TLR4 mRNA Expression Research Articles

Tenascin-C: Friend or Foe in Lung Aging?

Lung aging is characterized by lung function impairment, ECM remodeling and airspace enlargement. Tenascin-C (TNC) is a large extracellular matrix (ECM) protein with paracrine and autocrine regulatory functions on cell migration, proliferation and differentiation. This matricellular protein is highly expressed during organogenesis and morphogenetic events like injury repair, inflammation or cancer. We previously showed that TNC deficiency affected lung development and pulmonary function, but little is known about its role during pulmonary aging. In order to answer this question, we characterized lung structure and physiology in 18 months old TNC-deficient and wild-type (WT) mice. Mice were mechanically ventilated with a basal and high tidal volume (HTV) ventilation protocol for functional analyses. Additional animals were used for histological, stereological and molecular biological analyses. We observed that old TNC-deficient mice exhibited larger lung volume, parenchymal volume, total airspace volume and septal surface area than WT, but similar mean linear intercept. This was accompanied by an increase in proliferation, but not apoptosis or autophagy markers expression throughout the lung parenchyma. Senescent cells were observed in epithelial cells of the conducting airways and in alveolar macrophages, but equally in both genotypes. Total collagen content was doubled in TNC KO lungs. However, basal and HTV ventilation revealed similar respiratory physiological parameters in both genotypes. Smooth muscle actin (α-SMA) analysis showed a faint increase in α-SMA positive cells in TNC-deficient lungs, but a marked increase in non-proliferative α-SMA + desmin + cells. Major TNC-related molecular pathways were not up- or down-regulated in TNC-deficient lungs as compared to WT; only minor changes in TLR4 and TGFβR3 mRNA expression were observed. In conclusion, TNC-deficient lungs at 18 months of age showed exaggerated features of the normal structural lung aging described to occur in mice between 12 and 18 months of age. Correlated to the increased pulmonary function parameters previously observed in young adult TNC-deficient lungs and described to occur in normal lung aging between 3 and 6 months of age, TNC might be an advantage in lung aging.

Exercise alleviated intestinal damage and microbial disturbances in mice exposed to fluoride

Gastrointestinal reaction is an important symptom of fluorosis and is associated with intestinal morphological and functional impairment. Regular moderate exercise may reduce the incidence of infection and contribute to the maintenance of intestinal mucosal function and immune homeostasis. In this study, the mice were randomly divided to four groups: control group (C, distilled water), exercise group (E, distilled water and treadmill exercise), fluoride group (F, 100 mg/L NaF), and exercise plus fluoride group (EF, 100 mg/L NaF and treadmill exercise). The treadmill exercise was performed as 5 m/min, 5 min; 10 or 12 m/min, 20 min; 5 m/min, 5 min, with 5 consecutive days per week. After 6 months, exercise alleviated the intestinal morphological structure damage and restored the villus height (VH) and VH/crypt depth (VH/CD) in the duodenum of fluoride-exposed mice. Exercise decreased the mRNA expressions of IL-1β, IL-6, TNF-α, TLR2 and NF-κB (p65) in fluoride-exposed mice, and restored the gene levels of Occludin and ZO-1 in the duodenum, as well as Occludin, ZO-1, and Claudin-1 in the colon. Although there were no significant differences in the Occludin and ZO-1 protein expressions between F and EF, two proteins in EF presented statistical homogeneousness when compared with the C. The 16S rDNA high-throughput sequencing found that exercise restored the variations in intestinal microbiota composition and the abundances of specific bacteria in fluoride-exposed mice, including increasing the abundances of Epsilonbacteraenta and Firmicutes, reducing the Bacteroidetes abundance at the phylum level, and restoring the abundances of 13 bacterial genera. In conclusion, exercise improved intestinal morphological structure damage in fluoride-exposed mice, inhibited the secretion of duodenal inflammatory factors, increased the expression of tight junctions, and alleviated the microbial disorder in mice caused by fluoride exposure for 6 months through actively regulating the composition of intestinal microorganisms and the abundance of specific bacteria.

Total Saponin of Dioscorea collettii Attenuates MSU Crystal-Induced Inflammation by Inhibiting the Activation of the TLR4/NF-κB Signaling Pathway.

