Expression regulation of cold-inducible protein RBM3 by FAK/Src signaling for neuroprotection against rotenone under mild hypothermia.

Mild hypothermia is a well-established technique for alleviating neurological injuries in clinical surgery. RNA-binding protein motif 3 (RBM3) has been identified as a crucial factor in mediating hypothermic neuroprotection, providing its induction as a promising strategy for mimicking therapeutic hypothermia. However, little is known about molecular control of RBM3 and signaling pathways affected by hypothermia. In the present study, human SH-SY5Y neuroblastoma cells were used as a neural cell model. Screening of signaling pathways showed that cold exposure led to inactivation of ERK and AMPK pathways, and activation of FAK and PLCγ pathways, with activities of p38, JNK and AKT pathways moderately changed. Next, various small molecule inhibitors specific to these signaling pathways were applied. Interestingly, only FAK-specific inhibitor exhibited a significant inhibitory effect on hypothermia-induced RBM3 gene transcription and protein expression. Likewise, FAK silencing using siRNA technique significantly abrogated the induction of RBM3 by hypothermia. Moreover, FAK inhibition accounted for an inactivation of Src, a known kinase downstream of FAK. Next, either the silencing of Src by siRNA or its inactivation by a chemical inhibitor, strongly blocked the induction of RBM3 by cooling. Notably, in HEK293 and PC12 cells, FAK/Src activation was also shown to be indispensable for hypothermia-stimulated RBM3 expression. Lastly, the CCK8 and Western blot assays showed that both FAK/Src inacitivation and their knockdown substantially abrogate the neuroprotective effects of mild hypothermia against rotenone in SH-SY5Y cells. These data suggest that FAK/Src signaling axis regulates the transcription of Rbm3 gene and mediates neuroprotective effects of mild hypothermia.

Benzoylaconitine Inhibits Production of IL-6 and IL-8 via MAPK, Akt, NF-κB Signaling in IL-1ß-Induced Human Synovial Cells.

Benzoylaconitine (BAC), the main hydrolysate of aconitine, is a lower toxic monoester type alkaloid considered as the pharmacodynamic constituent in Aconitum species. In this study, the effects and mechanisms of BAC on production of inflammatory cytokines interleukin (IL)-6 and IL-8 were investigated in IL-1ß-stimulated human synovial SW982 cells. The SW982 cells were incubated with BAC (0, 5 and 10 µM) before stimulating with IL-1ß (10 ng/mL). The results revealed that BAC suppressed gene and protein expression of IL-6 and IL-8 induced by IL-1ß. BAC decreased activation of mitogen-activated protein kinase (MAPK) and phosphorylation of Akt. BAC also inhibited degradation of inhibitor of kappaB (IκB)-α, phosphorylation and nuclear transposition of p65 protein. The results demonstrate that BAC exerts an anti-inflammatory effect dependent on MAPK, Akt and nuclear factor-κB (NF-κB) pathways in human synovial cells stimulated with IL-1ß, suggesting that BAC may be exploited as a potential therapeutic agent for rheumatoid arthritis (RA) treatment.

Cold-inducible protein RBM3 mediates hypothermic neuroprotection against neurotoxin rotenone via inhibition on MAPK signalling.

Mild hypothermia and its key product, cold-inducible protein RBM3, possess robust neuroprotective effects against various neurotoxins. However, we previously showed that mild hypothermia fails to attenuate the neurotoxicity from MPP+ , one of typical neurotoxins related to the increasing risk of Parkinson disease (PD). To better understand the role of mild hypothermia and RBM3 in PD progression, another known PD-related neurotoxin, rotenone (ROT) was utilized in this study. Using immunoblotting, cell viability assays and TUNEL staining, we revealed that mild hypothermia (32°C) significantly reduced the apoptosis induced by ROT in human neuroblastoma SH-SY5Y cells, when compared to normothermia (37°C). Meanwhile, the overexpression of RBM3 in SH-SY5Y cells mimicked the neuroprotective effects of mild hypothermia on ROT-induced cytotoxicity. Upon ROT stimulation, MAPK signalling like p38, JNK and ERK, and AMPK and GSK-3ß signalling were activated. When RBM3 was overexpressed, only the activation of p38, JNK and ERK signalling was inhibited, leaving AMPK and GSK-3ß signalling unaffected. Similarly, mild hypothermia also inhibited the activation of MAPKs induced by ROT. Lastly, it was demonstrated that the MAPK (especially p38 and ERK) inhibition by their individual inhibitors significantly decreased the neurotoxicity of ROT in SH-SY5Y cells. In conclusion, these data demonstrate that RBM3 mediates mild hypothermia-related neuroprotection against ROT by inhibiting the MAPK signalling of p38, JNK and ERK.

