Toll-like Receptor 4 Protein Expression Research Articles

Effects of moxibustion on intestinal barrier function and TLR4/NF-κB p65 signaling pathway in obese rats.

To observe the effects of moxibustion on intestinal barrier function and Toll-like receptor 4 (TLR4)/nuclear factor-κB p65 (NF-κB p65) signaling pathway in obese rats and explore the mechanism of moxibustion in the intervention of obesity. Fifty-five Wistar rats of SPF grade were randomly divided into a normal group (10 rats) and a modeling group (45 rats). In the modeling group, the obesity model was established by feeding high-fat diet. Thirty successfully-modeled rats were randomized into a model group, a moxibustion group, and a placebo-control group, with 10 rats in each one. In the moxibustion group, moxibustion was applied at the site 3 cm to 5 cm far from the surface of "Zhongwan" (CV 12), with the temperature maintained at (46±1 ) ℃. In the placebo-control group, moxibustion was applied at the site 8 cm to 10 cm far from "Zhongwan" (CV 12), with the temperature maintained at (38±1) ℃. The intervention was delivered once daily for 8 weeks in the above two groups. The body mass and food intake of the rats were observed before and after intervention in each group. Using ELISA methool, the levels of serum triacylglycerol (TG), total cholesterol (TC) and lipopolysaccharide (LPS) were detected and the insulin resistance index (HOMA-IR) was calculated. HE staining was used to observe the morphology of colon tissue. The mRNA expression of zonula occludens-1 (ZO-1), Occludin, Claudin-1, TLR4 and NF-κB p65 in the colon tissue was detected by quantitative real-time PCR; and the protein expression of ZO-1, Occludin, Claudin-1, TLR4 and NF-κB p65 was detected by Western blot in the rats of each group. Compared with the normal group, the body mass, food intake, the level of HOMA-IR, and the serum levels of TC, TG and LPS were increased in the rats of the model group (P<0.01); those indexes in the moxibustion group were all reduced when compared with the model group and the placebo-control group respectively (P<0.01, P<0.05). Compared with the normal group, a large number of epithelial cells in the mucosa of colon tissue was damaged, shed, and the inflammatory cells were infiltrated obviously in the interstitium in the rats of the model group. When compared with the model group, in the moxibustion group, the damage of the colon tissue was recovered to various degrees and there were few infiltrated inflammatory cells in the interstitium, while, the epithelial injury of the colon tissue was slightly recovered and the infiltrated inflammatory cells in the interstitium were still seen in the placebo-control group. The mRNA and protein expressions of ZO-1, Occludin and Caudin-1 were decreased in the model group compared with those in the normal group (P<0.01). When compared with the model group and the placebo-control group, the mRNA and protein expressions of these indexes were increased in the moxibustion group (P<0.01, P<0.05). In the model group, the mRNA and protein expressions of TLR4 and NF-κB p65 were increased when compared with those in the normal group (P<0.01), and the mRNA and protein expressions of these indexes were reduced in the moxibustion group when compared with those in the model group and the placebo-control group (P<0.01). Moxibustion can reduce the body mass and food intake, regulate the blood lipid and improve insulin resistance in the rats of obesity. It may be related to alleviating inflammatory response through improving intestinal barrier function and modulating the intestinal TLR4/NF-κB p65 signaling pathway.

Hyperglycaemia Aggravates Oxidised Low-Density Lipoprotein-Induced Schwann Cell Death via Hyperactivation of Toll-like Receptor 4.

Increased low-density lipoprotein levels are risk factors for diabetic neuropathy. Diabetes mellitus is associated with elevated metabolic stress, leading to oxidised low-density lipoprotein formation. Therefore, it is important to investigate the mechanisms underlying the pathogenesis of diabetic neuropathy in diabetes complicated by dyslipidaemia with increased levels of oxidised low-density lipoprotein. Here, we examined the effects of hyperglycaemia and oxidised low-density lipoprotein treatment on Schwann cell death and its underlying mechanisms. Immortalised mouse Schwann cells were treated with oxidised low-density lipoprotein under normo- or hyperglycaemic conditions. We observed that oxidised low-density lipoprotein-induced cell death increased under hyperglycaemic conditions compared with normoglycaemic conditions. Moreover, hyperglycaemia and oxidised low-density lipoprotein treatment synergistically upregulated the gene and protein expression of toll-like receptor 4. Pre-treatment with TAK-242, a selective toll-like receptor 4 signalling inhibitor, attenuated hyperglycaemia- and oxidised low-density lipoprotein-induced cell death and apoptotic caspase-3 pathway. Our findings suggest that the hyperactivation of toll-like receptor 4 signalling by hyperglycaemia and elevated oxidised low-density lipoprotein levels synergistically exacerbated diabetic neuropathy; thus, it can be a potential therapeutic target for diabetic neuropathy.

[Mechanism of Fushen Granules treat peritoneal dialysis-related peritonitis by regulating TLR4/NF-κB pathway:based on network pharmacology and animal experiments].

