Levels Of Toll-like Receptor Research Articles

The therapeutic potential of chondroitin sulfate in Aspergillus fumigatus keratitis

PurposeTo explore the therapeutic effect of chondroitin sulfate (CS) on Aspergillus fumigatus (A. fumigatus) keratitis. MethodsThe nontoxic concentration of CS was determined using the Draize eye test and cell counting kit-8. Cell scratch test and cell proliferation test were evaluated the impact of CS on the proliferation and migration of human corneal epithelial cells (HCECs). Adherence assay and plate counting were used to detect fungal load in vivo and in vitro, respectively. Clinical score and hematoxylin-eosin (HE) staining were applied to assess the therapeutic effects of CS in an A. fumigatus keratitis mice model. The neutrophil infiltration and activity were detected by flow cytometry (FCM), immunofluorescence staining, and myeloperoxidase (MPO) assay. Toll-like receptor 4 (TLR-4) expression in RAW 264.7 cells and mouse cornea was detected by immunofluorescence staining. Real-time PCR (RT-PCR), western blot, and enzyme-linked immunosorbent assay (ELISA) were applied to examine the expression of inflammatory mediators. ResultsCS at 400 μg/mL (non-cytotoxic) significantly promoted proliferation and migration of HCECs. In an A. fumigatus keratitis mice model, CS treatment alleviated fungal keratitis (FK) severity by decreasing corneal fungal load and inhibiting neutrophil infiltration. In RAW 264.7 cells, the mRNA and protein levels of TLR-4, phosphorylated nuclear factor (NF)-κB (p-NF-κB), interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-a (TNF-α), cyclooxygenase 2 (COX-2), and macrophage inflammatory protein-2 (MIP-2) were remarkably lower in the siTLR-4 treated group, while higher in rTLR-4 treated group than in the corresponding control group. CS treatment suppressed rTLR-4 induced the mRNA and protein expression of TLR-4, p-NF-κB, IL-1β, IL-6, COX-2, TNF-α, and MIP-2 in RAW cells. ConclusionCS may ameliorate the prognosis of A. fumigatus keratitis by promoting corneal epithelial proliferation, inhibiting the recruitment and activity of neutrophils, and inhibiting the inflammatory response by down-regulation of the TLR-4/NF-κB signaling.

TLR4 promoted endoplasmic reticulum stress induced inflammatory bowel disease via the activation of p38 MAPK pathway.

Endoplasmic reticulum (ER) stress contribute to inflammatory bowel disease (IBD). However, the mechanistic link between toll-like receptor 4 (TLR4) and ER stress in IBD remains elusive. This study aimed to investigate the mechanism by which ER stress and TLR4 promote inflammation in IBD.IBD mouse model was established by the induction of TNBS, and Grp78 and TLR4 in intestine tissues were detected by immunohistochemistry. THP-1 cells were treated with lipopolysaccharides (LPS), ER stress inducer or inhibitor tauroursodeoxycholic acid (TUDCA), or p38 MAPK inhibitor. The activation of MAPK signaling was detected by Western blot, and the production and secretion of inflammatory factors were detected by PCR and ELISA. We found that the expression levels of TLR4 and GRP78 were significantly higher in the intestine of IBD model mice compared with control mice but were significantly lower in the intestine of IBD model mice treated with ER stress inhibitor TUDCA. ER stress inducer significantly increased while ER stress inhibitor TUDCA significantly decreased the expression and secretion of TNF-α, IL-1β and IL-8 in THP-1 cells treated by LPS. Only p38 MAPK signaling was activated in THP-1 cells treated by ER stress inducer. Furthermore, p38 inhibitor SB203580 inhibited the production and secretion of TNF-α, IL-1β and IL-8 in THP-1 cells treated with LPS. In conclusion, TLR4 promotes ER stress induced inflammation in IBD, and the effects may be mediated by p38 MAPK signaling. TLR4 and p38 MAPK signaling are novel therapeutic targets for IBD.

Elevated Expression of TLR2 in Aging Hearts Exacerbates Cardiac Inflammatory Response and Adverse Remodeling Following Ischemia and Reperfusion Injury.

