Effect of Coptidis Rhizoma-Zingiberis Rhizoma Extract on Colonic Epithelium TLR4/NF-κB Signaling Pathway in Mice with Ulcerative Colitis / 中国实验方剂学杂志

Objective::To observe the effect of berberine and 6-shogaol, main components of Coptiae Rhizoma and Zingiberis Rhizoma, on the inflammatory signaling pathway of Toll-like receptors 4 (TLR4)/nuclear factor kappa B (NF-κB) in colonic epithelial cells of mice with ulcerative colitis. Method::Fifty Kunming mice were randomly divided into normal group, model group, berberine group (100 mg·kg-1), 6-shogaol group (100 mg·kg-1), and 6-shogaol combined with berberine group (200 mg·kg-1), with 10 mice in each group. A mouse model of ulcerative colitis was established through oral administration with 2% dextroan sulfate for two weeks. Each group was given corresponding drugs by gavage, while normal group and model group were given equal amount of normal saline. Serum and colon tissue samples were taken 20 days after administration. Enzyme-linked immunosorbent method was used to detect serum interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) expressions, and real-time fluorescence quantitative polymerase chain reaction(Real-time PCR) and Western blot method were used to detect TLR4, NF-κB p65 mRNA and protein expressions in colon epithelial tissue. Result::Compared with the normal group, relative expressions of TLR4 and NF-κB p65 mRNA and protein were increased in the model group (P<0.01), and the contents of serum IL-1β and TNF-α were increased (P<0.01). Compared with the model group, relative expressions of TLR4 and NF-κB p65 mRNA and protein were significantly decreased in 6-shogaol group, berberine group and 6-shogaol combined with berberine group (P<0.01), and the contents of serum IL-1β and TNF-α were significantly decreased (P<0.01). Among the three groups, 6-shogaol combined with berberine group had the strongest effect (P<0.01). Conclusion::Both 6-shogaol and berberine can inhibit colonic inflammation, reduce inflammatory damage and treat ulcerative colitis. The combined application of 6-shogaol and berberine has a significant synergism effect. The mechanism is related to the excessive activation of TLR4/NF-κB pathway and the regulation of non-controllable intestinal inflammation.

TLR4/NF-κB signaling pathway regulates inflammatory response involved in pathophysiological mechanisms of type A aortic dissection / 中国胸心血管外科临床杂志

@#Objective     To investigate activated toll-like receptor-4 (TLR4) signaling pathway involved in pathophysiological mechanisms of type A aortic dissection (TAAD). Methods     Specimens of full-thickness ascending aorta wall from the TAAD patients (n=12) and the controlled donors (n=12) were collected. Western blotting was used to examine the associated proteins' expression of TLR4 signaling pathway. Blood samples from TAAD (n=43) and controlled patients (n=50) were examined by enzyme-linked immunosorbent assay (ELISA) to detect the circulating plasma cytokines levels of interleukin-1β (L-1β). Results     In the aortic wall of TAAD, expression levels of TLR4 and protein expression of major molecule significantly elevated, and activated macrophages increased. Furthermore, elevated IL-1β levels were observed in the TAAD patients’ plasma compared with the control plasma. Multiple logistic regression analysis and receiver operating characteristic (ROC) curve showed that elevated IL-1β could be a novel and promising biomarker with important diagnostic and predictive value in the identification of TAAD. Conclusion     Activated TLR4/NF-κB signaling pathway regulates inflammatory response to involve in pathophysiological mechanisms of type A aortic dissection and its regulated inflammatory products have important predictive value for patients with TAAD.

Effect of protein kinase D1 on myocardial inflammation and apoptosis in rats with myocardial infarction / 中国药理学通报

Aim To investigate the effect of protein kinase Dl (PKD1) on myocardial inflammation and apoptosis after myocardial infarction and to analyze its molecular mechanism. Methods Forty-five male Wistar rats were randomly divided into three groups: sham-operated group, model group and PKD1 group. Rat models of myocardial infarction were reproduced by classical left coronary artery ligation in model group and PKD1 group, while rats in sham group were operated without ligation of coronary artery. The parameters of hemodynamics in rats were assessed, the morphological changes of myocardial tissue were analyzed by HE staining, the apoptotic changes of myocardial cells were analyzed by TUNEL staining, immunohistochemical staining and Western blot, while the changes of myocardial inflammation were analyzed by immunoblotting and real-time quantitative PCR. Results Compared with model group, PKD1 could improve the hemodynamic indexes in rats with myocardial infarction, reduce the injury caused by myocardial infarction, inhibit apoptosis in myocardial tissue, up-regulate the protein expression of Bcl-2, down-regulate the protein expression of caspase-3, Bax, TLR4, TN-C, NF-ΚB p50, NF-ΚB p65 and decrease the mRNA expression of IL-1, NF-ΚB p50 and NF-ΚB p65, and all the differences were statistically significant (P <0. 01). Conclusion PKD1 might have the biological function of inhibiory effect on inflammation and apoptosis induced by myocardial infarction injury.

