Super-high procoagulant activity of gecko thrombin: A gift from sky dragon.

AIMS: Gecko, the "sky dragon" named by Traditional Chinese Medicine, undergoes rapid coagulation and scarless regeneration following tail amputation in the natural ecology, providing a perfect opportunity to develop the efficient and safe drug for blood clotting. Here, gecko thrombin (gthrombin) was recombinantly prepared and comparatively studied on its procoagulant activity. METHODS: The 3D structure of gthrombin was constructed using the homology modeling method of I-TASSER. The active gthrombin was prepared by the expression of gecko prethrombin-2 in 293 T cells, followed by purification with Ni2+ -chelating column chromatography prior to activation by snake venom-derived Ecarin. The enzymatic activities of gthrombin were assayed by hydrolysis of synthetic substrate S-2238 and the fibrinogen clotting. The vulnerable nerve cells were used to evaluate the toxicity of gthrombin at molecular and cellular levels. RESULTS: The active recombinant gthrombin showed super-high catalytic and fibrinogenolytic efficiency than those of human under different temperatures and pH conditions. In addition, gthrombin made nontoxic effects on the central nerve cells including neurons, contrary to those of mammalian counterparts, which contribute to neuronal damage, astrogliosis, and demyelination. CONCLUSIONS: A super-high activity but safe procoagulant candidate drug was identified from reptiles, which provided a promising perspective for clinical application in rapid blood clotting.

Macrophage Migration Inhibitory Factor Promotes Expression of Matrix Metalloproteinases 1 and 3 in Spinal Cord Astrocytes following Gecko Tail Amputation.

BACKGROUND: The matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that play a variety of physiological and pathological roles in development, remodeling of tissues and diseases, mainly through degradation of various components of the extracellular matrix (ECM). Particularly, the MMPs have increasingly been found to mediate neuropathology following spinal cord injury (SCI). Proinflammatory mediators are potent activators of the MMPs. However, how the spinal cord regenerative vertebrates circumvent MMPs-mediated neuropathogenesis following SCI remains unclear. METHODS: Following the establishment of gecko tail amputation model, the correlation of MMP-1 (gMMP-1) and MMP-3 (gMMP-3) expression with that of macrophage migration inhibitory factor in gecko (gMIF) was assayed by RT-PCR, Western blot and immunohistochemistry. Transcriptome sequencing of primary astrocytes was performed to analyze the intracellular signal transduction of macrophage migration inhibitory factor (MIF). The effects of MMP-1 and MMP-3 induced by MIF on astrocyte migration were assessed by transwell migration assay. RESULTS: The expression of gMIF significantly increased at lesion site of the injured cord, in parallel with those of gMMP-1 and gMMP-3 in the gecko astrocytes (gAS). Transcriptome sequencing and in vitro cell model revealed that gMIF efficiently promoted the expression of gMMP-1 and gMMP-3 in gAS, which in turn contributed to the migration of gAS. Inhibition of gMIF activity following gecko SCI remarkably attenuated astrocytic expression of the two MMPs, and further influenced gecko tail regeneration. CONCLUSIONS: Gecko SCI following tail amputation promoted production of gMIF, which induced the expression of gMMP-1 and gMMP-3 in gAS. The gMIF-mediated gMMP-1 and gMMP-3 expression was involved in gAS migration and successful tail regeneration.

D-dopachrome tautomerase drives astroglial inflammation via NF-κB signaling following spinal cord injury.

BACKGROUND: Reactive astrocytes are increasingly recognized as crucial regulators of innate immunity in degenerative or damaged central nervous system (CNS). Many proinflammatory mediators have been shown to drive inflammatory cascades of astrocytes through activation of NF-κB, thereby affecting the functional outcome of the insulted CNS. D-dopachrome tautomerase (D-DT), a newly described cytokine and a close homolog of proinflammatory macrophage migration inhibitory factor (MIF), has been revealed to share receptor and overlapping functional spectrum with MIF, but little is known about its roles in the neuropathological progression of the CNS and relevant regulatory mechanisms. RESULTS: D-DT protein levels were significantly elevated within neurons and astrocytes following SCI. Analysis of transcriptome profile revealed that D-DT was able to activate multiple signal pathways of astrocytes, which converged to NF-κB, a hub regulator governing proinflammatory response. Rat D-DT recombinant protein was efficient in inducing the production of inflammatory cytokines from astrocytes through interaction with CD74 receptor. Activation of mitogen-activated protein kinases (MAPKs) and NF-κB was observed to be essential for the transduction of D-DT signaling. Administration of D-DT specific inhibitor at lesion sites of the cord resulted in significant attenuation of NF-κB activation and reduction of the inflammatory cytokines following SCI, and accordingly improved the recovery of locomotor functions. CONCLUSION: Collectively, D-DT is a novel proinflammatory mediator of astrocytes following SCI. Insights of its cell-specific expression and relevant proinflammatory mechanisms will provide clues for the control of CNS inflammation.

