Effect and mechanism of Prunella vulgaris L. extract on alleviating lipopolysaccharide-induced acute mastitis in protecting the blood-milk barrier and reducing inflammation.

ETHNOPHARMACOLOGICAL RELEVANCE: According to ancient literature, Prunella vulgaris L. (P vulgaris) alleviates mastitis and has been used in China for many years; however, there are no relevant reports that confirm this or the mechanism of its efficacy. AIM OF THE STUDY: To explore the anti-acute mastitis effect and potential mechanism of P vulgaris extract. MATERIALS AND METHODS: First, the active ingredients and targets of P vulgaris against mastitis were predicted using network pharmacology. Next, the relevant active ingredients were enriched using macroporous resins and verified using UV and UPLC-Q-TOF-MS/MS. Lastly, a mouse model of acute mastitis was established by injecting lipopolysaccharides into the mammary gland and administering P vulgaris extract by oral gavage. The pathological changes in mammary tissue were observed by HE staining. Serum and tissue inflammatory factors were measured by ELISA method. MPO activity in mammary tissue was measured using colorimetry and MPO expression was detected by immunohistochemistry. The expression of tight junction proteins (ZO-1, claudin-3, and occludin) in mammary tissue was detected by immunofluorescence and Western blot. iNOS and COX-2 in mammary tissue were detected by Western blot. MAPK pathway and NF-κB pathway related proteins were also detected by Western blot. RESULTS: Network pharmacology predicted that phenolic acids and flavonoids in P vulgaris had anti-mastitis effects. The contents of total flavonoids and total phenolic acids in P vulgaris extract were 64.5% and 29.4%, respectively. UPLC-Q-TOF-MS/MS confirmed that P vulgaris extract contained phenolic acids and flavonoids. The results of animal experiments showed that P vulgaris extract reduced lipopolysaccharide-induced inflammatory edema, inflammatory cell infiltration, and interstitial congestion of mammary tissue. It also reduced the levels of serum and tissue inflammatory factors TNF-α, IL-6, and IL-1ß, and inhibited the activation of MPO. Furthermore, it downregulated the expression of MAPK and NF-κB pathway-related proteins. The expressions of ZO-1, occludin, and claudin-3 in mammary gland tissues were upregulated. CONCLUSIONS: P vulgaris extract can maintain the integrity of mammary connective tissue and reduce its inflammatory response to prevent acute mastitis. Its mechanism probably involves regulating NF-κB and MAPK pathways.

Chicoric acid ameliorates sepsis-induced cardiomyopathy via regulating macrophage metabolism reprogramming.

BACKGROUND: Sepsis-related cardiac dysfunction is believed to be a primary cause of high morbidity and mortality. Metabolic reprogramming is closely linked to NLRP3 inflammasome activation and dysregulated glycolysis in activated macrophages, leading to inflammatory responses in septic cardiomyopathy. Succinate dehydrogenase (SDH) and succinate play critical roles in the progression of metabolic reprogramming in macrophages. Inhibition of SDH may be postulated as an effective strategy to attenuate macrophage activation and sepsis-induced cardiac injury. PURPOSE: This investigation was designed to examine the role of potential compounds that target SDH in septic cardiomyopathy and the underlying mechanisms involved. METHODS/RESULTS: From a small molecule pool containing about 179 phenolic compounds, we found that chicoric acid (CA) had the strongest ability to inhibit SDH activity in macrophages. Lipopolysaccharide (LPS) exposure stimulated SDH activity, succinate accumulation and superoxide anion production, promoted mitochondrial dysfunction, and induced the expression of hypoxia-inducible factor-1α (HIF-1α) in macrophages, while CA ameliorated these changes. CA pretreatment reduced glycolysis by elevating the NAD+/NADH ratio in activated macrophages. In addition, CA promoted the dissociation of K(lysine) acetyltransferase 2A (KAT2A) from α-tubulin, and thus reducing α-tubulin acetylation, a critical event in the assembly and activation of NLRP3 inflammasome. Overexpression of KAT2A neutralized the effects of CA, indicating that CA inactivated NLRP3 inflammasome in a specific manner that depended on KAT2A inhibition. Importantly, CA protected the heart against endotoxin insult and improved sepsis-induced cardiac mitochondrial structure and function disruption. Collectively, CA downregulated HIF-1α expression via SDH inactivation and glycolysis downregulation in macrophages, leading to NLRP3 inflammasome inactivation and the improvement of sepsis-induced myocardial injury. CONCLUSION: These results highlight the therapeutic role of CA in the resolution of sepsis-induced cardiac inflammation.

