Yemazhui () ameliorates lipopolysaccharide-induced acute lung injury modulation of the toll-like receptor 4/nuclear factor kappa-B/nod-like receptor family pyrin domain-containing 3 protein signaling pathway and intestinal flora in rats.

OBJECTIVE: To investigate the impact of Yemazhui (Herba Eupatorii Lindleyani, HEL) against lipopolysaccharide (LPS)-induced acute lung injury (ALI) and explore its underlying mechanism in vivo. METHODS: The chemical constituents of HEL were analyzed by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry method. Then, HEL was found to suppress LPS-induced ALI in vivo. Six-week-old male Sprague-Dawley rats were randomly divided into 6 groups: control, LPS, Dexamethasone (Dex), HEL low dose 6 g/kg (HEL-L), HEL medium dose 18 g/kg (HEL-M) and HEL high dose 54 g/kg (HEL-H) groups. The model rats were intratracheally injected with 3 mg/kg LPS to establish an ALI model. Leukocyte counts, lung wet/dry weight ratio, as well as myeloperoxidase (MPO) activity were determined followed by the detection with hematoxylin and eosin staining, enzyme linked immunosorbent assay, quantitative real time polymerase chain reaction, western blotting, immunohistochemistry, and immunofluorescence. Besides, to explore the effect of HEL on ALI-mediated intestinal flora, we performed 16s rRNA sequencing analysis of intestinal contents. RESULTS: HEL attenuated LPS-induced inflammation in lung tissue and intestinal flora disturbance. Mechanism study indicated that HEL suppressed the lung coefficient and wet/dry weight ratio of LPS-induced ALI in rats, inhibited leukocytes exudation and MPO activity, and improved the pathological injury of lung tissue. In addition, HEL reduced the expression of tumor necrosis factor-alpha, interleukin-1beta (IL-1ß) and interleukin-6 (IL-6) in bronchoalveolar lavage fluid and serum, and inhibited nuclear displacement of nuclear factor kappa-B p65 (NF-κBp65). And 18 g/kg HEL also reduced the expression levels of toll-like receptor 4 (TLR4), myeloid differentiation factor 88, NF-κBp65, phosphorylated inhibitor kappa B alpha (phospho-IκBα), nod-like receptor family pyrin domain-containing 3 protein (NLRP3), IL-1ß, and interleukin-18 (IL-18) in lung tissue, and regulated intestinal flora disturbance. CONCLUSIONS: In summary, our findings revealed that HEL has a protective effect on LPS-induced ALI in rats, and its mechanism may be related to inhibiting TLR4/ NF-κB/NLRP3 signaling pathway and improving intestinal flora disturbance.

A novel mechanism for regulating lung immune homeostasis: Zukamu granules alleviated acute lung injury in mice by inhibiting NLRP3 inflammasome activation and regulating Th17/Treg cytokine balance.

ETHNOPHARMACOLOGICAL RELEVANCE: Acute lung injury (ALI) is a severely acute lung inflammation with high morbidity and mortality. Zukamu granules (ZKMG) is one of the Uygur patent drugs commonly used in clinic, which is included in the National Essential Drugs List (2018 edition). Clinical studies have shown that ZKMG has a significant effect on acute upper respiratory tract infection, and has better anti-inflammatory and antipyretic effects. However, the immunomodulatory mechanism of ZKMG on ALI is still not clear. AIM OF THE STUDY: The aim of this study is to investigate the lung protective effect and immunomodulatory mechanism of ZKMG on lipopolysaccharide (LPS) -induced ALI mice, and to provide an important basis for the treatment strategy and theoretical basis of ALI. MATERIALS AND METHODS: First, network pharmacology was used to predict the potential signaling pathways and biological processes of ZKMG related to immunology. Molecular docking technique was used to predict the possibility between the core components of ZKMG acting on NLRP3 protein. In addition, protein levels of F4/80 in lung tissues were assessed by Immunohistochemistry (IHC). The contents of IL-1ß, IL-18, IL-17A and IL-10 in the lung tissue and serum, MPO in the lung tissue were detected by enzyme-linked immunosorbent assay (ELISA). Real-time quantitative PCR analysis (RT-qPCR) was used to detect NLRP3 mRNA in lung tissue. Protein levels of NLRP3, Caspase-1, Cleaved caspase-1 p20, ASC, and GSDMD were detected by Western blot (WB). RESULTS: The results of network pharmacology showed that the immune pathways of ZKMG were mainly Th17 signaling pathway, IL-17 signaling pathway, NOD-like receptor signaling pathway, etc. Molecular docking results showed that the core components of ZKMG had good binding ability to NLRP3 protein. The verification experiments showed that ZKMG can reduce the degree of lung injury, and reduce the level of inflammatory infiltration of neutrophils and macrophages by reducing the content of MPO and F4/80. In addition, ZKMG can reduce NLRP3 mRNA, inhibit the expression of NLRP3/Caspase-1/GSDMD and other related pathway proteins, and reduce inflammatory factors such as IL-1ß and IL-18. It can also reduce the content of pro-inflammatory cytokine IL-17A, increase the content of anti-inflammatory cytokine IL-10 in lung tissue. CONCLUSION: ZKMG can reduce the degree of lung tissue injury in ALI by inhibiting NLRP3/Caspase-1/GSDMD signaling pathway and restoring the IL-17A/IL-10 cytokine balance, and its protective mechanism may be related to the regulation of lung immune homeostasis. It will provide a new strategy for studying the regulation of lung immune homeostasis.

