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.

Expression of Toll-like Receptor 4 in Neonatal Cord Blood Mononuclear Cells in Patients with Preeclampsia

The expression of Toll-like receptor 4 (TLR4) in neonatal cord blood mononuclear cells (MNCs) and serum TNF-α were investigated in order to explore the roles of TLR4 in the pathogenesis of preeclampsia.The study enrolled 27 patients suffering from preeclampsia (experimental group) and 21 normal pregnancy patients (control group).After MNCs were separated, the expression of TLR4 mRNA and protein was detected by using real-time quantitative PCR and Western blotting respectively, and the expression of TNF-α by using ELISA.The results showed the TLR4 mRNA level in cord blood MNCs (2-CT:0.07±0.17), TLR4 protein expression level (absorbance ratio:0.81%±0.15%) and TNF-α level (9.5±1.73 pg/mL) were all increased in experimental group as compared with control group with the differences being statistically significant (P【0.05).There was a positive correlation between the expression of TLR4 mRNA and TNF-α in both experimental group and control group (r=0.54 and 0.53, respectively, P【0.05).It was concluded that TLR4 expression in the experimental group of cord blood MNCs was increased and there was a positive correlation between the expression of TLR4 mRNA and TNF-α in both groups.TLR4-mediated release of inflammatory cytokines may be one of the important reasons leading to preeclampsia.

Expression and significance of toll-like receptor 2,4 in peripheral blood mononuclear cells in patients with systemic inflammatory response syndrome

To explore changes of toll-like receptor (TLR) 2,4 in peripheral blood mononuclear cells (PBMC) in acute abdomen patients with systemic inflammatory response syndrome (SIRS) and their significance.Methods A clinical study was done on 103 patients of which 65 were with SIRS.The mRNA expression of TLR2,4 were detected by RT-PCR;the expression of TNF-α and IL-6 were observed by ELISA;the correlation between TLR2,4 mRNA,the level of TNF-α and IL-6,and the clinical course was evaluated.Results TLR2 mRNA ,TNF-α and IL-6 were upregulated markedly on the first day of hospitalization,then decreased gradually;TLR2 mRNA maintained on high level till the 5th day.The expression of TLR2,4 mRNA was positive correlated with the level of TNF-α and IL-6,and the length of stay.TLR2,4 mRNA expression increased in patients with multiple organ failure.Conclusion In actue abdomen patients with SIRS,the expression of TLR2,4 of PBMC increased markedly,indicating its improtant role in the pathogenesis of SIRS.4 refs,2 figs,2 tabs.

Upregulation of Toll-like Receptor 4 on T Cells in PBMCs Is Associated with Disease Aggravation of HBV-related Acute-on-chronic Liver Failure

Summary： Immune-mediated inflammatory injury is an important feature of the disease aggravation of hepatitis B virus-related acute-on-chronic liver failure （ACLF）. Toll-like receptors （TLRs） have been shown previously to play a pivotal role in the activation of innate immunity. The purpose of this study was.to characterize the TLR4 expression in peripheral blood mononuclear cells （PBMCs） of ACLF pa- tients and its possible role in the disease aggravation. Twelve healthy subjects, 15 chronic HBV-infected （CHB） patients and 15 ACLF patients were enrolled in this study. The TLR4 expression in PBMCs and T cells of all subjects was examined by real-time PCR and flow cytometry. The correlation of TLR4 ex- pression on T cells with the markers of disease aggravation was evaluated in ACLF patients. The ability of TLR4 ligands stimulation to induce inflammatory cytokine production in ACLF patients was ana- lyzed by flow cytometry. The results showed that TLR4 mRNA level was upregulated in PBMCs of ACLF patients compared to that in the healthy subjects and the CHB patients. Specifically, the expres- sion of TLR4 on CD4＋ and CD8＋ T cells of PBMCs was significantly increased in ACLF patients. The TLR4 levels on CD4＋ and CD8＋T cells were positively correlated with serum total bilirubin （TBIL）, direct bilirubin （DBIL）, international normalized ratio （INR） levels and white blood cells （WBCs）, and negatively correlated with serum albumin （ALB） levels in the HBV-infected patients, indicating TLR4 pathway may play a role in the disease aggravation of ACLF. In vitro TLR4 ligand stimulation on PBMCs of ACLF patients induced a strong TNF-α production by CD4＋ T cells, which was also posi- tively correlated with the serum markers for liver injury severity. It was concluded that TLR4 expression is upregulated on T cells in PBMCs, which is associated with the aggravation of ACLF.

