[Mechanism of Xuebijing Injection in treatment of sepsis-associated ARDS based on network pharmacology and in vitro experiment].

The aim of this study was to investigate the effect and molecular mechanism of Xuebijing Injection in the treatment of sepsis-associated acute respiratory distress syndrome(ARDS) based on network pharmacology and in vitro experiment. The active components of Xuebijing Injection were screened and the targets were predicted by the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP). The targets of sepsis-associated ARDS were searched against GeneCards, DisGeNet, OMIM, and TTD. Weishengxin platform was used to map the targets of the main active components in Xuebijing Injection and the targets of sepsis-associated ARDS, and Venn diagram was established to identify the common targets. Cytoscape 3.9.1 was used to build the "drug-active components-common targets-disease" network. The common targets were imported into STRING for the building of the protein-protein interaction(PPI) network, which was then imported into Cytoscape 3.9.1 for visualization. DAVID 6.8 was used for Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment of the common targets, and then Weishe-ngxin platform was used for visualization of the enrichment results. The top 20 KEGG signaling pathways were selected and imported into Cytoscape 3.9.1 to establish the KEGG network. Finally, molecular docking and in vitro cell experiment were performed to verify the prediction results. A total of 115 active components and 217 targets of Xuebijing Injection and 360 targets of sepsis-associated ARDS were obtained, among which 63 common targets were shared by Xuebijing Injection and the disease. The core targets included interleukin-1 beta(IL-1ß), IL-6, albumin(ALB), serine/threonine-protein kinase(AKT1), and vascular endothelial growth factor A(VEGFA). A total of 453 GO terms were annotated, including 361 terms of biological processes(BP), 33 terms of cellular components(CC), and 59 terms of molecular functions(MF). The terms mainly involved cellular response to lipopolysaccharide, negative regulation of apoptotic process, lipopolysaccharide-mediated signaling pathway, positive regulation of transcription from RNA polyme-rase Ⅱ promoter, response to hypoxia, and inflammatory response. The KEGG enrichment revealed 85 pathways. After diseases and generalized pathways were eliminated, hypoxia-inducible factor-1(HIF-1), tumor necrosis factor(TNF), nuclear factor-kappa B(NF-κB), Toll-like receptor, and NOD-like receptor signaling pathways were screened out. Molecular docking showed that the main active components of Xuebijing Injection had good binding activity with the core targets. The in vitro experiment confirmed that Xuebijing Injection suppressed the HIF-1, TNF, NF-κB, Toll-like receptor, and NOD-like receptor signaling pathways, inhibited cell apoptosis and reactive oxygen species generation, and down-regulated the expression of TNF-α, IL-1ß, and IL-6 in cells. In conclusion, Xuebijing Injection can regulate apoptosis and response to inflammation and oxidative stress by acting on HIF-1, TNF, NF-κB, Toll-like receptor, and NOD-like receptor signaling pathways to treat sepsis-associated ARDS.

Arctigenin attenuates paraquat-induced human lung epithelial A549 cell injury by suppressing ROS/p38 mitogen-activated protein kinases-mediated apoptosis.

BACKGROUND: Paraquat (PQ)-induced acute lung injury (ALI) and pulmonary fibrosis are common diseases with high mortality but without effective antidotes in emergency medicine. Our previous study has proved that arctigenin suppressed pulmonary fibrosis induced by PQ. We wondered whether arctigenin could also have a protective effect on PQ-induced ALI. METHODS: A PQ-induced A549 cell injury model was used, and the effect of arctigenin was determined by a cell counting kit-8 (CCK-8) cell viability assay. In addition, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labelling (TUNEL) staining assays and mitochondrial membrane potential assays were performed to evaluate the level of cell apoptosis. The generation of reactive oxygen species (ROS) was reflected by dihydroethidium (DHE) staining and a 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) assay. Moreover, immunoblotting studies were used to assess the expression of mitogen-activated protein kinases (MAPKs) and p38 MAPK. RESULTS: Arctigenin attenuated PQ-induced inhibition of A549 cell viability in a dose-dependent manner. Arctigenin also significantly reduced PQ-induced A549 cell apoptosis, as reflected by the TUNEL assay and mitochondrial membrane potential assay, which may result from suppressed ROS/p38 MAPK signaling because we found that arctigenin dramatically suppressed ROS generation and p38 MAPK phosphorylation. CONCLUSION: Arctigenin could attenuate PQ-induced lung epithelial A549 cell injury in vitro by suppressing ROS/p38 MAPK-mediated cell apoptosis, and arctigenin might be considered a potential candidate drug for PQ-induced ALI.