This study tested the hypothesis that Toll-like receptor 2 (TLR2) augments the inflammatory responses and adverse remodeling in aging hearts to exacerbate myocardial injury and cardiac dysfunction.MethodsOld (20-22 months old) and adult (4-6 months old) mice of C57BL/6 wild-type and TLR2 knockout (KO) were subjected to coronary artery ligation (30 minutes) and reperfusion (3 or 14 days). Left ventricle function was assessed using a pressure-volume microcatheter. Cardiac infarct size was determined by histology. Levels of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), matrix metalloproteinase 9 (MMP 9), and collagen I in non-ischemic myocardium were assessed by immunoblotting. Monocyte chemoattractant protein-1 (MCP-1), keratinocyte chemoattractant (KC), and interleukin-6 (IL-6) levels in ischemic and non-ischemic myocardium were measured by enzyme-linked immunosorbent assay (ELISA). TLR2 expression in the myocardium of untreated wild type mice was also measured by immunoblotting.ResultsHigher levels of MCP-1, KC, IL-6 were induced in both ischemic and non-ischemic myocardium of old wild type mice at day 3 and 14 following ischemia/reperfusion (I/R) than those of adult wild type mice. The hyper-inflammatory responses to I/R in aging hearts were associated with elevated levels of myocardial TLR2. TLR2 KO markedly down-regulated the expression of MCP-1, KC, IL-6, ICAM-1 and VCAM-1 in aging hearts at day 3 and 14 following I/R. The down-regulated inflammatory activity in aging TLR2 KO hearts was associated with attenuated production of MMP 9 and collagen I at day 14 and resulted in reduced infarct size and improved cardiac function.ConclusionElevated expression of myocardial TLR2 contributes to the mechanism by which aging exacerbates the inflammatory responses, adverse remodeling and cardiac dysfunction following myocardial I/R in aging.

Effect of TLR4/MyD88/NF-kB axis in paraventricular nucleus on ventricular arrhythmias induced by sympathetic hyperexcitation in post-myocardial infarction rats.

Sympathetic activation after myocardial infarction (MI) leads to ventricular arrhythmias (VAs), which can result in sudden cardiac death (SCD). The toll‐like receptor 4 (TLR4)/myeloid differentiation primary response 88 (MyD88)/nuclear factor‐kappa B (NF‐kB) axis within the hypothalamic paraventricular nucleus (PVN), a cardiac‐neural sympathetic nerve centre, plays an important role in causing VAs. An MI rat model and a PVN‐TLR4 knockdown model were constructed. The levels of protein were detected by Western blotting and immunofluorescence, and localizations were visualized by multiple immunofluorescence staining. Central and peripheral sympathetic activation was visualized by immunohistochemistry for c‐fos protein, renal sympathetic nerve activity (RSNA) measurement, heart rate variability (HRV) analysis and norepinephrine (NE) level detection in serum and myocardial tissue measured by ELISA. The arrhythmia scores were measured by programmed electrical stimulation (PES), and cardiac function was detected by the pressure–volume loop (P‐V loop). The levels of TLR4 and MyD88 and the nuclear translocation of NF‐kB within the PVN were increased after MI, while sympathetic activation and arrhythmia scores were increased and cardiac function was decreased. However, inhibition of TLR4 significantly reversed these conditions. PVN‐mediated sympathetic activation via the TLR4/MyD88/NF‐kB axis ultimately leads to the development of VAs after MI.

Toll-Like Receptors/TNF-α Pathway Crosstalk and Impact on Different Sites of Recurrent Myocardial Infarction in Elderly Patients.

Background Recurrent myocardial infarction is associated with increased mortality. Risk and predictive factors of recurrent myocardial infarction in elderly patients after coronary stenting are not well known. This research sought to investigate the effects of proinflammatory cytokines and toll-like receptor on recurrent myocardial infarction after coronary stenting in elderly patients. Methods We measured the levels of toll-like receptor 2 (TLR2), toll-like receptor 3 (TLR3), toll-like receptor 4 (TLR4), tumor necrosis factor-α (TNF-α), soluble tumor necrosis factor-α receptor-1 (sTNFR-1), soluble tumor necrosis factor-α receptor-2 (sTNFR-2), endothelial progenitor cells (EPCs), and vascular endothelial growth factor (VEGF) in elderly patients with recurrent myocardial infarction and assessed the changes of proinflammatory cytokines and toll-like receptors in elderly patients with recurrent myocardial infarction after coronary stenting. Results Levels of TLR2, TLR3, TLR4, TNF-α, sTNFR-1, and sTNFR-2 were remarkably increased (P < 0.001), and EPCs and VEGF were remarkably lowered (P < 0.001) in the elderly patients with recurrent myocardial infarction after coronary stent implantation. Increased expressions of proinflammatory cytokines and toll-like receptors induced recurrent myocardial infarction after coronary stenting. Elevated expressions of proinflammatory cytokines and toll-like receptors may be used to identify elderly patients who have an increased risk of developing recurrent myocardial infarction after coronary stenting. Conclusion The increase levels of proinflammatory cytokines and toll-like receptors were associated with recurrent myocardial infarction after coronary stenting. Increased expressions of proinflammatory cytokines and toll-like receptors may be clinically useful biomarkers for predicting recurrent myocardial infarction in the elderly patients after coronary stent implantation.

Ethyl Pyruvate Alleviating Inflammatory Response after Diabetic Cerebral Hemorrhage.

