Effects of moxibustion on intestinal barrier function and TLR4/NF-κB p65 signaling pathway in obese rats. / è‰¾ç¸å¯¹è‚¥èƒ–å¤§é¼ è‚ é“å±éšœåŠŸèƒ½åŠTLR4/NF-κB p65信号通路的影响.

OBJECTIVES: 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. METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.

Anti-inflammatory effects of quinolinyl analog of resveratrol targeting TLR4 in MCAO/R ischemic stroke rat model.

BACKGROUND: Among adults, stroke is the main causes of mortality and permanent disability. Neuroinflammation is one of the main causes of stoke-mediated neuronal death. Our previous study revealed that (E)-5-(2-(Quinolin-4-yl) vinyl) benzene-1, 3-diol (RV01), a quinolinyl analog of resveratrol, inhibits microglia-induced neuroinflammation and safeguards neurons from inflammatory harm. The preventive role of RV01 in ischemic stroke and its underlying cellular mechanisms and molecular targets remain poorly understood. PURPOSE: To investigate whether RV01 alleviates ischemia-reperfusion (I/R) injury by inhibiting microglia-mediated neuroinflammation and determine the potential molecular mechanisms and targets by which RV01 inhibits the I/R-mediated microglia activation. METHODS: Rat middle cerebral artery occlusion and reperfusion (MCAO/R) and BV-2 or primary microglial cells oxygen-glucose deprivation and reperfusion (OGD/R) models were established. The neurological behavior scores, 2, 3, 5-triphenyl tetrazolium chloride staining and immunofluorescence were used to detect the neuroprotective effect of RV01 in the MCAO/R rats. In addition, the mRNA expression levels of IL-6, TNF-α, and IL-1ß were detected to reveal the antineuroinflammatory effect of RV01. Moreover, a western blot assay was performed to explore the protein expression changes in NF-κB-mediated neuroinflammation. Finally, we identified TLR4 as an RV01 target through molecular docking, drug sensitivity target stability analysis, cellular thermal shift analysis, and surface plasmon resonance techniques. RESULTS: RV01 reduced the infarct volume and neurological deficits, increased the rotarod duration, and decreased the number of rightward deflections in the MCAO/R rats. RV01 inhibited the NF-κB signaling pathway in vitro and in vivo, as demonstrated by the reduction in the transcription factor p65-mediated expression of several inflammatory factors including IL-6, TNF-α, and IL-1ß. Further studies showed that its protective effect was associated with targeting the TLR4 protein. Notably, the anti-inflammatory effect of RV01 was markedly reinforced by the TLR4 knockdown, but inhibited by the overexpression of TLR4. Results revealed that the conditioned medium derived from the RV01-treated BV-2 cells significantly decreased the OGD/R-mediated neuronal damage. CONCLUSION: Our results are the first to reveal the protective effects of RV01 on cerebral ischemia, depending on its inhibitory effect on the NF-κB pathway by targeting TLR4. RV01 could be a potential protective agent in ischemic stroke treatment.

Yanghepingchuan granule improves airway inflammation by inhibiting autophagy via miRNA328-3p/high mobility group box 1/Toll-like receptor 4 targeting of the pathway of signaling in rat models of asthma.

Background: As a type of traditional Chinese medicine, Yanghepingchuan granules (YHPCG) are used to treat inflammatory diseases of the lungs, including asthma. However, the underlying molecular mechanism of the ability of YHPCG to reduce airway inflammation remains unknown. Methods: By sensitizing rats to aluminum hydroxide and ovalbumin, an asthma model was established. During the 14-day treatment period, the rats received YHPCG, TAK242 (TLR4 inhibitor), and a combination of the two treatments. Histopathology and goblet cell hyperplasia were observed in rats with ovalbumin-induced asthma by using hematoxylin-eosin (HE) and periodic acid-Schiff (PAS) staining. Immunohistochemical, autophagy-related immunofluorescence, and western blotting analyses were performed to determine autophagic activity. The effects of YHPCG on high mobility group box 1 (HMGB1)-mediated Toll-like receptor 4 (TLR4)/nuclear factor κB (NF-κB) pathway-related proteins and inflammatory factors in rats were evaluated via western blotting, PCR analysis, and enzyme-linked immunosorbent assay. A dual luciferase method was used to detect the interaction between miRNA328-3p and HMGB1. Results: YHPCG inhibit the HMGB1/TLR4/NF-κB pathway by upregulating miR-328-3p, reducing autophagosome production, inhibiting autophagy, and effectively preventing the progression of lung inflammation. Conclusions: Asthma airway inflammation can be treated with YHPCG by inhibiting autophagy via miRNA328-3p/HMGB1/TLR4/NF-κB signaling pathways.

[Acacetin protects rats from cerebral ischemia-reperfusion injury by regulating TLR4/NLRP3 signaling pathway].