Astragalus polysaccharide restores insulin secretion impaired by lipopolysaccharides through the protein kinase B /mammalian target of rapamycin/glucose transporter 2 pathway.

BACKGROUND: Lipopolysaccharide (LPS)-induced dysfunction of pancreatic ß-cells leads to impaired insulin (INS) secretion. Astragalus polysaccharide (APS) is a bioactive heteropolysaccharide extracted from Astragalus membranaceus and is a popular Chinese herbal medicine. This study aimed to elucidate the mechanisms by which APS affects INS secretion from ß-cells under LPS stress. METHODS: Rat insulinoma (INS-1) cells were treated with LPS at a low, medium, or high concentration of APS. Glucose-stimulated insulin secretion (GSIS) was evaluated using an enzyme-linked immunosorbent assay (ELISA). Transcriptome sequencing was used to assess genome-wide gene expression. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was used to determine the signaling pathways affected by APS. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed to evaluate the gene expression of glucose transporter 2 (GLUT2), glucokinase (GCK), pancreatic duodenal homeobox-1 (PDX-1), and INS. Western blot analysis was used to detect the protein expression of phosphorylated protein kinase B (p-Akt), total Akt (t-Akt), phosphorylated mammalian target of rapamycin (p-mTOR), total mTOR (t-mTOR), and GLUT2. RESULTS: LPS decreased GLUT2, GCK, PDX-1, and INS expression and reduced GSIS. These LPS-induced decreases in gene expression and GSIS were restored by APS treatment. In addition, transcriptome sequencing in combination with KEGG enrichment analysis revealed changes in the INS signaling pathway following APS treatment. LPS decreased p-Akt and p-mTOR expression, which was restored by APS treatment. The restorative effects of APS on GSIS as well as on the expression of GLUT2, GCK, PDX-1, and INS were abolished by treatment with the Akt inhibitor MK2206 or the mTOR inhibitor rapamycin (RPM). CONCLUSIONS: APS restored GSIS in LPS-stimulated pancreatic ß-cells by activating the Akt/mTOR/GLUT2 signaling pathway.

Anti-inflammatory compounds from the rhizome of Acorus calamus var. angustatus Besser and their mechanism.

Seven compounds, named ß-sitosterol (1), daucosterol (2), (+)-pinoresinol-ß-D-glucoside (3), (-)-syringaresinol 4-O-ß-D-apiofuranosyl-(1→2)- ß-D-glucopyranoside (4), 4-hydroxybenzoic acid (5), 2-(3', 4'-dihydroxyphenyl)-1, 3-pepper ring-5-aldehyde (6) and spinosin (7) were isolated from the rhizome of Acorus calamus var. angustatus Besser. 3, 4, 6 and 7 were isolated from this medicinal plant for the first time. Structures were elucidated by physicochemical properties and extensive spectroscopic analysis, as well as by comparison with literature data. The anti-inflammatory activity and related mechanisms of the seven compounds showed that compounds 1-7 all increased the levels of GSH-PX and SOD and decreased the levels of MDA, TNF-α, IL-1ß and IL-6. Compound 4 showed the best effect of anti-inflammatory and Beclin-1 inhibition. These results suggest that compound 4 has stronger anti-inflammatory effect and provide preliminary evidence that the mechanism of action of compound 4 in attenuating LPS-induced inflammatory damage may be related to the inhibition of Beclin-1-dependent autophagy.

Insights into vitamin A in bladder cancer, lack of attention to gut microbiota?

Vitamin A has long been associated with bladder cancer, and many exogenous vitamin A supplements, vitamin A derivatives, and synthetic drugs have been investigated over the years. However, the effectiveness of these strategies in clinical practice has not met expectations, and they have not been widely adopted. Recent medical research on intestinal flora has revealed that bladder cancer patients exhibit reduced serum vitamin A levels and an imbalance of gut microbiota. In light of the close relationship between gut microbiota and vitamin A, one can speculate that a complex regulatory mechanism exists between the two in the development and occurrence of bladder cancer. As such, further exploration of their interaction in bladder cancer may help guide the use of vitamin A for preventive purposes. During the course of this review, attention is paid to the influence of intestinal microbiota on the vitamin A metabolism and the RA signaling pathway, as well as the mutual promotion relationships between them in the prevention of bladder cancer, In addition, it emphasizes the importance of intestinal microbiota for bladder cancer prevention and treatment.

Protective mechanisms of Tuina therapy against lipopolysaccharide-induced fever in young rabbits based on untargeted metabolomics analysis.

