Starring role of toll-like receptor-4 activation in the gutliver axis

Since the introduction of the term "gut-liver axis", many studies have focused on the functional links of intestinal microbiota, barrier function and immune responses to liver physiology. Intestinal and extra-intestinal diseases alter microbiota composition and lead to dysbiosis, which aggravates impaired intestinal barrier function via increased lipopolysaccharide translocation. The subsequent increased passage of gut-derived product from the intestinal lumen to the organ wall and bloodstream affects gut motility and liver biology. The activation of the toll-like receptor 4(TLR-4) likely plays a key role in both cases. This review analyzed the most recent literature on the gut-liver axis, with a particular focus on the role of TLR-4 activation. Findings that linked liver disease with dysbiosis are evaluated, and links between dysbiosis and alterations of intestinal permeability and motility are discussed. We also examine the mechanisms of translocated gut bacteria and/or the bacterial product activation of liver inflammation and fibrogenesis via activity on different hepatic cell types.

Mechanisms of Increased Expression of Toll-Like Receptor-4 in Human Monocyte/Macrophage-derived Foam Cells

Summary： The mechanisms of increased expression of toll-like receptor 4 （TLR-4） during the formation of foam cells were explored. Low density lipoprotein （LDL） was prepared by density gradient ultracentrifugation and oxidized by incubation with CuClz. The human monocytic leukemia cell line （THP-1） was cultured in RPMI1640. The differentiation of THP-1 cells into macrophages （MPs） was induced by using myristate acetate （PMA） for 48 h. The macrophages were then incubated with oxidized LDL （ox-LDL） to generate foam cells （FCs）. The mRNA and protein expression levels of human TLR-4 were detected by immunocytochemistry, Western blotting and reverse transcription polymerase chain reaction （RT-PCR）. The results showed that the TLR-4 mRNA and the protein expression levels were significantly increased during the differentiation of monocytes into macrophages （P〈0. 05） as well as during the formation of lipid-laden foam cells （P〈0.05）. It was concluded that the upregulation of human TLR-4 gene expression during the differentiation of monocytes into macrophages and the differentiation of macrophages into foam cells could increase TLR-4 protein synthesis dramatically, which may enhance the ability of foam cells inflammation reaction in atherosclerosis.

Transforming growth factor-β and toll-like receptor-4 polymorphisms are not associated with fibrosis in haemochromatosis

AIM:To investigate the role of genetic polymorphisms in the progression of hepatic fibrosis in hereditary haemochromatosis.METHODS:A cohort of 245 well-characterised C282Y homozygous patients with haemochromatosis was studied,with all subjects having liver biopsy data and DNA available for testing.This study assessed the association of eight single nucleotide polymorphisms(SNPs)in a total of six genes including toll-like receptor 4(TLR4),transforming growth factor-beta(TGF-β),oxoguanine DNA glycosylase,monocyte chemoattractant protein 1,chemokine C-C motif receptor 2 and interleukin-10 with liver disease severity.Genotyping was performed using high resolution melt analysis and sequencing.The results were analysed in relation to the stage of hepatic fibrosis in multivariate analysis incorporating other cofactors including alcohol consumption and hepatic iron concentration.RESULTS:There were significant associations between the cofactors of male gender(P=0.0001),increasing age(P=0.006),alcohol consumption(P=0.0001),steatosis(P=0.03),hepatic iron concentration(P<0.0001)and the presence of hepatic fibrosis.Of the candidate gene polymorphisms studied,none showed a significant association with hepatic fibrosis in univariate or multivariate analysis incorporating cofactors.We also specifically studied patients with hepatic iron loading above threshold levels for cirrhosis and compared the genetic polymorphisms between those with no fibrosis vs cirrhosis however there was no significant effect from any of the candidate genes studied.Importantly,in this large,well characterised cohort of patients there was no association between SNPs for TGF-βor TLR4and the presence of fibrosis,cirrhosis or increasing fibrosis stage in multivariate analysis.CONCLUSION:In our large,well characterised group of haemochromatosis subjects we did not demonstrate any relationship between candidate gene polymorphisms and hepatic fibrosis or cirrhosis.

