aconitine and Osteoarthritis

This study aims to investigate the therapeutic effects of alkaloids in Tibetan medicine Bangna(Aconiti Penduli et Aconiti Flavi Radix) on osteoarthritis(OA) rats in vitro and in vivo and the underlying mechanisms. Chondrocytes were isolated from 2-3 week-old male SD rats and lipopolysaccharide(LPS) was used to induce OA in chondrocytes in vitro. Methyl thiazolyl tetrazolium(MTT) assay was used to investigate the toxicity of seven alkaloids(12-epi-napelline, songorine, benzoylaconine, aconitine, 3-acetylaconitine, mesaconitine, and benzoylmesaconine) to chondrocytes. Chondrocytes were classified into the control group, model group(induced by LPS 5 μg·mL~(-1) for 12 h), and administration groups(induced by LPS 5 μg·mL~(-1) for 12 h and incubated for 24 h). The protein expression of inflammatory factors cyclooxygenase-2(COX-2), inducible nitric oxide synthetase(iNOS), tumor necrosis factor-α(TNF-α), and interleukin-1β(IL-1β) in each group were detected by Western blot, and the protein expression of matrix metalloprotease-13(MMP-13), aggrecan, collagen Ⅱ, fibroblast growth factor 2(FGF2) by immunofluorescence staining. For the in vivo experiment, sodium iodoacetate was used to induce OA in rats, and the expression of MMP-13, TNF-α, and FGF2 in cartilage tissues of rats in each group was detected by immunohistochemistry. The results showed that the viability of chondrocytes could reach more than 90% under the treatment of the seven alkaloids in a certain dose range. Aconitine, 12-epi-napelline, songorine, 3-acetylaconitine, and mesaconitine could decrease the protein expression of inflammatory factors COX-2, iNOS, TNF-α and IL-1β compared with the model group. Moreover, 12-epi-napelline, aconitine, and mesaconitine could down-regulate the expression of MMP-13 and up-regulate the expression of aggrecan and collagen Ⅱ. In addition, compared with the model group and other Bangna alkaloids, 12-epi-napelline significantly up-regulated the expression of FGF2. Therefore, 12-epi-napelline was selected for the animal experiment in vivo. Immunohistochemistry results showed that 12-epi-napelline could significantly reduce the expression of MMP-13 and TNF-α in cartilage tissues, and up-regulate the expression of FGF2 compared with the model group. In conclusion, among the seven Bangna alkaloids, 12-epi-napelline can promote the repair of OA in rats by down-regulating the expression of MMP-13 and TNF-α and up-regulating the expression of FGF2.

Topics: Aconitine; Aconitum; Aggrecans; Alkaloids; Animals; Cells, Cultured; Cyclooxygenase 2; Fibroblast Growth Factor 2; Interleukin-1beta; Iodoacetic Acid; Lipopolysaccharides; Male; Matrix Metalloproteinase 13; Medicine, Tibetan Traditional; NF-kappa B; Osteoarthritis; Rats; Rats, Sprague-Dawley; Tumor Necrosis Factor-alpha

A simple and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed and validated for the quantification of bulleyaconitine A (BLA) in human serum. Samples were extracted from human serum by protein precipitation. Domperidone was used as internal standard (I.S.). Chromatography separation was performed on a Waters Sunfire C18 column (2.1 x 50 mm, 5 microm) with a gradient mobile phase. Detection was performed in positive mode using multiple reaction monitoring (MRM) of the transition m/z 644.2>584.3 for BLA and m/z 426.3>175.3 for I.S. The proposed method was validated in a linear range of 0.0587-11.7 ng/ml. Intra- and inter-day precision was better than 9.88% and 7.76%. The recovery for BLA was 97.90%, and for I.S. 89.40%. This method provides a simple, rapid, specific, and sensitive tool for the quantitative determination of BLA in human serum, and can be used to monitor serum levels in patients treated with BLA.

Topics: Aconitine; Analgesics; Blood Chemical Analysis; Chemical Precipitation; Chromatography, Liquid; Humans; Linear Models; Osteoarthritis; Proteins; Reproducibility of Results; Sensitivity and Specificity; Tandem Mass Spectrometry; Time Factors

The central nervous system can regulate peripheral inflammation, but the efferent neuronal routes and the mediators remain poorly defined. One candidate is the cholinergic pathway, which releases acetylcholine (ACh). This neurotransmitter can bind to the alpha7 cholinergic receptor (alpha7R) expressed by nonneuronal cells and reduce inflammation. To test this possibility, we evaluated the expression of alpha7R and its potential role as a target in rheumatoid arthritis (RA).. The expression of alpha7R in human synovium and fibroblast-like synoviocytes (FLS) was determined using immunohistochemical, Western blot, and quantitative polymerase chain reaction (PCR) analyses. The effects of ACh in vitro were determined in interleukin-1 (IL-1)-stimulated FLS using immunoassays for protein, quantitative PCR for messenger RNA (mRNA), luciferase reporter constructs for IL-6 and NF-kappaB promoter activity, and electrophoretic mobility shift assays. Expression of alpha7R was knocked down with small interfering RNA (siRNA) or was inhibited with the selective alpha7R antagonist methyllycaconitine (MLA).. Protein and mRNA for alpha7R were demonstrated in RA and osteoarthritis synovium and cultured synoviocytes. Expression in synovium was mainly in the intimal lining. ACh significantly reduced the production of IL-6, CXCL8, CCL2, CCL3, CCL5, and granulocyte colony-stimulating factor by IL-1-stimulated FLS. This effect was blocked by the alpha7R antagonist MLA or by using alpha7R siRNA to knock down receptor expression. The selective alpha7R agonist PNU-282,987 decreased the production of IL-6 by IL-1-stimulated FLS. ACh did not reduce IL-6 transcription, but it decreased IL-6 mRNA half-life and reduced IL-6 mRNA steady-state levels.. The alpha7 receptor is expressed in the synovium and by synoviocytes. Receptor ligation inhibits cytokine expression in FLS through a posttranscriptional mechanism. Therefore, alpha7R is a potential therapeutic target for inflammatory diseases.

Topics: Acetylcholine; Aconitine; alpha7 Nicotinic Acetylcholine Receptor; Arthritis, Rheumatoid; Benzamides; Blotting, Western; Bridged Bicyclo Compounds; Cells, Cultured; Cytokines; Humans; Immunohistochemistry; Interleukin-6; NF-kappa B; Osteoarthritis; Polymerase Chain Reaction; Proteins; Receptors, Nicotinic; RNA, Messenger; Synovial Membrane