Neutrophil membrane-engineered Panax ginseng root-derived exosomes loaded miRNA 182-5p targets NOX4/Drp-1/NLRP3 signal pathway to alleviate acute lung injury in sepsis: experimental studies.

BACKGROUND: The purpose of this study was to prepare neutrophil membrane-engineered Panax ginseng root-derived exosomes (N-exo) and investigate the effects of N-exo microRNA (miRNA) 182-5p (N-exo-miRNA 182-5p) on acute lung injury (ALI) in sepsis. METHODS: Panax ginseng root-derived exosomes were separated by differential centrifugation. Neutrophil membrane engineering was performed on exo to obtain N-exo. miRNA182-5p was transmitted into N-exo by electroporation technology to obtain N-exo-miRNA 182-5p. LPS was used to establish an in-vivo and in-vitro model of ALI of sepsis to evaluate the anti-inflammatory effect of N-exo-miRNA 182-5p. RESULTS: The results of transmission electron microscope showed that exo was a double-layer membrane structure like a saucer. Nanoparticle size analysis showed that the average particle size of exo was 129.7 nm. Further, compared with exo, the level of miRNA182-5p was significantly increased in N-exo. The experimental results showed that N-exo-miRNA 182-5p significantly improved ALI via target regulation of NOX4/Drp-1/NLRP3 signal pathway in vivo and in vitro . CONCLUSION: In conclusion, this study prepared a novel engineered exosome (N-exo and N-exo-miRNA 182-5p significantly improved ALI in sepsis via target regulation of NOX4/Drp-1/NLRP3 signal pathway, providing new ideas and methods for treatment of ALI in sepsis.

[Mechanism of Qilongtian Capsules in treatment of acute lung injury based on network pharmacology prediction and experimental validation].

This study aimed to explore the mechanism of Qilongtian Capsules in treating acute lung injury(ALI) based on network pharmacology prediction and in vitro experimental validation. Firstly, UPLC-Q-TOF-MS/MS was used to analyze the main chemical components of Qilongtian Capsules, and related databases were used to obtain its action targets and ALI disease targets. STRING database was used to build a protein-protein interaction(PPI) network. Metascape database was used to conduct enrichment analysis of Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG). AutoDock software was used to perform molecular docking verification on the main active components and key targets. Then, the RAW264.7 cells were stimulated with lipopolysaccharide(LPS) for in vitro experiments. Cell viability was measured by MTT and ROS level was measured by DCFH-DA. NO content was measured by Griess assay, and IL-1ß, IL-6, and TNF-α mRNA expression was detected by RT-PCR. The predicted targets were preliminarily verified by investigating the effect of Qilongtian Capsules on downstream cytokines. Eighty-four compounds were identified by UPLC-Q-TOF-MS/MS. Through database retrieval, 44 active components with 589 target genes were screened out. There were 560 ALI disease targets, and 65 intersection targets. PPI network topology analysis revealed 10 core targets related to ALI, including STAT3, JUN, VEGFA, CASP3, and MMP9. KEGG enrichment analysis showed that Qilongtian Capsules mainly exerted an anti-ALI effect by regulating cancer pathway, AGE-RAGE, MAPK, and JAK-STAT signaling pathways. The results of molecular docking showed that the main active components in Qilongtian Capsules, including crenulatin, ginsenoside F_1, ginsenoside Rb_1, ginsenoside Rd, ginsenoside Rg_1, ginsenoside Rg_3, notoginsenoside Fe, notoginsenoside G, notoginsenoside R_1, notoginsenoside R_2, and notoginsenoside R_3, had good binding affinities with the corresponding protein targets STAT3, JUN, VEGFA, CASP3, and MMP9. Cellular experiments showed that Qilongtian Capsules at 0.1, 0.25, and 0.5 mg·mL~(-1) reduced the release of NO, while Qilongtian Capsules at 0.25 and 0.5 mg·mL~(-1) reduced ROS production, down-regulated mRNA expression of IL-1ß, IL-6, TNF-α, and inhibited the inflammatory cascade. In summary, Qilongtian Capsules may exert therapeutic effects on ALI through multiple components and targets.

