inflammation induced by lipopolysaccharides (LPS). Forty-two Wistar female rats (Rattus Novergicus) were used with 60 days old. The animals were feed for 60 days and divided in six groups (n=7): standard diet+water; standard diet+L. barbarum; palatable diet+water; palatable diet+L. barbarum; standard diet+water+LPS; standard diet+L. barbarum+LPS. A significant difference was shown between the analyzed groups concerning C-reactive protein, with the standard diet+water+LPS group presenting the highest inflammatory response in comparison to the other groups. Decreased inflammatory response was observed in the group administered a palatable diet along with the fruit extract when compared to the group that received only a palatable diet. Significant decrease in glutamic-oxaloacetic transaminase activity was observed in the standard diet+L. barbarum+LPS group compared to the standard diet+water group, as well as in the palatable diet+L. barbarum group compared to the palatable diet+water group. A significant increase in creatinine in the standard diet+water+LPS group was observed in according to the L. barbarum administration groups. The gene expression of the inflammatory markers genes in the liver showed a significant increase in TNF-α and IL-6 genes in the group treated with standard diet+L. barbarum+LPS when compared to the standard diet+LPS group. Thus, the administered L. barbarum extract displays the potential to reduce inflammatory responses induced by LPS and a palatable diet.

Acrocomia crispa fruits lipid extract prevents LPS-induced acute lung injury in mice / Extracto lipídico de los frutos de Acrocomia crispa previene el daño pulmonar agudo inducido por LPS en ratones

The aim of this study was to evaluate the effects of single oral doses of D-005 (a lipid extract obtained from the fruit oil of Acrocomia crispa) on LPS-induced acute lung injury (ALI) in mice. D-005 batch composition was: lauric (35.8%), oleic (28.4%), myristic (14.2%), palmitic (8.9%), stearic (3.3%), capric (1.9%), caprylic (1.2%), and palmitoleic (0.05%) acids, for a total content of fatty acids of 93.7%. D-005 (200 mg/kg) significantly reduced lung edema (LE) (≈ 28% inhibition) and Lung Weight/Body Weight ratio (LW/BW) (75.8% inhibition). D-005 (25, 50, 100 and 200 mg/kg) produced a significant reduction of Histological score (59.9, 56.1, 53.5 and 73.3% inhibition, respectively). Dexamethasone, as the reference drug, was effective in this experimental model. In conclusion, pretreatment with single oral doses of D-005 significantly prevented the LPS-induced ALI in mice.

El objetivo de este estudio fue evaluar los efectos de dosis orales únicas de D-005 (extracto lipídico obtenido del aceite de frutos de Acrocomia crispa) sobre el daño pulmonar agudo (DPA) inducido por LPS en ratones. La composición del lote de D-005 fue: ácido láurico (35.8%), oleico (28.4%), mirístico (14.2%), palmítico (8.9%), esteárico (3.3%), cáprico (1.9%), caprílico (1.2%) y palmitoleico (0.05%), con un contenido total de ácidos grasos de 93.7%. D-005 (200 mg/kg) redujo significativamente el edema pulmonar (EP) (≈ 28% de inhibición) y la relación peso pulmón/peso corporal (PP/PC) (75.8% de inhibición). D-005 (25, 50, 100 y 200 mg/kg) produjo una reducción significativa de la puntuación histológica (59.9, 56.1, 53.5 y 73.3% de inhibición, respectivamente). La dexametasona, fármaco de referencia, fue efectiva en este modelo experimental. En conclusión, el pretratamiento con dosis orales únicas de D-005 previno significativamente el DPA inducido por LPS en ratones.

Dexmedetomidine preconditioning protects against lipopolysaccharides-induced injury in the human alveolar epithelial cells / Pré-condicionamento com dexmedetomidina protege contra lesões induzidas por lipopolissacarídeos em células epiteliais alveolares humanas

