[Rosmarinic acid bolsters antibacterial immunity activity of macrophages by up-regulating PINK1/Parkin-mediated mitophagy].

This study aims to explore the molecular mechanism through which rosmarinic acid up-regulates mitophagy and enhances antibacterial immunity activity of macrophages. To be specific, RAW264.7 macrophages were treated with rosmarinic acid and then infected with Staphylococcus aureus. The total mRNA and proteins of the cells were then extracted. The mRNA and protein levels of phosphatase and tensin homolog(PTEN)-induced putative kinase 1(PINK1) were detected by q-PCR and Western blot, respectively. Cell mitochondria isolation kit was employed to isolate mitochondria in macrophages. Recruitment of E3 ubiquitin ligase Parkin to mitochondria and the phosphorylation of Parkin were detected by Western blot. Co-immunoprecipitation and laser confocal microscopy were employed to observe the co-localization of PINK1 and Parkin. Mitochondrial division inhibitor 1(Mdivi-1), small interfering RNA(siRNA)-directed gene knockdown, and plate-colony counting were used to detect the levels of inflammatory cytokines and the intracellular antibacterial ability, in an attempt to confirm that rosmarinic acid promotes antibacterial immunity activity of macrophages through strengthening PINK1/Parkin-mediated mitophagy. The results showed that rosmarinic acid up-regulated the mRNA and protein expression of PINK1, promoted the recruitment of Parkin from cytoplasm to mitochondria and the phosphorylation, and enhanced the interaction between PINK1 and Parkin and their co-localization in macrophages. Blocking mitophagy or knocking PINK1 significantly abrogated the promotion of macrophage antibacterial immune response by rosmarinic acid. In summary, rosmarinic acid enhances antibacterial immunity activity of macrophages through up-regulating PINK1/Parkin-mediated mitophagy.

A critical time window for the analgesic effect of central histamine in the partial sciatic ligation model of neuropathic pain.

BACKGROUND: It is known that histamine participates in pain modulation. However, the effect of central histamine on neuropathic pain is not fully understood. Here, we report a critical time window for the analgesic effect of central histamine in the partial sciatic nerve ligation model of neuropathic pain. METHODS: Neuropathic pain was induced by partial sciatic nerve ligation (PSL) in rats, wild-type (C57BL/6J) mice and HDC(-/-) (histidine decarboxylase gene knockout) and IL-1R(-/-) (interleukin-1 receptor gene knockout) mice. Histidine, a precursor of histamine that can increase the central histamine levels, was administered intraperitoneally (i.p.). Histidine decarboxylase (HDC) enzyme inhibitor α-fluoromethylhistidine was administered intracerebroventricularly (i.c.v.). Histamine H1 receptor antagonist mepyramine and H2 receptor antagonist cimetidine were given intrathecally (i.t.) and intracisternally (i.c.). Withdrawal thresholds to tactile and heat stimuli were measured with a set of von Frey hairs and infrared laser, respectively. Immunohistochemistry and Western blot were carried out to evaluate the morphology of microglia and IL-1ß production, respectively. RESULTS: Histidine (100 mg/kg, i.p.) administered throughout days 0-3, 0-7, or 0-14 postoperatively (PO) alleviated mechanical allodynia and thermal hyperalgesia in the hindpaw following PSL in rats. Intrathecal histamine reversed PSL-induced thermal hyperalgesia in a dose-dependent manner and intracisternal histamine alleviated both mechanical allodynia and thermal hyperalgesia. Moreover, α-fluoromethylhistidine (i.c.v.) abrogated the analgesic effect of histidine. However, histidine treatment initiated later than the first postoperative day (treatment periods included days 2-3, 4-7, and 8-14 PO) did not show an analgesic effect. In addition, histidine treatment initiated immediately, but not 3 days after PSL, inhibited microglial activation and IL-1ß upregulation in the lumbar spinal cord, in parallel with its effects on behavioral hypersensitivity. Moreover, the inhibitory effects on pain hypersensitivity and spinal microglial activation were absent in HDC(-/-) mice and IL-1R(-/-) mice. H1 receptor antagonist mepyramine (200 ng/rat i.t. or i.c.), but not H2 receptor antagonist cimetidine (200, 500 ng/rat i.t. or 500 ng/rat i.c.), blocked the effects of histidine on pain behavior and spinal microglia. CONCLUSIONS: These results demonstrate that central histamine is analgesic within a critical time window in the PSL model of neuropathic pain via histamine H1 receptors. This effect may partly relate to the inhibition of microglial activation and IL-1ß production in the spinal cord following nerve injury.