OBJECTIVE: To investigate the effect of Tuina on the plasma metabolites of lipopolysaccharide-induced febrile in infant rabbits. METHODS: Twenty-four infant New Zealand rabbits were selected and randomly divided into three groups: saline, model, and Tuina. The fever model was established by injecting LPS intravenously through the ear margin vein in the model group and Tuina group, respectively. The modeling was considered successful when the anal temperature increased by 0.5℃ or above within 1 h. In the Tuina group, six Tuina techniques (i.e., opening Tianmen / the heaven gate, pushing Kangong / the superciliary arch, kneading Taiyang and the prominent bone behind the ears, clearing Tianheshui, spine pinching) that alleviate fever were performed on the young rabbits 1 h after the modeling, whereas the model and saline groups were not given Tuina treatment, with the real-time anal temperature monitored during the experiment. The plasma was taken 3 h after the modeling for liquid chromatography-mass spectrometry (LC-MS) untargeted metabolomics study. RESULTS: Our results showed a fever-reducing effects of Tuina therapy on lipopolysaccharide-induced fever in young rabbits, as indicated by a significantly lower anal temperature, maximum rise in body temperature, and body response index at 2 and 3 h after modeling in the Tuina group compared to the model group, with reductions in the PGE2 expression observed in the blood and hypothalamus. The differential metabolites including riboflavin, nicotinamide N-oxide, porphobilinogen, 5-hydroxyindoleacetic acid, gamma-aminobutyric acid, and lysoPC (16:1 (9Z)/0:0) were found following the Tuina intervention. Tuina primarily involves glycine-serine-threonine, arginine-proline, porphyrin-chlorophyll, pyrimidine, primary bile acid biosynthesis, and cyanoamino acid metabolic pathways. CONCLUSION: Tuina therapy has proven to be effective in reducing body temperature and down-regulating PGE2 expression in LPS-induced febrile young rabbits, with its mechanism of fever-reducing action possibly associated with the changes in plasma metabolites and metabolic pathways.

Vitamin D Supplementation Impacts Calcium and Phosphorus Metabolism in Piglets Fed a Diet Contaminated with Deoxynivalenol and Challenged with Lipopolysaccharides.

Using alternative feed ingredients in pig diets can lead to deoxynivalenol (DON) contamination. DON has been shown to induce anorexia, inflammation, and-more recently-alterations in the vitamin D, calcium, and phosphorus metabolisms. Adding vitamin D supplementation in the form of vitamin D3 and 25-OH-D3 to the feed could modify the effects of DON in piglets. In this study, vitamin D3 or 25-OH-D3 supplementation was used in a control or DON-contaminated treatment. A repetitive exposure over 21 days to DON in the piglets led to disruptions in the vitamin D, calcium, and phosphorus metabolisms, resulting in a decreased growth performance, increased bone mineralization, and the downregulation of genes related to calcium and to phosphorus intestinal and renal absorption. The DON challenge also decreased blood concentrations of 25-OH-D3, 1,25-(OH)2-D3, and phosphate. The DON contamination likely decreased the piglets' vitamin D status indirectly by modifying the calcium metabolism response. Vitamin D supplementations did not restore vitamin D status or bone mineralization. After a lipopolysaccharide-induced inflammatory stimulation, feeding a 25-OH-D3 supplementation increased 25-OH-D3 concentration and 1,25-(OH)2-D3 regulations during the DON challenge. DON contamination likely induced a Ca afflux by altering the intestinal barrier, which resulted in hypercalcemia and hypovitaminosis D. The vitamin D supplementation could increase the calcitriol production to face the combined LPS and DON challenge.

The extracts of Dracaena cochinchinensis stemwood suppress inflammatory response and phagocytosis in lipopolysaccharide-activated microglial cells.