Abstract Background and objectives Dexmedetomidine (DEX) has demonstrated the preconditioning effect and shown protective effects against organize injury. In this study, using A549 (human alveolar epithelial cell) cell lines, we investigated whether DEX preconditioning protected against acute lung injury (ALI) in vitro. Methods A549 were randomly divided into four groups (n = 5): control group, DEX group, lipopolysaccharides (LPS) group, and D-LPS (DEX + LPS) group. Phosphate buffer saline (PBS) or DEX were administered. After 2 h preconditioning, the medium was refreshed and the cells were challenged with LPS for 24 h on the LPS and D-LPS group. Then the malondialdehyde (MDA), superoxide dismutase (SOD), Bcl-2, Bax, caspase-3 and the cytochrome c in the A549 were tested. The apoptosis was also evaluated in the cells. Results Compare with LPS group, DEX preconditioning reduced the apoptosis (26.43% ± 1.05% vs. 33.58% ± 1.16%, p < 0.05) in the A549, which is correlated with decreased MDA (12.84 ± 1.05 vs. 19.16 ± 1.89 nmoL.mg-1 protein, p < 0.05) and increased SOD activity (30.28 ± 2.38 vs. 20.86 ± 2.19 U.mg-1 protein, p < 0.05). DEX preconditioning also increased the Bcl-2 level (0.53 ± 0.03 vs. 0.32 ± 0.04, p < 0.05) and decreased the level of Bax (0.49 ± 0.04 vs. 0.65 ± 0.04, p < 0.05), caspase-3 (0.54 ± 0.04 vs. 0.76 ± 0.04, p < 0.05) and cytochrome c. Conclusion DEX preconditioning has a protective effect against ALI in vitro. The potential mechanisms involved are the inhibition of cell death and improvement of antioxidation.

Resumo Justificativa e objetivos Dexmedetomidina (DEX) demonstrou ter efeito pré-condicionante e também efeitos protetores contra lesão organizada. Neste estudo, com células A549 (células epiteliais alveolares humanas), investigamos se o pré-condicionamento com DEX proporcionaria proteção contra lesão pulmonar aguda (LPA) in vitro. Métodos Células A549 foram aleatoriamente distribuídas em quatro grupos (n = 5): controle, DEX, lipopolissacarídeos (LPS) e D-LPS (DEX + LPS). Administramos solução de PBS (tampão fosfato-alcalino) ou DEX. Após 2 h de pré-condicionamento, o meio foi renovado e as células desafiadas com LPS por 24 h nos grupos LPS e D-LPS. Em seguida, malondialdeído (MDA), superóxido dismutase (SOD), Bcl-2, Bax, caspase-3 e em A549 foram testados. Apoptose também foi avaliada nas células. Resultados Em comparação com o grupo LPS, o pré-condicionamento com DEX reduziu a apoptose (26,43% ± 1,05% vs. 33,58% ± 1,16%, p < 0,05) em células A549, o que está correlacionado com a diminuição de MDA (12,84 ± 1,05 vs. 19,16 ± 1,89 nmol.mg-1 de proteína, p < 0,05) e aumento da atividade de SOD (30,28 ± 2,38 vs. 20,86 ± 2,19 U.mg-1 de proteína, p < 0,05). O pré-condicionamento com DEX também aumentou o nível de Bcl-2 (0,53 ± 0,03 vs. 0,32 ± 0,04, p < 0,05) e diminuiu o nível de Bax (0,49 ± 0,04 vs. 0,65 ± 0,04, p < 0,05), caspase-3 (0,54 ± 0,04 vs. 0,76 ± 0,04, p < 0,05) e citocromo c. Conclusão O pré-condicionamento com DEX tem efeito protetor contra LPA in vitro. Os potenciais mecanismos envolvidos são inibição da morte celular e melhoria da antioxidação.

Neonatal lipopolysaccharide exposure induces sexually dimorphic sickness behavior in adult rats

The aim of the present study was to evaluate whether neonatal exposure to lipopolysaccharide (LPS; 50 µg/kg, i.p., on postnatal day 2) induces depressive- and/or anxiety-like effects and sexually dimorphic responses in rats challenged with LPS (100 µg/kg, i.p.) in adulthood. The results revealed that males presented a less depressive state in the forced swim test and exhibited no changes in general motor activity in the open field test. Females exhibited an increase in sickness behavior, revealing different behavioral strategies in response to a bacterial disease. The male rats also exhibited higher cell proliferation, reflected by bone marrow and peripheral blood counts, and female rats exhibited a decrease in corticosterone levels. No changes were observed in the elevated plus maze or peripheral cytokine levels (interleukin-1β and tumor necrosis factor-α). Neonatal exposure to LPS induced sexually dimorphic behavioral, neuroendocrine, and immune effects after an LPS challenge in adulthood, differentially affecting male and female susceptibility to disease later in life...