[Mechanism of modified Liangge San in treatment of acute respiratory distress syndrome based on network pharmacology and experimental validation].

A network pharmacology-based strategy combined with molecular docking and in vitro validation was employed to investigate potential targets and molecular mechanisms of modified Liangge San(MLGS) against acute respiratory distress syndrome(ARDS). Active ingredients and corresponding targets of MLGS were screened out on the Traditional Chinese Medicines Systems Pharmacology(TCMSP) database, and the disease targets of ARDS were obtained by integrating GeneCards and DisGeNET database. The two were intersected to obtain the potential targets of MLGS against ARDS. Cytoscape 3.7.2 was used to construct a "Chinese medicine-active ingredient-target network" of MLGS and a "regulatory network of MLGS against ARDS". The protein-protein interaction(PPI) network was created on the STRING database platform, and the Metascape database was used to carry out Gene Ontology(GO) function enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analysis. Subsequently, molecular docking and in vitro experiments were performed to further verify the above findings. A total of 211 active ingredients of MLGS and 54 key targets were obtained. The GO enrichment analysis obtained 709 GO entries(P<0.05), including 457 biological processes(BP), 50 cell components(CC), and 98 molecular functions(MF), mainly involved in lipopolysaccharides, response to reactive oxygen species, and apoptosis signal pathways. KEGG pathway enrichment analysis obtained 266 pathways, mainly involved in the cancer signaling pathways, advanced glycation end-products and their receptors(AGE-RAGE) signaling pathways, fluid shear stress, atherosclerosis, proteoglycan pathway in cancer, nuclear and factor kappa B(NF-κB) signaling pathway. Molecular docking showed that the main active ingredients bound steadily with the targets. The experiments proved that MLGS inhibited the generation of reactive oxygen species and the activation of NF-κB signaling pathway, thereby reducing apoptosis. The study shows that MLGS, through its multiple active ingredients including wogonin and luteolin, can treat ARDS by intervening in various signaling pathways such as NF-κB, inhibiting the inflammatory response and oxidative stress, and reducing apoptosis.

Pravastatin attenuates sepsis-induced acute lung injury through decreasing pulmonary microvascular permeability via inhibition of Cav-1/eNOS pathway.

BACKGROUND: Disruption of alveolar endothelial barrier caused by inflammation drives the progression of septic acute lung injury (ALI). Pravastatin, an inhibitor of HMG Co-A reductase, has potent anti-inflammatory effects. In the present study, we aim to explore the beneficial role of pravastatin in sepsis-induced ALI and its related mechanisms. METHODS: A septic ALI model was established by cecal ligation and puncture (CLP) in mice. The pulmonary microvascular endothelial cells (PMVECs) were challenged with lipopolysaccharide (LPS). The pathological changes in lung tissues were examined by HE staining. The pulmonary microvascular permeability was determined by lung wet-to-dry (W/D) weight ratio and Evans blue staining. The total protein concentration in bronchoalveolar lavage fluid (BALF) was detected by BCA assay. The levels of TNF-α, IL-1ß, and IL-6 were assessed by qRT-PCR and ELISA. Apoptosis was determined by flow cytometry and TUNEL. Western blotting was performed for detection of target protein levels. The expression of VE-Cadherin in lung tissues was evaluated by immunohistochemical staining. RESULTS: Pravastatin improved survival rate, attenuated lung pathological changes and reduced pulmonary microvascular permeability in septic mice. In addition, pravastatin restrained sepsis-induced inflammatory response and apoptosis in the lung tissues and PMVECs. Moreover, pravastatin up-regulated the levels of junction proteins ZO-1, JAM-C, and VE-Cadherin. Finally, pravastatin suppressed inflammation, apoptosis and enhanced the expression of junction proteins via regulating Cav-1/eNOS signaling pathway in LPS-exposed PMVECs. CONCLUSION: Pravastatin ameliorates sepsis-induced ALI through improving alveolar endothelial barrier disruption via modulating Cav-1/eNOS pathway, which may be an effective candidate for treating septic ALI.