Network pharmacology was employed to probe into the mechanism of Fushen Granules in treating peritoneal dialysis-rela-ted peritonitis(PDRP) in rats. The main active components of Fushen Granules were searched against the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform, and their targets were predicted. PDRP-related targets were retrieved from DisGeNET and other databases. The common targets shared by the drug and the disease were identified by the online tool, and protein-protein interaction(PPI) network of the common targets. The obtained 276 common targets were imported into DAVID for GO function enrichment and KEGG pathway enrichment. The main signaling pathway of Fushen Granules in the treatment of PDRP was predicted as Toll-like receptor 4(TLR4)/nuclear factor(NF)-κB. The rat model of uremia was induced by 5/6 nephrectomy. From two weeks after operation, the rat model of peritoneal dialysis(PD) was established by intraperitoneal injection of 20 mL dialysate with 1.25% glucose every day. The sham operation group and model group received 2 mL normal saline by gavage every day. The rats in Fushen Gra-nules groups were administrated with 2 mL solutions of low-(0.54 g·kg~(-1)), medium-(1.08 g·kg~(-1)) and high-dose(2.16 g·kg~(-1)) Fushen Granules every day. The bifico group received 2 mL(113.4 mg·kg~(-1)) of bifico solution every day. At the end of the 8th week, the levels of serum creatinine(Scr) and blood urea nitrogen(BUN) in each group were measured. The serum levels of hypersensitive C reactive protein(hs-CRP), tumor necrosis factor(TNF)-α, and interleukin(IL)-6 were measured, and the pathological changes in the colon tissue were observed by hematoxylin-eosin(HE) staining. The serum levels of lipopolysaccharide(LPS) and lipopolysaccharide-binding protein(LBP) of rats were measured, and the expression levels of LBP, TLR4, NF-κB p65, inhibitor of κB kinase α(IκBα), TNF-α, and IL-1β in the colon tissue were determined. Compared with sham operation group, the model group had abnormal structure of all layers of colon tissue, sparse and shorter intestinal villi, visible edema in mucosal layer, wider gap, obvious local inflammatory cell infiltration, significantly decreased body weight(P&lt;0.01), and significantly increased kidney function index(Scr, BUN) content(P&lt;0.01). Serum levels of inflammatory cytokines(hs-CRP, TNF-α, IL-6), LPS and LBP were significantly increased(P&lt;0.01), protein expressions of LBP, TLR4, NF-κB p65, TNF-α and IL-1β were significantly increased(P&lt;0.01), and protein expressions of IκBα were significantly decreased(P&lt;0.01). Compared with model group, intestinal villi damage in colonic tissue of rats in low-, medium-and high-dose Fushen Granules groups and bifico group were alleviated to different degrees, edema in submucosa was alleviated, space was narrowed, and inflammatory cell infiltration in lamina propria was reduced. The contents of renal function index(Scr, BUN) and serum inflammatory factors(hs-CRP, TNF-α, IL-6) were significantly decreased(P&lt;0.05 or P&lt;0.01) in medium-and high-dose Fushen Granules groups and bifico group(P&lt;0.05 or P&lt;0.01). Serum LPS and LBP contents in Fushen Granules group and bifico group were significantly decreased(P&lt;0.01), protein expressions of LBP, TLR4, NF-κB p65, TNF-α and IL-1β in Fushen Granules group were significantly decreased(P&lt;0.05 or P&lt;0.01), and protein expressions of IκBα were significantly increased(P&lt;0.01). The expression of LBP protein in bifico group was significantly decreased(P&lt;0.01). The results suggest that Fushen Granules can protect the residual renal function of PD rats, reduce the inflammatory response, and protect the colon tissue. Based on network pharmacology, TLR4/NF-κB pathway may be the main signaling pathway of Fushen granule in the treatment of PDRP. The results showed that Fushen Granules could improve intestinal inflammation and protect intestinal barrier to prevent PDRP by regulating the expression of key factors in TLR4/NF-κB pathway in colon of PD rats.

Epstein-Barr virus suppresses N6-methyladenosine modification of TLR9 to promote immune evasion

Epstein-Barr virus (EBV) is a human tumor virus associated with a variety of malignancies, including nasopharyngeal carcinoma, gastric cancers, and B-cell lymphomas. N6-methyladenosine (m6A) modifications modulate a wide range of cellular processes and participate in the regulation of virus-host cell interactions. Here, we discovered that EBV infection downregulates Toll-like receptor 9 (TLR9) m6A modification levels and thus inhibits TLR9 expression. TLR9 has multiple m6A modification sites. Knockdown of METTL3, an m6A "writer", decreases TLR9 protein expression by inhibiting its mRNA stability. Mechanistically, Epstein-Barr nuclear antigen 1 (EBNA1) increases METTL3 protein degradation via K48-linked ubiquitin-proteasome pathway. Additionally, YTHDF1 was identified as an m6A "reader" of TLR9, enhancing TLR9 expression by promoting mRNA translation in an m6A -dependent manner, which suggests that EBV inhibits TLR9 translation by "hijacking" host m6A modification mechanism. Using the METTL3 inhibitor STM2457 inhibits TLR9-induced B cell proliferation and Ig secretion, and opposes TLR9-induced immune responses to assist tumor cell immune escape. In clinical lymphoma samples, the expression of METTL3, YTHDF1 and TLR9 was highly correlated with immune cells infiltration. This study reveals a novel mechanism that EBV represses the important innate immunity molecule TLR9 through modulating the host m6A modification system.

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.

Spinal HMGB1 participates in the early stages of paclitaxel-induced neuropathic pain via microglial TLR4 and RAGE activation.