BACKGROUND: Neuroinflammation is a pivotal process in the brain that contributes to the development of neurodegenerative diseases, such as Alzheimer's disease (AD). During neuroinflammation, the over-activation of microglial cells can drive the pathological processes underlying AD, including an increase in amyloid ß (Aß) production and accumulation, ultimately leading to neuronal and synaptic loss. Dracaena cochinchinensis (Lour.) S.C. Chen, also known as "Chan-daeng" in Thai, belongs to the Asparagaceae family. In Thai traditional medicine, it has been used as an antipyretic, pain reliever, and anti-inflammatory agent. However, the effects of D. cochinchinensis on neuroinflammation are yet to be determined. PURPOSE: We aimed to evaluate the anti-neuroinflammatory activities of D. cochinchinensis stemwood extract in activated microglia. METHODS: In this study, lipopolysaccharide (LPS), a potent pro-inflammatory stimulus, was used to activate microglial BV2 cells, as a cell model of neuroinflammation. Our investigation included several techniques, including qRT-PCR, ELISA, Western blotting, phagocytosis, and immunofluorescence staining, to examine the potential anti-inflammatory effects of D. cochinchinensis stemwood. RESULTS: D. cochinchinensis stemwood, named DCS, was extracted with ethanol and water. The extracts of DCS showed dose-dependent anti-inflammatory effects, markedly suppressing the LPS-mediated mRNA expression of pro-inflammatory factors, including IL-1ß, TNF-α, and iNOS, while increasing expression of the anti-inflammatory biomarker Arg1 in both BV2 microglia and RAW264.7 macrophages. DCS extracts also decreased the protein levels of IL-1ß, TNF-α, and iNOS. These findings were correlated with the suppression of phosphorylated proteins of p38, JNK, and Akt in the LPS-activated microglia. Moreover, DCS extracts significantly attenuated excessive phagocytosis of beads and Aß fibrils during the LPS-mediated microglial activation. CONCLUSION: Taken together, our results indicated that DCS extracts had anti-neuroinflammatory properties by suppressing the expression of pro-inflammatory factors, increasing the expression of the anti-inflammatory biomarker Arg1, and modulating excessive phagocytosis in activated microglia. These findings suggested that DCS extract could be a promising natural product for the treatment of neuroinflammatory and neurodegenerative diseases, like AD.

Babao Dan decreases hepatocarcinogenesis by inhibiting hepatic progenitor cells malignant transformation via down-regulating toll-like receptor 4.

Background: Babao Dan (BBD) is a traditional Chinese medicine that has been widely used as a complementary and alternative medicine to treat chronic liver diseases. In this study, we aimed to observe the effect of BBD on the incidence of diethylnitrosamine (DEN)-initiated hepatocellular carcinoma formation in rats and explored its possible mechanism. Methods: To verify this hypothesis, BBD was administrated to rats at a dose of 0.5g/kg body weight per two days from the 9th to 12th week in HCC-induced by DEN. Liver injury biomarkers and hepatic inflammatory parameters were evaluated by histopathology as well as serum and hepatic content analysis. We applied immunohistochemical analysis to investigate the expression of CK-19 and SOX-9 in liver tissues. The expression of TLR4 was determined by immunohistochemical, RT-PCR, and western blot analysis. Furthermore, we also detected the efficacy of BBD against primary HPCs neoplastic transformation induced by LPS. Results: We observed that DEN could induce hepatocarcinogenesis, and BBD could obviously decrease the incidence. The biochemical and histopathological examination results confirmed that BBD could protect against liver injury and decrease inflammatory infiltration. Immunohistochemistry staining results showed that BBD could effectively inhibit the ductal reaction and the expression of TLR4. The results showed that BBD-serumcould obviously inhibit primary HPCs neoplastic transformation induced by regulating the TLR4/Ras/ERK signaling pathway. Conclusion: In summary, our results indicate that BBD has potential applications in the prevention and treatment of HCC, which may be related to its effect on hepatic progenitor cells malignant transformation via inhibiting the TLR4/Ras/ERK signaling pathway.

Protective effects of Bacillus licheniformis on growth performance, gut barrier functions, immunity and serum metabolome in lipopolysaccharide-challenged weaned piglets.

Bacillus licheniformis (B. licheniformis) is a well-accepted probiotic that has many benefits on both humans and animals. This study explored the effects of B. licheniformis on growth performance, intestinal mucosal barrier functions, immunity as well as serum metabolome in the weaned piglets exposed to lipopolysaccharide (LPS). One hundred and twenty piglets weaned at four weeks of age were separated into two groups that received a basal diet (the control group, CON), and a basal diet complemented with B. licheniformis (500 mg/kg, the BL group, BL). Twenty-four piglets were chosen from the above two groups and 12 piglets were injected with LPS intraperitoneally at a concentration of 100 µg/kg and the others were injected with sterile saline solution of the same volume. All the piglets were sacrificed 4 h after LPS challenge. Results showed that B. licheniformis enhanced the ADG and final body weight and lowered the F/G and diarrhea rate. Pre-treatment with B. licheniformis markedly attenuated intestinal mucosal damage induced by LPS challenge. Supplementation with B. licheniformis strengthened immune function and suppressed inflammatory response by elevating the concentrations of serum immunoglobulin (Ig) A and jejunum mucosal IgA and IgG and decreasing serum IL-6 and jejunum mucosal IL-1ß. In addition, B. licheniformis pretreatment prevented LPS-induced intestinal injury by regulating the NLRP3 inflammasome. Furthermore, pretreatment with B. licheniformis tended to reverse the reduction of acetate and propionic acids in the colonic contents that occurred due to LPS stress. B. licheniformis markedly modulated the metabolites of saccharopine and allantoin from lysine and purine metabolic pathways, respectively. Overall, these data emphasize the potentiality of B. licheniformis as a dietary supplement to overcome the challenge of bacterial LPS in the animal and to enhance the food safety.