Mechanism of Modified Erchentang on Signaling Pathway of TLR4/MyD88/NF-κB in Lung Tissue of Rats with Chronic Obstructive Pulmonary Disease / 中国实验方剂学杂志

Objective: To study the effect of modified Erchentang on expressions of Toll-like receptor 4 (TLR4), myeloid differentiation factor (MyD88) and nuclear factor-κB (NF-κB) genes in the lung tissue homogenate of rats with chronic obstructive pulmonary disease (COPD). Method: Forty SD rats were randomly divided into normal group, model group, modified Erchentang group and EVP4593 (NF-κB inhibitor) group. Rat COPD models were prepared through cigarette smoke and tracheal dripping with lipopolysaccharide (LPS). After the modeling, normal and model groups were intragastrically given normal saline solution, EVP4593 group was given EVP4593(1 mg · kg-1) through subcutaneous injection, and modified Erchentang group was given corresponding herbal drugs intragastrically (10 g · kg-1) for 14 days. The levels of high mobility group box 1(HMGB1), chemokines CXCL-2, CXCL-3 and monocyte chemoattractant protein-1 (MCP-1) in rats serum were detected by enzyme-linked immunosorbent assay in rats serum. The expressions of Toll-like receptors 4(TLR4), myeloid differentiation factor (MyD88) and nuclear factor-κB p65 (NF-κB p65) mRNA were detected by Real-time fluorescence quantitative PCR (Real-time PCR) method. Western blot were used to detect the levels of TLR4, MyD88, NF-κB p65 and p-NF-κB p65 protein. Immunohistochemistry (IHC) method was used to detect the localization and expressions of TLR4, MyD88 and p-NF-κB p65 protein in the lung tissue. Result: The mRNA and protein expressions of TLR4, MyD88 and NF-κB p65 were increased significantly (PPκB p65 mRNA and protein were decreased significantly (PConclusion: Modified Erchentang may inhibit the inflammatory response of COPD effectively. The mechanism may be related to the inhibition of the expressions of the signal molecule genes involved in the TLR4/MyD88/NF-κB pathway and the reduction of the release of HMGB1, CXCL-2, CXCL-3 and MCP-1.

Effect of Lumbricus Peptides on TLR4/NF-<i>κ</i>B Signaling Pathway in Spontaneous Hypertensive Rat Kidney / 中国实验方剂学杂志

@# <b>Objective:</b>to observe the effect and mechanism of lumbricus peptides on early renal injury in spontaneous hypertensive rats (SHR) based on Toll-like receptors 4 (TLR4)/nuclear factor-<i>κ</i>B(NF-<i>κ</i>B) signaling pathway. <b>Method:</b>The 40 SHRs were randomly divided into model group (equal volume of distilled water by intragastric administration), lumbricus peptides low, middle and high dose groups (126, 252, 504 mg·kg^-1), and Benazepril group (0.9 mg·kg^-1), <i>n</i>=8 in each group. 8 male rats with normal blood pressure at the same age were set as the normal control group,with equal volume of distilled water. After treatment for 60 consecutive days, enzyme-linked immunosorbent assay (ELISA) was used to determine microalbumin (mAlb)and <i>N</i>-acetyl-<i>β</i>-<i>D</i>-glucosaminidase (NAG) content in 24 h urine as well as the level of serum angiotensinⅡ (AngⅡ). Ultrastructural changes of rat kidneys were observed by transmission electron microscope. Western blot was used to detect renal TLR4, NF-<i>κ</i>B p65 protein levels. Immunohistochemical method was used to detect renal tumor necrosis factor-<i>α</i> (TNF-<i>α</i>) and interleukin 10 (IL-10) expression. <b>Result:</b>As compared with the normal control group, the levels of urine mAlb, NAG and serum Ang Ⅱ were increased in the model group (<i>P</i><0.05); the expression of TLR4 and NF-<i>κ</i>B p65 protein was increased (<i>P</i><0.05); expression of TNF-<i>α</i> was increased (<i>P</i><0.05), while IL-10 expression was decreased (<i>P</i><0.05). Transmission electron microscopy of kidney tissues showed that most of the glomeruli of the model group had podocyte foot process fusion, mesangial cells and mesangial matrix hyperplasia. As compared with the model group, the levels of urine mAlb, NAG, and serum Ang Ⅱ were decreased in the rats in lumbricus peptides groups and benazepril group (<i>P</i><0.05); the expression of TLR4 and NF-<i>κ</i>B p65 protein was decreased (<i>P</i><0.05); the positive expression of TNF-<i>α</i> in kidney was decreased to different extent (<i>P</i><0.05), but the expression of IL-10 was increased (<i>P</i><0.05). Transmission electron microscopy of kidney tissues showed that the damage of kidneys in rats after administration of high-dose lumbricus peptides and benazepril was improved in varying degrees. <b>Conclusion:</b>lumbricus peptides can reduce early renal damage in SHRs, and its mechanism may be achieved by regulating the AngⅡ-TLR4/NF-<i>κ</i>B pathway.

miR-195 targets TLR4 to inhibit proliferation and metastasis of liver cancer cells via blocking NF-κB pathway / 中国肿瘤生物治疗杂志

@# Objective: To explore the effect of miR-195/TLR4 axis on the proliferation, invasion and migration of liver cancer cells via regulating NF-κB pathway. Methods: Twenty-five pairs of liver cancer tissues and corresponding adjacent tissues surgically resected at the Second Affiliated Hospital of Kunming Medical University from March 2016 to January 2017 were collected for this study. Liver cancer HepG2 cells were cultured and then randomly divided into four groups: control group (NC), miR-195 mimic group (miR-195), TLR4 knockdown group (si-TLR4), and miR-195 inhibitor combined with TRL4 knockdown group (si-TLR4+miR-195 inhibitor). qRTPCR was used to detect the expression of miR-195 in liver cancer tissues and cell lines. CCK-8 assay was used to evaluate the cell viability of each group. Transwell and Wound healing assay were applied to detect the invasion and migration ability of HepG2 cells, respectively. Dual-luciferase reporter gene assay was used to verify the targeted regulation of TLR4 by miR-195. WB was applied to analyze the protein expressions of TLR4 and NF-κB p65. Results: miR-195 was down-regulated in the liver cancer tissues compared with adjacent tissues (P<0.01). Compared with human hepatic epithelial cells (THLE-3), the expression of miR-193 in liver cancer cell lines (HepG2 and Huh-7) was down-regulated (P<0.01), and the expression level in HepG2 cells was the lowest. The proliferation, invasion and migration of HepG2 cells was significantly suppressed after over-expression of miR-195 (all P<0.01). Moreover, over-expression of miR-195 significantly down-regulated TLR4 protein expression (P<0.05), and TLR4 was negatively correlated with miR-195 (R2= 0.602, P<0.0001). Furthermore, miR-195 over-expression inhibited proliferation, invasion and migration of HepG2 cells by targeting TLR4 expression and blocking NF-κB pathway (P<0.05 or P<0.01). Conclusion: miR-195 over-expression can inhibit the proliferation, invasion and migration of HepG2 cells. The mechanism may be related with targeting TLR4 and blocking the NF-κB pathway to affect cell biological behaviors.