Suppressors of cytokine signaling 1 protein in a regenerative model of the Gekko japonicus spinal cord.

Demyelination is one of the pathological outcomes that occur immediately following spinal cord injury. Protection of oligodendrocytes against death/apoptosis proves to be beneficial for the preservation of neurological functions. Suppressors of cytokine signaling 1 protein inhibit the harmful effects of several inflammatory cytokines on oligodendrocytes, but its roles in spinal cord injury (SCI) induced apoptosis of oligodendrocytes remain unclear. We cloned suppressors of cytokine signaling 1 cDNA from Gekko japonicus (Japanese gecko) and analyzed the protein structure revealing the conserved domains contained in other vertebrate suppressors of cytokine signaling 1 proteins. The gecko suppressors of cytokine signaling 1 protein expression were increased in the injured spinal cord following gecko tail amputation and displayed colocalization with oligodendrocytes. The enforced expression of gecko suppressors of cytokine signaling 1 by adenovirus in the Gsn3 gecko oligodendrocyte cell line demonstrated that gecko suppressors of cytokine signaling 1 significantly suppressed cell apoptosis-induced by glucose deprivation. Determination of apoptosis-related proteins revealed that gecko suppressors of cytokine signaling 1 was able to activate extracellular regulated protein kinases (ERK) and serine/threonine protein kinases (Akt). The results presented a distinct protective role of gecko suppressors of cytokine signaling 1 in the regenerative model of the spinal cord, which may provide new cues for central nervous system repair in mammals.

HSF1 is involved in suppressing A1 phenotype conversion of astrocytes following spinal cord injury in rats.

BACKGROUND: Two activation states of reactive astrocytes termed A1 and A2 subtypes emerge at the lesion sites following spinal cord injury (SCI). A1 astrocytes are known to be neurotoxic that participate in neuropathogenesis, whereas A2 astrocytes have been assigned the neuroprotective activity. Heat shock transcription factor 1 (HSF1) plays roles in protecting cells from stress-induced apoptosis and in controlling inflammatory activation. It is unknown whether HSF1 is involved in suppressing the conversion of A1 astrocytes following SCI. METHODS: A contusion model of the rat spinal cord was established, and the correlations between HSF1 expression and onset of A1 and A2 astrocytes were assayed by Western blot and immunohistochemistry. 17-AAG, the agonist of HSF1, was employed to treat the primary cultured astrocytes following a challenge by an A1-astrocyte-conditioned medium (ACM) containing 3 ng/ml of IL-1α, 30 ng/ml of TNF-α, and 400 ng/ml of C1q for induction of the A1 subtype. The effects of 17-AAG on the phenotype conversion of astrocytes, as well as underlying signal pathways, were examined by Western blot or immunohistochemistry. RESULTS: The protein levels of HSF1 were significantly increased at 4 days and 7 days following rat SCI, showing colocalization with astrocytes. Meanwhile, C3-positive A1 astrocytes were observed to accumulate at lesion sites with a peak at 1 day and 4 days. Distinctively, the S100A10-positive A2 subtype reached its peak at 4 days and 7 days. Incubation of the primary astrocytes with ACM markedly induced the conversion of the A1 phenotype, whereas an addition of 17-AAG significantly suppressed such inducible effects without conversion of the A2 subtype. Activation of HSF1 remarkably inhibited the activities of MAPKs and NFκB, which was responsible for the regulation of C3 expression. Administration of 17-AAG at the lesion sites of rats was able to reduce the accumulation of A1 astrocytes. CONCLUSION: Collectively, these data reveal a novel mechanism of astrocyte phenotype conversion following SCI, and HSF1 plays key roles in suppressing excessive increase of neurotoxic A1 astrocytes.

D-dopachrome tautomerase activates COX2/PGE2 pathway of astrocytes to mediate inflammation following spinal cord injury.

BACKGROUND: Astrocytes are the predominant glial cell type in the central nervous system (CNS) that can secrete various cytokines and chemokines mediating neuropathology in response to danger signals. D-dopachrome tautomerase (D-DT), a newly described cytokine and a close homolog of macrophage migration inhibitory factor (MIF) protein, has been revealed to share an overlapping function with MIF in some ways. However, its cellular distribution pattern and mediated astrocyte neuropathological function in the CNS remain unclear. METHODS: A contusion model of the rat spinal cord was established. The protein levels of D-DT and PGE2 synthesis-related proteinase were assayed by Western blot and immunohistochemistry. Primary astrocytes were stimulated by different concentrations of D-DT in the presence or absence of various inhibitors to examine relevant signal pathways. The post-injury locomotor functions were assessed using the Basso, Beattie, and Bresnahan (BBB) locomotor scale. RESULTS: D-DT was inducibly expressed within astrocytes and neurons, rather than in microglia following spinal cord contusion. D-DT was able to activate the COX2/PGE2 signal pathway of astrocytes through CD74 receptor, and the intracellular activation of mitogen-activated protein kinases (MAPKs) was involved in the regulation of D-DT action. The selective inhibitor of D-DT was efficient in attenuating D-DT-induced astrocyte production of PGE2 following spinal cord injury, which contributed to the improvement of locomotor functions. CONCLUSION: Collectively, these data reveal a novel inflammatory activator of astrocytes following spinal cord injury, which might be beneficial for the development of anti-inflammation drug in neuropathological CNS.