The social environment alters neural responses to a lipopolysaccharide challenge.

Sick animals display drastic changes in their behavioral patterns, including decreased activity, decreased food and water intake, and decreased interest in social interactions. These behaviors, collectively called "sickness behaviors", can be socially modulated. For example, when provided with mating opportunities, males of several species show reduced sickness behaviors. While the behavior is known to change, how the social environment affects neural molecular responses to sickness is not known. Here, we used a species, the zebra finch, Taeniopygia guttata, where males have been shown to decrease sickness behaviors when presented with novel females. Using this paradigm, we obtained samples from three brain regions (the hypothalamus, the bed nucleus of the stria terminalis, and the nucleus taeniae) from lipopolysaccharide (LPS) or control treated males housed under four different social environments. Manipulation of the social environment rapidly changed the strength and co-expression patterns of the neural molecular responses to the immune challenge in all brain regions tested, therefore suggesting that the social environment plays a significant role in determining the neural responses to an infection. In particular, brains of males paired with a novel female showed muted immune responses to LPS, as well as altered synaptic signaling. Neural metabolic activity in response to the LPS challenge was also affected by the social environment. Our results provide new insights into the effects of the social environment on brain responses to an infection, thereby improving our understanding of how the social environment can affect health.

Triptolide leads to hepatic intolerance to exogenous lipopolysaccharide and natural-killer-cell mediated hepatocellular damage by inhibiting MHC class I molecules.

BACKGROUND: Tripterygium wilfordii Hook. F (TWHF) is used as a traditional Chinese medicine, called thunder god vine, based on its efficacy for treating inflammatory diseases. However, its hepatotoxicity has limited its clinical application. Triptolide (TP) is the major active and toxic component of TWHF. Previous studies reported that a toxic pretreatment dose of TP leads to hepatic intolerance to exogenous lipopolysaccharide (LPS) stimulation, and to acute liver failure, in mice, but the immune mechanisms of TP-sensitised hepatocytes and the TP-induced excessive immune response to LPS stimulation are unknown. PURPOSE: To identify both the key immune cell population and mechanism involved in TP-induced hepatic intolerance of exogenous LPS. STUDY DESIGN: In vitro and in vivo experiments were conducted to investigate the inhibitory signal of natural killer (NK) cells maintained in hepatocytes, and the ability of TP to impair that signal. METHODS: Flow cytometry was performed to determine NK cell activity and hepatocyte histocompatibility complex (MHC) class I molecules expression; the severity of liver injury was determined based on blood chemistry values, and drug- or cell-mediated hepatocellular damage, by measuring lactate dehydrogenase (LDH) release. In vivo H-2Kb transduction was carried out using an adeno-associated viral vector. RESULTS: Interferon (IFN)-γ-mediated necroptosis occurred in C57BL/6N mice treated with 500 µg TP/kg and 0.1 mg LPS/kg to induce fulminant hepatitis. Primary hepatocytes pretreated with TP were more prone to necroptosis when exposed to recombinant murine IFN-γ. In mice administered TP and LPS, the intracellular IFN-γ levels of NK cells increased significantly. Subsequent study confirmed that NK cells were activated and resulted in potent hepatocellular toxicity. In vivo and in vitro TP administration significantly inhibited MHC class I molecules in murine hepatocytes. An in vitro analysis demonstrated the susceptibility of TP-pretreated hepatocytes to NK-cell-mediated cytotoxicity, an effect that was significantly attenuated by the induction of hepatocyte MHC-I molecules by IFN-α. In vivo induction or overexpression of hepatocyte MHC-I also protected mouse liver against TP and LPS-induced injury. CONCLUSION: The TP-induced inhibition of hepatocyte MHC-I molecules expression leads to hepatic intolerance to exogenous LPS and NK-cell mediated cytotoxicity against self-hepatocytes. These findings shed light on the toxicity of traditional Chinese medicines administered for their immunomodulatory effects.

Effect of tea catechins on gut microbiota in high fat diet-induced obese mice.

