Deficiency of leucine-rich repeat kinase 2 aggravates thioacetamide-induced acute liver failure and hepatic encephalopathy in mice.

BACKGROUND: Hepatic encephalopathy (HE) is closely associated with inflammatory responses. However, as a crucial regulator of the immune and inflammatory responses, the role of leucine-rich repeat kinase 2 (LRRK2) in the pathogenesis of HE remains unraveled. Herein, we investigated this issue in thioacetamide (TAA)-induced HE following acute liver failure (ALF). METHODS: TAA-induced HE mouse models of LRRK2 wild type (WT), LRRK2 G2019S mutation (Lrrk2G2019S) and LRRK2 knockout (Lrrk2-/-) were established. A battery of neurobehavioral experiments was conducted. The biochemical indexes and pro-inflammatory cytokines were detected. The prefrontal cortex (PFC), striatum (STR), hippocampus (HIP), and liver were examined by pathology and electron microscopy. The changes of autophagy-lysosomal pathway and activity of critical Rab GTPases were analyzed. RESULTS: The Lrrk2-/--HE model reported a significantly lower survival rate than the other two models (24% vs. 48%, respectively, p < 0.05), with no difference found between the WT-HE and Lrrk2G2019S-HE groups. Compared with the other groups, after the TAA injection, the Lrrk2-/- group displayed a significant increase in ammonium and pro-inflammatory cytokines, aggravated hepatic inflammation/necrosis, decreased autophagy, and abnormal phosphorylation of lysosomal Rab10. All three models reported microglial activation, neuronal loss, disordered vesicle transmission, and damaged myelin structure. The Lrrk2-/--HE mice presented no severer neuronal injury than the other genotypes. CONCLUSIONS: LRRK2 deficiency may exacerbate TAA-induced ALF and HE in mice, in which inflammatory response is evident in the brain and aggravated in the liver. These novel findings indicate a need of sufficient clinical awareness of the adverse effects of LRRK2 inhibitors on the liver.

Adipocyte-Derived Leptin Promotes PAI-1 -Mediated Breast Cancer Metastasis in a STAT3/miR-34a Dependent Manner.

The crosstalk between cancer cells and adipocytes is critical for breast cancer progression. However, the molecular mechanisms underlying these interactions have not been fully characterized. In the present study, plasminogen activator inhibitor-1 (PAI-1) was found to be a critical effector of the metastatic behavior of breast cancer cells upon adipocyte coculture. Loss-of-function studies indicated that silencing PAI-1 suppressed cancer cell migration. Furthermore, we found that PAI-1 was closely related to the epithelial-mesenchymal transition (EMT) process in breast cancer patients. A loss-of-function study and a mammary orthotopic implantation metastasis model showed that PAI-1 promoted breast cancer metastasis by affecting the EMT process. In addition, we revealed that leptin/OBR mediated the regulation of PAI-1 through the interactions between adipocytes and breast cancer cells. Mechanistically, we elucidated that leptin/OBR further activated STAT3 to promote PAI-1 expression via miR-34a-dependent and miR-34a-independent mechanisms in breast cancer cells. In conclusion, our study suggests that targeting PAI-1 and interfering with its upstream regulators may benefit breast cancer patients.

Endoplasmic Reticulum Stress Induces the Early Appearance of Pro-apoptotic and Anti-apoptotic Proteins in Neurons of Five Familial Alzheimer's Disease Mice.

BACKGROUND: Amyloid ß (Aß) deposits and the endoplasmic reticulum stress (ERS) are both well established in the development and progression of Alzheimer's disease (AD). However, the mechanism and role of Aß-induced ERS in AD-associated pathological progression remain to be elucidated. METHODS: The five familial AD (5×FAD) mice and wild-type (WT) mice aged 2, 7, and 12 months were used in the present study. Morris water maze test was used to evaluate their cognitive performance. Immunofluorescence and Western blot analyses were used to examine the dynamic changes of pro-apoptotic (CCAAT/enhancer-binding protein homologous protein [CHOP] and cleaved caspase-12) and anti-apoptotic factors (chaperone glucose-regulated protein [GRP] 78 and endoplasmic reticulum-associated protein degradation-associated ubiquitin ligase synovial apoptosis inhibitor 1 [SYVN1]) in the ERS-associated unfolded protein response (UPR) pathway. RESULTS: Compared with age-matched WT mice, 5×FAD mice showed higher cleaved caspase-3, lower neuron-positive staining at the age of 12 months, but earlier cognitive deficit at the age of 7 months (all P < 0.05). Interestingly, for 2-month-old 5×FAD mice, the related proteins involved in the ERS-associated UPR pathway, including CHOP, cleaved caspase-12, GRP 78, and SYVN1, were significantly increased when compared with those in age-matched WT mice (all P < 0.05). Moreover, ERS occurred mainly in neurons, not in astrocytes. CONCLUSIONS: These findings suggest that compared with those of age-matched WT mice, ERS-associated pro-apoptotic and anti-apoptotic proteins are upregulated in 2-month-old 5×FAD mice, consistent with intracellular Aß aggregation in neurons.