This study's purpose is to investigate the neuroprotective role of ethyl pyruvate (EP) in the pathogenesis of diabetic intracerebral hemorrhage. The present study used a mouse model of collagenase-induced intracerebral hemorrhage (ICH) and streptozotocin-induced diabetes. The C57BL/6 mice were randomly divided into 3 groups: sham operation, diabetic cerebral hemorrhage, and diabetic cerebral hemorrhage with EP. The EP (80 mg/kg) and EP (50 mg/kg) were injected intraperitoneally one day and one hour before modeling. The protein expression levels of high mobility group box 1 (HMGB1) and NOD-like receptors 3 (NLRP3) were detected with western blot. The mRNA levels of HMGB1 and toll-like receptor 4 (TLR4) were measured by quantitative real-time polymerase chain reaction (PCR). Immunofluorescence and ELISA were performed to confirm some inflammatory factors. Compared to the normal diabetic intracerebral hemorrhage group, the mRNA and protein expression levels of HMGB1 and TLR4 were downregulated in the EP-affected group with diabetic cerebral hemorrhage, together with the downregulation of the expression of inflammasomes, including NLRP3, apoptosis-associated speck-like protein containing CARD (ASC), and caspase 1. EP can reduce the inflammatory response after diabetic intracerebral hemorrhage and may inhibit the activation of inflammasomes by the HMGB1/TLR4 pathway.

Niloticin binds to MD-2 to promote anti-inflammatory pathway activation in macrophage cells.

Niloticin is an active compound isolated from Cortex phellodendri with uncharacterized anti-inflammatory activity. We assessed the drug potential of niloticin and examined its ability to target myeloid differentiation protein 2 (MD-2) to ascertain the mechanism for its anti-inflammatory activity. The Traditional Chinese Medicine Systems Pharmacology Database was used to evaluate niloticin. Bio-layer interferometry and molecular docking technologies were used to explore how niloticin targets MD-2, which mediates a series of toll-like receptor 4 (TLR4)-dependent inflammatory responses. The cytokines involved in the lipopolysaccharide (LPS)-TLR4/MD-2-NF-κB pathway were evaluated using ELISA, RT-qPCR, and western blotting. Niloticin could bind to MD-2 and had no evident effects on cell viability. Niloticin treatment significantly decreased the levels of NO, IL-6, TNF-α, and IL-1β induced by LPS (p < 0.01). IL-1β, IL-6, iNOS, TNF-α, and COX-2 mRNA expression levels were decreased by niloticin (all p < 0.01). Compared with that in the control group, the increase in TLR4, p65, MyD88, p-p65, and iNOS expression levels induced by LPS were suppressed by niloticin (all p < 0.01). Our results suggest that niloticin has therapeutic potential and binds to MD-2. Niloticin binding to MD-2 antagonized the effects of LPS binding to the TLR4/MD-2 complex, resulting in the inhibition of the LPS-TLR4/MD-2-NF-κB signaling pathway.

Novel insights into the SLC7A11-mediated ferroptosis signaling pathways in preeclampsia patients: identifying pannexin 1 and toll-like receptor 4 as innovative prospective diagnostic biomarkers

PurposeFerroptosis is associated with oxidative stress (OS) and is caused by iron-dependent lipid-peroxidative damage, but its role in PE is unclear. The aim of this study is to determine whether pannexin 1 (Panx1) and toll-like receptor 4 (TLR4) are key regulators of ferroptosis in PE.MethodsThe study included 65 patients with PE and 25 healthy pregnant women. In normal and PE placental tissues, OS and ferroptosis markers, including Fe2+, malondialdehyde (MDA), reduced glutathione (GSH) levels, heme oxygenase-1 (HO-1) and glutathione peroxidase 4 (Gpx4) activity, were estimated. Panx1 and solute carrier family 7 member 11 (SLC7A11) mRNA expression levels were relatively quantified in placental tissues using real‐time polymerase chain reaction (RT‐PCR), while serum Panx1, serum TLR4, and placental activating transcription factor 3 (ATF3) levels were measured by ELISA.ResultsIn placental tissues, Panx1 and TLR4 expression levels were significantly increased in patients with PE compared to controls and were positively correlated with pro-ferroptosis mediators such as placental Fe2+ and MDA levels and negatively correlated with anti-ferroptosis regulators such as placental GSH level, HO-1, and Gpx4 activity. Additionally, Panx1 and TLR4 had a positive correlation with ATF3 and a negative correlation with SLC7A11. Serum Panx1 and TLR4 levels were positively correlated with their placental tissue expression and showed good diagnostic capabilities for ferroptosis in PE.ConclusionTherefore, Panx1 and TLR4 are suggested to induce ferroptosis in PE via SLC7A11-mediated signaling pathways, offering a novel perspective on PE pathogenesis and novel diagnostic tools for PE.

