Systematic Pharmacology-Based Strategy to Explore the Mechanism of Bufei Huoxue Capsule in the Treatment of Chronic Obstructive Pulmonary Disease.

Objective: To explore the effects and mechanisms of Bufei Huoxue Capsule (BHC) on chronic obstructive pulmonary disease (COPD) based on network pharmacology. Methods: The effective components and related targets of BHC were collected by searching TCMSP, HERB, and ETCM databases, after which the related targets of COPD were obtained on GeneCards and OMIM databases. The common targets were imported into the STRING database and Cytoscape database to construct a target interaction network and screen core targets. Next, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed on the Metascape platform. According to the prediction results of network pharmacology, the action mechanism was further examined in an animal model of COPD. The pathological changes of lung tissue were observed by HE staining; goblet cells and mucus secretion in lung tissue were observed by AB-PAS staining, airway collagen deposition was observed by Masson staining, and the expression of NE, TGF-ß1, P-EGFR/EGFR, P-ERK1/2/ERK1/2, P-JNK/JNK, and P-P38/P38MAPK protein was detected by Western blot analysis. Results: A total of 379 targets related to BHC and 7391 targets related to COPD were obtained, including 313 potential targets of BHC in treating chronic obstructive pulmonary disease, with JUN, AKT1, TNF, IL6, EGFR, MAPK1, and MAPK14 as the core targets. Through enrichment analysis, BHC may interfere with COPD by regulating the MAPK signal pathway, HIF-1 signal pathway, NF-κB signal pathway, cAMP signal pathway, cGMP-PKG signal pathway, and so on. Animal experiments showed that the BHC could reduce airway inflammatory cell infiltration, inhibit airway epithelial goblet cell proliferation, reduce mucus secretion, and improve small airway collagen fiber deposition in COPD model rats. Besides, BHC could downregulate the protein expression of NE, TGF-ß1, P-EGFR, P-ERK1/2, and P-P38MAPK. Conclusion: BHC can reduce airway inflammation, inhibit mucus hypersecretion, and improve airway remodeling by regulating the MAPK signal transduction pathway.

Erratum to: Herbal decoctosome is a novel form of medicine.

Following the published article, we noticed an error duplication in Figure 5G "control" and "PGY-6" that was introduced during the revised process, with an attempt to replace it with higher-resolution images. Here we provide the original data in the first submitted manuscript (Figure 5G).

Herbal decoctosome is a novel form of medicine.

Traditionally, herbal medicine is consumed by drinking decoctions produced by boiling herbs with water. The functional components of the decoction are heat stable. Small RNAs (sRNAs) were reported as a new class of functional components in decoctions. However, the mechanisms by which sRNAs survive heat treatment of the decoction and enter cells are unclear. Previous studies showed that plant-derived exosome-like nanoparticles (ELNs), which we call botanosomes, could deliver therapeutic reagents in vivo. Here, we report that heat-stable decoctosomes (ELNs) from decoctions have more therapeutic effects than the decoctions in vitro and demonstrate therapeutic efficacy in vivo. Furthermore, sRNAs, such as HJT-sRNA-m7 and PGY-sRNA-6, in the decoctosome exhibit potent anti-fibrosis and anti-inflammatory effects, respectively. Decoctosome is comprised of lipids, chemical compounds, proteins, and sRNAs. A medical decoctosome mimic is called bencaosome. A single lipid sphinganine (d22:0) identified in the decoctosome was mixed and heated with the synthesized sRNAs to form the simplest bencaosome. This simple bencaosome structure was identified by critical micelle concentration (cmc) assay that sRNAs coassembled with sphinganine (d22:0) to form the lipid layers of vesicles. The heating process facilitates co-assembly of sRNAs and sphinganine (d22:0) until a steady state is reached. The artificially produced sphinganine-HJT-sRNA-m7 and sphinganine- PGY-sRNA-6 bencaosomes could ameliorate bleomycin-induced lung fibrosis and poly(I:C)-induced lung inflammation, respectively, following oral administration in mice. Our study not only demonstrates that the herbal decoctosome may represent a combinatory remedy in precision medicine but also provides an effective oral delivery route for nucleic acid therapy.

Louqin Zhisou Decoction Inhibits Mucus Hypersecretion for Acute Exacerbation of Chronic Obstructive Pulmonary Disease Rats by Suppressing EGFR-PI3K-AKT Signaling Pathway and Restoring Th17/Treg Balance.

