Therapy of traumatic brain injury by modern agents and traditional Chinese medicine.

Traumatic brain injury (TBI) is the leading cause of disability and death, and the social burden of mortality and morbidity caused by TBI is significant. Under the influence of comprehensive factors, such as social environment, lifestyle, and employment type, the incidence of TBI continues to increase annually. Current pharmacotherapy of TBI mainly focuses on symptomatic supportive treatment, aiming to reduce intracranial pressure, ease pain, alleviate irritability, and fight infection. In this study, we summarized numerous studies covering the use of neuroprotective agents in different animal models and clinical trials after TBI. However, we found that no drug has been approved as specifically effective for the treatment of TBI. Effective therapeutic strategies for TBI remain an urgent need, and attention is turning toward traditional Chinese medicine. We analyzed the reasons why existing high-profile drugs had failed to show clinical benefits and offered our views on the research of traditional herbal medicine for treating TBI.

Identification and expression of the elongator protein 2 (Ajelp2) gene, a novel regeneration-related gene from the sea cucumber Apostichopus japonicus.

Elongator proteins comprise six subunits (ELP1-ELP6) and form protein complexes. The elongator protein 2 gene (elp2) encodes a protein with a WD40 repeats domain that acts as a scaffold for complex assembly. It also plays an important role in growth and development. In this study, the full-length cDNA of elongator protein 2 (Ajelp2) was cloned from the sea cucumber Apostichopus japonicus (A. japonicus) using rapid amplification of cDNA ends PCR techniques and comprised 3,058 bp, including a 54 bp 5' untranslated (UTR), a 526 bp 3' UTR and a 2,478 bp open reading frame encoding a polypeptide of 825 amino acids. The Ajelp2 sequence showed high homology to 12 other species. The molecular weight and isoelectric of point the presumptive protein were 91.6 kDa and 5.84, respectively. In situ hybridization indicated that the gene is expressed in the body wall, intestine, respiratory tree and longitudinal muscle. The expression level of Ajelp2 increased in recovering of organs in sea cucumber and showed it's the highest expression level at the 15th day in the intestine and respiratory tree. Its expression then gradually decreased to normal levels. In the body wall, the expression level of Ajelp2 was up-regulated and then down-regulated. These results indicated that Ajelp2 is involved in protein regulation during the regeneration process in the sea cucumber A. japonicus.

Identification, expression pattern, cellular location and potential role of the caveolin-1 gene from Artemia sinica.

Caveolins are integral membrane proteins that serve as scaffolds to recruit numerous signaling molecules. Caveolins play an important role in membrane trafficking, signal transduction, substrate transport and endocytosis in differentiated cells. In this study, a caveolin-1 gene from Artemia sinica (As-cav-1) was successfully cloned for the first time. The full-length cDNA of As-cav-1 comprises 974 bp, with a 675 bp open reading frame (ORF) that encodes a polypeptide of 224 amino acids with a caveolin scaffolding domain (CSD) and two transmembrane domains. Multiple sequence alignment revealed that the putative As-CAV-1 protein sequence was relatively conserved across species, especially in the CSD domain. Real-time PCR revealed high levels of the As-cav-1 transcript at 0h of embryo development. Furthermore, As-cav-1 transcripts were highly upregulated under high salinity (200‰) and low temperature stresses (15°C). To further characterize As-cav-1, recombinant pET30a-cav-1 protein was expressed using a prokaryotic expression system. The recombinant protein comprised 290 amino acids with a theoretical molecular weight of 32kDa, and a predicted isoelectric point of 5.6. Western blotting of the expression levels of As-CAV-1 during different embryo development stages revealed that As-CAV-1 levels decreased gradually during development stages from 0 h to 40 h, and increased at 3d. Furthermore, western blotting showed that As-CAV-1 was upregulated to its highest expression level by low temperature stress (15°C) and high salinity. Confocal laser microscopy analysis, using antibodies generated against the recombinant As-CAV-1 protein, showed that As-CAV-1 was mostly located in the cell membrane. Our results suggested that As-cav-1 plays a vital role in protecting embryos from high salt damage and low temperature stress, especially during post-diapause embryonic development.

Cloning and expression analysis of the gene encoding fibrinogen-like protein A, a novel regeneration-related protein from Apostichopus japonicus.

