淋巴细胞特异蛋白质酪氨酸激酶/P38信号传递途径在小鼠肾小管上皮细胞的作用

目的 探讨小鼠肾上管上皮细胞是否存在淋巴细胞特异蛋白质酪氨酸激酶（lck）基因表达及lck/p38细胞分裂原活化蛋白信号传递分子经IL-12、IL-2、脂多糖（LPS）等刺激时在肾小管上皮细胞的变化。方法 无菌分离培养BALB/C小鼠肾小管上皮细胞,采用地高辛标记的lck寡核苷酸探针原位杂交检测肾小管上皮细胞中lck基因表达;应用放射自显影方法观察lck活性变化,利用免疫印迹分析lck蛋白表达及p38磷酸化。结果 IL-12、IL-2、LPS刺激肾小管上皮细胞活化,lck mRNA表达较对照组明显增强;经上述物质刺激后lck活性、p38磷酸化分别于5min、15min最强,其中LPS刺激最显著而lck、p38蛋白水平则无明显变化。加入lck抑制剂PP1,IL-12刺激时未发现p38磷酸化,而IL-2、LPS刺激只有轻度抑制。结论 IL-12、IL-2、LPS促进肾小管上皮细胞中lck基因表达,并只有IL-12唯一通过lck诱导p38磷酸化,它们皆可通过lck/p38信号传递途径参与对肾小皮细胞发挥生物作用。     

Mediation of inflammatory activation of renal tubular epithelial cells by high mobility group protein box 1 interacting with Toll-like receptor 4

Objective To observe functional changes of renal tubular epithelial cells stimulated by high mobility group protein box 1 (HMGB1) and associated mechanism. Methods Renal tubular epithelial cells (NRK52E) were divided into control group, HMGB1 group and HMGB1+lipopolysaccharide from Rhodobactersphaeroides (LPS RS) group. Toll-like receptor 4 (TLR4) expression was detected by immunofluorescence and Western blotting. Apoptosis rate and cell cycle arrest were identified with flow cytometry. The activation of MAPK signaling pathway and NF-κB were detected by Western blotting. The IL-1, IL-6 and tissue inhibitor of metalloproteinases 2 (TIMP2) mRNA levels were measured by real-time PCR. The secretion levels of IL-1, IL-6 and TIMP2 were measured by protein chips assay. Results TLR4 was expressed by NRK52E cells. Compared with the control group, there were increased cell cycle G1 arrest, MAPK signaling pathway and NF-κB activation in HMGB1 group. Furthermore, IL-1, IL-6 and TIMP2 mRNA levels were increased and IL-1, IL-6 and TIMP2 were secreted by NRK52E when stimulated with HMGB1 (all P＜0.05). However, effects mediated by HMGB1 stimulation could be inhibited by LPS RS (all P＜0.05). Conclusions Inflammatory activation of NRK52E cells can be mediated by the interaction of HMGB1 and TLR4.     

Cyclosporine-induced renal injury induces toll-like receptor and maturation of dendritic cells

BACKGROUND: The toll-like receptor (TLR) is stimulated by not only pathogen-associated molecular patterns but also endogenous TLR ligands provided by injured cells. The influence of cyclosporine A (CsA)-induced renal injury on TLR expression and subsequent signaling pathway was evaluated. METHODS: Induction of chronic CsA nephropathy was made by administering CsA (15 mg/kg/day) for 28 days in rats. The TLR2 and TLR4 mRNA and protein expression, TLR-signaling pathway (MYD88, NF-kappaB and AP-1), putative TLR ligand (heat shock protein 70 [HSP70]), and maturation of dendritic cells were evaluated in CsA-treated rat kidneys. RESULTS: Long-term CsA treatment upregulated TLR2 and TLR4 mRNA and protein expression on renal tubular cells, and these were accompanied by increased MYD88, NF-kappaB and AP-1 expression. Putative TLR ligand (HSP70) was also significantly increased in CsA-treated rat kidney compared with vehicle-treated rat kidney. CsA-treatment increased expression of TNF-alpha mRNA, the number of dendritic cells, and expression of MHC class II antigen. Double-labeling of markers of dendritic cells and MHC class II antigen revealed that matured dendritic cells increased in CsA-treated rat kidney. CONCLUSIONS: CsA-induced renal injury stimulates components of innate immunity, and this finding suggests close association between CsA-induced renal injury and activation of innate immunity.     