Dysfunctional monocytic toll-like receptor 4 signaling pathway and cognitive deficits in chronic schizophrenia patients with tardive dyskinesia

BackgroundMounting evidence suggests that the innate immune system is disrupted in schizophrenia patients with tardive dyskinesia (TD); however, the role of the toll-like receptor 4 (TLR4) signaling pathway remains unclear. MethodsIn this study, we quantified the expression of the monocytic TLR4 signaling pathway using flow cytometry, before and after lipopolysaccharide (LPS) stimulation, in chronic schizophrenia patients with (n = 61) and without TD (NTD, n = 61) and healthy controls (HCs, n = 74). Psychopathological symptoms, the severity of TD, and cognitive function were assessed using the Positive and Negative Syndrome Scale (PANSS), Abnormal Involuntary Movement Scale (AIMS), and MATRICS Consensus Cognitive Battery (MCCB), respectively. Results1) Both TD and NTD patients showed higher TLR4 signaling pathway activity at baseline than that in HCs, but their responses to LPS were weaker than those in HCs; 2) the alteration of the TLR4 signaling pathway was less severe in TD patients than in NTD patients; 3) TLR4 levels and MCCB scores were negatively correlated at baseline but positively correlated after LPS stimulation in TD patients; 4) there was no correlation between the TLR4 signals and PANSS or AIMS scores. ConclusionsOur findings suggested the TLR4 signaling pathway disturbance might be related to cognitive deficits in schizophrenia patients with TD.

The effects of vitamin B12 on the TLR-4/NF-κB signaling pathway in ovarian ischemia-reperfusion injury-related inflammation

Ovarian ischemia is a gynecological emergency case that occurs as a result of ovarian torsion. Oxidative stress and inflammation play central roles in the development of ischemia/reperfusion injuries. We investigated the effects of Vitamin B12, thought to possess antioxidant characteristics on oxidative stress and the toll-like receptor 4 (TLR-4)/nuclear factorkappa B (NF-κB) signaling pathway in the ovaries during ischemia-reperfusion. Forty-eight rats were randomly assigned into six groups and the groups are designed as follows: Control (C), Ischemia (I), Ischemia + Vitamin B12 (I + B12), Ischemia-Reperfusion (I/R), I/R + Vitamin B12 (I/R + B12) and Sham + Vitamin B12. Vitamin B12 was administered at a dose of 400 mcg/kg via the i.p. route once daily for three days before I/R procedure. Tissue interleukin-1β (IL-1β) and interleukin-6 (IL-6) and malondialdehyde (MDA) levels in ovarian tissue increased following I/R, while glutathione (GSH) levels decreased. Moreover, extensive congestion, edema, hemorrhage and defective follicle were observed. Both NF-κB and TLR-4 expression levels also increased in the group exposed to I/R. While GSH levels increased, IL-1β, IL-6, MDA, NF-κB and TLR-4 levels decreased with Vitamin B12 treatment. In addition, ovarian tissue without edema, mild congestion, and normal-appearing follicles were observed following Vitamin B12 administration. The findings showed that I/R in ovarian tissue resulted in significant tissue damage by increasing oxidative stress and inflammation. However, Vitamin B12 application was effective and alternative agent in reducing injury deriving from inflammation and oxidative stress developing in association with I/R in ovarian tissue.

LRRK2 Inhibition Mitigates the Neuroinflammation Caused by TLR2-Specific α-Synuclein and Alleviates Neuroinflammation-Derived Dopaminergic Neuronal Loss.

Evidence suggests that crosstalk occurs between microglial leucine-rich repeat kinase 2 (LRRK2)—a regulator of neuroinflammation—and neuron-released α-synuclein (αSyn)—a promoter of microglial activation and neuroinflammatory responses—in neuroinflammation-mediated Parkinson’s disease (PD) progression. Therefore, we examined whether LRRK2 inhibition reduces the responses of microglia to neuroinflammation caused by neuron-released αSyn. We examined the neuroinflammatory responses provoked by Toll-like receptor 2 (TLR2)-positive αSyn of neuronal cells using an LRRK2 inhibitor in the mouse glioma cells, rat primary microglia, and human microglia cell line; and the effects of LRRK2 inhibitor in the co-culture of ectopic αSyn-expressing human neuroblastoma cells and human microglia cells and in mouse models by injecting αSyn. We analyzed the association between LRRK2 activity and αSyn oligomer and TLR2 levels in the substantia nigra tissues of human patients with idiopathic PD (iPD). The TLR2-specific αSyn elevated LRRK2 activity and neuroinflammation, and the LRRK2 inhibitor ameliorated neuroinflammatory responses in various microglia cells, alleviated neuronal degeneration along with neuroinflammation in the co-culture, and blocked the further progression of locomotor failure and dopaminergic neuronal degeneration caused by TLR2-specific αSyn in mice. Furthermore, LRRK2 phosphorylation was increased in patients with iPD showing αSyn-specific high TLR2 level. These results suggest the application of LRRK2 inhibitors as a novel therapeutic approach against αSyn-mediated PD progression.