Microglia activation mediated by toll-like receptor-4 impairs brain white matter tracts in rats

Microglia activation and white matter injury coexist after repeated episodes of mild brain trauma and ischemic stroke. Axon degeneration and demyelination can activate microglia; however, it is unclear whether early microglia activation can impair the function of white matter tracts and lead to injury. Rat corpus callosum（CC） slices were treated with lipopolysaccharide（LPS） or LPS ＋ Rhodobacter sphaeroides（RS）-LPS that is a toll-like receptor 4（TLR-4） antagonist. Functional changes reflected by the change of axon compound action potentials（CAPs） and the accumulation of β-amyloid precursor protein（β-APP） in CC nerve fibers. Microglia activation was monitored by ionized calcium binding adaptor-1 immunofluorescent stain, based on well-established morphological criteria and paralleled proportional area measurement. Input-output（I/O） curves of CAPs in response to increased stimuli were significantly downshifted in a dose-dependent manner in LPS（0.2, 0.5 and 1.0μg/mL）-treated slices, implying that axons neurophysiological function was undermined. LPS caused significant β-APP accumulation in CC tissues,reflecting the deterioration of fast axon transport. LPS-induced I/O curve downshift and P-APP accumulation were significantly reversed by the pre-treatment or co-incubation with RS-LPS. RS-LPS alone did not change the I/O curve.The degree of malfunction was correlated with microglia activation, as was shown by the measurements of proportional areas. Function of CC nerve fibers was evidently impaired by microglia activation and reversed by a TLP-4 antagonist, suggesting that the TLP-4 pathway lead to microglia activation.

MicroRNA-630 alleviates inflammatory reactions in rats with diabetic kidney disease by targeting toll-like receptor 4

BACKGROUND Diabetic kidney disease(DKD)is a major complication of diabetes mellitus.Renal tubular epithelial cell(TEC)damage,which is strongly associated with the inflammatory response and mesenchymal trans-differentiation,plays a significant role in DKD;However,the precise molecular mechanism is unknown.The recently identified microRNA-630(miR-630)has been hypothesized to be closely associated with cell migration,apoptosis,and autophagy.However,the association between miR-630 and DKD and the underlying mechanism remain unknown.AIM To investigate how miR-630 affects TEC injury and the inflammatory response in DKD rats.METHODS Streptozotocin was administered to six-week-old male rats to create a hypergly cemic diabetic model.In the second week of modeling,the rats were divided into control,DKD,negative control of lentivirus,and miR-630 overexpression groups.After 8 wk,urine and blood samples were collected for the kidney injury assays,and renal tissues were removed for further molecular assays.The target gene for miR-630 was predicted using bioinformatics,and the association between miR-630 and toll-like receptor 4(TLR4)was confirmed using in vitro investigations and double luciferase reporter gene assays.Overexpression of miR-630 in DKD rats led to changes in body weight,renal weight index,basic blood parameters and histopathological changes.RESULTS The expression level of miR-630 was reduced in the kidney tissue of rats with DKD(P<0.05).The miR-630 and TLR4 expressions in rat renal TECs(NRK-52E)were measured using quantitative reverse transcription polymerase chain reaction.The mRNA expression level of miR-630 was significantly lower in the high-glucose(HG)and HG+mimic negative control(NC)groups than in the normal glucose(NG)group(P<0.05).In contrast,the mRNA expression level of TLR4 was significantly higher in these groups(P<0.05).However,miR-630 mRNA expression increased and TLR4 mRNA expression significantly decreased in the HG+miR-630 mimic group than in the HG+mimic NC group(P<0.05).Furthermore,the levels of tumor necrosis factor-alpha(TNF-α),interleukin-1β(IL-1β),and IL-6 were significantly higher in the HG and HG+mimic NC groups than in NG group(P<0.05).However,the levels of these cytokines were significantly lower in the HG+miR-630 mimic group than in the HG+mimic NC group(P<0.05).Notably,changes in protein expression were observed.The HG and HG+mimic NC groups showed a significant decrease in E-cadherin protein expression,whereas TLR4,α-smooth muscle actin(SMA),and collagen IV protein expression increased(P<0.05).Conversely,the HG+miR-630 mimic group exhibited a significant increase in E-cadherin protein expression and a notable decrease in TLR4,α-SMA,and collagen IV protein expression than in the HG+mimic NC group(P<0.05).The miR-630 targets TLR4 gene expression.In vivo experiments demonstrated that DKD rats treated with miR-630 agomir exhibited significantly higher miR-630 mRNA expression than DKD rats injected with agomir NC.Additionally,rats treated with miR-630 agomir showed significant reductions in urinary albumin,blood glucose,TLR4,and proinflammatory markers(TNF-α,IL-1β,and IL-6)expression levels(P<0.05).Moreover,these rats exhibited fewer kidney lesions and reduced infiltration of inflammatory cells.CONCLUSION MiR-630 may inhibit the inflammatory reaction of DKD by targeting TLR4,and has a protective effect on DKD.

