Zafirlukast in combination with pseudohypericin attenuates spinal cord injury and motor function in experimental mice.

BACKGROUND: Biosynthesis of leukotriene (LT) by arachidonic acid involves 5-lipoxygenase (5-LO) as an important precursor. Here, we evaluated the role of pseudohypericin (PHP) for its postulated 5-LO inhibitory activity along with a Cys-LT receptor antagonist zafirlukast (ZFL) against inflammatory response and tissue injury in mice. MATERIALS AND METHODS: The spinal injury was induced by two-level laminectomy of T6 and T7 vertebrae. The inflammation was assessed by histology, inflammatory mediators by enzyme-linked immunosorbent assay, apoptosis by Annexin-V, FAS staining, terminal deoxynucleoti-dyltransferase-mediated UTP end labeling (TUNEL) assay and expression of Bax and Bcl-2 by Western blot. Effect on motor recovery of hind limbs was evaluated for 10 days postinjury. RESULTS: The spinal injury resulted in tissue damage, apoptosis, edema, infiltration of neutrophils with increased expression of tumor necrosis factor-α (TNF-α) and cyclooxygenase-2 (COX-2). The spinal tissue showed elevated levels of prostaglandin E2 (PGE2), and LTB4 and increased phosphorylation of injured extracellular signal-regulated kinase-1/2 (ERK1/2). The PHP, ZFL and combination decreased inflammation, tissue injury and infiltration of neutrophils. Treatment also decreased the levels of PGE2, phosphorylation of extracellular signal-regulated kinase-1/2 (pERK 1/2), LT, TNF-α and COX-2 with a marked reduction in apoptosis and improved the motor function. CONCLUSION: The present study confirmed 5-LO antagonist activity of PHP and established its neuroprotective role along with ZFL.

Phosphodiesterase inhibitor ameliorates neuronal injury in spinal cord ischemia/reperfusion injured rat model.

This study investigated the mechanisms responsible for the neuroprotective effect of sildenafil citrate (SFC) on ischemia-reperfusion spinal cord (SC) injuries. Balloon occlusion of the thoracic aorta was used to induce SC ischemia. The animals (n=30) were separated into three groups: sham, SC injury with saline, and SC injury with 5mg/kg i.p. SFC treatment (SFC). The Basso, Beattie, and Bresnahan (BBB) score was determined to assess neurological function at different time intervals after reperfusion. After 48h, histopathology of the SC was assessed by triphenyltetrazolium chloride (TTC) and Nissl staining. Myeloperoxidase (MPO) activity was estimated using an MPO assay kit. Western blot and ELISA assays were performed to estimate interleukin 1 & 10 (IL-1 & IL-10), tumour necrosis factor α (TNF-α), and nuclear factor (NF-kB) levels in SC tissue homogenates. The study results suggest that treatment with SFC significantly increased neurological function compared with the SC group. In addition, SFC treatment reduced MPO activity compared with the SC group, which subsequently inhibited the infiltration of neutrophils into the SC. There was a significant (p<0.01) decrease in the expression of IL-1 and TNF-α, and an increase in the expression of IL-10 in SFC tissue homogenates compared with SC tissues. Moreover, SFC treatment inhibited the activation of NF-kB in the SC after injury. This study shows that SFC exerts a neuroprotective effect on the SC after ischemia/reperfusion injury by attenuating inflammatory mediators.

Iridium-catalyzed asymmetric hydrogenation of racemic α-substituted lactones to chiral diols.

We report a protocol for the highly efficient iridium-catalyzed asymmetric hydrogenation of racemic α-substituted lactones via dynamic kinetic resolution. Using Ir-SpiroPAP (R)-1d as a catalyst, a wide range of chiral diols were prepared in a high yield (80-95%) with a high enantioselectivity (up to 95% ee) under mild reaction conditions. This protocol was used for enantioselective syntheses of (-)-preclamol and a chiral 2,5-disubstituted tetrahydropyran.

Down-regulation apoptosis signal-regulating kinase 1 gene reduced the Litopenaeus vannamei hemocyte apoptosis in WSSV infection.

Apoptosis signal-regulating kinase 1 (ASK1), a mitogen-activated protein kinase kinase kinase, is crucial in various cellular responses. In the present study, we identified and characterized an ASK1 homolog from Litopenaeus vannamei (LvASK1). The full-length cDNA of LvASK1 was 5400 bp long, with an open reading frame encoding a putative 1420 amino acid protein. LvASK1 was highly expressed in muscle, hemocyte, eyestalk and heart. Real-time RT-PCR analysis showed that the expression of the LvASK1 was upregulated during the white spot syndrome virus (WSSV) challenge. The knocked-down expression of LvASK1 by RNA interference significantly reduced the apoptotic ratio of the hemocytes collected from WSSV-infected L. vannamei. Furthermore, the down-regulation of LvASK1 also decreased the cumulative mortality of WSSV-infected L. vannamei. These results suggested that down-regulation of LvASK1 decreased the apoptotic rate of hemocytes in WSSV-infected shrimp, and that it could contribute to the reduction of cumulative mortality in WSSV-infected L. vannamei.

