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.

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.

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.

[Effects of combined administration of Radix Angelicae Sinensis and hydrocortisone on the therapeutic action in the murine asthma model].

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 (n=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-1ß 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-1ßIL-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.

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.

TLR4 rs41426344 increases susceptibility of rheumatoid arthritis (RA) and juvenile idiopathic arthritis (JIA) in a central south Chinese Han population.

BACKGROUND: The aim of the study was to determine whether polymorphisms in toll-like receptor 4 (TLR4) confer susceptibility to rheumatoid arthritis (RA) and juvenile idiopathic arthritis (JIA) in a central south Chinese Han population. METHODS: Genotyping for six well studied polymorphisms (rs4986790, rs4986791, rs10759932, rs41426344, rs11536889 and rs7873784) in TLR4 gene were conducted in 1074 unrelated patients with RA and 1692 healthy control subjects, as well as in 217 unrelated patients with JIA and 378 healthy control subjects using direct sequencing technique. Comparisons between cases and controls in alleles, genotypes and haplotypes were carried out using Fisher's exact test. RESULTS: Significant genetic associations were detected between the 3'UTR rs41426344C and RA (p < 0.001, p adj < 0.001, OR = 2.24) and JIA (p < 0.001, p adj < 0.001, OR = 2.05). In addition, rs4986790G was found to be significantly associated with the susceptibility for RA (p = 0.005, p adj = 0.03, OR = 3.43), but not for JIA (p = 0.06, p adj = 0.36, OR = 2.65). Furthermore, significant increasing in the distributions of haplotypes H4 and H10 in RA (H4: p = 0.001, OR = 1.13; H10: p = 0.001, OR = 1.15) and JIA (H4: p = 0.04, OR = 2.06; H10: p = 0.02, OR = 2.47) were also found. Moreover, the frequency of rs41426344C significantly increased in RF-positive and anti-CCP positive subjects both in RA (RF+: p <0.0001, OR = 2.33; anti-CCP+: p =0.008, OR = 2.79) and JIA (RF+: p =0.02, OR = 2.91; anti-CCP+: p = 0.02, OR = 2.78). CONCLUSIONS: Our study suggested that rs41426344 and rs4986790 of TLR4 might contribute to RA, and rs41426344 might contribute to JIA pathogenesis in central south Chinese Han population.

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.