静脉注射脂多糖上调小鼠肺及肝CD14和Toll-like受体4表达

目的观察静脉注射脂多糖(lipopolysacchafide,LPS)致小鼠肺、肝组织及血液中LPS受体CD14、Toll-like receptor 4(TLR4)表达的变化.方法BALB/C小鼠随机分为5组,尾静脉注射LPS(7 mg/kg),分别于注后0、2、6、12和24 h取血及肺、肝组织.采用逆转录聚合酶链式反应(reverse transcription-polymerase chain reaction,RT-PCR)及Western Blot法分别检测肺和肝组织中CD14和TLR4 mRNA及蛋白表达,血清CD14(sCD14)蛋白表达.结果CD14和TLR4 mRNA在肺、肝组织中固有表达,LPS可使肺组织CD14和TLR4表达明显上调(P＜0.05),高峰出现在2-6 h(P＜0.05),24 h降至基础水平;而肝组织的变化明显滞后于肺,12 h达高峰(P＜0.05),24 h仍高于基础水平(P＜0.05).蛋白的变化与mRNA基本一致.血清CD14的表达则随时间延长而增加.结论LPS进入体内可诱导肺、肝和血液中内毒素受体CD14和TLR4的表达.不同组织中内毒素受体表达的时间差异表明肺是内毒素血症中较早受损的器官,肺组织损伤可能进一步引发肝损伤.

Effects of Propofol on The mRNA Expression of Toll-like Receptor 4 in BV-2 Cells During Mimic Ischemia-Reperfusion Injury In Vitro

This study investigated the effects of propofol on the mRNA expression of Toll-like receptor-4 （TLR4） in BV-2 cells during mimic ischemia-reperfusion （I/R） injury in vitro. BV-2 cells, a mouse microglia line, were cultured and divided into 4 groups at random： control group （group C）, ischemia/reperfusion group （group I/R）, low-dose propofol （25 μmol/L） intervention group （group PF25） and high-dose propofol （100 μmol/L） intervention group （group PF100）. The mRNA expression of TLR4 and NF-κB was measured by means of RT-PCR. TNF-α levels in the supernatants of BV-2 cells were detected by ELISA. The results showed that the mRNA expression of TLR4 and NF-κB was significantly higher in groups I/R, PF25 and PF100 than in group C （P〈0.01）. And the TNF-α level in the supernatants was elevated in groups I/R, PF25 and PF100 as compared with that in group C （P〈0.01）. After pre-treatment with propofol, the mRNA expressions of TLR4 and NF-κB and the TNF-α level were significantly decreased in groups PF25 and PF100 in comparison to those in group I/R （P〈0.01）. And the decrease in those indicators was more significant in group PF100 than in group PF25 （P〈0.01）. It was concluded that propofol exerted brain-protecting effects during I/R injury by suppressing the mRNA expressions of TLR4 and NF-κB and deceasing the TNF-α level.