[Effects of Sema3A derived from tumor cells on functions of dendritic cells].

OBJECTIVE: To investigate the effects of tumor cell-derived Sema3A on the immunological functions of murine dendritic cells (DCs). METHODS: Lung adenocarcinoma A549 cells were transfected with small interference RNA, Si-Sema and Si-mut, and the interference efficiency was determined by real-time PCR and Western-blot. The concentrated supernatants from cultured tumor cells, Si-Sema and Si-mut-infected tumor cells were subjected to DCs respectively. The immunophenotypes of DCs were analyzed by flow cytometry, the production of IL-12P70 and the ability of DCs to stimulate DO11. 10 T cells secreting IFN-gamma and IL-2 were detected by enzyme linked immunosorbent assay (ELISA). RESULTS: Knockdown with Si-Sema3A significantly decreased the secretion of Sema3A by A549 cells in comparison with the Si-mut cells. DCs exposed to supernatants from Si-Sema cells showed elevated levels of MHC, CD40 and CD80, more production of IL-12P70, and enhanced capability of activating antigen-specific T cells, as evidenced by the remarkably increased levels of IFN-gamma and IL-2. CONCLUSION: A549 cells secrete Sema3A to inhibit the maturation and functions of DCs, which might be associated with the unidentified mechanism of immune evasion by tumor cells.

[Adjuvant effect of co-stimulatory molecule CD137L on cellular responses to HBsAg DNA vaccination in mice].

OBJECTIVE: To investigate the adjuvant effect of co-stimulatory molecule CD137L on cellular responses to HBsAg DNA vaccination in mice. METHODS: Eukaryotic expression vector containing the full length of mouse CD137L cDNA sequence (pcD137L) was transfected into NIH3T3 cells, and then the expression of CD137L mRNA and protein in the transfected cells were detected by RT-PCR, flow cytometry and immunofluorescence method, respectively. The BALB/c mice were co-immunized with pcD137L and HBsAg DNA vaccine (pcDS) by intramuscular injection. HBsAg-specific activity of splenic cytotoxic T lymphocyte (CTL) in the immunized mice was measured by LDH release assay. The splenic memory CD8+ T cells, and intracellular IFN-gamma and IL-4 of splenic lymphocytes and CD8+ T cells after immunization were detected by flow cytometry. RESULTS: The NIH3T3 cells transfected with pcD137L efficiently expressed mouse CD137L mRNA and protein. HBsAg-specific CTL responses induced by the pcDS plus pcD137L group were much stronger than those induced by pcDS alone at a week after immunization (P<0.05). Compared to mice immunized with pcDS alone, CD44high and CD127(IL-7R) were all significantly up-regulated in memory CD8+ T cells from the mice immunized with pcDS combined CD137L both at a week and 12 weeks after immunization (P<0.05 and P<0.01). The pcDS plus CD137L group also elicited higher levels of IFN-gamma secreted by CD8+ T cells and splenic lymphocytes than pcDS alone at a week, 12 and 13 weeks after immunization, respectively (all P<0.01). CONCLUSION: DNA, viral/immunol; Co-stimulatory molecule CD137L can enhance the Tc1 (type I) cell-mediated immunity, HBsAg-specific CTL and memory responses induced by HBsAg DNA vaccine, and may be an efficient adjuvant in priming HBV-specific T cell response.

Dalestones A and B, two anti-inflammatory cyclopentenones from Daldinia eschscholzii with an edited strong promoter for the global regulator LaeA-like gene.

Replacement of the native promoter of theglobal regulator LaeA-like gene of Daldinia eschscholzii by a strong gpdA promoter led to the generation of two novel cyclopentenone metabolites, named dalestones A and B, whose structures were assigned by a combination of spectroscopic analysis, modified Mosher's reaction, and electronic circular dichroism (ECD). Dalestones A and B inhibit the gene expression of TNF-α and IL-6 in LPS-induced RAW264.7 macrophages.

Radiosensitization effect of hsa-miR-138-2-3p on human laryngeal cancer stem cells.