High-avidity antitumor T-cell generation by toll receptor 8-primed, myeloid- derived dendritic cells is mediated by IL-12 production

BACKGROUND: High-level production of heterodimeric p70 interleukin (IL)-12 by myeloid-derived dendritic cells (DCs) requires 2 signals: interferon gamma (IFN-gamma) and a maturation signal provided by CD40 ligation (CD40L) or lipopolysaccharide (LPS). METHODS: In the current study we demonstrate that signaling through toll-like receptor (TLR) 8, but not TLR3, TLR2, or TLR4, provides a priming signal to myeloid-derived DC for high IL-12 p70 heterodimer production. RESULTS: All the TLR agonists induced maturation of DC as evidenced by increased expression of CD83, CD80, and CD86. Both IFN-gamma and TLR7/8 agonist R848 increased expression of TLR8 in immature monocyte-derived DCs. The combination of TLR7/8 agonist R848 and maturation signals LPS or CD40L induced high-level expression of IL-12p35 and p40 similar to that induced by IFN-gamma plus LPS. In contrast, receptor agonists specific for TLR7 did not prime for IL-12 production. The p70 IL-12 produced by the TLR8-primed DC polarized CD4+ T for Th1 cytokine production and induced CD8+ T cells, displaying high functional avidity with enhanced tumor cell recognition. CONCLUSIONS: The data suggest that toll 8 receptor agonists are useful for inducing type-1 polarized DCs for vaccine design in treating cancer and infectious disease.     

Signaling through the interleukin-18 receptor α attenuates inflammation in cisplatin-induced acute kidney injury

Interleukin (IL)-18 is produced by leukocytes and renal parenchymal cells (tubular epithelial cells, podocytes, and mesangial cells). The IL-18 receptor (IL-18R) is expressed on these cells in cisplatin-induced acute kidney injury, but the role of IL-18R is unknown. To help define this, we compared IL-18Rα knockout with wild-type mice in cisplatin-induced acute kidney injury and found deteriorated kidney function, tubular damage, increased accumulation of leukocytes (CD4(+) and CD8(+) T-cells, macrophages, and neutrophils), upregulation of early kidney injury biomarkers (serum TNF, urinary IL-18, and KIM-1 levels), and increased expression of pro-inflammatory molecules downstream of IL-18. In vitro, leukocytes from the spleen and kidneys of the knockout mice produced greater amounts of pro-inflammatory cytokines upon stimulation with concanavalin A compared to that in wild-type mice. Levels of the suppressor of cytokine signaling 1 and 3 (negative regulators of cytokine signaling) were reduced in the spleen and kidneys of IL-18Rα-deficient compared to wild-type mice. Adoptive transfer of wild-type splenocytes by IL-18Rα-deficient mice led to decreased cisplatin nephrotoxicity compared to control IL-18Rα-deficient mice. In contrast, anti-IL-18Rα and anti-IL-18Rβ antibody treatment tended to increase cisplatin nephrotoxicity in wild-type mice. Thus, signaling through IL-18Rα activates both inflammation-suppressing and pro-injury pathways in cisplatin-induced acute kidney injury.     

In vitro and in vivo interleukin-8 production in human renal cortical epithelia.

The signals resulting in leukocytes infiltrating the tubulointerstitial compartment during renal inflammatory disease are not well understood. A recently described cytokine, interleukin-8 (IL-8), has been demonstrated to be chemotactic for lymphocytes and neutrophils at picomolar and nanomolar concentrations, respectively. Cytokeratin positive, renal cortical epithelial cells (RCEC) with tubular attributes were cultured from kidney tissue from six human subjects. We report that these human renal cortical epithelial cells in primary cell culture respond to either IL-1 beta, TNF or LPS in both a time- and dose-dependent manner by expressing IL-8 mRNA and secreting antigenic IL-8 peptide. In addition, RCEC were found to be strongly positive for cell-associated antigenic IL-8 peptide by immunostaining after 24 hour incubation with IL-1 beta, TNF and LPS. To ascertain whether IL-8 was present in renal disease associated with infiltrating leukocytes, we performed immunohistochemistry on renal biopsy specimens from patients with acute allograft rejection. Both proximal and distal tubular epithelial cells were found to be strongly positive for cell-associated antigenic IL-8. These findings suggest that the human renal tubule epithelial cell may actively participate in acute inflammatory processes in the kidney, including allograft rejection, by effecting and directing leukocyte chemotaxis via the production of IL-8.     