基于TLR4 NF-κB通路-神经相关因子探究依达拉奉对急性脑梗死患者炎症反应与神经损伤的保护机制

目的基于Toll样受体4(TLR4)核因子-κB(NF-κB)通路-神经相关因子探究依达拉奉对急性脑梗死(ACI)患者炎症反应与神经损伤的保护机制。方法选取2020-07—2023-07保定市第一中心医院收治的110例ACI患者,以随机数字表法分为观察组、对照组,各55例,对照组给予阿替普酶溶栓,观察组给予阿替普酶溶栓联合依达拉奉治疗。比较2组疗效、神经功能[美国国立卫生研究院卒中量表(NIHSS)评分、改良Rankin评分]、TLR4 NF-κB通路指标(TLR4、NF-κB)、神经损伤相关因子[神经元特异性烯醇化酶(NSE)、中枢神经特异性蛋白(S-100β)、脑源性神经营养因子(BDNF)]、TLR4 NF-κB通路相关炎症因子[白介素-1β(IL-1β)、超敏C反应蛋白(hs-CRP)、肿瘤坏死因子(TNF-α)、五聚素3(PTX3)、脂蛋白相关磷脂酶A2(Lp-PLA2)]。结果观察组总有效率96.36%,高于对照组的83.64%(P<0.05)。治疗1、2周观察组NIHSS评分、改良Rankin评分均低于对照组(P<0.05),观察组TLR4、NF-κB均低于对照组(P<0.05)。相较于治疗前,2组治疗1、2周后S-100β、NSE水平明显下降,BDNF水平明显升高,观察组S-100β、NSE水平均低于对照组,BDNF水平高于对照组(P<0.05)。相较于治疗前,2组治疗1、2周后IL-1β、hs-CRP、TNF-α、PTX3、Lp-PLA2水平均明显下降,观察组IL-1β、hs-CRP、TNF-α、PTX3、Lp-PLA2水平均低于对照组(P<0.05)。结论依达拉奉对ACI患者的疗效显著,有利于缓解炎症反应,改善神经损伤,其保护机制可能与TLR4 NF-κB通路调控神经损伤、炎症反应相关因子有关。

Betaine inhibits Toll-like receptor 4 expression in rats with ethanol-induced liver injury

AIM:To test whether ethanol feeding could induce Toll-like receptor 4(TLR4)responses,assess the hepatoprotective effect of betaine and its inhibitive effect on TLR4 in animal models of alcoholic liver injury.METHODS:Forty-eight female Sprague-Dawley rats were randomly divided into four groups as control,model,low and high dose betaine groups.Except control group,all rats were fed with high fat-containing diet plus ethanol and fish oil gavages for 8 wk.Betaine was administered intragastrically after exposure of ethanol for 4 wk.The changes of liver histology were examined.The expression of TLR4 mRNA and protein was detected by RT-PCR and Western blotting,respectively.The serum aminotransferase activity alanine transarninase(ALT),aspartate aminotransferase(AST),serum endotoxin,and liver inflammatory factors tumor necrosis factor-α(TNF-α),interferon-γ(IFN-γ),interleukin-18(IL-18)were also assayed.RESULTS:Compared with control group,rats of model group developed marked liver injury,accompanied by an increase of ALT(159.41±7.74 U/L vs 59.47± 2.34 U/L,P<0.0001),AST(248.25±1.40 U/L vs 116.89±3.48 U/L,P<0.0001),endotoxin(135.37± 30.17 ng/L vs 44.15±7.54 ng/L,P<0.0001),TNF-α(20.81±8.58 pg/mL vs 9.34±2.57 pg/mL,P=0.0003),IFN-γ(30.18±7.60 pg/mL vs 16.86±9.49 pg/mL,P= 0.0039)and IL-18(40.99±8.25 pg/mL vs 19.73±9.31 pg/mL,P=0.0001).At the same time,the expression of TLR4 mRNA and protein was markedly induced in the liver after chronic ethanol consumption(1.45±0.07 vs 0.44±0.04,P<0.0001;1.83±0.13 vs 0.56±0.08,P<0.0001).Compared with model group,betaine feeding resulted in significant decreases of ALT(64.93 ±6.06 U/L vs 159.41±7.74 U/L,P<0.0001),AST(188.73±1.11 U/L vs 248.25±1.40 U/L,P<0.0001),endotoxin(61.80±12.56 ng/L vs 135.37±30.17 ng/L,P<0.0001),TNF-α(9.79±1.32 pg/mL vs 20.81± 8.58 pg/mL,P=0.0003),IFN-γ(18.02±5.96 pg/mL vs 30.18±7.60 pg/mL,P=0.0008)and IL-18(18.23±7.01 pg/mL vs 40.99±8.25 pg/mL,P<0.0001).Betaine also improved liver steatosis.The expression levels of TLR4 mRNA or protein in liver tissues were significantly lowered(0.62±0.04 vs 1.45±0.07,P<0.0001;and 0.65±0.06 vs 1.83±0.13,P<0.0001).There was a statistical difference of TLR4 mRNA and protein expression between high-and low-dose betaine groups(0.62±0.04 vs 0.73±0.05,P<0.0001,and 0.65±0.06 vs 0.81±0.09,P<0.0001).CONCLUSION:Betaine can prevent the alcoholinduced liver injury effectively and improve the liver function.The expression of TLR4 increases significantly in ethanol-fed rats and betaine administration can inhibit TLR4 expression.

