Activated Toll-like receptors participate in chronic renal tubulointerstitial injury induced by cyclosporine A

AIM: To study the role of innate immunity in the pathogenesis of renal tubulointerstitial injury.METHODS: The model of nephrotoxic nephropathy was induced by chronic cyclosporine A (CsA) administration (15 mg·kg^-1·d^-1 for 4 weeks) in Sprague-Dawley rats. The tubulointerstitial injury, characterized by inflammatory cell infiltration and striped fibrosis, was examined by the methods of immunohistochemistry and trichrome staining. The expression of Toll-like receptors (TLR), TLR ligand heat-shock protein 70 (HSP70)and intrarenal complement elements (C3, C4d and C9) was evaluated in rat kidneys by the methods of in situ hybridization and immunohistochemistry.RESULTS: Compared with the normal rats, the rats exposed to CsA showed impaired renal function, ED-1-positive cell infiltration and striped tubulointerstitial fibrosis (all P<0.01). Concomitantly, CsA treatment up-regulated the expression of TLR2 and TLR4 at mRNA and protein levels in renal tubular cells, accompanied by increased putative TLR ligand (HSP70) and the immunoreactivity of intrarenal complements. These up-regulated innate immunity components were located in the areas of severe tubulinterstitial injury.CONCLUSION: CsA-induced renal tubulointerstitial injury is closely associated with the activation of intrarenal innate immunity.     

Role of Toll-like receptor 4/MAPKs pathway on monocyte chemoattractant protein-1 secretion induced by oxidized low density lipoprotein in vascular smooth muscle cells

AIM: To investigate the role of Toll-like receptor 4/MAPKs pathway on the secretion of monocyte chemoattractant protein-1 (MCP-1) induced by oxidized low density lipoprotein (ox-LDL) in the vascular smooth muscle cells (VSMCs). METHODS: mRNA and protein expressions of MCP-1 in VSMCs stimulated with oxidized low density lipoprotein were determined by RT-PCR and ELISA, respectively. The phosphorylated forms of ERK1/2 and p38MAPK were determined by Western blotting. TLR4 neutralizing antibodies (a specific TLR4 inhibitor), PD98059 (ERK1/2 specific inhibitor), SB23015 (p38MAPK specific inhibitor) and SP600125 (JNK specific inhibitor) were used to investigate the underlying mechanisms. RESULTS: The mRNA and protein expressions of MCP-1 in VSMCs were up-regulated by ox-LDL (P<0.05), while those were inhibited by TLR4 neutralizing antibodies, PD98059 or SB23015 (P<0.05), but not by SP600125 (P>0.05). TLR4 had regulatory effect on the phosphorylation of ERK1/2 and p38MAPK. CONCLUSION: ox-LDL is an endogenous ligand of TLR4. The secretion of MCP-1 induced by ox-LDL in VSMCs is at least in part via TLR4/ERK1/2 and TLR4/p38MAPKs pathways.     

Role of PARP and caspase-3 in the airway epithelial injury induced by peroxynitrite

AIM:To study the mechanism responsible for ONOO^--induced the airway epithelial injury. METHODS:Effects of 3-aminobenzamide(3-AB), a poly-(ADP-ribose) polymerase(PARP) inhibitor, and Ac-DEVD-CHO, a caspase-3 inhibitor, on LDH release and apoptosis of cultured rat tracheal epithelial (RTE) cells induced by ONOO^- were examined. The cleavage of PARP was analysed by Western blot. RESULTS:3-AB inhibited the release of LDH induced by ONOO^- partially, and had no effect on the apoptosis of RTE cells. Caspase-3 inhibitor Ac-DEVD-CHO obviously prevented the apoptosis of RTE cells induced by ONOO^- in a dose-dependent manner. The cleavage of PARP was observed in the process of apoptosis of RTE cells induced by ONOO^-. CONCLUSIONS:PARP activation represents one of the pathways of ONOO^--mediated epithelial injury, and the excessive activation of PARP contributes to the necrosis in RTE cells induced by ONOO^-. Cleavage of PARP by activated caspase-3 plays a crucial role in the apoptosis of RTE cells induced by ONOO^-.     