Background Rhizomes from Dioscorea collettii are extensively used in traditional medicine for the treatment of arthritic diseases, particularly gouty arthritis (GA). This study aims to investigate whether the total saponin of Dioscorea collettii (TSD) can attenuate monosodium urate (MSU) crystal-induced inflammatory effects by suppressing the activation of the TLR4/NF-κB signaling pathway in vivo and in vitro. Methods Seventy-two male Wistar rats and THP-1 cells were used in this study. Pathological examination was used to examine the ankle joints of rats. The expression levels of TLR4, NF-κB, MyD88, and IL-1β were detected by qRT-PCR, Western blotting, or immunofluorescence. Results Compared with those in the normal group, the ankle joints of rats in the model group exhibited significant swelling, synovial tissue hyperplasia, inflammatory cell infiltration, and increased expression of IL-1β protein. The joint swelling degree of rats in the TSD high- and medium-dose groups and the colchicine group was significantly decreased, and the histopathology was obviously improved. TSD and colchicine reduced the levels of IL-1β and TNF-α in synovial fluid. They also decreased the mRNA expression of TLR4, NF-κB, and IL-1β in rat joint synovial tissue and the protein expression of TLR4, MyD88, and NF-κB. NF-κB protein expression in both the cytoplasm and nuclei of THP-1 cells showed the opposite trend. Furthermore, immunofluorescence showed that TSD reduced the nuclear translocation of NF-κBp65 in the model group. Conclusion TSD exhibits an anti-inflammatory effect in the MSU-induced inflammation model, and the mechanism may be to reduce the production of cytokines by inhibiting the activation of the TLR4/NF-κB signaling pathway.

Protective effect of sinomenine against inflammation and oxidative stress in gestational diabetes mellitus in female rats via TLR4/MyD88/NF-κB signaling pathway.

Gestational diabetes mellitus (GDM) is a dangerous complication of pregnancy which is induced via dysfunction in glucose metabolism during pregnancy. Sinomenine (SM) has already proved an antidiabetic effect against streptozotocin (STZ)-induced diabetes mellitus (DM) in rats. In this protocol, we examined the protective effect of SM against STZ-induced GDM in rats. Wistar rats were divided into three groups and STZ (40mg/kg) was used to induce GDM. At the end of the experimental protocol, bodyweight, pub weight, and survival rate were estimated. Blood glucose level (BGL), fasting insulin (FINS), free fatty acid (FFA), Hemoglobin A1C (HbA1c), and C-peptide were measured. Lipid, antioxidant, inflammatory cytokines, and inflammatory mediators were also determined. RT-PCR was used for estimation of the role of TLR4/MyD88/NF-κB signaling pathway. SM treatment significantly (p<.001) reduced BGL, hepatic glycogen, and improved the levels of FINS, C-peptide, FFA, and HbA1c. SM significantly (p<.001) suppressed the levels of total cholesterol (TC), low-density lipoprotein (LDL), triglycerides (TG), coronary artery index (CAI), very low-density lipoprotein (VLDL), atherogenic index (AI), and boosted high-density lipoprotein (HDL) levels. SM significantly (p<.001) decreased the lipid peroxidation (LPO) level and enhanced glutathione peroxidase (GPx), total antioxidant capacity (TAC), glutathione S-transferase (GST), superoxide dismutase (SOD), respectively. It reduced the levels of inflammatory cytokines including interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and inflammatory mediators viz., nuclear kappa B factors (NF-κB). SM significantly (p<.001) reduced the mRNA expression of Myd88, NLRP3, TLR4, and NF-κB, which were boosted in the GDM group rats. These findings suggest that SM could be a probable drug to be used for treating GDM via inhibition of the TLR4 signaling pathway. PRACTICAL APPLICATIONS: It is well known that gestational diabetes mellitus (GDM) is a dangerous health problem during the pregnancy. SM reduced the glucose level; boosted the level of fasting insulin (FINS) and bodyweight. SM significantly improved the number of pubs and their survival rates. SM suppressed oxidative stress and inflammation via activation of TLR4/MyD88/NF-κB signaling pathway. According to our research, SM can be used as a preventive drug in the treatment of GDM during pregnancy.

Buddleoside-Rich Chrysanthemum indicum L. Extract has a Beneficial Effect on Metabolic Hypertensive Rats by Inhibiting the Enteric-Origin LPS/TLR4 Pathway.

As the number of patients with metabolic hypertension (MH) is increasing, there is an essential require for global measures to prevent and treat MH. Flavonoids such as buddleoside (BUD) from Chrysanthemum indicum L. are the main pharmacological components of cardiovascular activities. Previous studies have suggested that the buddleoside-rich Chrysanthemum indicum L. extract (BUDE) can reduce blood pressure in spontaneously hypertensive rats (SHR). However, its effect on MH and how it works remains to be researched. In this study, it was observed that BUDE could lower blood pressure, improve dyslipidemia, and decrease the level of plasma LPS in MH rats. Moreover, BUDE improved intestinal flora and increased the expression of occludin and claudin-1 in the colon, and improved the pathological injury of the colon. Western bolt and qRT-PCR experiments showed that BUDE could down-regulate TLR4 and MyD88 protein and mRNA expression and inhibit phosphorylation of IKKβ, IκBα and NF-κB p65 in vessels of MH rats. These results showed that BUDE could regulate intestinal flora, improve intestinal barrier function, reduce the production and penetration of LPS, thereby inhibiting the vascular TLR4/MyD88 pathway, improving vascular endothelial function, and ultimately lowering blood pressure in MH rats. This study provides a new mechanism of BUDE against MH by inhibiting the enteric-origin LPS/TLR4 pathway.

