MyD88 mRNA Research Articles

Curcumin attenuated neuroinflammation via the TLR4/MyD88/NF-κB signaling way in the juvenile rat hippocampus following kainic acid-induced epileptic seizures.

The study examined curcumin's impart on relieving neuroinflammation of juvenile rats in kainic acid (KA) induced epileptic seizures by inhibiting the TLR4/MyD88/NF-κB pathway. There were five groups: control, KA, KA + curcumin (KC), KA + oxcarbazepine (OXC) (KO), KA + curcumin + OXC (KCO) groups. KA was stereotactically injected into right hippocampus following intraperitoneal injection of curcumin or (and) OXC for seven days. The rats in the above groups were randomly divided into three subgroups (at 6h, 24h, and 72h of KA administration) following the seizure degree assessed. The number of NeuN (+) neurons and GFAP (+) astrocytes was counted. The gene and protein levels of TLR4, MyD88, and NF-κB were detected. Compared with the KA group, the seizure latency was longer, and the incidence of status epilepticus (SE) was lower in the KC, KO, and KCO groups. The most significant changes were in the KCO group. At 72h following KA injected, the number of neurons was the least, and the number of astrocytes was the most in the KA group. The number of neurons was the most and the number of astrocytes was the least in the KCO group. At 24h, the mRNA and protein levels of TLR4, MyD88, and NF-κB in the KA group were the most. The above valves were the least in the KCO group. Therefore, curcumin could enhance anti-epileptic effect of OXC, protect injured neurons and reduce proliferated glial cells of the hippocampus of epileptic rats by inhibiting inflammation via the TLR4/MyD88/NF-κB pathway.

Minthostachys verticillata essential oil modulates cytokine synthesis and Staphylococcus aureus internalization in MAC-T cells at least through TLR4/MyD88/NFkB pathway.

The aim of this study was to evaluate the activation pathway(s) triggered by Minthostachys verticillata essential oil (EO) in bovine mammary epithelial cells (MAC-T) challenged with a strain of bovine Staphylococcus aureus. MAC-T cells were stimulated with EO, S. aureus or pre-treated with EO and then challenged with S. aureus. Cytokine's release was measured by ELISA. The mRNA for TLR2, TLR4, NOD2, MyD88 and NFκB was quantified by RT-qPCR. S. aureus adherence and internalization was also evaluated. MAC-T cells stimulated with S. aureus synthesized high levels of IL-1ß and IL-6 were kept up to 48 h, while IL-4 levels were not altered.Cells pre-treated with EO for 2 and 6h and then challenged with S. aureus showed a significant increase of IL-1β and IL-6. However, in these cells, a decrease in IL-1ß and IL-6 levels and an increase of IL-4 values was observed from 24 h. No significant increase in the expression levels of TLR2 or NOD2 was detected in all stimulated cells. However, the expression of TLR4, MyD88 and NFκB was increased in cells stimulated with S. aureus at 2 and 6 h as well as in cells pre-treated with EO between 2 and 6h and then challenged with S. aureus. The NFκB expression levels was similar to control at 24h in all stimulated cells, although pro-inflammatory cytokine levels and TLR4 and MyD88 expression levels remained high in cells stimulated with S. aureus. This results suggested the activation of other pathways independent of MyD88 by the pathogen that involucrated the activation of others transcription factors. Pre-treatment with EO during 2, 6 and 24h did not affect S. aureus adherence but decreased its internalization. In conclusion, pre-treatment with EO increased the IL-1β and IL-6 synthesis during the first hours post-challenged with S. aureus up-regulating TLR4/MyD88/NFκB pathway. Furthermore, EO increased the IL-4 levels from 6 to 24 h down-regulating the NFκB and possibly other transcription factors activated by the pathogen, which decreased its internalization into MAC-T cells.

MiR-21 attenuated inflammation targeting MyD88 in human chondrocytes stimulated with Hyaluronan oligosaccharides

Inflammation is the body's response to injuries, which depends on numerous regulatory factors. Among them, miRNAs have gained much attention for their role in regulating inflammatory gene expression at multiple levels. In particular, miR-21 is up-regulated during the inflammatory response and reported to be involved in the resolution of inflammation by down-regulating pro-inflammatory mediators, including MyD88. Herein, we evaluated the regulatory effects of miR-21 on the TLR-4/MyD88 pathway in an in vitro model of 6-mer HA oligosaccharides-induced inflammation in human chondrocytes.The exposition of chondrocytes to 6-mer HA induced the activation of the TLR4/MyD88 pathway, which culminates in NF-kB activation. Changes in miR-21, TLR-4, MyD88, NLRP3 inflammasome, IL-29, Caspase1, MMP-9, iNOS, and COX-2 mRNA expression of 6-mer HA-stimulated chondrocytes were examined by qRT-PCR. Protein amounts of TLR-4, MyD88, NLRP3 inflammasome, p-ERK1/2, p-AKT, IL-29, caspase1, MMP-9, p-NK-kB p65 subunit, and IKB-a have been evaluated by ELISA kits. NO and PGE2 levels have been assayed by colorimetric and ELISA kits, respectively.HA oligosaccharides induced a significant increase in the expression of the above parameters, including NF-kB activity. The use of a miR-21 mimic attenuated MyD88 expression levels and the downstream effectors. On the contrary, treatment with a miR-21 inhibitor induced opposite effects. Interestingly, the use of a MyD88 siRNA confirmed MyD88 as the target of miR-21 action.Our results suggest that miR-21 expression could increase in an attempt to reduce the inflammatory response, targeting MyD88.