Chemotherapy-induced neuropathic pain (CINP) is one of the main adverse effects of chemotherapy treatment. At the spinal level, CINP modulation involves glial cells that upregulate Toll-like receptor 4 (TLR4) and signaling pathways, which can be activated by pro-inflammatory mediators as the high mobility group box-1 (HMGB1). To evaluate the spinal role of HMGB1 in the paclitaxel-induced neuropathic pain via receptor for advanced glycation end products (RAGE) and TLR4 activation expressed in glial cells. Male C57BL/6 Wild type and TLR4 deficient mice were used in the paclitaxel-induced neuropathic pain model. The nociceptive threshold was measured using the von Frey filament test. In addition, recombinant HMGB1 was intrathecally (i.t.) injected to confirm its nociceptive potential. To evaluate the spinal participation of RAGE, TLR4, NF-kB, microglia, astrocytes, and MAPK p38 in HMGB1-mediated nociceptive effect during neuropathic pain and recombinant HMGB1-induced nociception, the drugs FPS-ZM1, LPS-RS, PDTC, minocycline, fluorocitrate, and SML0543 were respectively administrated by i.t. rout. Microglia, astrocytes, glial cells, RAGE, and TLR4 protein expression were analyzed by Western blot. ELISA immunoassay was also used to assess HMGB1, IL-1β, and TNF-α spinal levels. The pharmacological experiments demonstrated that spinal RAGE, TLR4, microglia, astrocytes, as well as MAPK p38 and NF-kB signaling are involved with HMGB1-induced nociception and paclitaxel-induced neuropathic pain. Furthermore, HMGB1 spinal levels were increased during the early stages of neuropathic pain and associated with RAGE, TLR4 and microglial activation. RAGE and TLR4 blockade decreased spinal levels of pro-inflammatory cytokines during neuropathic pain. Taken together, our findings indicate that HMGB1 may be released during the early stages of paclitaxel-induced neuropathic pain. This molecule activates RAGE and TLR4 receptors in spinal microglia, upregulating pro-inflammatory cytokines that may contribute to neuropathic pain.

Quercetin Protects Against Global Cerebral ischemia‒reperfusion Injury by Inhibiting Microglial Activation and Polarization.

This study aims to investigate the protective effect of quercetin against global cerebral ischemia‒reperfusion (GCI/R) injury in rats and elucidate the underlying mechanism. A GCI/R injury rat model was established using a four-vessel occlusion (4-VO) method. An oxygen-glucose deprivation/reoxygenation (OGD/R) injury model was induced in BV2 cells. The extent of injury was assessed by evaluating neurological deficit scores (NDS) and brain water content and conducting behavioral tests. Pathomorphological changes in the prefrontal cortex were examined. Additionally, the study measured the levels of inflammatory cytokines, the degree of microglial activation and polarization, and the protein expression of Toll-like receptor 4 (TLR4) and TIR-domain-containing adaptor inducing interferon-β (TRIF). Quercetin pretreatment significantly ameliorated neurological impairment, improved learning and memory abilities, and reduced anxiety in rats subjected to GCI/R injury. Furthermore, quercetin administration effectively mitigated neuronal injury and brain edema. Notably, it suppressed microglial activation and hindered polarization toward the M1 phenotype. Simultaneously, quercetin downregulated the expression of TLR4 and TRIF proteins and attenuated the release of IL-1β and TNF-α. This study highlights the novel therapeutic potential of quercetin in alleviating GCI/R injury. Quercetin demonstrates its neuroprotective effects by inhibiting neuroinflammation and microglial activation while impeding their transformation into the M1 phenotype through modulation of the TLR4/TRIF pathway.

Physical activity levels, gut permeability, circulating inflammatory cytokines and TLR4 signalling in PBMCs of COVID-19 patients

Physical exercise could confer some protection against the inflammatory cytokine storm induced by SARS-CoV-2 infection and against severe COVID-19 symptomatology. However, the mechanisms underlying this protective effect of exercise have not been elucidated yet. Therefore, the aim of this study was to investigate the activation of the inflammatory pathway in peripheral blood mononuclear cells (PBMCs) and to test a possible association between the regulation of this pathway and the expression of plasma inflammatory cytokine markers as well as with physical activity levels. All patients (n = 31; 21 males and 10 females) had at least three symptoms associated with COVID-19 and were recruited in the emergency unit. They were immediately tested for COVID-19 by polymerase chain reaction (COVID-19 n = 18, amongst which 9 were hospitalized and 9 quarantined; Non-COVID-19 n = 13) and a blood sample was collected. Physical activity levels were determined using the international physical activity questionnaire (IPAQ). No differences in physical activity levels nor sitting time were found between the COVID-19 and the Non-COVID-19 group. However, moderate to vigorous physical activity was lower in the hospitalized compared to the quarantined group. Toll-like receptor 4 (TLR4) and MyD88 protein expression in PBMCs was lower in the COVID-19 than in the Non-COVID-19 group. Moreover, TLR4 protein expression was lower in the hospitalized than in the quarantined subgroup. While no difference was observed in plasma zonulin levels between COVID-19 and Non-COVID-19 patients, quarantined patients had lower plasma zonulin levels compared to hospitalized patients. In conclusion, patients hospitalized with severe COVID-19 were less physically active and presented greater gut permeability and a downregulation of the TLR4 pathway in PBMCs compared to quarantined and Non-COVID-19 individuals. The lower expression of TLR4 could be a mechanism to prevent, or at least to mitigate, the inflammatory cytokine storm related to the severe form of COVID-19.

