Integrated Network Pharmacology and Lipidomics to Reveal the Inhibitory Effect of Qingfei Oral Liquid on Excessive Autophagy in RSV-Induced Lung Inflammation

Background: Respiratory syncytial virus (RSV) can cause varying degrees of lung inflammation in children. Qingfei Oral Liquid (QF) is effective in treating childhood RSV-induced lung inflammation (RSV-LI) in clinics, but its pharmacological profiles and mechanisms remain unclear.Methods: This study combined network Pharmacology, lipidomics, pharmacodynamics, and pathway validation to evaluate the therapeutic mechanisms of QF. Using Cytoscape (v3.8.2) and enrichment analyses from the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO), a global view of the putative compound-target-pathway network was created. The corresponding lipidomic profiles were then used to detect differently activated lipids, revealing the metabolic pathway, using ultra-high-performance liquid chromatography linked to hybrid Quadrupole-Exactive Orbitrap mass spectrometry (UHPLC-Q-Exactive Orbitrap MS). Meanwhile, the in vivo efficiency of QF, the enrichment pathway, and the excessive autophagy inhibition mechanisms were validated in RSV-infected mice models.Results: The network pharmacology results demonstrated 117 active compounds acted directly upon 101 core targets of QF against RSV-LI. The most significantly enriched pathway was the PI3K/Akt/mTOR signaling pathway (p < 0.05). In addition, untargeted lipidomics were performed, and it was revealed that higher lung levels of DAG 30:0, DAG 30:5, DAG 32:0, DAG 16:0_18:0, DAG 17:0_17:0, DAG 34:1, DAG 36:0, DAG 36:1 in the RSV-LI group were decreased after QF administration (FDR < 0.05, FC > 1.2). Lipin-1, a key enzyme in DAG synthesis, was increased in the RSV-LI mouse model. Animal experiments further validated that QF inhibited the PI3K/Akt/mTOR signaling pathway, with lower lung levels of phosphorylated PI3K, AKT and mTOR, as well as its related proteins of lipin-1 and VPS34 (p < 0.01). Finally, pharmacodynamic investigations indicated that QF reduced airway inflammation caused by excessive autophagy by decreasing lung levels of RSV F and G proteins, Beclin-1, Atg5, and LC3B II, IL-1 and TNF-α (p < 0.05).Conclusion: Lipidomic-based network pharmacology, along with experimental validation, may be effective approaches for illustrating the therapeutic mechanism of QF in the treatment of RSV-LI.

Sequence Variation in the Bovine Lipin-1 Gene (LPIN1) and Its Association with Milk Fat and Protein Contents in New Zealand Holstein-Friesian × Jersey (HF × J)-cross Dairy Cows

Lipin-1 is known to play a regulatory role in tissues that function in lipid metabolism. In dairy cows, the lipin-1 gene (LPIN1) is highly expressed in the mammary gland, but its function in milk production is less understood. In this study, we used PCR-single strand conformation polymorphism analysis to investigate sequence variation in three regions of bovine LPIN1 in New Zealand Holstein-Friesian × Jersey (HF × J)-cross dairy cows, including part of the 5′ non-coding region, the region containing the LPIN1β-spliced exon, and the sixth coding exon that encodes the putative transcriptional activating domain of the protein. No variation was found in the LPIN1β-spliced exon, but two sequence variants containing one single nucleotide polymorphism (SNP) were identified in the 5′ non-coding region and four sequence variants containing four non-synonymous SNPs were identified in the sixth coding exon. Among the three common variants of the sixth coding exon, variant C was found to be associated with an increase in milk fat percentage (presence 4.96 ± 0.034% vs. absence 4.81 ± 0.050%; p = 0.006) and milk protein percentage (presence 4.09 ± 0.017% vs. absence 3.99 ± 0.025%; p = 0.001), but no associations (p > 0.01) were detected for milk yield. These results suggest that variation in LPIN1 affect the synthesis of fat and proteins in milk and has potential as a gene-marker to improve milk production traits.