Investigation the mechanism of iron overload-induced colonic inflammation following ferric citrate exposure

Iron overload occurs due to excessive iron intake compared to the body’s demand, leading to iron deposition and impairment of multiple organ functions. Our previous study demonstrated that chronic oral administration of ferric citrate (FC) caused colonic inflammatory injury. However, the precise mechanism underlying this inflammatory response remains unclear. The current study aims to investigate the mechanism by which iron overload induced by FC exposure leads to colonic inflammation. To accomplish this, mice were orally exposed to three different concentrations of FC (71 mg/kg/bw (L), 143 mg/kg/bw (M) and 286 mg/kg/bw (H)) for continuous 16 weeks, with the control group receiving ultrapure water (C). Exposure to FC caused disturbances in the excretory system, altered colonic flora alpha diversity, and enriched pathogenic bacteria, such as Mucispirillum, Helicobacter, Desulfovibrio, and Shigella. These changes led to structural disorders of the colonic flora and an inflammatory response phenotype characterized by inflammatory cells infiltration, atrophy of intestinal glands, and irregular thickening of the intestinal wall. Mechanistic studies revealed that FC-exposure activated the NF-κB signaling pathway by up-regulating TLR4, MyD88, and NF-κB mRNA levels and protein expression. This activation resulted in increased production of pro-inflammatory cytokines, further contributing to the colonic inflammation. Additionally, in vitro experiments in SW480 cells confirmed the activation of NF-κB signaling pathway by FC exposure, consistent with the in vivo findings. The significance of this study lies in its elucidation of the mechanism by which iron overload caused by FC exposure leads to colonic inflammation. By identifying the role of pathogenic bacteria and the NF-κB signaling pathway, this study could potentially offer a crucial theoretical foundation for the research on iron overload, as well as provide valuable insights for clinical iron supplementation.

Effects of chronic Cr and Ni co-exposure on liver inflammation and autophagy in mice by regulating the TLR4/mTOR pathway

Exposure to Cr and/or Ni can have widespread implications on the environment and health. However, the specific toxic effects of chronic Cr and Ni co-exposure on mice liver have not been reported. To ascertain the combined toxic effects of chronic Cr and Ni co-exposure on liver damage in mice, 80 6-week-old female C57BL/6 J mice were randomly divided into 4 groups: the Con group, Cr group (Cr+6 50 mg/L), Ni group (Ni+2 110 mg/L), and Cr + Ni group (Cr+6 50 mg/L + Ni+2 110 mg/L). The trial period lasted for 16 weeks. The results showed that Cr+6 and/or Ni+2 increased liver weight and liver index (P < 0.05) in mice, caused histological abnormality and ultrastructural damage, and micronutrients imbalance in mice liver. These findings serve as the basis for subsequent experiments. Compared with the individual exposure group, chronic Cr and Ni co-exposure resulted in decreased levels and activities of ALT, AST, MDA, T-AOC, and T-SOD (P < 0.05) in liver tissue, and decreased the mRNA expression levels of the TLR4/mTOR pathway related factors (TLR4, TRAM, TRIF, TBK-1, IRF-3, MyD88, IRAK-4, TRAF6, TAK-1, IKKβ, NF-κB, IL-1β, IL-6, TNFα, ULK1, Beclin 1, LC3) (P < 0.05) and decreased the protein expression levels of the factors (TLR4, MyD88, TRAF6, NF-κB p50, IL-6, TNFα, ULK1, LC3II/LC3I) (P < 0.05). Moreover, factorial analysis revealed the interaction between Cr and Ni, which was manifested as antagonistic effects on Cr concentration, Ni concentration, and TLR4, MyD88, NF-κB, mTOR, LC3, and p62 mRNA expression levels. In conclusion, the TLR4/mTOR pathway as a mechanism through which chronic Cr and Ni co-exposure induce liver inflammation and autophagy in mice, and there was an antagonistic effect between Cr and Ni. The above results provided a theoretical basis for understanding the underlying processes.

Three Artemisia pollens trigger the onset of allergic rhinitis via TLR4/MyD88 signaling pathway.