Syringic acid ameliorates experimental diabetic nephropathy in rats through its antiinflammatory, anti-oxidant and anti-fibrotic effects by suppressing Toll like receptor-4 pathway

Diabetic nephropathy (DN) is an important healthcare challenge because it occurs in about 40% of diabetic people and is a main cause end-stage renal disease. Toll like receptor-4 (TLR-4) signaling is involved in DN incidence and progression through its activation by hyperglycemia and hyperglycemia-induced alterations besides its crucial roles in initiating inflammation and fibrosis in kidney. Syringic acid is a natural phenolic acid. It is naturally available in many fruits and vegetables, including swiss chards, grapes and olives. In this study, Reno protective effects of syringic acid, as anti-inflammatory, anti-oxidant and anti-fibrotic natural compound, against experimental DN were investigated. Streptozotocin (45mg/kg) was used in inducing rat model of DN pathogenesis. In kidney homogenate, concentrations of TLR-4, interleukin-6 and transforming growth factor β1 (TGFβ1) were estimated by ELISA technique. Moreover, hydroxyproline and relevant collagen content in kidney tissues were evaluated using spectrophotometry. Oral administration of syringic acid (50 mg/kg) for 8 weeks succeeded in keeping kidney function evidenced by significant decrease in serum levels of creatinine, blood urea nitrogen (BUN) and decreased urinary protein content. Besides, it led to significant increase in renal superoxide dismutase (SOD) activity and significant decline in kidney malondialdehyde (MDA) content. Moreover, it showed significant anti-inflammatory and anti-fibrotic effects reflected by significant decrease in levels of TLR-4, interleukin-6, TGFβ1 and collagen in kidney homogenate as compared to DN group. Besides, it significantly attenuated DN-induced histopathological deteriorations. In conclusion, syringic acid can be proposed as an effective natural compound in halting DN progression through inhibiting TLR-4 pathway resulting in ameliorating oxidative stress, inflammation and fibrosis in kidney tissues.

The role of the novel small molecule QUIN-A in suppressing neuroinflammation and its target

Myeloid differentiation protein 88 (MyD88) is an important accessory protein of Toll-like receptor 4 (TLR4) Signal. Inhibiting MyD88 protein can inhibit the activation of TLR4 Signal, so it is particularly important to explore the small molecule inhibitor of MyD88.This study discovered that a kind of quinazoline derivative small molecule (QUIN-A) specifically suppressed MyD88. lipopolysaccharide（LPS） was used to induce inflammatory response in microglial cells BV2. Thereafter, cell apoptosis was detected by flow cytometry, propidium iodide（PI） staining, and Hoechst 33342 staining, while the levels of inflammatory factors were measured through enzyme linked immunosorbent assay (ELISA). In addition, the expression of superoxidedismutase（SOD）and Malondialdehyde（MDA）was detected by test kit, respectively, whereas that of TLR4 and MyD88 was observed by immunofluorescence (IF) staining, and the levels of TLR4 and MyD88 proteins were analyzed through Western blotting (WB) assay. The results suggested that QUIN-A suppressed the inflammatory injury in BV2 cells, decreased the cell apoptosis level and reduced the inflammatory factor levels in the culture medium. Besides, the expression levels of TLR4 and MyD88 in cells were down-regulated. In animal experiments, QUIN-A reduced the inflammatory factor levels in cerebrospinal fluid (CSF), but it did not significantly affect the serum inflammatory factors. At the same time, QUIN-A down-regulated the expression of TLR4 and MyD88 proteins. By pull-down assay, we discovered that QUIN-A specifically bound to MyD88 and served as the inhibitor of MyD88.QUIN-A bound to MyD88 and suppressed the transduction of TLR4 signal, thus exerting the anti-inflammation effect.

Investigation of the effects of the toll-like receptor 4 pathway on immune checkpoint vista in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most common malignant tumors of the pancreas. Preclinical studies show that it evades the immune system with immune checkpoints and promotes tumor development. V-domain Ig suppressor of T cell activation (VISTA) is a new immune-check point from the B7 family and is highly expressed in cancer cells. Overexpression of toll like receptor 4 (TLR4) in pancreatic adenocarcinoma is associated with induced tumorigenesis, tumor growth, resistancy to chemotherapy. Naloxone is an opioid and inhibits TLR4-ligand association. In this study, we investigated the relation of TLR4 and downstream pathways with immune-check point VISTA in pancreatic cancer proliferation. We initially collected pancreatic cancer-related datasets using the GEPIA2 and UALCAN databases. Based on this data obtained the effect of various concentrations and incubation times of naloxone were used on PANC-1 cells proliferation. A combination of naloxone and VISTA-siRNA were applied, and the effect of both naloxone and combined treatment on TLR4, Interleukin 1 receptor associated kinase 4 (IRAK4) and VISTA gene expression were analyzed in pancreatic cancer cells. As a result of analysis with Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR), gene expression levels of TLR4, IRAK4 and VISTA were significantly suppressed and cell proliferation was significantly reduced. We found that administration of naloxone and VISTA-siRNA in combination with PDAC cells suppressed signaling. Therefore, we considered that the relationship between VISTA and TLR4 signaling pathways and the other possible associated signal molecules may be an important marker in determining the response of immune checkpoint inhibitors in cancer treatment.

