(+)-Borneol enantiomer ameliorates epileptic seizure via decreasing the excitability of glutamatergic transmission.

Epilepsy is one common brain disorder, which is not well controlled by current pharmacotherapy. In this study we characterized the therapeutic potential of borneol, a plant-derived bicyclic monoterpene compound, in the treatment of epilepsy and elucidated the underlying mechanisms. The anti-seizure potency and properties of borneol were assessed in both acute and chronic mouse epilepsy models. Administration of (+)-borneol (10, 30, 100 mg/kg, i.p.) dose-dependently attenuated acute epileptic seizure in maximal-electroshock seizure (MES) and pentylenetetrazol (PTZ)-induced seizure models without obvious side-effect on motor function. Meanwhile, (+)-borneol administration retarded kindling-induced epileptogenesis and relieved fully kindled seizures. Importantly, (+)-borneol administration also showed therapeutic potential in kainic acid-induced chronic spontaneous seizure model, which was considered as a drug-resistant model. We compared the anti-seizure efficacy of 3 borneol enantiomers in the acute seizure models, and found (+)-borneol being the most satisfying one with long-term anti-seizure effect. In electrophysiological study conducted in mouse brain slices containing the subiculum region, we revealed that borneol enantiomers displayed different anti-seizure mechanisms, (+)-borneol (10 µM) markedly suppressed the high frequency burst firing of subicular neurons and decreased glutamatergic synaptic transmission. In vivo calcium fiber photometry analysis further verified that administration of (+)-borneol (100 mg/kg) inhibited the enhanced glutamatergic synaptic transmission in epilepsy mice. We conclude that (+)-borneol displays broad-spectrum anti-seizure potential in different experimental models via decreasing the glutamatergic synaptic transmission without obvious side-effect, suggesting (+)-borneol as a promising anti-seizure compound for pharmacotherapy in epilepsy.

Effect of Grilled Nux Vomica on Differential RNA Expression Profile of Gastrocnemius Muscle and Toll­Like Receptor 4 (TLR-4)/Nuclear Factor kappa B (NF-κB) Signaling in Experimental Autoimmune Myasthenia Gravis Rats.

BACKGROUND Myasthenia gravis (MG) is a progressive autoimmune disorder caused by the production of antibodies directed against acetylcholine receptors (AChRs), resulting in muscle weakness and fatigue. This study aimed to explore the effect and mechanism of grilled nux vomica (GNV) in experimental autoimmune myasthenia gravis (EAMG) rats. MATERIAL AND METHODS Rat 97-116 peptides were used to mediate disease in the EAMG model in SPF female Lewis rats. The treatment groups received grilled nux vomica (75 mg/kg, 150 mg/kg, and 225 mg/kg). The autoantibody and inflammatory cytokines levels were measured by enzyme-linked immunosorbent assay (ELISA). RNA profiling was performed on high-dose and model group rats. Profiling results and TLR-4/NF-kappaB signaling were validated by q-PCR and Western blot analysis. RESULTS The results showed that GNV could attenuate the symptoms of EAMG rats. There was a decreased level of AChR-ab, IFN-γ, TNF-alpha, IL-2, IL-4, and IL-17 levels, and an increased level of TGF-ß1. In total, 235 differentially expressed genes (DEGs), consisting of 175 upregulated DEGs and 60 downregulated DEGs, were identified. Functional annotation demonstrated that DEGs were largely associated with leukocyte cell-cell adhesion, NF-kappa B signaling pathway, muscle contraction, and cardiac muscle contraction pathway. Rac2, Itgb2, Lcp2, Myl3, and Tnni1 were considered as hub genes with a higher degree value in the protein-protein interaction (PPI) network. The q-PCR and Western blot results of hub genes were consistent with RNA profiles. GNV treatment also significantly reduced the TLR-4 and NF-kappaB p65 protein expression in EAMG rats. CONCLUSIONS These results indicate that grilled nux vomica ameliorates EAMG by depressing the TLR-4/NF-kappaB signaling pathway, and hub genes may serve as potential targets for MG treatment.

Emodin prevents intima thickness via Wnt4/Dvl-1/ß-catenin signaling pathway mediated by miR-126 in balloon-injured carotid artery rats.