The prevalence of allergic rhinitis is high, making it a relatively common chronic condition. Countless patients suffer from seasonal Allergic rhinitis (AR). The objective of this investigation is to examine the potential involvement of common pollen allergens in seasonal allergic rhinitis, and study the proposed mechanism of Toll-like receptor 4 (TLR4)/Myeloid differentiation primary response gene 88 (MyD88) signaling pathway in the induction of AR. A mouse AR model (sensitized group) was constructed with pollen extracts and ovalbumin(OVA) of Artemisia annua (An), Artemisia argyi (Ar) and Artemisia Sieversiana (Si), and thereafter, AR symptom score was performed. After successful modeling, mouse serum and nasal mucosa tissues were extracted for subsequent experiments. The expression levels of immunoglobulin E (IgE), Interleukin (IL)-4, IL-5, IL-13 and Tumor Necrosis Factor-α (TNF-α) in serum were detected using Enzyme-linked immunosorbent assay (ELISA); Hematoxylin-eosin (H&E) staining methods were used to observe the pathological changes of the nasal mucosal tissue; Utilizing immunohistochemistry (IHC) staining, the expression levels of TLR4, MyD88 and Nuclear factor kappa B (NF-κB) p65 in mouse nasal mucosa were quantified; The mRNA and protein expression levels of TLR4, MyD88 and NF-κB p65 in nasal mucosa of sensitized mice were detected with Quantitative reverse transcription PCR (qRT-PCR) and Western Blot. Finally, the in vitro culture of Human nasal mucosal epithelial cells (HNEpC) cells was conducted, and cells were treated with 200µg/ml Artemisia annua pollen extractand OVA for 24h. Western Blot assay was used to detect the expression level of TLR4, MyD88 and NF-κB p65 proteins before and after HNEpC cells were treated with MyD88 inhibitor ST-2825. On the second day after AR stimulation, the mice showed obvious AR symptoms. H&E results showed that compared to the control group, the nasal mucosal tissue in the sensitized group was significantly more inflamed. Furthermore, ELISA assay showed increased expression levels of IgE, IL-4, IL-5, IL-13 and TNF-α in serum of mice induced by OVA and Artemisia annua pollen, Artemisia argyi pollen and Artemisia Sieversiana pollen than those of the control group. However, the expression level of IL-2 was lower than that of the control group (P < 0.05). Using Immunohistochemistry staining visually observed the expression levels of TLR4, MyD88 and NF-κB p65 in mouse nasal mucosa tissues and quantitatively analyzed. The expression levels of TLR4, MyD88 and NF-κB p65 in the sensitized group were higher than those in the control group, and the differences were statistically significant (P < 0.05). The results from qRT-PCR and Western Blot showed that the mRNA and protein expression levels of TLR4, MyD88 and NF-κB p65 in nasal mucosa of the sensitized group were significantly higher than those in the control group (P < 0.05). Finally, HNEpC cells were cultured in vitro and analyzed using Western Blot. The expression levels of TLR4, MyD88 and NF-κB p65 in OVA and An groups were significantly increased (P < 0.05). After ST-2825 treatment, TLR4 protein expression was significantly increased (P < 0.05) and MyD88 and NF-κB p65 protein expression were significantly decreased (P < 0.05). To sum up, the occurrence and development of AR induced by OVA and pollen of Artemisia annua, Artemisia argyi and Artemisia Sieversiana were related to TLR4/MyD88 signal pathway.

Pleiotropic immunoregulation by growth-blocking peptide in Ostrinia furnacalis.

Insects rely on their innate immune system to eliminate pathogenic microbes. As a system component, cytokines transmit intercellular signals to control immune responses. Growth-blocking peptide (GBP) is a member of the stress-responsive peptide family of cytokines found in several orders of insects, including Drosophila. However, the physiological role of GBP in defence against pathogens is not thoroughly understood. In this study, we explored the functions of GBP in a lepidopteran pest, Ostrinia furnacalis. Injection of recombinant O. furnacalis GBP (OfGBP) precursor (proGBP) and chemically synthesised GBP significantly induced the transcription of antimicrobial peptides (AMPs) and other immunity-related genes including immune deficiency (IMD) and Dorsal. The level of OfGBP mRNA was upregulated after bacterial infection. Knockdown of OfGBP expression led to a decrease in IMD, Relish, MyD88 and Dorsal mRNA levels. OfGBP induced phenoloxidase activity and affected hemocyte behaviours in O. furnacalis larvae. In summary, GBP is a potent cytokine, effectively regulating AMP synthesis, melanization response and cellular immunity to eliminate invading pathogens.

Taohong Siwu decoction ameliorates atherosclerosis in rats possibly through toll-like receptor 4/myeloid differentiation primary response protein 88/nuclear factor-κB signal pathway.