Effect and mechanism of omega-3 polyunsaturated fatty acids on intestinal injury in rats with obstructive jaundice.

Obstructive jaundice (OJ) is a common clinical pathological syndrome in hepatobiliary surgery. High incidence of multiple organ injuries during perioperative period and its associated mortality remains challenging in clinical practice. Omega-3 polyunsaturated fatty acids (ω-3 PUFA) is an important enteral immune nutrition. This study investigated the protective role of ω-3 PUFA in the regulation of inflammatory response in OJ. Seventy-two rats were randomly divided into obstructive jaundice (OJ) group, obstructive jaundice + ω-3 PUFA group (OJPUFA) group, and sham group. OJ model was created by ligation of the bile duct. Abdominal thoracic catheter was placed to collect lymph. Body weight, liver function, serum and lymphatic levels of TNF-α, IL-1β, IL-10, HMGB1, and nitric oxide (NO) were measured on day 3, day 7, and day 14 after operation. Hematoxylin staining and Alcian blue-periodic acid-Shiff (AB-PAS) staining were performed on the ileum tissue. Protein and mRNA expression of HMGB1, TLR4, and NF-κB p65 were measured at the aforementioned time points. The general condition, including body weight and liver function, were worse in the OJ and the OJPUFA group compared to that in the sham group. On day 14, the body weight recovery and liver function were significantly better in the OJPUFA group than those in the OJ group were (p<0.05 for all). No marked change in the serum and lymphatic levels of TNF-α, IL-1β, IL-10, HMGB1 and NO was observed in the sham group after operation, while corresponding levels in the OJ and the OJPUFA groups were significantly higher. Compared with the OJPUFA group, serum and lymphatic levels of the above factors were consistently higher in the OJ group and were significantly higher on day 14 (p<0.05 for all). At the same time, ω-3 PUFA lowered the damage of intestinal villi and intestinal mucosal epithelium. It also improved the number and function of goblet cells in intestinal mucosal epithelium. The protein and mRNA expression of HMGB1, TLR4, and NF-κB p65 were significantly higher in the OJ group than those in the OJPUFA group (p<0.05 for all). ω-3 PUFA has protective effect in the management of obstructive jaundice. It can regulate the inflammatory response and reduce its damage to intestinal structure. Reducing the activation of HMGB1/TLR4/ NF-κB pathway might be a mechanism for its protective effect. We suggested that ω-3 PUFA and drugs targeted HMGB1/TLR4/NF-κB pathway might be potential treatment strategies in obstructive jaundice.

β-glucan modulates non-specific immune gene expression, thermal tolerance and elicits disease resistance in endangered Tor putitora fry challenged with Aeromonas salmonicida

An eight-week feeding trial was performed to assess the effect of different dietary levels (0, 0.5, 1.0, and 1.5%) of β-glucan (sourced from Saccharomyces cerevisiae) on growth, survival, immunological parameters (immune gene expression, lysozyme, and antiprotease), total antioxidant status, thermal tolerance, and disease resistance of Tor putitora fry. Feeding of moderate doses (0.5 and 1.0%) of β-glucan significantly improved survival but not weight gain percentage as compared to that received unsupplemented control and highest dose (1.5%) of glucan. Supplementation of β-glucan in diets differentially influenced the mRNA expression of cytokine and other immune genes. For instance, transcripts of cytokines such as tnf-α and il-1β were significantly upregulated, while ifn-γ and il-10 were unaffected by β-glucan intake. Also, the relative mRNA expression of tlr-5 and hepcidin1 along with lysozyme and antiprotease activities were remained largely unchanged by dietary glucan administration. In contrast, β-glucan induced mRNA expression of defensin1 and c3 while decreased the transcript level of mhc-1. On the other hand, dietary inclusion of β-glucan markedly improved total antioxidant levels and extended the thermal tolerance limits at both the ends, as shown by increased CTmax and lower CTmin than the control group. After feeding β-glucan for eight weeks, the fish were bath challenged with a bacterial pathogen, Aeromonas salmonicida. The challenge study results revealed that β-glucan intake improved most of the studied immune parameters, resulting in lower mortality. Overall, dietary inclusion of β-glucan (0.5–1.0%) was efficient in improving the immune responses, thermal tolerance, and disease resistance of T. putitora fry.