BACKGROUND: Tea catechins have been shown to have beneficial effects on the alleviation of obesity, the prevention of diabetes, and the amelioration of metabolic syndrome. The purpose of the present work is to explore the underlying mechanisms linking the intestinal microbiota and anti-obesity benefits of green tea, oolong tea, and black tea catechins in C57BL/6J mice fed with a high-fat diet (HFD). RESULTS: The results indicated that, after the dietary intake of three tea catechins, obesity and low-grade inflammation were significantly alleviated. Hepatic steatosis was prevented, and this was accompanied by the upregulation of the mRNA and protein expressions of hepatic peroxisome proliferator-activated receptor α (PPARα). Metagenomic analysis of fecal samples suggested that the three tea catechins similarly changed the microbiota in terms of overall structure, composition, and protein functions by regulating the metabolites, facilitating the generation of short-chain fatty acids (SCFAs), and repressing lipopolysaccharides. CONCLUSION: The anti-obese properties of three tea catechins were partially mediated by their positive effect on gut microbiota, hepatic steatosis alleviation, and anti-inflammatory activity. © 2023 Society of Chemical Industry.

Comparative effects of different bacterial lipopolysaccharides on modulation of immune levels to improve survival of the black tiger shrimp.

To prevent loss from disease, immunostimulants have been used as dietary supplements to improve immunity and survival of shrimps. Among the various types of immunostimulants, there is increasing evidence that a diet enriched with bacterial lipopolysaccharide can reduce the mortality rate of shrimp under exposure to pathogens. Here, the immunostimulatory effects of bacterial lipopolysaccharide (LPS) from various bacterial sources were explored. Bacterial LPS was extracted from a shrimp pathogen, Vibrio harveyi and its effects were compared with the commercially available LPS from the non-shrimp pathogen, Escherichia coli. Our results revealed that the LPS from V. harveyi was different in molecular size but contained similar functional groups to that from E. coli. To understand their molecular mechanisms, bacterial LPS from the two sources were applied as a supplementary diet and fed to juvenile shrimp for 4-week feeding period before tissue samples were collected for transcriptomic analysis by next generation sequencing. Gene expression profiling revealed that major immune-related genes such as pattern recognition proteins (PRPs), proteinases and proteinase inhibitors, prophenoloxidase systems (proPO system), antimicrobial peptides (AMPs), signaling transduction pathways, heat shock proteins (HSPs), oxidative stress responses, and other immune-related molecules such as mucins and peritrophins were modulated in the groups of shrimp fed with bacterial LPS from both sources, but at different levels. The results suggest that bacterial LPS could modulate shrimp immune system, and different LPS sources led to different activation of immune pathways. Additionally, metabolic-related genes were affected by LPS, suggesting that energy was required for immune stimulation. In the V. harveyi pathogen challenge trial, all shrimp groups fed with diets containing LPS from both bacterial sources showed better survival than the control group without LPS. When comparing groups fed with LPS supplemented diets, the higher concentration of LPS (8 µg/body weight) from E. coli resulted in a better survival rate than a lower concentration (4 µg/body weight). Conversely, shrimp fed with a diet containing LPS from V. harveyi showed a lower survival rate when a higher dose of LPS (8 µg/body weight) was administered than the group fed with a lower concentration of LPS (4 µg/body weight). This could be due to overstimulation of shrimp immune responses, especially by LPS derived from shrimp pathogens, resulting in a reverse effect. These results confirm that immunity in shrimp upon administration of bacterial LPS depends on the origin and dose of the LPS administered.

Chronic exposure to lipopolysaccharides as an in vitro model to simulate the impaired odontogenic potential of dental pulp cells under pulpitis conditions

Abstract Simulating a bacterial-induced pulpitis environment in vitro may contribute to exploring mechanisms and bioactive molecules to counteract these adverse effects. Objective To investigate the chronic exposure of human dental pulp cells (HDPCs) to lipopolysaccharides (LPS) aiming to establish a cell culture protocol to simulate the impaired odontogenic potential under pulpitis conditions. Methodology HDPCs were isolated from four healthy molars of different donors and seeded in culture plates in a growth medium. After 24 h, the medium was changed to an odontogenic differentiation medium (DM) supplemented or not with E. coli LPS (0 - control, 0.1, 1, or 10 µg/mL) (n=8). The medium was renewed every two days for up to seven days, then replaced with LPS-free DM for up to 21 days. The activation of NF-κB and F-actin expression were assessed (immunofluorescence) after one and seven days. On day 7, cells were evaluated for both the gene expression (RT-qPCR) of odontogenic markers (COL1A1, ALPL, DSPP, and DMP1) and cytokines (TNF, IL1B, IL8, and IL6) and the production of reactive nitrogen (Griess) and oxygen species (Carboxy-H2DCFDA). Cell viability (alamarBlue) was evaluated weekly, and mineralization was assessed (Alizarin Red) at 14 and 21 days. Data were analyzed with ANOVA and post-hoc tests (α=5%). Results After one and seven days of exposure to LPS, NF-κB was activated in a dose-dependent fashion. LPS at 1 and 10 µg/mL concentrations down-regulated the gene expression of odontogenic markers and up-regulated cytokines. LPS at 10 µg/mL increased both the production of reactive nitrogen and oxygen species. LPS decreased cell viability seven days after the end of exposure. LPS at 1 and 10 µg/mL decreased hDPCs mineralization in a dose-dependent fashion. Conclusion The exposure to 10 µg/mL LPS for seven days creates an inflammatory environment that is able to impair by more than half the odontogenic potential of HDPCs in vitro, simulating a pulpitis-like condition.

