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Houttuynia cordata polysaccharide (HCP) is extracted from Houttuynia cordata, a key traditional Chinese medicine. The study was to investigate the effects of HCP on intestinal barrier and microbiota in H1N1 virus infected mice. Mice were infected with H1N1 virus and orally administrated HCP at a dosage of 40 mg(kg−1(d−1. H1N1 infection caused pulmonary and intestinal injury and gut microbiota imbalance. HCP significantly suppressed the expression of hypoxia inducible factor-1α and decreased mucosubstances in goblet cells, but restored the level of zonula occludens-1 in intestine. HCP also reversed the composition change of intestinal microbiota caused by H1N1 infection, with significantly reduced relative abundances of Vibrio and Bacillus, the pathogenic bacterial genera. Furthermore, HCP rebalanced the gut microbiota and restored the intestinal homeostasis to some degree. The inhibition of inflammation was associated with the reduced level of Toll-like receptors and interleukin-1β in intestine, as well as the increased production of interleukin-10. Oral administration of HCP alleviated lung injury and intestinal dysfunction caused by H1N1 infection. HCP may gain systemic treatment by local acting on intestine and microbiota. This study proved the high-value application of HCP.

How cells drive the phospholipid signal response to heat stress (HS) to maintain cellular homeostasis is a fundamental issue in biology, but the regulatory mechanism of this fundamental process is unclear. Previous quantitative analyses of lipids showed that phosphatidylinositol (PI) accumulates after HS in Ganoderma lucidum, implying the inositol phospholipid signal may be associated with HS signal transduction. Here, we found that the PI-4-kinase and PI-4-phosphate-5-kinase activities are activated and that their lipid products PI-4-phosphate and PI-4,5-bisphosphate are increased under HS. Further experimental results showed that the cytosolic Ca2+ ([Ca2+ ]c ) and ganoderic acid (GA) contents induced by HS were decreased when cells were pretreated with Li+ , an inhibitor of inositol monophosphatase, and this decrease could be rescued by PI and PI-4-phosphate. Furthermore, inhibition of PI-4-kinases resulted in a decrease in the Ca2+ and GA contents under HS that could be rescued by PI-4-phosphate but not PI. However, the decrease in the Ca2+ and GA contents by silencing of PI-4-phosphate-5-kinase could not be rescued by PI-4-phosphate. Taken together, our study reveals the essential role of the step converting PI to PI-4-phosphate and then to PI-4,5-bisphosphate in [Ca2+ ]c signalling and GA biosynthesis under HS.

Bacterial lipopolysaccharide (LPS) contributes to airway inflammation and mucus hypersecretion in chronic airway inflammatory diseases, such as chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF). Neutrophil extracellular traps (NETs) are extracellular meshworks composed of DNA fibers and antimicrobial proteins. Although NET formation has been detected in COPD and CF patients, how NETs contribute to these diseases is poorly understood. This study was performed to clarify the effects and mechanisms of action of NETs in airway inflammation and mucus hypersecretion. We created a murine model of LPS-induced airway inflammation and mucus hypersecretion, and found that LPS-induced NET formation was degraded by aerosolized DNase I treatment in mice. Degradation of NETs by aerosolized DNase I reduced LPS-induced airway inflammation and mucus hypersecretion in mice, this reduction correlated with suppression of TLR4/NF-κB signaling pathway. More importantly, NETs promoted LPS-induced production of IL-1β, IL-6 and TNF-α in macrophages. These results suggest NET degradation using aerosolized DNase I is a potential new therapeutic strategy for treating COPD and CF.