Immunomodulatory Activity of Carboxymethyl Pachymaran on Immunosuppressed Mice Induced by Cyclophosphamide.

The effects of immunomodulatory activity of two types of carboxymethyl pachymaran (CMP-1 and CMP-2) on cyclophosphamide (CTX)-induced mice were investigated. Both CMP-1 and CMP-2 were found to restore the splenomegaly and alleviate the spleen lesions and the mRNA expressions of TLR4, MyD88, p65 and NF-κB in spleen were also increased. CMP-1 and CMP-2 could enhance the immunity by increasing the levels of TNF-α, IL-2, IL-6, IFN-γ, Ig-A and Ig-G in serum. In addition, CMP-1 could increase the relative abundance of Bacteroidetes and reduce the relative richness of Firmicutes at the phylum level. CMP-1 and CMP-2 could reduce the relative abundance Erysipelatoclostridum at the genus level. CMP-1 and CMP-2 might enhance the immune function of immunosuppression mice by regulating the gene expression in the TLR4/NF-κB signaling pathway and changing the composition and abundance of the intestinal microbiota. The results suggested that CMP-1 and CMP-2 would be as potential immunomodulatory agents in functional foods.

Pim-2 kinase inhibits inflammation by suppressing the mTORC1 pathway in atherosclerosis.

Background: Inflammatory immunity theory has raised considerable concern in the pathogenesis of atherosclerosis. Proviral integration site of murine 2 (Pim-2) kinases functions in apoptosis pathways and the anti-inflammatory response. Here, we investigated whether Pim-2 kinase inhibits atherosclerotic inflammation by suppressing the mTORC1 pathway.Methods: An atherosclerosis animal model was established by feeding ApoE -/- mice a high-fat diet. THP-1-derived macrophages were subjected to ox-LDL (50 μg/ml, 24h) conditions in vitro to mimic the in vivo conditions.Result: The protein expression of Pim-2 was upregulated in ox-LDL-treated THP-1-derived macrophages and an atherosclerotic mouse model. Additionally, ox-LDL upregulated the protein expression of p-mTOR, p-S6K1 and p-4EBP1, intracellular lipid droplets, free cholesterol and cholesterylester and the mRNA expression of inflammatory cytokines, including IL-6, MCP-1, TLR-4 and TNF-α, in THP-1-derived macrophages. Functionally, overexpressed Pim-2 (Pim-2 OE) attenuated atherosclerotic inflammation associated with the mTORC1 signaling pathway in vitro and in vivo, whereas knocked down Pim-2 (Pim-2 KD) markedly promoted atherosclerotic inflammation associated with upregulation of the mTORC1 signaling pathway. The plaque areas and lesions in the whole aorta and aortic root sections were alleviated in ApoE -/- mice with Pim-2 OE, but aggravated by Pim-2 KD. Additionally, an mTOR agonist (MHY1485) counteracted the anti-inflammatory effect of Pim-2 in ox-LDL-treated THP-1-derived macrophages after Pim-2 OE, whereas rapamycin rescued atherosclerotic inflammation in ox-LDL-treated THP-1-derived macrophages after Pim-2 KD. Furthermore, si-mTOR and si-Raptor alleviated the atherosclerotic proinflammatory effect in ox-LDL-treated THP-1-derived macrophages in a the background of Pim-2 KD.Conclusions:These results indicated that Pim-2 kinase inhibits atherosclerotic inflammation by suppressing the mTORC1 pathway.

Treprostinil alleviates hepatic mitochondrial injury during rat renal ischemia-reperfusion injury

BackgroundRenal ischemia-reperfusion injury (IRI) causes acute kidney injury as well as liver injury. Renal IRI depletes hepatic antioxidants, promotes hepatic inflammation and dysfunction through Tlr9 upregulation. There is no treatment available for liver injury during renal IRI. This study examines the hepatoprotective role of treprostinil, a prostacyclin analog, during renal IRI. MethodsMale Sprague-Dawley rats were divided into four groups: control, sham, IRI-placebo, or IRI-treprostinil and subjected to bilateral ischemia (45 min) followed by reperfusion (1–72 h). Placebo or treprostinil (100 ng/kg/min) was administered subcutaneously via an osmotic minipump. ResultsTreprostinil significantly reduced peak serum creatinine, BUN, ALT and AST levels vs. IRI-placebo. Treprostinil also restored hepatic levels of superoxide dismutase, glutathione, catalase, and Gclc expression to baseline, while reducing lipid peroxidation vs. IRI-placebo. Additionally, treprostinil significantly reduced elevated hepatic Tlr9, Il-1β, Ccl2, Vcam1, and Serpine1 mRNA expression. Renal IRI increased hepatic apoptosis which was inhibited by treprostinil through reduced cytochrome c and cleaved caspase-3 protein expression. Treprostinil enhanced hepatic ATP concentrations and mitochondrial DNA copy number and improved mitochondrial dynamics by restoring Pgc-1α expression and significantly upregulating Mfn1, Mfn2, and Sirt3 levels, while reducing Drp-1 protein vs. IRI-placebo. Non-targeted semi-quantitative proteomics showed improved oxidative stress indices and ATP subunits in the IRI-treprostinil group. ConclusionsTreprostinil improved hepatic function and antioxidant levels, while suppressing the inflammatory response and alleviating Tlr9-mediated apoptotic injury during renal IRI. Our study provides evidence of treprostinil’s hepatoprotective effect, which supports the therapeutic potential of treprostinil in reducing hepatic injury during renal IRI.