Melatonin affects hypoxia-inducible factor 1α and ameliorates delayed brain injury following subarachnoid hemorrhage via H19/miR-675/HIF1A/TLR4

ABSTRACT This study aimed to investigate the molecular mechanism of how melatonin (MT) interferes with hypoxia-inducible factor 1α (HIF1A) and toll-like receptor 4 (TLR4) expression, which is implicated in the management of delayed brain injury (DBI) after subarachnoid hemorrhage (SAH). Luciferase assay, real-time PCR, Western-blot analysis and immunohistochemistry (IHC) assays were utilized to explore the interaction among H19, miR-675, HIF1A and TLR4, and to evaluate the effect of MT on the expression of above transcripts in different groups. MT enhanced H19 expression by promoting the transcription efficiency of H19 promoter, and HIF1A was identified as a target of miR-675. HIF1A enhanced TLR4 expression via promoting the transcription efficiency of TLR4 promoter. Furthermore, administration of MT up-regulated miR-675 but suppressed the expressions of HIF1A and TLR4. Treatment with MT alleviated neurobehavioral deficits and apoptosis induced by SAH. According to the result of IHC, HIF1A and TLR4 protein levels in the SAH group were much higher than those in the SAH+MT group. Therefore, the administration of MT increased the levels of H19 and miR-675 which have been inhibited by SAH. In a similar way, treatment with MT decreased the levels of HIF1A and TLR4 which have been enhanced by SAH. MT could down-regulate the expression of HIF1A and TLR4 via the H19/miR-675/HIF1A/TLR4 signaling pathway, while TLR4 is crucial to the release of pro-inflammatory cytokines. Therefore, the treatment with MT could ameliorate post-SAH DBI.Running title: Melatonin ameliorates post-SAH DBI via H19/miR-675/HIF1A/TLR4 signaling pathways

Active components of Descurainia sophia improve lung permeability in rats with allergic asthma by regulating airway inflammation and epithelial damage

The present study investigated the effect of active components of Descurainia sophia on allergic asthma and explored the underlying mechanism. SD male rats were randomly divided into a normal group(NC), a model group(M), a D. sophia decoction group(DS), a D. sophia fatty oil group(FO), a D. sophia flavonoid glycoside group(FG), a D. sophia oligosaccharide group(Oli), and a positive drug dexamethasone group(Y). The allergic asthma model was induced in rats by intraperitoneal injection of ovalbumin(OVA) and aluminum hydroxide gel adjuvant(sensitization) and atomization of OVA solution(excitation). After modeling, asthma-related indicators, tracheal phenol red excretion, inflammatory cell levels in the peripheral blood, lung permeability index(LPI), and oxygenation index(OI) of rats were detected. The pathological changes of lung tissues were observed by HE staining. Enzyme-linked immunosorbent assay(ELISA) was used to detect the content of inflammatory factors immunoglobulin E(IgE), interleukin-4(IL-4), and interferon-γ(IFN-γ) in the bronchoalveolar lavage fluid(BALF) and the content of endothelin-1(ET-1) and angiotensin-converting enzyme(ACE) in lung tissue homogenate. The serum content of nitric oxide(NO) was detected by colorimetry. Western blot was employed to determine the protein expression of Toll-like receptor 4(TLR4), nuclear factor κB-p65(NF-κB-p65), phosphorylated NF-κB-p65(p-NF-κB-p65), myosin light chain kinase(MLCK), vascular endothelial cadherin(VE cadherin), connexin 43, and claudin 5, and the mechanism of active components of D. sophia on allergic asthma was explored. As revealed by the results, the M group showed extensive infiltration of inflammatory cells around the bronchus of the lung tissues of the allergic asthma rats, thickened bronchial wall, severely deformed alveolar structure, increased number of wheezes, the content of IgE, IL-4, ET-1, and ACE, inflammatory cells, and LPI, and reduced latency of asthma, tracheal phenol red excretion, IFN-γ, NO content, and OI. After the intervention of the active components of D. sophia, the DS, FO, FG, Oli, and Y groups showed improved asthma-related indicators, tracheal phenol red excretion, and lung tissue lesions in allergic asthma rats, and the effects in the FO and Oli groups were superior. The content of inflammatory factors in BALF was recovered in the DS, FO, and Y groups and the FG and Oli groups. The number of inflammatory cells in rats was reduced in the DS and FO groups, and the FG, Oli, and Y groups to varying degrees, and the effect in the FO group was superior. DS, FO, Oli, and Y reduced ET-1, ACE, and LPI and increased NO and OI. FG recovered NO, ET-1, ACE, LPI, and OI to improve lung epithelial damage and permeability. Further investigation of inflammation-related TLR4/NF-κB pathways, MLCK, and related skeleton protein levels showed that TLR4, NF-κB-p65, p-NF-κB-p65, and MLCK levels were increased, and VE cadherin, connexin 43, and claudin 5 were reduced in the M group. DS, FO, FG, Oli, and Y could reduce the protein expression related to the TLR4 pathway to varying degrees, and regulate the protein expression of MLCK, VE cadherin, connexin 43, and claudin 5. It is inferred that the active components of D. sophia improve lung permeability in rats with allergic asthma presumedly by regulating the TLR4/NF-κB signaling pathway to improve airway inflammation, mediating MLCK and connexin, and regulating epithelial damage.

