| 摘要: | 類胰島生長激素 II (insulin growth factor 2 (IGFII) 可在經過缺血後豬的心臟及人類心肌梗塞後表現出來。但是其接受器，IGFIIR，則在正常成人心臟中未曾發現。同時，在IGFII過度表現基因轉殖鼠中出現心臟及腎臟不呈比例之過度肥大生長，這個情形和人類的Beckwith-Wiedeman syndrome 相似。我們實驗室之前的研究亦指出：血管加壓素II (angiotensin II (AngII))在H9c2細胞中透過MEK及JNK活化IGFII及IGFIIR的表現，使得細胞質中鈣離子濃度增加、PLC-β在被Gαq活化後促進calcineurin活化並導致Bad蛋白插入粒線體膜中，進而活化caspase 9和3而引發凋亡。黨蔘 (Codonopsis pilosula)在傳統中草藥中使用於降血壓、增加紅白血球數目。本研究之目的在於探討黨蔘對於AngII加乘Leu27-IGFII於H9c2細胞粒線體膜電位不穩定及凋亡之影響。在我們Luciferase assay實驗結果中，AngII使得IGFIIR promoter活性明顯上升，但是黨蔘卻使其減少。Leu27-IGFII是一個IGFII的相似物並且會強力專一的和IGFIIR結合，引發IGFII//IGFIIR胞內訊號傳遞路線。在MTT assay結果顯示：H9c2細胞生存率明顯地被AngII及Leu27-IGFII所共同抑制；但黨蔘逆轉此情形。黨蔘恢復了AngII及Leu27-IGFII所共同引起之鈣離子湧入 (Fluo 4-AM staining)、粒線體膜電位不穩定(JC-1 staining) 及凋亡(TUNEL staining)現象.Western blotting及fluorescence immunomicroscopy結果顯示：AngII及Leu27-IGFII所加乘誘發之 IGFII/IGFIIR pathway 下游的IGFIIR、Gαq、p-PLC-β3Ser537及calcineurin，Bad的活性，cytochrome c的釋出及 caspase 9和3活性均被黨蔘所減低壓制。而p-BadSer136及Bcl-2減少之情形因黨蔘的加入而恢復則趨勢。所以，免疫沉澱 (co-immunoprecipitation)實驗中黨蔘同時抑制了AngII加Leu27-IGFII所誘發之IGFIIR和Gαq之結合能力。所以，黨蔘在H9c2細胞中可阻斷AngII及Leu27-IGFII所作用而達到減少IGFIIR promoter活性、鈣離子湧入、粒線體膜電位不穩定及凋亡的現象。
Insulin growth factor II (IGFII) is expressed after ischemic stress in pig hearts and after myocardial infarction in humans. However its receptor (IGFIIR) cannot be found in normal adult hearts. Moreover, a mouse IGFII overexpression model showed a heart and kidney hypertrophy phenomenon similar to Beckwith-Wiedeman syndrome in humans. The previous studies from our lab showed that increase in AngII in H9c2 cells causes an elevation in IGFII and IGFIIR through MEK and JNK activation, leading to rise in intracellular Ca2 + ions, activation of calcineurin by PLC-β3 via Gαq, insertion into mitochondrial membranes of Bad, and apoptosis via activation of caspases 9 and 3. Codonopsis pilosula (Dung-shen) is used in traditional Chinese medicine to lower blood pressure, increase red and white blood cell counts, etc. The purpose of our study is to investigate if the addition of C. pilosula will attenuate of the AngII plus Leu27-IGFII-induced mitochondrial outer-memembrane permeability (MOMP) and apoptosis in H9c2 cardiomyoblast cells. Although the luciferase assay showed an induction of IGFIIR promoter activity by AngII, the AngII effect was significantly attenuated by C. pilosula. Leu27-IGFII is an analog of IGFII and binds to IGFII receptor (IGFIIR) strongly, which was applied to enhance the AngII effect. From MTT (3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-tetrazolium bromide) results, it was revealed that AngII plus Leu27-IGFII significantly reduced cell viability, which was reversed by C. pilosula. C. pilosula also reversed Ca2+ influx (Fluo 4-AM), MOMP (JC-1 staining) and apoptosis (TUNEL staining) increased by AngII plus Leu27-IGFII. The induction of upstream factors (IGFIIR, Gαq, p-PLC-β3Ser537 and calcineurin), level of unphosphorylated Bad, cytochrome c release and caspases 9 and 3 activity were all attenuated by C. pilosula treatment, as shown in western blotting assay and immunofluorescence microscopy results. On the contrary, p-BadSer136 and Bcl-2 showed an increasing trend. Moreover, C. pilosula also decreased IGFIIR-Gαq association which was upregulated by AngII plus Leu27-IGFII in co-immunoprecipitation assay. Taken together, C. pilosula is able to suppress the IGFII/IGFIIR apoptotic pathway enhanced by AngII plus Leu27-IGFII in myocardial cells via reducing in Ca2+ influx and MOMP. |