Expression of Toll-like Receptor 9 in Peripheral Blood Mononuclear Cells from Patients with Different Hepatitis B and C Viral Loads

The aim of the present study was to investigate the expression of toll-like receptors （TLR） 9 in peripheral blood mononuclear cells （PBMC） of patients with chronic hepatitis B and C with different virus copies. The study group included 90 patients （60 with chronic hepatitis B, and 30 with chronic hepatitis C）, and 20 healthy people served as control group. The protein and mRNA levels of TLR9 were detected by using flow cytometry and real-time PCR. The serum viral copies of HBV and HCV were measured in all patients, and the correlation between HBV-DNA copies or HCV-RNA copies and the TLR9 expression was analyzed. Our results demonstrated that HBV or HCV infection led to a decreased expression of TLR9 mRNA and protein compared to the control group （P〈0.05）. The TLR9 protein and mRNA levels were negatively correlated with serum viral copies of HBV and HCV （r=-0.632, r=-0.909, P〈0.01）. It was concluded that TLR9 mRNA and protein are down-regulated in PBMC of HBV-infected or HCV-infected patients, and they are negatively correlated with serum viral copies and play an important role in detecting viral replication of HBV and HCV.

TGF-β1 treated murine dendritic cells are maturation resistant and down-regulate Toll-like receptor 4 expression

Objective: To explore the effects of transforming growth factor (51 (TGF-β1) on dendritic cells (DC). Methods: Murine bone marrow cells were cultured with GM-CSF and TGF-β1 to develop TGF-β1-treated DC (TGFβ-DC). Then they were stimulated by lipopolysaccharide (LPS). Their phenotypes were assessed by flow cytometry (FCM). The allogeneic stimulating capacity of DC was measured by mixed lymphocyte reaction (MLR) using BrdU ELISA method and IL-12p70 protein was detected by ELISA. The expression of Toll-like receptor 4 (TLR4) was analyzed by semi quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and FCM. Results: Compared to immature DC (imDC) cultured by GM-CSF alone, the TGFβ-DC express lower CD80, CD86,I-Ab and CD40. The TGFβ-DC were resistant to maturation with LPS. Maturation resistance was evident from a failure to up-regulate co-stimulatory molecules (CMs), to stimulate larger T cells proliferation and to enhance secretion of IL-12p70. We also found that TGF-β1 could down-regulate TLR4 expression on TGFβ-DC. Conclusion: TGFβ-DC are resistant to maturation stimulus (LPS) and might have some correlation with the down-modulation of TLR4 expression.

The implication of dendritic cells in lung diseases:Immunological role of toll-like receptor 4

The immune responses play a profound role in the progression of lung lesions in both infectious and non-infectious diseases.Dendritic cells,as the"frontline"immune cells responsible for antigen presentation,set up a bridge between innate and adaptive immunity in the course of these diseases.Among the receptors equipped in dendritic cells,Toll-like re-ceptors are a group of specialized receptors as one type of pattern recognition receptors,capable of sensing environmental signals including invading pathogens and self-antigens.Toll-like receptor 4,a pivotal member of the Toll-like receptor family,was formerly recognized as a receptor sensitive to the outer membrane component lipopolysaccharide derived from Gram-negative bacteria,triggering the subsequent response.Moreover,its other essential roles in immune responses have drawn significant attention in the past decade.A better under-standing of the implication of Toll-like receptor 4 in dendritic cells could contribute to the management of pulmonary diseases including pneumonia,pulmonary tuberculosis,asthma,acutelung injury,and lung cancer.