Effects of ω-3 fatty acids on toll-like receptor 4 and nuclear factor-κB p56 in lungs of rats with severe acute pancreatitis

AIM To determine the effects of ω-3 fatty acids(ω-3FA) on the toll-like receptor 4(TLR4)/nuclear factor κB p56(NF-κBp56) signal pathway in the lungs of rats with severe acute pancreatitis(SAP).METHODS A total of 56 Sprague-Dawley rats were randomly divided into 4 groups: control group, SAP-saline group, SAP-soybean oil group and SAP-ω-3FA group. SAP was induced by the retrograde infusion of sodium taurocholate into the pancreatic duct. The expression of TLR4 and NF-κBp56 in the lungs was evaluated by immunohistochemistry and Western blot analysis. The levels of inflammatory cytokines interleukin-6 and tumor necrosis factor-alpha in the lungs were measured by enzyme-linked immunosorbent assay. RESULTS The expression of TLR4 and NF-κBp56 in lungs and of inflammatory cytokines in serum significantly increased in the SAP group compared with the control group(P < 0.05), but was significantly decreased in the ω-3FA group compared with the soybean oil group at 12 and 24 h(P < 0.05).CONCLUSION During the initial stage of SAP, ω-3FA can efficiently lower the inflammatory response and reduce lung injury by triggering the TLR4/NF-κBp56 signal pathway.

Association of Toll-like receptor 4 Signaling Pathway with Steroid-induced Femoral Head Osteonecrosis in Rats

Osteonecrosis of the femoral head is frequently observed in patients treated with excessive corticosteroids. However, the pathogenesis of corticosteroid-induced osteonecrosis remains unclear. The purpose of this study was to investigate the role of Toil-like receptor 4 （TLR4） signaling pathway in steroid-induced femoral head osteonecrosis in rats. Male Sprague-Dawley rats were injected intramus- cularly with 20 mg/kg methylprednisolone （MP） for 8 weeks, twice per week. The animals were sacri- ficed at 2, 4 and 8 weeks after the last MP injection, respectively, and then allocated to the 2-, 4- and 8-week model groups （n=24 each）. Rats in the control group （n=12） were not given any treatment. Histopathological analysis was performed and the concentration of tartrate-resistant acid phosphatase （TRAP） in plasma was determined. The activation of osteoclasts in the femoral head was assessed by TRAP staining. The expression of TLR4, MyD88, TRAF6 and NF-Id3 p65 that are involved in TLR4 signaling, and MCP-1 production were detected by using real-time PCR （RT-PCR） and Western blotting. The results showed that the osteonecrosis in the femoral head was clearly observed and the concentra- tion of TRAP in the plasma was increased in the model rats. The femoral head tissues in MP-treated rats were positive for TRAP and the intensity of TRAP staining was greater in MP-treated rats than in con- trol rats. As compared with the control group, the mRNA expression of TLR4 signaling-related factors was enhanced significantly at 4 and 8 weeks, and the protein levels of these factors increased signifi- cantly with time. It was concluded that MP could induce the femoral head osteonecrosis in rats, which was associated with osteoclast activation via the TLR4 signaling pathway. These findings suggest that TLR4 signaling pathway plays a pivotal role in the pathogenesis of steroid-induced osteonecrosis.