This study aims to investigate the mechanism of acacetin in protecting rats from cerebral ischemia-reperfusion injury via the Toll-like receptor 4(TLR4)/NOD-like receptor protein 3(NLRP3) signaling pathway. Wistar rats were randomized into sham, model, low-and high-dose acacetin, and nimodipine groups, with 10 rats in each group. The rat model of middle cerebral artery occlusion(MCAO) was established with the improved suture method in other groups except the sham group. The neurological deficit score and cerebral infarction volume of each group were evaluated 24 h after modeling. Enzyme-linked immunosorbent assay(ELISA) was employed to measure the levels of interleukin-1ß(IL-1ß), IL-6, tumor necrosis factor-α(TNF-α), malondialdehyde(MDA), supe-roxide dismutase(SOD), and glutathione(GSH). Western blot was employed to determine the expression levels of B-cell lymphonoma-2(Bcl-2), Bcl-2-associated X protein(Bax), and TLR4/NLRP3 signaling pathway-related proteins(TLR4, p-NF-κB/NF-κB, NLRP3, pro-caspase-1, cleaved caspase-1, pro-IL-1ß, and cleaved IL-1ß) in the rat brain tissue. Hematoxylin-eosin(HE) staining was employed to reveal the histopathological changes in the ischemic area. Compared with the sham group, the modeling of MCAO increased the neurological deficit score and cerebral infarction volume, elevated the IL-1ß, IL-6, TNF-α, and MDA levels and lowered the SOD and GSH levels in the brain tissue(P<0.05). Compared with the MCAO model group, low-and high-dose acacetin and nimodipine decreased the neurological deficit score and cerebral infarction volume, lowered the IL-1ß, IL-6, TNF-α, and MDA levels and elevated the SOD and GSH levels in the brain tissue(P<0.05). Compared with the sham group, the model group showed up-regulated protein levels of Bax, TLR4, p-NF-κB/NF-κB, NLRP3, pro-caspase-1, cleaved caspase-1, pro-IL-1ß, and cleaved IL-1ß and down-regulated protein level of Bcl-2 in the brain tissue(P<0.05). Compared with the MCAO model group, the acacetin and nimodipine groups showed down-regulated protein levels of Bax, TLR4, p-NF-κB/NF-κB, NLRP3, pro-caspase-1, cleaved caspase-1, pro-IL-1ß, and cleaved IL-1ß and up-regulated protein level of Bcl-2 in the brain tissue(P<0.05). In conclusion, acacetin regulates the TLR4/NLRP3 signaling pathway to inhibit neuroinflammatory response and oxidative stress, thus exerting the protective effect on cerebral ischemia-reperfusion injury in rats.

Alcohol-induced liver injury in signalling pathways and curcumin's therapeutic potential.

Confronting the profound public health concern of alcohol-induced liver damage calls for inventive therapeutic measures. The social, economic, and clinical ramifications are extensive and demand a comprehensive understanding. This thorough examination uncovers the complex relationship between alcohol intake and liver damage, with a special emphasis on the pivotal roles of the Toll-like receptor 4 (TLR4)/NF-κB p65 and CYP2E1/ROS/Nrf2 signalling networks. Different alcohol consumption patterns, determined by a myriad of factors, have significant implications for liver health, leading to a spectrum of adverse effects. The TLR4/NF-κB p65 pathway, a principal regulator of inflammation and immune responses, significantly contributes to various disease states when its balance is disrupted. Notably, the TLR4/MD-2-TNF-α pathway has been linked to non-alcohol related liver disease, while NF-κB activation is associated with alcohol-induced liver disease (ALD). The p65 subunit of NF-κB, primarily responsible for the release of inflammatory cytokines, hastens the progression of ALD. Breakthrough insights suggest that curcumin, a robust antioxidant and anti-inflammatory compound sourced from turmeric, effectively disrupts the TLR4/NF-κB p65 pathway. This heralds a new approach to managing alcohol-induced liver damage. Initial clinical trials support curcumin's therapeutic potential, highlighting its ability to substantially reduce liver enzyme levels. The narrative surrounding alcohol-related liver injury is gradually becoming more intricate, intertwining complex signalling networks such as TLR4/NF-κB p65 and CYP2E1/ROS/Nrf2. The protective role of curcumin against alcohol-related liver damage marks the dawn of new treatment possibilities. However, the full realisation of this promising therapeutic potential necessitates rigorous future research to definitively understand these complex mechanisms and establish curcumin's effectiveness and safety in managing alcohol-related liver disorders.

