| 摘要: | 血管收縮素II (Angiotensin II ; Ang II ) 是心血管疾病中重要的影響因子，可引起心臟肥大與凋亡的相關蛋白表現，進而造成心臟功能的缺損。實驗室過去發表更證明血管收縮素II可藉活化IGF-IIR訊息途徑經Gαq及鈣離子，造成calcineurin及下游NFAT3之活化誘發心肌肥大。亦由活化Bad造成caspase 9及caspase 3活化，引起心肌細胞凋亡。本實驗中，欲探討當歸是否對於血管收縮素II 所引發的心臟病理性肥大與凋亡具有改善，甚至抑制的效果。當歸，性味甘辛溫，無毒，具活血化瘀之效用。本草綱目提及當歸具『治頭痛，心腹諸痛，潤腸胃筋骨皮膚。治癰疽，排膿止痛，和血補血』之功效。相關動物實驗報導，當歸確實對心肌缺血及心肌梗塞具有保護作用。且我們亦發現當歸萃取物可直接抑制血管收縮素II對IGF-IIR活化作用。因此，我們想進一步探討當歸抑制血管收縮素II 引發之心肌細胞肥大及凋亡的分子機轉及保護作用。
本研究將H9c2心肌細胞以血管收縮素II (5x10-7M)加入傷害，分別pre-treatment和post-treatment不同濃度當歸 (50、100 μg/ml)。再以actin staining、Western blotting assay、DAPI staining及JC-1staining等分析方法，探討當歸保護血管收縮素II引起之心肌細胞肥大及凋亡之機轉。
結果顯示當歸可有效的預防及治療因血管收縮素II 所造成的細胞型態肥大，Western blotting assay中更觀察到當歸對於血管收縮素II 引起的肥大相關蛋白ex:NFATc3、BNP、ANP，其蛋白表現量均有抑制的作用。在DAPI及Western blotting assay，亦發現當歸可降低因血管收縮素II所誘發之凋亡蛋白 (ex:IGF-IIR,caspase3及9)的表現量。同時抗凋亡相關蛋白 (ex:p-PI3K,p-Akt,p-bad及Bcl-xL)也隨當歸的加入，逆轉血管收縮素II 的壓制作用，其蛋白表現量有顯著增加的趨勢。進一步由JC-1 staining中也觀察到，當歸可回復因血管收縮素II所造成之粒線體膜電位不穩定。
本研究結果顯示，當歸能有效降低血管收縮素II 所引起心肌細胞的肥大與凋亡所造成的傷害，同時兼具預防及治療之效用，未來可在心臟保護方面提供一個嶄新的方向！
Angiotensin II is a risk factor for cardiovascular diseases ,and may cause cardiac hypertrophy and apoptosis. Our previous study showed the Angiotensin II can active IGF-IIR signaling pathway to trigger Gαq- calcineurin, and the phosphatase calcineurin will dephosphorylate the transcription factor NFAT3, and let it translocate to the nucleus. Then, the atrial natriuretic peptide (ANP) and b-type natriuretic peptide (BNP) will be overexpression.Active IGF-IIR signaling pathway also could induced cardiac apoptosis from Bad-caspase 9 apoptosis pathway. We applied a cardiovascular protective Traditional Chinese medicine, Radix angelicae sinensis (Danggui), to evaluate its therapeutic effect on Angiotensin II -induced cardiac hypertrophy and apoptosis. H9c2 cardiomyoblast cells were cultured in serum-free medium for 4 hr, then treated with Danggui (50,100 mg/ml) either 1hr before (pre- treatment) or 1hr after (post- treatment ) Angiotensin II treatment and followed another 23hr culture, to detect the protective and therapeutic mechanisms of Danggui against Angiotensin II effects. Cells were harvested for MTT assay, Actin staining, TUNEL assay, Immunofluorescence,western blotting.
In the results, we found Danggui could inhibit Angiotensin II -induced cyto-skeletal change,and prevent Angiotensin II-induced hypertrophy through IGF-IIR- calcineurin-NFAT3 pathway, and further down regulate the cardiac hypertrophy and decrease the pathological hypertrophic proteins ANP and BNP expression levels in myocardiac cells.
On addition, the induction of cardiomyocyte apoptosis, upregulation of pro-apoptotic Bad, instability of mitochondria membrane potential, cytochrome c release and caspase-9,3 activation were observed as well. However, as we applied Danggui pre- or post- Angiotensin II treatment to evaluate its preventive and therapeutic effects on Angiotensin II -induced H9c2 cardiomyoblast cells hypertrophy and apoptosis. The cell size change and hypertrophic markers induced by Angiotensin II were both significantly suppressed by Danggui. Treatment of Danggui reversed the Angiotensin II-induced mitochondria membrane potential instability on H9c2 cells was also observed by JC-1 stain assay. Moreover, Danggui suppressed the activities of caspase-9,3 induce by Angiotensin II to inhibit the apoptosis effect.
Chemical inhibitors JNK (SP600125),MEK (U0126) were applied to investigate who is mediator for Danggui attenuated Angiotensin II stimulated caspase 3 activation. JNK (SP600125) inhibitor totally block Danggui inhibited caspase 3 activation in Angiotensin II -treated H9c2 cells.
Taken together, we identified Danggui treated either prior or after Ang II both highly attenuated the Angiotensin II-induced cardiac hypertrophy and apoptosis effects in H9c2 cardiomyoblast cells. We believe that Danggui might used as a potential candidate for cardiac protective and therapeutic TCM. |