To investigate the effect of Taohong Siwu decoction (, TSD) on atherosclerosis in rats as well as investigate the underlying mechanism based on molecular docking. Sixty healthy male Sprague-Dawley rats were randomly divided into 6 groups with 10 rats in each group: control group, model group, atorvastatin group (AT, 2.0 mg/kg), and TSD groups (20, 10, 5 g/kg) after 7 d of acclimation. The model of atherosclerosis was successfully established except the control group by high fat diet (HFD) and vitamin D2. Biochemical analyzers were used to detect the levels of triglyceride (TG), total cholestero (TC), low density lipoprotein-cholesterol (LDL-C) and high density lipid-cholesterol (HDL-C) in blood lipid. The levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) were determined by enzyme-linked immunosorbent assay. Sudan IV staining and Hematoxylin and eosin staining (HE staining) were performed to observe the pathological changes in aortic tissue. Molecular docking technology was used to predict the best matching between the main components of TSD and the target proteins. The expression of target proteins was further detected by quantitative real time polymerase chain reaction (qRT-PCR) and Western blot analysis. The results showed that TSD restricted atherosclerosis development and decreased the inflammatory cytokines in plasma. Molecular docking results predicted that the main components of TSD showed a strong binding ability with toll-like receptor (TLR4), myeloid differentiation primary response protein 88 (MyD88), and nuclear factor kappa-B (NF-κB). The results of qRT-PCR and Western blot analysis showed that the mRNA and protein expressions of TLR4, MyD88 and NF-κB p65 in the aorta were reduced in atorvastatin group and TSD group. TSD can ameliorate atherosclerosis in rats, and the underlying mechanism is supposed be related to the suppression of inflammatory response by regulating TLR4/MyD88/NF-κB signal pathway.

Gastro-Intestinal Digested Bovine Milk Osteopontin Modulates Gut Barrier Biomarkers In Vitro.

Osteopontin (OPN) is a multifunctional protein naturally present in mammals' milk, associated with immune homeostasis and intestinal maturation. This study aims to investigate the protein digestion pattern and the cellular bioactivity of bovine milk OPN digesta in vitro. A modified INFOGEST static in vitro infant digestion protocol and a Caco-2/HT-29 co-culture cell model are employed to evaluate the digestion properties and the anti-inflammatory effects of OPN. OPN is resistant to gastric hydrolysis but degraded into large peptides during intestinal digestion. Its 10 kDa digesta permeate with predicted extensive bioactivities protects the co-culture cell model from the inflammation-induced dysfunction by dose-dependently recovering the expression of occludin, claudin-3, and ZO-1. Low dosage of OPN significantly decreases the production of IL-8 and IL-6, and downregulates the mRNA and protein expression of MyD88, NF-κB p65, and IκB-α, whereas a high dose evokes a mild pro-inflammatory response. Interestingly, anti-inflammatory effect of OPN digesta is stronger than lactoferrin and whey protein concentrate counterparts. The findings demonstrate that the bioactive peptides released from in vitro infant gastrointestinal digestion of bovine milk OPN alleviates intestinal epithelial cell inflammation by inhibiting NF-κB pathway activation and potentiates the barrier function of the intestinal epithelium.

Maillard-type glycated collagen with alginate oligosaccharide suppresses inflammation and oxidative stress by attenuating the expression of LPS receptors Tlr4 and Cd14 in macrophages.

Inflammation and oxidative stress contribute to noncommunicable diseases (NCDs), with macrophages playing pivotal roles. Glycated collagen through Maillard-type glycation holds promise for enhancing anti-inflammatory properties, but its mechanism remains unclear. This study investigates the cellular mechanism and aims to contribute to expanding collagen utilization. Collagen was glycated with alginate oligosaccharide (AO) and glucose (Glc: as a comparative case) at 60 °C and 35% relative humidity for up to 24 h (C-AO and C-Glc, respectively). The anti-inflammatory activities of both C-AO and C-Glc were evaluated using an LPS-stimulated macrophage model. 18 h AO-glycated collagen (C-AO18 h) was found to significantly reduce the production of nitric oxide and proinflammatory cytokines (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β). In contrast, C-Glc did not exhibit enhanced anti-inflammatory activity during any of the glycation periods. The enhanced anti-inflammatory activity of C-AO18 h was attributed to its downregulating effect on LPS receptors (toll-like receptor 4, Tlr4; cluster of differentiation 14, Cd14) and myeloid differentiation primary response 88 (Myd88) mRNA expression, with suppression in receptor expression resulting in decreased phagocytic ability of macrophages against E. coli. In addition, compared with intact collagen, C-AO18 h exhibited improved antioxidant activity in the LPS-stimulated macrophage model, as it significantly upregulated superoxide dismutase (SOD) and catalase (CAT) activities while reducing malondialdehyde (MDA) levels. Overall, this study contributes to the development of collagen-based functional foods for mitigating inflammation and oxidative stress in NCDs.

