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Traumatic brain injury (TBI) is one of the leading causes of neurological disability in young adults. Edaravone, a novel synthetic small-molecule free-radical scavenger, has been shown to have a neuroprotective effect in both animal models of cerebral ischemia and stroke patients; however, the underlying mechanism is poorly understood. In this report, we investigated the potential mechanisms of edaravone treatment in a rat model of TBI. TBI was induced in the right cerebral cortex of male adult rats using Feeney's weight-drop method. Edaravone (0.75, 1.5, or 3 mg/kg) or vehicle (normal saline) was intravenously administered at 2 and 12 h after TBI. Edaravone treatment significantly decreased hippocampal CA3 neuron loss, reduced oxidative stress, and decreased neuronal programmed cell death compared to vehicle treatment. The protective effects of edaravone treatment were also related to the pathology of TBI on non-neuronal cells, as edaravone decreased astrocyte and glial activation. Lastly, edaravone treatment significantly reduced the presence of inflammatory cytokines, cerebral edema, blood–brain barrier (BBB) permeability, and, importantly, neurological deficits following TBI. Our results suggest that edaravone exerts a neuroprotective effect in the rat model of TBI. The likely mechanism is via inhibiting oxidative stress, leading to a decreased inflammatory response and glial activation, and thereby reducing neuronal death and improving neurological function.

The effects of melatonin on the mitochondrial DNA (mtDNA) damage induced by 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) and 1-methyl-4-phenylpyridine ion (MPP(+)) were investigated both in vivo and in vitro. MPTP (24 mg/kg, s.c.) induced a rapid increase in the immunoreactivity of 8-hydroxyguanine (8-oxoG), a common biomarker of DNA oxidative damage, in the cytoplasm of neurons in the Substantia Nigra Compact of mouse brain. Melatonin preinjection (7.5, 15 or 30 mg/kg, i.p.) dose-dependently prevented MPTP-induced DNA oxidative damage. In SH-SY5Y cells, MPP(+) (1 mm) increased the immunoreactivity of 8-oxoG in the mitochondria at 1 hr and in the nucleus at 3 hr after treatment. Melatonin (200 microm) preincubation significantly attenuated MPP(+)-induced mtDNA oxidative damage. Furthermore, MPP(+) time-dependently increased the accumulation of mitochondrial oxygen free radicals (mtOFR) from 1 to 24 hr and gradually decreased the mitochondrial membrane potential (Psim) from 18 to 36 hr after incubation. At 72 hr after incubation, MPP(+) caused cell death in 49% of the control. However, melatonin prevented MPP(+)-induced mtOFR generation and Psim collapse, and later cell death. The present results suggest that cytoprotection of melatonin against MPTP/MPP(+)-induced cell death may be associated with the attenuation of mtDNA oxidative damage via inhibition of mtOFR generation and the prevention of Psim collapse.

Objectives: Hepatocellular carcinoma (HCC) is one of the most prevalent malignancies in China and, due to the limited efficacy of currently available therapies, is responsible for a large number of deaths. IFN-α therapy has shown promise in the treatment of various forms of human cancer and is considered in the treatment of HCC. Previous results from our group showed that high doses of IFN-α exert a significant antiproliferative effect on MHCC97 human xenografts in nude mice, but not on MHCC97 cells when tested in vitro. Here we present experiments designed to characterize the molecular mechanism underlying the defective response of MHCC97 cells to IFN-α. Elucidation of the mechanism underlying the defective response of MHCC97 to IFN-α may help to explain and possibly to overcome clinical failures of this form of tumor therapy. Methods: IFN-α2a was administered between 3,000 and 10,000 IU/ml, a range strongly inhibiting proliferation in other cell lines. Gene expression profiles of MHCC97 cells were obtained before and after treatment with IFN-α2a using cDNA microarray analysis. The transcriptional activity of relevant genes responding to IFN-α2a in the cDNA microarray experiments was confirmed by RT-PCR and Northern blot analysis. Transient transfection with an expression vector was used to restore p48-ISGFγ (IRF9) protein levels. Cell proliferation was evaluated using the MTT assay. Results: Although IFN-α treatment caused the activation of several signal transduction pathways in MHCC97 cells, the lack of an antiproliferative effect was found to mainly derive from a defect in the activation of the transcription factor ISGF3 required for Jak/STATS signaling. We show that the defect in ISGF3 activation is mainly caused by the absence of one of its essential components, the protein p48-ISGFγ from MHCC97 cells. Indeed, transient expression of p48-ISGFγ restores sensitivity to IFN-α2a. Although the mRNA levels of p48-ISGFγ were normal in MHCC97 cells, mutations could be detected in the gene coding for the protein. We hypothesize, therefore, that these mutations alter the message or protein stability, leading to the reduced protein levels observed. Conclusion: Our results confirm the important role of Jak/STATS signaling in the antiproliferative effects of IFN-α in tumor cells and indicate that defects in ISGF3 can cause resistance to IFN-α2a treatment.