ROLE OF TOLL-LIKE RECEPTOR 4 IN AIRWAY INFLAMMATION OF CHRONIC OBSTRUCTIVE PULMONARY DISEASES

Objective To confirm if pulmonary epithelial cells express Toll-like receptor 4 （TLR4） and investigate the role of TLR4 in airway inflammation of chronic obstructive pulmonary diseases （COPD）. Methods The expressions of TLR4, IL-8 mRNA and NF-KB activation stimulated by differen factors E lipopolysacharides （LPS）, interleukin-lβ, cigarette smoking extract （CSE）] in pulmonary epithelial cells were investigated. Results LPS, CSE and IL-lβ induced the production of IL-8 and activation of NF-KB. The levels of 1L-8 mRNA and NF-KB protein in E1A ＋ cell were markedly higher than E1A- cell and A549 cell （ P 〈0. 05）. The TLR4 mRNA of all the cells increased along with the increase of LPS＇ stimulated time. There was significant difference among different LPS＇ doses （ 12 h： P = O. 039 ; 24 h ： P = O. 013 ）. The TLR4 mRNA of E1A ＋ cell was higher than the other two groups （ P 〈0. 05）. IL-lβ induced all the cells expressing TLR4 mRNA. CSE had no effect on the expression of TLR4 mRNA. Conclusion Pulmonary epithelial cells express TLR4. LPS and IL-lβ up-regulate IL-8 mediated via the activation of NF-KB induced by TLR4. But CSE up-regulates IL-8 mediated via the activation of NF-KB, which has no relation to TLR4 and may have another signal transduction pathway.

SGLT2抑制剂联合新活素治疗高血压伴心力衰竭的疗效及对患者TLR4/NF-κB信号通路指标的影响

目的研究钠-葡萄糖协同转运蛋白2(SGLT2)抑制剂(达格列净)联合新活素治疗高血压伴心力衰竭(心衰)的临床效果及对患者外周血单核细胞Toll样受体4/核转录因子-κB(TLR4/NF-κB)信号通路指标的影响。方法前瞻性选取2021年5月至2023年6月郑州大学第一附属医院收治的138例高血压伴心衰患者作为研究对象,采用随机数表法分为对照组、单一组和联合组各46例。对照组接受常规基础治疗,单一组在此基础上接受新活素治疗,联合组在此基础上接受达格列净联合新活素治疗。治疗7 d后比较三组患者的临床疗效,以及治疗前后的心功能[左室收缩末期内径(LVESD)、左室射血分数(LVEF)、心率(HR)]、运动耐力[6 min步行试验距离(6 MWT)]、血压[收缩压(SBP)、舒张压(DBP)]、氧化应激指标[丙二醛(MDA)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-Px)]和TLR4/NF-κB信号通路相关指标[TLR4、NF-κB、白细胞介素-1β(IL-1β)、IL-6、肿瘤坏死因子-α(TNF-α)],同时比较三组患者的不良反应发生情况。结果联合组患者的治疗总有效率为95.65%,明显高于单一组的86.96%和对照组的73.91%,差异均有统计学意义(P<0.05);治疗后,联合组患者的LVEF、6 MWT分别为(43.28±3.16)%、(336.48±32.49)m,明显高于单一组的(40.54±2.68)%、(306.45±40.26)m和对照组的(38.07±2.89)%、(280.69±36.98)m,LVESD、HR分别为(35.29±4.26)mm、(71.34±3.62)次/min,明显低于单一组的(38.75±3.64)mm、(74.25±3.59)次/min和对照组的(42.36±4.05)mm、(76.46±3.98)次/min,且单一组变化幅度大于对照组,差异均有统计学意义(P<0.05);治疗后,联合组患者的SBP、DBP分别为(110.36±5.02)mmHg、(81.42±2.56)mmHg,明显低于单一组的(114.83±4.76)mmHg、(84.39±2.88)mmHg和对照组的(117.25±5.18)mmHg、(86.05±2.62)mmHg,且单一组低于对照组,差异均有统计学意义(P<0.05);治疗后,联合组患者的GSH-Px、CAT分别为(105.24±20.29)U/g·Hb、(148.35±32.46)U/mL,明显高于单一组的(90.35±19.45)U/g·Hb、(125.64±26.49)U/mL和对照组的(84.49±17.32)U/g·Hb、(103.29±25.38)U/mL,MDA为(7.39±2.56)mmol/L,明显低于单一组的(11.35±2.95)mmol/L和对照组的(13.08±2.72)mmol/L,且单一组变化幅度大于对照组,差异均有统计学意义(P<0.05);治疗后,联合组患者的TLR4 mRNA、NF-κB mRNA、IL-1βmRNA、IL-6 mRNA、TNF-αmRNA明显低于单一组和对照组,且单一组低于对照组,差异均有统计学意义(P<0.05);三组患者的不良反应总发生率比较差异无统计学意义(P>0.05)。结论SGLT2抑制剂联合新活素治疗高血压伴心衰能有效改善患者的氧化应激水平,调节TLR4/NF-κB信号通路,恢复患者血压与心功能,临床应用效果显著,且安全性较高。