Effects of combined administration of Radix Angelicae Sinensis and hydrocortisone on the therapeutic action in the murine asthma model / 中国应用生理学杂志

OBJECTIVE@#To investigate the effects of Radix Angelicae Sinensis (RASI) and hydrocortisone combination on the murine asthma model and the mechanism.@*METHODS@#BALB/c mice were randomly divided into control group, blood stasis model group, asthma model group, HSS group, RASI group and RASI+HSS group (=12). Ovalbumin (OVA) was used to replicate mice asthma model and hydrocortisone sodium succinate (HSS) to copy blood stasis model. Effects of RASI, HSS and their combination on hemorheology, anti-asthma (asthmatic behaviors, lung function, lung index and water content in lung tissue) were observed. and anti-asthma mechanisms The expression of relative cytokines, high-mobility group box 1 (HMGB1), toll-like receptor 4 (TLR4) and nuclear factor-κB (NF-κB) was detected by ELISA and immunohistochemistry respectively.@*RESULTS@#Eight g/kg RASI, 0.05 g/kg HSS and their combination could significantly relieve asthma behavioral indicators, improve lung function, reduce lung index and water content in lung tissue, decrease the levels of TNF-α, IL-1β and IL-6 in broncho-alveolar lavage fluid (BALF), and inhibit the high expression of HMGB1, TLR4 and NF-κB in lung tissue. The improvement of lung function and the decrease in level of relative cytokines (TNF-α、IL-1βIL-6) were better in RASI+HSS group than those in RASI group and HSS group, and the inhibition of protein expression of TLR4 and NF-κB was also too. Combined administration of RASI and hydrocortisone could decrease serum thromboxane B2 (TXB2) content and blood viscosity, which were increased induced by hydrocortisone.@*CONCLUSIONS@#Combined administration of RASI and hydrocortisone have obvious anti-asthma effects and one of the mechanisms is to inhibit protein synthetization of HMGB1, TLR4 and NF-κB.The combined administration of RASI and hydrocortisone has stronger improvement of lung function than that of RASI and hydrocortisone alone, and it may be related to the inhibition of TLR4 and NF-κB synthetization. The combined administration of RASI can alleviate abnormal changes of hemorheology induced by hydrocortisone in treatment of asthma.

Effect of ω-3 polyunsaturated fatty acids and ω-6 polyunsaturated acids on Toll-like receptor /nuclear fac-tor-κB signaling pathway and the inflammatory cytokines in neonatal rats with brain injury induced by lipopolysaccharide / 中华实用儿科临床杂志

Objective To investigate the effects of ω-3 polyunsaturated fatty acids(ω-3PUFAs)and ω-6 polyunsaturated fatty acids(ω-6PUFAs)on Toll-like receptor 4(TLR4)/nuclear factor-κB(NF-κB)signaling pathway,and the expressions of tumor necrosis factor-α(TNF-α),interleukin(IL)-1β and IL-6 in neonatal rats with brain injury induced by lipopolysaccharide (LPS). Methods Ninety-six neonatal rats were divided into control group,ω-3PUFAs group,ω-6PUFAs group,and LPS group by using random number table method. Intraperitoneal injection of LPS was performed in LPS group,ω-6PUFAs group and ω-3PUFAs group to establish models of rat brain injury. The rats in control group received 9 g/L saline. Twelve newborn rats were killed at 1 d or 5 d after intraperito-neal injection in each group for hippocampus selection. Real -time PCR and Western blot were used to detect the mRNA and protein expression levels of TLR4,NF-κB,TNF-α,IL-1β and IL-6. Results One day after mode-ling,TLR4,NF-κB,TNF-α,IL-1β and IL-6 mRNA expressions in ω-3PUFAs group (10. 63 ± 0. 07,5. 86 ± 1. 05,7. 65 ± 2. 29,5. 23 ± 1. 31,3. 36 ± 0. 72)were lower than those in ω-6PUFAs group (18. 83 ± 2. 10,8. 79 ± 2. 08,11. 95 ± 3. 23,10. 97 ± 2. 24,6. 37 ± 1. 17)and LPS group (15. 76 ± 1. 59,7. 13 ± 1. 10,9. 71 ± 2. 14,7. 83 ± 0. 85,4. 78 ± 0. 51),and the differences were all statistically significant(all P＜0. 05);which in ω-6PUFAs group were higher than those in LPS group,and the differences were all significant (all P＜0. 05). TLR4,NF-κB,TNF-α, IL-1β and IL-6 protein levels in ω-3PUFAs group (1. 57 ± 0. 11,1. 58 ± 0. 09,1. 55 ± 0. 09,1. 63 ± 0. 31,1. 36 ± 0. 12)were lower than those in ω-6PUFAs group (1. 96 ± 0. 17,2. 21 ± 0. 12,1. 95 ± 0. 23,1. 97 ± 0. 24,1. 77 ± 0. 17)and LPS group (1. 73 ± 0. 15,1. 87 ± 0. 10,1. 79 ± 0. 14,1. 83 ± 0. 15,1. 58 ± 0. 11)in 1 d,and the diffe-rences were all significant (all P＜0. 05),and those in ω-6PUFAs group were higher than those in LPS group (all P＜0. 05). Similarly,TLR,NF-κB,TNF-α,IL-1β and IL-6 mRNA and protein expression levels in ω-3PUFAs group (3. 78 ± 0. 88,3. 86 ± 0. 62,6. 26 ± 1. 94,3. 65 ± 1. 44,2. 11 ± 0. 87;1. 15 ± 0. 08,1. 32 ± 0. 10,1. 46 ± 0. 04, 1. 38 ± 0. 14,1. 21 ± 0. 09)were lower than those in ω-6PUFAs group (7. 76 ± 1. 65,5. 51 ± 0. 88,7. 96 ± 2. 13,5. 35 ± 1. 75,4. 88 ± 1. 35;1. 42 ± 0. 15,1. 51 ± 0. 36,1. 65 ± 0. 13,1. 72 ± 0. 23,1. 48 ± 0. 10)and LPS group (6. 21 ± 1. 87, 4. 98 ± 0. 73,7. 11 ± 2. 10,4. 84 ± 1. 75,4. 25 ± 0. 64;1. 35 ± 0. 13,1. 44 ± 0. 22,1. 59 ± 0. 10,1. 61 ± 0. 18,1. 35 ± 0. 07) in 5 d (all P＜0. 05),and which in ω-6PUFAs group were higher than those in LPS group,and the differences were sig-nificant (all P＜0. 05). Conclusion ω-6PUFAs can up-regulate the activity of TLR4,NF-κB,and reduce the re-lease of TNF-α,IL-1β and IL-6;and ω-3PUFAs can down-regulate the activity of TLR4,NF-κB,and reduce the release of TNF-α,IL-1β and IL-6,so it has a neural protective effect in brain injury induced by LPS.

