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OBJECTIVE@#To investigate the effect of Zuogui Jiangtang Jieyu Decoction (ZJJ) on Shh signaling and self-renewal of neural stem cells in the hippocampal dentate gyrus of diabetic rats with depression.@*METHODS@#Diabetic rat models with depression were randomly divided into model group, positive drug (metformin + fluoxetine) group, and low-, medium-, and high-dose ZJJ groups (n=16), with normal SD rats as the control group. The positive drugs and ZJJ were administered by gavage, and the rats in the control and model groups were given distilled water. After the treatment, blood glucose level was detected using test strips, and behavioral changes of the rats were assessed by forced swimming test and water maze test. ELISA was used to examine the serum level of leptin; The expressions of nestin and Brdu proteins in the dentate gyrus of the rats were detected using immunofluorescence assay, and the expressions of self-renewal marker proteins and Shh signaling proteins were detected using Western blotting.@*RESULTS@#The diabetic rats with depression showed significantly increased levels of blood glucose and leptin (P < 0.01) and prolonged immobility time in forced swimming test (P < 0.01) and increased stage climbing time with reduced stage seeking time and stage crossings in water maze test (P < 0.01). The expressions of nestin and Brdu in the dentate gyrus, the expressions of cyclin D1, SOX2, Shh, Ptch1, Smo in the hippocampus and the nuclear expression of Gli-1 were decreased (P < 0.01) while hippocampal Gli-3 expression was increased significantly (P < 0.01) in the rat models. Treatment of rat models with high-dose ZJJ significantly reduced the blood glucose (P < 0.01) and leptin level (P < 0.05) and improved their performance in behavioral tests (P < 0.01). The treatment also obviously increased the expressions of nestin, Brdu, cyclin D1, SOX2, Shh, Ptch1, and Smo and the nuclear expression of Gli-1 in the dentate gyrus (P < 0.01) and reduced hippocampal expression of Gli-3 (P < 0.05) in the rat models.@*CONCLUSION@#ZJJ can significantly improve the self-renewal ability of neural stem cells and activate Shh signaling in dentate gyrus of diabetic rats with depression.
Subject(s)
Animals , Rats , Blood Glucose , Bromodeoxyuridine , Cell Self Renewal , Cyclin D1 , Dentate Gyrus , Depression , Diabetes Mellitus, Experimental , Hippocampus , Leptin , Nestin , Rats, Sprague-DawleyABSTRACT
OBJECTIVE@#To investigate the effects of inhibiting Sonic Hedgehog (Shh) signaling on fibrous scar formation and functional outcome after ischemic brain injury.@*METHODS@#Adult SD rats were randomized into sham-operated group, middle cerebral artery occlusion (MCAO) and reperfusion (I/R) group, I/R with intraventricular empty adenoviral vector (rAd-NC) injection group, and I/R with adenovirus-mediated Shh knockdown (rAd-ShShh) group. After the treatments, the neurological deficits of the rats were assessed, and the protein and mRNA expressions of fibronectin (Fn), α-SMA, and Shh in the ischemic hemisphere were detected with immunofluorescence assay and qPCR; TUNEL staining was used for detecting neural cell apoptosis. In the cell experiment, primary meningeal fibroblasts isolated from neonatal SD rats were pretreated for 24 h with TGF-β1 or TGF-β1 plus cyclopamine (CYC) before oxygen-glucose deprivation for 150 min followed by reoxygenation for 72 h (OGD/R). CCK-8 assay and scratch test were performed to examine the changes in cell proliferation and migration, and immunofluorescence assay, qPCR and Western blotting were used for detecting cell transformation and the expressions of Shh, α-SMA, and Fn.@*RESULTS@#Cerebral I/R injury significantly increased the protein and mRNA expressions of Shh, α-SMA, and Fn in the ischemic hemisphere of the rats, but their expression levels were significantly lowered by intraventricular injection of rAd-Shshh (P < 0.05), which obviously increased cell apoptosis in the ischemic hemisphere (P < 0.05) and improved modified mNSS and modified Bederson scores of the rats (P < 0.05). In the cell experiment, pretreatment with TGF-β1 and TGF-β1+CYC both increased the viability of the primary meningeal fibroblasts after OGD/R. TGF-β1 significantly enhanced the migration ability and induced obvious transformation of the exposed cells (P < 0.05), but these effects were significantly attenuated by co-treatment with CYC (P < 0.05). The expressions of Shh, α-SMA and Fn in the TGF-β1 group were all significantly higher in TGF-β1-treated cells (P < 0.05) and were obviously lowered by co-treatment with CYC (P < 0.05).@*CONCLUSION@#Inhibition of Shh signaling may inhibit fibrous scar formation and functional recovery in rats after ischemic brain injury.