Toll-like receptor 4-mediated signaling participates in apoptosis of hippocampal neurons

The phosphatidylinositol 3 kinase （PI3K）/protein kinase B （AKT） signaling pathway is considered important for cell survival and has been shown to mediate various anti-apoptotic biological effects. This study explored the role of the Toll-like receptor 4 （TLR4）-mediated PI3K/AKT-glycogen syn-thase kinase 3β （GSK-3β） signaling pathways in lipopolysaccharide-induced apoptosis in a primary culture of hippocampal neurons. Results demonstrated that the apoptotic ratio of hippocampal neurons stimulated by lipopolysaccharide was significantly higher compared with the control group. Both the expression of P-AKTser473 and P-GSK-3βSserg in hippocampal neurons stimulated by lipopolysaccharide decreased compared with the control, while the level of active Caspase-3 and the ratio of Bax/Bcl-2 were significantly increased. The level of active Caspase-3 and the ratio of Bax/Bcl-2 in hippocampal neurons treated with TLR4 antibody or the GSK-3β inhibitor, LiCl, de-creased before intervention with lipopolysaccharide, but increased after treatment with the AKT in-hibitor, LY294002. These findings suggest that the TLR4-PI3K/AKT-GSKβ signaling pathway may be involved in lipopolysaccharide-induced apoptosis of hippocampal neurons.

Production of interleukin-1β related to mammalian target of rapamycin/Toll-like receptor 4 signaling pathway during Aspergillus fumigatus infection of the mouse cornea

AIM：To elucidate the effect of rapamycin on regulating the production of interleukin（IL）-1β in Aspergillus fumigatus（A.fumigatus）-induced keratitis and to verify whether the expression of IL-1β in A.fumigatus keratitis is associated with the mammalian target of rapamycin（mT OR）/Toll-like receptor 4（TLR4） signaling pathway.METHODS：Fungal keratitis mouse models of susceptible C57 BL/6 mice were established using A.fumigatus.The mice were subsequently treated with rapamycin.The protein levels of p-mT OR,TLR4,and IL-1β in normal and infected corneal tissue were measured by Western blot.The TLR4 and IL-1β m RNA levels were determined by real-time polymerase chain reaction（PCR）.RESULTS：In C57 BL/6 mice,rapamycin treatment decreased the clinical scores and production of the pro-inflammatory cytokine,IL-1β.The expression of TLR4,stimulated by A.fumigatus,was reduced as well when the mT OR signaling pathway was suppressed by rapamycin.CONCLUSION：Rapamycin is beneficial for the outcome of fungal keratitis and has an inhibitory effect expression of the inflammatory cytokine IL-1β.The inhibitory effect on IL-1β expression can be associated with the mT OR/TLR4 signaling pathway in A.fumigatus infection in mice.

Impact of Probiotics on Toll-like Receptor 4 Expression in an Experimental Model of Ulcerative Colitis

Toll-like receptors （TLRs） are key components of the innate immune system which trigger antimicrobial host defense responses. This study aimed to investigate the impact of probiotics （Lactoba- cillus, Bifiidobacterium） on the expression of TLR4 and tumor necrosis factor-alpha （TNF-α in the co- lon mucosa of rat experimental ulcerative colitis model induced by trinitrobenzene sulfonic acid （TNBS）/ethanol and immune complexes. The gross and histological changes of the colonic mucosa were observed and assessed by the means-standard deviation and independent samples t-test. The pro- tein expression levels of TLR4 and TNF-α were detected by using immunohistochemistry and Westem blotting, respectively. It was revealed that there was visible infiltration of inflammatory cells, formation of crypt abscess, and the reduction of goblet cells in the colon tissue of experimental models. As com- pared with the control group, the levels of TLR4 and TNF-α protein were significantly increased in the model group （P〈0.01 for both）. No significant difference was found in the expression of TLR4 and TNF-α between the two-week probiotics treatment group and the model group （P〉0.05）, whereas sig- nificant reductions were shown in rats which were treated with probiotics for four weeks as compared with the model group （P〈0.01）. There was no significant difference between two probiotics-treated groups. Our results implied that probiotics were likely to play a key role in protecting ulcerative colitis by reducing the inflammatory factor TNF-α expression through inhibiting the TLR4 expression in the colon tissue of experimental models.

