Protective Effect of Silibinin on Lipopolysaccharide-Induced Endotoxemia by Inhibiting Caspase-11-Dependent Cell Pyroptosis.

OBJECTIVE: To explore the protective effect and the underlying mechanism of silibinin (SIB), one of the active compounds from Silybum marianum (L.) Gaertn in endotoxemia. METHODS: Mouse peritoneal macrophage were isolated via intraperitoneally injection of BALB/c mice with thioglycolate medium. Cell viability was assessed using the cell counting kit-8, while cytotoxicity was determined through lactate dehydrogenase cytotoxicity assay. The protein expressions of interleukin (IL)-1 α, IL-1 ß, and IL-18 were determined by enzyme-linked immunosorbent assay. Intracellular lipopolysaccharide (LPS) levels were measured by employing both the limulus amoebocyte lysate assay and flow cytometry. Additionally, proximity ligation assay was employed for the LPS and caspase-11 interaction. Mice were divided into 4 groups: the control, LPS, high-dose-SIB (100 mg/kg), and low-dose-SIB (100 mg/kg) groups (n=8). Zebrafish were divided into 4 groups: the control, LPS, high-dose-SIB (200 εmol/L), and low-dose-SIB (100 εmol/L) groups (n=30 for survival experiment and n=10 for gene expression analysis). The expression of caspase-11, gasdermin D (GSDMD), and N-GSDMD was determined by Western blot and the expressions of caspy2, gsdmeb, and IL-1 ß were detected using quantitative real-time PCR. Histopathological observation was performed through hematoxylineosin staining, and protein levels in bronchoalveolar lavage fluid were quantified using the bicinchoninicacid protein assay. RESULTS: SIB noticeably decreased caspase-11 and GSDMD-mediated pyroptosis and suppressed the secretion of IL-1 α, IL-1 ß, and IL-18 induced by LPS (P<0.05). Moreover, SIB inhibited the translocation of LPS into the cytoplasm and the binding of caspase-11 and intracellular LPS (P<0.05). SIB also attenuated the expression of caspase-11 and N-terminal fragments of GSDMD, inhibited the relative cytokines, prolonged the survival time, and up-regulated the survival rate in the endotoxemia models (P<0.05). CONCLUSIONS: SIB can inhibit pyroptosis in the LPS-mediated endotoxemia model, at least in part, by inhibiting the caspase-11-mediated cleavage of GSDMD. Additionally, SIB inhibits the interaction of LPS and caspase-11 and inhibits the LPS-mediated up-regulation of caspase-11 expression, which relieves caspase-11-dependent cell pyroptosis and consequently attenuates LPS-mediated lethality.

Forsythoside A inhibits adhesion and migration of monocytes to type II alveolar epithelial cells in lipopolysaccharide-induced acute lung injury through upregulating miR-124.

Acute lung injury (ALI) is a severe disease for which effective drugs are still lacking at present. Forsythia suspensa is a traditional Chinese medicine commonly used to relieve respiratory symptoms in China, but its functional mechanisms remain unclear. Therefore, forsythoside A (FA), the active constituent of F. suspensa, was studied in the present study. Inflammation models of type II alveolar epithelial MLE-12 cells and BALB/c mice stimulated by lipopolysaccharide (LPS) were established to explore the effects of FA on ALI and the underlying mechanisms. We found that FA inhibited the production of monocyte chemoattractant protein-1 (MCP-1/CCL2) in LPS-stimulated MLE-12 cells in a dose-dependent manner. Moreover, FA decreased the adhesion and migration of monocytes to MLE-12 cells. Furthermore, miR-124 expression was upregulated after FA treatment. The luciferase report assay showed that miR-124 mimic reduced the activity of CCL2 in MLE-12 cells. However, the inhibitory effects of FA on CCL2 expression and monocyte adhesion and migration to MLE-12 cells were counteracted by treatment with a miR-124 inhibitor. Critically, FA ameliorated LPS-induced pathological damage, decreased the serum levels of tumor necrosis factor-α and interleukin-6, and inhibited CCL2 secretion and macrophage infiltration in lungs in ALI mice. Meanwhile, administration of miR-124 inhibitor attenuated the protective effects of FA. The present study suggests that FA attenuates LPS-induced adhesion and migration of monocytes to type II alveolar epithelial cells though upregulating miR-124, thereby inhibiting the expression of CCL2. These findings indicate that the potential application of FA is promising and that miR-124 mimics could also be used in the treatment of ALI.

Glycyrrhizin Ameliorates Radiation Enteritis in Mice Accompanied by the Regulation of the HMGB1/TLR4 Pathway.

