Growth Hormone Enhances Mitochondrial Function And Inhibits Ovarian Granulosa Cell Apoptosis

Objective Growth hormone(GH)has been used as an adjuvant in in vitro fertilization(IVF)for more than 20 years and it is effective in patients with poor ovarian responders(POR)in particular.However,the studies on the specific mechanism of GH functioning are scarce.In this study,cisplatin was used to establish a model of granulosa cell apoptosis,and the possible role of GH in promoting the growth of granulosa cells was explored,providing some theoretical basis for clinical application of GH.Methods Taking KGN cells as the research object,the experiment was divided into four groups.The control group: normal KGN cells were cultured for 24h;GH treatment group: normal KGN cells were added with 1ul / ml GH for 24h;cisplatin induced group: normal KGN cells were added with 10 ug / ml cisplatin cultured after 24 hours,washed and discarded with PBS,and then added to the culture solution for 24 hours;cisplatin + GH treatment group: 10 ug / ml cisplatin treatment for 24 h and then added GH culture for 24 h.The CCK-8 kit was used to detect the effects of KGN cell proliferation in the four groups;flow cytometry was used to detect the apoptosis of KGN cells;Western blot was used to detect the impact on the expression levels of apoptosis proteins Bcl-2,Bax and antioxidant proteins Sod2 and Sir T3;Cell ROX? Orange Reagent kit was used to measure the level of reactive oxygen species(ROS);JC-1 dye was used to assess the mitochondrial membrane potential of KGN cells and real-time quantitative PCR was performed to test the mitochondrial DNA copy number.Results 1.Cell proliferation: The proliferation of KGN cells decreased significantly after cisplatin treatment,while GH increased cell proliferation;2.Flow cytometry detection of apoptosis: Apoptosis of KGN cells increased significantly after cisplatin treatment,while apoptosis was reduced and survival rate increased after treatment with GH;3.Western blot detection of apoptosis and anti-oxidation related proteins: after cisplatin treatment of KGN cells,the expression of Bax was increased,and the expression of Bcl-2,Sir T3,Sod2 was reduced,and on this basis the expression level of Bax protein was down-regulated,and the expressions of Bcl-2,Sir T3,and Sod2 were up-regulated after adding GH;4.Cell ROX? Orange Reagent kit for detecting reactive oxygen: After cisplatin treatment,the ROS level of KGN cells was significantly increased,while was significantly reduced after the addition of GH;5.JC-1 kit for assessing mitochondrial membrane potential: cisplatin treatment significantly reduced the membrane potential,but after GH treatment,the decreased membrane potential increased again;6.RT-PCR for testing mitochondrial DNA copy number: After cisplatin inducing KGN cell damage,the mitochondrial DNA copy number was significantly reduced,and the addition of GH increased the mitochondrial DNA copy number.Conclusions 1.Growth hormone can promote the growth of granular cells.2.GH inhibits cisplatin-induced apoptosis of granulosa cells by activating Sir T3 / Sod2,regulating ROS levels,promoting Bcl-2 and simultaneously inhibiting Bax expression to enhance mitochondrial function.