Mesencephalic astrocyte-derived neurotrophic factor attenuates acute lung injury via inhibiting macrophages' activation.

Acute lung injury (ALI) is an urgent respiratory disease without effective treatment. Mesencephalic astrocyte-derived neurotrophic factor (MANF)has been demonstrated to play a suppressive role in some inflammatory conditions. However, the effect of MANF on ALI has not yet been reported. In this study, we collected bronchoalveolar lavage fluid (BALF) from the patients with or without pulmonary inflammation, and used lipopolysaccharide (LPS) to induce mice ALI model. Mono-macrophage-specific MANF knockout (MKO) mice were constructed and recombinant human MANF protein was used to ALI mice. We found that the endogenous MANF protein in both human BALF and mice lung tissues was increased in inflammatory conditions. MANF level in the macrophages of inflammatory lung was higher than that in normal controls in both human and mice. MANF deficiency in macrophages induced lung inflammation and aggravated LPS-induced lung injury. MANF lowered LPS-induced lung injury, inhibited macrophage polarization to M1 functional type. Meanwhile, MANF inhibited-LPS induced activation of NF-κB signal pathway by down regulating phosphorylated p65in lung tissue and macrophages. These results indicate that MANF acts as a suppressor in ALI via negatively regulating NF-κB activation and macrophages polarization, which may be a novel potential target and shed light on ALI therapy.

Xanthatin induces glioma cell apoptosis and inhibits tumor growth via activating endoplasmic reticulum stress-dependent CHOP pathway.

Xanthatin is a natural sesquiterpene lactone purified from Xanthium strumarium L., which has shown prominent antitumor activity against a variety of cancer cells. In the current study, we investigated the effect of xanthatin on the growth of glioma cells in vitro and in vivo, and elucidated the underlying mechanisms. In both rat glioma C6 and human glioma U251 cell lines, xanthatin (1-15 µM) dose-dependently inhibited cell viability without apparent effect on the cell cycle. Furthermore, xanthatin treatment dose-dependently induced glioma cell apoptosis. In nude mice bearing C6 glioma tumor xenografts, administration of xanthatin (10, 20, 40 mg·kg-1·d-1, ip, for 2 weeks) dose-dependently inhibited the tumor growth, but did not affect the body weight. More importantly, xanthatin treatment markedly increased the expression levels of the endoplasmic reticulum (ER) stress-related markers in both the glioma cell lines as well as in C6 xenografts, including glucose-regulated protein 78, C/EBP-homologous protein (CHOP), activating factor 4, activating transcription factor 6, spliced X-box binding protein-1, phosphorylated protein kinase R-like endoplasmic reticulum kinase, and phosphorylated eukaryotic initiation factor 2a. Pretreatment of C6 glioma cells with the ER stress inhibitor 4-phenylbutyric acid (4-PBA, 7 mM) or knockdown of CHOP using small interfering RNA significantly attenuated xanthatin-induced cell apoptosis and increase of proapoptotic caspase-3. These results demonstrate that xanthatin induces glioma cell apoptosis and inhibits tumor growth via activating the ER stress-related unfolded protein response pathway involving CHOP induction. Xanthatin may serve as a promising agent in the treatment of human glioma.

Activator protein­1 is a novel regulator of mesencephalic astrocyte­derived neurotrophic factor transcription.

Mesencephalic astrocyte­derived neurotrophic factor (MANF) is an endoplasmic reticulum stress­inducible protein, which has been suggested to be upregulated in inflammatory diseases; however, how inflammation regulates its transcription remains unclear. Activator protein­1 (AP­1), which is a transcription factor complex composed of c­Fos and c­Jun, is activated during the inflammatory process. The present study aimed to investigate whether the AP­1 complex regulates MANF transcription. The results of a luciferase reporter assay revealed that one of three putative AP­1 binding sites in the MANF promoter region is essential for enhancement of MANF transcription. Mechanistically, AP­1 was revealed to directly bind to the promoter region of the MANF gene by chromatin immunoprecipitation assay. Furthermore, MANF was strongly expressed in the liver tissues of patients with hepatitis B virus (HBV) infection, compared with in normal liver tissues from patients with hepatic hemangioma. Furthermore, c­Fos and c­Jun were also upregulated in the nuclei of hepatocytes from patients with HBV infection. In mice treated with carbon tetrachloride, the expression patterns of MANF, c­Fos and c­Jun were similar to those in patients with HBV. These results suggested that the AP­1 complex may be a novel regulator of MANF transcription, which may be involved in liver inflammation and fibrosis.