Radiation enteritis is a common side effect of radiotherapy for abdominal and pelvic malignancies, which can lead to a decrease in patients' tolerance to radiotherapy and the quality of life. It has been demonstrated that glycyrrhizin (GL) possesses significant anti-inflammatory activity. However, little is known about its anti-inflammatory effect in radiation enteritis. In the present study, we aimed to investigate the potential anti-inflammatory effects of GL on radiation enteritis and elucidate the possible underlying molecular mechanisms involved. The C57BL/6 mice were subjected to 6.5 Gy abdominal X-ray irradiation to establish a model of radiation enteritis. Hematoxylin and eosin staining was performed to analyze the pathological changes in the jejunum. The expression of TNF-α in the jejunum was analyzed by immunochemistry. The levels of inflammatory cytokines, such as TNF-α, IL-6, IL-1ß, and HMGB1 in the serum were determined by enzyme-linked immunosorbent assay. The intestinal absorption capacity was tested using the D-xylose absorption assay. The levels of HMGB1 and TLR4 were analyzed by western blotting and immunofluorescence staining. We found that GL significantly alleviated the intestinal damage and reduced the levels of inflammatory cytokines, such as TNF-α, IL-6, IL-1ß, and HMGB1 levels. Furthermore, the HMGB1/TLR4 signaling pathway was significantly downregulated by GL treatment. In conclusion, these findings indicate that GL has a protective effect against radiation enteritis through the inhibition of the intestinal damage and the inflammatory responses, as well as the HMGB1/TLR4 signaling pathway. Thereby, GL might be a potential therapeutic agent for the treatment of radiation enteritis.

Heat-Clearing Chinese Medicines in Lipopolysaccharide-Induced Inflammation.

Lipopolysaccharide (LPS)-induced inflammation causes massive threatening diseases, such as sepsis, acute lung injury and multiple organ dysfunction syndrome. Efficient treatment to prevent inflammation is crucial in LPS-induced inflammatory diseases. Heat-clearing Chinese medicines (CMs) have been used to ameliorate LPS-induced inflammation in China for centuries. Heat-clearing CMs regulate inflammatory pathways, thereby inhibiting the release of inflammatory factors. This review aimed to introduce promising heat-clearing CMs countering LPS-induced inflammation in the last 5 years, exploring the underlying molecular mechanisms.

Antioxidant and Antiapoptotic Polyphenols from Green Tea Extract Ameliorate CCl4-Induced Acute Liver Injury in Mice.

OBJECTIVE: To investigate the phenolic composition, antioxidant properties, and hepatoprotective mechanisms of polyphenols from green tea extract (GTP) in carbon tetrachloride (CCl4)-induced acute liver injury mouse model. METHODS: High-performance liquid chromatography was used to analyze the chemical composition of the extract. Antioxidant activity of GTP was assessed by O2∙-, OH∙, DPPH∙, and ferric-reducing antioxidant power (FRAP) assay in vitro. Sixty Kunming mice were divided into 6 groups including control, model, low-, medium-, and high-doses GTP (200, 400, 800 mg/kg) and vitamin E (250 mg/kg) groups, 10 in each group. GTP and vitamin E were administered at a level of abovementioned doses twice per day for 7 days prior to exposure to a single injection of CCl4. Hepatoprotective effects of GTP were evaluated in a CCl4-induced mouse model of acute liver injury, using commercial enzyme linked immunosorbent assay kits, histopathological observation, terminal deoxynucleotidyl transferase-mediated dUTPNick-end labeling (TUNEL) assay and Western blot. RESULTS: GTP contained 98.56 µg gallic acid equivalents per milligram extract total polyphenols, including epicatechingallate, epigallocatechin gallate, epicatechin, and epigallocatechin. Compared with the model group, low-, medium-, or high doses GTP significantly decreased serum levels of alanine aminotransferase and aspartate transaminase (P<0.01). Histopathological observation confirmed that pretreatment of GTP prevented swelling and necrosis in CCl4-exposed hepatocytes. Hepatoprotective effects of low-, medium-, and high-dose GTP were associated with eliminating free radicals and improving superoxide dismutase, catalase, and glutathione peroxidase activity in the liver. Additionally, low-, medium-, and high-dose GTP decreased cell apoptosis in the CCl4-exposed liver (P<0.01). Phosphorylated nuclear factor kappa-B (NF-κB), p53, Bcl-2 associated x protein/B-cell lymphoma/leukemia-2 gene, cytochrome C, and cleaved caspase-3 levels were downregulated compared with the model group (P<0.01). CONCLUSION: GTP achieves hepatoprotective effects by improving hepatic antioxidant status and preventing cell apoptosis through caspase-3-dependent signaling pathways.

[Protective effect of Sanhuangyinchi Fang drug serum on hydrogen peroxide-induced DNA oxidative damage in LO2 cells].

OBJECTIVE: To study the protective effect of Sanhuangyinchi Fang drug serum (SF) against hydrogen peroxide-mediated DNA oxidative damage in LO2 cells. METHODS: The LO2 cells were randomly divided into the control group, H(2)O(2) group, SF groups (5%, 10%, and 15%) and vitE group. The morphological features of the treated LO2 cells were observed under inverted microscope. The viability of the treated cells was assessed with CCK-8 method, and the activity of SOD, CAT and GSH-PX were detected biochemically. Reactive oxygen species (ROS) levels, the content of 8-OHdG, and DNA damage of the cells were evaluated by flow cytometry, ELISA, and Comet assay, respectively. RESULTS: Compared with H(2)O(2) group, the cells in SF groups (10% and 15%) and vitE group showed higher cell survival rate (P<0.05) and higher SOD, CAT, GSH-PX (P<0.05) and ROS scavenging activities (P<0.01) with markedly decreases the content of 8-OHdG (P<0.01) and reduced tailing ratio, tail length, tail moment and Olive tail moment (P<0.05). CONCLUSION: SF drug serum, especially at the concentration of 15%, can protect LO2 cells from H(2)O(2)-mediated DNA oxidative damage.