Renal cells express a functional interleukin-15 receptor.

BACKGROUND: Interleukin (IL)-15 is a pleiotropic cytokine known to be involved in graft rejection and to serve as a survival factor for leukocytes and epithelial cells, including renal cells. It utilizes a heterotrimeric receptor complex that consists of the IL-2 receptor betagammac subunits (IL-2/15Rbetagammac) and a unique high-affinity alpha-chain responsible for IL-15 specificity. METHODS: The cDNA of IL-15Ralpha main mRNA product was isolated from primary human tubular epithelial cells (TEC) and sequenced. IL-15R expression in TEC and in murine renal tissue was demonstrated using western blotting, ligand binding and flow cytometry. TEC were activated with combinations of IL-15, IL-2 and interferon-gamma (IFN-gamma), and mRNA and protein levels of IL-15R were determined. Jak-STAT tyrosine phosphorylation was assayed following IL-15 exposure. RESULTS: The full-length alpha-chain mRNA bearing exons 1-7 is expressed in TEC. IL-15Ralpha protein was detected on intact cells by flow cytometry and in extracts of human and mice renal cells using a specific anti-IL-15Ralpha antibody and by ligand-binding assay. The three subunits of the IL-15R were similarly expressed in cortex and medulla of mice kidney. Stimulation of TEC with IFN-gamma upregulated the alpha-chain while IL-2 and IL-15 had no effect on its expression. A short IL-15 stimulation of TEC induced tyrosine phosphorylation of the main IL-15 signalling molecules (Jak-1, Jak-3, STAT-3 and STAT-5). CONCLUSIONS: Our data demonstrate the presence of a functional IL-15 receptor in the kidney. Since renal cells produce IL-15, this cytokine may have an autocrine/paracrine regulatory role in the kidney.     

TLR4介导大鼠肾脏缺氧复氧炎症反应依赖于MyD88

目的：探讨MyD88是否参与了TLR介导大鼠肾脏缺氧复氧（hypoxia/reoxygenation,H/R）引起的炎症反应.方法：体外分离获得Wistar大鼠原代肾小管上皮细胞（proximal tubule epithelial cells,PTECs）,建立H/R模型.采用TLR4siRNA干扰PTECs,检测白细胞介素8（interleukin-8,IL-8）和肿瘤坏死因子α（tumor necrosis factor α,TNF-α）的表达.之后,加入髓样分化因子88（myeloid differentiation factor 88,MyD88）的抑制剂ST2825后,检测IL-8和TNF-α的表达.结果：TLR4表达沉默后,IL-8、TNF-α和MyD88的表达均显著下降（P〈0.05）;加入ST2825后,IL-8、TNF-α和MyD88的表达均显著下降（P〈0.05）,且抑制MyD88活性后显著降低细胞的凋亡水平（P〈0.05）.结论：MyD88参与了TLR4介导的大鼠肾脏缺氧复氧引起的炎症反应.     

Toll-like receptors 2 and 4 in renal ischemia/reperfusion injury

Toll-like receptors (TLRs) are an evolutionarily conserved family of cell membrane receptors that are part of the innate immunity system playing an important role as a first response to tissue injury. TLR2 and TLR4 are constitutively expressed on renal epithelium, and their expression is enhanced following renal ischemia/reperfusion (I/R) injury. Genetic deletion of either TLR2 or TLR4 protects from renal I/R injury. However, it is not known whether deletion of both combined protects the kidney more than a deletion of either one alone. Therefore, we performed renal I/R injury in mice lacking TLR2, TLR4, and TLR2/4, respectively. Our results demonstrate that there are no significant differences regarding protection from renal I/R injury in TLR2/4^(?/?) compared with either TLR2^(?/?) or TLR4^(?/?) gene-targeted mice as determined by histological evaluation and renal functional parameters. Furthermore, there was no difference in the number of apoptotic tubular cells and in nuclear translocation of nuclear factor kappa-B (NF-κB) between the TLR-gene-targeted groups. In parallel, in vitro experiments did not demonstrate an additional effect of the double genetic deletion compared with the single gene deletion with respect to tumor necrosis factor (TNF)-alpha and interleukin (IL)-8 production in hypoxic isolated proximal tubular epithelial cells of the respective animals. In conclusion, a double genetic deletion of TLR2 and TLR4 confers a similar protection following renal I/R injury compared with single deletions of TLR2 and TLR4.     