Tripchlorolide ameliorates experimental autoimmune encephalomyelitis by down-regulating ERK1/2-NF-κB and JAK/STAT signaling pathways.

Tripchlorolide (T4), an extract of the natural herb Tripterygium wilfordii Hook F, has been found to possess anti-inflammatory and immunosuppressive actions. In the current study, these actions were evaluated in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis by scoring the clinical signs, observing the infiltration of inflammatory cells and myelin sheath in the lumbar spinal cord of EAE mice. The results demonstrated that T4 (at a dose of 40 µg/kg) significantly reduced the severity of EAE and slowed down the ongoing EAE. Further analysis showed that T4 suppressed the mRNA and protein levels of the transcription factors T-bet and RoRrt and mRNA levels of IFN-γ and IL-17 in the spinal cords. Furthermore, T4 down-regulated the ERK1/2-NF-κB and JAK/STAT signaling pathways. At 40 µg/kg, T4 did not induce side effects on hematological parameters. These findings suggest that T4 ameliorates EAE by immunosuppression, providing a new insight into T4 application in multiple sclerosis treatment.

[JNK/p38 MAPK involves in ginsenoside Rb1 attenuating beta-amyloid peptide (25-35) -induced tau protein hyperphosphorylation in embryo rat cortical neurons].

To explore the effect of ginsenoside Rb1 on JNK/p38 MAPK in the process of beta-amyloid peptide (25-35) -induced tau protein hyperphosphorylation, Western blotting and immunocytochemical stain were performed to observe the tau protein phosphorylation and the expression of JNK/p38 MAPK. The level of tau protein phosphorylation in the sites of Ser396 , Ser199/202 and Thr205 increased after rat cortical neurons exposed to 20 micromol x L(-1) Abeta25-35, meanwhile the level of JNK/p38 MAPK also increased after Abeta25-35 treatment for 12 h. Pretreatment with several doses of ginsenoside Rbl markedly attenuated tau protein hyperphosphorylation and the expression of JNK/p38 MAPK. Ginsenoside Rbl markedly attenuated tau protein hyperphosphorylation through JNK/p38 MAPK pathway.

[Inhibitory action of propyl gallate on the activation of SAPK/JNK and p38MAPK induced by cerebral ischemia-reperfusion in rats].

AIM: To explore the protective effect of propyl gallate against neuronal injury in the boundary zone of the infarction area in the rat cerebral ischemia-reperfusion model and its possible mechanism. METHODS: Transient focal ischemia induced by middle cerebral artery occlusion in the rats was established by ligation of the left internal carotid artery for 2 h. Rats were treated by propyl gallate with different doses (23.5, 47 and 94 micromol x kg(-1)) for three days before operation. Coronal brain sections were collected after 1 , 2, 4, 6, 12 and 24 h of reperfusion, neuronal injury in the boundary zone of the infarction area was evaluated by TUNEL and Nissl staining. The expression of activated Caspase-3, total SAPK/JNK, p38MAPK and their phosphorylation (Thr183/Tyr185, Thr180/Tyr182) was investigated by immunohistochemistry and Western blotting with corresponding antibodies. RESULTS: Although SAPK/JNK immunoreactivity did not increase at each time point in the boundary zone of the infarction area after reperfusion, p-SAPK/JNK immunoreactivity increased significantly at 1 h and then decreased gradually, and p38MAPK immunoreactivity was enhanced at each time point, peaked at 6 h. Expression of p-p38MAPK peaked at 6 h. Activated Caspase-3 immunoreactivity appeared at 6 h in the boundary zone of the infarction area and peaked at 12 h. TUNEL positive neurons were observed at 12 h and became more abundant at 24 h. The number of Nissl positive neurons decreased gradually and apoptosis ratio of neurons peaked at 24 h. Propyl gallate reduced the immunoreactivity of SAPK/JNK, p-SAPK/JNK, p38MAPK and p-p38MAPK markedly at 1 and 6 h. Propyl gallate with doses of 47 and 94 micromol x kg(-1) were more effective. CONCLUSION: Inhibition on the activation of SAPK/JNK and p38MAPK is the possible protective mechanism of propyl gallate against neuronal injury induced by cerebral ischemia-reperfusion.

[Ginsenoside Rbl attenuates beta-amyloid peptide25-35 -induced tau hyperphosphorylation in cortical neurons].

AIM: To explore the effect and the possible mechanism of ginsenoside Rb1 on beta-amyloid peptide (beta-AP)(25-35) -induced tau protein hyperphosphorylation in cortical neurons. METHODS: Western blotting and immunocytochemical staining were used to detect tau phosphorylation level, total tau and glycogen synthase kinase-3beta (GSK-3beta) in cortical neurons. RESULTS: After exposure to beta-AP(25-35) (20 micromol x L(-1)) for 12 h, the levels of tau protein phosphorylation in the sites of Ser 396, Ser 199/202, Thr 231 and total tau were raised. Meanwhile, the expression of GSK-3beta also increased. Pretreatment with ginsenoside Rbl or lithium chloride, a specific inhibitor of GSK-3beta, markedly reduced beta-AP(25-35)-induced tau hyperphosphorylation and the expression of GSK-3beta. CONCLUSION: Ginsenoside Rb1 can attenuate beta AP(25-35)-induced tau protein hyperphosphorylation in cortical neurons by inhibiting the expression of GSK-3beta.