Traditional Chinese Medicine Intervention in Ulcerative Colitis Based on TLR4 Signaling Pathway： A Review / 中国实验方剂学杂志

Ulcerative colitis （UC） is a disease that affects the mucosal and submucosal layers of the colon and is characterized by inflammation of the intestinal mucosa. The incidence of UC is increasing year by year， and it is complex and refractory， severely impacting the physical and mental health of patients. The pathological mechanism of this disease is complex， with immune responses and uncontrollable inflammatory reactions in the intestine being important physiopathologic mechanisms. Toll-like receptor 4 （TLR4）， as a transmembrane signaling receptor， plays a key role in mediating immune responses and inflammatory reactions in the development of UC. Currently， the treatment of UC mainly relies on salicylic acids， glucocorticoids， and other agents to reduce intestinal inflammation. While these drugs can partially inhibit the progression of the disease， they often come with significant adverse effects and the potential for relapse upon discontinuation. Traditional Chinese medicine （TCM） offers multiple pathways， effects， and targets for regulating the TLR4 pathway， suppressing inflammatory responses， and effectively intervening in the progression of UC. This approach has become a hot topic in the prevention and treatment of UC. Numerous studies have shown that TCM treatment of UC has unique advantages. TCM can enhance immune defenses， suppress inflammatory responses， promote intestinal mucosal healing， and maintain the balance of the intestinal microbiota by regulating the TLR4 signaling pathway， thereby effectively treating UC， with substantial progress achieved. However， there is currently a lack of comprehensive reviews on the role of TCM in regulating the TLR4 signaling pathway for the treatment of UC. Therefore， this article systematically summarized the relationship between the TLR4 signaling pathway and UC， as well as the role of TCM in this context， by reviewing relevant literature from recent years， aiming to provide new insights into the potential treatment and new drug development for UC.

Effect of Chuanshanlong Granule on TLR4/MyD88/NF-κB Signaling Pathway in Rats with Autoimmune Thyroiditis / 中国实验方剂学杂志

ObjectiveTo investigate the effect of Chuanshanlong granule on Toll-like receptor 4 （TLR4）/myeloid differentiation protein 88 （MyD88）/nuclear transcription factor -κB （NF-κB） signaling pathway， and to explore the mechanism of its treatment of autoimmune thyroiditis （AIT） in rats. MethodForty AIT models were established following excess iodine and injection of porcine thyroglobulin and Freund's adjuvant into Lewis rats for six weeks. Then the rats were randomly divided into the model group， Chuanshanlong granule low-， medium- and high-dose group （0.52， 1.03， 2.06 g·kg-1·d-1）， with ten in each group. Rats in the Chuanshanlong granule low-， medium- and high-dose groups were separately given 0.01 mL·g-1·d-1 Chuanshanlong granule， and those in the normal group and the model group were given the same volume of deionized water for eight weeks. Serum of rats was taken to measure thyroglobulin antibody （TgAb） and thyroid peroxidase antibody （TPOAb） by enzyme-linked immunosorbent assay （ELISA）， and the concentrations of free triiodothyronine （FT3）， free thyroxine （FT4） and thyroid stimulating hormone （TSH） were detected. The rat thyroid lobes were stained with hematoxylin and eosin （HE）， and the pathological changes were observed under light microscope. In addition， the relative expression of TLR4， MyD88， NF-κB protein and mRNA was determined by immunohistochemistry and real-time polymerase chain reaction （Real-time PCR）. ResultCompared with the conditions in the normal group， the serum concentrations of TPOAb and TgAb （P<0.01） and FT3 and FT4 （P<0.01） increased and TSH decreased （P<0.01） in the model group. Compared with the conditions in the model group， the concentrations of TPOAb and TgAb in the Chuanshanlong granule treatment groups reduced （P<0.01）， and the concentrations of FT3 and FT4 were lowered （P<0.01） while TSH increased （P<0.01） in the Chuanshanlong granule high-dose group. HE staining showed that there was lymphocyte infiltration in the thyroid follicular space， a large number of destroyed or diminished follicular cavities， decreased colloid content， and thinned or destroyed follicular wall in the model group， while the thyroid lymphocyte infiltration in the Chuanshanlong granule treatment groups was significantly less and the structure of thyroid follicles was more complete than those in the model group. Compared with the normal group， the model group had up-regulated relative expression of TLR4， MyD88 and NF-κB protein （P<0.01） and mRNA （P<0.01）. Compared with the model group， the Chuanshanlong granule high-dose group had down-regulated relative expression of TLR4 protein and mRNA （P<0.05）， MyD88 protein （P<0.01） and mRNA （P<0.05）， and NF-κB protein and mRNA （P<0.01）. ConclusionChuanshanlong granule may play a therapeutic role in AIT by inhibiting the activation of TLR4/MyD88/NF-κB signaling pathway.

[Effect of acupuncture on intestinal flora and TLR4 in brain and intestinal tissues in rats with stress gastric ulcer].