Efecto de los antagonistas de los receptores AT1 de la angiotensina II sobre la enfermedad periodontal inducida por inyecciones de lipopolisacáridos en ratas / Effect of ang II AT1 receptor antagonists, on periodontal disease induced by injections of lipopolysaccharide in rats

La Enfermedad Periodontal (EP) es una condición inflamatoria progresiva que afecta los tejidos que soportan y rodean a los dientes. Las endotoxinas bacterianas como los lipopolisacáridos (LPS), inducen una cascada inflamatoria causando resorción ósea mediante la producción y modulación de la red de citoquinas del tejido periodontal, del sistema RANK-RANKL-OPG y de la producción de especies reactivas del oxígeno (ERO). Siendo la vía final la activación del factor de transcripción NFκB para el control de la infección. Se sabe que el Sistema Renina Angiotensina (SRA) esta involucrado en la inflamación. Estas acciones pro-inflamatorias de la Ang II son producidas por la activación de NFkB mediante los receptores AT1, y por la generación de ERO. Nuestro objetivo fue determinar si la inhibición del receptor AT1 con el uso del valsartán reduciría la respuesta inmunitaria innata inflamatoria en un modelo de EP inducida por las inyecciones de LPS en la encía de las ratas. Nuestros resultados demuestran que el Valsartán disminuyó la leucocitosis sistémica, la movilidad dentaria, atenuó la pérdida de peso corporal de las ratas, disminuyó la formación de enzimas antioxidantes y NOS, redujo la producción y liberación de citocinas pro inflamatorias y aumentó las citocinas antinflamatorias, disminuyó la activación de p-p38, p-NFkB y la expresión de los receptores TLR4. El Valsartán también revirtió los efectos del LPS sobre la resorción ósea ya que disminuyó el número de osteoclastos, la expresión de los receptores RANK/RANKL/OPG y la relación RANKL/OPG y aumentó los depósitos de calcio y colágeno. Los mecanismos por los cuales el Valsartán reduce los efectos inflamatorios producidos por el LPS no están claras, pero la interferencia del ensamblaje de la NAD(P)H oxidasa con apocinina y el Tempol, indica que el Valsartán puede interferir con los pasos para el reconocimiento de LPS y su asociación con TLR4. Concluyéndose que las ERO participan en la señalización intracelular de la ANG II, vía el AT1R. Este estudio ayuda a dilucidar el papel del SRA en procesos inflamatorios. Además contribuye con sus resultados a ofrecer una alternativa terapéutica en el tratamiento de la EP.

Effects of prenatal lipopolysaccharide exposure on reproductive activities and serum concentrations of pituitary-gonadal hormones in mice offspring

Maternal infection during pregnancy is a risk factor for some behavioral problems with neurodevelopmental origin. This study aimed to evaluate the effects of exposure of pregnant mice to the bacterial lipopolysaccharide [LPS] on sexual behaviour and serum level of pituitary-gonadal hormones of offspring in adulthood. In this Expremental study, pregnant NMRI mice [n=7/group] were treated with intra-peritoneal administration of LPS [1, 5 and 10 micro g/kg] at day 10 of gestation. Induction of the pro-inflammatory cytokines, Tumor necrosis factor-alpha [TNF-alpha], interleukin-1beta [IL-1beta] and interleukin-6 [IL-6] were measured in maternal serum 2 hours following the maternal LPS challenge. Behavior in the adult male offspring reproductive activity was investigated using receptive female mice. Concentrations of testosterone, luteinizing hormone [LH] and follicle-stimulating hormone [FSH] in adult offspring serum were measured using the enzyme-linked immunosorbent assay [ELISA] method [at postnatal day 60, n=10/group]. One-way ANOVA showed that LPS administration induces a significant increase in TNF-alpha, IL-1beta and IL-6 levels of maternal serum. Prenatal LPS exposure reduces sexual behavior and serum concentration of LH and testosterone in adult male offspring. The overall results suggest that prenatal exposure to LPS increases pro-inflammatory cytokine levels, affects development of neuroendocrine systems and results in the inhibition of reproductive behaviors and reactivity of hypothalamic-pituitary-gonadal [HPG] axis in adult male offspring