Autoantigen La Regulates MicroRNA Processing from Stem-Loop Precursors by Association with DGCR8.

In humans, primary microRNA (pri-miRNA) processing starts from precise cleavage of the stem loop, which is catalyzed by the Drosha-DGCR8 complex. However, the significant inconsistencies in the expression levels among primary, precursor, and mature miRNAs clearly indicate that many other factors may be involved in this regulation. Here, we utilize a newly developed RNA affinity technique to isolate such factors. In this study, a tRNA-scaffolded aptamer (tRSA)-based RNA affinity tag, by directly fusing primary let-7 miRNA to the 3'-end of tRSA, is employed to pull down the protein components specifically binding to pri-let-7. We show that La protein binds to pri-let-7 via its La motif and significantly promotes the processing efficiency of pri-let-7 in vitro and in cells. In addition, we demonstrate that La protein is associated with DGCR8, but not Drosha, in an RNA-dependent manner. Interestingly, the RNA binding capacity of La motif is important for miRNA processing. Hence, we propose that La protein is an important microprocessor component regulating miRNA processing efficiency by association with DGCR8 to regulate formation of the DGCR8-Drosha complex for miRNA processing.

NCAM affects directional lamellipodia formation of BMSCs via ß1 integrin signal-mediated cofilin activity.

The neural cell adhesion molecule (NCAM), a key member of the immunoglobulin-like CAM family, was reported to regulate the migration of bone marrow-derived mesenchymal stem cells (BMSCs). However, the detailed cellular behaviors including lamellipodia formation in the initial step of directional migration remain largely unknown. In the present study, we reported that NCAM affects the lamellipodia formation of BMSCs. Using BMSCs from Ncam knockout mice we found that Ncam deficiency significantly impaired the migration and the directional lamellipodia formation of BMSCs. Further studies revealed that Ncam knockout decreased the activity of cofilin, an actin-cleaving protein, which was involved in directional protrusions. To explore the molecular mechanisms involved, we examined protein tyrosine phosphorylation levels in Ncam knockout BMSCs by phosphotyrosine peptide array analyses, and found that the tyrosine phosphorylation level of ß1 integrin, a protein upstream of cofilin, was greatly upregulated in Ncam-deficient BMSCs. Notably, by blocking the function of ß1 integrin with RGD peptide or ROCK inhibitor, the cofilin activity and directional lamellipodia formation of Ncam knockout BMSCs could be rescued. Finally, we found that the effect of NCAM on tyrosine phosphorylation of ß1 integrin was independent of the fibroblast growth factor receptor. These results indicated that NCAM regulates directional lamellipodia formation of BMSCs through ß1 integrin signal-mediated cofilin activity.

Hierarchical chrysanthemum-flower-like carbon nanomaterials grown by chemical vapor deposition.

Novel hierarchical chrysanthemum-flower-like carbon nanomaterials (CFL-CNMs) were synthesized by thermal chemical vapor deposition based on acetylene decomposition. A scanning electron microscope and a transmission electron microscope were employed to observe the morphology and structure of the unconventional nanostructures. It is found that the CFL-CNMs look like a blooming chrysanthemum with a stem rather than a spherical flower. The carbon flower has an average diameter of 5 µm, an average stem diameter of 150 nm, branch diameters ranging from 20 to 70 nm, and branch lengths ranging from 0.5 to 3 µm. The morphologies of the CFL-CNMs are unlike any of those previously reported. Fishbone-like carbon nanofibers with a spindle-shaped catalyst locating at the tip can also be found. Furthermore, the catalyst split was proposed to elucidate the formation mechanism of CFL-CNMs. A large and glomerate catalyst particle at the tip of the carbon nanofiber splits into smaller catalyst particles which are catalytic-active points for branch formation, resulting in the formation of CFL-CNMs.