Effects of Honokiol on airway inflammation in asthmatic mice exposed to PM2.5 and its mechanism / 中华实用儿科临床杂志

Objective To investigate the protective effect of Honokiol on the airway inflammation induced by particulate matter 2.5(PM2.5)in the asthmatic mice and its mechanism.Methods Fifty male specific pathogen free (SPF)Balb/c mice were randomly divided into 5 groups.Group A:normal control group;group B:asthmatic model group;group C:PM2.5 exposure asthmatic group;group D:TAK -242 group;group E:Honokiol group. Asthmatic mouse models were established by ovalbumin(OVA)sensitization and challenge.On days 0 and 7,the mice in B-E groups were injected intraperitoneally with injection 100 mg/L OVA and aluminum hydroxide for sensitization;on days 14 to 21,10 g/L OVA solution was given 30 min per day to challenge.During challenge phrase,the mice in C -E groups received intratracheal injection of PM2.5,every other day,4 times totally.On this basis,the mice in group D re-ceived TAK-242 intraperitoneal injection,and the mice in group E received honokiol intragastric administration.Group A was given saline instead of OVA.Animals were sacrificed 24 h after the final inhalation challenge,and the bronchoal-veolar lavage fluid(BALF)of the left lung was used for differential inflammatory cell counts.The expressions of Toll-like receptors 4(TLR4)and nuclear factor(NF)-κB at mRNA level were detected by real-time quantitative PCR. Flow cytometry analysis was performed to measure the levels of Th17 and Treg cells.Results Compared with group A,mice in group B and group C expressed more serious disorders of bronchial epithelial cells,alveolar wall congestion and edema,increased mucus secretion in the airway and infiltration of inflammatory cells in the lung,and those in group C were more obvious than those of group B and group E significantly reduced respiratory inflammation;compared with group A[(4.15 ± 1.35)×108/L,0.012 0 ± 0.002 3],the total number of inflammatory cell counts[(16.79 ± 5.62)×108/L and(24.58 ± 13.46)×108/L],eosinophils proportions(0.113 8 ± 0.022 3 and 0.197 8 ± 0.084 9)in group B and group C,were significantly higher,and the differences were statistically significant(all P<0.05);The total number of inflammatory cell counts and eosinophils proportion in group E(8.56 ± 3.28)×108/L and 0.041 5 ± 0.013 5)were significantly lower than those in group C,and the differences were statistically significant(all P <0.05);The expressions of TLR4 mRNA and NF-κB mRNA in group B and C(1.85 ± 0.56,1.82 ± 0.28 and 2.97 ± 0.41,2.83 ± 0.32)were significantly higher,and the differences were statistically significant(all P <0.05);The expressions of TLR4 mRNA and NF-κB mRNA in group E(1.60 ± 0.28,1.54 ± 0.25)was significantly lower than those in group C,and the differences were statistically significant(all P<0.05);the expressions of Th17 in group B and C[(2.89 ± 0.61)% and(4.96 ± 0.27)%]were significantly higher than those of group A[(1.03 ± 0.35)%] (all P<0.05);The expression of Th17 in group E[(1.83 ± 0.23)%]was significantly lower than that of group C,and the differences were statistically significant(P<0.05);the expressions of Treg in group B and C[(4.96 ± 0.35)%and(2.27 ± 0.41)%]were significantly lower than those of group A[(7.37 ± 0.56)%],and the differences were sta-tistically significant(all P<0.05);The expression of Treg in group E was significantly increased[(6.45 ± 0.38)%] compared with that in group C,and the difference were statistically significant(P<0.05);and those of group D and E were improved remarkably.Conclusions Honokiol can relieve PM2.5 exposure of asthmatic airway inflammation through down-regulating the expression of TLR4 and NF-κB and Th17 and regulating the balance of Th17 and Treg cells.