Transcriptome analysis of the unfolded protein response in hemocytes of Litopenaeus vannamei.

In this study, Litopenaeus vannamei was injected with double-stranded RNA (dsRNA) against L. vannamei immunoglobulin heavy chain binding protein (LvBip) to activating UPR in the hemocytes, shirmps injected dsRNA against enhanced green fluorescence protein (eGFP) as control group. And genes expression in hemocytes of then were analyzed using Illumina Hiseq 2500 (PE100). By comparing the analyzed results, 1418 unigenes were significantly upregulated, and 596 unigenes were significantly down-regulated upon UPR. Analysis of the differentially expressed genes against known databases indicated that the distribution of gene pathways between the upregulated and down-regulated genes were substantially different. A total of 208 genes of UPR system were obtained, and 69 of them were differentially expressed between the two groups. Results also showed that L. vannamei UPR was involved in various metabolic processes, such as glycometabolism, lipid metabolism, amino acid metabolism, and nucleic acid metabolism. In addition, UPR was emgaged in immune-assicoated signaling pathways, such as NF-κB signaling pathway, NOD-like receptor signaling pathway, Hippo signaling pathway, p38 MAPK signaling pathway and Wnt signaling pathway in L. vannamei. These results improved our current understanding of the L. vannamei UPR, and highlighted its importance in cell homeostasis upon environmental stress.

Identification and functional characterization of an endoplasmic reticulum oxidoreductin 1-α gene in Litopenaeus vannamei.

In the current study, full-length sequence of endoplasmic reticulum oxidoreductin 1-α (LvERO1-α) was cloned from Litopenaeus vannamei. Real-time RT-PCR results showed that LvERO1-α was highly expressed in hemocytes, gills, and intestines. White spot syndrome virus (WSSV) challenge was performed, and the expression of LvERO1-α and two other downstream genes of the double-stranded RNA-activated protein kinase-like ER kinase-eIF2α (PERK-α) pathway, namely, homocysteine-induced endoplasmic reticulum protein (LvHERP) and acylamino-acid-releasing enzyme (LvAARE), strongly increased in the hemocytes. Flow cytometry assay results indicated that the apoptosis rate of L. vannamei hemocytes in the LvERO1-α knockdown group was significantly lower than that of the controls. Moreover, shrimps with knockdown expression of LvERO1-α exhibited decreased cumulative mortality upon WSSV infection. Downregulation of L. vannamei immunoglobulin-binding protein (LvBip), which had been proven to induce unfolded protein response (UPR) in L. vannamei, did not only upregulate LvERO1-α, LvHERP, and LvAARE in hemocytes, but also increased their apoptosis rate, as well as the shrimp cumulative mortality. Furthermore, reporter gene assay results showed that the promoter of LvERO1-α was activated by L. vannamei activating transcription factor 4, thereby confirming that LvERO1-α was regulated by the PERK-eIF2α pathway. These results suggested that LvERO1-α plays a critical role in WSSV-induced apoptosis, which likely occurs through the WSSV-activated PERK-eIF2α pathway.

Identification and characterization of a mitochondrial unfolded protein response transcription factor ATFS-1 in Litopenaeus vannamei.

A mitochondrial specific stress response termed mitochondrial unfolded protein response (UPR(mt)) is activated in responding to disturbance of protein homeostasis in mitochondria. The activating transcription factor associated with stress-1 (designated as ATFS-1) is the key regulator of UPR(mt). To investigating the roles of ATFS-1 (LvATFS-1) in Litopenaeus vannamei mitochondrial stress remission and immunity, it's full length cDNA was cloned. The open reading frame of LvATFS-1 was 1, 557 bp in length, deducing to a 268 amino acids protein. LvATFS-1 was highly expressed in muscle, hemocytes and eyestalk. Subcellular location assays showed that N-terminal of LvATFS-1 contained a mitochondrial targeting sequence, which could directed the fused EGFP located to mitochondria. And the C-terminal of LvATFS-1, which had a nuclear localization signal, expressed in nucleus. The in vitro experiments verified that LvATFS-1 could reduced the level of intracellular reactive oxygen species (ROS). And results of real-time RT-PCR indicated that LvATFS-1 might scavenge excess ROS via ROS-eliminating genes regulation. Reporter gene assays showed that LvATFS-1 could upregulated the expression of the antimicrobial peptide genes in Drosophila Schneider 2 cells. Results of real-time RT-PCR showed that Vibrio alginolyticus or white spot syndrome virus (WSSV) infection induced the expression of LvATFS-1. And knocked-down LvATFS-1 by RNAi resulted in a higher cumulative mortality of L. vannamei upon V. alginolyticus or WSSV infection. These results suggested that LvATFS-1 not only rolled in mitochondrial specific stress responding, but also important for L. vannamei immunologic defence.

Identification and functional characterization of a solute carrier family 15, member 4 gene in Litopenaeus vannamei.

