Bu-Shen-Tian-Jing formulas alleviate the mitochondrial damage induced by oxidative stress in ovarian granulosa cells exposed to DEHP through the HDAC3-HSP90AA pathway

CONTEXT: di-(2-Ethylhexyl) phthalate (DEHP) has potential reproductive toxicity. Bu-Shen-Tian-Jing formulations (BSTJFs) are beneficial for female reproductive capacity. However, BSTJF2 has much lower cytotoxicity than BSTJF1. OBJECTIVE: To investigate the effects of BSTJFs on ovarian granulosa cells exposed to DEHP and determine the potential molecular mechanisms. METHODS AND MATERIALS: Human granulosa-like tumor cell line (KGN) cells were divided into control, DEHP, BSTJF1 and BSTJF2 groups. The DEHP group were given 1 μM DEHP for 24 h. They were then given BSTJF1 at 200 μg/mL or BSTJF2 at 100 μg/mL for 24 h. The control group was treated with the same concentration of DMSO (0.1%). Oxidative stress and mitochondrial function were measured. The mRNA and protein expression levels of HDAC3 and HSP90AA were determined. Integrative network pharmacology analysis of BSTJF2 was also performed. RESULTS: DEHP (1 μM) significantly suppressed the proliferation of KGN cells by 17%, significantly increased ROS levels by 28% and MDA levels by 47%, significantly decreased MMP levels by 22% and mtDNA copy by 30%. DEHP significantly increased protein expression of HDAC3 by 21%and HSP90AA by 64%. All these changes were significantly reversed by BSTJFs. Integrative network pharmacology analysis revealed HSP90AA was a key target (degree = 8). Both RGFP966 and BSTJF2 significantly reversed the increased expression of HDAC3 and HSP90AA, attenuated oxidative stress, and mitochondrial damage which were induced by DEHP. CONCLUSION: BSTJFs might have therapeutic potential on oxidative stress and mitochondrial damage through the HDAC3/HSP90AA pathway which encourages further clinical trials.