Dipeptedyl peptidase-4 (DPP-4) inhibitor downregulates HMGB1/TLR4/NF-κB signaling pathway in a diabetic rat model of non-alcoholic fatty liver disease

Context Inflammatory and immune pathways play a crucial role in the pathophysiology of non-alcoholic fatty liver disease (NAFLD). Sitagliptin blocks the dipeptidyl peptidase-4 (DPP-4) enzyme, mechanisms that alter inflammatory pathways and the innate immune system, and by which Sitagliptin affects the pathogenesis of NAFLD weren’t previously discussed. Objective This study aims to understand the interaction between Sitagliptin and innate immune response in order to meliorate NAFLD. Methods Thirty- two Wistar male albino rats were categorised into four groups. Rats have received a standard diet or a high-fat diet either with or without Sitagliptin. Serum HMGB1, protein and mRNA expressions of hepatic TLR4 and NF-κB, inflammatory cytokines, and histopathological changes were analysed. Results An ameliorative action of Sitagliptin in NAFLD was demonstrated via decreasing HMGB1-mediated TLR4/NF-κB signalling in order to suppress inflammation and reduce insulin resistance. Conclusion Sitagliptin may in fact prove to be a beneficial therapeutic intervention in NAFLD.

Involvement of oxidative stress and toll-like receptor-4 signaling pathways in gentamicin-induced nephrotoxicity in male Sprague Dawley rats

Gentamicin (GM) is an antibiotic belonging to an aminoglycoside family that might induce nephrotoxicity in human and animal models via oxidative stress. Toll-like receptors (TLRs) are part of innate immune systems that participate in inflammatory responses. In this regard, we investigated the effect of GM on kidney functional and structural parameters, enzymatic antioxidant levels, and mRNA expression of TLR4 and IL6 in the rat kidney. Adult male Sprague Dawley rats were randomly divided into two groups (n = 10): Control and Gentamicin (100 mg/kg, i.p.). After ten days of GM administration, a blood sample was taken, and the kidneys were removed. The serum levels of creatinine (Cr) and blood urea nitrogen (BUN) were measured. Furthermore, the right kidney was preserved in formalin 10% for hematoxylin and eosin (H&E) staining, and the left kidney was kept at −80 °C for molecular and oxidative indexes analysis. Administration of GM caused tubular damages and functional disturbance. So that, Cr and BUN values in the GM group were higher than Control group. Furthermore, molecular findings showed upregulation of TLR4 and IL-6 mRNA expression in renal tissue of the GM-received group. In this study, superoxide dismutase (SOD) activity was slightly increased as a compensatory mechanism in response to elevated malondialdehyde (MDA) levels in the GM-treated group. On the other hand, the activity of catalase (CAT) and glutathione peroxidase (GPx) were significantly declined. Our results demonstrated that oxidative stress and subsequent TLR4 upregulation signaling pathways are involved in GM-induced nephrotoxicity.

Chitosan-chelated zinc modulates ileal microbiota, ileal microbial metabolites, and intestinal function in weaned piglets challenged with Escherichia coli K88.

This study was to investigate the effects of chitosan-chelated zinc on ileal microbiota, inflammatory response, and barrier function in weaned piglets challenged with Escherichia coli K88. Piglets of the chitosan-chelated zinc treatment (Cs-Zn; 100mg zinc + 766mg chitosan/kg basal diet, from chitosan-chelated zinc) and the chitosan treatment (CS, 766mg chitosan/kg basal diet) had significantly increased ileal villus height and the ratio of villi height to crypt depth. CS-Zn group piglets had a higher abundance of Lactobacillus in the ileal digesta, while the abundance of Streptococcus, Escherichia shigella, Actinobacillus, and Clostridium sensu stricto 6 was significantly decreased. The concentrations of propionate, butyrate, and lactate in the CS-Zn group piglets were significantly increased, while the pH value was significantly decreased. Furthermore, the concentrations of IL-1β, TNF-α, MPO, and INF-γ in the ileal mucosa of the CS-Zn and the H-ZnO group (pharmacological dose of 1600mg Zn/kg basal diet, from ZnO) were significantly lower than those of the control group fed with basal diet, and the mRNA expression of TLR4, MyD88, and NF-κB of the CS-Zn group was also reduced. In addition, the mRNA expression of IGF-1 was increased, the protein expression of occludin and claudin-1 was enhanced, while the mRNA expression of caspase 3 and caspase 8 was decreased in the CS-Zn group. These results suggest CS-Zn treatment could help modulate the composition of ileal microbiota, attenuate inflammatory response, and maintain the intestinal function in weaned piglets challenged with Escherichia coli K88. KEY POINTS: • Chitosan-chelated zinc significantly modulated ileal microbiota. • Chitosan-chelated zinc can improve ileal health. •The ileal microbiota plays an important role in host health.