Ameliorative effects of supranutritional selenium on TLR-4-NF-kB-TNF-α-mediated hepatic oxidative injury and inflammation in goats fed high concentrate diet.

We examined whether surplus dietary selenium (Se) supply could alleviate high concentrate (HC) diet-induced hepatic oxidative stress (OS) and inflammation. Eighteen young goats were distributed into three groups; were fed low (LC, concentrate: forage; 35: 65), high concentrate (HC, 65: 35), or Se-supplemented HC (HCSe, 65: 35 + 0.5 mg Se kg-1 diet) diets for 10 weeks. Short chain fatty acids, OS markers and immunoinflammatory genes expressions were assessed through gas chromatograph, kits, and RT-qPCR, respectively. Compared with LC, HC diet increased (p < .05) colonic and serum lipopolysaccharide (LPS) levels and induced hepatic oxidative injury by increasing (p < .05) malondialdehyde (MDA) levels and decreasing (p < .05) activities of glutathione peroxidase, superoxide dismutase, and catalase. HC diet altered hepatic mRNA expressions of toll-like receptor-4 (TLR-4), cluster of differentiation-14 (CD-14), tumor necrosis factor-α (TNF-α), TNF receptor-associated factor-6 (TRAF-6), nuclear factor kappa B (NF-κB), interleukin-1ß (IL-1ß), IL-10, IL-13, LPS-binding protein (LBP), serum amyloid A (SAA), α-acid glycoprotein (AGP), and albumin (ALB). Conversely, extra-Se supply lowered LPS and attenuated antioxidant status and inflammation in liver. In conclusion, HC diet induced oxidative lesions and TLR-4 pathway-mediated inflammation, whereas supranutritional Se alleviated oxidative and inflammatory lesions through TLR-4 pathway regulation in goat liver.

Identification of Bcl2 as a Stably Expressed qPCR Reference Gene for Human Colon Cancer Cells Treated with Cottonseed-Derived Gossypol and Bioactive Extracts and Bacteria-Derived Lipopolysaccharides.

Cottonseed contains many bioactive molecules including plant polyphenols. Cottonseed value might be increased by providing high-value bioactive polyphenols for improving nutrition and health. However, there was a lack of molecular evidence for cottonseed bioactivity in mammalian cells. One widely used method for evaluating the bioactivity of natural products is quantitative real-time-PCR (qPCR). The selection of stably expressed internal reference genes is a crucial task of qPCR assay for data analysis. The rationale for reference gene selection is that a lower standard deviation of the cycle of threshold (Cq) among the treatments indicates a more stable expression of the gene. The objective of this study was to select reference genes in human colon cancer cells (COLO 205) treated with cottonseed-derived gossypol and bioactive extracts along with bacterial endotoxin lipopolysaccharides (LPS). SYBR Green qPCR was used to analyze the mRNA levels of a wide range of biomarkers involved in glucose transport, lipid biosynthesis, inflammatory response, and cancer development. qPCR data (10,560 Cq values) were generated from 55 genes analyzed from 64 treatments with triplicate per treatment for each gene. The data showed that B-cell lymphoma 2 (Bcl2) mRNA was the most stable among the 55 mRNAs analyzed in the human colon cancer cells. Glyceraldehyde 3 phosphate dehydrogenase (Gapdh) and ribosome protein L32 (Rpl32) mRNAs were not good qPCR references for the colon cancer cells. These observations were consistent regardless of the treatment comparison between gossypol and LPS, glanded and glandless seed extracts, seed coat and kernel extracts, or treatment for 8 and 24 h. These results suggest that Bcl2 is a preferable reference gene for qPCR assays in human colon cancer cells treated with cottonseed-derived gossypol and bioactive extracts as well as LPS. The extensive qPCR results firmly support the conclusion that the Bcl2 gene is stably expressed at the mRNA level in the human colon cancer cells regardless of the treatment, suggesting that Bcl2 gene expression is not regulated at the mRNA level but at the post-transcriptional level. These results should facilitate studies designated to evaluate bioactivity on gene expression regulation by cottonseed molecules and other natural and synthetic molecules for nutrition and health uses.