缺血性脑卒中患者TLR2 TLR4与血小板神经功能的关系

目的探究缺血性脑卒中患者外周血单核细胞(PBMC)中Toll样受体(TLR)2、TLR4表达水平与血小板活化及神经功能的关系。方法选取2017年5月至2020年6月邢台医学高等专科学校第二附属医院收治的134例缺血性脑卒中患者为研究对象(研究组),同期140例健康体检者为对照(对照组),收集两组对象一般资料。采用实时荧光定量PCR(RT-qPCR)检测两组对象PBMC中TLR2、TLR4 mRNA水平;利用酶联免疫吸附试验(ELISA)法检测两组对象血浆中血小板活化因子(PAF)、血小板活化标志物α颗粒膜糖蛋白140(GMP-140)、β-血小板球蛋白(β-TG)、血小板第4因子(PF4)水平;采用美国国立卫生院卒中量表(NIHSS)评分评估研究组患者神经功能;Spearman分析研究组患者PBMC中TLR2、TLR4与糖尿病史、高血压史的相关性;Pearson分析研究组患者PBMC中TLR2、TLR4与低密度脂蛋白胆固醇(LDL-C)、空腹血糖(FBG)、高密度脂蛋白胆固醇(HDL-C)水平,血浆中PAF、GMP-140、β-TG、PF4水平,NIHSS评分的相关性。结果与对照组相比,研究组糖尿病史、高血压史比例,LDL-C、FBG水平,PBMC中TLR2、TLR4水平,血浆中PAF、GMP-140、β-TG、PF4水平升高(P<0.05),HDL-C水平降低(P<0.05)。研究组NIHSS评分为(14.59±7.48)分。相关性分析发现,PBMC中TLR2、TLR4与PAF、β-TG、PF4、NIHSS评分,TLR4与LDL-C、FBG、GMP-140呈正相关(P<0.05)。结论缺血性脑卒中患者PBMC中TLR2、TLR4表达水平升高,其与血小板活化及神经功能评分具有一定相关性。

Hepatocellular carcinoma and macrophage interaction induced tumor immunosuppressionvia Treg requires TLR4 signaling

AIM: To investigated the interaction between toll-like receptor 4 (TLR4)-activated hepatoma cells and macrophages in the induction of tumor-immune suppression mediated by CD4+CD25high family of transcription factor P3 (FOXP3) regulatory T cells (Tregs). METHODS: The proportion of FOXP3+ Tregs was identified in peripheral blood and tumor tissues of 60 hepatocellular carcinoma (HCC) patients. TLR4 expression was examined in tumor tissues and cell lines. The correlation was examined between FOXP3+ Tregs in peripheral blood and TLR4 expression of HCC tissues. Following activation of TLR4 in H22 murine hepatoma cells pre-incubated with lipopolysaccharide (LPS) and co-cultured with macrophage cell line RAW246.7, the synthesis of cytokines tumor necrosis factor-α, CCL22, and interleukin (IL)-10 by the two cell lines was detected and analyzed. RESULTS: FOXP3+ Tregs were enriched in tumor sites, and circulating FOXP3+ Tregs were increased in HCC patients in correlation with multiple tumor foci and up-regulated TLR4 expression in HCC tissues. Semi-quantitative analysis indicated that TLR4 was over-expressed in HCC compared with the matched normal tissues. Cell cultivation experiments indicated that the mRNAs of IL-10 and CCL22 were significantly up-regulated in the RAW246.7 cell line when co-cultured with LPS preincubated H22 cells. CONCLUSION: In hepatoma cell lines, TLR4 may indirectly facilitate the recruitment of Tregs to the tumor site and promote intrahepatic metastasis through its interaction with macrophages.

