Xiangrui Wang

Dose-dependent neuroprotection of delta opioid peptide [d-Ala2, d-Leu5] enkephalin in neuronal death and retarded behavior induced by forebrain ischemia in rats

Cerebral ischemic insult, mainly induced by cardiovascular disease, is one of the most severe neurological diseases in clinical. Theres mounting evidence showing that delta opioid agonist [D-Ala2, D-Leu5] enkephalin (DADLE) has a tissue-protective effect. However, whether this property is effective to prevent neuronal death induced by forebrain ischemia is not clear. This study was aimed to investigate whether intracerebroventricular (ICV) administration of DADLE has a neuroprotective effect against forebrain ischemia in rats. We found in our study that administration of DADLE 45 min before forebrain ischemia had significant protective effect against CA1 neuronal lose. Further more, we found that DADLE had a dose-dependent protection for improving behavioral retardation revealed by Morris water maze and motor score test, while naltrindole, the antagonist of delta opioid receptor, partially abolished neuroprotective effect of DADLE, which implicated that both opioid and non-opioid systems are involved in ischemic insults and neuroprotection.

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.

Astroglial reaction to delta opioid peptide [D-Ala2, D-Leu5] enkephalin confers neuroprotection against global ischemia in the adult rat hippocampus.

Delta opioid receptor (DOR) is essential for neuronal survival against hypoxic/ischemic damages. However, current understanding on how DOR activation affects astrocytic functions under ischemia remains incomplete. The present study investigated the astroglial responses to [d-Ala2, d-Leu5] enkephalin (DADLE) (a selective DOR agonist)-induced DOR activation after global cerebral ischemia. Adult male rats were preimplanted with intracerebral cannula and subjected to global ischemia for 10 min. The rats were divided into four groups: normal group (without any procedure), sham group (sham procedure with intracerebroventricular injection of ACSF), I/R group (ischemia procedure with intracerebroventricular injection of ACSF) and DAD-treated group (ischemia procedure with intracerebroventricular injection of DADLE). Hippocampal CA1 neuronal survival and activation of astrocytes were measured in the animals at 72 h post-ischemia. The distribution and phenotypes of p-Akt and active caspase-3 were also determined. The ischemic injury resulted in a significant neuronal loss and an increase in the dying astrocytes in the hippocampal CA1 region as compared with those in the sham animals (200.7±22.7/mm(2) vs. 6.6±3.1/mm(2), P<0.001). Improved neuronal survival in the DAD-treated animals was evident, which was accompanied by less dying astrocytes and enhanced astrocytes reaction with more active astrocytes than that in the I/R group (267.6±13.2/mm(2) vs. 157.0±18.1/mm(2), P<0.01) and a significantly increased immunoreactivity of p-Akt. However, the active caspase-3 positive cells were also evident in DAD-treated group (313.0±23.1/mm(2)) and significantly increased as compared with those of the sham group (159.0±15.8/mm(2), P<0.001) or I/R group (193.6±26.2/mm(2), P<0.01). Most of the active caspase-3-expressing cells were colabeled with glial fibrillary acidic protein (GFAP), an astrocytes marker. We conclude that the post-ischemic treatment with DADLE promotes beneficial astrocytes activation and induces astroglial apoptosis 72 h after reperfusion which may be involved in reducing their harmful effect to neurons survival.

Attenuating phosphorylation of p38 MAPK in the activated microglia: a new mechanism for intrathecal lidocaine reversing tactile allodynia following chronic constriction injury in rats.

Increasing evidences approve the long-term analgesia effects of intrathecal lidocaine in patients with chronic pain and in animal peripheral nerve injury models, but the underlying mechanism remains elusive. Previous evidences suggest that the activation of the p38 MAPK signaling pathway in hyperactive microglia in the dorsal horn of spinal cord involves in nerve injury-induced neuropathic pain. In this study, we demonstrate that attenuating phosphorylation of p38 MAPK in the activated microglia of spinal cord, at least partly, is the mechanism of intrathecal lidocaine reversing established tactile allodynia in chronic constriction injury model of rats. This finding not only provides a new insight into the mechanisms underlying long-term therapeutic effects of lidocaine on neuropathic pain, but also reveals one more specific drug target for analgesia.

Isoflurane-induced spatial memory impairment in mice is prevented by the acetylcholinesterase inhibitor donepezil.