Involvement of TLR4/NLRP3 inflammasome in contrast medium-induced inflammation and injury in renal tubular epithelial cells

AIM: To investigate whether Toll-like receptor 4 (TLR4) and Nod-like receptor protein 3 (NLRP3) inflammasome were involved in contrast medium (CM)-induced inflammation and injury in renal tubular epithelial cells. METHODS: Iopromide was used to injure NRK-52E cells in the study. The cell viability was measured by CCK-8 assay. The protein levels of TLR4, NLRP3, apoptosis-associated speckle-like protein (ASC), caspase-1 and cleaved caspase-3 were determined by Western blot. The releases of interleukin (IL)-1β and IL-18 were detected by ELISA. The apoptotic rate was evaluated by Hoechst staining, and mitochondrial membrane potential (MMP) was analyzed by JC-1 staining. siRNA was transfected into the NRK-52E cells to silence NLRP3 expression. RESULTS: CM decreased the viability of NRK-52E cells (P<0.05). CM also elevated the protein levels of cleaved caspase-3, TLR4, NLRP3, IL-1β and IL-18 (P<0.05). Silencing NLRP3 attenuated CM-induced releases of inflammatory cytokines. Moreover, treatment with TLR4 inhibitor TAK-242 or knockdown of NLRP3 by siRNA transfection both attenuated cell apoptosis and loss of MMP caused by CM. CONCLUSION: TLR4/NLRP3 inflammasome takes part in the pathogenesis of CM-induced acute kidney injury, and mediates CM-induced injury and inflammation in renal tubular epithelial cells.     

Toll-like receptor 4 promotes migration and invasion abilities of human non-small-cell lung cancer cells

AIM:To evaluate the expression and biological role of Toll-like receptor 4 (TLR4) in human non-small-cell lung cancer (NSCLC) cells. METHODS:The mRNA and protein levels of TLR4 in NSCLC tissue were exa-mined by RT-qPCR, Western blot, and immunohistochemistry. After treating the A549 cells and SPC-A-1 cells with TLR4 stimulator lipopolysaccharide (LPS) and inhibitor TAK-242, RT-qPCR, Western blot and flow cytometry were performed to detect the expression of TLR4. The migration and invasion abilities were detected by Transwell assay, and the mRNA expression of matrix metalloproteinase (MMP)-2, MMP-9, and vascular endothelial growth factor (VEGF) was also detected. RESULTS:The mRNA and protein levels of TLR4 were higher in the NSCLC tissue than those in the noncancerous tissue (P<0.01). LPS stimulation significantly increased the mRNA and protein expression levels of TLR4 in the NSCLC cell lines A549 and SPC-A-1 (P<0.01). The LPS-induced TLR4 activation enhanced the migration and invasion abilities of A549 cells and SPC-A-1 cells (P<0.01). LPS increased the expression levels of MMP-2, MMP-9 and VEGF in the A549 cells and SPC-A-1 cells (P<0.01). Moreover, the expression levels of TLR4, MMP-2, MMP-9 and VEGF, as well as the migration and invasion abilities of the cells were blocked by TAK-242 (P<0.01). CONCLUSION:TLR4 might be involved in the migration and invasion of NSCLC cells, and TLR4 inhibition might be considered as a therapeutic target for treatment of NSCLC.     

Toll-like receptor 4 expression on mast cells in human gingival tissues with chronic periodontitis

AIM: To observe the expression of Toll-like receptor 4 (TLR4) on mast cells in human gingival tissues with chronic periodontitis. METHODS: A total of 68 volunteers, including 23 cases of mild chronic periodontitis, 25 cases of severe chronic periodontitis and 20 healthy controls, were involved in this study, and their gingival specimens were taken and fixed in 4% neutral formalin. The histological changes of gingival tissues were observed by HE staining. The expression of TLR4 in gingival tissues was detected by immunohistochemical staining, and TLR4 expression on mast cells was detected by immunofluorescence double staining. RESULTS: The expression of TLR4 in gingival tissues and on mast cells in chronic periodontitis groups was significantly higher than that in normal control group (P<005), and that in severe chronic periodontitis group was significantly higher than that in mild chronic periodontitis group (P<005). CONCLUSION: The expression of TLR4 in gingival tissues and on mast cells is increased with the severity of chronic periodontitis, suggesting that TLR4, especially TLR4 on mast cells, may play an important role in human chronic periodontitis.     