Lipopolysaccharide upregulates the expression of Toll-like receptor 4 in human vascular endothelial cells

OBJECTIVE: To investigate the expression of Toll-like receptor 2 (TLR2) and Toll-like receptor 4 (TLR4) and the effect of lipopolysaccharide (LPS) on their expression in cultured endothelial cells. METHODS: Total RNA was extracted from ECV304 cells and isolated human umbilical vein endothelial cells (HUVECs) exposed to LPS, respectively. The quantification of TLR2 and TLR4 mRNA in HUVECs and EVC304 cells was carried out by reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: ECV304 cells and HUVECs were able to express TLR2 and TLR4 mRNA, but the expression levels of TLR4 appeared to be stronger than those of TLR2. LPS could upregulate the expression levels of TLR4 obviously, whereas it had no effect on the expression level of TLR2. CONCLUSIONS: Our data indicate that TLR4 may be the LPS signal transducer in endothelial cells and plays important roles in the cell activation of LPS. The ECV304 cell line is a better experimental model than isolated HUVECs in the research of endothelial cells.

不同肝组织炎症分级和纤维化分期的自身免疫性肝炎患者外周血单个核细胞TLR2和CTLA-4水平变化研究

目的 调查不同肝组织炎症分级和纤维化分期的自身免疫性肝炎(AIH)患者外周血单个核细胞(PBMCs)Toll样受体2(TLR2)和细胞毒性T淋巴细胞相关抗原4(CTLA-4)水平变化。方法 2018年6月~2022年6月我院诊治的47例AIH患者和同期50例健康人,所有AIH患者接受肝活检。抽取外周血,分离PBMCs,采用PCR法检测PBMCs TLR2和CTLA-4 mRNA水平,使用流式细胞仪检测TLR2和CTLA-4阳性的PBMCs百分比。结果 AIH患者PBMCs TLR2 mRNA水平和细胞表面TLR2阳性的PBMCs百分比分别为(1.7±0.4)和(34.2±7.9)%,均显著高于健康人【分别为(0.9±0.2)和(21.4±3.5)%,P<0.05】,而PBMCs CTLA-4 mRNA水平和细胞表面CTLA-4阳性的PBMCs百分比分别为(0.5±0.2)和(2.3±0.8)%,均显著低于健康人【分别为(1.1±0.2)和(13.7±3.9)%,P<0.05】;21例肝组织G3/G4级患者TLR2 mRNA水平和细胞表面TLR2阳性的PBMCs百分比分别为(1.9±0.3)和(37.2±5.7)%,均显著高于26例G1/G2级患者【分别为(1.5±0.3)和(30.5±6.2)%,P<0.05】,而PBMCs CTLA-4 mRNA水平和细胞表面CTLA-4阳性的PBMCs百分比分别为(0.3±0.1)和(1.9±0.6)%,均显著低于G1/G2级患者【分别为(0.7±0.2)和(2.6±0.8)%,P<0.05】;17例肝组织F3/F4期患者TLR2 mRNA水平和细胞表面TLR2阳性的PBMCs百分比分别为(1.9±0.3)和(37.6±5.3)%,均显著高于25例F1/F2期患者【分别为(1.6±0.3)和(32.9±4.2)%,P<0.05】或5例F0期患者【分别为(1.3±0.2)和(28.7±1.9)%,P<0.05】,而PBMCs CTLA-4 mRNA水平和细胞表面CTLA-4阳性的PBMCs百分比分别为(0.3±0.1)和(1.8±0.6)%,均显著低于F1/F2期患者【分别为(0.6±0.2)和(2.3±0.7)%,P<0.05】或F0期患者【分别为(0.9±0.2)和(4.2±0.4)%,P<0.05】。结论 AIH患者PBMCs TLR2 mRNA和TLR2阳性的PBMCs百分比升高,而PBMCs CTLA-4 mRNA水平和CTLA-4阳性的PBMCs百分比降低,并与肝组织炎症活动度和纤维化程度相关,值得进一步研究。