OBJECTIVE: To observe the effect of acupuncture at "Guanyuan" (CV 4) and "Xiajuxu" (ST 39) on intestinal flora and Toll-like receptors-4 (TLR4) in brain and intestinal tissue in rats with stress gastric ulcer (SGU), and to explore the possible mechanism of acupuncture for SGU. METHODS: Thirty-one male SD rats were randomly divided into a blank group (n=7), a model group (n=8), an acupuncture group (n=8) and a drug group (n=8). The rats in the model group, acupuncture group and drug group were treated with modified restraint plus water-immersion stress method to establish SGU model. After modeling, the rats in the acupuncture group were treated with acupuncture at "Guanyuan" (CV 4) and "Xiajuxu" (ST 39), 20 min each time, and the needles were twirled for 30 s every 5 min. The rats in the drug group were treated with intragastric administration of 2 mL omeprazole enteric-coated tablets (20 mg/mL). Both the treatments were given once a day for 5 days. After the intervention, the gastric mucosal damage index was calculated by Guth method; the morphological changes of gastric mucosa were observed by HE staining; the diversity of intestinal flora was detected by 16S rDNA high-throughput sequencing; TLR4 contents in brain and intestinal tissues were determined by ELISA. RESULTS: Compared with the blank group, the gastric mucosal damage index was significantly increased in the model group (P<0.01); the morphological changes of gastric mucosa were obvious; the Observed Species index and Shannon index of α diversity index of intestinal flora were decreased (P<0.05); the ß diversity showed that the spatial distance between the model group and the blank group was far; the TLR4 contents in the brain and intestinal tissue were increased (P<0.05, P<0.01). Compared with the model group, the gastric mucosal damage index was decreased in the acupuncture group and the drug group (P<0.05); the morphology of gastric mucosa was improved; the Observed Species index and Shannon index of α diversity index of intestinal flora in the acupuncture group was increased (P<0.05), and the Shannon index in the drug group was increased (P<0.05); the ß diversity showed that the spatial distance between the acupuncture group and the blank group was close; the TLR4 contents in the brain and intestinal tissues of the acupuncture group were decreased (P<0.01). Compared with the drug group, the contents of TLR4 in the intestinal tissue of the acupuncture group were decreased (P<0.05). CONCLUSION: Acupuncture at "Guanyuan" (CV 4) and "Xiajuxu" (ST 39) could alleviate SGU in rats, and its mechanism may be related to increasing the diversity of intestinal flora, promoting the disorder of intestinal flora to normal, and reducing the overexpression of TLR4 in brain and intestinal tissues.

Effect of acupuncture on intestinal flora and TLR4 in brain and intestinal tissues in rats with stress gastric ulcer / 中国针灸

OBJECTIVE@#To observe the effect of acupuncture at "Guanyuan" (CV 4) and "Xiajuxu" (ST 39) on intestinal flora and Toll-like receptors-4 (TLR4) in brain and intestinal tissue in rats with stress gastric ulcer (SGU), and to explore the possible mechanism of acupuncture for SGU.@*METHODS@#Thirty-one male SD rats were randomly divided into a blank group (@*RESULTS@#Compared with the blank group, the gastric mucosal damage index was significantly increased in the model group (@*CONCLUSION@#Acupuncture at "Guanyuan" (CV 4) and "Xiajuxu" (ST 39) could alleviate SGU in rats, and its mechanism may be related to increasing the diversity of intestinal flora, promoting the disorder of intestinal flora to normal, and reducing the overexpression of TLR4 in brain and intestinal tissues.

Adjunct Therapy With Glycyrrhiza Glabra Rapidly Improves Outcome in Depression-A Pilot Study to Support 11-Beta-Hydroxysteroid Dehydrogenase Type 2 Inhibition as a New Target.

Mineralocorticoid-receptor (MR) dysfunction as expressed by low systolic blood pressure and a high salivary aldosterone/cortisol ratio predicts less favorable antidepressant treatment outcome. Inhibition of peripheral 11-beta-hydroxysteroid-dehydrogenase type 2 (11betaHSD2) reverses these markers. We therefore tested the hypothesis that the 11betaHSD2 inhibitor glycyrrhizin affects treatment outcome via this mechanism. We administered Glycyrrhiza glabra (GG) extract containing 7-8 % of glycyrrhizin at a dose of 2 × 700 mg daily adjunct to standard antidepressants in hospitalized patients with major depression. These subjects were compared in an open-label fashion with patients, who did not receive GG (treatment as usual, TAU). Assessments were done at baseline and approximately 2 weeks after. Twelve subjects were treated with GG and compared to 55 subjects with TAU. At week 2, the Hamilton Depression Rating Scale (HAMD-21) change from baseline as well as the CGI-S change showed a significant time × treatment interaction (p < 0.03), indicating a possible therapeutic benefit of GG. Clinical benefit seems to be more pronounced in subjects with lower systolic blood pressure and significantly correlated with reduced sleep duration in the GG group. Our preliminary data show that treatment with the 11betaHSD2 inhibitor glycyrrhizin may possess a beneficial effect on antidepressant response, which may be specific to a defined depression subtype.

Symptomatic Protective Action of Glycyrrhizin (Licorice) in COVID-19 Infection?