Study on the effect of ginaton on reducing cerebral vasospasm and early brain injury after hemorrhagic stroke by inhibiting inflammatory response

Objective: The objective of this study was to investigate the effects and possible mechanisms of action of ginseng on cerebral vasospasm and early brain injury (EBI) following hemorrhagic stroke. Materials and Methods: Sprague-Dawley (SD) rats (n = 48) were randomly divided into sham operation (sham group), subarachnoid hemorrhage (SAH) model (SAH group), normal saline (NS group), and Ginaton (Extract of Ginkgo Biloba Leaves Drops) intervention (gin group) groups. MCP-1 mRNA and tumor necrosis factor levels were detected using reverse transcription–polymerase chain reaction. The relative expression of mRNA was detected by Western blotting. Results: (1) Compared with the sham group, the SAH, NS, and gin groups had different degrees of neurological dysfunction. Compared with the SAH and NS groups, the neurological deficits in the gin group were significantly improved (P &lt; 0.05). (2) Compared with the sham group, the relative expression levels of MCP-1 mRNA in the SAH, NS, and gin groups were 5.1 ± 0.9, 3.4 ± 0.6, and 2.5 ± 0.4, respectively; the relative expression levels of mRNA were 13.3 ± 2.4, 11.2 ± 1.8, and 3.8 ± 0.6, respectively. (3) The apoptosis rates of brain tissue in the sham, SAH, NS, and gin groups were 4.8 ± 0.7, 54.2 ± 10.3, 50.1 ± 7.4, and 28.4 ± 4.5, respectively. (4) Western blot showed that the relative expression levels of toll-like receptor-4 (TLR-4) protein in the sham, SAH, NS, and gin groups were 0.29 ± 0.03, 0.87 ± 0.15, 0.65 ± 0.13, and 0.41 ± 0.17, respectively; the relative expression levels of B protein were 0.21 ± 0.04, 0.96 ± 0.14, 0.73 ± 0.18, and 0.30 ± 0.05, respectively. Gin treatment could inhibit TLR-4 and nuclear factor-κB (NF-κB) protein expression. Conclusions: Dona tablets may inhibit activation of the NF-κB signaling pathway, and SAH-induced inflammatory response, so as to reduce cerebral vasospasm and EBI.

Myeloid Differentiation Factor 2 Inhibition Protects Against Doxorubicin‐induced Microglial Activation and Senescence

AbstractBackgroundDoxorubicin, an anti‐tumor drug, generates neuroinflammation that leads to microglial senescence and chemo‐brain (1, 2). Toll‐like receptor 4 (TLR4) signaling pathway is mainly expressed in microglia and it plays a key role in developing doxorubicin‐induced neuroinflammation, microglial activation and chemo‐brain (3). Myeloid differentiation factor 2 (MD‐2) is known as an essential protein in activation of TLR4 signaling pathway (1). However, the effect of blocking TLR4 signaling via targeting MD‐2 on doxorubicin‐induced microglial activation and senescence has never been investigated.MethodThe human microglial clone 3 cell line (HMC3) was treated with three different doses of MD‐2 inhibitor (L6H21: 5μM, 10μM, 20μM) for 30 minutes, then with doxorubicin hydrochloride (DOX) (50nM) for 24 hours. After 24 hours treatment with L6H21 and DOX, the microglial cells were assessed for the TLR4 signaling, microglial activation, cell death and cellular senescence pathways.ResultDOX treatment activated TLR4 signaling pathway as well as microglia, as evidenced by significantly increased protein expressions of TLR4, MD‐2, tumor necrosis factor alpha and ionized calcium‐binding adapter molecule 1. In addition, DOX treatment induced microglial cell death and senescence, as shown by significantly increased protein expressions of Bcl‐2 associated X‐protein to B‐cell lymphoma 2 ratio and p16Ink4a. Treatment with L6H21 (5μM) attenuated not only TLR4 signaling along with microglial activation, but also apoptosis and cellular senescence in DOX‐treated microglia. L6H21 (10μM and 20μM) protected against TLR4 signaling along with microglial activation, apoptosis as well as cellular senescence in DOX‐treated microglia.ConclusionThese findings imply that MD‐2 inhibition could be the potential therapeutic strategy for attenuating DOX‐induced microglial activation and senescence.

Salvia miltiorrhiza Extract Prevents the Occurrence of Early Atherosclerosis in Apoe -/- Mice via TLR4/ NF-kB Pathway.

Salvia miltiorrhiza (SM) contains four major aqueous active ingredients, which have been isolated, purified and identified as danshensu (DSS), salvianolic acid A (Sal-A), salvianolic acid B (Sal-B) and protocatechuic aldehyde (PAL), A mixture of these four ingredients is called SABP. Although aqueous extract from Salvia miltiorrhiza has been traditionally used to treat cardiovascular diseases, the efficacy and function of the optimal ratio of SABP in preventing and treating cardiovascular diseases remain unknown. This study aims to explore the antiinflammatory mechanisms underlying the attenuation of atherosclerosis development by aqueous extract from Salvia miltiorrhiza. Male ApoE-/- mice (6 weeks) were randomly allocated into three groups: the model group (Model), the SABP group (SABP), and the rosuvastatin calcium group (RC). Male C57BL/6 mice (6 weeks) were used as a control group. All mice were fed with an ordinary diet. After 8 weeks of treatment, the lipid profiles in serum and the lactate dehydrogenase (LDH) and creatine kinase (CK) in heart tissue were measured using an automatic biochemical analyzer. Alterations of the thoracic aorta and the heart were assessed using Hematoxylin and eosin staining. The protein expression of Toll-like receptor 4 (TLR4), TGF beta-activated kinase 1 (TAK1), nuclear factor kappa-B (NF-κB), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in the heart tissue were determined though immunohistochemistry and western blotting analysis. The serum low-density lipoprotein cholesterol (LDL-C), triglyceride (TG), and total cholesterol (TC) levels were increased, and the high-density lipoprotein cholesterol (HDL-C) level was decreased in ApoE-/- mice. SABP significantly decreased serum lipid levels and improved histopathology in the thoracic aorta. In addition. SABP treatment inhibited the expression of TLR4, TAK1, NF-κB, IL-6 and TNF-α in the heart in ApoE-/- mice. The LDH and CK in the heart did not differ significantly among different groups, and the heart did not have obvious pathological changes. These findings indicated that SABP may exert an anti-atherosclerotic effect by lowering blood lipids and inhibiting inflammatory response via TLR4/ NF-κB signaling pathway.