Effects of Combination of 1,25(OH)_(2)D_(3) and TLR-4 Inhibitor on the Damage to HaCaT Cells Caused by UVB Irradiation

Objective Vitamin D and Toll-like receptor-4(TLR-4)inhibition are involved in the protection of keratinocytes.The effects of combination of 1,25(OH)_(2)D_(3) and TLR-4 inhibitor on the protection of keratinocytes against ultraviolet radiation B(UVB)irradiation remain unclear.This study was undertaken to explore the effects of combination of 1,25(OH)_(2)D_(3) and TAK-242(TLR-4 inhibitor)on the damage to HaCaT cells caused by UVB irradiation.Methods In vitro,HaCaT cells were treated with 1,25(OH)_(2)D_(3) or/and TAK-242 prior to UVB irradiation at the intensity of 20 mJ/cm^(2),then the production of reactive oxygen species(ROS),cell migration,apoptosis of cells,and the expression of oxidative stress,endoplasmic reticulum stress,and apoptosis related proteins were determined.Results Compared with the HaCaT cells treated with 1,25(OH)_(2)D_(3) or TAK-242,the cells treated with both 1,25(OH)_(2)D_(3) and TAK-242 showed,1)significantly lower production of ROS(P<0.05);2)significantly less apoptosis of HaCaT cells(P<0.05);3)significantly lower expression of NF-κB,Caspase-8,Cyto-C,Caspase-3(P<0.05).Conclusion The combination of 1,25(OH)_(2)D_(3) and TAK-242 could produce a better protection for HaCaT cells via inhibiting the oxidative stress,endoplasmic reticulum stress and apoptosis than 1,25(OH)_(2)D_(3) or TAK-242 alone.

Multiomics reveal human umbilical cord mesenchymal stem cells improving acute lung injury via the lung-gut axis

BACKGROUND Acute lung injury(ALI)and its final severe stage,acute respiratory distress syndrome,are associated with high morbidity and mortality rates in patients due to the lack of effective specific treatments.Gut microbiota homeostasis,including that in ALI,is important for human health.Evidence suggests that the gut microbiota improves lung injury through the lung-gut axis.Human umbilical cord mesenchymal cells(HUC-MSCs)have attractive prospects for ALI treatment.This study hypothesized that HUC-MSCs improve ALI via the lung-gut microflora.AIM To explore the effects of HUC-MSCs on lipopolysaccharide(LPS)-induced ALI in mice and the involvement of the lung-gut axis in this process.METHODS C57BL/6 mice were randomly divided into four groups(18 rats per group):Sham,sham+HUC-MSCs,LPS,and LPS+HUC-MSCs.ALI was induced in mice by intraperitoneal injections of LPS(10 mg/kg).After 6 h,mice were intervened with 0.5 mL phosphate buffered saline(PBS)containing 1×10^(6) HUC-MSCs by intraperitoneal injections.For the negative control,100 mL 0.9%NaCl and 0.5 mL PBS were used.Bronchoalveolar lavage fluid(BALF)was obtained from anesthetized mice,and their blood,lungs,ileum,and feces were obtained by an aseptic technique following CO_(2) euthanasia.Wright’s staining,enzyme-linked immunosorbent assay,hematoxylin-eosin staining,Evans blue dye leakage assay,immunohistochemistry,fluorescence in situ hybridization,western blot,16S rDNA sequencing,and non-targeted metabolomics were used to observe the effect of HUC-MSCs on ALI mice,and the involvement of the lung-gut axis in this process was explored.One-way analysis of variance with post-hoc Tukey’s test,independent-sample Student’s t-test,Wilcoxon rank-sum test,and Pearson correlation analysis were used for statistical analyses.RESULTS HUC-MSCs were observed to improve pulmonary edema and lung and ileal injury,and decrease mononuclear cell and neutrophil counts,protein concentrations in BALF and inflammatory cytokine levels in the serum,lung,and ileum of ALI mice.Especially,HUC-MSCs decreased Evans blue concentration and Toll-like receptor 4,myeloid differentiation factor 88,p-nuclear factor kappa-B(NF-κB)/NF-κB,and p-inhibitorαof NF-κB(p-IκBα)/IκBαexpression levels in the lung,and raised the pulmonary vascular endothelial-cadherin,zonula occludens-1(ZO-1),and occludin levels and ileal ZO-1,claudin-1,and occludin expression levels.HUC-MSCs improved gut and BALF microbial homeostases.The number of pathogenic bacteria decreased in the BALF of ALI mice treated with HUCMSCs.Concurrently,the abundances of Oscillospira and Coprococcus in the feces of HUS-MSC-treated ALI mice were significantly increased.In addition,Lactobacillus,Bacteroides,and unidentified_Rikenellaceae genera appeared in both feces and BALF.Moreover,this study performed metabolomic analysis on the lung tissue and identified five upregulated metabolites and 11 downregulated metabolites in the LPS+MSC group compared to the LPS group,which were related to the purine metabolism and the taste transduction signaling pathways.Therefore,an intrinsic link between lung metabolite levels and BALF flora homeostasis was established.CONCLUSION This study suggests that HUM-MSCs attenuate ALI by redefining the gut and lung microbiota.