The role of the ACE2 enzyme in the COVID-19 infection is 2-fold, with opposing implications for the disease development. 1. The membrane bound angiotensin converting enzyme 2 (ACE2) serves as the entry point of COVID-19 2. Conversely, it supports an anti-inflammatory pathway. This led to the controversy of the impact of medications, which influence its expression. ACE2 is part of the wider renin-angiotensin-aldosterone system (RAAS) and is upregulated via compounds, which inhibits the classical ACE, thereby plasma aldosterone and aldosterone receptor (MR) activation. MR activation may therefore protect organs from binding the COVID-19 by reducing ACE2 expression. Glycyrrhizin (GL) is a frequent component in traditional Chinese medicines, which have been used to control COVID-19 infections. Its systemically active metabolite glycyrrhetinic acid (GA) inhibits 11beta hydroxysteroid dehydrogenase(11betaHSD2) and activates MR in organs, which express this enzyme, including the lungs. Does this affect the protective effect of ACE2? Importantly, GL has anti-inflammatory properties by itself via toll like receptor 4 (TLR4) antagonism and therefore compensates for the reduced protection of the downregulated ACE2. Finally, a direct effect of GL or GA to reduce virus transmission exists, which may involve reduced expression of type 2 transmembrane serine protease (TMPRSS2), which is required for virus uptake. Glycyrrhizin may reduce the severity of an infection with COVID-19 at the two stages of the COVID-19 induced disease process, 1. To block the number of entry points and 2. provide an ACE2 independent anti-inflammatory mechanism.

Farrerol attenuates MPP+ -induced inflammatory response by TLR4 signaling in a microglia cell line.

Farrerol was found to possess neuroprotective effect; however, the mechanism remains unknown. The aim of the present study was to explore the effect of farrerol on MPP+ -induced inflammation in mouse microglial BV-2 cells and to elaborate the underlying mechanism. MTT assay was performed to measure the cell viability. The pro-inflammatory mediators and cytokines including interleukin (IL)-6, IL-1ß, and tumor necrosis factor-α (TNF-α); inducible nitric oxide synthase; and cyclooxygenase 2 were measured. The expression of p-p65, p-IκBα, toll-like receptor 4 (TLR4), and myeloid differentiation primary response 88 were analyzed by western blot. We found that farrerol treatment improved cell viability in MPP+ -induced BV-2 cells. MPP+ -induced upregulation of IL-6, IL-1ß, and TNF-α was inhibited by farrerol treatment. Farrerol treatment also attenuated MPP+ -induced expression of inducible nitric oxide synthase and cyclooxygenase 2 as well as the activation of NF-κB in BV-2 cells. MPP+ -induced TLR4 signaling was markedly diminished by farrerol treatment. Knockdown of TLR4 attenuated MPP+ -induced inflammatory response in BV-2 cells. In conclusion, farrerol treatment attenuated MPP+ -induced inflammatory response by inhibiting the TLR4 signaling pathway in BV-2 cells. The results indicated that farrerol could be used as a therapeutic agent for preventing or alleviating the neuroinflammation-related diseases, such as Parkinson's disease.

Structural insights into pharmacophore-assisted in silico identification of protein-protein interaction inhibitors for inhibition of human toll-like receptor 4 - myeloid differentiation factor-2 (hTLR4-MD-2) complex.

Toll-like receptor 4 (TLR4) is a member of Toll-Like Receptors (TLRs) family that serves as a receptor for bacterial lipopolysaccharide (LPS). TLR4 alone cannot recognize LPS without aid of co-receptor myeloid differentiation factor-2 (MD-2). Binding of LPS with TLR4 forms a LPS-TLR4-MD-2 complex and directs downstream signaling for activation of immune response, inflammation and NF-κB activation. Activation of TLR4 signaling is associated with various pathophysiological consequences. Therefore, targeting protein-protein interaction (PPI) in TLR4-MD-2 complex formation could be an attractive therapeutic approach for targeting inflammatory disorders. The aim of present study was directed to identify small molecule PPI inhibitors (SMPPIIs) using pharmacophore mapping-based approach of computational drug discovery. Here, we had retrieved the information about the hot spot residues and their pharmacophoric features at both primary (TLR4-MD-2) and dimerization (MD-2-TLR4*) protein-protein interaction interfaces in TLR4-MD-2 homo-dimer complex using in silico methods. Promising candidates were identified after virtual screening, which may restrict TLR4-MD-2 protein-protein interaction. In silico off-target profiling over the virtually screened compounds revealed other possible molecular targets. Two of the virtually screened compounds (C11 and C15) were predicted to have an inhibitory concentration in µM range after HYDE assessment. Molecular dynamics simulation study performed for these two compounds in complex with target protein confirms the stability of the complex. After virtual high throughput screening we found selective hTLR4-MD-2 inhibitors, which may have therapeutic potential to target chronic inflammatory diseases.

