Zhengyu He

Lipopolysaccharide induces lung fibroblast proliferation through Toll-like receptor 4 signaling and the phosphoinositide3-kinase-Akt pathway.

Pulmonary fibrosis is characterized by lung fibroblast proliferation and collagen secretion. In lipopolysaccharide (LPS)-induced acute lung injury (ALI), aberrant proliferation of lung fibroblasts is initiated in early disease stages, but the underlying mechanism remains unknown. In this study, we knocked down Toll-like receptor 4 (TLR4) expression in cultured mouse lung fibroblasts using TLR4-siRNA-lentivirus in order to investigate the effects of LPS challenge on lung fibroblast proliferation, phosphoinositide3-kinase (PI3K)-Akt pathway activation, and phosphatase and tensin homolog (PTEN) expression. Lung fibroblast proliferation, detected by BrdU assay, was unaffected by 1 mug/mL LPS challenge up to 24 hours, but at 72 hours, cell proliferation increased significantly. This proliferation was inhibited by siRNA-mediated TLR4 knockdown or treatment with the PI3K inhibitor, Ly294002. In addition, siRNA-mediated knockdown of TLR4 inhibited the LPS-induced up-regulation of TLR4, down-regulation of PTEN, and activation of the PI3K-Akt pathway (overexpression of phospho-Akt) at 72 hours, as detected by real-time PCR and Western blot analysis. Treatment with the PTEN inhibitor, bpV(phen), led to activation of the PI3K-Akt pathway. Neither the baseline expression nor LPS-induced down-regulation of PTEN in lung fibroblasts was influenced by PI3K activation state. PTEN inhibition was sufficient to exert the LPS effect on lung fibroblast proliferation, and PI3K-Akt pathway inhibition could reverse this process. Collectively, these results indicate that LPS can promote lung fibroblast proliferation via a TLR4 signaling mechanism that involves PTEN expression down-regulation and PI3K-Akt pathway activation. Moreover, PI3K-Akt pathway activation is a downstream effect of PTEN inhibition and plays a critical role in lung fibroblast proliferation. This mechanism could contribute to, and possibly accelerate, pulmonary fibrosis in the early stages of ALI/ARDS.

Toll-Like Receptor 4 Mediates Acute Lung Injury Induced by High Mobility Group Box-1

Background Acute lung injury (ALI) is considered to be the major cause of respiratory failure in critically ill patients. Clinical studies have found that in patients with sepsis and after hemorrhage, the elevated level of high mobility group box-1(HMGB-1) in their circulation is highly associated with ALI, but the underlying mechanism remains unclear. Extracellular HMGB-1 has cytokine-like properties and can bind to Toll-like Receptor-4 (TLR4), which was reported to play an important role in the pathogenesis of ALI. The aim of this study was to determine whether HMGB-1 directly contributes to ALI and whether TLR4 signaling pathway is involved in this process. Methods Recombinant human HMGB-1 (rhHMGB-1) was used to induce ALI in male Sprague-Dawley rats. Lung specimens were collected 2 h after HMGB-1 treatment. The levels of TNF-α, IL-1β, TLR4 protein, and TLR4 mRNA in lungs as well as pathological changes of lung tissue were assessed. In cell studies, the alveolar macrophage cell line, NR8383, was collected 24 h after rhHMGB-1 treatment and the levels of TNF-α and IL-1β in cultured medium as well as TLR4 protein and mRNA levels in the cell were examined. TLR4-shRNA-lentivirus was used to inhibit TLR4 expression, and a neutralizing anti-HMGB1 antibody was used to neutralize rhHMGB-1 both in vitro and in vivo. Results Features of lung injury and significant elevation of IL-1β and TNF-α levels were found in lungs of rhHMGB-1-treated animals. Cultured NR8383 cells were activated by rhHMGB-1 treatment and resulted in the release of IL-1β and TNF-α. TLR4 expression was greatly up-regulated by rhHMGB-1. Inhibition of TLR4 or neutralization of HMGB1 with a specific antibody also attenuated the inflammatory response induced by HMGB-1 both in vivo and in vitro. Conclusion HMGB-1 can activate alveolar macrophages to produce proinflammatory cytokines and induce ALI through a mechanism that relies on TLR-4.