Subject(s)
Animals , Rats , Brain Injuries , Cicatrix , Hedgehog Proteins , RNA, Messenger , Rats, Sprague-Dawley , Transforming Growth Factor beta1ABSTRACT
OBJECTIVE@#To investigate the effect of TGF-β1 on Shh signaling pathway during the transformation of meningeal fibroblasts into myofibroblasts.@*METHODS@#Primary meningeal fibroblasts were isolated from neonatal (24 h) SD rats and purified using type Ⅳ collagenase. The isolated cells were treated with 10 ng/mL TGF-β1 alone or in combination with 20 μmol/L SB-431542 (a TGF-β1 receptor inhibitor) for 72 h, and the changes in proliferation and migration abilities of the fibroblasts were assessed with CCK-8 assay and cell scratch test. The expression of fibronectin (Fn) was detected with immunofluorescence assay, and Western blotting was performed to examine the expressions of Fn, α-SMA and Shh protein in the cells; the expression of Shh mRNA was detected with real-time fluorescence quantitative PCR.@*RESULTS@#TGF-β1 treatment obviously enhanced the proliferation and migration of primary meningeal fibroblasts (P < 0.05), and promoted the transformation of meningeal fibroblasts into myofibroblasts and the secretion of Fn (P < 0.05). TGF-β1 treatment also upregulated the expression of Shh at both protein and mRNA levels (P < 0.05). Treatment with SB-431542 partially blocked the effect of TGF-β1 on the transformation of meningeal fibroblasts (P < 0.05).@*CONCLUSION@#TGF-β1 can induce the transformation of meningeal fibroblasts into myofibroblasts by up-regulating Shh expression in Sonic Hedgehog signaling pathway.
Subject(s)
Animals , Rats , Fibroblasts/metabolism , Hedgehog Proteins , Myofibroblasts/metabolism , Rats, Sprague-Dawley , Transforming Growth Factor beta1/metabolismABSTRACT
Sonic Hedgehog gene (SHH) plays a vital role in embryogenesis through its secreted protein sonic hedgehog protein (Shh). During embryogenesis, Shh acts as a morphogen controlling proximal and distant signaling in the specific development of tissue lineages, patterning, regulation of cell proliferation and suppression of tissue apoptosis. Shh also exerts its role in odontogenesis by determining the site of tooth bud formation, in tooth morphogenesis and root formation. The difference in the specific development of a region by Shh can be explained by its [a] 'Spatial gradient [b] the 'form' [c] Concentration gradient and [d] Temporal gradient. Shh signaling pathway has an extracellular and an intracellular component. A disruption of Shh pathway contributes to tumorigenesis of several cell types including those arising from odontogenic structures. This article reviews Shh from its formation in embryonic stages, its role in development and odontogenesis, to its reactivation in tumorigenesis and in specific to odontogenic pathologies.
