Purified extract from Clematis mandshurica prevents adenoviral-TRAIL induced apoptosis on rat articular chondrocytes.

Since TNF-related apoptosis inducing ligand (TRAIL) is one of several apoptotic stimuli on articular chondrocytes, the modulation of the mechanism mediated by TRAIL could be considered as a novel strategy for the treatment of osteoarthritis (OA). Previous studies demonstrated that Clematis mandshurica prevents staurosporin-induced apoptosis in articular chondrocytes. This study was undertaken to examine whether Clematis mandshurica could prevent TRAIL-induced apoptosis in articular chondrocytes. Our data show that Clematis mandshurica prevents adenoviral TRAIL (Ad-TRAIL)-induced apoptosis in primary cultured articular chondrocytes. Clematis mandshurica prevents Ad-TRAIL-induced down-regulation of 14-3-3 and phosphorylated Akt. In addition, Clematis mandshurica treatment prevents the Ad-TRAIL-induced reduction of the interactions between 14-3-3 with phospho-ser112-Bad and phospho-ser136-Bad, and BcL-xL with phospho-ser155-Bad. A better understanding of the mechanism underlying inhibition of apoptosis in OA chondrocytes by Clematis mandshurica might lead to the development of a new therapeutic strategy for OA.

A purified extract from Clematis mandshurica prevents staurosporin-induced downregulation of 14-3-3 and subsequent apoptosis on rat chondrocytes.

OBJECTIVE: To dissect the mechanism of the protection of staurosporin-induced apoptosis on rat chondrocytes by a purified extract from Clematis mandshurica. DESIGN: Primary cultured rat articular chondrocytes as well as RCJ3.1C.18 cells were incubated with 1 microM staurosporin and 300 microg/ml purified extract from Clematis mandshurica. Western blot assay, silencing 14-3-3 gene and immunoprecipitation were conducted. RESULTS: Clematis mandshurica prevented staurosporin-induced downregulation of several antiapoptotic bcl-2 family proteins Bcl-xL and Bcl-2, and staurosporin-induced upregulation of an apoptotic bcl-2 family protein Bax. Clematis mandshurica also prevented staurosporin-induced downregulation of a premitochondrial antiapoptotic protein 14-3-3. It is noticeable that siRNA to 14-3-3 abolished the prevention of caspase-3 activation by Clematis mandshurica. Furthermore viability assay corroborated that silencing of 14-3-3 gene abolished this apoptosis protection efficacy by Clematis mandshurica. Immunoprecipitation assay elucidated that Clematis mandshurica prevented the staurosporin-induced reduction of the interactions between 14-3-3 with phospho-ser112-Bad and Bcl-xL to phospho-ser155-Bad. CONCLUSIONS: Clematis mandshurica prevents staurosporin-induced apoptosis of rat chondrocytes via 14-3-3.

Transgenic ginseng cell lines that produce high levels of a human lactoferrin.

In order to produce a human lactoferrin (hLf) protein in cultured plant cells, we developed Korean ginseng (Panax ginseng) cell line using an oxidative stress-inducible peroxidase (SWPA2) promoter and characterized the production of human lactoferrin in cultured cells. A construct containing a targeting signal peptide from tobacco endoplasmic reticulum fused to human lactoferrin cDNA under the control of SWPA2 promoter was engineered. Transgenic Korean ginseng cell lines that produced a recombinant hLf protein were successfully generated and confirmed by PCR and Southern blot analyses. Western blot and ELISA analyses showed that hLf protein was synthesized in the transgenic cells. The production of hLf showed a maximal level (up to 3.0% of total soluble protein) in the stationary phase of callus cultures. These results suggest that the transgenic cell lines in this study will be biotechnologically useful for the commercial production of hLf protein in cell cultures, with no need for purification.