Tetramethylpyrazine inhibits keratitis and neovascularization induced by corneal alkali burn by suppressing the TLR4/NF-κB pathway activation and NLRP1/NLRP3 inflammasomes in rats

The role and related mechanisms of tetramethylpyrazine (TMP) in corneal alkali burn in rats were expected to be explored in this article. After construction of corneal alkali burn rat models, TMP eye drops were given four times daily for consecutive 7 days. H&E staining was utilized for observing the histopathological changes of corneas on the 3rd and 7th days of treatment; immunohistochemistry for detecting the Nestin protein expression changes; qRT-PCR for determining the expression changes of genes correlated with neovascularization [C-X-C Motif Chemokine Ligand 1 (CXCL-1), vascular endothelial growth factor A (VEGFA) and CD31] and inflammation-related factors [monocyte chemoattractant protein-1 (MCP-1), interleukin-1β (IL-1β), tumour necrosis factor α (TNF-α), and IL-6]; Western blot for testing NLR Family Pyrin Domain Containing 1 (NLRP1)/NLRP3 inflammasomes and toll-like receptor 4 (TLR4)/nuclear factor kappa-B (NF-κB) pathway-related protein expression changes. All above trials were completed based on rat corneal tissue. TMP ameliorated the pathological damage in alkali-burned rat corneal tissue. Specifically, TMP treatment decreased Nestin-positive cell expression and the CXCL-1, VEGFA and CD31 mRNA expression in alkali-burned rat corneal tissue dose-dependently. TMP also down-regulated the IL-1β, TNF-α, MCP-1 and IL-6 mRNA expression and inhibited the NLRP1, caspase-1, NLRP3, pro-IL-1β and mature IL-1β protein expression in the alkali-burned rat corneal tissue. In addition, TMP treatment down-regulated the myeloid differentiation factor 88 (MyD88) and TLR4 protein expression and decreased the p–NF–κB p65/NF-κB p65 ratio in the alkali-burned rat corneal tissue. The mechanism of TMP relieving the inflammatory response and inhibiting neovascularization caused by corneal alkali burn in rats might have a correlation with the suppression of acitivation of TLR4/NF-κB pathway and NLRP1/NLRP3 inflammasomes in rats.

Role and mechanism of MiR-542-3p in regulating TLR4 in nonylphenol-induced neuronal cell pyroptosis

BackgroundThis study aimed to investigate the spatial learning/memory and motor abilities of rats and the alteration of miR-542-3p and pyroptosis in the midbrain nigrostriatal area in vivo after nonylphenol (NP) gavage and to explore the mechanism of miR-542-3p regulation of Toll-like receptor 4 (TLR4) in NP-induced pyroptosis in BV2 microglia in vitro. MethodsIn vivo: Thirty-six specific-pathogen-free-grade Sprague–Dawley rats were divided into three equal groups: blank control group (treated with pure corn oil), NP group (treated with NP, 80 mg/kg body weight per day for 90 days), and positive control group [treated with lipopolysaccharide (LPS), 2 mg/kg body weight for 7 days]. In vitro: The first part of the experiment was divided into blank group (control, saline), LPS group [1 µg/ml + 1 mM adenosine triphosphate (ATP)], and NP group (40 µmol/L). The second part was divided into mimics NC (negative control) group, miR-542-3p mimics group, mimics NC + NP group, and miR-542-3p mimics + NP group. ResultsIn vivo: Behaviorally, the spatial learning/memory and motor abilities of rats after NP exposure declined, as detected via Y-maze, open field, and rotarod tests. Some microglia in the substantia nigra of the NP-treated rats were activated. The downregulation of miR-542-3p was observed in rat brain tissue after NP exposure. The mRNA/protein expression of pyroptosis-related indicators (TLR4), NOD-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein (ASC), gasdermin-D (GSDMD), cysteinyl aspartate–specific proteinase-1 (caspase-1), and interleukin-1β (IL-1β) in the substantia nigra of the midbrain increased after NP exposure. In vitro: ASC fluorescence intensity increased in BV2 cells after NP exposure. The mRNA and/or protein expression of pyroptosis-related indicators (TLR4, NLRP3, GSDMD, caspase-1, and IL-1β) in BV2 cells was upregulated after NP exposure. The transfection of miR-542-3p mimics inhibited NP-induced ASC expression in BV2 cells. The overexpression of miR-542-3p, followed by NP exposure, significantly reduced TLR4, NLRP3, ASC, caspase-1, and IL-1β gene and/or protein expression. ConclusionsThis study suggested that NP exposure caused a decline in spatial learning memory and whole-body motor ability in rats. Our study was novel in reporting that the upregulation of miR-542-3p targeting and regulating TLR4 could inhibit NLRP3 inflammatory activation and alleviate NP-induced microglia pyroptosis.