Effect of paeoniflorin on distal survival of random flaps

BackgroundDistal ischemic necrosisis a common complication of orthopedic random skin flaps surgery. Paeoniflorin, a natural compound extracted from Paeonia lactiflora, can enhances angiogenesis and alleviates excessive inflammatory response. We investigated the changes of ischemic extra-long flaps with paeoniflorin and its possible mechanism. MethodsWe raised dorsal McFarlane flaps in 54 Sprague-Dawley rats. We designed three groups of rats: high-paeoniflorin group (HP, 50 mg/kg/d), low-paeoniflorin group (LP, 20 mg/kg/d), and control group. The flap survival rate was calculated, seven days after flap construction.Blood perfusion was detected by laser Doppler flow imaging, and angiogenesis wasdetected by Lead oxide/gelatin angiography.Oxidative stress levels of flaps were determined by detecting superoxide dismutase (SOD) and malondialdehyde (MDA). The histopathological status of flap was evaluated by hematoxylin and eosin (H&E) staining.Immunohistochemistry was used to determine the expression of high mobility group protein B1 (HMGB1), nuclear factor-kappa B (NF-κB), Toll-like receptor 4 (TLR4), tumor necrosis factor-α (TNF-α), interleukin (IL)-6, IL-1β, IL-18, vascular endothelial growth factor (VEGF), cysteine protease-1 (caspase-1) and NLPR3. ResultsThe flap survival rates and SOD activity in the experimental groups were significantly higher, while MDA activity was lower. Experimental groups showed significantly improved microcirculatory blood flow to the flap and increased angiogenesis. Immunohistochemistry revealed that paeoniflorin was associated with significantly increased VEGF expression, and decreased level of HMGB1, TLR4, TNF-α, NF-κB, IL-6, IL-1β, caspase-1, NLPR3, and IL-18. ConclusionsPaeoniflorin effectively enhanced the survival of rat random skin flaps by promoting vascular hyperplasia, inhibiting pyroptosis, and down-regulating inflammation.

Clinical significance of high mobility group box 1/toll-like receptor 4 in obese diabetic patients.

High mobility group box 1 (HMGB1) is an alarmin that may link to obesity and type 2 diabetes mellitus (T2DM). The present study analyzed the correlation between HMGB1/ Toll-like receptor 4 (TLR4) and certain biochemical parameters in obese (OB) diabetic patients. 40 normal glucose tolerant subjects (NGT) and 40 patients with newly diagnosed T2DM were enrolled. All patients were further divided into non-obese NGT (NGT-NOB), obese NGT (NGT-OB), non-obese T2DM (T2DM-NOB) and obese T2DM (T2DM-OB) groups according to body mass index (BMI).The levels of HMGB1 in serum were quantified using ELISA, whereas the mRNA expression levels of TLR4 in peripheral blood mononuclear cells were assessed using reverse transcription-quantitative PCR. The results suggested that the levels of HMGB1 and TLR4 were higher in NGT-OB and T2DM-NOB groups compared with those in NGT-NOB group. Similarly, the levels of these two markers were higher in T2DM-OB group compared with those in NGT-OB group. Correlation analysis indicated that the levels of HMGB1 and TLR4 were positively correlated with triglyceride (TG), fasting plasma glucose (FPG) levels and BMI, whereas a negative correlation between HMGB1 and high density lipoprotein (HDL) was noted. Linear regression analysis suggested that HMGB1 was associated with FPG and TG levels, whereas TLR4 was strongly associated with TG levels and BMI. The results demonstrated that the expression levels of HMGB1 and TLR4 in patients with T2DM or obesity were increased, which were associated with glycolipid metabolism disorders. Therefore, the HMGB1/TLR4 may serve a role in inflammatory process associated with obesity and T2DM.