The Terminalia chebula retz extract treats hyperuricemic nephropathy by inhibiting TLR4/myd88/NF-κb axis

Ethnopharmacological relevanceHyperuricemic nephropathy (HN) is a renal injury caused by hyperuricemia and is the main cause of chronic kidney disease and end-stage renal disease. ShiWeiHeZiSan, which is composed mainly of components of Terminalia chebula Retz. And is recorded in the Four Medical Tantras, is a typical traditional Tibetan medicinal formula for renal diseases. Although T. chebula has been reported to improve renal dysfunction and reduce renal cell apoptosis, the specific mechanism of the nephroprotective effects of T. chebula on HN is still unclear. Aim of the studyThis study was conducted to evaluate the effects and specific mechanism of T. chebula extract on HN through network pharmacology and in vivo and in vitro experiments. Materials and methodsPotassium oxalate (1.5 g/kg) and adenine (50 mg/kg) were combined for oral administration to establish the HN rat model, and the effects of T. chebula extract on rats in the HN model were evaluated by renal function indices and histopathological examinations. UPLC-Q-Exactive Orbitrap/MS analysis was also conducted to investigate the chemical components of T. chebula extract, and the potential therapeutic targets of T. chebula in HN were predicted by network pharmacology analysis. Moreover, the activation of potential pathways and the expression of related mRNAs and proteins were further observed in HN model rats and uric acid-treated HK-2 cells. ResultsT. chebula treatment significantly decreased the serum uric acid (SUA), blood urea nitrogen (BUN) and serum creatinine (SCr) levels in HN rats and ameliorated renal pathological injury and fibrosis. A total of 25 chemical components in T. chebula extract were identified by UPLC-Q-Exactive Orbitrap/MS analysis, and network pharmacology analysis indicated that the NF-κB pathway was the potential pathway associated with the therapeutic effects of T. chebula extract on HN. RT‒PCR analysis, immunofluorescence staining and ELISA demonstrated that the mRNA and protein levels of TLR4 and MyD88 were significantly decreased in the renal tissue of HN rats after treatment with T. chebula extract at different concentrations, while the phosphorylation of P65 and the secretion of TNF-α and IL-6 were significantly inhibited. The results of in vitro experiments showed that T. chebula extract significantly decreased the protein levels of TLR4, MyD88, p-IκBα and p-P65 in uric acid-treated HK-2 cells and inhibited the nuclear translocation of p65 in these cells. In addition, the expression of inflammatory factors (IL-1β, IL-6 and TNF-α) and fibrotic genes (α-SMA and fibronectin) was significantly downregulated by T. chebula extract treatment, while E-cadherin expression was significantly upregulated. ConclusionT. chebula extract exerts nephroprotective effects on HN, such as anti-inflammatory effects and fibrosis improvement, by regulating the TLR4/MyD88/NF-κB axis, which supports the general use of T. chebula in the management of HN and other chronic kidney diseases.

Sophora flavescens-Angelica sinensis in the treatment of eczema by inhibiting TLR4/MyD88/NF-κB pathway

Ethnopharmacological relevanceSophora flavescens Ait.-Angelica sinensis(Oliv.) Diels drug pairing (SA) is a transformed drug pairing from Shengui pill, a traditional Chinese medicine prescription in the ninth volume of Traditional Chinese Medicine classic “Gu Jin Yi Jian”, which is famous for clearing heat, moistening dryness, and promoting blood circulation. It is commonly used in the treatment of eczema, a skin condition that causes itching and inflammation. Despite its widespread use, there is still limited research on the mechanism of how SA treats eczema. This paper aims to fill this gap by conducting animal experiments to uncover the mechanism behind SA's therapeutic effects on eczema. Our findings provide a solid foundation for the clinical use of this TCM prescription. Aim of the studyThe basic purpose of this study is to clarify the therapeutic mechanism of Sophora flavescens-Angelica sinensis (SA) in the treatment and control of eczema. Materials and methodsThe chemical compositions of SA were analyzed using HPLC-Q-Orbitrap-MS. In vivo, a mouse model of eczema was created, and the serum levels of TNF-α and IL-1β were quantified using an enzyme-linked immunosorbent assay (ELISA). Hematoxylin and eosin (HE) staining was performed to assess the pathological state of the mouse skin, and immunohistochemical technique (IHC) was employed to estimate the contents of TNF-α, TLR4, and NF-κB semi-quantitatively. The expression levels of TLR4, MyD88, and NF-κB mRNA were determined through real-time quantitative polymerase chain reaction (qRT-PCR). Western Blotting was utilized to identify the protein levels of TLR4, MyD88, and NF-κB in mouse skin tissue. ResultsSA identified 18 active chemicals, some of which were shown in vivo to inhibit the TLR4/MyD88/NF-κB signaling pathway while reducing serum levels of TNF-α and IL-1β, making them ideal agents for the treatment of eczema. ConclusionsSA's anti-inflammatory properties are attributed to its ability to reduce serum levels of TNF-α and IL-1β, likewise inhibit the TLR4/MyD88/NF-κB signaling pathway.

