Estrogen receptors and estetrol-dependent neuroprotective actions: a pilot study

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   Figure 1

   Effect of E4 in combination with different receptor inhibitors on LDH activity in primary hippocampal neuronal cultures subjected to the H₂O₂-induced oxidative stress. Primary hippocampal cell cultures were exposed to 3.25 mM E4 alone or in combination with MPP, PHTTP, G15 and/or 2-BR after induction of oxidative stress. (A) LDH activity was significantly decreased by treatment with E4 alone or in combination with ER-α inhibitor MPP compared to the H₂O₂-treated cell cultures or cultures combinedly treated by E4 + MPP + PHTTP. Combined use of MPP and PHTTP significantly increased the LDH activity compared to the cells treated by E4 alone or in combination with MPP. (B) LDH activity was significantly decreased by treatment with E4 alone or in combination with ER-β inhibitor PHTTP compared to the H₂O₂-treated cell cultures or cultures combinedly treated by E4 + MPP + PHTTP. Combined use of MPP and PHTTP significantly increased the LDH compared to the cell cultures treated by E4 alone or in combination with PHTTP. (C) Inhibition of palmitoylation alone or in combination with MPP significantly downregulated LDH activity compared to the H₂O₂-treated cells or to those treated by E4 alone. Combination of E4 with 2-BR, MPP and PHTTP significantly upregulated LDH activity compared to the cell cultures treated by E4 or 2-BR alone or in combination with MPP. (D) Cell cultures treated by E4 alone or in combination with GPR30 inhibitor G15 had significantly lower LDH activity compared to the cultures treated by H₂O₂ alone. No significant difference was observed between the cells treated by E4 alone or in combination with G15.
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   Figure 2

   Effect of E4 in combination with different receptor inhibitors on cell survival in primary hippocampal neuronal cultures subjected to the H₂O₂-induced oxidative stress. Primary hippocampal cell cultures were exposed to 3.25 mM E4 alone or in combination with MPP, PHTTP, G15 and/or 2-BR after induction of oxidative stress. (A) Cell survival rate was significantly upregulated in cells treated by E4 alone or in combination either with MPP or MPP + PHTTP compared to cells solely treated by H₂O₂. (B) Cultures treated either by E4 alone or with PHTTP with/without MPP had significantly upregulated cell survival rate compared to cells treated by H₂O₂ alone. Cells combinedly treated by E4 with PHTTP had significantly lower cell survival rate than the cell cultures treated by E4 alone. (C) Cells treated either by E4 alone or in combination with 2-BR, MPP and/or PHTTP had significantly higher cell survival rate compared to the cells solely treated by H₂O₂. Treatment of cultures by E4 and 2-BR along with MPP resulted in significant upregulation of cell survival compared to the cultures treated by 2-BR alone or in combination with MPP and PHTTP. No significant difference was observed between the cells treated by E4 alone or those treated by different combinations of E4, 2-BR, MPP and/or PHTTP. (D) Treatment of cell cultures by E4 alone or in combination with G15 significantly upregulated the cell survival rate compared to cell cultures treated by H₂O₂. No significant difference was observed between cells treated by E4 alone or in combination with G15.
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   Figure 3

   Myelin basic protein (MBP) staining of brain coronal sections in rat pups pretreated with estetrol. (A) MBP staining of brain coronal sections (scale bar: 2 mm) is shown. (B) MBP staining of cingulum of the left hemisphere is shown (scale bar: 2 mm). (C) The ratio of the MBP-positive areas OD ratio was calculated as the MBP-positive area OD of the ipsilateral hemisphere divided by the MBP-positive area OD of the contralateral hemisphere. 10 samples from each study group were analyzed. The ratio of the MBP-positive area OD in the Sham group was considered by default as 1.0. The MBP-positive area OD ratio was significantly higher in sham-operated animals and the 5 mg/kg/day and 50 mg/kg/day E4-pretreated groups compared to the vehicle group. All measurements are expressed as mean ± s.e.m. *P < 0.05.
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   Figure 4

   Myelin basic protein (MBP) staining of brain coronal sections in rat pups treated with estetrol. (A) MBP staining of brain coronal sections (scale bar: 2 mm) is shown. (B) MBP staining of cingulum of the left hemisphere is shown (scale bar: 2 mm). (C) The ratio of the MBP-positive areas OD ratio was calculated as the MBP-positive area OD of the ipsilateral hemisphere divided by the MBP-positive area OD of the contralateral hemisphere. 10 samples from each study group were analyzed. The ratio of the MBP-positive area OD in the sham group was considered by default as 1.0. The MBP-positive area OD ratio was significantly higher in sham-operated animals and the 1 mg/kg/day, 5 mg/kg/day, 10 mg/kg/day and 50 mg/kg/day E4-treated groups compared to the vehicle group. All measurements are expressed as mean ± s.e.m. *P < 0.05.

• © 2017 The authors