A 5'-flanking region polymorphism in toll-like receptor 4 is associated with gastric cancer in a Chinese population

Objective： Inflammation induced by H.pylori colonization in the stomach is related to the development of gastric cancer and the genetic variations of the genes involved in the immune responses modify the host response to the infection. The aim of this study was to evaluate whether polymorphisms in the toll-like receptor 4 （TLR4） gene, a key regulator of both innate and adaptive immunity, were related to the susceptibility to

Expression and Immune Effect of Toll-Like Receptor 4 in Human Trophoblast Cells

This study investigated the expression and immune effect of TLR4 in human trophoblast cells. The expression level of TLR4 mRNA in normal and LPS-stimulated human term trophoblast cells （1 mg/L LPS, 12 h） was detected by RT-PCR. In LPS-stimulated human term trophoblast cells of TLR4-blocked group and non-TLR4-blocked group, and normal term trophoblast cells of blank control group, apoptosis rate was measured by flow cytometry （FCM）, and the level of TNF-α determined by using enzyme linked immunosorbent assay （ELISA） respectively. RT-PCR results showed that the expression level of TLR4 mRNA in LPS-stimulated human trophoblast cells was significantly higher than that in normal cells （P〈0.01）. FCM revealed that there was significant difference in apoptosis rate of LPS-stimulated human term trophoblast cells between TLR4-blocked group and non-TLR4-blocked group （P〈0.05）, or between TLR4 antibody-blocked group and blank control group. ELISA indicated that the level of TNF-α in LPS-stimulated human trophoblast cells also had statistical differences between TLR4 antibody-blocked group and non-TLR4 antibody-blocked group （P〈0.05）. Our results suggest that TLR4 plays an important role in the immunological mechanism of apoptosis and secretion of TNF-α of human term trophoblast cells stimulated by LPS.

Role of Toll-like receptor 4 in inflammatory reactions of hippocampal neurons

Lipopolysaccharide stimulates Toll-like receptor 4 on immune cells to produce immune mediators. Toll-like receptor 4 is also expressed by non-immune cells, which can be stimulated by lipopolysaccharide. However, whether Toll-like receptor 4 is expressed by primary cultured hippocampal neurons and its specific role in lipopolysaccharide-induced neuroinflammation is currently undefined, in this study, Toll-like receptor 4 antibody blocking was used to analyze the Toll-like receptor 4 signaling pathway and changes in inflammation of lipopolysaccharide stimulated hippocampal neurons. Immunofluorescence showed that Toll-like receptor 4 protein was mainly located in the membrane of hippocampal neurons. Quantitative reverse transcription-PCR and western blot assay showed that after stimulation of lipopolysaccharide, the mRNA and protein levels of Toll-like receptor 4 and the mRNA levels of interleukin-ll3 and tumor necrosis factor-（] were significantly increased. In addition, there was increased phosphorylation and degradation of kappa B a inhibitor in the cytosol and increased nuclear factor-KB p65 expression in the nuclei. Pretreatment with Toll-like receptor 4 antibody could almost completely block this increase. These experimental findings indicate that lipopolysaccharide participates in neuroinflammation by stimulating Toll-like receptor 4/nuclear factor-KB pathway in hippocampal neurons, which may be both ＂passive victims＂ and ＂activators＂ of neuroinflammation.

Toll-like receptor 4 polymorphisms and bacterial infections in patients with cirrhosis and ascites

AIM To assess the relationship between the presence of toll-like receptor 4(TLR4) polymorphisms and bacterial infections in cirrhotic patients with ascites. METHODS We prospectively included consecutive patients with cirrhosis and ascites hospitalized during a 6-year period. Patients with human immunodeficiency virus(HIV) infection or any other immunodeficiency, patients with advanced hepatocellular carcinoma(beyond Milan's criteria) or any other condition determining poor short-term prognosis, and patients with a permanent urinary catheter were excluded. The presence of D299 G and/or T399 I TLR4 polymorphisms was determined by sequencing and related to the incidence and probability of bacterial infections, other complications of cirrhosis, hepatocellular carcinoma, and mortality during follow-up. A multivariate analysis to identify predictive variables of mortality in the whole series was performed. RESULTS We included 258 patients: 28(10.8%) were carriers of D299G and/or T399I TLR4 polymorphisms(polymorphism group) and 230 patients were not(wildtype group). The probability of developing any bacterial infection at one-year follow-up was 78% in the polymorphism group and 69% in the wild-type group(P = 0.54). The one-year probability of presenting infections caused by gram-negative bacilli(51% vs 44%, P = 0.68), infections caused by gram-positive cocci(49% vs 40%, P = 0.53), and spontaneous bacterial peritonitis(29% vs 34%, respectively, P = 0.99) did not differ between the two groups. The oneyear probability of transplant-free survival was 55% in the polymorphism group and 66% in the wild-type group(P = 0.15). Multivariate analysis confirmed that age, Child-Pugh score, active alcohol intake, previous hepatic encephalopathy, hepatocellular carcinoma and serum creatinine were associated with a higher risk of death during follow-up. CONCLUSION Genetic polymorphisms D299 G and/or T399 I of TLR4 do not seem to play a relevant role in the predisposition of cirrhotic patients with ascites to bacterial infections.

