Tanshinone IIA improves degranulation of mast cells and allergic rhinitis induced by ovalbumin by inhibiting the PLCγ1/PKC/IP3R pathway

Allergic rhinitis (AR) is a common allergic inflammatory and chronic reactive disease caused by allergen-induced immunoglobulin E (IgE). Tanshinone IIA (Tan IIA) is one of the active ingredients in Salvia miltiorrhiza Bunge (Danshen) and plays a vital role in inhibiting inflammation. Thus, we hypothesized that Tan IIA has anti-allergic effects and studied the function of Tan IIA in mast cells and an AR animal model. We induced RBL-2H3 cell sensitization with monoclonal anti-2,4,6-dinitrophenyl-immunoglobulin (Ig) E/human serum albumin (DNP-IgE/HSA) and constructed an ovalbumin (OVA)-induced AR model in mice. The role of Tan IIA in AR progression was studied using the MTT assay, ELISA, western blot, toluidine blue staining, HE staining, and Alcian blue and safranin O (A&S) staining. Tan IIA treatment significantly increased IgE/HSA-induced cell viability. However, Tan IIA treatment markedly downregulated the expression levels of β-hexosaminidase, histamine, tumor necrosis factor (TNF-α), interleukin 1β (IL-1β), IL-4, and IL-5 in IgE/HSA-induced cells. Furthermore, Tan IIA improved typical symptoms in the OVA-induced AR model mice by inhibiting the phospholipase Cγ1 (PLCγ1)/protein kinase C (PKC)/IP3R pathway. Additionally, Tan IIA effectively improved the degranulation of RBL-2H3 cells and OVA-induced AR in mice. Together, these results suggest that Tan IIA may be a potential drug for the treatment of AR in the future.

Tryptophan Ameliorates Barrier Integrity and Alleviates the Inflammatory Response to Enterotoxigenic Escherichia coli K88 Through the CaSR/Rac1/PLC-γ1 Signaling Pathway in Porcine Intestinal Epithelial Cells

BackgroundImpaired intestinal barrier integrity plays a crucial role in the development of many diseases such as obesity, inflammatory bowel disease, and type 2 diabetes. Thus, protecting the intestinal barrier from pathological disruption is of great significance. Tryptophan can increase gut barrier integrity, enhance intestinal absorption, and decrease intestinal inflammation. However, the mechanism of tryptophan in decreasing intestinal barrier damage and inflammatory response remains largely unknown. The objective of this study was to test the hypothesis that tryptophan can enhance intestinal epithelial barrier integrity and decrease inflammatory response mediated by the calcium-sensing receptor (CaSR)/Ras-related C3 botulinum toxin substrate 1 (Rac1)/phospholipase Cγ1 (PLC-γ1) signaling pathway.MethodsIPEC-J2 cells were treated with or without enterotoxigenic Escherichia coli (ETEC) K88 in the absence or presence of tryptophan, CaSR inhibitor (NPS-2143), wild-type CaSR overexpression (pcDNA3.1-CaSR-WT), Rac1-siRNA, and PLC-γ1-siRNA.ResultsThe results showed that ETEC K88 decreased the protein concentration of occludin, zonula occludens-1 (ZO-1), claudin-1, CaSR, total Rac1, Rho family member 1 of porcine GTP-binding protein (GTP-rac1), phosphorylated phospholipase Cγ1 (p-PLC-γ1), and inositol triphosphate (IP3); suppressed the transepithelial electrical resistance (TEER); and enhanced the permeability of FITC-dextran compared with the control group. Compared with the control group, 0.7 mM tryptophan increased the protein concentration of CaSR, total Rac1, GTP-rac1, p-PLC-γ1, ZO-1, claudin-1, occludin, and IP3; elevated the TEER; and decreased the permeability of FITC-dextran and contents of interleukin-8 (IL-8) and TNF-α. However, 0.7 mM tryptophan+ETEC K88 reversed the effects induced by 0.7 mM tryptophan alone. Rac1-siRNA+tryptophan+ETEC K88 or PLC-γ1-siRNA+tryptophan+ETEC K88 reduced the TEER, increased the permeability of FITC-dextran, and improved the contents of IL-8 and TNF-α compared with tryptophan+ETEC K88. NPS2143+tryptophan+ETEC K88 decreased the TEER and the protein concentration of CaSR, total Rac1, GTP-rac1, p-PLC-γ1, ZO-1, claudin-1, occludin, and IP3; increased the permeability of FITC-dextran; and improved the contents of IL-8 and TNF-α compared with tryptophan+ETEC K88. pcDNA3.1-CaSR-WT+Rac1-siRNA+ETEC K88 and pcDNA3.1-CaSR-WT+PLC-γ1-siRNA+ETEC K88 decreased the TEER and enhanced the permeability in porcine intestine epithelial cells compared with pcDNA3.1-CaSR-WT+ETEC K88.ConclusionTryptophan can improve intestinal epithelial barrier integrity and decrease inflammatory response through the CaSR/Rac1/PLC-γ1 signaling pathway.

