Fig 2: Effects of HSP25 knockdown on heat shock protein 25 (HSP25) and lysosomal-associated membrane protein 1 (LAMP1) expressions in CA1 astrocytes of control and epileptic rats. As compared to control animals (Cont), HSP25 and LAMP1 expressions are upregulated in CA1 astrocytes in epileptic rats. HSP25 siRNA reduces the increased HSP25 and LAMP1 expressions in CA1 astrocytes, as compared to control siRNA (cont siRNA). (A,B) Representative photos demonstrating astroglial HSP25 (A) and LAMP1 (B) expressions in CA1 astrocytes. (C–E) Quantifications of the fraction of clasmatodendritic astrocytes in total CA1 astrocytes (C), HSP25 expression (D) and LAMP1 fluorescent intensity (E) in CA1 astrocytes. Open circles indicate each value. Horizontal bars indicate the mean value. Error bars indicate SEM (*, # p < 0.05 vs. control and control siRNA-treated epileptic rats, respectively; n = 7, respectively).

Fig 3: Effects of 3CAI on heat shock protein 25 (HSP25) and lysosomal-associated membrane protein 1 (LAMP1) expressions in CA1 astrocytes of control and epileptic rats. As compared to control animals (Cont), HSP25 and LAMP1 expressions are upregulated in CA1 astrocytes in epileptic rats. 3CAI reduces the increased LAMP1, but not HSP25, expression in CA1 astrocytes, as compared to vehicle (Veh). (A,B) Representative photos demonstrating astroglial HSP25 (A) and LAMP1 (B) expressions in CA1 astrocytes. (C–E) Quantifications of the fraction of clasmatodendritic astrocytes in total CA1 astrocytes (C), HSP25 expression (D) and LAMP1 fluorescent intensity (E) in CA1 astrocytes. Open circles indicate each value. Horizontal bars indicate the mean value. Error bars indicate SEM (*, # p < 0.05 vs. control and vehicle-treated epileptic rats, respectively; n = 7, respectively).

Fig 4: Effects of CDDO-Me and SN50 on clasmatodendritic changes in CA1 astrocytes of epilepsy rats. Compared to control animals (Cont), lysosomal-associated membrane protein 1 (LAMP1) and microtubule-associated protein 1A/1B light-chain 3 (LC3) expressions are upregulated in CA1 astrocytes in epilepsy rats. LC3 signal is undetectable in reactive astrocyte (arrowhead). Compared to vehicle (Veh), CDDO-Me (CDDO) and SN50 reduce the increased LAMP1 expression in CA1 astrocytes. In addition, they decrease the LC3 expression in some vacuolized CA1 astrocytes (arrows). (A) Representative photos demonstrating clasmatodendritic degeneration in CA1 astrocytes. Inserts are high magnification of rectangles (bar = 7.5 µm). (B–D) Quantifications of the protective effects of CDDO-Me and SN50 on clasmatodendrosis (B), LAMP1 upregulation (C), and LC3 induction in CA1 astrocytes (D). Open circles and horizontal bars indicate the individual value and the mean value, respectively. Error bars are S.D. (*, # p < 0.05 vs. control rats and vehicle-treated epilepsy rats; n = 7).

Fig 5: Effects of CDDO-Me and SN50 on autophagy process in the epileptic hippocampus. In vehicle-treated epilepsy rats, LC3-positive puncta are clearly observed in vacuolized astrocytes. Compared to control animals, LAMP1, LC3-I, and LC3-II densities are increased in the epileptic animals concomitant with the elevated LC3-II/LC3-I ratio, which are ameliorated by CDDO-Me and SN50. (A) Representative photos demonstrating LC3 immunoreactivity in clasmatodendritic CA1 astrocytes. (B) Representative Western blot images demonstrating LAMP1, LC3-I, and LC3-II bands in the epileptic hippocampus. (C,D) Quantifications of the effects of CDDO-Me and SN50 on LAMP1 (C), LC3-I (D), LC3-II (E), and LC3-II/LC3-I ratio (F). Open circles and horizontal bars indicate the individual value and the mean value, respectively. Error bars are S.D. (*, # p < 0.05 vs. control rats and vehicle-treated epilepsy rats; n = 7).