TLR4 as receptor for HMGB1-mediated acute lung injury after liver ischemia/reperfusion injury

Acute lung injury (ALI) frequently occurs after liver transplantation and major liver surgery. Proinflammatory mediators released by damaged liver after liver ischemia/reperfusion (I/R) injury might contribute to this form of ALI, but the underlying mechanisms have not been well characterized. High-mobility group box protein 1 (HMGB1), a recently identified proinflammatory cytokine, was found to be significantly higher in the serum after liver I/R injury. This study investigated whether HMGB1 was involved as a stimulating factor, and whether its downstream Toll-like receptor 4 (TLR4), p38 mitogen-activated protein kinase (p38MAPK), and activator protein-1 (AP-1) signaling pathways act as mediators in the development of liver I/R injury-induced ALI. Extensive ALI and lung inflammation was induced in a rat model of liver I/R injury. Serum HMGB1 was significantly higher after liver I/R injury, and more importantly, expression of HMGB1 mRNA and protein in the lung tissue was also significantly increased. We further found that liver I/R injury enhanced the expression of TLR4 mRNA and protein, and the activity of p38MAPK and AP-1 in the lung tissue. Inhibition of TLR4 expression in the lung tissue by infection with pGCSIL-GFP-lentivirus-expressing small hairpin RNAs targeting the TLR4 gene (TLR4-shRNA lentivirus) significantly attenuated ALI, lung inflammation, and activity of p38MAPK and AP-1 in the lung tissue. These findings indicate that HMGB1 might contribute to the underlying mechanism for liver I/R injury-induced ALI and that its downstream TLR4, p38MAPK, and AP-1 signaling pathways are potentially important mediators in the development of ALI.

Overexpression of PTEN suppresses lipopolysaccharide-induced lung fibroblast proliferation, differentiation and collagen secretion through inhibition of the PI3-K-Akt-GSK3beta pathway

BackgroundAbnormal and uncontrolled proliferation of lung fibroblasts may contribute to pulmonary fibrosis. Lipopolysaccharide (LPS) can induce fibroblast proliferation and differentiation through activation of phosphoinositide3-Kinase (PI3-K) pathway. However, the detail mechanism by which LPS contributes to the development of lung fibrosis is not clearly understood. To investigate the role of phosphatase and tensin homolog (PTEN), a PI3-K pathway suppressor, on LPS-induced lung fibroblast proliferation, differentiation, collagen secretion and activation of PI3-K, we transfected PTEN overexpression lentivirus into cultured mouse lung fibroblasts with or without LPS treatment to evaluate proliferation by MTT and Flow cytometry assays. Expression of PTEN, alpha-smooth muscle actin (alpha-SMA), glycogen synthase kinase 3 beta (GSK3beta) and phosphorylation of Akt were determined by Western-blot or real-time RT-PCR assays. The PTEN phosphorylation activity was measured by a malachite green-based assay. The content of C-terminal propeptide of type I procollagen (PICP) in cell culture supernatants was examined by ELISA.ResultsWe found that overexpression of PTEN effectively increased expression and phosphatase activity of PTEN, and concomitantly inhibited LPS-induced fibroblast proliferation, differentiation and collagen secretion. Phosphorylation of Akt and GSK3beta protein expression levels in the LPS-induced PTEN overexpression transfected cells were significantly lower than those in the LPS-induced non-transfected cells, which can be reversed by the PTEN inhibitor, bpV(phen).ConclusionsCollectively, our results show that overexpression and induced phosphatase activity of PTEN inhibits LPS-induced lung fibroblast proliferation, differentiation and collagen secretion through inactivation of PI3-K-Akt-GSK3beta signaling pathways, which can be abrogated by a selective PTEN inhibitor. Thus, expression and phosphatase activity of PTEN could be a potential therapeutic target for LPS-induced pulmonary fibrosis. Compared with PTEN expression level, phosphatase activity of PTEN is more crucial in affecting lung fibroblast proliferation, differentiation and collagen secretion.