Harnessing the Role of Bacterial Plasma Membrane Modifications for the Development of Sustainable Membranotropic Phytotherapeutics.

Membrane-targeted molecules such as cationic antimicrobial peptides (CAMPs) are amongst the most advanced group of antibiotics used against drug-resistant bacteria due to their conserved and accessible targets. However, multi-drug-resistant bacteria alter their plasma membrane (PM) lipids, such as lipopolysaccharides (LPS) and phospholipids (PLs), to evade membrane-targeted antibiotics. Investigations reveal that in addition to LPS, the varying composition and spatiotemporal organization of PLs in the bacterial PM are currently being explored as novel drug targets. Additionally, PM proteins such as Mla complex, MPRF, Lpts, lipid II flippase, PL synthases, and PL flippases that maintain PM integrity are the most sought-after targets for development of new-generation drugs. However, most of their structural details and mechanism of action remains elusive. Exploration of the role of bacterial membrane lipidome and proteome in addition to their organization is the key to developing novel membrane-targeted antibiotics. In addition, membranotropic phytochemicals and their synthetic derivatives have gained attractiveness as popular herbal alternatives against bacterial multi-drug resistance. This review provides the current understanding on the role of bacterial PM components on multidrug resistance and their targeting with membranotropic phytochemicals.

Dietary Isoflavones Alter Gut Microbiota and Lipopolysaccharide Biosynthesis to Reduce Inflammation.

The etiopathogenesis of multiple sclerosis (MS) is strongly affected by environmental factors such as diet and the gut microbiota. An isoflavone-rich (ISO) diet was previously shown to reduce the severity of MS in the animal model experimental autoimmune encephalomyelitis (EAE). Translation of this concept to clinical trial where dietary isoflavones may be recommended for MS patients will require preliminary evidence that providing the isoflavone-rich diet to people with MS (PwMS) who lack phytoestrogen-metabolizing bacteria has beneficial effects. We have previously shown that the gut microbiota of PwMS resembles the gut microbiota of mice raised under a phytoestrogen-free (phyto-free) diet in that it lacks phytoestrogen-metabolizing bacteria. To investigate the effects of phytoestrogens on the microbiota inflammatory response and EAE disease severity we switched the diet of mice raised under a phyto-free (PF) diet to an isoflavone-rich diet. Microbiota analysis showed that the change in diet from one that is ISO to one that is PF reduces beneficial bacteria such as Bifidobacterium species. In addition we observed functional differences in lipopolysaccharide (LPS) biosynthesis pathways. Moreover LPS extracted from feces of mice fed an ISO diet induced increased production of anti-inflammatory cytokines from bone marrow-derived macrophages relative to fecal-LPS isolated from mice fed a PF diet. Eventually mice whose diet was switched from a PF diet to an ISO diet trended toward reduced EAE severity and mortality. Overall we show that an isoflavone-rich diet specifically modulates LPS biosynthesis of the gut microbiota imparts an anti-inflammatory response and decreases disease severity.

Yajieshaba prevents lipopolysaccharide-induced intestinal barrier injuryanti-inflammatory and anti-apoptosis.

OBJECTIVE: To investigate the protective effect of Yajieshaba (YJSB) on the intestinal barrier dysfunction induced by lipopolysaccharide (LPS). METHODS: C57BL/6 mice and rat intestinal epithelial cells were treated with LPS. Thiazolyl Blue Tetrazolium Bromide assay were used to detect cell viability. D-Lactate, diamine oxidase and myeloperoxidase and cytokines were determined by enzyme-linked immunosorbent assay. Western blot was used to detect apoptosis-related proteins and tight junction (TJ) proteins. Real-time quantitative polymerase chain reaction was used to quantify the levels of mRNA expression of cytokines. Histological analysis was performed by hematoxylin and eosin staining. An immunofluorescence staining assay was performed to determine the expression level of TJ protein. RESULTS: YJSB increased cell viability and decreased apoptosis, maintained intestinal permeability after LPS-induced. YJSB inhibited LPS-induced decrease of TJ protein expression, pro-inflammatory cytokine levels and neutrophil infiltration. CONCLUSION: YJSB protect against LPS-induced intestinal barrier dysfunction anti-inflammatory and anti-apoptosis, suggesting its therapeutic potential against intestinal barrier injury-related diseases.