The role of TLR4/MyD88/NF-κB in the protective effect of ulinastatin on the intestinal mucosal barrier in mice with sepsis

ObjectiveTo investigate the effect of the TLR4/MyD88/NF-κB (Toll-like receptor 4/myeloid differentiation factor/nuclear factor kappa B) signalling pathway on the protective effect of ulinastatin on the intestinal mucosal barrier in mice with sepsis.MethodsA mouse model of sepsis was established by classical caecal ligation and perforation. Forty-four SPF C57BL/6 mice were randomly divided into the following four groups with 11 mice in each group: the control group (Con group), ulinastatin group (Uti group), Uti + LPS (lipopolysaccharide, LPS) group (Uti + LPS group) and LPS group. Mice in the Con group and Uti group received saline or ulinastatin injected 2 h after modelling; Mice in the Uti + LPS group received LPS injected 0 h after modelling, other procedures were the same as in the Uti group; Mice in the LPS group received LPS only. At 48 h after surgery, the levels of TNF-α (tumour necrosis factor-α, TNF-α), IL-6 (interleukin-6, IL-6) and IL-1β (interleukin-1β, IL-1β) in vein, and the expression of TLR4, MyD88 and NF-κB mRNA in small intestinal mucosa tissues using ELISA and RT‒PCR.ResultsThe pathological specimens showed increased inflammatory injury in the Con and LPS groups, while these injuries and changes improved in the Uti group. The scores of intestinal mucosal injury at 48 h of Uti injection were significantly lower than those of the Con group (P < 0.001), while the scores of intestinal mucosal injury of Uti + LPS were significantly higher than those of the Uti group (P = 0.044). The expression of TNF-α, IL-6 and IL-1β in the Uti decreased significantly at 48 h after surgery than that in the Con group (P = 0.001, P = 0.014, P = 0.004), while the expression of TNF-α, IL-6 and IL-1β in the Uti + LPS group increased significantly after surgery than that in the Uti group (P = 0.026, P = 0.040, P = 0.039). The expression of TLR4, MyD88 and NF-κB mRNA in the Uti group decreased significantly compared with that in the Con group (P = 0.001, P = 0.021, P = 0.007), while the expression of TLR4, MyD88 and NF-κB mRNA in the Uti + LPS group was higher than that in the Uti group (P = 0.023, P = 0.040, P = 0.045).ConclusionThese findings indicate that the protective effect of ulinastatin on the intestinal mucosal barrier against sepsis may be mediated through the TLR4/MyD88/NF-κB pathway.

Cordyceps militaris Solid Medium Extract Alleviates Lipoteichoic Acid-Induced MH-S Inflammation by Inhibiting TLR2/NF-κB/NLRP3 Pathways.

The aim of this study was to investigate the inhibitory effects of Cordyceps militaris solid medium extract (CME) and cordycepin (COR) on LTA-induced inflammation in MH-S cells and their mechanisms of action. In this study, the establishment of an LTA-induced MH-S inflammation model was determined, the CCK-8 method was used to determine the safe concentration range for a drug for COR and CME, the optimal concentration of COR and CME to exert anti-inflammatory effects was further selected, and the expression of inflammatory factors of TNF-α, IL-1β, IL-18, and IL-6 was detected using ELISA. The relative expression of TNF-α, IL-1β, IL-18, IL-6, IL-10, TLR2 and MyD88 mRNA was detected using RT-PCR, and the IL-1β, IL-18, TLR2, MyD88, NF-κB p-p65, NLRP3, pro-caspase-1, Caspase-1 and ASC protein expression in the cells were detected using Western blot; immunofluorescence assay detected the expression of Caspase-1 in MH-S cells. The results revealed that both CME and COR inhibited the levels of IL-1β, IL-18, IL-6, and TNF-α in the supernatants of LTA-induced MH-S cells and the mRNA expression levels of IL-1β, IL-18, IL-6, TNF-α, TLR2 and MyD88, down-regulated the LTA-induced IL-1β, IL-18, TLR2 in MH-S cells, MyD88, NF-κB p-p65/p65, NLRP3, ASC, pro-caspase-1, and caspase-1 protein expression levels, and inhibited LTA-induced caspase-1 activation in MH-S cells. In conclusion, CME can play a therapeutic role in LTA-induced inflammation in MH-S cells via TLR2/NF-κB/NLRP3, and may serve as a potential drug for bacterial pneumonia caused by Gram-positive bacteria.

Puerarin Alleviates Oxidized Oil-Induced Oxidative Injury and Inflammation via Inhibition of the Nrf2/Keap1 and HMGB1/TLR4/MAPK Signaling Pathways: An Investigation in a Chicken Model.

Puerarin has possessed a wide range of pharmacological activities. However, little is known about the protective effects of puerarin on the oxidized oil-induced injury. Here, the antioxidant and anti-inflammatory effects of puerarin are described using a chicken model. A total of 360 broilers are arranged in four treatments. Diets include two types of soybean oil (fresh or oxidized) and two levels of puerarin (0 or 750 mgkg-1 ). Results show that puerarin alleviates oxidized soybean oil-induced hepatic and thymic oxidative injury. This effect is observed by increasing the SOD activity and the expressions of Nrf2 signaling pathway-related genes and reducing the MDA content in the liver and thymus. Moreover, puerarin supplementation decreases the concentrations and mRNA levels of pro-inflammatory factors in the liver and thymus. The potential mechanism responsible for this is the decrease in the mRNA or protein levels of HMGB1, TLR4, MyD88, and p65 in the liver or thymus. Western blotting results indicate that puerarin also decreases the phosphorylation of JNK1/2, ERK1/2, and p38 in the liver and thymus. This study demonstrates puerarin may be a potential nutrient supplement in the treatment of oxidized oil-induced damage, and the Nrf2/Keap1 and HMGB1/TLR4/MAPK signaling pathways might be its important target.