Impact of Toll-like Receptor 4 Deficiency on Cerebrocardiac Syndrome

In order to investigate the role of Toll-like receptor 4 （TLR4） in cerebrocardiac syndrome （CCS）, the partial cerebral ischemia/reperfusion （I/R） models in mice with different TLR4 genotypes were established in the present study. TLR4 wild-type （C3H/HeN） and mutant （C3H/HeJ） mice of 6-8 weeks of age were divided into 4 groups at random： C3H/HeN sham group （n=10）, C3H/HeJ sham group （n=10）, C3H/HeN model group （n=10） and C3H/HeJ model group （n=10）. Partial cerebral I/R was caused by the middle cerebral artery occlusion （MCAO） to duplicate CCS models in mice. After the operation, the electrocardiogram （ECG）, the level of tumor necrosis factor-alpha （TNF-c0 in myocardial tissue and the cardiac pathological changes were observed in each group. It was shown that the brain infarct volume in C3H/HeN model group was larger than that in C3H/HeJ model group （P〈0.01）. The ST segment change and T wave inversion occurred frequently in model groups. Moreover, the TNF-ct level in C3H/HeN model group was higher than that in C3H/HeJ model group （P〈0.01）. The myocar- dial injury was aggravated in C3H/HeN group as compared with C3H/HeJ group. It was concluded that TLR4 was implicated in the development of CCS.

PAI-1和TLR4在烧伤后增生性瘢痕组织中的表达水平及意义

目的探究纤溶酶原激活物抑制剂-1(PAI-1)和Toll样受体4(TLR4)在烧伤后增生性瘢痕(HS)组织中的表达水平及临床意义。方法选取河南科技大学第一附属医院2020年7月至2022年7月收治的94例HS病人为研究对象,术中取病人HS组织为HS组,并选取HS病人正常皮肤组织为对照组。实时荧光定量逆转录聚合酶链反应(qRT-PCR)法对组织中PAI-1、TLR4 mRNA表达水平进行测定;免疫组化法对组织中PAI-1、TLR4阳性表达情况进行测定;采用蛋白质印迹法测定PAI-1、TLR4的蛋白表达水平;使用温哥华瘢痕量表(VSS)对瘢痕组织严重情况进行评价,使用跨皮水分丢失(TEWL)对HS病人的表皮屏障功能进行评估;Pearson相关性分析病人HS组织中PAI-1、TLR4表达水平与VSS及TEWL净丧失量之间的关系。结果HS组PAI-1表达水平1.54±0.32、TLR4 mRNA表达水平1.61±0.38高于对照组1.11±0.24、1.06±0.21,HS组PAI-1、TLR4阳性表达率及蛋白表达水平也显著高于对照组(P<0.05);病人HS组织VSS评分为(12.01±3.48)分,HS组织TEWL值显著高于对照组(P<0.05),TEWL净丧失量为(7.87±2.37)g·m^(−2)·h^(−1);Pearson相关性分析结果表明,HS组织中PAI-1、TLR4表达水平分别与VSS及TEWL净丧失量呈显著正相关(P<0.05)。结论烧伤后病人HS组织中PAI-1、TLR4 mRNA表达水平、阳性表达率及蛋白表达水平均显著升高,且PAI-1、TLR4与HS组织严重程度,表皮屏障功能障碍有关,因此PAI-1、TLR4有望成为临床治疗HS的新靶点。