Lipin-1-derived diacylglycerol activates intracellular TRPC3 which is critical for inflammatory signaling

Exposure to Gram-negative bacterial LPS exacerbates host immune responses and may lead to sepsis, a life-threatening condition. Despite its high mortality and morbidity, no drugs specifically directed to treating sepsis are currently available. Using human cell genetic depletion, pharmacological inhibition, live-cell microscopy and organelle-targeted molecular sensors we present evidence that the channel TRPC3 is activated intracellularly during macrophage exposure to LPS and is essential for Ca2+ release from internal stores. In this manner, TRPC3 participates in cytosolic Ca2+ elevations, activation of the transcription factor NF-κB and cytokine upregulation. We also report that TRPC3 is activated by diacylglycerol generated by the phosphatidic acid phosphatase lipin-1. In accord with this, lipin-1-deficient cells exhibit reduced Ca2+ responses to LPS challenge. Finally, pharmacological inhibition of TRPC3 reduces systemic inflammation induced by LPS in mice. Collectively, our study unveils a central component of LPS-triggered Ca2+ signaling that involves intracellular sensing of lipin-1-derived DAG by TRPC3, and opens new opportunities for the development of strategies to treat LPS-driven inflammation.

NLIP and HAD-like Domains of Pah1 and Lipin 1 Phosphatidate Phosphatases Are Essential for Their Catalytic Activities

Saccharomyces cerevisiae Pah1 phosphatidate phosphatase (PAP) catalyzes the dephosphorylation of phosphatidate to yield diacylglycerol, controlling phospholipids and triacylglycerol metabolisms. Pah1 and human Lipin 1 are intrinsically disordered proteins with 56% and 43% unfolded regions, respectively. Truncation analysis of the conserved and non-conserved regions showed that N- and C-conserved regions are essential for the catalytic activity of Pah1. PAP activities can be detected in the conserved N-terminal Lipin (NLIP) domain and C-terminal Lipin (CLIP)/haloacid dehalogenase (HAD)-like domain of Pah1 and Lipin 1, suggesting that the evolutionarily conserved domains are essential for the catalytic activity. The removal of disordered hydrophilic regions drastically reduced the protein solubility of Pah1. Thioredoxin is an efficient fusion protein for production of soluble NLIP–HAD recombinant proteins in Escherichia coli.

The middle lipin domain adopts a membrane-binding dimeric protein fold

Phospholipid synthesis and fat storage as triglycerides are regulated by lipin phosphatidic acid phosphatases (PAPs), whose enzymatic PAP function requires association with cellular membranes. Using hydrogen deuterium exchange mass spectrometry, we find mouse lipin 1 binds membranes through an N-terminal amphipathic helix, the Ig-like domain and HAD phosphatase catalytic core, and a middle lipin (M-Lip) domain that is conserved in mammalian and mammalian-like lipins. Crystal structures of the M-Lip domain reveal a previously unrecognized protein fold that dimerizes. The isolated M-Lip domain binds membranes both in vitro and in cells through conserved basic and hydrophobic residues. Deletion of the M-Lip domain in lipin 1 reduces PAP activity, membrane association, and oligomerization, alters subcellular localization, diminishes acceleration of adipocyte differentiation, but does not affect transcriptional co-activation. This establishes the M-Lip domain as a dimeric protein fold that binds membranes and is critical for full functionality of mammalian lipins.

Lipin-1, a Versatile Regulator of Lipid Homeostasis, Is a Potential Target for Fighting Cancer

The rewiring of lipid metabolism is a major adaptation observed in cancer, and it is generally associated with the increased aggressiveness of cancer cells. Targeting lipid metabolism is therefore an appealing therapeutic strategy, but it requires a better understanding of the specific roles played by the main enzymes involved in lipid biosynthesis. Lipin-1 is a central regulator of lipid homeostasis, acting either as an enzyme or as a co-regulator of transcription. In spite of its important functions it is only recently that several groups have highlighted its role in cancer. Here, we will review the most recent research describing the role of lipin-1 in tumor progression when expressed by cancer cells or cells of the tumor microenvironment. The interest of its inhibition as an adjuvant therapy to amplify the effects of anti-cancer therapies will be also illustrated.