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@#Choroid neovascularization is the characteristic pathological change of many fundus diseases and is the most common cause for severe vision loss and metamorphopsia. Among the pathogenic factors, VEGF is considered to be the most important and treatment targeting VEGF showed promising results. However, anti-VEGF agents need to be administrated frequently and they are usually expensive. Also, some patients got no response to this treatment. These facts force us to find other pathway that involves in the formation of CNV. This article reviews the latest research on CNV-related signaling pathways so as to provide a deeper look into CNV and hopefully point out new directions for treating diseases that share similar pathogenesis.
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Objective: To investigate the effect of activation of SHH signaling pathway on the apoptosis and invasion of the trophoblast cells in the preeclampsia (PE) patients, and to clarify its mechanism. Methods: The HTR8/SVneo cells were divided into normal control group, hypoxia/reoxygenation (H/R) treatment group and purmorphamine+H/R treatment group. The expression levels of SHH signaling pathway related proteins SHH, Ptchl, Smo, Glil, and Gli3 in the placenta tissues of PE and late trimester of normal pregnancy were detected by Western blotting method; the apoptotic rates of HTR8/SVneo cells in various groups were detected by flow cytometry (FCM); Transwell assay was used to detect the number of transmembrane cells; the expression levels of matrix metalloproteinase-2 (MMP-2) and metalloproteinase-9 (MMP-9) proteins in the HTR8/SVneo cells in various groups were detected by Western blotting method. Results: The expression levels of SHH, Ptchl, Smo, Glil and Gli3 proteins in the placenta tissue of PE were significantly lower than those in normal pregnancy placenta tissue (P<0. 05); the expression levels of SHH, Ptchl, Smo, Glil, Gli3, MMP-2, and MMP-9 proteins and the number of transmembrane cells in H/R treatment group were significantly lower than those in normal control group (P
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Pituitary stalk interruption syndrome(PSIS) is a developmental defect,characterized by a thin or absent pituitary stalk. PSIS is a rare disease with combined pituitary hormone deficiencies, the pathogenesis of which is related to genetic mutations and environmental factors. It is also a genetically and clinically heterogeneous disease. Nine genes were found to be related to PSIS,including HESX1,LHX4,OTX2,SOX3,CDON,GPR161, PROKR2,TGIF and Wnt,Notch,SHH signalling pathways. In recent years,the intensive genetic studies show that four novel genes(CDON,GPR161,PROKR2,TGIF)and SHH pathway are related to PSIS,which provides a brand-new etiopathogenesis of PSIS.
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To study the effect of primary cilia-mediated sonic hedgehog (Shh) signaling on differentiation of rat bone marrow stromal cells (MSCs) into neuron-like cells. Methods Rat MSCs were isolated and cultured. The study was divided into normal resveratrol-cultured group, resveratrol-induced group, SAG (Smoothened [Smo] agonist) group, and cyclopamine (Smo inhibitor) group. Cell morphology was observed under inverted microscope; the expressions of Ac-Tu, Ptc, Smo, and Glil were examined by immunofluorescence method. Western blotting analysis was used to detect the protein expressions of Smo and Gli, and RT-PCR was applied to detect mRNA expressions of Smo and Glil. Results No expression of primary cilia was found in the normally cultured MSCs. After pre-induction or 24 h starvation, MSCs expressed primary cilia, Ptc, Smo and Glil proteins, with Ptc protein in the primary cilia and Smo, Glil in the cytoplasm. In normal cultivation, resveratrol did not promote the translocation of Smo or induce differentiation of MSCs into neuronal-like cells. When MSCs expressed primary cilia, resveratrol and SAG led to translocation of Smo from the cytoplasm into primary cilia, accompanied by MSCs differentiation into neuronal-like cells and significantly increased expression of Smo and Glil mRNA and protein (P< 0. 05). When cyclopamine was added, Smo remained expressed in the cytoplasm, the expression of Smo and Glil mRNA and protein was significantly decreased (P < 0. 05), and MSCs differentiation into neuronal-like cells was inhibited. Conclusion MSCs express primary cilia and have Shh signaling. Primary cilia-mediated Shh signaling participates in MSCs differentiation into neuronal-like cells.