Caveolin-1 mediates gene transfer and cytotoxicity of polyethyleneimine in mammalian cell lines.

Polyethyleneimine (PEI) is a cost-effective and non-viral vector for gene transfer, but the factors determining gene transfer efficiency and cytotoxicity of PEI in different mammalian cell lines remain largely unknown. In the present study, three different cell lines were chosen for investigation. Using pEGFP DNA and PEI, 21.5, 29.2, and 92.1 % of GFP-positive cells were obtained in BMSC, Hela, and 293T, respectively. In luciferase reporter assay, similar results were obtained (for luciferase activity, BMSC < Hela < 293T cells). By MTT test and cell apoptotic marker analysis, we demonstrated that high gene transfer efficiency is accompanied with high cytotoxicity of PEI. Moreover, we found that high expression level of caveolin-1 was accompanied with high gene transfer efficiency and cytotoxicity of PEI in 293T cells. More convincingly, caveolin-1 silencing in 293T could reduce both gene transfer efficiency and cytotoxicity of PEI. In contrast, caveolin-1 overexpression in BMSCs increases both gene transfer efficiency and cytotoxicity of PEI. Taken together, our study suggests that caveolin-1 may at least in part determine gene transfer efficiency and cytotoxicity of PEI in mammalian cell lines, providing caveolin-1 as a potential target for improving gene transfer efficiency when applying positively charged polyplexes to cell transfection.

A novel role for neural cell adhesion molecule in modulating insulin signaling and adipocyte differentiation of mouse mesenchymal stem cells.

Neural cell adhesion molecule (NCAM) has recently been found on adult stem cells, but its biological significance remains largely unknown. In this study, we used bone-marrow-derived mesenchymal stem cells (MSCs) from wild-type and NCAM knockout mice to investigate the role of NCAM in adipocyte differentiation. It was demonstrated that NCAM isoforms 180 and 140 but not NCAM-120 are expressed on almost all wild-type MSCs. Upon adipogenic induction, Ncam(-/-) MSCs exhibited a marked decrease in adipocyte differentiation compared with wild-type cells. The role of NCAM in adipocyte differentiation was also confirmed in NCAM-silenced preadipocyte 3T3-L1 cells, which also had a phenotype with reduced adipogenic potential. In addition, we found that Ncam(-/-) MSCs appeared to be insulin resistant, as shown by their impaired insulin signaling cascade, such as the activation of the insulin-IGF-1 receptor, PI3K-Akt and CREB pathways. The PI3K-Akt inhibitor, LY294002, completely blocked adipocyte differentiation of MSCs, unveiling that the reduced adipogenic potential of Ncam(-/-) MSCs is due to insulin resistance as a result of loss of NCAM function. Furthermore, insulin resistance of Ncam(-/-) MSCs was shown to be associated with induction of tumor necrosis factor α (TNF-α), a key mediator of insulin resistance. Finally, we demonstrated that re-expression of NCAM-180, but not NCAM-140, inhibits induction of TNF-α and thereby improves insulin resistance and adipogenic potential of Ncam(-/-) MSCs. Our results suggest a novel role of NCAM in promoting insulin signaling and adipocyte differentiation of adult stem cells. These findings raise the possibility of using NCAM intervention to improve insulin resistance.

Neural cell adhesion molecule potentiates the growth of murine melanoma via ß-catenin signaling by association with fibroblast growth factor receptor and glycogen synthase kinase-3ß.