Hydroxyethyl Starch (130 kD) Inhibits Toll-Like Receptor 4 Signaling Pathways in Rat Lungs Challenged with Lipopolysaccharide

BACKGROUND: A number of studies have shown that hydroxyethyl starch (HES) solutions are able to down-regulate the expression of inflammatory mediators and inhibit neutrophil-mediated tissue injuries when they are used in patients with sepsis or other diseases with severe inflammatory responses. However, our knowledge about the underlying mechanisms is limited. Toll-like receptor 4 (TLR4) signaling has a pivotal role in inflammatory processes. In this study, we examined the possible involvement of TLR4 signaling in the antiinflammatory effects of HES. METHODS: Male Sprague-Dawley rats were exposed to lipopolysaccharide (LPS) (10 mg/kg, IV) and received IV saline (30 mL/kg) or HES 130/0.4 (15 or 30 mL/kg). Six hours after LPS challenge, rats were killed and their lungs harvested. Lung injury was examined by hematoxylin and eosin staining. TLR4 mRNA expression, p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinases 1/2 MAPK activation, and activator protein 1 (AP-1) activity in the lungs were detected with quantitative polymerase chain reaction, Western blotting, and electrophoretic mobility shift assay, respectively. RESULTS: Compared with saline, HES profoundly attenuated the histological changes induced by LPS in the lungs at both dose levels. Molecular analysis showed that both 15 and 30 mL/kg HES significantly decreased TLR4 mRNA levels and inhibited activation of p38 MAPK and AP-1 in rats challenged with LPS, whereas activation of extracellular signal-regulated kinases 1/2 MAPK was not affected by either dose of HES. CONCLUSIONS: These findings indicate that the beneficial effects of HES 130/0.4 on inflammation are mediated at least in part by inhibiting the TLR4/p38 MAPK/AP-1 pathway in lungs from rats challenged with LPS.

High-mobility group box 1 accelerates lipopolysaccharide-induced lung fibroblast proliferation in vitro: involvement of the NF-|[kappa]|B signaling pathway

The mechanism underlying lipopolysaccharide (LPS)-induced aberrant proliferation of lung fibroblasts in Gram-negative bacilli-associated pulmonary fibrosis is unknown. High-mobility group box 1 (HMGB1) is a ubiquitous nuclear protein that is released from the nuclei of lung fibroblasts after LPS stimulation. It can exasperate LPS-induced inflammation and hasten cell proliferation. Thus, this study investigated the effects of LPS- and/or HMGB1-stimulating murine lung fibroblasts on gene expression using various assays in vitro. Thiazolyl-diphenyl-tetrazolium bromide (MTT) assay data showed that either LPS or HMGB1 could induce lung fibroblast proliferation. Endogenous HMGB1 secreted from lung fibroblasts was detected by enzyme-linked immunosorbent assay (ELISA) 48 h after LPS stimulation. Pretreatment with an anti-HMGB1 antibody inhibited the proliferative effects of LPS on lung fibroblasts. DNA microarray data showed that the NF-κB signaling genes were upregulated in cells after stimulated with LPS, HMGB1, or both. Secretion of matrix metalloproteinase (MMP)-2 and MMP-9, and tissue inhibitor of metalloproteinase 2 (TIMP-2) was significantly upregulated after treatment with LPS, HMGB1, or their combination. However, an NF-κB inhibitor was able to downregulate levels of these proteins. In addition, levels of Toll-like receptor 4 (TLR4), Toll-like receptor 2 (TLR2), and receptors for advanced glycation end products (RAGE) mRNA and proteins were also upregulated in these cells after LPS treatment and further upregulated by LPS plus HMGB1. In conclusion, the data from the current study demonstrate that LPS-induced lung fibroblast secretion of endogenous HMGB1 can augment the proproliferative effects of LPS and, therefore, may play a key role in exacerbation of pulmonary fibrosis. The underlying molecular mechanisms are related to the activation of the TLR4/NF-κB signaling pathway and its downstream targets.