Protective effects of esmolol on lung function in septic rat / 中华急诊医学杂志

Objective To explore the protective effects of short-acting β31 receptor blocker esmolol on lung injury in septic rats and explore its mechanism.Methods Fifty-four male SpragueDawley rats were randomly(random number) divided into three groups (n=18 in each),namely sham operation group(Sham),sepsis group(Spesis),esmolol group(ES).Each group were further divided into three subgroups of 6 h,12 h and 24 h(n=6 in each subgroup).The sepsis models were established with cecal ligation and puncture (CLP) in rats of Sepsis group and ES group,while rats of SH group were treated with laparotomy without CLP.Dwelled cannulation in the left internal jugular vein was performed after the model establishment.Saline in the rate of 1 mL/h was continuously pumped intravenously into the rats of SH group and Sepsis group by micro pump for 6 h,and esmolol solution in the rate of 1 mL/h [15 mg/(kg·h)]was continuously pumped into the rats of ES group for 6h.Rats in each subgroup were sacrificed at 6h,12h and 24 h after modeling,separately.The levels of catecholamine (CA) in plasma and levels of NF-κB in lung tissue were measured by ELISA.The protein levels of TLR4 and NF-κB in lung tissue were detected by Western Blot.The protein content of alveolar lavage fluid,wet/dry weight ratio(W/D) of lung tissue,lung coefficient and lung water content were measured.The pathological changes of lung tissues were observed under an optical microscope.Results (①) The levels of CA in plasma in Sepsis and ES groups at 6 h after modeling was significantly increased compared with Sham group (P＜0.05),and the level of CA in plasma increased significantly in ES group compared with Sham and Sepsis groups (P＜0.05).At 12 h after modeling,level of CA in plasma was still significantly increased in ES group compared with SH group (P＜0.05);At 24 h after modeling,level of CA in plasma was increased significantly in ES group compared with Sepsis group (P＜0.05).(②) At 6 h,12 h and 24 h after modeling,levels ofNF-κ B in lung tissue were significantly increased in Sepsis group and ES group compared with SH group (P＜0.05),and levels of NFκ B in lung tissue in ES group were also significantly lower than those in Sepsis group at given intervals (P＜0.05).③ At 6 h,12 h and 24 h after modeling,protein content of alveolar lavage fluid,wet/dry weight ratio (W/D) in lung tissue,lung coefficient and lung water content in Sepsis group and ES group were significantly increased compared with SH group (P＜0.05),and protein content of alveolar lavage fluid,wet/ dry weight ratio (W/D) in lung tissue,lung coefficient and lung water content in ES group also significantly lower than those in Sepsis group at given intervals (P＜0.05).④ Western Blot revealed at 6 h,12 h and 24 h after modeling,the protein levels of TLR4 and NF-κ B in lung tissue in Sepsis group and ES group were significantly increased compared with SH group (P＜0.05),and the protein levels of TLR4 and NF-κ B in lung tissue of ES group were also significantly lower than those in Sepsis group at all given intervals (P＜0.05).⑤ The lightest degree of pathological change was observed in SH group,the most serious degree of pathological change was found in Sepsis group,and the pathological change was significantly alleviated in ES group compared with Sepsis group whereas the pathological change was significantly increased compared with SH group at all given intervals.Conclusions Esmolol can promote the secretion of catecholamine,inhibit the release of inflammatory cytokines,reduce lung permeability,reduce pulmonary edema,and thus play an important role in protecting the lungs.The mechanism may be that esmolol improves the responsiveness of β-adrenergic receptor to catecholamine,and regulates the level of inflammatory cytokines by inhibiting TLR4-NF-κB-TNF-α signaling pathways,thus exerting protective effect on the lungs.

Angiotensin 1-7 antagonizes cerebral ischemia-reperfusion injuries in mice through the TLR4/NF-κB signaling pathway / 国际生物医学工程杂志

Objective To investigate the protective effect and mechanism of angiotensin-(1-7) on cerebral ischemia-reperfusion injury in rats.Methods A rat model of middle cerebral artery occlusion (MCAO) reperfusion was established by non-invasive arteriolar clamping of bilateral common carotid artery in SD rats.The sham operation group and the model group were given artificial cerebrospinal fluid,and the low,medium and high dose treatment groups were given Ang-(1-7),with a dose of 1 pmol/0.5 μl/h,100 pmol/0.5 μl/h and 10 nmol/0.5 μl/h,respectively.After 24 h of arterial occlusion and reperfusion,Garcia JH standard was used to evaluate the neurological function of rats.The water content of brain tissue was determined by gravimetric method.The volume of cerebral infarction was determined by TIC staining method.The expression of Toll-like receptors 4 (TLR4) and nuclear factor-κB (NF-κB)p65 protein in ischemic parietal cortex were determined by Western Blot.The content of serum intercellular cell adhesion molecule-1(ICAM-1) and tumor necrosis factor-α (TNF-α) were detected by ELISA.Results Compared with the model group,the low,medium and high Ang-(1-7) dose groups significantly improved the neurological function scores of MACO rats (all P＜0.05).Compared with the model group,the brain water content of the high-dose Ang-(1-7) treatment group was significantly decreased (P＜0.05).Compared with the model group,the cerebral infarction volume of the rats in the low,medium and high Ang-(1-7) dose groups was significantly reduced (all P＜0.05).Compared.with the model group,the expression of TLR4 and NF-κB p65 protein in the middle and high-dose Ang-(1-7) treatment group was decreased (all P＜0.05),and the levels of ICAM-1 and TNF-α in serum was decreased (all P＜0.05).Conclusions Ang-(1-7) has a protective effect on cerebral ischemia-reperfusion injury of MACO rats,and its mechanism may be related with the inhibition of TLR4/NF-κB pathway.