MiR-128-3p alleviates TNBS-induced colitis through inactivating TRAF6/NF-κB signaling pathway in rats.

miR-128-3p is reported to involve in pathogenesis of several autoimmune diseases, yet the role of miR-128-3p in inflammatory bowel disease (IBD) remains unknown. To investigate miR-128-3p in IBD, experimental colitis animal model was generated by 2,4,6-Trinitrobenzenesulfonic acid solution (TNBS). miR-128-3p agomir was used to overexpress miR-128-3p in rats. Histological assessment and myeloperoxidase activity were conducted to evaluate the TNBS-induced colitis. Effect of miR-128-3p overexpression on levels of TNF-α, IL-1ß, ICAM-1, and MCP-1 was tested by ELISA assay. The target of miR-128-3p was predicted and further confirmed by dual-luciferase reporter assay. The expressions of TRAF6, p-NF-κB, and NF-κB were determined by western blot. The miR-128-3p level was significantly decreased in rats with TNBS-induced colitis. miR-128-3p could alleviate TNBS-induced colitis and inhibit production of inflammatory factors. We found TRAF6 was a direct target of miR-128-3p using bioinformatics and luciferase assay. By western blot, we discovered miR-128-3p activates NF-κB by targeting TRAF6. Our data reveal a novel mechanism that a decreased miR-128-3p level in TNBS-induced colitis could inhibit production of inflammatory factors, which activates NF-κB signaling by targeting TRAF6. Our findings might provide a novel therapeutic target for drug design and development for IBD therapeutics.

Pretreatment with intestinal trefoil factor alleviates stress-induced gastric mucosal damage via Akt signaling.

BACKGROUND: Stress-related gastric mucosal damage or ulcer remains an unsolved issue for critically ill patients. Stress ulcer prophylaxis has been part of routine intensive care, but uncertainty and controversy still exist. Co-secreted with mucins, intestinal trefoil factor (ITF) is reported to promote restitution and regeneration of intestinal mucosal epithelium, although the mechanism remains unknown. AIM: To elucidate the protective effects of ITF on gastric mucosa and explore the possible mechanisms. METHODS: We used a rat model of gastric mucosal damage induced by water immersion restraint stress and lipopolysaccharide-treated human gastric epithelial cell line to investigate the potential effects of ITF on damaged gastric mucosa both in vivo and in vitro. RESULTS: ITF promoted the proliferation and migration and inhibited necrosis of gastric mucosal epithelia in vitro. It also preserved the integrity of gastric mucosa by upregulating expressions of occludin and zonula occludens-1. In the rat model, pretreatment with ITF ameliorated the gastric mucosal epithelial damage and facilitated mucosal repair. The protective effects of ITF were confirmed to be exerted via activation of Akt signaling, and the specific inhibitor of Akt signaling LY249002 reversed the protective effects. CONCLUSION: ITF might be a promising candidate for prevention and treatment of stress-induced gastric mucosal damage, and further studies should be undertaken to verify its clinical feasibility.

[Effects of Rg_1 on LPS-induced apoptosis and autophagy of lung epithelial cells].

