Molecular Mechanism of Wendantang Against Schizophrenia in Rats： An Exploration Based on miRNA-219， NR2B， DISC1 and CaMKⅡγ / 中国实验方剂学杂志

ObjectiveTo study the effects of Wendantang on the expression of miRNA-219， N-methyl-D-aspartate receptor 2B （NR2B）， disrupted in schizophrenia 1 （DISC1）， and Ca2+/calmodulin-dependent protein kinase Ⅱγ （CaMKⅡγ） in the frontal lobe of rats with schizophrenia. MethodSixty rats were randomly divided into six groups， namely normal group， model group， high-， medium-， and low-dose Wendantang groups， and clozapine group， with 10 rats in each group. Rats in high-， medium-， and low-dose Wendantang groups were intragastric with 40， 20， and 10 g·kg-1 Wendantang， and the ones in clozapine group were intragastric with 0.02 g·kg-1 clozapine， those in normal and model group were intragastric with equal volume of normal saline， once a day. After 21 days of administration， rats in all groups except for the normal group were injected with 0.6 mg·kg-1 dizocilpine maleate （MK-801） into the left abdominal cavity for inducing acute schizophrenia. The stereotypic behavior and ataxia in rats were scored according to SAMS and HOFFMAN criteria. The morphological changes in the prefrontal cortex were observed by hematoxylin-eosin （HE） staining. The protein expression levels of NR2B, DISC1 and CaMKⅡγ in the frontal lobe was detected by Western blot. The mRNA expression levels of miRNA-219 was detected by real-time fluorescence quantitative polymerase chain reaction（Real-time PCR）. ResultCompared with normal group， the model group exhibited significantly increased stereotypic behavior and ataxia scores （P<0.01）， karyopyknosis and karyolysis in most neurons of the prefrontal cortex， and down-regulated NR2B， DISC1， and CaMKⅡγ protein expression （P<0.01） and miRNA-219， NR2B， DISC1， and CaMKⅡγ mRNA expression （P<0.01）. Compared with model group， Wendantang high-， medium-， and low-doses group lowered the scores of stereotypic behavior and ataxia at 50， 60 mmin（P<0.05，P<0.01）. In high- and medium-dose Wendantang groups， the neurons in the prefrontal cortex were densely arranged. The karyopyknosis and karyolysis were alleviated to varying degrees. The NR2B protein expression in the frontal lobe was up-regulated （P<0.01）. In the medium- and low-dose Wendantang groups， the DISC1 protein expression in the frontal lobe was up-regulated （P<0.05，P<0.01）. Wendantang at each dose significantly increased the CaMKⅡγ protein expression （P<0.05） and miRNA-219， NR2B， DISC1， and CaMKⅡγ mRNA expression in the frontal lobe （P<0.05，P<0.01）. ConclusionWendantang improves the scores of stereotypical behavior and ataxia， relieves the karyopyknosis and karyolysis of neurons in the prefrontal cortex， and increases the expression levels of miRNA-219， NR2B， DISC1， and CaMKⅡγ of rats with schizophrenia， so as to alleviate the schizophrenic-like symptoms and schizophrenia.

Low frequency pulsed electromagnetic field promotes differentiation of oligodendrocyte precursor cells through upregulation of miR-219-5p in vitro.

AIM: Spinal cord injury (SCI) is a common demyelinating disorder of the central nervous system. The differentiation of oligodendrocyte precursor cells (OPCs) into mature oligodendrocytes (OLs), which induce myelination, plays a critical role in the functional recovery following SCI. In this study, the effect of low frequency pulsed electromagnetic field (PEMF) on the differentiation of OPCs and the potential underlying mechanisms were investigated. MAIN METHODS: OPCs were randomly divided into the PEMF and non-PEMF (NPEMF) groups. Immunofluorescence and western blot assays were performed to assess the expression levels of OLs stage-specific markers after 3, 7, 14, and 21 days of PEMF or NPEMF exposure. qRT-PCR was used to further assess the expression levels of miR-219-5p, miR-338, miR-138, and miR-9, which are associated with OPCs differentiation, and the expression levels of genes associated with miR-219-5p. Finally, following PEMF or NPEMF exposure, qRT-PCR and western blot assays were performed to explore the relationship between miR-219-5p and Lingo1 and between miR-219-5p and PEMF in promoting OPCs differentiation. KEY FINDINGS: PEMF promoted the differentiation of OPCs. PEMF upregulated the expression level of miR-219-5p and downregulated the expression level of Lingo1 during the differentiation of OPCs. Under PEMF exposure, miR-219-5p targeted Lingo1 and reversed the inhibitory effect of miR-219-5p inhibitor on OPCs differentiation. In addition, PEMF synergized with miR-219-5p to promote OPCs differentiation. SIGNIFICANCE: Our results, for the first time, indicated that PEMF promoted OPCs differentiation by regulating miR-219-5p activity in vitro.

microRNA-219 Reduces Viral Load and Pathologic Changes in Theiler's Virus-Induced Demyelinating Disease.

