Cytoprotective effects of Rxa in inhibiting apoptosis induced by H_2O_2 via regulating Ca~(2+) proton flux in cultured human hepatocytes L02 cell line

AIM To evaluate the cytoprotective effect of Rxa, a kind of extract of Radix salviae miltiorrhizae (RSM), in inhibiting apoptosis induced by H 2O 2 via Ca 2+ signal pathway in cultured human hepatocytes L02 cell line. METHODS L02 cells were divided into three groups. ① control group (L group); ② Rxa pre treated group (LaH group, Rxa 2*!mmol/*!L , H 2O 2 10*!μmol/*!L ); and ③ Rxa non treated group (LH group, H 2O 2 10*!μmol/*!L ). The indexes of normal cells, earlier apoptotic cells (Annexin V + cells), and necrotic cells (PI + cells) were measured with flow cytometry (FCM) combined with fluorescence microscopy; the intracellular free calcium concentration ([Ca 2+ ]i) was measured with amp combined with single cell microfluorescence [Ca 2+ ]i measurement, and the morphological changes of cell membrane surface untrastructure was observed by scanning electron microscopy during the periods of Rxa and/*!or H 2O 2 incubated with L02 cells for 0,0 5, 1, 2, 4, 6, 8 hours. RESULTS The increase of [Ca 2+ ]i and Ca 2+ spiking induced for 0 5*!h in LH group, and Ca 2+ oscillation of L02 cells appeared for 1*!h when [Ca 2+ ]i 400*!nmol/*!L , probably; the early phenomenon of PS transformation was showed for 2*!h - 4*!h ; and the [Ca 2+ ]i and index of Annexin V + cells increased for 4*!h - 8*!h significantly; the membrane protuberance, budding and atrophy were observed subsequently. By contrast, [Ca 2+ ]i and index of Annexin V + cells in LaH group were all lower than that in LH group ( a P 0 01, a F =245 30; b P 0 01, b F =72 12), the membrane injury was also weaker in that one. CONCLUSION H 2O 2 induced apoptosis in L02 cells is mediated through an increase of [Ca 2+ ]i; Rxa has cytoprotective effect, which may relate to its effect of calcium blocking.