[Effects of total ginsenosides from Panax ginseng stems and leaves on gut microbiota and short-chain fatty acids metabolism in acute lung injury mice].

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-1ß(IL-1ß), interleukin-6(IL-6), and tumor necrosis factor-α(TNF-α) in BALF were detected. The mRNA expression levels of IL-1ß, 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.

Deciphering the molecular mechanisms of Maxing Huoqiao Decoction in treating pulmonary fibrosis via transcriptional profiling and circRNA-miRNA-mRNA network analysis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, fibrotic interstitial lung condition with unknown etiology and high mortality. Chinese herbal medicine has been used for more than a thousand years to treat various lung diseases. PURPOSE: The current study aimed to examine whether Chinese herbal Maxing Huoqiao Decoction (MXHQD) exerts therapeutic effects on IPF and to further uncover its underlying molecular mechanisms. METHODS: Mouse model of acute lung injury (ALI) or IPF was induced by intratracheal instillation of LPS or bleomycin, respectively. ALI mice were treated with MXHQD for 7 days, and lung tissues were taken for test after modeling 24 h. IPF mice were gavaged for 21 days after modeling. Lung tissues were subjected to whole transcriptome detection, and the differential RNAs were experimentally verified. RESULTS: The results showed that MXHQD alleviated the computed tomography (CT) and the pathological degree changes in mice with IPF, improved changes in the expression of fibrosis related genes and reduced the hydroxyproline expression in IPF mice. MXHQD also decreased the cell numbers in bronchoalveolar lavage fluids, and the expression levels of the inflammatory factors in the ALI mice lung tissues were significantly inhibited. By applying whole transcriptome analysis, results showed that MXHQD acted on 40 mRNAs, 15 miRNAs, 25 novel lncRNAs and 17 circRNAs to resist pulmonary fibrosis. The competing endogenous RNA (ceRNA) network diagram showed that the multiple components of MXHQD against fibrosis through a network of multiple targets. The differential mRNAs were mainly related to the innate immune response and the defense response to virus. Then the expression of mRNAs in the differential mRNA-miRNA-differential circRNA network in the lung tissue of IPF was verified. The expression of ZBP1 and ISG15 related to immune system and anti virus was verified at both gene and protein expressions. MXHQD could significantly inhibit the elevation of ZBP1 and ISG15 factors induced by the fibrosis model. CONCLUSION: Overall, our findings provide compelling evidence that MXHQD can alleviate IPF by modulating innate immunity. This is the first study to reveal the molecular mechanism underlying the multi-components, multi-channels and multi-targets anti-IPF immune injury of MXHQD, and supports its potential clinical application for IPF.

Orally administered BZL-sRNA-20 oligonucleotide targeting TLR4 effectively ameliorates acute lung injury in mice.

The global COVID-19 pandemic emerged at the end of December 2019. Acute respiratory distress syndrome (ARDS) and acute lung injury (ALI) are common lethal outcomes of bacterial lipopolysaccharide (LPS), avian influenza virus, and SARS-CoV-2. Toll-like receptor 4 (TLR4) is a key target in the pathological pathway of ARDS and ALI. Previous studies have reported that herbal small RNAs (sRNAs) are a functional medical component. BZL-sRNA-20 (Accession number: B59471456; Family ID: F2201.Q001979.B11) is a potent inhibitor of Toll-like receptor 4 (TLR4) and pro-inflammatory cytokines. Furthermore, BZL-sRNA-20 reduces intracellular levels of cytokines induced by lipoteichoic acid (LTA) and polyinosinic-polycytidylic acid (poly (I:C)). We found that BZL-sRNA-20 rescued the viability of cells infected with avian influenza H5N1, SARS-CoV-2, and several of its variants of concern (VOCs). Acute lung injury induced by LPS and SARS-CoV-2 in mice was significantly ameliorated by the oral medical decoctosome mimic (bencaosome; sphinganine (d22:0)+BZL-sRNA-20). Our findings suggest that BZL-sRNA-20 could be a pan-anti-ARDS ALI drug.

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.

Tanreqing Injection Attenuates Macrophage Activation and the Inflammatory Response via the lncRNA-SNHG1/HMGB1 Axis in Lipopolysaccharide-Induced Acute Lung Injury.