This paper aimed to study the protective effect of ginsenoside Rg_1 on endotoxin(LPS)-induced apoptosis of lung epithelial cells and its mechanism of action. Mouse lung epithelial cells(MLE-12) were first treated with LPS. The autophagy changes and apoptosis and the relationship with concentration and time of LPS were observed. Then,the level of autophagy in MLE-12 was regulated at a specific concentration and action time of LPS,and the changes of apoptosis were observed. Secondly,ginsenoside Rg_1 and autophagy inhibitor 3-MA were added respectively at the same concentration and action time of LPS. The lung epithelial cells were grouped to observe the effect of ginsenoside Rg_1 on LPS-induced apoptosis of lung epithelial cells and its mechanism. In the animal experiment,the mice were grouped and tested by apoptosis protein,lung injury score and HE staining section to verify whether ginsenoside Rg_1 has a protective effect on LPS-induced lung injury. The results showed that apoptosis and autophagy increased as the rise of concentration after treatment with LPS for 12 h. The apoptosis increased gradually,and the autophagy increased first and then decreased over time at the LPS concentration of 25 g·L-1. The apoptosis of LPS group was higher than that of control group,and LPS+3-MA group increased further,while apoptosis decreased significantly in LPS+RAM(rapamycin,autophagy promoter) group. The autophagy increased in LPS group,decreased in LPS+3-MA group and increased in LPS+RAM group. The apoptosis of LPS group was higher than that of control group,and the apoptosis of LPS+Rg_1 group decreased. The apoptosis of LPS+Rg_1+3-MA group increased again. The autophagy of LPS group further increased after administration of ginsenoside Rg_1,but decreased after administration of 3-MA. In the in vivo experiments in mice,the apoptosis of LPS group increased significantly compared with the control group,while LPS + ginsenoside Rg_1 group decreased. Lung injury score and HE staining also conformed to the above trend. LPS can induce the apoptosis of lung epithelial cells in a time-dependent and concentration-dependent manner. The autophagy of lung epithelial cells increases with the rise of LPS concentration. At the specific concentration of LPS,autophagy increases first and then decreases after 12-16 hours. Proper increase of autophagy in lung epithelial cells within a certain period of time can reduce the apoptosis induced by LPS,while inhibition of autophagy can increase apoptosis. Ginsenoside Rg_1 has a protective effect on lung cancer epithelial cell apoptosis induced by autophagy.

The role of p38 MAPK in acute paraquat-induced lung injury in rats.

CONTEXT: Paraquat (PQ; 1,1'-dimethyl-4,4'-bipyridinium dichloride) is highly toxic and accounts for a large proportion of the herbicide poisonings seen in clinic. The major cause of mortality is respiratory failure. The p38 mitogen-activated protein kinase (MAPK) signal transduction pathway coordinates various cellular stress responses that have been shown to participate in the pathogenesis of PQ-induced lung injury. OBJECTIVE: To evaluate the effect of the specific p38 MAPK inhibitor SB203580 on PQ-induced lung injury and cytokine secretion. METHODS: In groups of 24, rats were treated with PQ, PQ and SB203580 (SB + PQ), SB203580 alone (SB) or normal saline (control group). Six rats from each group were euthanized at 1, 3, 5 or 7 d. Pathology of lung specimens was scored through hematoxylin and eosin staining. Edema in the lung was quantified from wet-to-dry weight ratios. p38 and p-p38MAPK proteins were measured via electrochemiluminescent Western blots. tumor necrosis factor (TNF)-alpha and interleukin-1 beta (IL-1ß) concentrations in lung specimens and bronchoalveolar lavage fluid (BALF) were quantified via enzyme-linked immunosorbent assay. RESULTS: The mortality rate of the SB + PQ group (16.7%) was significantly lower than that of the PQ group (33.3%; p < 0.05). The PQ group had significantly higher pulmonary histology scores, wet-to-dry weight ratios and phosphorylated p-p38 MAPK levels, as well as higher IL-1ß and TNF-alpha levels in BALF and lung tissues, that did the SB + PQ and control groups (p < 0.05, all). CONCLUSION: The data suggest that the p38 MAPK signaling pathway has an important role in regulating the production of IL-1ß and TNF-alpha in PQ-induced lung injury in rats.