Toll-like receptor 4 and damage of central nervous system

As a receptor mediating the transmembrane signal transduction in the innate immunity, Toll-like receptor 4 (TLR4) is a bridge between innate immunity and required immunity, and plays an important role when signal-transducing of some cells are activated. Recent reports show that TLR4 expresses in the different glial cells and strongly links to the innate immune activation and inflammatory response in the central nervous system (CNS). TLR4 plays a key role in the processes of brain damage by infection of the CNS, stroke, cerebral hemorrhage and trauma. In this review, we concentrate on recent findings regarding the progress of function and mechanism of TLR4 in the processes of the CNS damage in various diseases.     

Unfractionated heparin ameliorates lipopolysaccharide-induced expression of interleukin-8 in human endothelial cells through Toll-like receptor 4 signaling

AIM:To determine the effect of unfractionated heparin(UFH) on lipopolysaccharide(LPS)-induced expression of interleukin-8(IL-8) and the role of Toll-like receptor 4(TLR4) signaling. METHODS:Human pulmonary microvascular endothelial cells(HPMECs) were either exposed to LPS alone(10 mg/L) or in combination with 100 U/L or 10³ U/L UFH. UFH was added to the cells 15 min prior to stimulation with LPS. Those samples not receiving LPS or UFH received an equal volume of phosphate-buffered saline. The concentrations of IL-8 in the cell culture supernatants were detected by ELISA at 2, 6 and 12 h. The mRNA expression of IL-8, CD14 and TLR4 in HPMECs was detected by real-time fluorescence quantitative polymerase chain reaction at 2, 6 and 12 h. RESULTS:Compared with normal control group, the mRNA expression of IL-8 in LPS group was increased, and reached the peak at 6 h. The protein level of IL-8 reached the peak at 12 h. The mRNA expression of TLR4 in LPS group reached the peak at 6 h. They were down-regulated in UFH group. The mRNA expression of CD14 was not detected. CONCLUSION:The expression of IL-8 is obviously increased in LPS-treated HPMECs. UFH might exert its therapeutic effect through TLR4 signaling.     

Effect of VDUP-1 on apoptosis of renal tubular epithelial cells induced by high glucose

AIM: To investigate the effect of vitamin D3 up-regulated protein 1 (VDUP-1) on apoptosis of renal tubular epithelial cells induced by high glucose and its mechanism. METHODS: Human renal proximal tubular epithelial cell line HK-2 was treated with high glucose. The mRNA and protein levels of VDUP-1 in HK-2 cells were detected by real-time PCR and Western blot. HK-2 cells were transfected with VDUP-1 small interfering RNA (siRNA). Real-time PCR and Western blot were used to detect the inhibitory effect. The HK-2 cells were treated with high glucose, and the change of VDUP-1 expression was detected. The apoptosis was analyzed by flow cytometry. The activities of caspase-3 and caspase-9 in the cells were measured. The tumor necrosis factor-α (TNF-α) content in the culture supernatant was examined by ELISA. The key proteins of Sonic hedgehog (Shh) signaling pathway, Patched 1 (Ptch1), Smoothened (Smo), zinc finger protein Gli2 and Shh, were determined by Western blot. The HK-2 cells were treated with exogenous Shh, and the levels of Ptch1, Smo and Gli2 were detected by Western blot. After the HK-2 cells with VDUP-1 silencing were treated with exogenous Shh and high glucose, the apoptosis was analyzed by flow cytometry, the activities of caspase-3 and caspase-9 in the cells were examined, and the TNF-α content in culture supernatant was measured by ELISA. RESULTS: High levels of VDUP-1 mRNA and protein were observed in the HK-2 cells treated with high glucose. The mRNA and protein levels of VDUP-1 were decreased in the HK-2 cells transfected with VDUP-1 siRNA(P<0.05). Compared with the normally cultured cells, the apoptotic rate of HK-2 cells was increased after high glucose treatment, and the activities of caspase-3 and caspase-9 and the content of TNF-α were also significantly increased (P<0.05). After down-regulation of VDUP-1 expression by siRNA transfection, the apoptotic rate of HK-2 cells decreased after high glucose treatment, and the activities of caspase-3 and caspase-9, and the content of TNF-α were also significantly decreased (P<0.05). The protein levels of Ptch1, Smo, Gli2 and Shh were decreased after high glucose culture, while down-regulation of VDUP-1 partly antagonized the effect of high glucose on the expression of Ptch1, Smo, Gli2 and Shh in the HK-2 cells. Exogenous Shh promoted the expression of Ptch1, Smo and Gli2, and inhibited the apoptosis of the HK-2 cells induced by high glucose. Exogenous Shh and down-regulation of VDUP-1 synergistically inhibited high glucose-induced apoptosis of the HK-2 cells. CONCLUSION: Down-regulation of VDUP-1 expression inhibits high glucose-induced apoptosis and release of TNF-α in renal tubular epithelial cells by activating Shh signaling pathway.     

