加味芍药甘草汤对卵巢颗粒细胞凋亡自噬及p38 AMPK信号通路的影响

Effects of modified Shaoyao Gancao Decoction on the apoptosis,autophagy and p38 AMPK signaling pathway of ovarian granulosa cells

目的探究加味芍药甘草汤对卵巢颗粒细胞凋亡、自噬及p38 AMPK信号通路的影响。方法培养SD雌性大鼠,分为对照组、模型组、低剂量组、中剂量组及高剂量组,除对照组外其余各组均使用来曲唑灌胃大鼠用于制备大鼠模型,造模成功后灌胃加味芍药甘草汤12.5、25.0、50.0 g/kg,连续14 d,对照组、模型组灌胃等体积蒸馏水。处死大鼠,体外培养卵巢颗粒细胞,采用MTT检测细胞活性;流式细胞术检测细胞凋亡;Western Blot检测Bcl-2、Bax、Beclin-1、LC3Ⅱ/Ⅰ、p-p38 AMPK及p38 AMPK蛋白表达。将通路抑制剂SB203580处理高剂量组的卵巢颗粒细胞,按照上述方法检测卵巢颗粒细胞增殖活性、凋亡及自噬的作用。结果与对照组[48 h(102.23±6.45)%,72 h(98.24±8.71)%,(0.72±0.05)]相比,模型组细胞增殖活性[48 h(56.87±4.22)%,72 h(47.62±4.16)%]、Bcl-2蛋白表达(0.25±0.04)降低,差异均有统计学意义(t=10.220、9.080及12.170,均P<0.05);凋亡率、Bax、Beclin-1、LC3Ⅱ/Ⅰ及p-p38 AMPK蛋白表达增加,差异均有统计学意义(均P<0.05)。与模型组相比,低剂量组、中剂量组及高剂量组细胞增殖活性、Bcl-2蛋白表达增加,差异均有统计学意义(均P<0.05);凋亡率、Bax、Beclin-1、LC3Ⅱ/Ⅰ及p-p38 AMPK蛋白表达降低,差异均有统计学意义(均P<0.05)。加入通路抑制剂SB203580增加细胞活性、Bcl-2蛋白表达,差异均有统计学意义(均P<0.05),降低凋亡率、Bax、Beclin-1及LC3Ⅱ/Ⅰ蛋白表达,差异均有统计学意义(均P<0.05)。结论加味芍药甘草汤可能通过抑制p38 AMPK信号通路抑制多囊卵巢综合征卵巢颗粒细胞凋亡、自噬,促进细胞增殖。

Objective To explore the effect of modified Shaoyao Gancao Decoction on the apoptosis,autophagy,and p38 AMPK signal pathway of ovarian granulosa cells.Methods SD female rats were cultured and divided into control group,model group,low-dose group,middle-dose group,and high-dose group.In addition to the rats in the control group,rats in other groups were administrated letrozole to generate rat models.After the model was successfully established,rates were administrated with 12.5,25.0,and 50.0 g/kg of modified Shaoyao Gancao Decoction for 14 days.The rates in the control group and the model group were given intragastrically equal volumes of distilled water.The rats were sacrificed,and ovarian granulosa cells were harvested and cultured in vitro.Cell viability was detected by MTT.Cell apoptosis was detected by flow cytometry.The protein expression of Bcl-2,Bax,Beclin-1,LC3Ⅱ/Ⅰ,p-p38 AMPK,and p38 AMPK were detected by Western blot.The pathway inhibitor SB203580 was treated with ovarian granulosa cells in the high-dose group,and cell proliferative activity,apoptosis,and autophagy were detected according to the above method.Results Compared with the control group[48 h(102.23±6.45)%,72 h(98.24±8.71)%,(0.72±0.05)],cell proliferation activity[48 h(56.87±4.22)%,72 h(47.62±4.16)%]and Bcl-2(0.25±0.04)protein expression decreased(t=10.220,9.080 and 12.170,P<0.05),but cell apoptosis rate,Bax,Beclin-1,LC3Ⅱ/Ⅰ,and p-p38 AMPK protein expression increased in the model group(P<0.05).Compared with the model group,cell proliferation activity and Bcl-2 protein expression were increased(P<0.05),but cell apoptosis rate,Bax,Beclin-1,LC3Ⅱ/Ⅰ,p-p38 AMPK protein expression were decreased in the low,middle,and high-dose groups(P<0.05).The addition of pathway inhibitor SB203580 increased cell viability,Bcl-2 protein expression(P<0.05),and decreased cell apoptosis rate,Bax,Beclin-1,and LC3Ⅱ/Ⅰprotein expression(P<0.05).Conclusion Modified Shaoyao Gancao Decoction may inhibit the apoptosis and autophagy and promote the proliferation of ovarian granulosa cells by inhibiting the p38 AMPK signaling pathway in polycystic ovary syndrome.