Effects of Buyang Huanwu Decoction and Astragali Radix-Angelicae Sinensis Radix combination on inflammatory responses in atherosclerotic mice

The study aims to observe the effects and explore the mechanisms of Buyang Huanwu Decoction and Astragali Radix-Angelicae Sinensis Radix combination in the treatment of the inflammatory response of mice with atherosclerosis(AS) via the Toll-like receptor 4(TLR4)/myeloid differentiation primary response protein 88(MyD88)/nuclear factor-κB(NF-κB) signaling pathway. Male ApoE~(-/-) mice were randomly assigned into a model group, a Buyang Huanwu Decoction group, an Astragali Radix-Angelicae Sinensis Radix combination group, and an atorvastatin group, and male C57BL/6J mice of the same weeks old were used as the control group. Other groups except the control group were given high-fat diets for 12 weeks to establish the AS model, and drugs were administrated by gavage. Aortic intimal hyperplasia thickness, blood lipid level, plasma inflammatory cytokine levels, M1/M2 macrophage markers, and expression levels of proteins in TLR4/MyD88/NF-κB pathway in the vessel wall were measured to evaluate the effects of drugs on AS lesions and inflammatory responses. The results showed that the AS model was successfully established with the ApoE~(-/-) mice fed with high-fat diets. Compared with the control group, the model group showed elevated plasma total cholesterol(TC), triglyceride(TG), and low-density lipoprotein cholesterol(LDL-c) levels(P&lt;0.05), thickened intima(P&lt;0.01), and increased plasma tumor necrosis factor-α(TNF-α) and interleukin-6(IL-6) levels(P&lt;0.01). Moreover, the model group showed increased expression of vascular cell adhesion molecule-1(VCAM-1) and inducible nitric oxide synthase(iNOS)(P&lt;0.01), inhibited expression of endothelial nitric oxide synthase(eNOS) and cluster of differentiation 206(CD206)(P&lt;0.01), and up-regulated mRNA and protein levels of TLR4, MyD88, NF-κB inhibitor alpha(IκBα), and NF-κB in the vessel wall(P&lt;0.05). Compared with the model group, Buyang Huanwu Decoction and Astragali Radix-Angelicae Sinensis Radix combination lowered the plasma TC and LDL-c levels(P&lt;0.01), alleviated the intimal hyperplasia(P&lt;0.01), and reduced the plasma TNF-α and IL-6 levels(P&lt;0.05). Moreover, the two interventions promoted the expression of eNOS and CD206(P&lt;0.05), inhibited the expression of VCAM-1 and iNOS(P&lt;0.01), and down-regulated the mRNA and protein levels of TLR4, MyD88, IκBα, and NF-κB(P&lt;0.05) in the vessel wall. This study indicated that Buyang Huanwu Decoction and Astragali Radix-Angelicae Sinensis Radix combination could delay the progression of AS, inhibit the polarization of vascular wall macrophages toward M1 type, and attenuate vascular inflammatory response by inhibiting the activation of TLR4/MyD88/NF-κB signaling pathway in the vascular wall. Astragali Radix and Angelicae Sinensis Radix were the main pharmacological substances in Buyang Huanwu Decoction for alleviating the AS vascular inflammatory response.

Study on the correlation of the TLR4/MyD88 axis with the degree of inflammatory response in patients with synovitis of the knee joint.

This work aims to provide a novel reference for future diagnosis and treatment of synovitis of the knee joint (SKJ) by analyzing the correlation of the TLR4/MyD88 axis with the degree of inflammatory response in SKJ patients. First, this study retrospectively analyzed the clinical data of 46 SKJ patients (research group, RG) treated in our hospital from January 2021 to December 2022 and 52 concurrent healthy controls (control group, CG). Concentrations of TLR4, MyD88 and inflammatory factors (IFs) in peripheral blood were measured, and differences in TLR4 and MyD88 between groups were observed to explore the diagnostic performance of the two for SKJ. Additionally, the correlation of TLR4 and MyD88 with IFs and Western Ontario Mac Master (WOMAC) scores in SKJ patients was discussed. Through the above experiment, we found that TLR4 and MyD88 presented higher mRNA levels in RG than in CG (P<0.05), both of which had excellent diagnostic efficiency for SKJ. Pearson correlation coefficients identified a positive correlation of TLR4 and MyD88 mRNA with IFs and WOMAC scores (P<0.05). Therefore, The TLR4/MyD88 axis is activated in SKJ patients and is strongly related to the intensification of inflammatory responses.