[Ginsenoside Rb1 attenuates okadaic acid-induced Tau protein hyperphosphorylation in rat hippocampal neurons].

The present study was aimed to investigate the effects of ginsenoside Rb1 on okadaic acid (OA)-induced Tau hyperphosphorylation in hippocampal neurons of Sparague-Dawley rat and to explore its possible mechanism. Animals were randomly divided into four groups. Group 1 received dimethysulphoxide (DMSO) injection (vehicle group), group 2 only received OA injection (OA group), group 3 was pretreated with Rb1 and then received OA injection (Rb1 pretreatment group), and the group 4 was an intact control group. The animals in group 3 were injected intraperitoneally with various doses of Rb1 at 5, 10, and 20 mg/kg (once a day for 14 d). On the thirteen day of pretreatment, animals in Rb1 pretreatment group as well as animals in OA group received a bolus injection of 0.483 microg of OA (1.5 microl of solution in DMSO) at right dorsal aspect of hippocampus to induce Tau hyperphosphrylation. The brains were harvested one day after the last treatment. In all groups, the morphology of neurofibrils, phosphorylation of Tau protein, and the activity of phosphatase 2A (PP2A) were investigated. In OA group, the Bielschowski's assay revealed darkened and uneven neurofibrils staining in the hippocampus. The immunohistochemistry results showed a significant increase in Thr(231) phosphorylation of Tau protein in OA group relative to the control group (P<0.01). OA injection also markedly decreased PP2A activity (P<0.01). Western blot confirmed Thr(231) phosphorylation of Tau protein and it also detected phosphorylation of Ser(396) of Tau protein. The animals with Rb1 pretreatment displayed even staining of neurofibrils and normal pattern of fiber organization. Rb1 pretreatment also attenuated Thr(231) and Ser(396) hyperphosphorylations of Tau protein, and restored PP2A activity compared to the OA group (P<0.01). These results indicate that OA-induced hyperphosphorylation of Tau protein in rat hippocampal neurons can be attenuated by the pretreatment of ginsenoside Rb1. These data also implicate that Rb1 has potential neuroprotective effects on Tau-related neuropathology.

[Effect of ginsenoside Rg1 on expression of p21, cyclin E and CDK2 in the process of cell senescence].

AIM: To explore the possible role of p21, cyclin E and cyclin-dependent kinase 2 (CDK2) in the protection of ginsenoside Rg1 against tert-butylhydroperoxide (t-BHP)-induced senescence in WI-38 cells. METHODS: The cellular ultrastructure, cytometric assay and beta-galactosidase (beta-gal) cytochemistry staining were used to evaluate cell senescence. The levels of of p21, cyclin E and CDK2 protein were detected by Western blot. RESULTS: Pretreatment with Rg1 significantly attenuated t-BHP-induced senescence in WI-38 cells. Simultaneously, compared with cells treated with t-BHP alone, Rg1 pretreatment markedly decreased the level of p21 protein and increased the levels of CDK2 and cyclin E. CONCLUSION: p21, cyclin E and CDK2 may be involved in the process of ginsenoside Rg1 protection against t-BHP-induced senescence in WI-38 cells.

Curcumin protects mitochondria from oxidative damage and attenuates apoptosis in cortical neurons.

AIM: To investigate the effect of curcumin on tert-butyl hydroperoxide (t-BHP)-induced oxidative damage in rat cortical neurons and to explore the possible mechanism. METHODS: Primary cultured rat cortical neurons were performed in vitro and cell viability was measured by MTT assay. DNA fragmentation was used to evaluate cell apoptosis. Intracellular reactive oxygen species (ROS) and mitochondrial membrane potential (Deltapsim) was determined by flow cytometric assay. Cellular glutathione (GSH) content was measured by spectrophotometer. Bcl-2 family proteins, cytochrome c, cleaved caspase-3, and poly (ADP-ribose) polymerase (PARP) were detected by Western blot. RESULTS: Exposure of tBHP 100 micromol/L to neurons for 60 min resulted in DYm loss and cytochrome c release from mitochondria and subsequent activation of caspase-3 and PARP cleavation, and cell apoptosis. After removal of tBHP and then further treatment with curcumin (2.5-20 micromol/L) for 18 h, curcumin abrogated Deltapsim loss and cytochrome c release, blocked activation of caspase 3, and altered the expression of Bcl-2 family. Further curcumin treatment also prevented cellular GSH and decreased intracellular ROS generation markedly. Curcumin eventually attenuated tBHP-induced apoptosis in cortical neurons. CONCLUSION: Curcumin may attenuate oxidative damages in cortical neurons by reducing intracellular production of ROS and protecting mitochondria from oxidative damage.