The expression and significance of Toll-like receptors-4 in peripheral blood and placental tissuein gestational diabetes mellitus patients / 国际检验医学杂志

Objective To investigate the expression and significance of Toll-like receptors-4 in peripheral blood and placental tissue in gestational diabetes mellitus (GDM) patients.Methods From February 2013 to February 2015.a total of 30 cases of gestational diabetes mellitus patients(GDM group) and 30 cases of normal pregnant people(health group)were selected as research objects.Peripheral blood and placental tissue of two groups were collected.The expression of TLR4 mRNA in peripheral blood mononuclear cells was detected by RT-PCR,the expression of TLR4 protein in placenta tissue was detected by immunohistochemistry.Results The expression of TLR4 mRNA in peripheral blood mononuclear cells of the observation group was significantly higher than that of the health group[(0.63±0.12) vs.(0.32±0.07)],the difference was statistically significant(t=12.223,P<0.05).The expression of TLR4 in villous trophoblast cells,decidual cells and amniotic epithelial cells in GDM group was significantly higher than that in the health group(Z=2.325,2.374,2.162,P<0.05).Conclusion The expression of TLR4 in peripheral blood mononuclear cells,villous trophoblast cells and decidual cells in gestational diabetes mellitus patients significantly enhanced,suggesting that TLR4 might be related to the e gestational diabetes mellitus.

Effects of vinpocetine on inflammation of brain in rats with intracerebral hemorrhage / 中国病理生理杂志

AIM:To investigate the effects of vinpocetine on inflammation of brain in intracerebral hemorrhage ( ICH) rats and to explore the underlying mechanisms .METHODS:All rats were randomly divided into sham group , ICH group, ICH with low dose of vinpocetine treatment group , ICH with medium dose of vinpocetine treatment group , and ICH with high dose of vinpocetine treatment group .Except sham group , the rats in other groups were injected with type VII col-lagenase to establish ICH model , and then the rats in vinpocetine treatment groups were received vinpocetine at 0.5, 1.0 or 1.5 mg/kg by intraperitoneal injection once a day for 7 days.After corresponding treatment , the impairment of neurological function in the rats was scored and the water content of the brain tissues was measured .Moreover, the activity of myeloper-oxidase (MPO) was determined by ELISA, and the protein expression of Toll-like receptors 4 (TLR4), nuclear factor-κB (NF-κB), intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molcule-1 (VCAM-1) in the brain tissues was determined by Western blot .RESULTS: Compared with ICH group , vinpocetine treatment significantly de-creased the scores of the impairment of neurological function and the water content of the brain tissues .Moreover, the activ-ity of MPO and the protein expression of TLR4, NF-κB, ICAM-1 and VCAM-1 were also reduced after vinpocetine treat-ment (P<0.05).CONCLUSION:Vinpocetine improves neurological function in ICH rats via suppression of inflamma -tion by inhibiting NF-κB signaling and expression of ICAM-1 and VCAM-1.

The effect of Toll-like receptor 4 in nicotine suppressing the osteogenic potential of periodontal ligament stem cells / 华西口腔医学杂志

Objective To explore the impact of nicotine on proliferation and osteogenic capability of periodontal ligament stem cells (PDLSCs), and the role of Toll-like receptor 4 (TLR4) in nicotine, suppressing the osteogenic capability of PDLSCs. Methods PDLSCs were cultured in vitro, and the flow cytometer was used to identify the surface antigen markers of PDLSCs. WST-1 was used to detect the proliferation ability of PDLSCs, which were stimulated by different concentrations of nicotine. Alizarin red staining was used to observe the formation of mineralized nodules after PDLSCs stimulation with different concentrations of nicotine. Real-time polymerase chain reaction (RT-PCR) and Western blot were used to detect the change in osteogenic potential of PDLSCs stimulated by nicotine, after TAK-242, and with the inhibitor of TLR4. Results PDLSCs expressed mesenchymal stem cell-associated markers CD90 and CD105. When the concentration of nicotine was 10⁻⁴ mol·L⁻¹, the PDLSC proliferation could be suppressed after 3 d compared with the control group (P<0.05). The amount of mineralized nodules reduced after osteogenic differentiation at 21 d by alizarin red staining. RT-PCR and Western blot showed the expression levels of alkaline phosphatase (ALP), and osteocalcin (OCN), and the Runt-related transcription factor-2 (Runx-2) were lower than in the control group when nicotine suppressed the PDLSCs (P<0.05). This effect was attenuated after TAK-242 was added. Conclusion Nicotine suppresses the proliferation and osteogenic capability of PDLSCs, which may be regulated by TLR4.

The expression of HMGB1 and TLR4 in pancreatic tissue of rats with severe acute pancreatitis and the intervention effects of Ulinastatin / 重庆医学

Objective To explore the mechanism of HMGB1 and TLR4 in pancreatic tissue of rats with severe acute pancreatitis and the intervention effect of Ulinastatin .Methods The 54 SD rats were completely random divided into control group ,SAP group and Ulinastatin treatment group ,and each group was divided into three groups :6 ,12 h and 24 h groups (each group n=6) .In con‐trol group ,we turned the pancreatic tissue ,in SAP group ,the SAP model was made with 5% taurocholic acid ;and in the treatment group ,and intravenous injection of ulinastatin was conducted after the SAP model was successfully made .Then we observed the pancreatic tissue pathology in the three groups .The amylase in serum was detected by EPS‐G7 assay ,the HMGB1 in serum and pancreatic tissue was detected by ELISA assay ,the expression levels of HMGB1 and TLR4 in pancreatic tissue were detected by Envision two‐step immunoassay .Results Compared with control group ,the amylase of each time point in SAP group and treatment group were significantly higher ,and the pathology changed obviously (P<0 .05) ,and the SAP model was successfully made .The HMGB1 expression in pancreatic tissue and serum started increase at 6 h ,increased quickly at 12 h and maintained the increasing trend to 24 h in SAP group and it was significantly higher at the same time point compared with that of control group (P<0 .05);at the same time point ,the HMGB1 in treatment group was significantly lower than that of SAP group (P<0 .05);in SAP group , the expression of TLR4 in pancreatic tissue started increasing at 6 h ,reached its peak at 12 h and started decreasing at 24 h ,it was significantly higher than the control group at the same time point (P<0 .05) .At the same time point ,the TLR4 was significantly lower in the treatment group than SAP group (P<0 .05) .Conclusion The proinflammatory effect of HMGB1 in SAP rats pancre‐atic could be partly combine its receptor TLR4 and MyD88‐dependent pathway through implementation ,and the protecting mecha‐nism of Ulinastatin could be interrupt the HMGB1 and TLR4 signaling pathway in SAP rats pancreatic tissue .