Ischemia-reperfusion injury activates innate immunity in rat kidneys

BACKGROUND: There is growing evidence of a role of the immune system in the pathophysiology of ischemia-reperfusion (I/R) injury, but the influence of I/R injury on innate immunity is still undetermined. METHODS: Sprague-Dawley rats were used. I/R injury was induced by clamping both renal arteries for 45 min, and the rats were killed 1, 3, 5, and 7 days later. Activation of innate immunity was evaluated in terms of the expression of toll-like receptor (TLR) 2 or TLR4 mRNAs and protein, by the level of the TLR ligand (heat shock protein [HSP] 70), and maturation of dendritic cells by double-label immunohistochemistry of dendritic cells for major histocompatibility complex (MHC) class II antigen. RESULTS: I/R injury increased TLR2 and TLR4 mRNA and protein expression, and they were mainly observed on renal tubular cells. I/R injury also produced endogenous TLR ligand (HSP70) on renal tubular cells. I/R injury increased not only the numbers of dendritic cells but also the production of MHC class II antigen in dendritic cells, suggesting maturation of these cells. Activation of innate immunity was observed at day 1, peaked at days 3 to 5 after I/R injury, and thereafter gradually decreased. CONCLUSIONS: I/R injury rapidly activates the innate immune response.     

外源性硫化氢抑制TLR2和TLR4表达并减轻大鼠肾脏缺血再灌注损伤

目的观察外源性硫化氢(H₂S)供体硫氢化钠(NaHS)能否抑制Toll样受体2(TLR2)和Toll样受体4(TLR4)表达、减轻大鼠肾脏缺血再灌注损伤(IRI)。 方法24只6~8周龄雄性SD大鼠随机分为3组：假手术(Sham)组、肾脏缺血再灌注(I/R)组、NaHS+I/R组。采用右肾切除联合左肾动脉夹闭45 min后再灌注24 h的方法诱导肾IRI。夹闭左肾动脉前，NaHS+I/R组给予NaHS(300 nmol/min)连续输注10 min，Sham组和I/R组则给予等体积生理盐水。分别留取各组腹主动脉血及肾组织标本。Western印迹法检测肾组织TLR2、TLR4蛋白的表达；免疫组织化学法检测肾组织白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)的表达；比色法检测血尿素氮(BUN)、血肌酐(Scr)。HE染色观察肾脏组织学改变；TUNEL法检测肾组织细胞凋亡。 结果与Sham组比较，I/R组的TLR2、TLR4、IL-6、TNF-α表达均增加(P<0.05)，BUN、Scr亦明显升高(P<0.05)，肾小管上皮损伤评分较高(P<0.05)，肾组织凋亡细胞增加(P<0.05)。与I/R组比较，NaHS+I/R组的TLR2、TLR4、IL-6、TNF-α表达均减少(P<0.05)，BUN、Scr亦明显下降(P<0.05)，肾小管上皮损伤评分较低(P<0.05)，肾组织凋亡细胞减少(P<0.05)。 结论外源性H₂S可以抑制TLR2、TLR4途径，减少炎症因子释放及细胞凋亡，减轻大鼠肾脏IRI。     

Histones from dying renal cells aggravate kidney injury via TLR2 and TLR4

In AKI, dying renal cells release intracellular molecules that stimulate immune cells to secrete proinflammatory cytokines, which trigger leukocyte recruitment and renal inflammation. Whether the release of histones, specifically, from dying cells contributes to the inflammation of AKI is unknown. In this study, we found that dying tubular epithelial cells released histones into the extracellular space, which directly interacted with Toll-like receptor (TLR)-2 (TLR2) and TLR4 to induce MyD88, NF-κB, and mitogen activated protein kinase signaling. Extracellular histones also had directly toxic effects on renal endothelial cells and tubular epithelial cells in vitro. In addition, direct injection of histones into the renal arteries of mice demonstrated that histones induce leukocyte recruitment, microvascular vascular leakage, renal inflammation, and structural features of AKI in a TLR2/TLR4-dependent manner. Antihistone IgG, which neutralizes the immunostimulatory effects of histones, suppressed intrarenal inflammation, neutrophil infiltration, and tubular cell necrosis and improved excretory renal function. In summary, the release of histones from dying cells aggravates AKI via both its direct toxicity to renal cells and its proinflammatory effects. Because the induction of proinflammatory cytokines in dendritic cells requires TLR2 and TLR4, these results support the concept that renal damage triggers an innate immune response, which contributes to the pathogenesis of AKI.     