Effect of Traditional Chinese Medicine on Hepatic Disease by Inhibiting TLR4 Signaling Pathway / 中国实验方剂学杂志

Liver disease is the general term for all diseases that occur in the liver. In recent years, traditional Chinese medicine has certain advantages in the treatment of liver diseases. With a high experimental study value, good clinical efficacy and less adverse reactions, it has broad prospects. Toll-like receptor 4 (TLR4) signaling pathway is closely related to liver diseases. Its mechanism is to activate nuclear factor-κB (NF-κB) through tlr4-mediated signaling pathway, inhibit the secretion of such inflammatory factors as interleukin-1(IL-1), interleukin-6(IL-6) and tumor necrosis factor-alpha (TNF-α), and the inflammatory damage of liver cells, so as to further inhibit the effect of IL-1,IL-6, TNF-α in activating Hepatic Stellate Cell(HSC). The ways of blocking TRL4 pathway are as follows:Inhibiting the expression of TLR4,Inhibiting the dimerization of TLR4. Blocking intracellular signal transduction:①acting on the binding protein; ②acting on the kinase IRAKs; ③acting on TLRAFst. In these ways, the TRL4 pathway is blocked, the inflammation is inhibited, and the anti-liver disease effect is achieved. Therefore, inhibiting or enhancing TLR4 signaling pathway or intervening in some links of TLR4 signaling pathway has become a new strategy for the treatment of liver diseases. Toll-like receptor 4 signaling pathway has become one of the targets of traditional Chinese medicine (TCM) against liver diseases. In this paper, the recent literatures on the effect of TCM in resisting activation of TLR4 signaling pathway and the effect of anti-liver diseases through monomers and effective parts of TCM, extracts of TCM and compound prescriptions of TCM were collected and summarized to provide important guiding significance and direction for the treatment of liver diseases by TCM and WM in the next step.

Efficacy and Immune Mechanism of Baitouweng Tang on Ulcerative Colitis in Rats / 中国实验方剂学杂志

Objective: To investigate the therapeutic effect and immune mechanism of Baitouweng Tang on ulcerative colitis(UC) rats. Method: The mode of UC rats was made of 2, 4-dinitrochlorobenzene (DNCB)/ethanol enema. Rats were randomly divided into control group, model group, mesalazine group, and high-dose, middle-dose and low-dose Baitouweng Tang groups. The mesalazine group were administered with mesalazine (0.5 g·kg-1). Baitouweng Tang groups were given Baitouweng Tang (10, 5, 2.5 g·kg-1), while the other groups were given double steaming water. After 7 days of continuous administration, the general condition and disease activity index of rats in each group were observed. After anesthesia in rats, blood was taken from the abdominal aorta. Then the rats were put to death, and the length and morphological observation of the colon were measured. Ultraviolet spectrophotometry detection was used to detect the activities of myeloperoxidase (MPO) in blood and colon tissue. The levels of P-selectin, macrophage migration inhibitory factor (MIF) and thromboxane B2 (TXB2) in blood and colon tissues were detected by enzyme-linked immunosorbent assay(ELISA). Immunohistochemistry and Western blot methods were undertaken to determine the expressions of Toll-like receptor 4 (TLR4) and nuclear transcription factor-κB (NF-κB) proteins in colon tissue. Result: Compared with the model group, the rats in model group showed severe symptoms, such as loose stools, diarrhea and bloody stools, while Baitouweng Tang obviously ameliorated them. Moreover, Baitouweng Tang significantly reduced DAI, colon general and pathological scores, which were high in model group(P2 in the serum and colon tissues of the model group were obviously increased(PκB in colons of model group were markedly higher than those in control group(PPConclusion: Baitouweng Tang could inhibit TLR4/NF-κB signaling pathway in treatment of ulcerative colitis, and reduce the expressions of P-selectin, MPO, MIF and TXB2, and thus promoting intestinal mucosal repair and improving intestinal function.

Effect of Whenshen Prescription on Hepatic Encephalopathy in Patients with HBV-related Hepatic Cirrhosis and Content of HMGB1/TLR4 / 中国实验方剂学杂志

Objective:To investigate the effect of Whenshen prescription on hepatic encephalopathy in patients with HBV-related hepatic cirrhosis and kidney Yang deficiency syndrome and the content of high-mobility group protein B1 (HMGB1) and Toll-like receptor 4 (TLR4). Method:The 66 patients treated in the Hospital of Chengdu University of Traditional Chinese Medicine from August 2013 to January 2015 were included. A prospective, parallel controlled design was used. The 66 cases were randomly divided into treatment group and control group according to 1:1 ratio, with 33 cases in each group.The control group was treated with comprehensive therapy, colon dialysis and placebo enema, while treatment group was treated with comprehensive therapy, colon dialysis and Whenshen prescription enema for 10 days. Finally, liver function, number connect test (NCT), digit-symbol test (SDT), awake time, the total effective rate and content of HMGB1 and TLR4 were analyzed. Result:There was no significant difference between two groups at baseline. The total effective rate in treatment group was 93.9%, which was higher than 72.7% in control group (PP1 and TLR4 in treatment group were lower than those in the control group. Conclusion:Whenshen prescription can significantly improve the clinical efficacy by inhibiting the contents of HMGB1 and TLR4.