GDF-15 Suppresses Atherosclerosis by Inhibiting oxLDL-Induced Lipid Accumulation and Inflammation in Macrophages.

The growth differentiation factor-15 (GDF-15) may be involved in atherosclerosis. However, the role of GDF-15 in atherosclerosis remains unclear. The main goal of this study was to verify the role and mechanism of GDF-15 in atherogenesis. We first compared the serum GDF-15 level between patients with coronary atherosclerosis and healthy people. And then one ApoE−/− mouse model of atherosclerosis was used to explore the effects of GDF-15 on oxidized low-density lipoprotein (oxLDL) accumulation, atherosclerosis-related gene expression, and lipid accumulation-related protein expression in mouse macrophages. As a result, the level of serum GDF-15 in patients with coronary atherosclerosis was significantly higher than that in healthy people. In the mouse model, GDF-15 expression was elevated in the core of plaque, and it was secreted mainly by the macrophages. In addition, GDF-15 decreased oxLDL-induced lipid accumulation and inflammation activation in macrophages. GDF-15 decreased the mRNA expressions of CD36, LOX1, and TLR4 that are associated with lipoprotein accumulation in macrophages. Further study showed that GDF-15 might suppress oxLDL-induced lipoprotein accumulation via inhibiting CD36 and LOX1 and decrease inflammation in macrophages by inhibiting TLR4. Thus, GDF-15 may suppress atherosclerosis and plaque formation by inhibiting lipoprotein accumulation and inflammation activation.

Paeoniflorin Protects against ANIT-Induced Cholestatic Liver Injury in Rats via the Activation of SIRT1-FXR Signaling Pathway.

Paeoniflorin (PF), a water-soluble monoterpene glycoside, is initially isolated from the dried roots of Paeonia lactiflora Pall., which has effects on ameliorating cholestasis in our previous study. However, comprehensive approaches for understanding the protective effects and mechanisms underlying cholestatic liver injury from the regulating of bile acid metabolism have not been sufficiently elucidated. This study was aimed to explore the effectiveness as well as potential mechanism of PF on alpha-naphthylisothiocyanate (ANIT)-induced cholestatic liver injury. Rats with cholestasis induced by ANIT was used to evaluate the protective effects and mechanism of PF by regulating SIRT1/FXR and NF-κB/NLRP3 signaling pathway. Rats were intragastrically administrated with ANIT to establish cholestatic liver injury model. Serum levels of ALT, AST, TBA, TBIL, ALP, γ-GT and ALB in rats were detected. The histopathology of the liver of rats was analyzed in vivo. The relative mRNA expression and protein expression levels of IL-18, IL-1β, TNF-α, HO-1, Nrf2, TLR4, NLRP3, Caspase-1, ASC, NF-κB, FXR, and SIRT1 in liver of rats were investigated. The results showed that the serum indexes and the liver histopathology were significantly improved by PF. The overexpression of IL-18, IL-1β, TNF-α, NLRP3, ASC, and Caspase-1 in liver was markedly reduced by PF. Furthermore, PF dramatically increased the mRNA and protein expressions of SIRT1, FXR, HO-1, and Nrf2, but decreased NF-κB p65 and TLR4 levels in liver of rats. Taken together, the protective effects of PF on cholestatic liver injury were possibly related to the activation of the SIRT1/FXR and inhibition of NF-κB/NLRP3 inflammasome signaling pathway. These findings might provide a potential protection for cholestatic liver injury.