Analysis of microRNA (miR) expression in the central nervous system white matter of SJL mice infected with the BeAn strain of Theiler's murine encephalomyelitis virus (TMEV) revealed a significant reduction of miR-219, a critical regulator of myelin assembly and repair. Restoration of miR-219 expression by intranasal administration of a synthetic miR-219 mimic before disease onset ameliorates clinical disease, reduces neurogliosis, and partially recovers motor and sensorimotor function by negatively regulating proinflammatory cytokines and virus RNA replication. Moreover, RNA sequencing of host lesions showed that miR-219 significantly downregulated two genes essential for the biosynthetic cholesterol pathway, Cyp51 (lanosterol 14-α-demethylase) and Srebf1 (sterol regulatory element-binding protein-1), and reduced cholesterol biosynthesis in infected mice and rat CG-4 glial precursor cells in culture. The change in cholesterol biosynthesis had both anti-inflammatory and anti-viral effects. Because RNA viruses hijack endoplasmic reticulum double-layered membranes to provide a platform for RNA virus replication and are dependent on endogenous pools of cholesterol, miR-219 interference with cholesterol biosynthesis interfered virus RNA replication. These findings demonstrate that miR-219 inhibits TMEV-induced demyelinating disease through its anti-inflammatory and anti-viral properties.

[ROLE OF Smad4 ON FIBROSIS OF TENDON DERIVED FIBROBLASTS INDUCED BY TRANSFORMING GROWTH FACTOR ß1 BY TARGETED REGULATION OF miRNA219-5P].

OBJECTIVE: To investigate the effect of Smad4 on the fibrosis of tendon derived fibroblasts (TDFs) induced by transforming growth factor ß1(TGF-ß1) by targeted regulation of miRNA219-5P (miR219-5P). METHODS: The tendons donated by the volunteers were harvested to isolate and culture TDFs. The 3rd generation cells were used for experiment. Chemically synthesized miR219-5P mimics, miR219-5P inhibitor, and negative control sequences were transfected into TDFs. The gene expression of miR219-5P in TDFs was detected by real-time PCR, and the protein expression of Smad4 in TDFs was detected by Western blot at 48 hours after transfection. The combining sites of miR219-5P and Smad4 in 3'UTR district were predicted by informatics software. Wild type and mutant type reporter gene expression vectors were constructed and then targeted verification was carried out by the luciferase reporter gene test. Transfected TDFs were then induced by TGF-ß1. The proliferation activity of the cells were measured by the cell counting kit 8 after culturing for 24, 48, and 72 hours. The expressions of fibrosis related proteins in TDFs were detected by Western blot at 72 hours. RESULTS: After TDFs were transfected by miR219-5P mimics, miR219-5P expression was significantly up-regulated, but the expressions of Smad4 was decreased subsequently (P<0.05). Intracellular expression of miR219-5P was inhibited by miR219-5P mimics inhibitor, however, the protein expression of Smad4 was significantly increased (P<0.05). Luciferase reporter gene test showed that luciferase activities were significantly decreased in pGL3-WT-Smad4+mimics group, but were significantly increased in pGL3-WT-Smad4+inhibitor group when compared with pGL3-WT-Smad4 transfected group (P<0.05), but no significant difference was found between GL3-MT-Smad4+mimics and pGL3-MT-Smad4+inhibitor groups (P>0.05). Cell proliferation and the fibrosis related proteins were increased in TGF-ß1 induced TDFs, however, decreased in TGF-ß1 induced TDFs after transfected by miR219-5P inhibitor (P<0.01). CONCLUSIONS: miR219-5P can significantly inhibit fibrosis of TDFs induced by TGF-ß1 by down-regulating Smad4 expression.

ROLE OF Smad4 ON FIBROSIS OF TENDON DERIVED FIBROBLASTS INDUCED BY TRANSFORMING GROWTH FACTOR β1 BY TARGETED REGULATION OF miRNA219-5P / 中国修复重建外科杂志

OBJECTIVE: To investigate the effect of Smad4 on the fibrosis of tendon derived fibroblasts (TDFs) induced by transforming growth factor β1(TGF-β1) by targeted regulation of miRNA219-5P (miR219-5P). METHODS: The tendons donated by the volunteers were harvested to isolate and culture TDFs. The 3rd generation cells were used for experiment. Chemically synthesized miR219-5P mimics, miR219-5P inhibitor, and negative control sequences were transfected into TDFs. The gene expression of miR219-5P in TDFs was detected by real-time PCR, and the protein expression of Smad4 in TDFs was detected by Western blot at 48 hours after transfection. The combining sites of miR219-5P and Smad4 in 3'UTR district were predicted by informatics software. Wild type and mutant type reporter gene expression vectors were constructed and then targeted verification was carried out by the luciferase reporter gene test. Transfected TDFs were then induced by TGF-β1. The proliferation activity of the cells were measured by the cell counting kit 8 after culturing for 24, 48, and 72 hours. The expressions of fibrosis related proteins in TDFs were detected by Western blot at 72 hours. RESULTS: After TDFs were transfected by miR219-5P mimics, miR219-5P expression was significantly up-regulated, but the expressions of Smad4 was decreased subsequently (P0.05). Cell proliferation and the fibrosis related proteins were increased in TGF-β1 induced TDFs, however, decreased in TGF-β1 induced TDFs after transfected by miR219-5P inhibitor (P<0.01). CONCLUSIONS: miR219-5P can significantly inhibit fibrosis of TDFs induced by TGF-β1 by down-regulating Smad4 expression.