The etiology of acute lung injury (ALI) is not clear, and the treatment of ALI presents a great challenge. This study aimed to investigate the pathogenesis and potential therapeutic targets of ALI and to define the target gene of Tanreqing (TRQ), which is a traditional Chinese medicine formula composed of five medicines, scutellaria baicalensis, bear bile powder, goat horn powder, honeysuckle and forsythia. Macrophage activation plays a critical role in many pathophysiological processes, such as inflammation. Although the regulation of macrophage activation has been extensively investigated, there is little knowledge of the role of long noncoding RNAs (lncRNAs) in this process. In this study, we found that lncRNA-SNHG1 expression is distinctly regulated in differently activated macrophages in that it is upregulated in LPS. LncRNA-SNHG1 knockdown attenuates LPS-induced M1 macrophage activation. The SNHG1 promoter was bound by NF-κB subunit p65, indicative of SNHG1 being a direct transcriptional target of LPS-induced NF-κB activation. SNHG1 acts as a proinflammatory driver that leads to the production of inflammatory cytokines and the activation of macrophages and cytokine storms by physically interacting with high-mobility group box 1 (HMGB1) in ALI. TRQ inhibited NF-κB signaling activation and binding of NF-κB to the SNHG1 promoter. In conclusion, this study defined TRQ target genes, which can be further elucidated as mechanism(s) of TRQ action, and provides insight into the molecular pathogenesis of ALI. The lncRNA-SNHG1/HMGB1 axis is an ideal therapeutic for ALI treatment.

Baicalin inhibits inflammation of lipopolysaccharide-induced acute lung injury toll like receptor-4/myeloid differentiation primary response 88/nuclear factor-kappa B signaling pathway.

OBJECTIVE: To explore the effect and mechanism of baicalin in the treatment of acute lung injury (ALI) by and experiments. METHODS: ALI was induced by instilling 10 mg/mL lipopolysaccharide (LPS) into the airway of rats. Different doses of baicalin (50 and 100 mg·kg ·d) were administered by gavage one day before modeling. RESULTS: Baicalin significantly reduced the permeability of the alveolocapillary membrane, alleviated tissue injury and inflammatory infiltration, and inhibited the secretion of inflammatory factors and the infiltration of neutrophils. The decline in these inflammations was related to the inhibition of the toll like receptor-4 (TLR4)/myeloid differentiation factor 88 (MyD88)/nuclear factor-kappa B (NF-κB)/nod-like receptor pyrin containing 3 (NLRP3) signaling pathway and the mitogen-activated protein kinase (MAPK) signaling pathway. CONCLUSIONS: Baicalin inhibits the secretion of inflammatory factors by inhibiting the TLR4-MyD88-NF-κB/NLRP3 pathway and the MAPK signaling pathway. Thus, it reduces lung bronchial epithelial layer, alveolar damage, and pulmonary edema as detected in the and experiments. Therefore, baicalin may be a potential preventive and therapeutic drug for ALI.

[Mechanism of lung and intestine combination therapy in treatment of acute lung injury by inhibiting inflammatory response based on NF-κB/NLRP3 signaling pathway and alveolar macrophage activation].

Lung and intestine combination therapy(LICT) is effective in the treatment of acute lung injury(ALI). In this study, the combination of Mahuang Decoction and Dachengqi Decoction(hereinafter referred to as the combination), a manifestation of LICT, was employed to explore the effect of nuclear factor kappaB(NF-κB)/nucleotide binding oligomerization domain-like receptors-3(NLRP3) pathway and alveolar macrophage activation on the lung inflammation in rats with ALI, for the purpose of elucidating the mechanism of LICT in treating ALI. After the modeling of ALI with limpolysaccharide(LPS, ip), rats were respectively given(ig) the combination at 10, 7.5, and 5 g·kg~(-1)(high-dose, medium-dose, and low-dose LICT groups, separately), once every 8 h for 3 times. Haematoxylin-eosin(HE) staining was used to observe the histopathological changes of lung tissue, followed by the scoring of inflammation. Immunohistochemistry was applied to detect alveolar macrophage activation, enzyme-linked immunosorbent assay(ELISA) was applied to detect the serum content of tumor necrosis factor-α(TNF-α) and interleukin-18(IL-18), Western blot was applied to detect the protein expression of phosphorylated-nuclear factor kappaB p65(p-NF-κB p65), nuclear factor kappaB p65(NF-κB p65), phosphorylated-inhibitor kappaB alpha(p-IκBα), inhibitor kappaB alpha(IκBα), and NLRP3 in lung tissue, and quantitative reverse transcription-PCR(qRT-PCR) was applied to detect the mRNA expression of TNF-α, IL-18, NLRP3, and NF-κB p65 in lung tissue. The results showed that LICT groups demonstrated lung injury relief, decrease in inflammation score, alleviation of alveolar macrophage activation, significant decline in serum content of inflammatory factors TNF-α and IL-18, and decrease of the protein expression of p-NF-κB p65/NF-κB p65, p-IκBα/IκBα, and NLRP3, and mRNA expression of TNF-α, IL-18, NLRP3, and NF-κB p65 in lung tissue. In summary, LICT has definite therapeutic effect on ALI. The mechanism is that it inhibits alveolar macrophage activation by suppressing NF-κB/NLRP3 signaling pathway, thereby reducing the activation and release of inflammatory factors and finally inhibiting inflammation.