表面活性蛋白A在脂多糖诱导的人肾小管上皮细胞中的表达

目的 研究表面活性蛋白A（SP-A）及其亚型在人肾组织和人肾小管上皮细胞（HK-2）的表达和分布,同时分析脂多糖（LPS）对HK-2细胞中SP-A亚型的 mRNA和蛋白表达的影响。 方法 收集10例人的肾组织,以5例人的肺组织作为对照,同时培养HK-2细胞。免疫组化法检测SP-A在人肾组织的表达部位;RT-PCR法检测SP-A mRNA在人肾组织和HK-2细胞中的表达;限制性内切酶片段长度多态性（RFLP）和测序的方法分析SP-A亚型在HK-2细胞的表达;实时定量PCR法比较SP-A mRNA在人肾组织和在人肺组织中的相对含量;Western印迹法检测人肾组织和HK-2细胞中SP-A蛋白的表达;Western印迹和ELISA法检测人的尿液和HK-2细胞培养上清液中SP-A的分泌量。RT-PCR和Western印迹检测LPS在不同浓度（0、0.1、1、2、5、10 mg/L）作用8 h及5 mg/L LPS在不同时间（0、2、4、8、16、24 h）作用HK-2细胞后,SP-A mRNA和蛋白表达的变化情况。 结果 免疫组化结果显示SP-A主要表达在肾皮质的远曲和近曲小管的肾小管上皮细胞。RFLP和测序的方法均证实HK-2细胞可同时表达SP-A1和SP-A2亚型。实时定量PCR证实SP-A mRNA在人肾组织的表达量仅为肺组织的30%（n = 5）。Western印迹检测到人肾组织和HK-2细胞可表达SP-A蛋白。同时在人的尿液和HK-2细胞培养上清液中也检测到SP-A的分泌,其分泌量分别为（106.614±72.772） nmol/L（n = 30）和（85.533± 58.622） nmol/L（n = 10）。应用1、2、5、10 mg/L的 LPS刺激HK-2细胞8 h后,SP-A1和SP-A2 mRNA及SP-A蛋白表达较0、0.1 mg/L显著升高（P < 0.05）;同时,应用5 mg/L的LPS作用HK-2细胞4、8、16、24 h后, SP-A1和SP-A2 mRNA及SP-A蛋白表达较LPS作用0、2 h显著升高（P < 0.05）。 结论 HK-2细胞能同时表达SP-A1和SP-A2亚型,能产生和分泌SP-A蛋白。SP-A1和SP-A2可能在肾脏的天然免疫和炎性反应调节方面起重要作用。     

TLR2 and NODs1 and 2 cooperate in inflammatory responses associated with renal ischemia reperfusion injury

Pattern recognition receptors (PRRs) are potent triggers of tissue injury following renal ischemia/reperfusion injury (IRI). Specific PRRs, such as the toll-like receptor 2 (TLR2) and the nucleotide-binding oligomerization domain-like receptors (NLRs) NOD1 and NOD2 are promising targets to abrogate inflammatory injury associated with renal IRI. Several recent reports have shown there is crosstalk between TLRs and NODs, which might boost inflammatory responses to tissue injury. This study examined the relative roles of TLR2 and NODs 1 and 2 in activation of myeloid cells that contribute to inflammation after renal IRI. We found that TLR2 and NOD1 and 2 signaling induces neutrophil, macrophage and dendritic cell migration in vitro, however their blockade only decreases neutrophil infiltration into ischemic kidneys. The results of this study suggest that future therapies targeted to innate immune blockade should consider that either TLR2 or NOD1/2 blockade could decrease neutrophil inflammation following an ischemic insult to the kidney, however blockade of these PRRs would not likely impact infiltration of dendritic cells or macrophages. Developing rational approaches that target innate immunity in IRI-induced acute kidney injury requires an understanding of the relative role of PRRs in directing inflammation in the kidney.     