The neural cell adhesion molecule (NCAM) was recently shown to be involved in the progression of various tumors with diverse effects. We previously demonstrated that NCAM potentiates the cellular invasion and metastasis of melanoma. Here we further report that the growth of melanoma is obviously retarded when the expression of NCAM is silenced. We found that the proliferation of murine B16F0 melanoma cells, their colony formation on soft agar, and growth of transplanted melanoma in vivo are clearly inhibited by the introduction of NCAM siRNA. Interestingly, change of NCAM expression level is shown to regulate the activity of Wnt signaling molecule, ß-catenin, markedly. This novel machinery requires the function of FGF receptor and glycogen synthase kinase-3ß but is independent of the Wnt receptors, MAPK-Erk and PI3K/Akt pathways. In addition, NCAM is found to form a functional complex with ß-catenin, FGF receptor, and glycogen synthase kinase-3ß. Moreover, up-regulation of NCAM140 and NCAM180 appears more potent than NCAM120 in activation of ß-catenin, suggesting that the intracellular domain of NCAM is required for facilitating the ß-catenin signaling. Furthermore, the melanoma cells also exhibit distinct differentiation phenotypes with the NCAM silencing. Our findings reveal a novel regulatory role of NCAM in the progression of melanoma that might serve as a new therapeutic target for the treatment of melanoma.

Clonal analysis for elucidating the lineage potential of embryonic NG2+ cells.

BACKGROUND AIMS: The widespread NG2-expressing neural progenitors in the central nervous system (CNS) are considered to be multifunctional cells with lineage plasticity, thereby possessing the potential for treating CNS diseases. Their lineages and functional characteristics have not been completely unraveled. The present study aimed to disclose the lineage potential of clonal NG2(+) populations in vitro and in vivo. METHODS: Twenty-four clones from embryonic cerebral cortex-derived NG2(+) cells were induced for oligodendrocyte, astrocyte, neuronal and chondrocyte differentiation. The expression profiles of neural progenitor markers chondroitin sulfate proteoglycan 4 (NG2), platelet-derived growth factor-α receptor (PDGFαR); nestin and neuronal cell surface antigen (A2B5) were subsequently sorted on cells with distinct differentiation capacity. Transplantation of these NG2(+) clones into the spinal cord was used to examine their lineage potential in vivo. RESULTS: In vitro differentiation analysis revealed that all the clones could differentiate into oligodendrocytes, and seven of them were bipotent (oligodendrocytes and astrocytes). Amazingly, one clone exhibited a multipotent capacity of differentiating into not only neuronal-glial lineages but also chondrocytes. These distinct subtypes were further found to exhibit phenotypic heterogeneity based on the examination of a spectrum of neural progenitor markers. Transplanted clones survived, migrated extensively and differentiated into oligodendrocytes, astrocytes or even neurons to integrate with the host spinal cord environment. CONCLUSIONS: These results suggest that NG2(+) cells contain heterogeneous progenitors with distinct differentiation capacities, and the immortalized clonal NG2(+) cell lines might provide a cell source for treating spinal cord disorders.

[Corrigendum] Transforming growth factor­ß1 induces fibrosis in rat meningeal mesothelial cells via the p38 signaling pathway.

Subsequently to the publication of this paper, while performing a careful re­examination of the scientific integrity of the data included in their publications, the authors have realized that they inadvertently used the incorrect western blotting images in Fig. 2B of this article, However, still having access to their original data, the authors were able to reassemble Fig. 2 correctly, and the corrected version of this figure is shown below. Note that this error did not significantly affect the results or the conclusions reported in this paper, and all the authors agree to this Corrigendum. The authors thank the Editor of Molecular Medicine Reports for granting them the opportunity to publish this corrigendum, and apologize to the readership for any inconvenience caused. [the original article was published on Molecular Medicine Reports 14: 1709­1713, 2016; DOI: 10.3892/mmr.2016.5411].

Neural Cell Adhesion Molecule Regulates Osteoblastic Differentiation Through Wnt/ß-Catenin and PI3K-Akt Signaling Pathways in MC3T3-E1 Cells.