Epigenetic regulation of Thy‐1 gene expression by histone modification is involved in lipopolysaccharide‐induced lung fibroblast proliferation

Lipopolysaccharide (LPS)‐induced pulmonary fibrosis is characterized by aberrant proliferation and activation of lung fibroblasts. Epigenetic regulation of thymocyte differentiation antigen 1 (Thy‐1) is associated with lung fibroblast phenotype transformation that results in aberrant cell proliferation. However, it is not clear whether the epigenetic regulation of Thy‐1 expression is required for LPS‐induced lung fibroblast proliferation. To address this issue and better understand the relative underlying mechanisms, we used mouse lung fibroblasts as model to observe the changes of Thy‐1 expression and histone deacetylation after LPS challenge. The results showed that cellular DNA synthesis, measured by BrdU incorporation, was impacted less in the early stage (24 hrs) after the challenge of LPS, but significantly increased at 48 or 72 hrs after the challenge of LPS. Meanwhile, Thy‐1 expression, which was detected by real‐time PCR and Western blot, in lung fibroblasts decreased with increased time after LPS challenge and diminished at 72 hrs. We also found that the acetylation of either histone H3 or H4 decreased in the LPS‐challenged lung fibroblasts. ChIP assay revealed that the acetylation of histone H4 (Ace‐H4) decreased in the Thy‐1 promoter region in response to LPS. In addition, all the above changes could be attenuated by depletion of TLR4 gene. Our studies indicate that epigenetic regulation of Thy‐1 gene expression by histone modification is involved in LPS‐induced lung fibroblast proliferation.

HDAC is essential for epigenetic regulation of Thy-1 gene expression during LPS/TLR4-mediated proliferation of lung fibroblasts

Lipopolysaccharide (LPS)-induced proliferation of lung fibroblasts is closely correlated with loss of gene expression of thymocyte differentiation antigen-1 (Thy-1), accompanied with deacetylation of histones H3 and H4 at the Thy-1 gene promoter region; however, the mechanism remains enigmatic. We report here that LPS downregulates Thy-1 gene expression by activating histone deacetylases (HDACs) via Toll-like receptor 4 (TLR4) signaling. Treatment of primary cultured mouse lung fibroblasts with LPS resulted in significant upregulation of TLR4 and enhanced cell proliferation that was abolished by silencing TLR4 with lentivirus-delivered TLR4 shRNA. Interestingly, LPS increased the mRNA and protein levels of HDAC-4, -5, and -7, an effect that was abrogated by HDAC inhibitor trichostatin A (TSA) or TLR4-shRNA-lentivirus. Consistent with these findings, Ace-H3 and Ace-H4 were decreased by LPS that was prevented by TSA. Most importantly, chromosome immunoprecipitation (ChIP) analysis demonstrated that LPS decreased the association of Ace-H4 at the Thy-1 promoter region that was efficiently restored by pretreatment with TSA. Accordingly, LPS decreased the mRNA and protein levels of Thy-1 that was inhibited by TSA. Furthermore, silencing the Thy-1 gene by lentivirus-delivered Thy-1 shRNA could promote lung fibroblast proliferation, even in the absence of LPS. Conversely, overexpressing Thy-1 gene could inhibit lung fibroblast proliferation and reduce LPS-induced lung fibroblast proliferation. Our data suggest that LPS upregulates and activates HDACs through TLR4, resulting in deacetylation of histones H3 and H4 at the Thy-1 gene promoter that may contribute to Thy-1 gene silencing and lung fibroblast proliferation.