外周血单个核细胞Toll样受体4和B淋巴细胞刺激因子表达水平与IgA肾病患者临床指标的关系

目的 探讨外周血单个核细胞Toll样受体4(TLR4)和B淋巴细胞刺激因子(BLyS)表达水平与免疫球蛋白(Ig)A肾病患者临床指标的关系。方法 回顾性分析122例IgA肾病患者的临床资料,比较不同外周血单个核细胞TLR4和BLyS表达水平患者临床指标,并分析外周血单个核细胞TLR4和BLyS表达水平与临床指标的相关性。结果 低TLR4水平组和高TLR4水平组间、低BLyS水平组和高BLyS水平组间性别、收缩压、高血压史、低密度脂蛋白(LDL)、血尿酸、血肌酐和尿素氮差异均有统计学意义;Pearson相关性分析结果显示,IgA肾病患者外周血单个核细胞TLR4、BLyS表达水平与收缩压、LDL、血尿酸和血肌酐水平均呈正相关(均P<0.05)。结论 外周血单个核细胞TLR4和BLyS表达水平与IgA肾病患者收缩压、LDL、血尿酸和血肌酐水平均呈正相关。

Mannan-binding lectin directly interacts with Toll-like receptor 4 and suppresses lipopolysaccharide-induced inflammatory cytokine secretion from THP-1 cells

Mannan-binding lectin(MBL)plays a key role in the lectin pathway of complement activation and can influence cytokine expression.Toll-like receptor 4(TLR4)is expressed extensively and has been demonstrated to be involved in lipopolysaccharide(LPS)-induced signaling.We first sought to determine whether MBL exposure could modulate LPS-induced inflammatory cytokine secretion and nuclear factor-kB(NF-kB)activity by using the monocytoid cell line THP-1.We then investigated the possible mechanisms underlying any observed regulatory effect.Using ELISA and reverse transcriptase polymerase chain reaction(RT-PCR)analysis,we found that at both the protein andmRNAlevels,treatment withMBLsuppresses LPS-induced tumor-necrosis factor(TNF)-a and IL-12 production in THP-1 cells.An electrophoretic mobility shift assay and western blot analysis revealed that MBL treatment can inhibit LPS-induced NF-kB DNA binding and translocation in THP-1 cells.While the binding of MBL to THP-1 cells was evident at physiological calcium concentrations,this binding occurred optimally in response to supraphysiological calcium concentrations.This binding can be partly inhibited by treatment with either a soluble form of recombinant TLR4 extracellular domain or anti-TLR4 monoclonal antibody(HTA125).Activation of THP-1 cells by LPS treatment resulted in increased MBL binding.We also observed that MBL could directly bind to the extracellular domain of TLR4 in a dose-dependent manner,and this interaction could attenuate the binding of LPS to cell surfaces.Taken together,these data suggest that MBL may affect cytokine expression through modulation of LPS-/TLR-signaling pathways.These findings suggest that MBL may play an important role in both immune regulation and the signaling pathways involved in cytokine networks.