Mechanism of lung and intestine combination therapy in treatment of acute lung injury by inhibiting inflammatory response based on NF-κB/NLRP3 signaling pathway and alveolar macrophage activation / 中国中药杂志

Lung and intestine combination therapy(LICT) is effective in the treatment of acute lung injury(ALI). In this study, the combination of Mahuang Decoction and Dachengqi Decoction(hereinafter referred to as the combination), a manifestation of LICT, was employed to explore the effect of nuclear factor kappaB(NF-κB)/nucleotide binding oligomerization domain-like receptors-3(NLRP3) pathway and alveolar macrophage activation on the lung inflammation in rats with ALI, for the purpose of elucidating the mechanism of LICT in treating ALI. After the modeling of ALI with limpolysaccharide(LPS, ip), rats were respectively given(ig) the combination at 10, 7.5, and 5 g·kg~(-1)(high-dose, medium-dose, and low-dose LICT groups, separately), once every 8 h for 3 times. Haematoxylin-eosin(HE) staining was used to observe the histopathological changes of lung tissue, followed by the scoring of inflammation. Immunohistochemistry was applied to detect alveolar macrophage activation, enzyme-linked immunosorbent assay(ELISA) was applied to detect the serum content of tumor necrosis factor-α(TNF-α) and interleukin-18(IL-18), Western blot was applied to detect the protein expression of phosphorylated-nuclear factor kappaB p65(p-NF-κB p65), nuclear factor kappaB p65(NF-κB p65), phosphorylated-inhibitor kappaB alpha(p-IκBα), inhibitor kappaB alpha(IκBα), and NLRP3 in lung tissue, and quantitative reverse transcription-PCR(qRT-PCR) was applied to detect the mRNA expression of TNF-α, IL-18, NLRP3, and NF-κB p65 in lung tissue. The results showed that LICT groups demonstrated lung injury relief, decrease in inflammation score, alleviation of alveolar macrophage activation, significant decline in serum content of inflammatory factors TNF-α and IL-18, and decrease of the protein expression of p-NF-κB p65/NF-κB p65, p-IκBα/IκBα, and NLRP3, and mRNA expression of TNF-α, IL-18, NLRP3, and NF-κB p65 in lung tissue. In summary, LICT has definite therapeutic effect on ALI. The mechanism is that it inhibits alveolar macrophage activation by suppressing NF-κB/NLRP3 signaling pathway, thereby reducing the activation and release of inflammatory factors and finally inhibiting inflammation.

[Multiple components of Mahuang Shengma Decoction on prevention and treatment of acute lung injury based on RAGE/NF-κB signaling pathway].

To investigate the potential molecular markers and drug-compound-target mechanism of Mahuang Shengma Decoction(MHSM) in the intervention of acute lung injury(ALI) by network pharmacology and experimental verification. Databases such as TCMSP, TCMIO, and STITCH were used to predict the possible targets of MHSM components and OMIM and Gene Cards were employed to obtain ALI targets. The common differentially expressed genes(DEGs) were therefore obtained. The network diagram of DEGs of MHSM intervention in ALI was constructed by Cytoscape 3. 8. 0, followed by Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment analyses of target genes. The ALI model was induced by abdominal injection of lipopolysaccharide(LPS) in mice. Bronchoalveolar lavage fluid(BALF) was collected for the detection of inflammatory factors. Pathological sectioning and RT-PCR experiments were performed to verify the therapeutic efficacy of MHSM on ALI. A total of 494 common targets of MHSM and ALI were obtained. Among the top 20 key active compounds of MHSM, 14 from Ephedrae Herba were found to be reacted with pivotal genes of ALI [such as tumor necrosis factor(TNF), tumor protein 53(TP53), interleukin 6(IL6), Toll-like receptor 4(TLR4), and nuclear factor-κB(NF-κB)/p65(RELA)], causing an uncontrolled inflammatory response with activated cascade amplification. Pathway analysis revealed that the mechanism of MHSM in the treatment of ALI mainly involved AGE-RAGE, cancer pathways, PI3 K-AKT signaling pathway, and NF-κB signaling pathway. The findings demonstrated that MHSM could dwindle the content of s RAGE, IL-6, and TNF-α in the BALF of ALI mice, relieve the infiltration of inflammatory cells in the lungs, inhibit alveolar wall thickening, reduce the acute inflammation-induced pulmonary congestion and hemorrhage, and counteract transcriptional activities of Ager-RAGE and NF-κB p65. MHSM could also synergically act on the target DEGs of ALI and alleviate pulmonary pathological injury and inflammatory response, which might be achieved by inhibiting the expression of the key gene Ager-RAGE in RAGE/NF-κB signaling pathway and downstream signal NF-κB p65.