Effects total glucosides paeony on downregulating the expression ERKI/2, TLR4 and TRL9 in rats with nonalcoholic fatty liver disease / 中国药学杂志

OBJECTIVE: To investigate total glucosides paeony (TGP) on the expression phosphorylated extracellelar signal regulated kinase 1/2(p-ERK1/2), Toll-like receptors 4(TLR4) and Toll-like receptors 9(TLR9) in rats with nonalcoholic fatty liver disease (NAFLD) induced by fructose and high-fat feed. METHODS: SD rats were divided into normal group and test groups. The rats in test groups were fed with fructose and high-fat feed for 10 weeks totally to induce the test model. After 6 weeks the model was established, the rats were divided into four groups randomly, the model group (NAFLD), the metformin group (Met, 200 mg · kg-1), the low-dose TGP group(TGP-L, 100 mg · kg-1) and the high-dose TGP group(TGP-H, 200 mg · kg-1) (n=10). Four weeks later all the rats were killed and checked the indexes such as serum fasting blood glucose(FBG), fasting insulin(Fins), insulin sensitivity index (ISI), total cholesterol (TC), low-density lipoprotein (LDL-C), high-density lipoprotein (HDL-C), triglyceride (TG), free fatty acids (FFA), alanine aminotransferase (ALT), aspartate aminotransferase (AST), glutathione S-transferase (GST) and liver index. The expression p-ERK1/2, TLR4 and TLR9 were inspected by Western-blot. RESULTS: Compared with the normal group, the rats in test groups were with the high levels serum FBG, insulin, TC, LDL-C, TG, FFA, ALT, AST, GST, liver index, p-ERK1/2, TLR4 and TLR9 (P 0.05). CONCLUSION: By downregulating the expression ERK1/2, TLR4 and TRL9, TGP can improve abnormal glucose and lipid metabolism and insulin resistance, enhance insulin sensitivity and ameliorate liver function in rats with NAFLD induced by fructose and high-fat feed.

Expression of Toll-like receptor 4 and protein of PI3 K/AKT signal pathway in cervical lesion and their relationship with HPV 16 infection / 临床与实验病理学杂志

Purpose To explore the expression of Toll-like receptor 4 (TLR4), PI3K, AKT and NF-κB in cervical lesions, and to in-vestigate their association with human papillomavirus ( HPV) 16 infection. Methods Immunohistochemical SP staining was performed to detect the expression of TLR4, PI3K, AKT, NF-κB in paraffin-embedded cervical tissue specimens from Uighur women with chroni-cal cervicitis, cervical intraepithelial neoplasia ( CIN) and cervical squamous cell carcinoma ( CSCC) . The HPV 16 DNA was detected by PCR. Results The positive expression rates of TLR4, PI3K, AKT, NF-κB in chronical cervicitis, CIN and cervical cancer were 32. 0%, 59. 4%, 77. 8%, 28. 0%, 56. 3%, 73. 0%, 24. 0%, 56. 3%, 79. 4%, and 8. 0%, 48. 4%, 81. 0%, respectively. The expression of them was higher in cervicitis than in CIN and cevical cancer ( P<0. 05 ) . The positive expression rates of HPV 16 in three groups were 8. 0%, 48. 4% and 81. 0% (P<0. 05). The expression of TLR4, PI3K, NF-κB and HPV 16 was related to cervi-cal cancer differentiation (P<0. 05). PI3K and AKT were significantly correlated with FIGOs’ stages (P<0. 05). NF-κB was corre-lated with lymph node metastasis. The expression of TLR4 was significantly associated with HPV 16 infection in CIN and CSCC ( r=0. 303, P=0. 015, r=0. 633, P=0. 000), and correlation with PI3K in CIN and CSCC (r=0. 254, P=0. 045, r=0. 386, P=0. 003). PI3K was associated with AKT only in CSCC (r=0. 298, P=0. 018). Conclusions The expression of TLR4 can be up-regulated by HPV 16 infection. High expression of PI3K/AKT signal pathway mediated by TLR4 may play important roles in the devel-opment and progression of CIN and CSCC, and HPV 16 infection may be a trigger factor affecting the molecular signal pathway.

Triptolide inhibited the invasion ability of pancreatic cancer cells through TLR4/NF-kB signaling pathway / 中华胰腺病杂志