BACKGROUND: Treatments that target cancer stem cells play an important role in the controlling and eliminating of tumor initiation as well as in development, progression, and chemotherapy/radiotherapy resistance. In our previous study, we cultured and harvested human laryngeal cancer stem cells (CSCs) and applied microRNA biochips to screen differentially expressed miRNAs that were related to radiation tolerance in irradiated human laryngeal CSCs. According to the predicted genes and pathways of differential miRNAs target, down-regulated expression of hsa-miR-138-2-3p under radiation was thought to play a key role in enhancing the radio-sensitivity in human laryngeal squamous cancer stem cells. METHOD: To investigate the radiational enhancement of hsa-miR-138-2-3p, we transfected hsa-miR-138-2-3p mimics that were synthesized based on the sequences of hsa-miR-138-2-3p in vitrointo human laryngeal CSCs (Hep-2, M2e, and TU212 cell lines) to make hsa-miR-138-2-3p overexpressed, and the tumorous specialities of CSCs, like cell proliferation, invasion, apoptosis, cell cycle arrest, and DNA damage were evaluated by CCK-8 assay, clone formation assay, invasion assay, flow cytometry, and comet assay. Furthermore, we explored the signal transduction pathways that regulated the cancer stem cell initiation, development, invasion, apoptosis and cell cycle arrest, which were controlled by hsa-miR-138-2-3p. RESULT: Overexpressed hsa-miR-138-2-3p played a key role in many anti-cancer biological processes in human laryngeal CSCs: (1) it decreased laryngeal CSCs proliferation and invasion in response to radiotherapy; (2) it increased the proportion of early and late apoptosis in laryngeal CSCs after radiation, raised G1 phase arrest in laryngeal CSCs after radiation, and decreased the proportion of S stage cells of cell cycle that were related to radio-resistance in laryngeal CSCs; (3) it down-regulated the expression of ß-catenin in Wnt signal pathway that was related to the tolerance of laryngeal CSCs to radiotherapy; (4) it down-regulated the expression of YAP1 in Hippo signal pathway that regulated cell proliferation, invasion and apoptosis; (5) it up-regulated the expression of p38 and JNK1 in MAPK signal pathway that was concerned to radio-sensitivity. CONCLUSION: In the present study, it was found that hsa-miR-138-2-3p regulated the Wnt/ß-catenin pathways, the Hippo/YAP1 pathways, and the MAPK/p38/JNK1 pathways that were involved in cell proliferation, invasion, apoptosis, cell cycle arrest, radio-resistance and radio-sensitivity in laryngeal CSCs. These results will be useful for a better understanding of the cell biology of hsa-miR-138-2-3p in laryngeal CSCs, and for serving hsa-miR-138-2-3p as a promising biomarker and as a target for diagnosis and for novel anti-cancer therapies for laryngeal cancers.

Daphnetin Alleviates Experimental Autoimmune Encephalomyelitis via Regulating Dendritic Cell Activity.

AIMS: Daphnetin, a coumarin derivative extracted from Daphne odora var. marginata, has been reported to have antiinflammatory and immunosuppressive properties. Our previous study indicated that it was able to remarkably suppress the neuroinflammation and suggested its potential application in treating neuroinflammatory diseases. Multiple sclerosis (MS), a Th cell-mediated autoimmune disease, is the most common inflammatory demyelinating disease of the central nervous system (CNS). We examined whether daphnetin treatment can protect mice against experimental autoimmune encephalomyelitis (EAE), an animal model for MS. METHODS: To assess the effect of daphnetin in neuroinflammatory diseases, the EAE mice were established and treated with daphnetin at 8 mg/kg for 28 days. The severity of neuroinflammation and demyelination in the spinal cords was examined histopathologically. Infiltration of CD4(+) T cells into the CNS was assessed by immunohistochemistry, and the cytokine production was determined by ELISA. Meanwhile, the effect of daphnetin on the activity of dendritic cells (DCs) was evaluated, as assessed by DCs' capability to express surface markers, secrete cytokines, and activate naïve CD4(+) T cells. Furthermore, we explored the molecular mechanisms whereby DAPH regulated DCs' activity and thereby CD4(+) T cell responses. RESULTS: The administration of daphnetin markedly alleviated the clinical symptoms of EAE and reduced the CNS inflammation and demyelination in experimental mice. Th1 and Th17 cell responses were profoundly repressed in daphnetin-treated EAE mice. Mechanistically, daphnetin treatment significantly repressed the activation, maturation, and antigen-presenting capability of DCs. NF-κB signaling was significantly reduced in daphnetin-treated DCs, along with a concomitant induction of heme oxygenase-1, a negative regulator of inflammatory signaling. CONCLUSIONS: Our findings for the first time demonstrate the property of daphnetin in regulating DCs' function and subsequently Th development. Given the low or absent toxicity associated with daphnetin, our data may suggest a novel safe and effective approach to control autoimmune neuroinflammation.

