The protective effects of progesterone on PC12 cells have been revealed under OGD/R condition

To investigate the function of progesterone on OGD/R-induced PC12 cells, we first selected the effective working concentration of progesterone. Under normal condition, 0–500 nM progesterone has no obvious effect on PC12 cells viability. However, 1000 nM progesterone treatment significantly decreased the viability of PC12 cells (Fig. 1A, p  = 0.0169, F = 35.082). Therefore, 500 nM progesterone was selected for the subsequent research. After OGD/R stimulation, the OD values of PC12 cells decreased (Fig. 1B, p  < 0.01, F = 342.172), the production of MDA and ROS increased (Fig. 1C, p  < 0.01, F = 901.084; Fig. 1D, p  < 0.01, F = 291.538), the concentration of SOD decreased (Fig. 1E, p  < 0.01, F = 202.015), the invasion number of PC12 cells decreased (Fig. 1F, p  < 0.01, F = 132.399), the protein patterns of pro-apoptotic genes Bax, cleaved caspase3 increased, the protein patterns of anti-apoptotic gene Bcl-2 decreased (Fig. 1G), and the protein patterns of pro-inflammatory genes IL-1, IL-6 and TNF-α was also increased (Fig. 1H). Moreover, the relative migratory distance of PC12 cells reduced after OGD/R treatment (Fig. 2A, p  < 0.01, F = 63.034). However, with progesterone stimulation, the above injury of PC12 cells induced by OGD/R was diminished and tended to the normal level (Figs. 1B-H and 2A). Thus, the protective effects of progesterone on OGD/R-caused PC12 cells were demonstrated.

Fig. 1

The protective effects of progesterone on PC12 cells have been demonstrated under OGD/R stimulation. A. The working concentration of progesterone has been identified. B. The effect of progesterone on PC12 cells viability has been revealed by CCK-8 assay. C-E. The levels of MDA, ROS, and SOD in PC12 cells have been detected by ELISA kits after the co-treatment of progesterone and OGD/R. F. The invasion number of PC12 cells was examined by transwell assay after the co-treatment of progesterone and OGD/R. G-H. The protein levels of Bax, Bcl-2, cleaved caspase3, IL-1, IL-6, and TNF-α in PC12 cells have been detected by western blot after the co-treatment of progesterone and OGD/R. **p < 0.01 vs. control, ##p < 0.01 vs. OGD/R

Fig. 2

Progesterone affects the migration ability of PC12 cells and regulates FABP5 expression. A. The migration distance of PC12 cells was examined by wound healing assay after the co-treatment of progesterone and OGD/R. B-C. The mRNA and protein levels of FABP5 were examined by qPCR and western blot assays under OGD/R condition. D-E. The mRNA and protein levels of FABP5 were examined by qPCR and western blot assays after si-FABP5 treatment. F. The effect of progesterone and FABP5 on PC12 cells viability has been revealed by CCK-8 assay

FABP5 was identified as a specific target of progesterone

Information from CTD suggested that FABP5 was regarded as a target of progesterone. According to the prediction, FABP5 expression was measured in OGD/R-induced PC12 cells. Data from Fig. 2B-C showed that the expression patterns of FABP5 remarkably increased in OGD/R-induced PC12 cells (Fig. 2B, p  < 0.01, F = 108.001; Fig. 2C, p  < 0.01, F = 128.371), however, progesterone treatment suppressed the expression of FABP5. These results insinuated that FABP5 was a potential target of progesterone. To further detect the function of progesterone/FABP5 in OGD/R-induced PC12 cells, we used small interference RNA technique to knock down FABP5. As presented in Fig. 2D-E, the levels of FABP5 reduced after si-FABP5 treatment (Fig. 2D, p  < 0.01, F = 23.48; Fig. 2E, p  < 0.01, F = 2.862).