Airway mucus hypersecretion is the main pathogenic factor in acute exacerbation of chronic obstructive pulmonary disease (AECOPD) and the control of mucus secretion is closely associated with survival. Louqin Zhisou decoction (LQZS) has been found to improve lung function and reduce sputum in AECOPD patients, but the mechanism remains unclear. This study aimed to explore the mechanism of LQZS against mucus hypersecretion in lung tissues of rat AECOPD model. Wistar rats were used to establish AECOPD model by intratracheal instillation of LPS in combination with the continuous cigarette smoking. Rats were administrated LQZS/clarithromycin (CAM)/distilled water via gavage every day and all rats were sacrificed after 30 days. BALF and lung tissues were obtained. Lung morphology, cytokines levels, MUC5AC mRNA transcription and protein expression, phosphorylation of the EGFR-PI3K-AKT signaling pathway, and molecules involved in Th17/Treg balance were evaluated. The results demonstrated that LQZS protected rats from decline in pulmonary function and ameliorated lung injury. LQZS treatment decreased the number of goblet cells in airway and suppressed MUC5AC mRNA and protein expression of lung tissues. Furthermore, LQZS attenuated the level of phospho-EGFR, phospho-PI3K and phospho-AKT in AECOPD rats. In addition, LQZS could inhibit the production of proinflammatory cytokines in BALF, including IL-6 and IL-17A and downregulate the secretion of NE and MCP-1, indicating that LQZS could limit inflammatory responses in AECOPD. Moreover, LQZS reversed RORγt and Foxp3 expression, the key transcription factors of Th17 and Treg, respectively. In conclusion, this research demonstrated the inhibitory effects of LQZS against mucus hypersecretion in AECOPD via suppressing EGFR-PI3K-AKT signaling pathway and restoring Th17/Treg balance.

Plant-derived phosphocholine facilitates cellular uptake of anti-pulmonary fibrotic HJT-sRNA-m7.

Pulmonary fibrosis, a progressive chronic disease with a high mortality rate, has limited treatment options. Currently, lung transplantation remains the only effective treatment. Here we report that a small RNA, HJT-sRNA-m7, from a Chinese herbal medicine Hong Jing Tian (HJT, RHODIOHAE CRENULATAE RADIX ET RHIZOMA, Rhodiola crenulata) can effectively reduce the expressions of fibrotic hallmark genes and proteins both in alveolar in vitro and in mouse lung tissues in vivo. We also discovered over one hundred oil-soluble chemicals from HJT decoctions, most of which are found in lipid extracts from other Chinese herbals decoctions, including Pu Gong Ying (PGY, TARAXACI HERBA, Taraxacum mongolicum), Chuan Xin Lian (CXL, changed to "ANDROGRAPHIS HERBA, Andrographis paniculata"), and Jin Yin Hua (JYH, lonicera japonica or Honeysuckle). We identified the active component in these decoctions as two forms of phosphocholines, PC (18:0/18:2) and PC (16:0/18:2). These PCs potentially could form liposomes with small RNAs to enter human alveolar and gastric cells. Our experimental results suggest an unprecendent lipid complex route through which botanic small RNA can enter human bodies. Our results provide an innovative treatment strategy for oral delivery of siRNAs as therapeutic medication.

Large-scale analysis of small RNAs derived from traditional Chinese herbs in human tissues.

Plant-derived microRNAs have recently been reported to function in human blood and tissues. Controversy was immediately raised due to possible contamination and the lack of large sample sizes. Here, we report thousands of unique small RNAs derived from traditional Chinese medicine (TCM) herbs found in human blood cells and mouse lung tissues using a large-scale analysis. We extracted small RNAs from decoctions of 10 TCM plants (Ban Zhi Lian, Chai Hu, Chuan Xin Lian, Di Ding Zi Jin, Huang Qin, Jin Yin Hua, Lian Qiao, Pu Gong Ying, Xia Ku Cao, and Yu Xing Cao) and obtained millions of RNA sequences from each herb. We also obtained RNA-Seq data from the blood cells of humans who consumed herbal decoctions and from the lung tissues of mice administered RNAs from herbal decoctions via oral gavage. We identified thousands of unique small RNA sequences in human blood cells and mouse lung tissues. Some of these identified small RNAs from Chuan Xin Lian and Hong Jing Tian could be mapped to the genomes of the herbs, confirming their TCM plant origin. Small RNAs derived from herbs regulate mammalian gene expression in a sequence-specific manner, and thus are a superior novel class of herbal drug components that hold great potential as oral gene-targeted therapeutics, highlighting the important role of herbgenomics in their development.

miRNA-200c-3p is crucial in acute respiratory distress syndrome.

Influenza infection and pneumonia are known to cause much of their mortality by inducing acute respiratory distress syndrome (ARDS), which is the most severe form of acute lung injury (ALI). Angiotensin-converting enzyme 2 (ACE2), which is a negative regulator of angiotensin II in the renin-angiotensin system, has been reported to have a crucial role in ALI. Downregulation of ACE2 is always associated with the ALI or ARDS induced by avian influenza virus, severe acute respiratory syndrome-coronavirus, respiratory syncytial virus and sepsis. However, the molecular mechanism of the decreased expression of ACE2 in ALI is unclear. Here we show that avian influenza virus H5N1 induced the upregulation of miR-200c-3p, which was then demonstrated to target the 3'-untranslated region of ACE2. Then, we found that nonstructural protein 1 and viral RNA of H5N1 contributed to the induction of miR-200c-3p during viral infection. Additionally, the synthetic analog of viral double-stranded RNA (poly (I:C)), bacterial lipopolysaccharide and lipoteichoic acid can all markedly increase the expression of miR-200c-3p in a nuclear factor-κB-dependent manner. Furthermore, markedly elevated plasma levels of miR-200c-3p were observed in severe pneumonia patients. The inhibition of miR-200c-3p ameliorated the ALI induced by H5N1 virus infection in vivo, indicating a potential therapeutic target. Therefore, we identify a shared mechanism of viral and bacterial lung infection-induced ALI/ARDS via nuclear factor-κB-dependent upregulation of miR-200c-3p to reduce ACE2 levels, which leads increased angiotensin II levels and subsequently causes lung injury.

