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四物汤含药血清对多囊卵巢综合征卵巢颗粒细胞自噬的影响
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| 篇名： | 四物汤含药血清对多囊卵巢综合征卵巢颗粒细胞自噬的影响 |
| TITLE： | Effects of medicated serum of Siwutang on autophagy of ovarian granulosa cells in polycystic ovarian syndrome |
| 摘要： | 目的 探讨四物汤含药血清对多囊卵巢综合征（PCOS）卵巢颗粒细胞自噬的影响及潜在机制。方法取3月龄雌性SD大鼠，分别以生理盐水和不同剂量四物汤灌胃，制备空白血清和不同浓度[0.52、1.04、2.08g/（kg·d）]四物汤含药血清。筛选四物汤含药血清干预浓度后，将人卵巢颗粒细胞KGN分为对照组（细胞不进行任何处理）、脱氢表雄酮（DHEA）组（以50μmol/L的DHEA处理细胞48h）、空白血清组（以50μmol/L的DHEA处理细胞48h后，再用10%空白血清处理72h）、中浓度四物汤含药血清组（以50μmol/L的DHEA处理细胞48h后，再用10%中浓度四物汤含药血清处理72h），观察各组细胞中的自噬体数量以及通路相关蛋白[果糖-1，6-二磷酸酶1（FBP1）、哺乳动物雷帕霉素靶蛋白（mTOR）、磷酸化mTOR（p-mTOR）]、自噬相关蛋白[p62、微管相关蛋白1轻链3（LC3）]和FBP1mRNA的表达水平。将（转染）细胞分为四物汤组（以50μmol/L的DHEA处理细胞48h后，再用10%中浓度四物汤含药血清处理72h）、四物汤+si-NC组（以50μmol/L的DHEA处理阴性对照小干扰RNA转染细胞48h后，再用10%中浓度四物汤含药血清处理72h）、四物汤+si-FBP1组（以50μmol/L的DHEA处理FBP1小干扰RNA转染细胞48h后，再用10%中浓度四物汤含药血清处理72h），考察敲低FBP1对四物汤上述作用的影响。结果与对照组比较，DHEA组细胞的自噬体数量明显增加，LC3-Ⅱ/LC3-Ⅰ、p-mTOR/mTOR、FBP1蛋白及mRNA的表达水平均显著升高，p62蛋白的表达水平显著降低（P＜0.05）。与DHEA组和空白血清组比较，中浓度四物汤含药血清组细胞的自噬体数量明显减少，LC3-Ⅱ/LC3-Ⅰ显著降低，p-mTOR/mTOR和p62蛋白、FBP1蛋白及mRNA的表达水平均显著升高（P＜0.05）。敲低FBP1后，与四物汤+si-NC组比较，四物汤+si-FBP1组细胞的存活率、p62蛋白的表达水平、FBP1蛋白及mRNA的表达水平、p-mTOR/mTOR均显著降低，LC3-Ⅱ/LC3-Ⅰ显著升高（P＜0.05）。结论四物汤可通过上调KGN细胞中FBP1蛋白及mRNA的表达来促进mTOR蛋白的磷酸化，从而抑制其自噬。 |
| ABSTRACT： | OBJECTIVE To investigate the effects of medicated serum of Siwutang on autophagy of ovarian granulosa cells （KGN cells） in polycystic ovarian syndrome （PCOS） and its underlying mechanism. METHODS Blank serum and different- concentration medicated serum of Siwutang were prepared by intragastric administration of normal saline and different doses of Siwutang [0.52， 1.04， 2.08 g/（kg·d）] in 3-month-old female SD rats. After screening the intervention concentration of Siwutang medicated serum， KGN cells were divided into control group （without any treatment）， dehydroepiandrosterone （DHEA） group （treated with 50 μmol/L DHEA for 48 h）， blank serum group （treated with 50 μmol/L DHEA for 48 h and with 10% blank serum for 72 h） and medium-concentration of Siwutang medicated serum group （treated with 50 μmol/L DHEA for 48 h and with 10% medium-concentration Siwutang medicated serum for 72 h）. The number of autophagosomes was observed in each group， and protein expressions of pathway-related proteins [fructose-1， 6-bisphosphatase 1 （FBP1），mammalian target of rapamycin （mTOR）， phosphorylated mTOR （p-mTOR）]， autophagy-related proteins [p62， microtubule-associated protein 1 light chain 3 （LC3）] and mRNA expression of FBP1 were also detected. The （transfected） cells were further divided into Siwutang group （treated with 10% medium dose of Siwutang medicated serum for 72 h after 48 h intervention with 50 μmol/L DHEA）， Siwutang+si-NC group [negative control small interfering RNA （siRNA） transfected cells treated with 50 μmol/L DHEA for 48 h， and then with 10% medium-concentration of Siwutang medicated serum for 72 h] and Siwutang+si-FBP1 group （FBP1 siRNA transfected cells treated with 50 μmol/L DHEA for 48 h， and then with 10% medium-concentration Siwutang medicated serum for 72 h）. The effects of knocking down FBP1 on the above-mentioned effects of Siwutang were detected. RESULTS Compared with control group， DHEA group exhibited an increase in the number of autophagosomes， an elevated LC3-Ⅱ/LC3-Ⅰ and p-mTOR/mTOR， as well as increases in protein and mRNA expressions of FBP1， and decreased protein expression of p62 （P＜0.05）. Compared to both DHEA group and blank serum group， the medium-concentration of Siwutang medicated serum group showed a decrease in the number of autophagosomes， a decrease in LC3-Ⅱ/LC3-Ⅰ， and increases in p-mTOR/mTOR， protein expression of p62， protein and mRNA expressions of FBP1 （P＜0.05）. After knocking down FBP1， compared with Siwutang+si-NC group， Siwutang+si-FBP1 group showed a significant decrease in cell viability， protein expression of p62 ， protein and mRNA expressions of FBP1 as well as p-mTOR/mTOR， and an increase in LC3-Ⅱ/LC3-Ⅰ （P＜0.05）. CONCLUSIONS Siwutang can promote the phosphorylation of mTOR protein by up- regulating the protein and mRNA expressions of FBP1 in KGN cells， thus inhibiting autophagy of KGN cells. |
| 期刊： | 2025年第36卷第02期 |
| 作者： | 邵艳社；许雪梅；杨宝芹；李会娟；姬霞 |
| AUTHORS： | SHAO Yanshe，XU Xuemei，YANG Baoqin，LI Huijuan，JI Xia |
| 关键字： | 四物汤；含药血清；多囊卵巢综合征；卵巢颗粒细胞；FBP1/mTOR信号通路 |
| KEYWORDS： | Siwutang； medicated serum； polycystic ovarian syndrome； ovarian granulosa cells； FBP1/mTOR signaling pathway |
| 阅读数： | 4 次 |
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