Neural cell adhesion molecule (NCAM) is involved in cell multi-directional differentiation, but its role in osteoblast differentiation is still poorly understood. In the present study, we investigated whether and how NCAM regulates osteoblastic differentiation. We found that NCAM silencing inhibited osteoblast differentiation in pre-osteoblastic MC3T3-E1 cells. The function of NCAM was further confirmed in NCAM-deficient mesenchymal stem cells (MSCs), which also had a phenotype with reduced osteoblastic potential. Moreover, NCAM silencing induced decrease of Wnt/ß-catenin and Akt activation. The Wnt inhibitor blocked osteoblast differentiation, and the Wnt activator recovered osteoblast differentiation in NCAM-silenced MC3T3-E1 cells. We lastly demonstrated that osteoblast differentiation of MC3T3-E1 cells was inhibited by the PI3K-Akt inhibitor. In conclusion, these results demonstrate that NCAM silencing inhibited osteoblastic differentiation through inactivation of Wnt/ß-catenin and PI3K-Akt signaling pathways.

Insulin-like growth factor 1 protects human neuroblastoma cells SH-EP1 against MPP+-induced apoptosis by AKT/GSK-3ß/JNK signaling.

Parkinson's disease (PD) is primarily caused by severe degeneration and loss of dopamine neurons in the substantia nigra pars compacta. Thus, preventing the death of dopaminergic neurons is thought to be a potential strategy to interfere with the development of PD. In the present work, we studied the effect of insulin-like growth factor-1 (IGF-1) on 1-methyl-4-phenylpyridinium (MPP+)-induced apoptosis in human neuroblastoma SH-EP1 cells. We found that the PI3K/AKT pathway plays a central role in IGF-mediated cell survival against MPP+ neurotoxicity. Furthermore, we demonstrated that the protective effect of AKT is largely dependent on the inactivation of GSK-3ß, since inhibition of GSK-3ß by its inhibitor, BIO, could mimic the protective effect of IGF-1 on MPP+-induced cell death in SH-EP1 cells. Interestingly, the IGF-1 potentiated PI3K/AKT activity is found to negatively regulate the JNK related apoptotic pathway and this negative regulation is further shown to be mediated by AKT-dependent GSK-3ß inactivation. Thus, our results demonstrated that IGF-1 protects SH-EP1 cells from MPP+-induced apoptotic cell death via PI3K/AKT/GSK-3ß pathway, which in turn inhibits MPP+-induced JNK activation.

Cloning and characterization of three novel WSSV recognizing lectins from shrimp Marsupenaeus japonicus.

C-type lectins (CTLs) acting as pattern recognition receptors play essential roles in shrimp innate immune responses. Using WSSV envelope proteins (VP26, VP28, and VP281) to screen a phage display library of Marsupenaeus japonicus, three lectins (termed as MjLecA, MjLecB, and MjLecC) were found to interact with WSSV. Sequence analysis revealed that these MjLecs shared low similarities with each other. Phylogenetic analysis indicated MjLecA and MjLecB are likely to belong to the same lectin sub-family, while MjLecC belongs to another sub-family. These MjLecs showed broad, unique carbohydrate binding spectra. Also, the three MjLecs could interact with several envelope proteins of WSSV and could recognize a wide range of microorganisms. Moreover, binding of MjLecA or MjLecB to WSSV reduced the viral infection rate in vitro. These results suggest that various kinds of CTLs with structural and functional diversities may constitute a recognizing network against invading pathogens such as bacteria and virus, and play essential roles in the defence system of shrimp.

Neural cell adhesion molecule regulates chondrocyte hypertrophy in chondrogenic differentiation and experimental osteoarthritis.