Effect of netrin-1 on high glucose induced injury of renal tubular epithe-lial cells

AIM:To study the effect of netrin-1 on the damage of renal tubular epithelial cells induced by high glucose. METHODS:Human renal tubular epithelial HK-2 cells were treated with high glucose. Real-time PCR and Western blot were used to detect the expression level of netrin-1 in the cells. HK-2 cells were infected with netrin-1-over-expressing lentivirus, and the effect of netrin-1 over-expression on the HK-2 cells treated with high glucose was observed. The apoptosis rate was analyzed by flow cytometry. The protein level of cleaved caspase-3 was determined by Western blot. lactate dehydrogenase (LDH) activity in the culture medium was measured by 2,4-binitrobenzene hydrazine method. The content of malondialdehyde (MDA) in the culture medium was detected by thiobarbituric acid method. The concentrations of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the culture medium were measured by ELISA. RESULTS:The expression of netrin-1 at mRNA and protein levels in the HK-2 cells after high glucose treatment was significantly lower than that in the control cells (P<0.05). Infection with netrin-1-over-expressing lentivirus up-regulated the expression of netrin-1 in the HK-2 cells treated with high glucose. High glucose promoted the secretion of IL-1β and TNF-α, decreased the levels of LDH and MDA in the cell culture supernatant, and induced apoptosis and activation of caspase-3 in renal tubular epithelial cells (P<0.05). After the HK-2 cells with up-regulation of netrin-1 were induced by high glucose, the IL-1β and TNF-α secretion, the levels of LDH and MDA in the culture medium, the apoptosis, and the level of activated caspase-3 protein in the cells were all decreased, as compared with the control cells (P<0.05). CONCLUSION:Up-regulation of netrin-1 expression attenuates oxidative damage and inflammatory injury, and reduces apoptosis induced by high glucose in renal tubular epithelial cells.     

Role of autophagy inhibitor 3-methyladenine in the injury of U251 cells induced by H2O2

AIM: To investigate the role of autophagy inhibitor 3-methyladenine(3-MA) in the injury of U251 glioma cells induced by H₂O₂. METHODS: The following groups in this study were set up: control group, 10 mmol/L 3-MA group, 1 mmol/L H₂O₂ group and 1 mmol/L H₂O₂ +10 mmol/L 3-MA group. The viability of U251 cells in each group was detected by MTT assay. Autophagic vacuoles in the cells were observed by staining with MDC. The cells were stained with Hoechst 33342 to determine the chromatin condensation. Cell apoptotic ratio was measured by flow cytometry analysis. RESULTS: Compared with control group, no effect of 3-MA on the viability of U251 cells was observed. In H₂O₂ group, the cell viability decreased and cell apoptotic ratio increased.The autophagic vacuoles and nuclear chromatin condensation in the cells were also detected. Compared with H₂O₂ group, addition of 3-MA inhibited the increase in autophagic vacuoles but exacerbated the apoptosis. CONCLUSION: Autophagy inhibitor 3-MA inhibits autophagy partially, but exacerbates apoptosis in U251 cells, indicating that autophagy exerts protective effect in the process of injury in U251 cells induced by H₂O₂.     