Objective To investigate the role of TLR4/NF-kB signaling pathway in inhibited invasion ability of pancreatic cancer cells caused by triptolide (TP).Methods PANC1 cells were divided into parental cells group,TP group,lipopolysaccharide (LPS) group and TP + LPS group.50 ng/ml of TP was added in culture medium in TP group,and 1 μg/ml of LPS was added in culture medium in LPS group,while 50 ng/ml of TP was pretreated for 2 h and 1 μg/ml of LPS was added in culture medium in TP + LPS group.All the ceils were cultured for 24 h.The TLR4 and matrix metalloproteinase-9 (MMP-9) mRNA and protein expression were evaluated by real-time PCR and Western blot.The NF-kB activity was determined by dual-luciferase reporter assay system.The invasion ability of pancreatic cancer cells was evaluated by transwell invasion chamberassay.Results The TLR4 mRNA expressions in parental cells group,TP group,LPS group and TP + LPS group were 0.41 ± 0.06,0.46 ± 0.10,0.20 ± 0.04,0.25 ± 0.06 ; the TLR4 protein expressions were 0.55 ±0.06,0.55 ±0.06,0.18 ±0.04,0.13 ±0.00; the activities of NF-kB were 13.0 ±3.0,31.6 ±4.3,7.3 ±1.5 and 10.8 ± 2.1,and the numbers of invasion cell were (56.8 ± 8.6),(104.5 ± 12.8),(32.0 ± 5.7) and (46.8 ± 7.0) ; the MMP-9 mRNA expressions were 0.36 ± 0.05,0.58 ± 0.07,0.18 ± 0.03,0.30 ± 0.004 ;the MMP-9 protein expressions were 0.31 ± 0.04,0.53 ± 0.08,0.11 ± 0.02,0.15 ± 0.00.In LPS group,TLR4 mRNA and protein expressions were not statistic significant when compared with those in parental cells group,but the activities of NF-kB,the numbers of invasion cell,MMP 9 mRNA and protein expressions were statistically increased when compared with those in parental cells group (t =8.654,7.593,6.655,4.982,P ＜0.01).TLR4 mRNA and protein expressions,activities of NF-kB,the numbers of invasion cell,MMP 9 mRNA and protein expressions in TP group were significantly lower than those in parental cells group (t =-7.609,-9.948,-4.176,-5.915,-8.179,-9.948,P＜ 0.01).TLR4 mRNA and protein expressions,activities of NF-kB,the numbers of invasion cell,MMP 9 mRNA and protein expressions in TP +LPS group were significantly lower than those in LPS group (t =-4.437,-14.805,-10.506,-9.700,-9.055,-8.932,P＜ 0.01).Conclusions TP can inhibit pancreatic cancer cell invasion,and the mechanism is related to the inhibition of TLR4/NF-kB signaling pathway and down-regulation of MMP-9 expression.

Effects of preconditioning with different concentrations of sevoflurane on the expression of Toll-like receptor 4 in rats with acute lung injury induced by lipopolysaccharide / 中华麻醉学杂志

Objective To investigate the effects of preconditioning with different concentrations of sevoflurane on the expression of Toll-like receptor 4 (TLR4) in rats with acute lung injury (ALI) induced by lipopolysaccharide (LPS).Methods Thirty adult male Sprague-Dawlcy rats,wcighing 200-250 g,were randomly divided into 5 groups (n =6 each):normal saline group (group NS),group ALI,and preconditioning with different concentrations of sevoflurane groups (groups S1-3).ALI was induced by intratracheal instillation of LPS 5 mg/kg.Groups S1-3 inhaled 1.2％,2.4 ％ and 4.8 ％ sevoflurane for 30 min respectively,and ALI was induced 30 min later.The rats were sacrificed at 12 h after administration of LPS or normal saline and lungs were removed for microscopic examination and for determination of TLR4 mRNA and protein expression in lung tissues and concentrations of TNF-α and IL-1β in broncho-alveolar lavage fluid (BALF).W/D lung weight ratio was calculated.Results Compared with group NS,W/D lung weight ratio and concentrations of TNF-a and IL-1β in BALF were significantly increased and the expression of TLR4 mRNA and protein was up-regulated in groups ALI and S1-3 (P ＜ 0.01).Compared with group ALI,the parameters mentioned above were significantly decreased in groups S2 and S3,and the concentrations of TNF-α and IL-1 β in BALF were significantly decreased and the expression of TLR4 mRNA was down-regulated in group S1 (P ＜ 0.05 or 0.01).The parameters mentioned above were significantly lower in groups S2 and S3 than in group S1 (P ＜ 0.05).The pathologic changes were significantly attenuated in groups S1-3 compared with group ALI.Conclusion Preconditioning with sevoflurane can concentration-dependently reduce LPS-induced ALI in rats through inhibiting the up-regulation of TLR4 expression in lung tissues and reducing the inflammatory response.

The role of TLR2, TLR4 and its signal pathway in gouty arthritis / 中华风湿病学杂志

objective The roles of TLRs and their signal pathway in gouty arthritis(GA)were explored.Methods TLR2 and TLR4 mRNA was measured using real-time quantitative polymerase chain reaction(RT-PCR)in PBMCs,IL-1β level was detected using ELISA in plasma,and NF-κB p65 protein level in PBMCs was measured using Western blot.Level of TLR2 mRNA,ILR4 mRNA,IL-1β,NF-κB p65protein was compared among acute GA,non-acute GA and healthy controls.Correlation between TLR2mRNA,TLR4 mRNA and serum uric acid,IL-1β level in GA patients was analyzed.One-way ANOVA was used to analyze data between multiple groups and q-test was used for two-two comparison.Spearman's analysis was applied for correlation analysis.Resuits The expression of TLR4 mRNA,NF-KB p65 protein,IL-1β arid serum uric acid level in patients with acute GA [(5.0±1.2), (7.11±0.18), (283±83)pg/ml,[585±123)μmol/L] was significantly increased compared to non-acute GA[(2.3±0.4),(0.63±0.06),(134±29)pg/ml,(493±107)μmol/Lj and healthy controls(1.1±0.6),(0.52±0.12),(97±17)pg/ml,(326±65)μmol/L](P<0.01,respectively).Significant diffefence was also observed between non-acute GA patients and healthy controls(P<0.05,respectively).The level of TLRR4 mRNA was positively correlated with uric acid and IL-1β level in GA patients(rs=0.876,0.779;P<0.05,respectively).Conclusion Innate immunity are activated by membrane-type pattern recognition receptors in primary GA.TLR4-NFκB p65-IL-1β signat transduction may participate in the inflammatory mechanisms of gout.Urate crystals in patients with gout may:be involved in the activation of TLR4 and its signal pathway.