Chondrocyte hypertrophy-like change is an important pathological process of osteoarthritis (OA), but the mechanism remains largely unknown. Neural cell adhesion molecule (NCAM) is highly expressed and involved in the chondrocyte differentiation of mesenchymal stem cells (MSCs). In this study, we found that NCAM deficiency accelerates chondrocyte hypertrophy in articular cartilage and growth plate of OA mice. NCAM deficiency leads to hypertrophic chondrocyte differentiation in both murine MSCs and chondrogenic cells, in which extracellular signal-regulated kinase (ERK) signaling plays an important role. Moreover, NCAM expression is downregulated in an interleukin-1ß-stimulated OA cellular model and monosodium iodoacetate-induced OA rats. Overexpression of NCAM substantially inhibits hypertrophic differentiation in the OA cellular model. In conclusion, NCAM could inhibit hypertrophic chondrocyte differentiation of MSCs by inhibiting ERK signaling and reduce chondrocyte hypertrophy in experimental OA model, suggesting the potential utility of NCAM as a novel therapeutic target for alleviating chondrocyte hypertrophy of OA.

Helicobacter pylori promotes hepatic fibrosis in the animal model.

Helicobacter pylori infection has been reported to be very common in patients with chronic liver diseases, including cirrhosis. To elucidate the pathological effect of H. pylori infection on the progression of hepatic fibrosis, C57BL/6 mice and Sprague-Dawley rats were orally inoculated with H. pylori, and hepatic fibrosis was induced with carbon tetrachloride (CCl(4)) administration. We observed the histopathological changes and the presence of H. pylori genes by PCR in the liver. Significant increase in the fibrotic score as well as in serum alanine aminotransferase and aspartate aminotransferase levels was shown in the CCl(4)+H. pylori group compared with that in the CCl(4)-treated group. Compared with the CCl(4)-treated group, alpha-smooth muscle actin and transforming growth factor-beta1 were enhanced; however, senescence marker protein-30, a multifunctional protein protecting hepatocytes against oxidative stress and apoptosis, was suppressed in the CCl(4)+H. pylori group. The 16S rRNA (400 bp) was demonstrated by PCR for H. pylori genes from genomic DNA extracted from the liver, and H. pylori-infected mice showed 93.8% (15 of 16) seropositivity by contrast with seronegativity in all H. pylori-noninfected mice. In addition, immunohistochemical study against H. pylori showed positive antigen fragments in the liver of the infected groups. Consequently, our data suggest that H. pylori infection could be an important contributing infectious factor to the development of liver cirrhosis.

Multiple urogenital abnormalities in a Persian cat.

A 1.5-year-old female Persian cat was presented for inappetence and azotemia. Ultrasonography and urography revealed multiple abnormalities involving the genitourinary tract, including agenesis of the right kidney and ureter. Gross examination of the abnormal uterus revealed segmental aplasia of right caudal uterine horn causing cranial distension with fluid, a normal left uterine horn, and both normal ovaries. Microscopically, endometrial glands of the right uterine horn were markedly decreased in number. The right uterine horn was hemorrhagic suggesting estrus. This is the first report of this combination of urinary and uterus abnormalities in the veterinary literature.

Mild Hypothermia Prevents NO-Induced Cytotoxicity in Human Neuroblastoma Cells Via Induction of COX-2.

The cold-inducible protein RBM3 mediates hypothermic neuroprotection against nitric oxide (NO)-induced cell death. Meanwhile, it is well-known that cyclooxygenase-2 (COX-2) is upregulated by RBM3 in several types of cells; however, it is still unclear whether COX-2 contributes to the neuroprotective effects of mild hypothermia/RBM3 against NO-induced cell death. Using human SH-SY5Y neuroblastoma cells, it was revealed that NO remarkably downregulates the expression of COX-2 at both mRNA and protein levels. When COX-2 was silenced using siRNA technique, cells became more sensitive to NO-induced cell death. Conversely, the overexpression of COX-2 significantly prevented NO-induced cell death in SH-SY5Y cells, indicating a pro-survival role of COX-2. Upon mild hypothermia pre-treatment, COX-2 was notably induced at both mRNA and protein levels; however, COX-2 silencing abrogated hypothermia-related neuroprotection against NO-induced cell death. Furthermore, it was revealed that either silencing or overexpression of RBM3 had no effects on the expression of COX-2 in SH-SY5Y cells. These findings suggest that mild hypothermia could protect neuroblastoma cells against NO-induced cell death by inducing COX-2 in a RBM3-independent manner.