Neointimal proliferation after vascular injury is a key mechanism of restenosis, a major cause of percutaneous transluminal angioplasty failure and artery bypass occlusion. Emodin, an anthraquinone with multiple physiological activities, has been reported to inhibit proliferation of vascular smooth muscle cells (VSMCs) that might cause intimal arterial thickening. Thus, in this study, we established a rat model of balloon-injured carotid artery and investigated the therapeutic effect of emodin and its underlying mechanism. Intimal thickness was analyzed by hematoxylin and eosin staining. Expression of Wnt4, dvl-1, ß-catenin and collagen was determined by immunohistochemistry and/or western blotting. The proliferation of VSMC was evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and electron microscopy. MicroRNA levels were quantified by real-time quantitative PCR. Emodin relieved injury-induced artery intimal thickness. Results of western blots and immunohistochemistry showed that emodin suppressed expression of signaling molecules Wnt4/Dvl-1/ß-catenin as well as collagen protein in the injured artery. In addition, emodin enhanced expression of an artery injury-related microRNA, miR-126. In vitro, MTT assay showed that emodin suppressed angiotensin II (AngII)-induced proliferation of VSMCs. Emodin reversed AngII-induced activation of Wnt4/Dvl-1/ß-catenin signaling by increasing expression of miR-126 that was strongly supported by transfection of mimic or inhibitor for miR-126. Emodin prevents intimal thickening via Wnt4/Dvl-1/ß-catenin signaling pathway mediated by miR-126 in balloon-injured carotid artery of rats.

[Effect of tetramethylpyrazine on the proliferation and collagen synthesis of vascular smooth muscle cells].

OBJECTIVE: To study the action mechanism of tetramethylpyrazine (TMP) on the proliferation of vascular smooth muscle cells (VSMCs), thus providing experimental evidence for Chinese medicine to effectively prevent restenosis. METHODS: Rats' thoracic aorta VSMCs in vitro cultured (cell line A7r5) were divided into five groups, i.e., the negative control group, the angiotensin II (Ang II, 10(-6) mol/L) group, the low dose TMP (20 micromol/L) plus Ang II group, the middle dose TMP (40 micromol/L) plus Ang II group, the high dose TMP (80 micromol/L) plus Ang II group. The proliferation ratio was detected by MTT. Gene and protein expressions of Wnt4, Dvl-1, beta-catenin, CyclinD1, and collagen I and III were detected with real-time fluorescent quantitative PCR and Western blot respectively. RESULTS: Compared with the negative control group, the proliferation ratio of VSMCs obviously increased in the Ang II group (P < 0.05). Compared with the Ang II group, the proliferation ratio of VSMCs obviously decreased in the middle dose TMP plus Ang II group and the high dose TMP plus Ang II group (P < 0.05). Compared with the negative control group, gene and protein expressions of Wnt4, Dvl-1, beta-catenin, CyclinD1, Col I, and Col III were obviously up-regulated in the Ang II group (P < 0.05). Compared with the Ang II group, mRNA and protein expressions of Wnt4, Dvl-1, beta-catenin, CyclinD1, Col I, and Col III were obviously down-regulated in the middle dose TMP plus Ang II group and the high dose TMP plus Ang II group (P < 0.05). The aforesaid indices were dose-dependent in the low, middle, and high dose TMP plus Ang II groups. CONCLUSION: TMP inhibited Ang II induced proliferation and collagen secretion of VSMCs through down-regulating Wnt signal pathway.

[Relationship between endothelial-to-mesenchymal transition and cardiac fibrosis in acute viral myocarditis].

OBJECTIVE: To investigate the relationship between endothelial-to-mesenchymal transition (EndMT) and myocardial fibrosis in acute viral myocarditis (VMC). METHODS: Twenty-eight Balb/c mice were randomized into 3 groups: control group (n=8), VMC group(n=10) and intervention group(n=10). Mice in VMC and intervention groups were injected intraperitoneally(i.p) with single dose of coxsackievirus B3, mice in control group were injected with equal amount of viral-free vehicle. In the following day, mice in control and VMC groups were injected i.p with 0.1 ml of saline and intervention group with 0.1 ml of recombinant human bone morphogenetic protein 7(rh-BMP7) at a concentration of 300 µg/kg. The mice hearts were harvested after 7 d, cardiac collagen volume fraction (CVF) was calculated on picrosirius red-stained sections. mRNA and protein expression of TGF-ß1, CD31, VE-cadherin, fibroblast special protein 1 (FSP-1) and α-smooth muscle actin (α-SMA) and collagen 1α1 in myocardiac tissues were detected by real-time RT-PCR and Western blot analysis, respectively. RESULTS: Compared to controls, overt fibrosis was presented in necrotic area of myocardium in VMC group. Meanwhile, marked increase of TGF-ß1 expression accompanied with EndMT characterized by loss of endothelial phenotype (decreased expression of CD31 and VE-cadherin), gain of mesenchymal proteins (overexpression of FSP-1 and α-SMA) and increased synthesis of collagen was also demonstrated. Both EndMT and cardiac fibrosis were simultaneously reversed by TGF-ß1 inhibition. CONCLUSION: EndMT is involved in cardiac fibrosis in acute viral myocarditis, TGF-ß1 might be a main mediator.