Although many studies have shown that isoflurane exposure impairs spatial memory in aged animals, there are no clinical treatments available to prevent this memory deficit. The anticholinergic properties of volatile anesthetics are a biologically plausible cause of cognitive dysfunction in elderly subjects. We hypothesized that pretreatment with the acetylcholinesterase inhibitor donepezil, which has been approved by the Food and Drug Administration (FDA) for the treatment of Alzheimers disease, prevents isoflurane-induced spatial memory impairment in aged mice. In present study, eighteen-month-old mice were administered donepezil (5 mg/kg) or an equal volume of saline by oral gavage with a feeding needle for four weeks. Then the mice were exposed to isoflurane (1.2%) for six hours. Two weeks later, mice were subjected to the Morris water maze to examine the impairment of spatial memory after exposure to isoflurane. After the behavioral test, the mice were sacrificed, and the protein expression level of acetylcholinesterase (AChE), choline acetylase (ChAT) and α7 nicotinic receptor (α7-nAChR) were measured in the brain. Each group consisted of 12 mice. We found that isoflurane exposure for six hours impaired the spatial memory of the mice. Compared with the control group, isoflurane exposure dramatically decreased the protein level of ChAT, but not AChE or α7-nAChR. Donepezil prevented isoflurane-induced spatial memory impairments and increased ChAT levels, which were downregulated by isoflurane. In conclusions, pretreatment with the AChE inhibitor donepezil prevented isoflurane-induced spatial memory impairment in aged mice. The mechanism was associated with the upregulation of ChAT, which was decreased by isoflurane.

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.

Effects of intrathecal lidocaine on hyperalgesia and allodynia following chronic constriction injury in rats

The present study investigated the effects of different doses of intrathecal lidocaine on established thermal hyperalgesia and tactile allodynia in the chronic constriction injury model of neuropathic pain, defined the effective drug dose range, the duration of pain‐relief effects, and the influence of this treatment on the body and tissues. Male Sprague–Dawley rats were divided into five groups and received intrathecal saline or lidocaine (2, 6.5, 15, and 35mg/kg) 7 days after loose sciatic ligation. Respiratory depression and hemodynamic instability were found to become more severe as doses of lidocaine increased during intrathecal therapy. Two animals in the group receiving 35mg/kg lidocaine developed pulmonary oedema and died. Behavioral tests indicated that 6.5, 15, and 35mg/kg intrathecal lidocaine showed different degrees of reversal of thermal hyperalgesia, and lasted for 2–8 days, while 2mg/kg lidocaine did not. The inhibition of tactile allodynia was only observed in rats receiving 15 and 35mg/kg lidocaine, and the anti‐allodynic effects were identical in these two groups. Histopathologic examinations on the spinal cords revealed mild changes in rats receiving 2–15mg/kg lidocaine. However, lesions were severe after administration of 35mg/kg lidocaine. These findings indicate that intrathecal lidocaine has prolonged therapeutic effects on established neuropathic pain. The balance between sympathetic and parasympathetic nervous activities could be well preserved in most cases, except for 35mg/kg. Considering the ratio between useful effects and side effects, doses of 15mg/kg are suitable for intrathecal injection for relief of neuropathic pain.

Lidocaine attenuates proinflammatory cytokine production induced by extracellular adenosine triphosphate in cultured rat microglia.

BACKGROUND: Our previous studies demonstrated that intrathecal lidocaine treatment could produce prolonged reversal of established hyperalgesia or allodynia, both induced by chronic constriction injury. Indeed, intrathecal lidocaine treatment remarkably suppressed the activation of p38 mitogen-activated protein kinase (MAPK) in hyperactive microglia. In the present study we suggest that lidocaine may act directly on the microglia and attenuate the release of cytokines. METHODS: We assessed the influence of lidocaine on the levels of phospho-p38 MAPK, tumor necrosis factor-&agr; (TNF-&agr;), interleukin-1beta (IL-1&bgr;), IL-6, and intracellular calcium triggered by extracellular adenosine triphosphate (ATP) in cultured rat microglia. Our experimental methods included Western blot, real-time reverse transcription–polymerase chain reaction, enzyme-linked immunosorbent assay, and calcium imaging. RESULTS: We found that lidocaine (in a dose-dependent manner) significantly attenuated p38 MAPK activation triggered by 1 mM ATP, by inhibiting the transcription of 3 cytokine messenger RNAs and causing a decrease in their respective protein concentrations (TNF-&agr;, IL-1&bgr;, and IL-6, P < 0.05, vs. the ATP group). SB203580, an antagonist of P38, attenuated ATP-activated elevation in protein levels of TNF-&agr;, IL-1&bgr;, and IL-6 in the microglia. The high level of intracellular calcium ([Ca2+]i) that is induced by ATP was decreased by the addition of 10 mM lidocaine (P < 0.05 vs. the ATP group). CONCLUSIONS: These findings indicate that lidocaine can directly act on microglia. Lidocaine, by inhibiting the increase of intracellular calcium, also inhibited p38 MAPK activation and attenuated the production of proinflammatory cytokines (including TNF-&agr;, IL-1&bgr;, and IL-6), which were triggered by extracellular ATP in cultured rat microglia.