丹酚酸B干预PI3K/AKT通路对STZ诱导大鼠胰岛细胞凋亡的影响

Effect of Salvianolic acid B on PI3K/Akt pathway on STZ induced apoptosis of rat islets cells

关键词:  丹酚酸B  胰岛团  凋亡  Akt

Key words:  Salvianolic acid B  islets  apoptosis  Akt

摘要:

[目的] 研究丹酚酸B是否对受损的大鼠胰岛团有保护作用。[方法] 纯化的大鼠胰岛团分为5组：空白对照组（Control），链脲佐菌素（STZ）处理组，丹酚酸B（SalB）低、中、高剂量组（STZ+SalB 15 μmol/L、STZ+SalB 30 μmol/L、STZ+SalB 60 μmol/L）。空白对照组的胰岛团以柠檬酸-柠檬酸钠缓冲溶液处理；STZ处理组的胰岛团以8 μmol/L的STZ处理4 h后换成普通完全培养基；STZ+SalB各剂量组先以STZ处理4 h，换掉STZ溶液，以不同浓度的SalB处理72 h。TUNEL染色法检测各组胰岛团凋亡情况。ELISA法检测各组胰岛团上清液中的胰岛素水平。SalB低、中、高剂量直接处理胰岛团72 h，荧光二乙脂/碘丙啶（FDA/PI）双染法检测胰岛团活细胞和晚期凋亡细胞比例。蛋白免疫印迹法（Western blot）检测各组胰岛团中PI3K、Akt和p-Akt蛋白的表达。[结果] 与空白对照组比较，STZ处理组的TUNEL阳性率显著升高（P<0.01）；与STZ处理组比较，中、高剂量SalB组TUNEL阳性率显著下降（P<0.01），表明中、高剂量SalB有效抑制STZ诱导的大鼠胰岛细胞凋亡。与空白对照组比较，低、中、高剂量SalB组的FDA阳性（活细胞）的百分比无统计学差异（P>0.05），表明低、中、高剂量SalB对正常的胰岛细胞没有损伤作用。与空白对照组比较，STZ处理组胰岛团的胰岛素分泌水平显著下降（P<0.01）；与STZ处理组比较，中、高剂量SalB促进胰岛团胰岛素释放（P<0.05）。与STZ处理组比较，中、高剂量SalB升高PI3K蛋白水平，对Akt蛋白表达无影响，显著升高p-Akt的水平。[结论] SalB抑制STZ诱导的胰岛细胞凋亡，且SalB本身对胰岛细胞无损伤，其抑制凋亡的机制可能与促进Akt磷酸化激活PI3K/Akt信号通路有关。

Abstract:

[Objective] To study whether salvianolic acid B (SalB) exert protective effects on damaged rat islets.[Methods] The purified rat islets were divided into 5 groups:control group,streptozocin (STZ)-treated group,low dose of SalB (STZ+SalB15),medium (STZ+SalB30) and high dose of SalB (STZ+SalB60) groups. The pancreatic islets in Control group were treated with citric acid-sodium citrate buffer solution. The islets in STZ-treated group were treated with 8 μmol/L of STZ for 4 h and then replaced with ordinary complete medium. The STZ+SalB groups were treated with STZ for 4 h. The STZ solution was removed and islets were treated with different concentrations of SalB(15 μmol/L,30 μmol/L,60 μmol/L) for 72 h. The apoptosis of each group of islets was detected by TUNEL staining. Insulin levels in the supernatant of islets were detected by ELISA. Then,islets were directly treated with low(15 μmol/L),medium (30 μmol/L) and high (60 μmol/L) doses of SalB,and the ratio of live cells/late apoptotic cells were detected by FDA/PI double staining. Western blot was used to detect the expression of PI3K,Akt and p-Akt protein in each group of islets.[Results] Compared with Control group,TUNEL positive rate of islets increased significantly in STZ group(P<0.01). Medium and high doses of SalB significantly decreased the TUNEL positive rate by comparing with STZ group(P<0.01),indicated that medium and high doses of SalB effectively inhibited STZ-induced apoptosis of rat islet cells. Compared with Control group,there was no significant difference in the percentage of FDA-positive (live cells) in the low,medium and high doses of SalB groups (P>0.05),indicated that the low,medium and high doses of SalB have no damaging effect on normal islet cells. Compared with Control group,insulin release of islets significantly decreased in STZ group (P<0.01). Compared with STZ group,medium and high doses of SalB promoted insulin release of the islets (P<0.05). Compared with STZ group,the medium and high doses of SalB increased the levels of PI3K protein,and had no effects on the expression of Akt protein,whereas significantly increased the levels of p-Akt.[Conclusion] SalB inhibits STZ-induced apoptosis of islet cells,and SalB itself has no damage on islet cells. The mechanism of its inhibition of apoptosis may be related to the activation of PI3K/Akt signaling pathway by enhancing Akt phosphorylation.