Dictyophora polysaccharides alleviate intestinal-hepatic injury exposed to low-arsenic by regulating the imbalance of gut microbiota and LPS/TLR4 pathway in rats

Intestinal and liver damage caused by low arsenic exposure exists in worldwide, but the pathogenic mechanism is not clear at present.Dictyophora is a characteristic edible and medicinal fungus in China, and research has shown that its polysaccharides have good prebiotic effects.Therefore,we explored whether volva polysaccharide from Dictyophora has a certain therapeutic effect and its mechanism on intestinal and hepatic injury caused by low arsenic exposure.Firstly,we constructed the SD rat model of enteritis caused by low arsenic-induced.Then, from the perspectives of inflammatory responses capacity,histological alterations and serum biochemical levels of rats by arsenic-exposed with DIP intervention are analyzes the possible mechanism of intestinal and liver injury.Besides, the caecal microbiomes patterns were analysed using 16 S rDNA amplicon sequencing.Results showed that dictyophora polysaccharide increased the abundance of beneficial bacteria such as Turicibacter, Oscillospiraceae_UCG-005,and reduced the abundance of Desulfovibrionia and Lachnospiraceae_NK4A136_group proinflammatory bacteria.After dictyophora polysaccharide intervention, the pathological changes of liver and colon were alleviated,and the levels of triglyceride, total cholesterol,and lipopolysaccharide in hepatic portal vein, total arsenic contents in the liver and colon tissues were reduced. Finally,we also found after dictyophora polysaccharide intervention,the gene and protein expression of Toll-like receptor 4 was returned to normal level,and the expression levels of pro-inflammatory factors in liver and colon were reduce. In summary, our study found that dictyophora polysaccharide alleviated intestinal and hepatic injury in rats exposed to low arsenic by regulated intestinal microbiota and reduced TLR4 activation, which inhibited downstream proteins MyD88 to reduce the nuclear translocation of the NF-κB p65 genes and protein.

Effect of Kangfuxiaomi suppository on pelvic inflammatory disease in rats

Pelvic inflammatory disease (PID) is commonly encountered in gynecological practice. Kangfuxiaomi suppository, made from the compound extract of Periplaneta Americana, is a Traditional Chinese Medicine remedy widely used for the treatment of gynecological disorders. This study aimed to preliminarily explore the therapeutic effect of Kangfuxiaomi suppository in a rat model of PID established by chemical injury and pathogen infection. The key parameters assessed were vulvar inflammation score, vaginal + uterine organ index, and serum levels of interleukin (IL)− 8; tumor necrosis factor (TNF)-α; C-reactive protein (CRP); superoxide dismutase (SOD); and malondialdehyde (MDA). In addition, levels of IL-6, cyclooxygenase (COX)− 2, and IL-2 in cervical tissues as well as that of IL-1β and prostaglandin E-2 (PGE2) in uterine tissues were measured. The expression levels of nuclear factor-kappa B (NF-κB) p65 and Toll-like receptor 4 (TLR4) in uterine tissues were detected by immunohistochemical method. After Kangfuxiaomi suppository treatment, the vulva inflammation score and histopathological score of PID rats showed a tendency to decrease. Serum IL-8, TNF-α, CRP, and MDA levels were reduced, while SOD levels were significantly increased. Levels of IL-6, IL-2, and COX-2 in cervical tissues were somewhat decreased, and PGE2 and IL-1β levels in uterine tissue were significantly decreased. Moreover, the levels of NF-κB p65 and TLR4 protein expression were also decreased. These findings demonstrated the therapeutic effect of Kangfuxiaomi suppository in PID rats. The underlying mechanism may involve enhanced antioxidant capacity and decreased secretion of proinflammatory factors via the NF-κB/TLR4 signaling pathway.

Role of activating the nuclear factor kappa B signaling pathway in the development of septic cardiomyopathy in rats with sepsis.