Phospholipid-mediated signal transduction plays a key role in responses to environmental changes, but little is known about the role of phospholipid signalling in microorganisms. Heat stress (HS) is one of the most important environmental factors. Our previous study found that HS could induce the biosynthesis of the secondary metabolites, ganoderic acids (GA). Here, we performed a comprehensive mass spectrometry-based analysis to investigate HS-induced lipid remodelling in Ganoderma lucidum. In particular, we observed a significant accumulation of phosphatidic acid (PA) on HS. Further genetic tests in which pld-silencing strains were constructed demonstrated that the accumulation of PA is dependent on HS-activated phospholipase D (PLD) hydrolysing phosphatidylethanolamine. Furthermore, we determined the role of PLD and PA in HS-induced secondary metabolism in G. lucidum. Exogenous 1-butanol, which decreased PLD-mediated formation of PA, reverses the increased GA biosynthesis that was elicited by HS. The pld-silenced strains partly blocked HS-induced GA biosynthesis, and this block can be reversed by adding PA. Taken together, our results suggest that PLD and PA are involved in the regulation of HS-induced secondary metabolism in G. lucidum. Our findings provide key insights into how microorganisms respond to heat stress and then consequently accumulate secondary metabolites by phospholipid remodelling.

Ganoderma lucidum has become a potential model system for evaluating how environmental factors regulate the secondary metabolism of basidiomycetes. Heat stress (HS) is one of the most important environmental factors. It was previously reported that HS could induce the biosynthesis of ganoderic acids (GA). In this study, we found that HS increased GA biosynthesis and also significantly increased cell membrane fluidity. Furthermore, our results showed that addition of the membrane rigidifier dimethylsulfoxide (DMSO) could revert the increased GA biosynthesis elicited by HS. These results indicate that an increase in membrane fluidity is associated with HS-induced GA biosynthesis. Further evidence showed that the GA content was decreased in D9des-silenced strains and could be reverted to WT levels by addition of the membrane fluidizer benzyl alcohol (BA). In contrast, GA content was increased in D9des-overexpression strains and could be reverted to WT levels by the addition of DMSO. Furthermore, both membrane fluidity and GA biosynthesis induced by HS could be reverted by DMSO in WT and D9des-silenced strains. To the best of our knowledge, this is the first report demonstrating that membrane fluidity is involved in the regulation of heat stress induced secondary metabolism in filamentous fungi.

Ceramide synthases (CERSs) catalyse an N-acyltransferase reaction using long-chain base (LCB) and fatty acyl-coenzyme A (CoA) as substrates to synthesize ceramide (Cer), which is the backbone of all complex sphingolipids. In the present study, three CERSs (LAG1, LAG2 and LAG3) form Ganoderma lucidum were analysed. The silencing of lag1 by RNA interference reduced ganoderic acid biosynthesis and Cer and complex sphingolipids contents, which contain long-chain-fatty-acids (LCFAs, including C16 and C18). In contrast, the silencing of lag2 or lag3 did not result in obvious phenotypic and sphingolipid homeostasis changes, although the lag2/lag3 double-silenced mutants exhibited increased ganoderic acid biosynthesis as well as reduced growth, reduced Cer and complex sphingolipids contents, which contain very-long-chain fatty acids (VLCFAs, including C22, C24 and C26). The results of the present study indicate that the three assayed CERSs have distinct physiological functions and substrate specificities in G. lucidum.

This study aimed to identify suitable reference genes under three chemical inducers, methyl jasmonate (MeJA), salicylic acid (SA) and hydrogen peroxide (H2O2) in Ganoderma lucidum. In this study, expression stabilities of 14 candidate reference genes had been validated. Four algorithms were used: geNorm, NormFinder, BestKeeper, and RefFinder. Our results showed that, in short time, UCE2 (ubiquitin conjugating enzyme) was the most stable gene both in MeJA and H2O2 treatments, ACTIN (beta-actin) was the most suitable reference gene for SA treatment. ACTIN/UCE2 were considered the most suitable genes to normalize in MeJA, SA and H2O2 conditions. In long time, PP2A (protein phosphatase 2A regulatory subunit) was the most stable gene in MeJA and SA treatments, UCE2 was the most suitable reference gene for H2O2 treatment. PP2A/UBQ1 (polyubiquitin 1) were considered the most suitable genes to normalize in MeJA, SA and H2O2 conditions. Furthermore, target gene, oxidosqualene cyclase (osc), was selected to validate the most and least stable reference genes under different treatments. Our work provided a better support to study the regulatory mechanism of MeJA, SA and H2O2 on biological functions.

