Qiangli Wuhu mixture alleviates LPS-induced pneumonia by inhibiting the TLR4/NF-κB/NLRP3 pathway: a study based on network pharmacology.

CONTEXT: Qiangli Wuhu (QLWH) mixture is a concoction approved and registered by Ningxia Medical Products Administration. It has therapeutic effects on various types of pneumonia. OBJECTIVE: To clarify the mechanisms of QLWH in treating pneumonia. MATERIALS AND METHODS: The potential targets of QLWH in the treatment of pneumonia were predicted by network pharmacology. Male, Institute of Cancer Research (ICR) mice were randomly divided into five groups of 12 mice, control, vehicle, QLWH (10 and 20 mg/kg) and dexamethasone (DXM), and orally treated twice daily with normal saline, QLWH or DXM. The pneumonia model was established by tracheal instillation of lipopolysaccharide (LPS). After treatment five days, ELISA, H&E staining and Western blot were used to investigate protective effects of QLWH. RESULTS: Nine hundred and ninety-four active ingredients were found through network pharmacology, corresponding to 135 targets for the treatment of pneumonia; compared to the vehicle group, QLWH (10 and 20 mg/kg) significantly decreased the levels of TNF-α (14.3% and 28.8%), IL-1ß (23.9% and 42.8%) and IL-6 (13.2% and 16.1%), increased the levels of IL-10 (134.3% and 172.9%); in terms of mechanism, QLWH down-regulated TLR4/NF-κB/NLRP3 axis related proteins in lung tissue of pneumonia model mice (p < 0.05). DISCUSSION AND CONCLUSIONS: This study combined network pharmacology and animal experiments, providing effective evidence for the clinical promotion of QLWH. Meanwhile, it is of significance for further development.

The truncated MyD88s negatively regulates TLR2 signal on expression of IL17-1 in oyster Crassostrea gigas.

Toll like receptor (TLR) signaling plays a key role in the innate immune recognition and inflammatory regulation in both vertebrates and invertebrates. The expanded TLR signaling components, including 83 TLRs and 10 MyD88s, have been reported in the genome of the Pacific oyster Crassostrea gigas. In the present study, one endogenous TLR (designated CgTLR2) and two MyD88s (including a full-length CgMyD88-2 containing intact TIR domain and Death-domain, and a truncated CgMyD88s with only TIR domain) were identified from oyster C. gigas. CgTLR2 was highly expressed in haemocytes, especially in granulocytes. The recombinant protein of the extracellular LRR domains of CgTLR2 recognized and bound a variety of PAMPs with the strongest binding capability to LPS. The recombinant protein of intracellular TIR domain of CgTLR2 was able to bind the recombinant proteins of rCgMyD88-2 (KD = 1.96 × 10-9 M) and rCgMyD88s (KD = 4.84 × 10-8 M), with higher affinity towards rCgMyD88-2. After Vibrio splendidus stimulation, the mRNA expression levels of CgTLR2 and CgMyD88-2 were rapidly up-regulated at early stage of immune response (from the 3rd hours after V. splendidus stimulation), while that of CgMyD88s did not change until 24 h post stimulation. When CgTLR2 was knocked-down by siRNA interference, the expression levels of CgMyD88-2 and CgMyD88s decreased significantly, concomitant with the down-regulation of expression of CgIL17-1. After the expression of CgMyD88-2 was interfered, the expressions of CgMyD88s and CgIL17-1 were all decreased. In contrast, after the expression of CgMyD88s was interfered, the expressions of CgMyD88-2 and CgIL17-1 all increased. The results showed that CgMyD88s played a negative role in the regulation of CgTLR2 on inflammatory factor CgIL17-1.

The Elevated Expressions of Anti-lipopolysaccharide Factors After Priming Stimulation Confer Lastingly Humoral Protection in Crab Eriocheir sinensis.

Evidence of immune memory in invertebrates (immune priming) has accumulated in various organisms, and both cellular and humoral immune reactions are speculated to be involved in immune priming. However, there is a lack of understanding of the molecular mechanisms involved. In the present study, the protective effect of primed haemolymph was further validated by the increased survival rate of naïve crabs receiving a transfusion of primed haemolymph. By proteomic analysis, there were 474 proteins identified from the primed haemolymph, and most of them were functionally annotated in transport and metabolism classes. A total of 70 proteins were found to be differentially expressed in haemolymph at 12 hours and 7 days after priming stimulation with Aeromonas hydrophila, among which anti-lipopolysaccharide factor 1 (EsALF-1) and 3 (EsALF-3) were identified as the most significant (p < 0.05). After being challenged with A. hydrophila, EsALF-1 and EsALF-3 were highly expressed at both mRNA (in haemocytes) and protein (in haemolymph) levels compared with blank crabs, and the mRNA expressions of components in the EsTLR1-EsMyd88-EsPelle-EsALF pathway also increased significantly (p < 0.05). The EsALF-3 and EsMyd88 were even significantly higher expressed in response to the second A. hydrophila challenge, but their expressions all decreased (p < 0.05) when EsTLR1 was knocked down by RNAi. After the naïve crabs received an injection with the recombinant protein of EsALF-1 (rEsALF-1) or EsALF-3 (rEsALF-3), their survival rate increased significantly (p < 0.05) upon A. hydrophila stimulation. In contrast, the survival rate of the primed crabs reduced significantly (p < 0.05) after they received an injection with the antibody of EsALF-1 or EsALF-3. The enhanced expressions of EsALF-1 and EsALF-3 after A. hydrophilap riming stimulation could sustain for four weeks. All the results suggested that the EsTLR1-mediated productions of EsALF-1 and EsALF-3 in haemolymph played an indispensable role in the month-long humoral immune protection induced by A. hydrophila, which provides solid evidence of immune priming in crabs and a valuable reference for further understanding immune memory in invertebrates.

Enhanced immune response of BMDCs pulsed with H9N2 AIV and CpG.

Dendritic cells (DCs), professional antigen presenting cells, have demonstrated effective in controlling the initial of innate immune, while CpG could improve the performance of immune system. To explore the mechanism of CpG enhancing the immune response, we compared different stimulated mouse DCs with systemic approach microarrays. Analysis revealed 1840 differentially expressed genes in H9N2 stimulated group, more than 1728 altered genes in inactive H9N2 group. Investigation also proved that CpG/inactive H9N2 co-stimulation changed 2140 genes, more than that in H9N2 group, strongly demonstrated that CpG improved the performance of inactive H9N2 vaccination. Pathways analysis founded that DCs response rapid to shift in their maturation state, which involved Toll-like receptor (TLR) pathway significantly. Microarrays results were also verified by qRT-PCR with 14 elected representative genes. Further analysis proved that co-stimulatory molecules (CD40, CD80, CD86 and MHC-II), regulatory protein (IRF-7 and TRAF-6) and pro-inflammatory cytokines (IL-1, IL-6 and IL-12) were all changed and involved in DCs maturation. At last we demonstrated TLR signalling pathway in chicken bone marrow-derived dendritic cells (chBM-DCs) stimulated with CpG. The distinct transcriptional profiles of DCs pulsed with various stimuli expanded our understanding of how DCs respond and recognize influenza.