[Toll-like Receptor 4/Nuclear Factor-κB Signaling in Synovial Tissue Is Involved in the Anti-inflammatory Effect of Moxibustion in Rats with Rheumatoid Arthritis].

OBJECTIVE: To observe the effect of moxibustion on Toll-like receptor 4/nuclear factor-κB (TLR 4/NF-κB) signaling in the synovial tissue of the ankle joint in rats with rheumatoid arthritis (RA), so as to analyze its biological mechanism underlying improvement of RA. METHODS: Fifty male SD rats were randomly divided into normal control, model, moxibustion, moxibustion ＋ TLR 4 agonist, and moxibustion ＋ TLR 4 antagonist groups (n＝10 rats in each). The RA model was established by subcutaneous injection of Freund's complete adjuvant (FCA, 0.1 mL/rat) at the right hind-paw and by being raised in a wind (air fan blowing), cold (about 10 ℃) and wet (purling) environment for 20 days. After 3 days of modeling, mild moxibustion was applied to bilateral "Shenshu" (BL 23) and "Zusanli" (ST 36) for 20 minutes, once daily for successive 10 days. The TLR 4 agonist (lipopolysaccharide) or TLR 4 antagonist (TAK-242) (1 mg/mL) was separately administered via the tail vein 30 min before performing moxibustion every time in the agonist group and the antagonist group. The expression of NF-κB inhibitory factor ɑ (IκBɑ), IκB kinase complex ß(IκKß), myeloid differentiation factor 88 (MyD 88), TLR 4, and NF-κB p 65 proteins in the synovial tissue of the ankle joint was detected by using Western blot. RESULTS: Following modeling, the girth of the swollen ankle joint was obviously bigger (P<0.01), and the expression levels of IκBɑ， IκKß， TLR 4, MyD 88, and NF-κB p 65 proteins in the synovial tissue were considerably increased in the model group relevant to the normal control group (P<0.01). After moxibustion intervention, the girth of the swollen ankle joint and the expression levels of IκBɑ， IκKß， TLR 4, MyD 88, and NF-κB p 65 proteins were significantly down-regulated in the moxibustion, moxibustion ＋TLR 4 agonist, moxibustion＋TLR 4 antagonist groups compared with the model group (P<0.01, P<0.05). Comparison among the 3 moxibustion groups showed that the lowered levels of ankle-joint girth, and IκBɑ， IκKß， TLR 4, MyD 88, and NF-κB p 65 expression were significantly smaller in the moxibustion＋TLR 4 agonist group than in the simple moxibustion and moxibustion＋TLR 4 antagonist groups (P<0.05，P<0.01). No significant differences were found between the moxibustion and moxibustion＋TLR 4 antagonist groups in the decreased ankle joint girth and IκBɑ， IκKß， TLR 4, MyD 88, and NF-κB p 65 expression levels (P>0.05), suggesting that activation of TLR 4 reduced the anti-inflammatory effect of moxibustion intervention. CONCLUSION: Moxibustion can reduce the ankle joint swelling in RA rats, which may be closely associated with its effect in down-regulating the expression of IκBɑ， IκKß， TLR 4, MyD 88, and NF-κB p 65 proteins and in inhibiting TLR 4/NF-κB signaling in the synovial tissue of the ankle joint.

[Effects of Herbal-cake-separated Moxibustion on Blood Lipid Protein Levels and Expression of Toll-like Receptor and Nuclear Factor Genes in Atherosclerotic Plaques in Hyperglycemia Rabbits].