Melanin of Sporothrix globosa affects the function of THP-1 macrophages and modulates the expression of TLR2 and TLR4

BackgroundMelanin is an important virulence factor for Sporothrix globosa, the causative agent of sporotrichosis, a subcutaneous mycosis that occurs worldwide. Although previous research suggests that melanin is involved in the pathogenesis of sporotrichosis, little is known about its influence on the macrophages that represent the frontline components of innate immunity. ObjectivesTo evaluate the effects of melanin on phagocytic activity and the expression of Toll-like receptor (TLR)2 and TLR4 during S. globosa infection of macrophages in vitro. MethodsTo compare phagocytic activity and survival rates, THP-1 macrophages and primary mouse peritoneal macrophages were co-cultured with a wild-type S. globosa strain (Mel+), an albino mutant strain (Mel˗), a tricyclazole-treated Mel + strain (TCZ-Mel+), or melanin ghosts extracted from S. globosa conidia. Reactive oxygen species (ROS), nitric oxide (NO) generation, tumor necrosis factor (TNF)-α and interleukin (IL)-6 were assayed in THP-1 cells infected with S. globosa conidia. Quantitative PCR and western blotting were used to observe the effect of melanin on TLR2 and TLR4 expression. Knockdown of TLR2/4 expression with small interfering RNA was performed to further verify the role of these receptors during infection. ResultsMacrophages infected with Mel + conidia showed a lower phagocytosis index and a higher survival rate than TCZ-Mel+ and Mel˗ in vitro. After incubation with S. globosa, the release of ROS, NO, TNF-α and IL-6 by THP-1 were decreased in the presence of melanin. Increased mRNA and protein expression of TLR2 and TLR4 occurred upon S. globosa infection in THP-1, whereas the presence of melanin suppressed TLR2 and TLR4. Moreover, TLR2 or TLR4 knockdown showed a trend toward reducing the pernicious effect of S. globosa conidia on THP-1 cells in vitro. ConclusionsCollectively, our results indicated that melanin inhibits the phagocytosis of S. globosa and guards against macrophage attack by providing protection from oxygen- and nitrogen-derived radicals, as well as suppressing the host pro-inflammatory cytokine response (TNF-α and IL-6). Melanin was also involved in modulating TLR2 and TLR4 receptor expression, weakening the killing efficiency of S. globosa.

Effect of Tea Tree Oil on the Expression of Genes Involved in the Innate Immune System in Goat Rumen Epithelial Cells.

Simple SummarySubacute rumen acidosis (SARA) often causes significant losses on commercial farms. SARA is mainly caused by endotoxin (LPS) produced by the lysis of Gram-negative bacteria, which causes an inflammatory response. To alleviate the inflammatory response mediated by LPS, it is important to improve animal production performance. Tea tree oil (TTO) is a plant extract that possesses good bactericidal and anti-inflammatory effects. According to this study, LPS can significantly induce inflammatory responses in goat rumen epithelial cells (GRECs), while the addition of TTO could markedly mitigate inflammatory responses mediated by LPS in GRECs. Therefore, it may be useful for the treatment of SARA.In subacute rumen acidosis (SARA), the rumen epithelium is frequently attacked by endotoxin (LPS), which is caused by the lysis of dead Gram-negative bacteria. However, the rumen epithelium innate immune system can actively respond to the infection. Previous studies have demonstrated that tea tree oil (TTO) has good bactericidal and anti-inflammatory effects. Therefore, the aim of this study was to investigate the effect of TTO on the expression of genes involved in the inflammatory cytokines in goat rumen epithelial cells (GRECs) triggered by LPS. Our study shows that rumen epithelial cells isolated from goat rumen tissue can be cultured in vitro in 0.25% trypsin for a long time. These cells were identified as epithelial cells by the expression of cytokeratin 18, monocarboxylate transporter 4 (MCT4), Na[+]/H[+] hydrogen exchanger 1 (NHE1), putative anion transporter 1 (PAT1), vH+ ATPase B subunit (vH+ ATPase), and anion exchanger 2 (AE2). The mRNA expression of IL-1β, IL-6, TNF-α, TLR-2, NF-κB, CXCL6 and CXCL8 genes was significantly increased when LPS was used compared to untreated controls. In addition, mRNA expression of IL-1β, IL-6, TNF-α, TLR-2, NF-κB, CXCL8, CXCL6 and interferon-induced protein with tetratricopeptide repeats 3 (IFIT3) genes was also significantly higher in the LPS group compared to the 0.05% TTO group. However, the expression of IL-1β, IL-6, TNF-α, TLR-2, CXCL6 and IFIT3 genes was significantly lower in the LPS and 0.05% TTO group compared to the 1 μg/mL LPS group. These results suggest that TTO can inhibit LPS-induced inflammatory cytokines expression in GRECs.