Cissus subtetragona Planch. Ameliorates Inflammatory Responses in LPS-induced Macrophages, HCl/EtOH-induced Gastritis, and LPS-induced Lung Injury via Attenuation of Src and TAK1.

Several Cissus species have been used and reported to possess medicinal benefits. However, the anti-inflammatory mechanisms of Cissus subtetragona have not been described. In this study, we examined the potential anti-inflammatory effects of C. subtetragona ethanol extract (Cs-EE) in vitro and in vivo, and investigated its molecular mechanism as well as its flavonoid content. Lipopolysaccharide (LPS)-induced macrophage-like RAW264.7 cells and primary macrophages as well as LPS-induced acute lung injury (ALI) and HCl/EtOH-induced acute gastritis mouse models were utilized. Luciferase assays, immunoblotting analyses, overexpression strategies, and cellular thermal shift assay (CETSA) were performed to identify the molecular mechanisms and targets of Cs-EE. Cs-EE concentration-dependently reduced the secretion of NO and PGE2, inhibited the expression of inflammation-related cytokines in LPS-induced RAW264.7 cells, and decreased NF-κB- and AP-1-luciferase activity. Subsequently, we determined that Cs-EE decreased the phosphorylation events of NF-κB and AP-1 pathways. Cs-EE treatment also significantly ameliorated the inflammatory symptoms of HCl/EtOH-induced acute gastritis and LPS-induced ALI mouse models. Overexpression of HA-Src and HA-TAK1 along with CETSA experiments validated that inhibited inflammatory responses are the outcome of attenuation of Src and TAK1 activation. Taken together, these findings suggest that Cs-EE could be utilized as an anti-inflammatory remedy especially targeting against gastritis and acute lung injury by attenuating the activities of Src and TAK1.

[Molecular mechanism of Fagopyri Dibotryis Rhizoma in treatment of acute lung injury based on network pharmacology and in vitro experiments].

The present study explored the mechanism of Fagopyri Dibotryis Rhizoma(FDR) and its main active components in the treatment of acute lung injury(ALI) based on the network pharmacology and the in vitro experiments. The main active components of FDR were obtained from the TCMSP database and screened by oral bioavailability and drug-likeness. The related target proteins of FDR were retrieved from the PubChem database, and the target genes related to ALI were screened out from the GeneCards database. A protein-protein interaction(PPI) network of compound target proteins and ALI target genes was constructed using STRING 11.0. Ingenuity Pathway Analysis(IPA) platform was used to analyze the common pathways of the potential compound target proteins of FDR and ALI target genes, thereby predicting the key targets and potential signaling pathways of FDR for the treatment of ALI. Finally, the potential pathways and key targets were verified by the in vitro experiments of lipopolysaccharide-induced RAW264.7 cells intervened by epicatechin(EC), the active component of FDR. The results of network pharmacology showed that 15 potential active components such as EC, procyanidin B1, and luteolin presumedly functioned in the treatment of ALI through nuclear transcription factor-κB(NF-κB) signaling pathway, transforming growth factor-ß(TGF-ß) signaling pathway, and adenosine 5'-monophosphate(AMP)-activated protein kinase(AMPK) signaling pathway through key targets, such as RELA(P65). The results of in vitro experiments showed that 25 µmol·L~(-1) EC had no toxicity to cells and could inhibit the expression of the p65-phosphorylated protein in the NF-κB signaling pathway to down-regulate the expression of downstream inflammatory cytokines, including tumor necrosis factor-α(TNF-α), IL-1ß and nitric oxide(NO), and up-regulate the expression of IL-10. These results suggested that the therapeutic efficacy of FDR on ALI was achieved by inhibiting the phosphorylation of p65 protein in the NF-κB signaling pathway and down-regulating the level of proinflammatory cytokines downstream of the signaling pathways.

[Mechanism of Tanreqing Injection in treatment of acute lung injury based on network pharmacology and molecular docking].

This study aimed to explore the mechanism of Tanreqing Injection in the treatment of acute lung injury(ALI) based on network pharmacology and molecular docking. The active components and action targets of Tanreqing Injection were retrieved from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP), PubChem, and SwissTargetPrediction databases, as well as available literature reports. The ALI-related targets were obtained from the GeneCards database and then mapped with Tanreqing Injection targets. Following the construction of "drug-component-potential target" network with Cytoscape 3.6.1, the potential targets were input into STRING to yield the protein-protein interaction(PPI) network, which was plotted using Cytoscape 3.6.1. Then the screened key targets were subjected to gene ontology(GO) and Kyoto encyclopedia of genes and genomes(KEGG) enrichment analysis based on DAVID database. The top three key targets RAC-alpha serine/threonine-protein kinase(AKT1), albumin(ALB) and interleukin-6(IL6) were docked to the top three key compounds by PyMOL and AutoDock vina. A total of 58 active components of Tanreqing Injection, 597 corresponding targets and 503 common targets shared by Tanreqing Injection and ALI were fi-gured out, with the key targets AKT1, ALB and IL6 involved. GO and KEGG enrichment analysis yielded 1 445 biological processes and 148 signaling pathways, respectively. Molecular docking verified a good binding ability of the top three key targets to the top three key compounds. The analysis based on network pharmacology and molecular docking uncovered that Tanreqing Injection directly or indirectly regulated the pulmonary capillary endothelial cells and alveolar epithelial cells via anti-inflammation, thus alleviating ALI.