Despite advances in the treatment of sepsis over time, this condition remains both a serious threat and a cause of death among critical patients. This study aimed to explore the role of the nuclear factor kappa B (NF-κB) signaling pathway in the development of septic cardiomyopathy in rats with sepsis. A total of 32 Sprague Dawley rats were randomized into a sham operation group and three groups with sepsis, which were tested at one of the following time-points: 3, 6, or 12 h. Each group included eight rats. Sepsis models were created via cecal ligation and puncture procedures. All the study rats had the following cardiac parameters and serum levels measured at either 3, 6, or 12 h after the operation (according to their assigned group): heart rate, left ventricular systolic pressure (LVSP), maximum rate of left ventricular pressure rise (+dP/dtmax) and fall (-dP/dtmax), tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1β), interleukin 6 (IL-6), and cardiac troponin I (cTnI). The myocardium of the left ventricle was collected and subjected to hematoxylin and eosin staining to observe the changes in pathological morphology. The expression of toll-like receptor 4 (TLR4) and NF-κB in the myocardium were detected by western blot analysis. Compared with the sham operation group, the rats in the sepsis subgroups exhibited significantly lower values for all the cardiac parameters measured, including the heart rate (sham operation group = 386.63 ± 18.62 beats per minute [bpm], sepsis 3-h group = 368.38 ± 12.55 bpm, sepsis 6-h group = 341.75 ± 17.05 bpm, sepsis 12-h group = 302.13 ± 21.15 bpm), LVSP (sham operation group = 125.50 ± 11.45 mmHg, sepsis 3-h group = 110.88 ± 7.51 mmHg, sepsis 6-h group = 100.00 ± 15.06 mmHg, sepsis 12-h group = 91.38 ± 14.73 mmHg), +dp/dtmax (sham operation group = 7137.50 ± 276.44 mm Hg/sec, sepsis 3-h group = 5745.00 ± 346.16 mm Hg/sec, sepsis 6-h group = 4360.00 ± 312.04 mm Hg/sec, sepsis 12-h group = 2871.25 ± 443.99 mm Hg/sec), and -dp/dtmax (sham operation group = 6363.75 ± 123.86 mm Hg/sec, sepsis 3-h group = 6018.75 ± 173.49 mm Hg/sec, sepsis 6-h group = 5350.00 ± 337.89 mm Hg/sec, sepsis 12-h group = 4085.00 ± 326.76 mm Hg/sec). They also displayed significantly higher levels of serum cytokines, including TNF-α (sham operation group = 14.72 ± 2.90 pg/mL, sepsis 3-h group = 34.90 ± 4.79 pg/mL, sepsis 6-h group = 24.91 ± 2.57 pg/mL, sepsis 12-h group 22.06 ± 3.11 pg/mL), IL-1β (sham operation group = 42.25 ± 16.91, 3-h group = 112.25 ± 13.77, sepsis 6-h group = 207.90 ± 22.64, sepsis 12-h group = 157.18 ± 23.06), IL-6 (sham operation group = 39.89 ± 5.74, sepsis 3-h group = 78.27 ± 9.31, sepsis 6-h group = 123.75 ± 13.11, sepsis 12-h group = 93.21 ± 8.96), and cTnI (sham operation group = 0.07 ± 0.03 ng/mL, sepsis 3-h group = 0.18 ± 0.06 ng/mL, sepsis 6-h group = 0.67 ± 0.19 ng/mL, sepsis = 12-h group 1.28 ± 0.10 ng/mL). The rats in the sepsis groups exhibited pathological changes in the myocardium, which deteriorated gradually over time. The animals in all the sepsis groups exhibited significantly higher levels of TLR4 and NF-κB protein expression compared with the sham group. The TLR4 protein expressions were 0.376 in the sham operation group, 0.534 in the sepsis 3-h group, 0.551 in the sepsis 6-h group, and 0.719 in the sepsis 12-h group. The NF-κB protein expressions were 0.299 in the sham operation group, 0.488 in the sepsis 3-h group, 0.516 in the sepsis 6-h group, and 0.636 in the sepsis 12-h group. Sepsis can lead to myocardial injury and cardiac dysfunction. This may be related to the activation of the NF-κB intracellular signal transduction pathway and the release of inflammatory factors as a result of lipopolysaccharides acting on TLR4 during the onset of sepsis.

Lactiplantibacillus plantarum L47 and inulin alleviate enterotoxigenic Escherichia coli induced ileal inflammation in piglets by upregulating the levels of α-linolenic acid and 12,13-epoxyoctadecenoic acid.

Alternatives to antibiotics for preventing bacteria-induced inflammation in early-weaned farm animals are sorely needed. Our previous study showed that Lactiplantibacillus plantarum L47 and inulin could alleviate dextran sulfate sodium (DSS)-induced colitis in mice. To explore the protective effects of L.plantarum L47 and inulin on the ileal inflammatory response in weaned piglets challenged with enterotoxigenic Escherichia coli (ETEC), 28 weaned piglets were assigned into four groups, namely, CON group-orally given 10mL/d phosphate buffer saline (PBS), LI47 group-orally given a mixture of 10mL/d L.plantarum L47 and inulin, ECON group-orally given 10mL/d PBS and challenged by ETEC, and ELI47 group-orally given 10mL/d L.plantarum L47 and inulin mixture and challenged by ETEC. The results demonstrated that the combination of L.plantarum L47 and inulin reduced inflammatory responses and relieved the inflammatory damage caused by ETEC, including ileal morphological damage, reduced protein expression of ileal tight junction, decreased antioxidant capacity, and decreased anti-inflammatory factors. Transcriptome analysis revealed that L.plantarum L47 and inulin up-regulated the gene expression of phospholipase A2 group IIA (PLA2G2A) (P<0.05) as well as affected alpha-linolenic acid (ALA) metabolism and linoleic acid metabolism. Moreover, L.plantarum L47 and inulin increased the levels of ALA (P<0.05), lipoteichoic acid (LTA) (P<0.05), and 12,13-epoxyoctadecenoic acid (12,13-EpOME) (P<0.05) and the protein expression of Toll-like receptor 2 (TLR2) (P=0.05) in the ileal mucosa. In conclusion, L.plantarum L47 and inulin together alleviated ETEC-induced ileal inflammation in piglets by up-regulating the levels of ALA and 12,13-EpOME via the LTA/TLR2/PLA2G2A pathway.

Herbal melanin modulates PGE2 and IL-6 gastroprotective markers through COX-2 and TLR4 signaling in the gastric cancer cell line AGS

We reported a gastric anti-ulcerogenic effect of the Nigella sativa (L.)-derived herbal melanin (HM) using rat models. However, the molecular mechanisms underlying this HM gastroprotective effect remain unknown. Cyclooxygenase-2 (COX-2)-catalyzed prostaglandin E2 (PGE2) and toll-like receptor 4 (TLR4)-mediated interleukin-6 (IL-6) production and secretion play major roles in gastric mucosal protection. In the current study, the human gastric carcinoma epithelial cell line AGS was used as a model to investigate the effect of HM on TLR4, COX-2, glycoprotein mucin 4 protein and gene expression using immuno-cyto-fluorescence staining, Western blot technology, and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Gastroprotective markers PGE2 and IL-6 production and secretion were also assessed using an enzyme-linked immunosorbent assay (ELISA). Bacterial lipopolysaccharides (LPS), well-known inducers of TLR4, COX-2, PGE2 and IL-6 expression, were used as a positive control. We showed that HM upregulated its main receptor TLR4 gene and protein expression in AGS cells. HM increased, in a dose- and time-dependent manner, the secretion of PGE2 and the expression of COX-2 mRNA and protein, which was detected in the nucleus, cytoplasm and predominantly at the intercellular junctions of the AGS cells. In addition, HM enhanced IL-6 production and secretion, and upregulated the mucin 4 gene expression, the hallmarks of gastroprotection. To check whether HM-induced PGE2 and IL-6 through TLR4 signaling and COX-2 generated, AGS cells were pre-treated with a TLR4 signaling inhibitor TAK242 and the COX-2 inhibitor NS-398. A loss of the stimulatory effects of HM on COX-2, PGE2 and IL-6 production and secretion was observed in TAK242 and NS-398-pre-treated AGS cells, confirming the role of TLR4 signaling and COX-2 generated in the HM gastroprotective effects. In conclusion, our results showed that HM enhances TLR4/COX-2-mediated secretion of gastroprotective markers PGE2 and IL-6, and upregulates mucin 4 gene expression in the human gastric epithelial cell line AGS, which may contribute to the promising beneficial gastroprotective effect of HM for human gastric prevention and treatment.