Chemokine CCL2-CCR2 Signaling Induces Neuronal Cell Death via STAT3 Activation and IL-1ß Production after Status Epilepticus.

Elevated levels of chemokine C-C motif ligand 2 (CCL2) and its receptor CCR2 have been reported in patients with temporal lobe epilepsy and in experimental seizures. However, the functional significance and molecular mechanism underlying CCL2-CCR2 signaling in epileptic brain remains largely unknown. In this study, we found that the upregulated CCL2 was mainly expressed in hippocampal neurons and activated microglia from mice 1 d after kainic acid (KA)-induced seizures. Taking advantage of CX3CR1GFP/+:CCR2RFP/+ double-transgenic mice, we demonstrated that CCL2-CCR2 signaling has a role in resident microglial activation and blood-derived monocyte infiltration. Moreover, seizure-induced degeneration of neurons in the hippocampal CA3 region was attenuated in mice lacking CCL2 or CCR2. We further showed that CCR2 activation induced STAT3 (signal transducer and activator of transcription 3) phosphorylation and IL-1ß production, which are critical for promoting neuronal cell death after status epilepticus. Consistently, pharmacological inhibition of STAT3 by WP1066 reduced seizure-induced IL-1ß production and subsequent neuronal death. Two weeks after KA-induced seizures, CCR2 deficiency not only reduced neuronal loss, but also attenuated seizure-induced behavioral impairments, including anxiety, memory decline, and recurrent seizure severity. Together, we demonstrated that CCL2-CCR2 signaling contributes to neurodegeneration via STAT3 activation and IL-1ß production after status epilepticus, providing potential therapeutic targets for the treatment of epilepsy.SIGNIFICANCE STATEMENT Epilepsy is a global concern and epileptic seizures occur in many neurological conditions. Neuroinflammation associated with microglial activation and monocyte infiltration are characteristic of epileptic brains. However, molecular mechanisms underlying neuroinflammation in neuronal death following epilepsy remain to be elucidated. Here we demonstrate that CCL2-CCR2 signaling is required for monocyte infiltration, which in turn contributes to kainic acid (KA)-induced neuronal cell death. The downstream of CCR2 activation involves STAT3 (signal transducer and activator of transcription 3) phosphorylation and IL-1ß production. Two weeks after KA-induced seizures, CCR2 deficiency not only reduced neuronal loss, but also attenuated seizure-induced behavioral impairments, including anxiety, memory decline, and recurrent seizure severity. The current study provides a novel insight on the function and mechanisms of CCL2-CCR2 signaling in KA-induced neurodegeneration and behavioral deficits.

Activated macrophage-like synoviocytes are resistant to endoplasmic reticulum stress-induced apoptosis in antigen-induced arthritis.

OBJECTIVE: To explore the characteristic expression of endoplasmic reticulum (ER) stress protein in antigen-induced arthritis models and the role of ER stress in arthritis. METHODS: Effective animal models of rheumatoid arthritis in rabbits and rats were induced by methylated bovine serum albumin and Freund's complete adjuvant. Pathological changes were assessed by magnetic resonance imaging and histological analysis. The expression and localization of ER stress proteins in synovium and peritoneal macrophages (PMΦ) were analyzed by double immunofluorescence staining. RT-PCR was performed to detect mRNA expression of ER stress-related genes. Tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1ß) levels in synoviocytes were measured by RT-PCR and radioimmunoassay. RESULTS: We found that the ER stress marker BiP was highly up-regulated in arthritis synovium and extensively expressed in fibroblast-like synoviocytes (FLS) and macrophage-like synoviocytes (MLS). The expression of the pro-apoptotic factor CHOP/GADD153 was slightly elevated in inflammatory synovium and mainly localized in FLS, but insignificant in MLS. Unexpectedly, increased expression of CHOP was observed in PMΦ in arthritis rats. Likewise, cleaved caspase-3 was rarely expressed in MLS. In addition, induction of ER stress by tunicamycin resulted in significantly increased expression of pro-inflammatory molecules such as IL-1ß and TNF-α in cultured inflammatory FLS. CONCLUSION: Differential activation of the ER stress proteins in synovium MLS may contribute to the resistance of synoviocytes to ER stress-induced apoptosis. Furthermore, ER stress is a potential mediator of arthritis inflammation.