Ethnopharmacological relevance Houttuynia cordata (HC) has been used as a folk therapy to treat pulmonary infections. This study aimed to determine the role and mechanism of action of polysaccharides isolated from HC (HCP) in lipopolysaccharide (LPS)-induced ALI in the mice. Materials and methods LPS was delivered by the intratracheal route to Balb/c mice 2 h before HCP (40, 80 and 160 mg/kg) administration. Results The number of total cells, protein and tumor necrosis factor-α (TNF-α) concentrations in bronchoalveolar lavage fluid, the wet/dry weight ratio (w/d) of lungs and pulmonary pathology of each mouse were analyzed, it was found that HCP significantly alleviated ALI induced by LPS. Moreover, in lungs of mice, it was found that the infiltration of inflammatory cells, the expression of Toll-like receptor 4 and complement deposition were significantly decreased by HCP treatment. In vitro assays showed that C5a, a complement activation product, induced significant macrophage migration and treatment with HCP prevented it. The in vitro results also proved that LPS increased nitric oxide and pro-inflammatory cytokines (TNF-α, interleukin-6, and interleukin-1β) production, and HCP antagonized these effects of LPS. It was also found that HCP alone augmented secretion of some pro-inflammatory cytokines. Conclusion These results indicate that HCP may alleviate LPS induced lung inflammatory injury, which may be associated with its inhibitory effect on the over activation of complement and macrophages. This suggests a potential role to treat ALI., Graphical abstract

Lipopolysaccharides (LPS) contamination in herbal crude polysaccharides is inevitable. The present study was performed to explore the effect of polymyxin B on abolishing the influence of LPS contamination in mononuclear cells. LPS was pretreated with polymyxin B sulfate (PB) at different concentrations for 1, 5 or 24 h, and then used to stimulate RAW264.7 and mouse peritoneal macrophages (MPMs). The nitric oxide (NO) and tumor necrosis factor-α (TNF-α) in cell culture supernatant, as the indications of cell response, were assayed. Bupleurum chinensis polysaccharides (BCPs) with trace amount contamination of LPS was treated with PB. 30 μg·mL-1 of PB, treating LPS (10 and 1 000 ng·mL-1 in stimulating RAW264.7 and MPMs respectively) at 37 °C for 24 h, successfully abolished the stimulating effect of LPS on the cells. When the cells were stimulated with LPS, BCPs further promoted NO production. However, pretreated with PB, BCPs showed a suppression of NO production in MPMs and no change in RAW264.7. In the in vitro experiments, LPS contamination in polysaccharide might bring a great interference in assessing the activity of drug. Pretreatment with PB (30 μg·mL-1) at 37 °C for 24 h was sufficient to abolish the effects of LPS contamination (10 and 1 000 ng·mL-1).

Diabetes mellitus is related to low-grade chronic inflammation and oxidative stress. Bupleurum Polysaccharides (BPs), isolated from Bupleurum smithii var. parvifolium has anti-inflammatory and anti-oxidative properties. However, little is known about its therapeutic effects on diabetes. In this experiment, the effects of BPs on alleviation of diabetes and the underlying mechanisms were investigated. Diabetic mice model was established via successive intraperitoneal injections of streptozotocin (100 mg/kg body weight) for two days. Mice with blood glucose levels higher than 16.8mmol/L were selected for experiments. The diabetic mice were orally administered with BPs (30 and 60 mg/kg) once a day for 35 days. BPs not only significantly decreased levels of blood glucose, but also increased those of serum insulin and liver glycogen in diabetic mice compared to model mice. Additionally, BPs adminstration improved the insulin expression and suppressed the apoptosis in pancreas of the diabetic mice. Histopathological observations further demonstrated that BPs protected the pancreas and liver from oxidative and inflammatory damages. These results suggest that BPs protect pancreatic β cells and liver hepatocytes and ameliorate diabetes, which is associated with its anti-oxidative and anti-inflammatory properties.