Machanism Exploration Of Mesenchymal Stem Cell-conditioned Medium Ameliorating Triptolide Induced Granulosa Cells Damage

Granulosa cells are considered very essential for their role in maintaining the microenvironment of oocytes development and maturation in mammalian ovary. The functional status of granulosa cells impacts on the quality and subsequent embryo potential development of oocytes. Triptolide is an extract and principal bioactive component of the Chinese herb Tripterygium wilfordii Hook F (TWHF). TWHF were found having reproduction toxicity during the treatment of autoimmune and inflammatory diseases, can cause female menstrual disorders and amenorrhea, and may harm the ovarian granulosa cells. As a main component of TWHF, triptolide could directly hurt human ovarian granulosa cells, but the machanism is not very clear, neither is the strategy in improving these damages entirety. MSCs can produce various of growth factors and cytokines, thus contributes to the treatment of tissue repair and some immunological diseases. We speculated that MSCs might have ameliorative effect on granulosa cells damage induced by triptolide. Thus, in this study, we mainly discussed the ameliorative effect and machanism of MSCs conditioned medium on the ovarian granulosa cells damage induced by triptolide, and tried to propose a strategy to improve triptolide induced ovarian granulosa cells damage.Mesenchymal stem cell-conditioned medium could ameliorated the triptolide induced damage in KGN cellsKGNs are human granulosa-like cell line that can be used as a model for understanding granulosa cell growth. We analysed the cell vitality and cell cycle distribution of KGNs under the treatment of triptolide. The results showed that triptolide inhibited the cell vitality, and induced S-phase arrest of KGNs. We later used Real-time PCR method to evaluate the expression of CDKN1A, which was a negative regulate gene. The results showed that the expression of CDKN1A was abnormally up-regulated by triptolide. MSCs from umbilical cord were successfully isolated and cultured in order to obtaining MSCs conditioned medium. The effect of MSCs conditioned medium on the KGNs damage induced by triptolide was analysed, and the results showed that MSCs conditioned medium improved the triptolide-induced vitality inhibition and extent of S-phase arrest in KGNs. Further more, the abnormal upregulation of CDKN1A was decreased moderately by MSCs conditioned medium. These results suggested that MSCs conditioned medium ameliorated the KGN cell damage induced by triptolide probably through down-regulation of the CDKN1A expression.Mesenchymal stem cell-conditioned medium influenced on the vitality and proliferation of mouse ovarian granulosa cellsWe have confirmed the cell damage of triptolide on the human granulosa-like cell line KGN. However, KGN is a aneuploid granulosa cell line, which is different to normal granulosa cells (GCs). It was reported that there were differences on the aspect of functional regulation and drug susceptibility between KGNs and GCs. Therefore, we speculated that triptolide might impact on KGNs and mouse GCs differently, and the machanism of ameliorating the triptolide induced primary ovarian GCs damage may also different. Thus, the isolation and culture of the mouse primary GCs were performed to analyse the influence of triptolide on them and to discuss the effect of MSCs conditioned medium on mouse ovarian GCs growth. Results showed that triptolide can directly inhibited the vitality and induced the apotosis of GCs, while it had no obvious effect on GCs cell cycle distribution. However, MSCs conditioned medium did not show ameliorative effects on GCs vitality inhibition and apoptosis induced by triptolide. Notably, however, we found that normal GCs vitality was improved by MSCs conditioned medium. Further more, normal GCs proliferation was promoted with the up-regulated expression of PCNA and CCND2. These results suggested that MSCs conditioned medium could promote mouse GCs proliferation through up-regulating the expression of PCNA and CCND2, but it was not effective enough to ameliorate the damage induced by triptolide.In conclusion, triptolide can inhibit the vitality and proliferation of KGNs, and MSCs conditioned medium ameliorated the triptolide induced cell damage in KGNs probably by the inhibition of CDKN1A expression. Triptolide can also inhibit the vitality, and induced the apoptosis of mouse GCs. MSCs conditioned medium can promoted the vitality and proliferation of normal GCs by up-regulating PCNA and CCND2 expression. The different effects between MSCs conditioned medium on the triptolide-induced KGN and mouse granulosa cell damage need to be studied further.