MiR-15a-5p up-regulates TLR4 and induces the formation of hypertrophic scars and keloids.

The formation of hypertrophic scar and keloid is considered to be a very complex pathological process. Our previous studies have shown that miR-15a-5p is an important miRNA in HTS tissues, and its expression level is significantly increased. Therefore, the potential mechanism of action of miR-15a-5p in scarring arouses our interest. This study preliminarily investigated the expression level of miR-15a-5p in HTS tissue and normal skin tissue and further explored the molecular mechanism. The results of this study once again confirmed that the expression level of miR-15a-5p was increased in HTS tissues and cells, and the closely related mRNA and protein levels of MyD88 and TGF-β were also highly expressed. The relative expression levels of fibrosis-related indicators in HTsFb cells were up-regulated, such as collagen-Ⅰ, collagen-III and α-SMA. We constructed the HTS cell model and BALB/c nude animal model, and down-regulating miR-15a-5p, the HTsFb cells proliferation was inhibited, and qRT-PCR results showed that the fibrosis index mRNA was also reduced, and significantly reduce the pathological state of scar tissue. In conclusion, miR-15a-5p may participate in the formation and development of HTS through TLR/MyD88 signaling pathway and TGF-β1 signaling pathway.

Si Jun Zi decoction inhibits the growth of lung cancer by reducing the expression of PD-L1 through TLR4/MyD88/NF-κB pathway

Ethnopharmacological relevanceSi Jun Zi decoction (SJZT) is a traditional Chinese medicine (TCM) formula with the effect of invigorating the spleen qi and replenishing qi. TCM believes that a strong spleen qi helps to strengthen lung qi. Lung cancer is often caused by a deficiency of lung qi. Based on this theory, TCM often applies SJZT to the treatment of lung cancer and has achieved remarkable results. However, the mechanism of SJZT in the treatment of lung cancer remains unclear and requires further study. Aim of the studyThe main purpose of this study is to explore the mechanism of SJZT against lung cancer. Materials and methodsIn this study, the chemical constituents in SJZT were analyzed by UPLC-Q-Exactive-MS/MS. MTT and cell scratch test were used to determine the cell viability and inhibition of migration in vitro. The effect of SJZT on the expression of PD-L1 protein in A549 cells was detected by Western Blotting (WB). Apoptosis was detected by crystal violet staining. The mouse model of Lewis lung cancer was established in vivo, and the levels of serum TNF-α and IL-2 were detected by enzyme linked immunosorbent assay (ELISA). The protein levels of TLR4, MyD88, NF-κB and PD-L1 in tumor tissues of mice were detected by WB. Quantitative real-time PCR (qRT-PCR) was used to detect the levels of TLR4, MyD88, NF-κB and PD-L1 mRNA. Finally, hematoxylin and eosin (H&E) staining were used to detect the pathological status of tumor tissues in mice. ResultsA total of 16 active chemical constituents were identified in SJZT. In vitro experiments showed that SJZT could inhibit the growth of A549, induce apoptosis and reduce the expression of PD-L1. In vivo experiments showed that SJZT regulated TLR4/MyD88/NF-κB signaling pathway, decreased the expression of PD-L1, and inhibited tumor growth. ConclusionsSJZT inhibits the growth of lung cancer by regulating TLR4/MyD88/NF-κB signal pathway and reducing the expression of PD-L1.

Arsenic exposure induces small intestinal toxicity in mice by barrier damage and inflammation response via activating RhoA/ROCK and TLR4/Myd88/NF-κB signaling pathways

Numerous studies have shown that arsenic (As) is an important hazardous metalloid that is commonly considered to have systemic toxicity. The main pathway of arsenic exposure is oral; however, many of the events that occur during its passage through the gastrointestinal tract are unclear, and there are few reports on the effect of arsenic on small intestinal mucosal barrier. This study aimed to investigate arsenic-induced mucosal barrier damage in the small intestine of mice induced by oral exposure and its potential mechanisms. In the present study, histomorphometric and immunohistochemical analyses showed that arsenic-treated mice exhibited signs of irregularly arranged and atrophied small intestinal villi, reduced villus lengths, inflammatory cells infiltration, along with up-regulated expression of inflammatory factors TNF-α, IL-6 and IL-1β in the small intestine of mice. The myeloperoxidase (MPO) activity was also increased in As-exposed mice. Transmission electron microscopy (TEM) analysis demonstrated that intestinal epithelial tight junctions (TJs) were impaired in the small intestines of mice in As group. In addition, arsenic down-regulated mRNA levels of TJ-related genes (ZO-1, ZO-2, occludin, claudin-1, and claudin-7) and protein levels of ZO-1, occludin and claudin-1 were significantly reduced in arsenic-treated groups, while arsenic also increased levels of TLR4, Myd88, NF-κB, RhoA, and ROCK mRNA and protein expression. In summary, these results indicate that the small intestine toxicity in mice evoked by arsenic was correlated with the activation of TLR4/Myd88/NF-κB and RhoA/ROCK pathways.