Progesterone exerts protective effects on PC12 cells under OGD/R condition by targeting FABP5

Subsequently, the OD value and the concentrations of MDA, ROS and SOD in PC12 cells were measured after progesterone or/and si-FABP5 treatment under OGD/R condition. The data from Fig. 2F (p < 0.01, F = 519.996) showed that the decrease in OD values caused by OGD/R in PC12 cells could be alleviated by treatment with progesterone or si-FABP5. Moreover, the co-treatment of progesterone and si-FABP5 led to more significant remission of PC12 cells viability. Results from Fig. 3A-C showed that the augment of MDA and ROS, and the reduction of SOD induced by OGD/R in PC12 cells could be alleviated by stimulation with progesterone or si-FABP5, which were more obvious after the co-treatment of progesterone and si-FABP5 (Fig. 3A, p  < 0.01, F = 165.016; Fig. 3B, p  < 0.01, F = 206.763; Fig. 3C, p   < 0.01, F = 149.184). Analysis from Fig. 3D (p < 0.01, F = 128.459) showed that progesterone or si-FABP5 treatment could alleviate the decrease in the number of PC12 cells invasion caused by OGD/R, and the effects of combined treatment with progesterone and si-FABP5 were more obvious. In PC12 cells, the protein expression of Bax and cleaved caspase3 was increased and Bcl-2 protein expression was decreased induced by OGD/R, which could be suppressed by progesterone or si-FABP5 treatment alone or in combination (Fig. 3E). Results from Fig. 3F showed that the elevated protein expression of IL-1, IL-6 and TNF-α caused by OGD/R could be suppressed by progesterone or si-FABP5 treatment alone or in combination. Moreover, results from wound healing assay suggested that the reduction of the relative migration distance of PC12 cells induced by OGD/R stimulation was mitigated by the addition of progesterone or si-FABP5, which were more obvious after the co-treatment of progesterone and si-FABP5 (Fig. 4A, p  < 0.01, F = 72.688).

Fig. 3

The beneficial effects of progesterone on OGD/R-induced PC12 cells were realized by targeting FABP5. A-C. The levels of MDA, ROS, and SOD in OGD/R-induced PC12 cells have been detected by ELISA kits after the co-treatment of progesterone and si-FABP5. D. The invasion number of PC12 cells was examined by transwell assay after the co-treatment of progesterone and si-FABP5 under OGD/R stimulation. E–F. The protein levels of Bax, Bcl-2, cleaved caspase3, IL-1, IL-6, and TNF-α in PC12 cells have been detected by western blot after the co-treatment of progesterone and si-FABP5 under OGD/R stimulation. **p < 0.01 vs. control, ##p < 0.01 vs. OGD/R, @@p < 0.01 vs. OGD/R + si-FABP5, & &p < 0.01 vs. OGD/R + Prog

Fig. 4

The beneficial effects of progesterone/FABP5 on PC12 cells were realized by regulating TLR4/NF-κB pathway. A. The migration distance of PC12 cells was examined by wound healing assay after the co-treatment of progesterone and si-FABP5 under OGD/R stimulation, **p < 0.01 vs. control, ##p < 0.01 vs. OGD/R, @@p < 0.01 vs. OGD/R + si-FABP5, & &p < 0.01 vs. OGD/R + Prog. B. The protein levels of FABP5, TLR4, p-P65 NF-κB, and P65 NF-κB in PC12 cells have been detected by western blot under OGD/R stimulation, **p < 0.01 vs. control, ##p < 0.01 vs. OGD/R

The beneficial effects of progesterone/FABP5 on PC12 cells were realized by regulating TLR4/NF-κB pathway

Previous study suggested that progesterone administration could inhibit TLR4/NF-κB signaling pathway in the rat brain following subarachnoid hemorrhage(Wang et al. 2011). In our study, the data from Fig. 4B showed that the patterns of FABP5, TLR4 and p-P65 NF-κB were obviously elevated under OGD/R treatment in PC12 cells. However, progesterone treatment significantly diminished the patterns of FABP5, TLR4 and p-P65 NF-κB in OGD/R caused PC12 cells. Therefore, our results suggested that progesterone may ameliorate OGD/R-caused damage of PC12 cells by inactivating FABP5/TLR4/NF-κB pathway.