Comparative protein profiling reveals the inhibitory role of curcumin on IL-17A mediated minichromosome maintenance (MCM) proteins as novel putative markers for acute lung injury in vivo.

The Pro-inflammatory cytokine, Interleukin 17A (IL-17A) plays a vital role in the pathogenesis of inflammatory-induced acute lung injury (ALI). But, the mechanisms of this pro-inflammatory cytokine in response to activation after replication stress are not yet known. Control on DNA replication (DR) is vital for maintaining genome stability. Minichromosome maintenance (MCM) proteins play essential roles in various cancers, but their involvement during ALI is not yet been discussed. The present study was carried out to assess the participation of IL-17A during replication stress and to evaluate the contribution of curcumin on this. Mass spectrometry-based proteomic approach has been used on mice lung tissues treated with IL-17A, as a prime mediator to cause injury and curcumin a natural polyphenol as an intervention. Several trends were identified from the proteomic subset which revealed that IL-17A induces expressions of proteins like MCM2, MCM3, and MCM6 along with other proteins involved in DR. Interestingly, curcumin was found in suppressing the expression levels of these proteins. This was also confirmed via validating LC-MS/MS data using appropriate molecular techniques. Pathway and gene ontology analysis were performed with DAVID GO databases. Apart from this, the present study also reports the unique contribution of curcumin in suppressing the mRNA levels of other MCMs like MCM4, MCM5, and MCM7 as well as of ORC1 and ORC2. Hence, the present study revolves around linking the replication stress by pro-inflammatory effects, highlighting the implications for ALI and therapies. This study, therefore, enhances our capacity to therapeutically target DR-specific proteins.

Mechanism of protective effect of xuan-bai-cheng-qi decoction on LPS-induced acute lung injury based on an integrated network pharmacology and RNA-sequencing approach.

Xuan-bai-cheng-qi decoction (XCD), a traditional Chinese medicine (TCM) prescription, has been widely used to treat a variety of respiratory diseases in China, especially to seriously infectious diseases such as acute lung injury (ALI). Due to the complexity of the chemical constituent, however, the underlying pharmacological mechanism of action of XCD is still unclear. To explore its protective mechanism on ALI, firstly, a network pharmacology experiment was conducted to construct a component-target network of XCD, which identified 46 active components and 280 predicted target genes. Then, RNA sequencing (RNA-seq) was used to screen differentially expressed genes (DEGs) between ALI model rats treated with and without XCD and 753 DEGs were found. By overlapping the target genes identified using network pharmacology and DEGs using RNA-seq, and subsequent protein-protein interaction (PPI) network analysis, 6 kernel targets such as vascular epidermal growth factor (VEGF), mammalian target of rapamycin (mTOR), AKT1, hypoxia-inducible factor-1α (HIF-1α), and phosphoinositide 3-kinase (PI3K) and gene of phosphate and tension homology deleted on chromsome ten (PTEN) were screened out to be closely relevant to ALI treatment. Verification experiments in the LPS-induced ALI model rats showed that XCD could alleviate lung tissue pathological injury through attenuating proinflammatory cytokines release such as tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1ß. Meanwhile, both the mRNA and protein expression levels of PI3K, mTOR, HIF-1α, and VEGF in the lung tissues were down-regulated with XCD treatment. Therefore, the regulations of XCD on PI3K/mTOR/HIF-1α/VEGF signaling pathway was probably a crucial mechanism involved in the protective mechanism of XCD on ALI treatment.

Ginsenoside Rb1 protects from Staphylococcus aureus-induced oxidative damage and apoptosis through endoplasmic reticulum-stress and death receptor-mediated pathways.