萝卜硫素通过Traf6/TAK1通路调节肾小管上皮细胞间充质转分化

目的探讨萝卜硫素对脂多糖(LPS)诱导的肾小管上皮细胞(HK-2)间充质转分化的影响及其对Traf6/TAK1信号通路活化的作用。方法体外培养HK-2细胞,并将其分为正常组、LPS诱导组(100 ng/mL)、LPS+萝卜硫素低剂量组(10μmol/L)、LPS+萝卜硫素中剂量组(20μmol/L)、LPS+萝卜硫素高剂量组(40μmol/L)。培养12、24、48 h后,采用CCK8法检测细胞增殖情况,ELISA法检测细胞上清中IL-6、IL-1β及TNF-α含量,Western blot方法检测E-cadherin、α-SMA、Traf6、TAK1及p-TAK1蛋白的表达量。结果LPS诱导组相对于正常组HK-2细胞增殖能力、炎症因子(IL-6、IL-1β和TNF-α)水平、蛋白(α-SMA、Traf6及p-TAK1)表达量均升高,而E-cadherin蛋白表达水平降低;然而不同浓度萝卜硫素处理细胞后细胞增殖能力、炎症因子分泌量、蛋白(α-SMA、Traf6及p-TAK1)表达量均降低,E-cadherin蛋白表达水平升高,表现一定剂量依赖性,差异有统计学意义(P<0.05)。结论萝卜硫素能够抑制LPS诱导的HK-2细胞增殖能力及炎症因子的产生,发挥抑制肾小管上皮细胞-肌成纤维细胞转变的纤维化过程,其机制可能与抑制Traf6/TAK1信号通路活化有关。     

Dysregulated toll-like receptor-induced interleukin-1β and interleukin-6 responses in subjects at risk for the development of type 1 diabetes

We tested the hypothesis that altered Toll-like receptor (TLR) signaling may be involved in early stages of type 1 diabetes (T1D). To do so, we analyzed TLR-induced interleukin (IL)-1β and IL-6 responses in freshly isolated peripheral blood mononuclear cells (PBMNCs) from seropositive compared with seronegative subjects. Similar frequencies of myeloid dendritic cells (mDCs), plasmacytoid DCs (pDCs), and monocytes were observed in seropositive and seronegative subjects. Subjects with autoantibodies had increased proportions of monocytes expressing IL-1β ex vivo. Activating PBMNCs with TLR3, TLR4, or TLR7/8 agonists in vitro led to increased percentages of IL-1β-expressing monocytes and mDCs from seropositive versus seronegative subjects. TLR ligation also resulted in a diminished IL-6 response in seropositive individuals as lower frequencies of IL-6-expressing monocytes and mDCs were induced. The dysregulated TLR-induced IL-1β and IL-6 pathways were more readily detectable in children aged <11 years and from 11 to <21 years, respectively, and did not involve altered HbA(1c) or the presence of one or more autoantibodies. Finally, subjects with autoantibodies had lower amounts of serum chemokine (C-X-C motif) ligand 10 compared with autoantibody-negative subjects. Our data may imply that alterations in innate immune pathways are detectable in genetically susceptible individuals and could be linked with the early course of T1D.     