OBJECTIVE: To observe the effect of herbal cake-separated moxibustion on blood lipid-apoprotein levels and the expression of Toll-like receptor 2 (TLR 2), TLR 4 and nuclear factor kappa B(NF-κB) mRNAs in atherosclerotic (AS) vulnerable plaques of hyperglycemia rabbits, so as to explore its mechanism underlying improvement of atherosclerosis. METHODS: Sixty New Zealand rabbits were randomly divided into 5 groups: control, model, direct moxibustion, herbal-cake-separated moxibustion and medication groups(n＝12 rabbits in each group). The AS vulnerable plaque model was established by high-fat forage feeding plus balloon-induced abdominal aorta injury and gene transfection of Ad 5-p 53 recombinant vector. Direct moxibustion or herbal-cake-separated moxibustion was applied to "Juque" (CV 14) and bilateral "Tianshu" (ST 25), "Fenglong" (ST 40), or bilateral "Xinshu" (BL 15), "Pishu" (BL 20) and "Ganshu" (BL 18) for 15－20 min every time. The medication group was treated by feeding Atorvastatin. All the treatments were conducted once daily for 8 weeks. Plasma total cholesterol(TC) and triglyceri-de(TG) contents were detected by enzyme method, high-density lipoprotein (HDL) and low-density lipoprotein (LDL) contents detected by colorimetric determination, and plasma apolipoprotein A(Apo-A) and apolipoprotein B(Apo-B) levels determined by electrophoretic method. The pathological changes of vulnerable plaque and the aortic intima and media thickness were observed under light microscope after H．E. staining. The expression levels of TLR 2, TLR 4 and NF-κB mRNAs in AS plaques were determined by quantitative real-time PCR. RESULTS: After modeling, the levels of plasma TC, TG, LDL and Apo-B in the model group were remarkably increased (P<0.01), and Apo-A and HDL/LDL were significantly decreased in comparison with the control group (P<0.01). Additionally, the aortic intima and media thickness and the expression levels of TLR 2, TLR 4 and NF-κB mRNAs in AS plaques were significantly increased (P<0.01). After the treatment, the elevated levels of plasma TC, TG, LDL and Apo-B, the aortic intima thickness and media thickness, and the expression levels of TLR 2, TLR 4 and NF-κB mRNAs in the 3 treatment groups were significantly down-regulated in comparison with the model group (P<0.05, P<0.01), while the decreased levels of Apo-A and HDL/LDL were considerably increased (P<0.01). Comparison among the 3 treatment groups showed that the therapeutic effects of the herbal-cake-separated moxibustion and medication were significantly superior to those of the direct moxibustion in down-regulating the levels of TC, TG, LDL, Apo-B, TLR 2 mRNA, TLR 4 mRNA and NF-κB mRNA, and reducing the thickness of the aortic intima and media, as well as in up-regulating the levels of Apo-A and HDL/LDL (P<0.05, P<0.01). No significant differences were found between the herbal-cake-separated moxibustion and medication groups in the above-mentioned indexes (P>0.05). CONCLUSION: Herbal-cake-separated moxibustion has a positive role in stabilizing AS vulnerable plaque in hyperglycemia rabbits, which may be associated with its effects in regulating blood lipid-apolipoprotein levels and inhibiting the expression of TLR 2, TLR 4 and NF-κB mRNAs in vulnerable plaques.

The Effect of Inhalant Organic Dust on Bone Health.

PURPOSE OF REVIEW: Agriculture remains a major economic sector globally, and workers experience high rates of chronic inflammatory lung and musculoskeletal diseases. Whereas obstructive pulmonary diseases are known risk factors for bone loss, the underlying relationship between lung inflammation and bone health is not well known. RECENT FINDINGS: An agriculture organic dust extract inhalation animal model has recently linked lung injury-induced inflammation to systemic bone loss. This process is dependent upon lipopolysaccharide and the toll-like receptor 4 (TLR4) signaling pathway. Downstream systemic interleukin-6 is a key mediator that subsequently activates osteoclastogenesis. Age is a host factor that impacted bone disease with younger mice demonstrating increased susceptibility to bone loss following inhalant exposures as compared to older mice. Supplemental dietary vitamin D was shown to prevent organic dust-induced bone loss, but not lung disease, in animals. Recent animal studies provide new mechanistic insight into the lung-bone inflammatory axis. Host factors, diet, and lipopolysaccharide/TLR4 signaling pathways play a significant role in explaining how inhalant organic dust exposures impact bone health. These investigations might lead to specific targeted therapeutic approaches.

Rice Bioactive Peptide Binding with TLR4 To Overcome H2O2-Induced Injury in Human Umbilical Vein Endothelial Cells through NF-κB Signaling.

Reactive oxygen species-induced vessel endothelium injury is crucial in cardiovascular diseases progression. Rice-derived bran bioactive peptides (RBAP) might exert antioxidant effect through unknown mechanisms. Herein, we validated the antioxidant effect and mechanism of RBAP on H2O2-induced oxidative injury in human umbilical vein endothelial cells (HUVECs). Here, HUVECs were treated with RBAP under H2O2 stimulation; the effects of RBAP on HUVECs oxidative injury were evaluated. H2O2 injury-induced cell morphology changes were ameliorated by RBAP. The effect of H2O2- on HUVEC apoptosis (percentage of apoptotic cell: 38.00 ± 2.00 in H2O2 group vs 21.07 ± 2.06 in RBAP + H2O2 group, P = 0.0013 compared to H2O2 group), the protein levels of cleaved caspase-3 (relative protein expression: 2.90 ± 0.10 in H2O2 group vs 1.82 ± 0.09 in RBAP + H2O2 group, P < 0.0001 compared to H2O2 group) and p-p65 (relative protein expression: 1.86 ± 0.09 in H2O2 group vs 1.35 ± 0.08 in RBAP + H2O2 group, P < 0.0001 compared to H2O2 group) could be attenuated by RBAP. RBAP exerts its protective function through binding with Toll-like receptor 4 (TLR4). Taken together, RBAP protects HUVECs against H2O2-induced oxidant injury, which provided the theoretical basis for the molecular mechanism of rice deep processing and exploitation of functional peptides.