The Pro-inflammatory Cytokine Interleukin-1ß is a Key Regulatory Factor for the Postictal Suppression in Mice.

AIMS: The postictal suppression (PS) is a common and important period following an epileptic seizure but has not been well studied. This study was designed to determine whether interleukin-1ß (IL-1ß) is involved in the PS. METHODS: The effects of IL-1ß on the PS were tested in three independent seizure models induced by hippocampal kindling, maximal electroshock seizure (MES), and 4-aminopyridine, respectively. RESULTS: IL-1R1 knockout or IL-1RA enhanced the seizure refractory phenomenon without influencing the baseline seizure threshold in intermittent MES model. IL-1ß attenuated the seizure refractory phenomenon without affecting the severity of the preceding seizures in hippocampal kindling model, while IL-1RA enhanced it. Besides, IL-1ß reduced the postictal EEG suppression period, while IL-1RA prolonged it. And IL-1ß showed no further effect on the postictal EEG suppression and seizure refractory phenomenon in IL-1R1 knockout mice. In addition, 30 min after intrahippocampal injection of 4-aminopyridine, IL-1ß increased the incidence of SE, while IL-1RA prolonged the intervals between recurrent seizures. CONCLUSIONS: This study provides the first direct evidence that IL-1ß is key regulatory factor for the PS, and its receptor IL-1R1 may be a potential target for adjuvant treatment of postictal problems.

[Construction and expression of the eukaryotic expression vector containing human soluble transforming growth factor beta receptor II].

OBJECTIVE: To construct a eukaryotic expression vector encoding the gene of extracellular region of type II transforming growth factor beta receptor (sTGFbetaR II), to express the protein in CHO cell line and to determine its biological activity. METHODS: The extracellular region (amino acids 1-159) of the human TGFbetaR II cDNA was amplified by PCR from a TGFbetaR II chimeric plasmid,and the eukaryotic expression plasmid pCDNA3.1/myc-his(-)B-sTGFbetaR II(pCDNA-sTGFbetaR II) was constructed by inserting the sTGFbetaR II cDNA into the EcoR I/Hind III-digested pCDNA. The DNA sequence was confirmed by double digestion and the pCDNA-sTGFbetaR II plasmid was transfected into the CHO cell line. The sTGFbetaR II protein was confirmed by Western blotting analysis and its biological function was determined. RESULTS: The specific protein was observed in western blotting, and the protein abrogated the growth-inhibitory effects of TGF-beta1 on mink lung epithelial cells (Mv1Lu). CONCLUSION: The eukaryotic expression plasmid pCDNA-sTGFbetaR II has been successfully constructed and the sTGFsTGFbetaR II protein with biological activity obtained.

Anti-inflammatory and protective properties of daphnetin in endotoxin-induced lung injury.

Uncontrolled inflammatory responses cause tissue injury and severe immunopathology. Pharmacological interference of intracellular pro-inflammatory signaling may confer a therapeutic benefit under these conditions. Daphnetin, a natural coumarin derivative, has been used to treat inflammatory diseases including bronchitis. However, the protective effect of daphnetin in inflammatory airway disorders has yet to be determined, and the molecular basis for its anti-inflammatory properties is unknown. This paper shows that daphnetin treatment conferred substantial protection from endotoxin-induced acute lung injury (ALI), in parallel with reductions in the production of inflammatory mediators, symptoms of airway response, and infiltration of inflammatory cells. Further studies indicate that activation of macrophage and human alveolar epithelial cells in response to lipopolysaccharide (LPS) was remarkably suppressed by daphnetin, which was related to the down-regulation of NF-κB-dependent signaling events. Importantly, this study demonstrates that TNF-α-induced protein 3 (TNFAIP3), also known as A20, was significantly induced by daphnetin, which appeared to be largely responsible for the down-regulation of NF-κB activity through modulation of nondegradative TRAF6 ubiquitination. Accordingly, the deletion of TNFAIP3 in primary macrophages reversed daphnetin-elicited inhibition of immune response, and the beneficial effect of daphnetin in the pathogenesis of ALI was, partially at least, abrogated by TNFAIP3 knockdown. These findings demonstrate the anti-inflammatory and protective functions of daphnetin in endotoxin-induced lung inflammation and injury and also reveal the key mechanism underlying its action in vitro as well as in vivo.