Acute lung injury (ALI) is acute uncontrolled inflammation of lung tissue that leads to high fatality both in human and animals. Staphylococcus aureus (S. aureus) could be an opportunistic, versatile bacterial etiology of ALI. Ginsenoside Rb1 (Rb1) is extracted from the Panax ginseng, which displays a wide range of biological and pharmacological effects. However, protective effects of Rb1 in S. aureus-induced ALI though endoplasmic reticulum (ER) stress and death receptor-mediated pathways have not yet been reported. Therefore, present study was planned with the aims to investigate the antioxidant and anti-apoptotic properties of Rb1 through regulation of ER stress as well as death receptor-mediated pathways in ALI induced by S. aureus in mice. In this study, four groups of healthy Kunming mice (n = 48) were used. The S. aureus (80 µl; 1 ×107 CFU/10 µl) was administered intranasally to establish mice model of ALI. After 24 h of onset of S. aureus-induced ALI, the mice were injected thrice with Rb1 (40 mg/kg) intraperitoneally six hours apart. Histopathology, enzyme linked immunosorbent assay (ELISA), real time quantitative polymerase chain reaction (RT-qPCR), Immunohistochemistry and western blotting assay were employed in the current study. Our results suggested that Rb1 administration save lungs from pulmonary injury by reducing wet to dry (W/D) ratio, protein levels, total cells, neutrophilic count, reactive oxygen species (ROS), myeloperoxidase (MPO), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (Gpx)1 depletion. Meanwhile, Rb1 therapy ameliorated histopathology alteration of lung tissue and pro-inflammatory cytokines secretion. The gene expression of ER stress marker (PERK, AFT-6, IRE1 and CHOP) were upregulated markedly (P < .05) in S. aureus-instilled groups, which was reduced by Rb1 administration that is reveled from the result findings of the RT-qPCR and immunoblot assay. The results of immunohistochemistry for CHOP indicated the increased expression in S. aureus groups which in turn ameliorated by Rb1 treatment. The mRNA expression demonstrated that death receptor-associated genes (FasL, Fas, FADD and caspase-8) showed up-regulation in S. aureus group. The similar findings were observed for the protein expression of caspase-8, FADD and Fas. Rb1 treatment markedly (P < .05) reversed protein and mRNA expression levels of these death receptor-associated genes when compared to the S. aureus group. Taken together, Rb1 attenuated S. aureus-induced oxidative damage via the ER stress-mediated pathway and apoptosis through death receptor-mediated pathway. Conclusively, our findings provide an insight into preventive mechanism of Rb1 in ALI caused by S. aureus and hence proven a scientific baseline for the therapeutic application of Rb1.

An integrated network pharmacology and RNA-Seq approach for exploring the preventive effect of Lonicerae japonicae flos on LPS-induced acute lung injury.

ETHNOPHARMACOLOGICAL RELEVANCE: Lonicerae japonicae flos (LJF, the dried flower bud or newly bloomed flower of Lonicera japonica Thunb.), a typical herbal medicine, targets the lung, heart and stomach meridian with the function of clearing heat and detoxication. It ameliorated inflammatory responses and protected against acute lung inflammation in animal models. Acute lung injury (ALI) is a kind of inflammatory disease in which alveolar cells are damaged. However, a network pharmacology study to thoroughly investigate the mechanisms preventing ALI has not been performed. AIM OF THE STUDY: In this study, we examined the main active ingredients in LJF and the protective effects of LJF on LPS-induced ALI in rats. MATERIALS AND METHODS: First, the main active ingredients of LJF were screened in the TCMSP database, and the ALI-associated targets were collected from the GeneCards database. Then, we used compound-target and target-pathway networks to uncover the preventive mechanisms of LJF. Furthermore, we assessed the preventive effects of LJF in an LPS-induced rat model with the RNA-Seq technique to validate the possible molecular mechanisms of the effects of LJF in the treatment of ALI. RESULTS: The network pharmacology results identified 28 main active compounds in LJF, and eight chemical components highly related to the potential targets, which were potential active compounds in LJF. In all, 94 potential targets were recognized, including IL6, TNF, PTGS2, APP, F2, and GRM5. The pathways revealed that the possible targets of LJF involved in the regulation of the IL-17 signalling pathway. Then, in vivo experiments indicated that LJF decreased the levels of proinflammatory cytokines (TNF-, IL-1, and IL-6) in serum and bronchoalveolar lavage fluid, decreased the levels of oxidative stress factors (MDA and MPO) and increased the activities of SOD and GSH-Px in lung tissue. The RNA-Seq results revealed that 7811, 775 and 3654 differentially expressed genes (DEGs) in Ctrl (control group), ALI-LJF (Lonicerae japonicae flos group) and ALI-DXSM (dexamethasone group), respectively. KEGG pathway analysis showed that the DEGs associated with immune response and inflammation signalling pathways and the IL-17 signalling pathway were significantly enriched in LJF. Compared with those in ALI, the expression of CXCL2, CXCL1, CXCL6, NFKBIA, IFNG, IL6, IL17A, IL17F, IL17C, MMP9 and TNFAIP3, which are involved in the IL-17 signalling pathway, were significantly decreased in the LJF group according to the qRT-PCR analyses. CONCLUSIONS: In view of the network pharmacology and RNA-Seq results, the study identified the main active ingredient and potential targets of LJF involved in protecting against ALI, which suggests directions for further research on LJF.