Fibrinogen-like protein A (FGLA), a member of the fibrinogen-related protein superfamily, exists in different tissues of vertebrates and invertebrates. FGLA plays crucial roles including innate immune response, blood clotting and regeneration. In this study, the fibrinogen-like protein A (fglA) was cloned from Apostichopus japonicus using rapid amplification of cDNA ends PCR techniques. The cDNA sequence of fglA is 1,524 bp with a 849 bp open reading frame encoding a polypeptide of 282 amino acids, with an N-terminal signal peptide and a conserved C-terminal domain. Bioinformatic analysis revealed that the predicted molecular weight of the whole protein is 31.9 kDa and it has an isoelectric point of 5.64. In-situ hybridization demonstrated that fglA is widely distributed in body wall, intestines, longitudinal muscles and respiratory tree. The expression levels of fglA during different regeneration stages in the body wall, intestine and respiratory trees were analyzed by real-time PCR. The expression of fglA gradually increased within 1 h in body wall, and reached a plateau before decreasing to the basal level. This indicates that fglA is associated with the regeneration of Apostichopus japonicus.

The potential role of As-sumo-1 in the embryonic diapause process and early embryo development of Artemia sinica.

During embryonic development of Artemia sinica, environmental stresses induce the embryo diapause phenomenon, required to resist apoptosis and regulate cell cycle activity. The small ubiquitin-related modifier-1 (SUMO), a reversible post-translational protein modifier, plays an important role in embryo development. SUMO regulates multiple cellular processes, including development and other biological processes. The molecular mechanism of diapause, diapause termination and the role of As-sumo-1 in this processes and in early embryo development of Artemia sinica still remains unknown. In this study, the complete cDNA sequences of the sumo-1 homolog, sumo ligase homolog, caspase-1 homolog and cyclin B homolog from Artemia sinica were cloned. The mRNA expression patterns of As-sumo-1, sumo ligase, caspase-1, cyclin B and the location of As-sumo-1 were investigated. SUMO-1, p53, Mdm2, Caspase-1, Cyclin B and Cyclin E proteins were analyzed during different developmental stages of the embryo of A. sinica. Small interfering RNA (siRNA) was used to verify the function of sumo-1 in A. sinica. The full-length cDNA of As-sumo-1 was 476 bp, encoding a 92 amino acid protein. The As-caspases-1 cDNA was 966 bp, encoding a 245 amino-acid protein. The As-sumo ligase cDNA was 1556 bp encoding, a 343 amino acid protein, and the cyclin B cDNA was 739 bp, encoding a 133 amino acid protein. The expressions of As-sumo-1, As-caspase-1 and As-cyclin B were highest at the 10 h stage of embryonic development, and As-sumo ligase showed its highest expression at 0 h. The expression of As-SUMO-1 showed no tissue or organ specificity. Western blotting showed high expression of As-SUMO-1, p53, Mdm2, Caspase-1, Cyclin B and Cyclin E at the 10 h stage. The siRNA caused abnormal development of the embryo, with increased malformation and mortality. As-SUMO-1 is a crucial regulation and modification protein resumption of embryonic diapause and early embryo development of A. sinica.

Identification of the glycerol kinase gene and its role in diapause embryo restart and early embryo development of Artemia sinica.

Glycerol kinase (GK) catalyzes the rate-limiting step in glycerol utilization by transferring a phosphate from ATP to glycerol, yielding glycerol 3-phosphate, which is an important intermediate for both energy metabolism and glycerolipid production. Artemia sinica has an unusual diapause process under stress conditions of high salinity, low temperature and lack of food. In the process, diapause embryos of A. sinica (brine shrimp) accumulate high concentrations of glycerol as a cryoprotectant to prevent low temperature damage to embryos. Upon embryo restart, glycerol is converted into glucose and other carbohydrates. Therefore, GK plays an important role in the diapause embryo restart process. However, the role of GK in diapause termination of embryo development in A. sinica remains unknown. In the present study, a 2096 bp full-length cDNA of gk from A. sinica (As-gk) was obtained, encoding putative 551 amino acids, 60.6 kDa protein. As a crucial enzyme in glycerol uptake and metabolism, GK has been conserved structurally and functionally during evolution. The expression pattern of As-gk was investigated by quantitative real-time PCR and Western blotting. Expression locations of As-gk were analyzed using in situ hybridization. As-gk was widely distributed in the early embryo and several main parts of Artemia after differentiation. The expression of As-GK was also induced by stresses such as cold exposure and high salinity. This initial research into the expression pattern and stress response of GK in Artemia provides a sound basis for further understanding of the function and regulation of genes in early embryonic development in A. sinica and the stress response.