The Effect of Chicken Spleen Transfer Factor on Intestinal Mucosa Immunity Barrier of Laying Hens

Chicken spleen Transfer Factor (TF) is a low-molecular-weight lymphocyte extract composed of polypeptide and nucleotide. However, its role in regulating intestinal structure and function in laying hens has remained largely unknown. 100 one-day-old laying hens were randomly divided into five groups and administered with different doses of TF (0.00 [control], 0.05mL, 0.10mL, 0.25mL and 1.00mL). The results showed that the high dose of TF (1.00mL) improved the intestinal mucosa morphology and strengthened the digestive and absorption function. Furthermore, the histology analysis revealed an increase in the number of intraepithelial lymphocytes and goblet cells. Similarly, the results from ELISA demonstrated an increase in the content of IL-10 in the intestinal tract, while the content of TNF-a showed a decrease in this regard. The RT-PCR assay also demonstrated the upregulation of the relative mRNA expressions of Muc2, TLR-2, and TLR-4 genes. The intestinal antioxidant function was significantly enhanced. In conclusion, high-dose of TF can improve the intestinal mucosa morphology and structure, enhance digestion and absorption functions, enhance the intestinal mucosal barrier immune function and antioxidant function, and up-regulate Muc2, TLR-2 and TLR-4 gene relative expression.

High Mobility Group Box 1 in Pig Amniotic Membrane Experimentally Infected with E. coli O55.

Intra-amniotic infections (IAI) are one of the reasons for preterm birth. High mobility group box 1 (HMGB1) is a nuclear protein with various physiological functions, including tissue healing. Its excessive extracellular release potentiates inflammatory reaction and can revert its action from beneficial to detrimental. We infected the amniotic fluid of a pig on the 80th day of gestation with 1 × 104 colony forming units (CFUs) of E. coli O55 for 10 h, and evaluated the appearance of HMGB1, receptor for glycation endproducts (RAGE), and Toll-like receptor (TLR) 4 in the amniotic membrane and fluid. Sham-infected amniotic fluid served as a control. The expression and release of HMGB1 were evaluated by Real-Time PCR, immunofluorescence, immunohistochemistry, and ELISA. The infection downregulated HMGB1 mRNA expression in the amniotic membrane, changed the distribution of HMGB1 protein in the amniotic membrane, and increased its level in amniotic fluid. All RAGE mRNA, protein expression in the amniotic membrane, and soluble RAGE level in the amniotic fluid were downregulated. TLR4 mRNA and protein expression and soluble TLR4 were all upregulated. HMGB1 is a potential target for therapy to suppress the exaggerated inflammatory response. This controlled expression and release can, in some cases, prevent the preterm birth of vulnerable infants. Studies on suitable animal models can contribute to the development of appropriate therapy.

Artemisia ordosica Polysaccharide Alleviated Lipopolysaccharide-induced Oxidative Stress of Broilers via Nrf2/Keap1 and TLR4/NF-κB Pathway

Artemisia ordosica is one of the main shrubby perennials belonging to Artemisia species of Asteraceae and could be used in folk Chinese/Mongolian medicine to treat symptoms of various inflammatory ailments. The present study was conducted to investigate the protective effects of dietary Artemisia ordosica polysaccharide (AOP) against lipopolysaccharide (LPS) induced oxidative stress in broilers via Nrf2/Keap1 and TLR4/NF-κB pathway. A total of 192 1-day-old Arbor Acres male broilers were randomly allotted to four treatments with 6 replicates (n = 8): (1) CON group, non-challenged broilers fed basal diet; (2) LPS group, LPS-challenged broilers fed basal diet; (3) AOP group, non-challenged broilers fed basal diet supplemented with 750 mg/kg AOP; (4) LPS+AOP group, LPS-challenged broilers fed basal diet supplemented with 750 mg/kg AOP. The trial included starter phase (d 1–14), stress period Ⅰ (d 15–21), convalescence Ⅰ (d 22–28), stress period Ⅱ (d 29–35) and convalescence Ⅱ (d 36–42). During stress period Ⅰ (on d 15, 17, 19 and 21) and stress period Ⅱ (on d 29, 31, 33 and 35), broilers were injected intra-abdominally either with LPS solution or with an equal amount of sterile saline. The results showed that dietary AOP supplementation alleviated LPS-induced reduction in antioxidant enzyme activity and excessive production of ROS, 8-OHdG and PC in serum of broilers challenged with LPS. Moreover, dietary AOP supplementation alleviated the decrease of T-AOC and activities of SOD, CAT and GPx in liver of broilers challenged with LPS by increasing expression of Nrf2, and inhibiting over-expression of Keap1 both at gene and protein level. Additionally, dietary AOP supplementation decreased the over-production of IL-1β and IL-6 in liver of broilers challenged by LPS through decreasing mRNA expression of TLR4, MyD88, NF-κB P65, IL-1β and IL-6, and alleviating the increase of protein expression of TLR4, IKKβ, NF-κB P65, IL-1β, IL-6, and the decrease of protein expression of IkBα. In conclusion, dietary AOP supplementation could alleviate LPS-induced oxidative stress through Nrf2/Keap1 and TLR4/NF-κB pathway.