Effects of Modified Duhuo Jisheng Decoction Combined with Arthroscopic Surgery on Bone Metabolism, Oxidative Stress, and Serum TLR4 and TGF-β1 in Patients with Knee Osteoarthritis.

Objective To analyze the effects of modified Duhuo Jisheng Decoction combined with arthroscopic surgery on bone metabolism, oxidative stress, and serum TLR4 and TGF-β1 in patients with knee osteoarthritis (KOA). Methods Prospectively select 82 patients with KOA from January 2020 to January 2022 in our hospital and divide them into the control group and observation group according to the random number table method, with 41 patients in each group. The control group was treated with arthroscopic surgery alone and routine anti-infection after operation. The observation group was treated with Duhuo Jisheng Decoction on the basis of the treatment of the control group. The patients in the two groups were treated continuously for 4 weeks. The improvement of patients' symptoms was evaluated by the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC). Before treatment and 4 weeks after treatment, the scores of traditional Chinese medicine (TCM) symptoms, bone metabolism indicators (cartilage oligomeric matrix protein (COMP), collagen type II carboxy terminal peptide (ctx-II), and matrix metalloproteinase-3 (MMP-3)), oxidative stress indicators (superoxide dismutase (SOD), glutathione peroxidase (GSHPx), malondialdehyde (MDA), nitric oxide (NO)), serum Toll-like receptor 4 (TLR4), and transforming growth factor β (TGF-β) level were compared between the two groups. Results After treatment, the WOMAC score of the two groups decreased (42.45 ± 10.83) in the observation group and (67.81 ± 14.63) in the control group. The WOMAC score of the observation group was lower than that of the control group (P < 0.05). After treatment, the levels of COMP, CTX-II, and MMP-3 in the two groups decreased, and the levels of COMP, CTX-II, and MMP-3 in the observation group were lower than those in the control group (P < 0.05). After treatment, the levels of SOD and GSHPx increased, while the levels of MDA and NO decreased in the two groups. The levels of SOD and GSHPx in the observation group were higher than those in the control group, while the levels of MDA and NO were lower than those in the control group (P < 0.05). After treatment, the TLR4 level in the observation group was lower than that of the control group, and the level of TGF-β in the observation group was higher than that of the control group (P < 0.05). Conclusion Compared with arthroscopic surgery alone, combined with modified Duhuo Jisheng Decoction can better alleviate the clinical symptoms of patients with KOA, improve their bone metabolism, oxidative stress indicators, and serum TLR4 and TGF-β 1 level, and reduce the inflammatory injury of knee joint.

KDM2B overexpression prevents myocardial ischemia-reperfusion injury in rats through regulating inflammatory response via the TLR4/NF-κB p65 axis.

Histone modifier lysine-specific demethylase 2B (KDM2B) has been previously reported to activate the inflammatory response by transcription initiation of the IL-6 gene. However, the effects of KDM2B on the inflammatory response during myocardial ischemia-reperfusion (I/R) injury and corresponding mechanisms remain poorly understood. The present study aimed to investigate the role and mechanism of KDM2B in myocardial I/R injury. Therefore, a myocardial I/R injury model was established in rats through coronary artery ligation. Adeno-associated virus-encoding KDM2B and small interfering RNA-KDM2B were designed to determine the effects of KDM2B on myocardial I/R injury using H&E staining and a TUNEL assay in the myocardial tissues. Reverse transcription-quantitative PCR and western blotting were performed to detect the mRNA and protein expression levels of KDM2B, toll-like receptor 4 (TLR4), NF-κB p65 and NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3). ELISA was used to detect the levels of TNF-α, IL-6 and IL-1β in the peripheral blood samples. Pathological analysis demonstrated that the cells in the model group were disordered, with a large area of necrosis and neutrophil infiltration. Knocking down KDM2B expression significantly upregulated the mRNA and protein expression levels of TLR4, NLRP3, NF-κB p65 and the ratio of phosphorylated (p)-p65 to p65. KDM2B knockdown also significantly increased the levels of IL-1β, IL-6 and TNF-α in the peripheral blood, which aggravated myocardial injury and promoted the apoptosis of myocardial cells. However, overexpression of KDM2B downregulated the mRNA and protein expression levels of TLR4, NLRP3, NF-κB P65, the ratio of p-p65 to p65 whilst reducing the levels of IL-1β, IL-6 and TNF-α in the peripheral blood, which markedly improved myocardial injury and significantly inhibited the apoptosis of cells in myocardial tissue. In conclusion, the results indicated that overexpression of KDM2B may prevent myocardial I/R injury in rats by reducing the inflammatory response through regulation of the TLR4/NF-κB p65 axis.