Primary culture of adult cortical neurons from reptile Gekko japonicus.

Adult neurons of several reptiles still retain the ability of axonal regeneration in contrast to the low intrinsic regenerative capacity of those in the central nervous system (CNS) in mammals. This feature of the reptilian neurons has provided a perfect model for elucidating the regenerative mechanism lost in the mammalian counterparts. However, little information is available on the primary culture method of adult reptilian neurons, which greatly limits their valuable applications. In the present study, we introduced a simple and easy method for the isolation, culture, and identification of neurons from the cerebral cortex using the adult geckos. The cultured cells were further identified by immunofluorescence using antibodies against neuron-specific markers ß-Ⅲ-tubulin and NeuN. The cortical neurons from adult gecko displayed spindle-shaped, bipolar, or multipolar morphology with a plump soma. This primary culture method for adult reptilian neurons will be beneficial for comparative studies of neuronal biology in various vertebrates.

Adult astrocytes from reptiles are resistant to proinflammatory activation via sustaining Vav1 expression.

Adult mammalian astrocytes are sensitive to inflammatory stimuli in the context of neuropathology or mechanical injury, thereby affecting functional outcomes of the central nervous system (CNS). In contrast, glial cells residing in the spinal cord of regenerative vertebrates exhibit a weak astroglial reaction similar to those of mammals in embryonic stages. Macrophage migration inhibitory factor (MIF) participates in multiple neurological disorders by activation of glial and immune cells. However, the mechanism of astrocytes from regenerative species, such as gecko astrocytes (gAS), in resistance to MIF-mediated inflammation in the severed cords remains unclear. Here, we compared neural stem cell markers among gAS, as well as adult (rAS) and embryonic (eAS) rat astrocytes. We observed that gAS retained an immature phenotype resembling rat eAS. Proinflammatory activation of gAS with gecko (gMIF) or rat (rMIF) recombinant protein was unable to induce the production of inflammatory cytokines, despite its interaction with membrane CD74 receptor. Using cross-species screening of inflammation-related mediators from models of gMIF- and rMIF-induced gAS and rAS, we identified Vav1 as a key regulator in suppressing the inflammatory activation of gAS. The gAS with Vav1 deficiency displayed significantly restored sensitivity to inflammatory stimuli. Meanwhile, gMIF acts to promote the migration of gAS through regulation of CXCL8 following cord lesion. Taken together, our results suggest that Vav1 contributes to the regulation of astrocyte-mediated inflammation, which might be beneficial for the therapeutic development of neurological diseases.

High mobility group box 1 mediates inflammatory response of astrocytes via cyclooxygenase 2/prostaglandin E2 signaling following spinal cord injury.

High mobility group box 1 (HMGB1) interacts with pattern-recognition receptors of immune cells to activate the inflammatory response. Astrocytes play a positive role in the inflammatory response of the central nervous system by expressing a broad range of pattern-recognition receptors. However, the underlying relationship between HMGB1 and the inflammatory reaction of astrocytes remains unclear. In this study, we established rat models of spinal cord injury via laminectomy at the T8-10 level, and the injured spinal cord was subjected to transcriptome sequencing. Our results showed that the HMGB1/Toll-like receptor 4 (TLR4) axis was involved in the activation of astrocyte inflammatory response through regulation of cyclooxygenase 2 (COX2)/prostaglandin E2 (PGE2) signaling. Both TLR4 and COX2 were distributed in astrocytes and showed elevated protein levels following spinal cord injury. Stimulation of primary astrocytes with recombinant HMGB1 showed that COX2 and microsomal PGE synthase (mPGES)-1, rather than COX1, mPGES-2, or cytosolic PGE synthase, were significantly upregulated. Accordingly, PGE2 production in astrocytes was remarkably increased in response to recombinant HMGB1 challenges. Pharmacologic blockade of TLR2/4 attenuated HMGB1-mediated activation of the COX2/PGE2 pathway. Interestingly, HMGB1 did not impact the production of tumor necrosis factor-α or interleukin-1ß in astrocytes. Our results suggest that HMGB1 mediates the astrocyte inflammatory response through regulating the COX2/PGE2 signaling pathway. The study was approved by the Laboratory Animal Ethics Committee of Nantong University, China (approval No. 20181204-001) on December 4, 2018.