Effect of D-003, a mixture of high molecular weight aliphatic acids, on glucocorticoid- and lipopolysaccharides [LPS]-induced osteonecrosis

Osteonecrosis [ON] is characterized through the impairment of osseous blood flow that leads to the collapse of femur head. Corticoid-induced ON in rats and lipopolysaccharide [LPS]-induced in rabbits are useful models to assess the efficacy of potential treatments on this disease. D-003 inhibits the mevalonate pathway, lipid peroxidation and prevents osteoporosis in rats through increasing the osteoclast apoptosis. This study investigated the effects of D-003 on corticoid- and LPS-induced ON in rats and rabbits. Corticoid-induced ON: Rats were randomized into five groups. A negative control and four groups treated with prednisolone 6 mg/Kg: a positive control and three treated with D-003 [5, 25 and 200 mg/Kg] for 80 days. All positive controls presented ON areas. D-003 significantly reduced the numbers and proportions of ON lesions, as compared to the positive control group. LPS-induced ON in rabbits: Rabbits were randomized into five groups: a negative control and four injected with a single intra-venous injection of LPS [10 micro g/Kg] including a positive control and three with D-003 [5, 25 and 200 mg/Kg] for 30 days. ON was seen in all positive controls. The incidence of ON and the number of ON lesions in the groups treated with D-003 [25 and 200 mg/Kg] was significantly lower compared to the positive controls. LPS injection significantly increased the size of bone marrow fat cells in positive controls and such increase was significantly decreased by D-003. In conclusion, D-003 reduced ON lesions in corticoid-and LPS-induced ON and also the size of bone marrow fat cells in rabbits with LPS

Acellular and "low" pertussis vaccines: adverse events and the role of mutations / Vacinas pertussis acelular e pertussis "low": eventos adversos e o papel das mutações

Objective: to discuss the current PAHO recommendation that does not support the substitution of traditional cellular DTP vaccine by acellular DTP, and the role of mutations, in humans, as the main cause of rare adverse events, such as epileptic-like convulsions, triggered by pertussis vaccine. Data review: the main components related to toxic effects of cellular pertussis vaccines are the lipopolysaccharide of bacterial cell wall and pertussis toxin. The removal of part of lipopolysaccharide layer has allowed the creation of a safer cellular pertussis vaccine, with costs comparable to the traditional cellular vaccine, and which may be a substitute for the acellular vaccine. Conclusion: The new methodology introduced by Instituto Butantan allows for the development of a new safer pertussis vaccine with low LPS content (Plow), and the use of the lipopolysaccharide obtained in the process in the production of monophosphoryl lipid A. This component has shown potent adjuvant effect when administered together with influenza inactivated vaccine, making possible to reduce the antigen dose, enhancing the production capacity and lowering costs.

Objetivo: Discutir as recomendações da WHO-OPAS que não consideram indicada a substituição da vacina DTP celular clássica pela DTP acelular e o papel de mutações, em humanos, como principal causa dos raros eventos de convulsões epileptiformes desencadeadas pela vacina pertussis. Revisão dos dados: Os principais componentes relacionados aos efeitos tóxicos da vacina pertussis celular são o lipopolissacarídio da parede celular da bactéria e a toxina pertussis. A remoção de parte da camada lipopolissacarídica permitiu a criação de uma vacina pertussis celular, mais segura e de custo comparável ao da vacina celular tradicional, podendo substituir a vacina pertussis acelular. Conclusão: A nova vacina pertussis, com baixo teor de LPS (Plow) desenvolvida pelo Instituto Butantan, além de oferecer uma vacina mais segura, permite o aproveitamento do lipopolissacarídeo para a produção de monofosforil lipídeo A. Esse componente mostrou-se potente como adjuvante e altamente eficiente quando administrado com a vacina de influenza, levando à possibilidade de se reduzir a dose de antígeno, aumentando a capacidade de produção e redução dos custos.