ALPHA7 Nicotine Acetylcholine Receptor Agonist PNU-282987 Attenuates Acute Lung Injury in a Cardiopulmonary Bypass Model in Rats.

Objective: Cardiopulmonary bypass (CPB) carries a risk of lung ischemia-reperfusion, leading to acute lung injury (ALI). Alpha7 nicotinic acetylcholine receptor (alpha7nAChR) has been implicated in the release of high mobility group box1 (HMGB1), which promotes systemic inflammation in response to ischemia-reperfusion injury. However, the specific role of alpha7nAChR in CPB is poorly understood. This study employed the alpha7nAChR agonist PNU-282987 and a rat model of CPB to determine whether alpha7nAChR was associated with CPB-induced lung damage. Methods: Thirty Sprague–Dawley rats were randomly divided into five groups as follows: normal group, sham group, CPB group, PNU-282987 plus CPB group, and PNU-282987 plus sham group. Rats were subjected to CPB under anesthesia for 60 min. PNU-282987 (4.8 mg/kg) was administered via arterial inflow. Two hours post-CPB, samples of blood, bronchoalveolar lavage fluid (BALF), and lung tissues were processed for investigations. Results: In CPB rats, structural damage in the lung was marked. Density of alpha7nAChR of the lung in the CPB group was significantly less than all other groups, while lung edema, inflammatory markers in serum and lung, protein concentrations in BALF were significantly higher. In the PNU-282987 plus CPB group, by all the above measures the CPB-associated effects were significantly ameliorated but were not identical to the control groups. Conclusion: Our results suggest that PNU-282987 affords protective effect against CPB-induced ALI, and inhibits HMGB1 release.

Effects of mild hypoalbuminemia on the pharmacokinetics and pharmacodynamics of dexmedetomidine in patients after major abdominal or thoracic surgery.

STUDY OBJECTIVE To explore the effects of mild hypoalbuminemia on pharmacokinetics and pharmacodynamics of dexmedetomidine in patients after major abdominal or thoracic surgery. DESIGN A prospective cohort study. SETTING University-affiliated teaching hospital. PATIENTS The study was performed in 30 consecutive patients undergoing major abdominal or thoracic surgery. They were aged 18 to 65 years and graded as American Society of Anesthesiologists physical status I and II. All patients were scheduled to require more than 6 hours of postoperative sedation and mechanical ventilation. Nine of the patients had low plasma albumin levels (<35 g/L but >24 g/L; male/female, 6/3) after the operation, who were assigned to hypoalbuminemia group, and the remainder with normoalbuminemia (>35 g/L; male/female, 15/6) were assigned to normoalbuminemia group. INTERVENTIONS All patients were administered a loading dose of dexmedetomidine 1.0 μg/kg infused over 10 minutes after admitted into intensive care unit and a maintenance dose of 0.4 μg/kg per hour followed for 6 hours. MEASUREMENTS Plasma dexmedetomidine levels were determined by high performance liquid chromatography - mass spectrum. Sedation was evaluated using Ramsay sedation score. Heart rate and arterial pressures were monitored. Adverse events were recorded. MAIN RESULTS Compared with patients with normoalbuminemia, maximum plasma concentration of dexmedetomidine decreased by 21.2% in patients with hypoalbuminemia (P < .01). Its volume of distribution at steady state increased by 40.5%; elimination half-life decreased by 33.5% (P < .01). However, heart rates, arterial pressures, and Ramsay sedation scores did not differ significantly between the 2 groups. No serious adverse events occurred in either the patients with hypoalbuminemia or normoalbuminemia. CONCLUSIONS Sedation and adverse reactions of dexmedetomidine infusion did not differ significantly between patients with mild hypoalbuminemia and normoalbuminemia, although its volume of distribution at steady state increased and elimination half-life shortened in patients with hypoalbuminemia. This suggests that dexmedetomidine infusion can safely be used in mild hypoalbuminemia patients after major abdominal or thoracic surgery.