Cytotoxic effects of the conjugated linoleic acid isomers t10c12, c9t11-CLA and mixed form on rat hepatic stellate cells and CCl4-induced hepatic fibrosis.

Rat hepatic stellate cells (HSC-T6) were incubated for 24 h with 10-180 microM of t10c12 (98%), c9t11 (96%) and a mixed form (c9,t11:t10,c12; 41%:44%) of conjugated linoleic acid (CLA). The MTS dye reduction was measured to verify cell viability in a dose-dependent manner. Among the three CLAs, c9,t11-CLA exhibited the most intense cytotoxic effect on HSCs, the survival rate of which was reduced to 60% under 80 microM of treatment, while cell survival was slightly affected by the mixed form. Three CLA-induced cell deaths were determined by measuring DNA fragmentation using 4',6-diamidino-2-phenylindole staining. The degrees of DNA fragmentation were the most severe in HSC treated with 80 microM of c9,t11-CLA. The mitogen-activated protein kinase/extracellular signal-regulated kinase-kinase and mitogen-activated or extracellular signal-regulated protein kinase (MEK) 1 and 2 were not activated in the t10,c12-CLA treatment. This suggests that the MEK-dependent apoptosis signal is crucial in HSC, which is induced by c9,t11 and mixed CLA. In order to evaluate the protective effect of CLA on carbon tetrachloride (CCl4)-induced hepatic fibrosis in vivo, animals were treated with 10% CCl4 to induce hepatic fibrosis during all experimental periods. Rats were divided into two treatment groups: (1) control diet with tap water ad libitum (n=15) and (2) 1% CLA diet with tap water ad libitum (n=15). In the CLA-supplemented rat livers, alpha-smooth muscle actin-positive cells were significantly reduced around the portal vein. In addition, collagen fibers were not detected in the CLA-treated group. These results suggest that 9c,11t-CLA influences cytotoxic effect on HSC in an MEK-dependent manner and preserving liver from fibrosis.

Hepatoprotective effect of Arazyme on CCl4-induced acute hepatic injury in SMP30 knock-out mice.

Arazyme is a novel protease produced by the HY-3 strain of Aranicola proteolyticus, which is a Gram-negative aerobic bacterium that has been isolated from the intestine of the spider Nephila clavata. This study focused on the hepatoprotective effect of Arazyme on carbon tetrachloride (CCl4)-induced acute hepatic injury in senescence marker protein 30 (SMP30) knock-out (KO) mice and SMP30 wild-type (WT) mice. WT mice and SMP30 KO mice were divided into eight groups as follows: (i) two negative control groups (G1, G5) which were treated with a single intraperitoneal (i.p.) olive oil injection. (ii) Two positive control groups (G2, G6) which received a single i.p. CCl4 (0.4mL/kg) injection. (iii) Two vitamin C-treated groups (G3, G7) which received a single oral administration of vitamin C (100mg/kg) and were injected with a single i.p. CCl4 (0.4mL/kg). (iv) Two Arazyme-treated groups (G4, G8) which received a single oral administration of Arazyme (500mg/kg) and were injected with a single i.p. CCl4 (0.4mL/kg). Through present study, we could find that Arazyme-treated groups showed decreased degree of liver injury, increased expression of SMP30, decreased expression of phospho-Smad3 (p-Smad3), elevated expression of antioxidant proteins including sorbitol dehydrogenase, dihydropteridine reductase (DHPR), dehydrofolate reductase (DHFR), NADH dehydrogenase, glutathione S-transferase kappa 1 (GSTK1) and phospholipid hydroperoxide glutathione peroxidase (PHGPx) compared with non-Arazyme-treated groups. Therefore, it is concluded that Arazyme plays a significant role in protecting injured hepatocytes by increasing the expression of SMP30, inhibiting the transforming growth factor-beta (TGF-beta)/Smad pathway and elevating the expression of antioxidant proteins.