Protective Effect of a Derivative of MNQ Against LPS-Induced Inflammatory Injury in Bovine Ovarian Follicular Granulosa Cells in Vitro
The aim of this experiment was to eliminate the inflammatory response of bovine ovarian follicular granulosa cells(GCs)exposed to lipopolysaccharide(LPS)in vitro and to attenuate LPS-induced functional disorders of follicular GCs using D19,a derivative of 2-methoxy-1,4-naphthoquinone(MNQ)from the extract of Impatiens balsamina L.Ovarian tissues were collected from sexually mature and healthy Holstein cattle,and follicular GCs were isolated and cultured.The effect of D19 and LPS on the survival of follicular GCs was determined by MTT assay.The experiment was divided into 3 groups:control,LPS-treated and D19-combined LPS-treated groups,with 3 replicates in each group.The relative expression of inflammatory factors and genes related to steroid synthesis was determined by qRT-PCR.TEM was performed to observe the protective effect of D19 against cellular inflammatory injury.ELISA was performed to measure the levels of estradiol(E2)and progesterone(P4)in the supernatant of the culture fluid.The results showed that the maximum no-cytotoxic concentration of D19 acting on follicular GCs for 12 h was 64 μmol·L-1.LPS concentration of 10 μg·mL-1 had little effect on the survival of follicular GCs after 12 h of action,but the relative expression of the inflammatory factors IL-6,IL-1β,and TNF-α was significantly higher(P＜0.01),whereas the relative expression of the inflammatory factors in the D19+L group was significantly lower compared with the LPS group(P＜0.01).The protective effect of D19 against LPS-induced structural damage to organelles was demonstrated by TEM observation.ELISA results showed that the levels of E2 and P4 in the culture broth were significantly reduced after LPS treatment(P＜0.01),and the relative expression of steroid synthesis-related genes CYP19A1,CYP11A1,3β-HSD,and STAR as detected by qRT-PCR was also significantly reduced(P＜0.01).However,the amount of E2 and P4 secretion as well as the relative expression of steroid synthesis-related genes were significantly higher in the D19 combined with LPS treatment than that in the LPS group(P＜0.01).In the present study,we demonstrated that D19,a derivative of MNQ,has the function of eliminating the inflammatory response of follicular GCs induced by LPS in vitro,and it can alleviate the functional disorders of follicular GCs caused by LPS to a certain extent.
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