Wip1 phosphatase involved in lipopolysaccharide-induced neuroinflammation.

Wild type p53-induced phosphatase 1 (Wip1) is a phosphatase which belongs to protein phosphatase type 2C family, which have been predominantly linked to cell growth and to cellular stress signaling. Numerous downstream targets of Wip1 have been identified, and genetic studies confirm that some play a part in tumorigenesis. Recent evidence highlights a new role for Wip1 in the regulation of NF-κB p65, which indicated that it might play a critical role in immune system. However, its regulation role in central nervous system (CNS) remains poorly understood. To elaborate whether Wip1 was involved in CNS injury, we performed a neuroinflammatory model by lipopolysaccharide (LPS) lateral-ventral injection in adult rats.Wip1 expression was strongly upregulated in active astrocytes in inflamed brain cortex. In vitro studies indicated that the upregulation of Wip1 may be involved in the subsequent astrocytic activation following LPS exposure, and knockdown of Wip1 in primary astrocytes by siRNA showed that Wip1 inhibited the synthesis of TNF-α. Collectively, these results suggested that Wip1 may be important in host defense in CNS immune response, which might provide a potent therapeutic target of neuroinflammation.

Increased expression of actin filament-stabilizing protein tropomyosin after rat traumatic brain injury.

Tropomyosin (TM), is a coiled-coil dimmer which modulates actin filament properties, has been implicated in the control of actin filament dynamics during cell migration, morphogenesis, and cytokinesis. However, the expressions and possible functions of tropomyosin in central nervous system (CNS) lesion remain unknown. In this study, we found the expression of tropomyosin gradually increased in rat brains subjected to traumatic brain injury (TBI). Double immunofluorescence staining showed tropomyosin was expressed in neurons and reactive astrocytes following TBI but not in quiescent astrocytes in normal brains. Furthermore, we detected that proliferating cell nuclear antigen (PCNA) had the co-localization with GFAP, and tropomyosin. In conclusion, this was the first description of tropomyosin expression in rat traumatic brain. Our date suggested that tropomyosin might be involved in the astrocytes proliferation following TBI.

[Ginkgolides induced ischemic tolerance and its possible molecular mechanism: experiment with rat pheochromocytoma cell line PC12].

OBJECTIVE: To explore the ischemic tolerance induced by Ginkgolides in PC12 cells and its possible molecular mechanism. METHODS: An ischemic model was developed in PC12 cell line with deprivation of oxygen-glucose (OGD). PC12 cells was randomly divided into four groups: 9 hours ischemia group, 1.5 hours ischemic preconditioning + 9 hours ischemia group, Ginkgolides preconditioning + 9 hours ischemia group and control group. Cells viability was examined by MTT assay and cellular morphology was analyzed under the phase-contrast microscope. The molecular mechanism of Ginkgolides induced ischemic tolerance was pinpointedby analyzing the expression of hypoxia-inducible factor-1 alpha (HIF-1alpha) and erythropoietin (EPO). The DNA binding activities of HIF-1 in PC12 cells were examined by electrophoretic mobility shift assay. RESULTS: In ischemic model, the viability of PC12 cells was decreased (49.3 +/- 2.8)% after OGD for 9 hours. However, Ginkgolides pretreatment could remarkably increase the viability of PC12 cells (65.9 +/- 2.8)% (P < 0.01). Pretreatment of Ginkgolides for 24 hours could largely rescue the morphology of PC12 cells to the damage of subsequent exposure to 9 hours ischemia insult, many cellular bodies were intact and many neurites and network of PC12 cells were still exist. At molecular level, the expression of HIF-1alpha was greatly induced by Ginkgolides treatment after compared with the control group (P < 0.01). The DNA binding activities of HIF-1 in PC12 cells pretreated with Ginkgolides was also increased. And it activates its downstream target EPO, the protein expression (P < 0.01). CONCLUSION: The pretreatment of Ginkgolides could induce tolerance against ischemia in PC12 cells. The molecular mechanism of this process may involve in the activation of HIF-1alpha and the DNA binding activity of HIF-1 and its downstream target EPO.

[Acanthopanax Senticosus Saponins induced tolerance to ischemia and its possible molecular mechanism in PC12 cells].

OBJECTIVE: To study the tolerance to ischemia induced by Acanthopanax Senticosus Saponins (ASE) in PC12 cells and the involved mechanism. METHODS: An ischemic model was developed in PC12 cell line by treatment with oxygen-glucose deprivation. The effects of ASE pretreatment on tolerance of PC12 cells to ischemia were evaluated by MTT assay and analysis of cellular morphology. The expression of hypoxia-inducing factor (HIF)-1alpha, erythropoietin (EPO) after the pretreatment with ASE was detected by Western blotting. The DNA binding activities of HIF-1 in PC12 cells with the pretreatment of ASE were demonstrated by using electrophoretic mobility shift assay (EMSA). RESULTS: In ischemia model, the viability of PC12 cells was decreased to (49.12 +/- 3.22)% after oxygen-glucose deprivation for 9 hours. However, ASE (50 microg/ml) pretreatment could remarkably increase the viability of PC12 cells by (67.97 +/- 2.92)%. There were significant differences between the experimental group and control group (F = 473.67, P < 0.01). The cellular morphology showed that PC12 cells exposed for 7 days to nerve growth factor (NGF) exhibited round, smooth cell bodies with normal processes and that processes formed extensive network. At 9 hour after ischemia, cell bodies of many PC12 cells were found shrinken, the processes were disrupted and network disappeared. However, pretreatment with ASE (50 microg/ml) could largely prevent the morphological damage to PC12 cells that would have caused by subsequent exposure to 9 h ischemic insult, many cellular bodies were intact and many processes and network of PC12 cells still existed. The expression of HIF-1alpha increased after pretreatment with ASE shown by Western blot. There were significant differences between the experimental group and control group (F = 167.18, P < 0.01). The DNA binding activities of HIF-1 in PC12 cells after pretreatment with ASE was significantly increased, and it could activate the expression of EPO (F = 128.37, P < 0.01). CONCLUSIONS: The pretreatment with ASE could induce tolerance against ischemia in PC12 cells. The elevated expression and increased DNA binding activity of HIF-1alpha, the overexpression of its downstream target EPO may be the molecular mechanism in tolerance of PC12 cells to ischemia induced by ASE pretreatment.