Total glucosides of Danggui Buxue Tang attenuates bleomycin-induced pulmonary fibrosis via inhibition of extracellular matrix remodelling.

OBJECTIVES: This study was designed to investigate the antifibrosis effects and possible mechanism of action of total glucosides of Danggui Buxue Tang (DBTG) on bleomycin-induced pulmonary fibrosis in rats. METHODS: DBTG was extracted from Radix Astragali and Radix Angelicae Sinensis. Pulmonary fibrosis was induced by intratracheal instillation of bleomycin (5 mg/kg) in Wistar rats. Subsequently, the rats received daily intragastric administration of DBTG (16, 32 or 64 mg/kg per day) or cortisone (3 mg/kg) 1 day after bleomycin instillation for 4 weeks. Histological changes in the lung were evaluated by hematoxylin and eosin and Masson's trichrome staining. Markers of fibrosis in serum were determined by radioimmunoassay. The mRNA expression of metalloproteinases 1 and 9 (MMP-1, MMP-9) and tissue inhibitor of metalloproteinase 1 (TIMP-1) in lung tissue were detected by reverse transcription PCR. KEY FINDINGS: DBTG administration attenuated the degree of alveolitis and lung fibrosis, and markedly reduced the elevated levels of hyaluronic acid, laminin, type III procollagen and type IV collagen in serum. DBTG decreased the mRNA levels of MMP-9 and TIMP-1. MMP-1 expression was only moderately decreased by DBTG. CONCLUSIONS: DBTG had an inhibitory effect on bleomycin-induced pulmonary fibrosis and its effect may be associated with the ability of DBTG to inhibit the synthesis of extracellular matrix and balance the MMP/TIMP-1 system.

[Effects of low-level laser irradiation on rat mesenteric microcirculatory disturbance during early stage of endotoxemia].

OBJECTIVE: To observe the effects of low-level laser irradiation on mesenteric microcirculation of rats in vivo in the early stage of endotoxemia (ETM). METHODS: The experimental model of ETM was reproduced by injection of lipopolysaccharide (LPS). Sixty healthy male Sprague-Dawley (SD) rats were divided into three groups used random number table: control group, LPS group and low-level laser irradiation group, each group included 20 rats which were subdivided into four temporal subgroups (1, 2, 4, 6 hours, respectively). In low-level laser irradiation group, the rats were irradiated by type SLT semiconductor laser (650 nm, 5 mW) on unilateral femoral artery and vein, and blood vessel of the ear concurrently for 30 minutes. The interference course was vertical irradiation taken at 30 minutes after the injection of LPS. At 1, 2, 4, 6 hours after the injection of LPS, changes in mesenteric microcirculation and microcirculatory blood flow were recorded with the laser Doppler flowmeter, the velocity of red blood cells in venules was observed, and the number of open capillaries and adherent leukocytes were recorded. RESULTS: The blood flow velocity (mm/s) of the mesenteric microcirculation in LPS group was accelerated at 1 hour and 2 hours after LPS injection (1 hour: 0.190+/-0.007 vs. 0.174+/-0.009, 2 hours: 0.200+/-0.010 vs. 0.172+/-0.015, both P<0.05, respectively), but decelerated at 6 hours (0.116+/-0.015 vs. 0.164+/-0.011, P<0.05). The blood flow volume in the mesenteric vessels and the number of open capillaries did not show any significant change at that time. Significant increase in number of adherent leukocytes was observed at 2, 4, 6 hours after injury (2 hours: 2.60+/-1.14 vs. 0.40+/-0.55, 4 hours: 5.40+/-0.89 vs. 0.40+/-0.55, 6 hours : 5.40+/-1.52 vs. 0.60+/-0.90, all P<0.05, respectively). The state of blood flow in the microcirculation became abnormal. After irradiated with laser in low dose, the blood flow velocity was smooth and stable (mm/s, 1 hour: 0.174+/-0.011, 2 hours: 0.180+/-0.023, 4 hours: 0.168+/-0.013, 6 hours: 0.162+/-0.023), and the number of adherent leukocytes was reduced significantly at 4 hours and 6 hours than that in LPS group (4 hours: 2.00+/-0.71 vs. 5.40+/-0.89, 6 hours: 2.60+/-1.52 vs. 5.40+/-1.52, both P<0.05) and the microcirculatory flow state was improved obviously. CONCLUSION: Low-level laser irradiation may ameliorate the local mesenteric microcirculation, alleviate the microcirculatory disorder in early stage of ETM.