PLCγ1 promotes phase separation of T cell signaling components

The T cell receptor (TCR) pathway receives, processes, and amplifies the signal from pathogenic antigens to the activation of T cells. Although major components in this pathway have been identified, the knowledge on how individual components cooperate to effectively transduce signals remains limited. Phase separation emerges as a biophysical principle in organizing signaling molecules into liquid-like condensates. Here, we report that phospholipase Cγ1 (PLCγ1) promotes phase separation of LAT, a key adaptor protein in the TCR pathway. PLCγ1 directly cross-links LAT through its two SH2 domains. PLCγ1 also protects LAT from dephosphorylation by the phosphatase CD45 and promotes LAT-dependent ERK activation and SLP76 phosphorylation. Intriguingly, a nonmonotonic effect of PLCγ1 on LAT clustering was discovered. Computer simulations, based on patchy particles, revealed how the cluster size is regulated by protein compositions. Together, these results define a critical function of PLCγ1 in promoting phase separation of the LAT complex and TCR signal transduction.

Compound Cocktail Inhibits Influenza Viral Pneumonia via Phospholipase Cγ1 Phosphorylation-Related Necroptosis and Partial Autophagy in Natural Killer Cells

Influenza viral infections are prone to global outbreaks and cause pneumonia in affected populations. High morbidity and mortality caused by pneumonia occur during an influenza pandemic. Antivirals or control of inflammation is the primary means of influenza treatment. A compound cocktail composed of arctiin, daidzein, glycyrrhizic acid, and liquiritin inhibited mouse pneumonia resulting from a PR8 viral infection and caused a weight gain after oral administration. Natural killer cell activating receptors, both Ly49D and Ly49H in the lungs, were increased in the treatment in mice. In H3N2 virus-infected natural killer-92MI cells, the cocktail treatment had different effects on phosphorylation sites of phospholipase Cγ1 (PLCγ1) and killed infected cells through necroptosis or late apoptosis, in which RIP3 was increased and both caspase-3 and phosphorylated-JNK in the cells were downregulated. Acid phosphatase activity in viral-infected natural killer-92MI cells was induced by the compound cocktail treatment, which could be related to the p62 decrease in natural killer-92MI cells. In addition, an autophagic flux induction was observed in alveolar basal epithelial cells (A549). Protein p65, but not phosphorylated-p65, was significantly decreased by the treatment. Our results indicate that the compound cocktail strengthened the phosphorylation of PLCγ1-related necroptosis and partial autophagy in natural killer cells, which could yield an inhibitory effect on viral pneumonia in influenza.

PhospholipaseCγ1/calcium-dependent membranous localization of Gsdmd-N drives endothelial pyroptosis, contributing to lipopolysaccharide-induced fatal outcome

Our study newly reveals that phospholipase Cγ1 (PLCγ1) contributes to gasdermin D (Gsdmd)-mediated endothelial pyroptosis in a calcium-dependent fashion. Cytosolic calcium signaling promotes activated NH2-terminal fragment of Gsdmd (Gsdmd-N) to translocate to the plasma membrane, enhancing endothelial pyroptosis induced by cytoplasmic LPS. Genetic or pharmacologic inhibition of endothelial PLCγ1 attenuated breakdown of endothelial barrier, reduced vascular leakage, improve perfusion disturbances, and decrease mortality of mice in endotoxemia.