Efficacy and significance of various scores for pneumonia severity in the management of patients with community-acquired pneumonia in China.

BACKGROUND: Community-acquired pneumonia (CAP) remains one of the leading causes of death from infectious diseases around the world. Most severe CAP patients are admitted to the intensive care unit (ICU), and receive intense treatment. The present study aimed to evaluate the role of the pneumonia severity index (PSI), CURB-65, and sepsis score in the management of hospitalized CAP patients and explore the effect of ICU treatment on prognosis of severe cases. METHODS: A total of 675 CAP patients hospitalized in the Second Affiliated Hospital of Zhejiang University School of Medicine were retrospectively investigated. The ability of different pneumonia severity scores to predict mortality was compared for effectiveness, while the risk factors associated with 30-day mortality rates and hospital length of stay (LOS) were evaluated. The effect of ICU treatment on the outcomes of severe CAP patients was also investigated. RESULTS: All three scoring systems revealed that the mortality associated with the low-risk or intermediate-risk group was significantly lower than with the high-risk group. As the risk level increased, the frequency of ICU admission rose in tandem and LOS in the hospital was prolonged. The areas under the receiver operating characteristic curve in the prediction of mortality were 0.94, 0.91 and 0.89 for the PSI, CURB-65 and sepsis score, respectively. Compared with the corresponding control groups, the mortality was markedly increased in patients with a history of smoking, prior admission to ICU, respiratory failure, or co-morbidity of heart disease. The differences were also identified in LOS between control groups and patients with ICU treatment, heart, or cerebrovascular disease. Logistic regression analysis showed that age over 65 years, a history of smoking, and respiratory failure were closely related to mortality in the overall CAP cohort, whereas age, ICU admission, respiratory failure, and LOS at home between disease attack and hospital admission were identified as independent risk factors for mortality in the high-risk CAP sub-group. The 30-day mortality of patients who underwent ICU treatment on admission was also higher than for non-ICU treatment, but much lower than for those patients who took ICU treatment subsequent to the failure of non-ICU treatment. CONCLUSIONS: Each severity score system, CURB-65, sepsis severity score and especially PSI, was capable of effectively predicting CAP mortality. Delayed ICU admission was related to higher mortality rates in severe CAP patients.

[Construction and expression of recombinant adenovirus containing human thymic stromal lymphopoietin gene].

AIM: To construct and express recombinant adenovirus bearing human thymic stromal lymphopoietin (TSLP) gene. METHODS: TSLP gene amplified from human fetal lung cells was first cloned into eukaryotic expression vector pcDNA3.1, and then subcloned into shuttle vector pShuttle. The resultant plasmid was subsequently cotransformed into E. coli BJ5183 cells with adenoviral backbone plasmid pAdEasy-1. The recombinant adenovirus plasmid containing TSLP was digested with Pac I and transfected into HEK293 cells to package recombinant adenovirus particles. The TSLP gene of the recombinant virus was detected by PCR, and its expression was analyzed by Western blot. RESULTS: Recombinant adenovirus vector bearing human TSLP gene was constructed by homologous recombination in E.coli, and recombinant adenovirus was obtained by transfecting HEK293 cells with this infectious DNA. PCR test indicated that TSLP gene was successfully integrated into the adenoviral genome, and the titer of the recombinant Ad reached 1 x 10(11) pfu/L. Meanwhile, expression of TSLP in the infected Hela cells was confirmed by Western blot. CONCLUSION: The construction of recombinant adenovirus bearing human TSLP gene and its expression mediated by this adenovirus pave a foundation for the study on the biological function of this novel cytokine.

[Preliminary observation of CXCR3 expression on peripheral blood lymphocytes from patients with chronic hepatitis B infection].

AIM: To explore the role of chemokine receptor CXCR3 in pathogenesis of chronic hepatitis B. METHODS: The expression of CXCR3 on peripheral blood lymphocytes from chronic hepatitis B patients with various degrees of inflammation was detected, and the distributions of CXCR3 on CD4(+) and CD8(+) T lymphocytes were also evaluated by FACS. RESULTS: As compared with healthy control, the percentages of CXCR3(+) lymphocytes, monocytes and CD8(+) T cells were increased significantly in chronic hepatitis B patients. CONCLUSION: Above data suggest that chemokine receptor CXCR3 may play an important role in the recruitment of lymphocytes, especially CTLs recruited to inflammation sites.