Neuraminidase of Influenza A Virus Binds Lysosome-Associated Membrane Proteins Directly and Induces Lysosome Rupture.

UNLABELLED: As a recycling center, lysosomes are filled with numerous acid hydrolase enzymes that break down waste materials and invading pathogens. Recently, lysosomal cell death has been defined as "lysosomal membrane permeabilization and the consequent leakage of lysosome contents into cytosol." Here, we show that the neuraminidase (NA) of H5N1 influenza A virus markedly deglycosylates and degrades lysosome-associated membrane proteins (LAMPs; the most abundant membrane proteins of lysosome), which induces lysosomal rupture, and finally leads to cell death of alveolar epithelial carcinoma A549 cells and human tracheal epithelial cells. The NA inhibitors peramivir and zanamivir could effectively block the deglycosylation of LAMPs, inhibit the virus cell entry, and prevent cell death induced by the H5N1 influenza virus. The NA of seasonal H1N1 virus, however, does not share these characteristics. Our findings not only reveal a novel role of NA in the early stage of the H5N1 influenza virus life cycle but also elucidate the molecular mechanism of lysosomal rupture crucial for influenza virus induced cell death. IMPORTANCE: The integrity of lysosomes is vital for maintaining cell homeostasis, cellular defense and clearance of invading pathogens. This study shows that the H5N1 influenza virus could induce lysosomal rupture through deglycosylating lysosome-associated membrane proteins (LAMPs) mediated by the neuraminidase activity of NA protein. NA inhibitors such as peramivir and zanamivir could inhibit the deglycosylation of LAMPs and protect lysosomes, which also further interferes with the H5N1 influenza virus infection at early stage of life cycle. This work is significant because it presents new concepts for NA's function, as well as for influenza inhibitors' mechanism of action, and could partially explain the high mortality and high viral load after H5N1 virus infection in human beings and why NA inhibitors have more potent therapeutic effects for lethal avian influenza virus infections at early stage.

Hsa-miR-1246, hsa-miR-320a and hsa-miR-196b-5p inhibitors can reduce the cytotoxicity of Ebola virus glycoprotein in vitro.

Ebola virus (EBOV) causes a highly lethal hemorrhagic fever syndrome in humans and has been associated with mortality rates of up to 91% in Zaire, the most lethal strain. Though the viral envelope glycoprotein (GP) mediates widespread inflammation and cellular damage, these changes have mainly focused on alterations at the protein level, the role of microRNAs (miRNAs) in the molecular pathogenesis underlying this lethal disease is not fully understood. Here, we report that the mi-RNAs hsa-miR-1246, hsa-miR-320a and hsa-miR-196b-5p were induced in human umbilical vein endothelial cells (HUVECs) following expression of EBOV GP. Among the proteins encoded by predicted targets of these miRNAs, the adhesion-related molecules tissue factor pathway inhibitor (TFPI), dystroglycan1 (DAG1) and the caspase 8 and FADD-like apoptosis regulator (CFLAR) were significantly downregulated in EBOV GP-expressing HUVECs. Moreover, inhibition of hsa-miR-1246, hsa-miR-320a and hsa-miR-196b-5p, or overexpression of TFPI, DAG1 and CFLAR rescued the cell viability that was induced by EBOV GP. Our results provide a novel molecular basis for EBOV pathogenesis and may contribute to the development of strategies to protect against future EBOV pandemics.

Curcumin loaded mixed micelles composed of Pluronic P123 and F68: preparation, optimization and in vitro characterization.

In this study, curcumin (Cur) loaded mixed micelles (Cur-PF), composed of Pluronic P123 (P123) and Pluronic F68 (F68), was prepared using the thin-film hydration method and evaluated in vitro. The preparation process was optimized with a central composite design (CCD). The average size of the mixed micelles was 68.2 nm, and the encapsulating efficiency for Cur was 86.93%, and 6.996% for drug-loading. Compared with the Cur propylene glycol solution, the in vitro release of Cur from Cur-PF presented the sustained-release property. The in vitro cytotoxicity assay showed that the IC(50) values on MCF-7 cells for Cur-PF and free Cur in DMSO solution were 5.04 µg/mL and 8.35 µg/mL, while 2.52 µg/mL and 8.27 µg/mL on MCF-7/ADR cells. It could be concluded from the results that P123/F68 mixed micelles might serve as a potential nanocarrier to improve the solubility and biological activity of Cur.