Influence of bacterial endotoxins on bone marrow and blood components

Endotoxin [Lipopolysaccharide, LPS] a component of the bacterial wall of gram-negative bacteria, has been recognized as one of the most potent bacterial products in the induction of host inflammatory responses and tissue injury and was used in this study to mimic infections. LPS induces production and release of several cytokines. In response to these cytokines, different effects of endotoxins are seen. The effect of three types of endotoxins [Escherichia coli, Klebsiella pneumoniae and Salmonella typhimurium] on bone marrow, differential counts and peripherial blood parameters were investigated in adult rats. Male spraguo Dawely albino rats weighing 220 - 250 g were used. They were injected i.p. [1 mg/kg body weight] with single dose of 3 types of endotoxins. Blood samples were collected from the experimental animals at 24 and 72 hours of the injection. At 72 hours the bone marrow aspirations were harvested from the femur of the rats for microscopic examination. Endotoxins induced different changes in the cells of bone marrow. Also, lipopolysaccharide caused significant decreases in red blood cells, white blood cells and platelets counts, hemoglobin content and hematocrit percent. Data of the present study point out to the dose of these toxins according to suitable pharmacopeia. Lemulus amebocyte lysate [LAL] test is specificly used for determination of the endotoxin limit. This recommendation should be observed to avoid the toxic effects of endotoxins

protective effect of some polyphenolic compounds on lipopolysaccharide-induced hepatitis in D-galactosamine sensitized rats

To study the hepatoprotective effect of teas and cocoa extracts against liver injury and to know the potent effect of each in protecting the liver from Lipopolysaccharide-induced hepatitis in D-galactosamine sensitized rats. Rats were divided into eight groups; group I received saline, groups II, III and IV received black tea, green tea and cocoa extracts respectively orally for one month; group V received saline, groups VI, VII and VIII received black tea, green tea and cocoa extracts respectively orally for one month before induction of hepatitis. Indicated that the prophylactic study significantly improved serum hepatic enzymes; liver oxidants/antioxidants profile and serum tumor necrosis factor-a levels compared to DGa1N / LPS group. Green tea extract showed the maximum improvement in liver enzymes and oxidants/antioxidants profile in prophylactic groups. Also cocoa extract showed the maximum improvement in tumor necrosis factor-a levels compared to green and black tea prophylactic groups. The antihepatotoxic effect of teas and cocoa was attributed to their free radical scavenging antioxidants [catechins, epicatechins and procyanidins] which protected the liver from oxidative damage

insight into the possible protective effect of pyrrolidine dithiocarbamate against lipopolysaccharide- induced oxidative stress and acute hepatic injury in rats

Lipopolysaccharide [LPS] is a major cell wall molecule of Gram-negative bacteria known to stimulate the synthesis and secretion of several toxic metabolites, such as reactive oxygen species. In this study, the effect of pyrrolidine dithiocarbamate [PDTC], an antioxidant with nuclear factor- kappa B inhibitor activity, was evaluated in LPS-induced oxidative stress and acute hepatic injury in rats. Animals were pre-treated for 3 consecutive days with PDTC[200 mg/kg/day, i.p.] or saline and animals were then challenged with LPS[6 mg/kg, i.p.] or saline. Six hours after LPS injection, animals were decapitated and blood and liver samples were collected to assess the chosen biochemical parameters. Saline-pretreated animals challenged with LPS revealed extensive liver damage, as evidenced by increase in serum levels of alanine aminotransferase [ALT], aspirate aminotransferase [AST] and gamma glutamyl transferase [gamma -GT]. Also, LPS treatment resulted in significant increases in serum lactate dehydrogenase [LDH], tumor necrosis factor-alpha [TNF- alpha] and nitrite levels. Furthermore, LPS challenge caused oxidative stress as indicated by an increase in hepatic lipid peroxidation measured as thiobarbituric acid reactive substances [TBARS] and a decrease in hepatic reduced glutathione concentration [GSH] as well as decreased activities of superoxide dismutase [SOD] and [GSH] as well as decreased activities of superoxide dismutase [SOD] and catalase in hepatic tissues. The administration of PDTC prior to LPS challenge resulted in improved liver functions as evidenced by the decline in serum AST, ALT, gamma-GT levels and reduction in serum LDH, TNF- alpha and nitrite levels. Moreover, PDTC reduced the chosen lipid peroxidation marker, TBARS and increased GSH concentration, and SOD and catalase activities in hepatic tissues. These results indicate that PDTC may be a useful pharmacological agent in alleviating LPS-induced oxidative stress and acute hepatic injury