[Analysis of symptom checklist 90 of relief crew during critical period after Wenchuan earthquake in Sichuan province].

OBJECTIVE: To investigate the psychological features of relief crew in critical period after Wenchuan earthquake and to improve their mental health. METHODS: On May 12 of 2008, severe earthquake of a magnitude of 8.0 on Richter scale occurred in Wenchuan county, Sichuan province of China. Within 20 days after the earthquake, a research group conducted a study of psychological features of 600 relief crew with symptom checklist 90 (SCL-90), and the general symptom index and factorial scores were compared with Chinese norm (results from 1 388 personnel, published in 1986) and army norm (results from 19,662 servicemen, published in 1999). The positive items were measured and the relative factors were evaluated. RESULTS: (1) In the critical period after the earthquake, the relief workers had lower general symptomatic index than Chinese normal personnel or normal servicemen (both P<0.01). (2) Obsessive-compulsive symptoms (factorial score: 1.29+/-0.36) and somatization symptoms (factorial score: 1.22+/-0.29) were the main psychologic problems in this period. (3) Psychology of relief workers was correlated positively with working zones and education (r1=0.29, r2=0.15, both P<0.01), while negatively with working period (r=-0.28 , P<0.01). CONCLUSION: In immediate post-tremor period, the mental health of relief crew is predominantly fine, with obsessive-compulsive symptoms and somatization symptoms as outstanding mental disorders. Working zone, working period and education are main factors which affect their mental health.

Blockage of the formation of new blood vessels by recombinant human endostatin contributes to the regression of rat adjuvant arthritis.

The formation of new blood vessels permits a supply of nutrients and oxygen to the proliferating synovial cells and augmented inflammatory cell mass in rheumatoid arthritis (RA). Angiogenesis inhibition is not dependent on a down-regulated immune system. Therefore, angiogenesis is an attractive target in treating rheumatoid arthritis. To confirm the effect of recombinant human endostatin, an angiogenesis inhibitor, on inflammatory angiogenesis and to elucidate the related mechanisms, rat adjuvant arthritis model induced by Freund's complete adjuvant was used. The secondary arthritis was evaluated by using clinical scores and determining the volume of hind paw swelling. The number of new blood vessels was counted under microscope based on HE (hematoxylin and eosin) staining and positive immunoreactivity of factor VIII related antigen. factor VIII related antigen and vascular endothelial growth factor (VEGF) expressions in synovial tissue were determined by using immunohistochemistry. It was found that endostatin attenuated rat secondary paw swelling induced by Freund's complete adjuvant in a dose-dependent manner. Meanwhile, the number of new blood vessels in synovial tissue stained with HE was reduced after treatment with endostatin, which was proved by the positive immunostaining of factor VIII related antigen. Further, endostatin decreased the expression of VEGF in both cartilage and synovial tissue. These suggest that endostatin inhibiting VEGF expression contributes to the regression of rat adjuvant arthritis.

Selected ion chromatograms and tandem mass spectrometry for detection of trace polycyclic aromatic hydrocarbons in diesel exhaust.

Polycyclic aromatic hydrocarbon (PAH) measurements are essential for scientists and engineers who investigate these anthropogenic compounds. Diesel engines contribute to the problem, so analysts are measuring PAHs from these sources. However, diesel exhaust presents special problems for precise analytical measurements. The exhaust matrix is very complex; consequently, PAH detection sensitivity deteriorates, especially for trace PAHs in the exhaust. Yet, these are conditions and amounts that exist in real samples. Nonetheless, selected ion chromatogram (SIC) and tandem mass spectrometry (MS/MS) techniques improve trace PAH detection; ion trap technology makes both mass techniques possible. The purpose of this investigation was to evaluate SIC and MS/MS for applications to measure PAHs in diesel exhaust samples. The signal-to-noise ratio for accurate quantitation improves, relative to traditional mass techniques, because these techniques ignore or eliminate interfering components. On a VF-5MS chromatographic column, these techniques improve sensitivity and reproducibility. They produce a superior limit of detection in the useful range for PAH samples extracted from actual engine exhaust, 10-30 pg for the smaller PAHs and 1-6 ng for the larger PAHs. The results with SIC and MS/MS are reproducible, so analysts can report PAH amounts with defined statistical confidence intervals. SIC and MS/MS improve detection for trace PAHs in convoluted diesel exhaust samples.