ABSTRACT
ObjectiveTo observe the effect of Zhuluan decoction on the ovarian reserve function of rats with cyclophosphamide-induced premature ovarian insufficiency， and explore the protective mechanism of Zhuluan decoction in the rat model of premature ovarian insufficiency based on the phosphatidylinositol-3-kinases/protein kinase B/mammalian target of rapamycin （PI3K/Akt/mTOR） signaling pathway. MethodSixty female SD rats were randomly divided into normal group （n=10） and model group （n=50）. The model group was given intraperitoneal injection of cyclophosphamide （50 mg·kg-1 loading dose on the 1st day+8 mg·kg-1 low-dose maintenance on the 2nd–15th days）. After successfully modeling， the rats were randomly divided into model group， positive drug （progynova） group （0.1 mg·kg-1·d-1）， and low-， medium-， and high-dose Zhuluan decoction groups （14， 28， 56 g·kg-1·d-1 ）， with 10 rats in each group. The model group and the normal group were given equal volume of distilled water by gavage， once a day， continuous administration for 21 d. The estrous cycle and body weight of rats in each group were detected， and the ovarian organ index and uterine organ index were calculated. The ovarian tissue pathology and ovarian follicle counts at all levels were determined by hematoxylin-eosin （HE） staining. The content of the serum antimullerian hormone （AMH）， follicle-stimulating hormone （FSH）， luteinizing hormone （LH）， and inhibin-B （INH-B） of rats was determined by enzyme-linked immunosorbent assay （ELISA）， and the protein expression levels of PI3K， Akt， mTOR in the rat ovarian tissue were determined by Western blot. The microtubule-associated protein 1 light chain 3B （LC3B） protein expression in the rat ovarian tissue was determined by immunohistochemistry. ResultAs compared with the blank group， the estrous cycle of rats in the model group was disordered， the body weight， ovarian organ index， and uterine organ index decreased， the number of primordial follicles decreased， and the number of secondary follicles and atretic follicles increased. In the model group， FSH increased （P<0.01）， LH increased （P<0.05）， AMH level decreased （P<0.05）， the protein expression levels of PI3K， Akt， and mTOR in the ovarian tissue decreased （P<0.01）， and the protein expression level of LC3B increased significantly （P<0.01）. As compared with the model group， the above indexes were improved in the progynova group and different doses of Zhuluan decoction groups， the content of AMH increased （P<0.05）， and FSH decreased （P<0.05）. In the progynova group and different doses of Zhuluan decoction groups， the protein expression level of LC3B decreased obviously （P<0.01）， and the protein expression levels of PI3K， Akt， and mTOR all showed an increasing trend. Moreover， there was a statistically significant difference in the progynova group and low- and medium-dose Zhuluan decoction groups （P<0.05）. ConclusionZhuluan decoction may inhibit the occurrence of excessive autophagy in ovarian granulosa cells by activating the PI3K/Akt/mTOR pathway， thereby reversing the effect of modeling on ovarian reserve in rats.
ABSTRACT
ObjectiveTo investigate the regulatory effect of Shoutaiwan on oxidative stress and pyroptosis in lipopolysaccharide （LPS）-induced human extravillous trophoblast （HTR-8/SVneo） cells and provide a new direction for deciphering the mechanism of action of Shoutaiwan. MethodLPS （100 μg∙L-1） was used to induce the injury of HTR-8/SVneo cells （modeling）. Five groups were designed in this study， including a blank group， a model group， a Shoutaiwan （10% Shoutaiwan-containing serum） group， an antioxidant （1 mmol·L-1 NAC） group， and NOD like receptor thermoprotein domain 3 （NLRP3） inhibitor （50 μmol·L-1 MCC950） group. Cell viability was detected by cell counting kit-8 （CCK-8） kit. Hochest 33342/PI double fluorescence staining and flow cytometry were employed to observe cell death. The levels of interleukin-18 （IL-18）， interleukin-1β （IL-1β）， malondialdehyde （MDA）， and superoxide dismutase （SOD） in cell supernatant was determined by enzyme-linked immunosorbent assay （ELISA）. DCFH-DA probe was used to measure the level of intracellular reactive oxygen species （ROS）. Western blot was employed to determine the protein levels of NLRP3， Caspase-1， gastermin D （GSDMD）， and IL-1β in cells， and Real-time fluorescence quantitative polymerase chain reaction（Real-time PCR） to measure the mRNA levels of NLRP3 and Caspase-1 in cells. ResultCompared with the blank group， the modeling decreased the cell viability （P<0.01）， elevated the levels of IL-1β， IL-18， ROS， and MDA， and weakened the activity of SOD （P<0.01）. Furthermore， it up-regulated the protein levels of NLRP3， Caspase-1， GSDMD， and IL-1β and the mRNA levels of NLRP3 and Caspase-1 （P<0.01）. Compared with the model group， Shoutaiwan， NAC， and MCC950 increased the cell viability （P<0.01）. Further， Shoutaiwan and NAC lowered the levels of MDA and ROS and increased the activity of SOD （P<0.01）. Shoutaiwan and MCC950 reduced the IL-1β and IL-18 in cell supernatant （P<0.01）， and down-regulated the protein levels of NLRP3， Caspase-1， GSDMD， and IL-1β and the mRNA levels of NLRP3， Caspase-1， and IL-1β （P<0.05，P<0.01）. ConclusionShoutaiwan can regulate oxidative stress and pyroptosis to attenuate the LPS-induced damage of HTR-8/SVneo cells， which may be the mechanism of Shoutaiwan in preventing recurrent spontaneous abortion.