Role of Toll-like receptor 4 and human defensin 5 in primary endocervical epithelial cells

Background Endocervical epithelial cells play early roles in the defense of upper female genital tract to pathogens. Toll-like receptors （TLRs） and human defensins （HD） have recently been identified as fundamental components of the innate immune responses to bacterial pathogens. We aimed to use in vitro model of human primary endocervical epithelial cells （HPECs） to investigate their roles in innate immune response of the endocervix. Methods TLR4 expression and distribution in HPECs and endocervix were investigated by immunofluorescence （IF）. Cultured HPECs were divided into lipopolysaccharide （LPS） group which were treated by LPS for 0, 24 and 48 hours, and control group without treatment. At each time point, the levels of HD5, IL-6 and TNF-a in supernants were determined by ELISA. TLR4 and HD5 expressions of cells were detected by Western blotting simultaneously. HD5 expression pattern was also compared between the HeLa cell line and HPECs. Results Endocervix tissue surface and HPECs expressed TLR4. After incubated with LPS, HPECs expressed significantly higher levels of TLR4 than control group, especially after 24 hours （P 〈0.01）, however decreased after 48 hours with a similar level of TLR4 expression compared with control group. LPS could upregulate the secretion of HD5, IL-6 and TNF-a in a time-dependent manner （24 hours： P 〈0.05; 48 hours： P 〈0.01, compared with control group）. Intracellular HD5 expression levels decreased over time. HD5 expression patterns in HPECs were different from HeLa cell line. Conclusions To respond to LPS stimulation, HPECs may function in the mucosal immune defense through TLR4 activation and HD5 secretion. HPEC is considered a significant model for immunological study.

类风湿性关节炎患者早期外周血单个核细胞Toll样受体2、4表达及意义

目的探讨类风湿性关节炎(RA)早期患者外周血单个核细胞(PBMC)Toll样受体(TLR)2、TLR4对其配体双糖链蛋白多糖(BGN)、脂多糖(LPS)的刺激反应性,阐明PBMC TLR2、TLR4在RA疾病早期中的作用。方法采用流式细胞术、实时荧光定量逆转录(RT)-聚合酶链反应(PCR)、酶联免疫吸附试验(ELISA)分别检测BGN和LPS刺激前后,RA组和健康对照组外周血CD14+单核细胞TLR4+细胞频率、PBMC TLR2 mRNA和TLR4mRNA及上清液白细胞介素6(IL-6)和肿瘤坏死因子α(TNFα)的含量变化。结果 RA早期患者TLR2 mRNA明显升高、TLR4 mRNA下降(P<0.01);经LPS刺激后,RA患者TLR4 mRNA升高3.50倍,而健康对照组下降到0.11倍;LPS和BGN促进了各组PBMC产生IL-6、TNFα,但RA早期患者组上升的倍数明显高于健康对照组。结论 PBMC TLR2、TLR4参与早期RA的发生、发展。

乙型肝炎肝硬化患者外周血中单个核细胞Toll样受体4的表达及其与血清内毒素的关系

目的:通过检测乙型肝炎肝硬化患者外周血单个核细胞(PMBCs)Toll样受体4(TLR4)mRNA的表达、血清内毒素水平及肿瘤坏死因子α(TNF-α);探索TLR4 mRNA表达与血清内毒素及TNF-α的关系。方法:抽取29例乙型肝炎肝硬化患者外周静脉血,分离单个核细胞,用RT-PCR半定量分析TLR4 mRNA水平;同时用鲎试剂测定血清内毒素水平和用放免法测定血清TNF-α水平。结果:正常对照组和肝硬化患者血清内毒素水平分别为0.050±0.020$复旦大学附属中山医院消化科!上海200032 EU·mL-1和0.107±0.080 EU·mL-1(P<0.05),血清TNF-α水平分别为1.282pg·mL-1±0.249pg·mL-1和2.076±0.358pg·mL-1(P<0.05),后者均明显升高,而且肝功能损害越严重内毒素水平升高越明显;肝硬化患者PMBCs中TLR4 mRNA的表达(0.900±0.263)与正常对照(0.923±0.461)无显著差异(P>0.05)。结论:在肝硬化患者,尤其主要是Child-Pugh C级患者,血清内毒素及TNF-α水平较正常对照组明显升高,但PMBC的TLR4 mRNA的表达水平无变化,这可能与肝硬化患者同时存在内毒素耐受有关。