Innate immunity in shrimp is important in resisting bacterial infection. The NF-κB pathway is pivotal in such an immune response. This study cloned and functionally characterized the solute carrier family (SLC) 15 member A 4 (LvSLC15A4) gene in Litopenaeus vannamei. The open reading frame of LvSLC15A4 is 1, 902 bp long and encodes a putative 633-amino acid protein, which is localized in the plasma membrane and intracellular vesicular compartments. Results of the reporter gene assay showed that LvSLC15A4 upregulated NF-κB target genes, including the immediate-early gene 1 of white spot syndrome virus, as well as several antimicrobial peptide genes, such as pen4, CecA, AttA, and Mtk in S2 cells. Moreover, knocked-down expression of LvSLC15A4 reduced pen4 expression in L. vannamei. LvSLC15A4 down-regulation also increased the cumulative mortality of Vibrio parahemolyticus-infected L. vannamei. Furthermore, LvSLC15A4 expression was induced by unfolded protein response (UPR) in L. vannamei hematocytes. These results suggest that LvSLC15A4 participates in L. vannamei innate immunity via the NF-κB pathway and thus may be related to UPR.

Divergent enantioselective synthesis of hapalindole-type alkaloids using catalytic asymmetric hydrogenation of a ketone to construct the chiral core structure.

A divergent enantioselective approach to hapalindole-type alkaloids is described. The route features a ruthenium-catalyzed asymmetric hydrogenation of a ketone via DKR to construct the chiral trans-1-indolyl-2-isopropenylcyclohexane skeleton and a switchable sequence of methylation and acetylation/aldol reaction to access a chiral quaternary stereocenter. (+)-Hapalindole Q (1, 13 steps, 5.9% overall yield), (-)-12-epi-hapalindole Q isonitrile (2, 15 steps, 5.5% overall yield), (-)-hapalindole D (3, 14 steps, 2.3% overall yield), and (+)-12-epi-fischerindole U isothiocyanate (4, 14 steps, 3.0% overall yield) were synthesized in 13-15 steps from a commercially available material to demonstrate the application of this approach.

An activating transcription factor of Litopenaeus vannamei involved in WSSV genes Wsv059 and Wsv166 regulation.

Members of activating transcription factor/cyclic adenosine 3', 5'-monophosphate response element binding protein (ATF/CREB) family are induced by various stress signals and function as effector molecules. Consequently, cellular changes occur in response to discrete sets of instructions. In this work, we found an ATF transcription factor in Litopenaeus vannamei designated as LvATFß. The full-length cDNA of LvATFß was 1388 bp long with an open reading frame of 939 bp that encoded a putative 313 amino acid protein. The protein contained a basic region-leucine zipper (bZip) domain that was a common feature among ATF/CREB transcription factors. LvATFß was highly expressed in intestines, gills, and heart. LvATFß expression was dramatically upregulated by white spot syndrome virus (WSSV) infection. Pull-down assay revealed that LvATFß had strong affinity to promoters of WSSV genes, namely, wsv059 and wsv166. Dual-luciferase reporter assay showed that LvATFß could upregulate the expression of wsv059 and wsv166. Knocked down LvATFß resulted in decreased expression of wsv059 and wsv166 in WSSV-challenged L. vannamei. Knocked down expression of wsv059 and wsv166 by RNA interference inhibited the replication and reduce the mortality of L. vannamei during WSSV challenge inoculation. The copy numbers of WSSV in wsv059 and wsv166 knocked down group were significant lower than in the control. These results suggested that LvATFß may be involved in WSSV replication by regulating the expression of wsv059 and wsv166.

Activating transcription factor 4 and X box binding protein 1 of Litopenaeus vannamei transcriptional regulated white spot syndrome virus genes Wsv023 and Wsv083.

In response to endoplasmic reticulum (ER) stress, the signaling pathway termed unfolded protein response (UPR) is activated. To investigate the role of UPR in Litopenaeus vannamei immunity, the activating transcription factor 4 (designated as LvATF4) which belonged to a branch of the UPR, the [protein kinase RNA (PKR)-like ER kinase, (PERK)]-[eukaryotic initiation factor 2 subunit alpha (eIF2α)] pathway, was identified and characterized. The full-length cDNA of LvATF4 was 1972 bp long, with an open reading frame of 1299 bp long that encoded a 432 amino acid protein. LvATF4 was highly expressed in gills, intestines and stomach. For the white spot syndrome virus (WSSV) challenge, LvATF4 was upregulated in the gills after 3 hpi and increased by 1.9-fold (96 hpi) compared to the mock-treated group. The LvATF4 knock-down by RNA interference resulted in a lower cumulative mortality of L. vannamei under WSSV infection. Reporter gene assays show that LvATF4 could upregulate the expression of the WSSV gene wsv023 based on the activating transcription factor/cyclic adenosine 3', 5'-monophosphate response element (ATF/CRE). Another transcription factor of L. vannamei, X box binding protein 1 (designated as LvXBP1), has a significant function in [inositol-requiring enzyme-1(IRE1) - (XBP1)] pathway. This transcription factor upregulated the expression of the WSSV gene wsv083 based on the UPR element (UPRE). These results suggest that in L. vannamei UPR signaling pathway transcription factors are important for WSSV and might facilitate WSSV infection.