Huoxin Pill Reduces Myocardial Ischemia Reperfusion Injury in Rats via TLR4/NFκB/NLRP3 Signaling Pathway.

To explore the protective effect of Huoxin Pill (HXP) on acute myocardial ischemia-reperfusion (MIRI) injury in rats. Seventy-five adult SD rats were divided into the sham-operated group, model group, positive drug group (diltiazem hydrochloride, DH), high dose group (24 mg/kg, HXP-H) and low dose group (12 mg/kg, HXP-L) of Huoxin Pill (n=15 for every group) according to the complete randomization method. After 1 week of intragastric administration, the left anterior descending coronary artery of the rat's heart was ligated for 45 min and reperfused for 3 h. Serum was separated and the levels of creatine kinase (CK), creatine kinase isoenzyme (CK-MB) and lactate dehydrogenase (LDH), superoxide dismutase (SOD), and malondialdehyde (MDA), hypersensitive C-reactive protein (hs-CRP) and interleukin-1β (IL-1β) were measured. Myocardial ischemia rate, myocardial infarction rate and myocardial no-reflow rate were determined by staining with Evans blue and 2,3,5-triphenyltetrazolium chloride (TTC). Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) and Bioinformatics Analysis Tool for Molecular mechANism of Traditional Chinese Medicine (BATMAN) databases were used to screen for possible active compounds of HXP and their potential therapeutic targets; the results of anti-inflammatory genes associated with MIRI were obtained from GeneCards, Drugbank, Online Mendelian Inheritance in Man (OMIM), and Therapeutic Target Datebase (TTD) databases was performed; Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment were used to analyze the intersected targets; molecular docking was performed using AutoDock Tools. Western blot was used to detect the protein expression of Toll-like receptor 4 (TLR4)/nuclear factor kappa-B (NFκB)/NOD-like receptor protein 3 (NLRP3). Compared with the model group, all doses of HXP significantly reduced the levels of LDH, CK and CK-MB (P<0.05, P<0.01); HXP significantly increased serum activity of SOD (P<0.05, P<0.01); all doses of HXP significantly reduced the levels of hs-CRP and IL-1β (P<0.05, P<0.01) and the myocardial infarction rate and myocardial no-reflow rate (P<0.01). GO enrichment analysis mainly involved positive regulation of gene expression, extracellular space and identical protein binding, KEGG pathway enrichment mainly involved PI3K-Akt signaling pathway and lipid and atherosclerosis. Molecular docking results showed that kaempferol and luteolin had a better affinity with TLR4, NFκB and NLRP3 molecules. The protein expressions of TLR4, NFκB and NLRP3 were reduced in the HXP group (P<0.01). HXP has a significant protective effect on myocardial ischemia-reperfusion injury in rats, and its effect may be related to the inhibition of redox response and reduction of the inflammatory response by inhibiting the TLR4NFκB/NLRP3 signaling pathway.

Erythropoietin Suppresses the Hepatic Fibrosis Caused by Thioacetamide: Role of the PI3K/Akt and TLR4 Signaling Pathways.

Erythropoietin (EPO) is recognized for its function in erythropoiesis; however, its potential antifibrotic effect against liver fibrosis remains unknown. This study examined whether EPO affects thioacetamide (TAA)-induced liver fibrosis by concentrating on the Toll-like receptor 4 (TLR4) cascade and the phosphatidylinositol 3-kinase (PI3K)/Akt pathway as possible pathways. Male Wistar rats were randomized into four groups, which included: the negative control group, the TAA group (intraperitoneal; TAA 100 mg/kg three times per week for 2 weeks), and EPO-treated groups (150 and 300 IU/kg, i.p.) for 2 weeks after TAA injections. EPO attenuated hepatic fibrosis in a dosage-dependent way, as manifested by the diminution in serum alanine aminotransferase and aspartate aminotransferase activities, as well as the increase in albumin level. EPO inhibited the increase in tissue levels of tumor necrosis factors-α, interleukin-1β, transforming growth factor-β1, and TLR4 and raised tissue levels of PI3K and p-PI3K. EPO antioxidant properties were demonstrated by restoring hepatic glutathione and superoxide dismutase by preventing the accumulation of hepatic malondialdehyde. Further, EPO increased the protein expression of PI3K and Akt and decreased TLR4 protein expression. Immunohistochemically, EPO treatment altered tissue histology and downregulated mitogen-activated protein kinase protein expression. Overall, the research suggested that EPO could prevent TAA-induced hepatic fibrosis through upregulating the PI3K/Akt signaling cascade and downregulation the TLR4 downstream axis.