Effect of TLR4 on regulation of autophagy in renal tubular epithelial cells by soluble uric acid

AIM To investigate the role of Toll-like receptor 4 (TLR4) in autophagy induced by soluble uric acid in renal tubular epithelial HK-2 cells. METHODS After HK-2 cells were co-stimulated with soluble uric acid or/and chloroquine (CQ), the protein expression of LC3-Ⅱ and P62 was determined by Western blot. Autophagosomes and autophagolysosomes were observed by transmission electron microscopy. Next, HK-2 cells were co-stimulated with soluble uric acid and TLR4 inhibitor TAK242. The mRNA expression of TLR4 was detected by RT-qPCR, the protein expression of TLR4, LC3-Ⅱ and P62 was determined by Western blot, and the autophagic flux was observed by the method of mRFP-GFP-LC3. RESULTS The expression of LC3-Ⅱand P62 in the HK-2 cells was up-regulated by soluble uric acid. The expression of LC3-Ⅱ and P62 was further increased after co-stimulated with uric acid and CQ,and the number of autophagic body was increased while the number of autophagolyososome was decreased as observed by TEM, indicating that the autophagic flux was blocked. The expression levels of TLR4, LC3-Ⅱ and P62 in soluble uric acid group were higher than those in control group. Meanwhile, TAK242 inhibited the up-regulation of TLR4, LC3-Ⅱ and P62 by soluble uric acid. The results of mRFP-GFP-LC3 experiment showed that the levels of autophagosomes were significantly higher in soluble uric acid group than that in control group, and the levels of autophagolysosomes were lower. Compared with soluble uric acid group, the level of autophagosomes was decreased, and autophagolysosomes was increased in TAK242+soluble uric acid group, indicating that the fusion of autophagosomes and lysosomes was increased, and the process of autophagolysosome formation was smoother. CONCLUSION Soluble uric acid leads blocked autophagic flux in renal tubular epithelial cells. Soluble uric acid mediates abnormal tubular autophagic flux through TLR4.     

A model of necroptosis in human renal tubular epithelial cells

AIM:To make a model of necroptosis in human renal tubular epithelial HK-2 cells. METHODS:To induce necroptosis, HK-2 cells were treated with tumor necrosis factor α (TNF-α) followed by ATP depletion, and benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk) was added to block the activity of caspase-8. The morphological changes of the cells were observed under light microscope and electronic microscope.The cell viability was detected by CCK-8 assay, and the marker of necroptosis was analyzed by Western blotting. RESULTS:In the cells treated with TNF-α followed by zVAD-fmk and antimycin A for 1 h, the morphological changes including the cell and organelle inflation, and membrane fragmentation, with a large amount of autophagysome, were observed.However, these abnormalities were markedly attenuated after treatment with Nec-1. Meanwhile, the cell viability was also significantly improved after using Nec-1. No similar variation was observed in other groups. In addition, the expression of LC3-II was significantly decreased in Nec-1+TNF-α+zVAD-fmk+ antimycin A (1 h) group compared with control group. CONCLUSION: TNF-α stimulation and energy depletion induce necroptosis in renal tubular epithelial cells.Nec-1 inhibits necroptosis in a caspase-independent pathway, and may have therapeutic potential to prevent and treat renal ischemia injury.     

Role of glucocorticoid receptor in hepatic injury in early stage after severe multiple injury

AIM: To investigate the functions of GR in the course of hepatic secondary injury after severe multiple injury. METHODS: Rat model was produced by adopting severe thoracic impact injury accompanied with mono-side femur fracture, and glucocorticoid receptor was blocked before severe multiple injury. Hepatic macropathology and alterations under light microscope were examined. Maximal binding volume of glucocorticoid receptor (GR) in hepatic tissue was assayed by radio-ligand binding assay and protein content was assayed by Western blot. RESULTS: Maximal binding volume and protein content of GR were gradually decreased in hepatic tissue after severe multiple injury, obviously lower than that in normal control at 4 h after trauma (P＜0. 01), and the lowest value was at 12 h after trauma. Maximal binding volume was 12. 9% of normal control (P＜0. 01), and protein content was 21. 9% of normal control (P＜0. 01). In addition, no significant pathological alteration in liver was found after trauma. However, the pathological analysis showed that the administration of GR blocker before severe multiple injury could cause severe hepatic congestion and infiltration of inflammatory cells in hepatic sinusoid in a dose-dependent manner. CONCLUSION: GR insufficiency could cause secondary hepatic injury in early stage after severe multiple injury, implying an important role of GR in injury and anti-injury mechanism of hepatic tissue cells.