右美托咪啶对脂多糖诱导大鼠外周血单核细胞Toll样受体4mRNA表达的影响

目的 探讨右美托咪啶对脂多糖(LPS)诱导大鼠外周血单核细胞Toll样受体4(TLR4)mRNA表达的影响.方法 健康雄性Wistar大鼠40只,取外周血分离培养单核细胞,采用随机数字表法,将其随机分为5组(n=8),A组:阴性对照;B组:单核细胞中加入LPS(终浓度为1μg/ml);C组:单核细胞中加入LPS(终浓度为1μg/ml)+右美托咪啶(终浓度为0.5 ng/ml);D组:单核细胞中加入LPS(终浓度为1μg/ml)+右美托咪啶(终浓度为5.0 ng/ml);E组:单核细胞中加入LPS(终浓度为1μg/ml)+右美托咪啶(终浓度为50.0 ng/ml).孵育24 h后,收集上清液,采用ELISA法测定TNF-α、IL-1β和IL-6的浓度,采用RT-PCR法测定TLR4 mRNA的表达.结果 与A组比较,B组TNF-α、IL-1p、IL-6的浓度升高,TLR4 mRNA表达上调(P＜0.01);与B组比较,C组、D组和E组TNF-α、IL-1β、IL-6的浓度降低,TLR4 mRNA表达下调(P＜0.05或0.01);与C组比较,D组和E组TNF-α、IL-1β、IL-6的浓度降低(P＜0.01),TLR4 mRNA表达差异无统计学意义(P＞0.05);D组和E组各指标比较差异无统计学意义(P＞0.05).结论 右美托咪啶可通过下调TLR4 mRNA表达,抑制TLR4的合成,从而抑制LPS诱导大鼠外周血单核细胞TNF-α、IL-1β和IL-6的生成与释放.

Abstract：

Objective To investigate the effects of different concentrations of dexmedetomidine on the expression of Toll-like receptor 4 (TLR4) mRNA in rat peripheral blood monocytes exposed to lipopolysaccharide ( LPS ). Methods Peripheral blood monocytes isolated from male Wistar rats were seeded in 24-well plate in RPMI 1640 liquid culture medium in CO2 incubator at 37 ℃ and 5% CO2 for 2 h, and were randomly divided into 5 groups ( n = 8 each): group A negative control; group B was exposed to LPS 1 μg/ml and C, D and E groups were exposed to LPS 1 μg/ml + dexmetomidine 0.5, 5.0 and 50.0 ng/ml respectively. The monocytes were then incubated for 24 h. The concentrations of TNF-α, IL-1β and IL-6 in the supernatant of the cultured monocytes were detected by ELISA. The expression of TLR4 mRNA in the monocytes was detected by RT-PCR.Results Exposure to LPS significantly increased the expression of TLR4 mRNA and the concentrations of TNF-α, IL-1β and IL -6 in group B as compared with group A ( P ＜ 0.01 ). Dexmedetomidine attenuated the LPS-induced increase in the expression of TLR 4 mRNA and the concentrations of TNF-α, IL-1β and IL-6 in a dose-dependent manner ( P ＜0.05or 0.01 ). Conclusion Dexmedetomidine can inhibit the synthesis of TLR4 and inhibit the secretion and dilivery of TNF-α, IL-1β and IL-6 by down-regulating the gene expression of TLR4 in rat peripheral blood monocytes exposed to LPS.     

Toll-like receptor 4 promotes tubular inflammation in diabetic nephropathy

Inflammation contributes to the tubulointerstitial lesions of diabetic nephropathy. Toll-like receptors (TLRs) modulate immune responses and inflammatory diseases, but their role in diabetic nephropathy is not well understood. In this study, we found increased expression of TLR4 but not of TLR2 in the renal tubules of human kidneys with diabetic nephropathy compared with expression of TLR4 and TLR2 in normal kidney and in kidney disease from other causes. The intensity of tubular TLR4 expression correlated directly with interstitial macrophage infiltration and hemoglobin A1c level and inversely with estimated glomerular filtration rate. The tubules also upregulated the endogenous TLR4 ligand high-mobility group box 1 in diabetic nephropathy. In vitro, high glucose induced TLR4 expression via protein kinase C activation in a time- and dose-dependent manner, resulting in upregulation of IL-6 and chemokine (C-C motif) ligand 2 (CCL-2) expression via IκB/NF-κB activation in human proximal tubular epithelial cells. Silencing of TLR4 with small interfering RNA attenuated high glucose-induced IκB/NF-κB activation, inhibited the downstream synthesis of IL-6 and CCL-2, and impaired the ability of conditioned media from high glucose-treated proximal tubule cells to induce transmigration of mononuclear cells. We observed similar effects using a TLR4-neutralizing antibody. Finally, streptozotocin-induced diabetic and uninephrectomized TLR4-deficient mice had significantly less albuminuria, renal dysfunction, renal cortical NF-κB activation, tubular CCL-2 expression, and interstitial macrophage infiltration than wild-type animals. Taken together, these data suggest that a TLR4-mediated pathway may promote tubulointerstitial inflammation in diabetic nephropathy.