To elucidate the roles of JAK/STATs signal pathway on anti-proliferative effects induced by IFN-alpha in MHCC97.An IRF9 expression vector was transfected into MHCC97 with Dosper. The expression of IRF9, cycle regulating proteins and the forming of ISGF3 complex were detected using Western blot and EMSA, respectively. Cell proliferation and distribution were monitored using MTT and flow cytometry.High expression of IRF9 restored the anti-proliferative response of MHCC97 on IFN-alpha treatment and delayed the cell transition from S phase to G2 phase induced by IFN-alpha.The integrity and functions of JAK/STATs signal pathway played an important role in mediating the anti-proliferative effects of IFN-alpha in MHCC97.

To detect the resistin expression of white adipose tissue in diet-induced obese (DIO) versus diet-resistant (DR) rats, and to investigate the relationship of mutated resistin and 3T3-L1 preadipocytes differentiation.RT-PCR and Western Blot were used to detect gene /protein expression. 3T3-L1 cells were cultured, transfected, and induced to differentiation using 0.5 mmol/L 3-isobutyl-1-methylxanthine (MIX), 1 mg/L insulin, and 1 micromol/L dexamethasone. Oil red O staining was applied to detect the degree of preadipocytes differentiation.Expression of resistin mRNA was upregulated in DIO rats and downregulated in DR rats. However, the expression levels varied greatly within the groups. Sequencing of the resistin genes from DIO and DR rats revealed a Leu9Val (C25G) missense mutation within the signal peptide in one DR rat. The mutant resistin inhibited preadipocyte differentiation. Local experiments and Western blotting with tagged resistin fusion proteins identified both mutant and wild type proteins in the cytoplasm and secreted into the culture medium. Computer predictions using the Proscan and Subloc programs revealed four putative phosphorylation sites and a possible leucine zipper motif within the rat resistin protein.Resistin-increased differentiation may be inhibited by the mutation-containing precursor protein, or by the mutant non-secretory resistin isoform.

This paper was designed in middle cerebral artery occlusion (MCAO) model of rats, to explore the role of transient receptor potential channel 4 (TRPC4) as Ca(2+) selective channel by detecting the changes of the expression of TRPC4 in different parts of cerebral tissues under the condition of focal cerebral ischemia. The rats were sacrificed after MCAO surviving time 6 h, 12 h, 1 d, 3 d. As determined by Western blot, the expressions of TRPC4 in striatum and hippocampus of 12 h, 1 d, 3 d groups were significant higher than that in the control group (P0.05). Immunohistochemical staining showed that the TRPC4 immunoreactive substances were present in the membrane of neurons. Compared with the control group, immunostaining positive cells increased in hippocampus and striatum of cerebral ischemia groups. The TRPC4 immunostaining positive cells increased significantly in 1d-group and 3d-group (P0.05). It suggests that as a Ca(2+) selective channel, the variance of the expression of TRPC4 may play a role in acute and delayed neuronal injury in focal cerebral ischemia.

Hepatocellular carcinoma (HCC) is one of the most prevalent malignancies in China and, due to the limited efficacy of currently available therapies, is responsible for a large number of deaths. IFN-alpha therapy has shown promise in the treatment of various forms of human cancer and is considered in the treatment of HCC. Previous results from our group showed that high doses of IFN-alpha exert a significant antiproliferative effect on MHCC97 human xenografts in nude mice, but not on MHCC97 cells when tested in vitro. Here we present experiments designed to characterize the molecular mechanism underlying the defective response of MHCC97 cells to IFN-alpha. Elucidation of the mechanism underlying the defective response of MHCC97 to IFN-alpha may help to explain and possibly to overcome clinical failures of this form of tumor therapy.IFN-alpha(2a) was administered between 3,000 and 10,000 IU/ml, a range strongly inhibiting proliferation in other cell lines. Gene expression profiles of MHCC97 cells were obtained before and after treatment with IFN-alpha(2a) using cDNA microarray analysis. The transcriptional activity of relevant genes responding to IFN-alpha(2a) in the cDNA microarray experiments was confirmed by RT-PCR and Northern blot analysis. Transient transfection with an expression vector was used to restore p48-ISGFgamma (IRF9) protein levels. Cell proliferation was evaluated using the MTT assay.Although IFN-alpha treatment caused the activation of several signal transduction pathways in MHCC97 cells, the lack of an antiproliferative effect was found to mainly derive from a defect in the activation of the transcription factor ISGF3 required for Jak/STATS signaling. We show that the defect in ISGF3 activation is mainly caused by the absence of one of its essential components, the protein p48-ISGFgamma from MHCC97 cells. Indeed, transient expression of p48-ISGFgamma restores sensitivity to IFN-alpha(2a). Although the mRNA levels of p48-ISGFgamma were normal in MHCC97 cells, mutations could be detected in the gene coding for the protein. We hypothesize, therefore, that these mutations alter the message or protein stability, leading to the reduced protein levels observed.Our results confirm the important role of Jak/STATS signaling in the antiproliferative effects of IFN-alpha in tumor cells and indicate that defects in ISGF3 can cause resistance to IFN-alpha(2a) treatment.