Inhibition of Hepatic Stellate Cell Proliferation and Extracellular Ca2+ Influx by Leptin-siRNA / 华中科技大学学报(医学版)

Objective To evaluate the effect of silencing leptin by small interfering RNA(siRNA)on the expression of lep‐tin ,and apoptosis ,proliferation and intracellular Ca2+ concentration([Ca2+ ]i )of hepatic stellate cells(HSCs)and to provide evi‐dence for liver fibrosis gene therapy.Methods HSCs were divided into normal control group ,blank vector group ,siRNA nega‐tive control group and leptin‐siRNA group.After transfection of the leptin‐siRNAs into HSCs ,cell proliferation was measured by MTT assay.Cell cycle and apoptosis were measured by flow cytometry.Expression of leptin was detected by immunocyto‐chemistry and Western blot. [Ca2+ ]i was measured by Fura‐2/AM loading.Results Compared with the normal control group , the blank vector group and the siRNA control group ,the protein expression of leptin and the cell growth were significantly in‐hibited in the leptin‐siRNA group(P<0.05). The proliferation rate of HSCs was significantly different at different time points (24 ,48 and 72 h)(P<0.05).The cell apoptosis rate was increased significantly in the leptin‐siRNA group(P<0.01).At the same time ,Leptin‐siRNA‐induced [Ca2+ ]i was also significantly reduced(P<0.05).Conclusion The leptin gene may play an important role in liver fibrosis progression and is potentially a novel predictive and prognostic marker for liver fibrosis.

Toosendanin Modifies K~+-and Ca~(2+)-Channel Activity and Intracellular Ca~(2+) Concentration / 生物化学与生物物理进展

Usage of the fruit and bark of a Melia-family plant as a digestive tract-parasiticide and agricultural insecticide was recorded about two thousant years ago in ancient China. Toosendanin (TSN), a triterpenoid, is an effectual ingredient extracted from the plant. Studies have demonstrated that TSN selectively affects neurotransmitter release, effectively antagonizes botulism, induces cell differentiation and apoptosis and inhibits proliferation of various human cancer cells, inhibits feeding and dovelopment in insects and modifies K+- and Ca2+-channel activity. The research data to demonstrate that TSN inhibits K+-channel and facilitates L-type Ca2+-channel are summarized, and the mechanism of action of TSN is discussed.

Buffering Contribution of Mitochondria to the Ca2+i Increase by Ca2+ Influx through Background Nonselective Cation Channels in Rabbit Aortic Endothelial Cells

To prove the buffering contribution of mitochondria to the increase of intracellular Ca2+ level ([Ca2+]i) via background nonselective cation channel (background NSCC), we examined whether inhibition of mitochondria by protonophore carbonylcyanide m-chlorophenylhydrazone (CCCP) affects endothelial Ca2+ entry and Ca2+ buffering in freshly isolated rabbit aortic endothelial cells (RAECs). The ratio of fluorescence by fura-2 AM (R340/380) was measured in RAECs. Biological state was checked by application of acetylcholine (ACh) and ACh (10microM) increased R340/380 by 1.1+/-0.15 (mean+/-S.E., n=6). When the external Na+ was totally replaced by NMDG+, R340/380 was increased by 1.19+/-0.17 in a reversible manner (n=27). NMDG+-induced [Ca2+]i increase was followed by oscillatory decay after [Ca2+]i reached the peak level. The increase of [Ca2+]i by NMDG+ was completely suppressed by replacement with Cs+. When 1microM CCCP was applied to bath solution, the ratio of [Ca2+]i was increased by 0.4+/-0.06 (n=31). When 1microM CCCP was used for pretreatment before application of NMDG+, oscillatory decay of [Ca2+]i by NMDG+ was significantly inhibited compared to the control (p < 0.05). In addition, NMDG+-induced increase of [Ca2+]i was highly enhanced by pretreatment with 2microM CCCP by 320+/-93.7%, compared to the control (mean+/-S.E., n=12). From these results, it is concluded that mitochondria might have buffering contribution to the [Ca2+]i increase through regulation of the background NSCC in RAECs.