Transient receptor potential vanilloid 4 blockage attenuates pyroptosis in hippocampus of mice following pilocarpine‑induced status epilepticus

Abstract

Pyroptosis contributes to the neuronal damage that occurs during epilepsy. Calcium-activated neutral protease (calpain) dissociates cysteinyl aspartate specific proteinase-1 (caspase-1, cas-1) from the cytoskeleton, and the activated cas-1 is responsible for the production of N-terminus of gasdermin D (N-GSDMD), the final executor of pyroptosis. Blocking transient receptor potential vanilloid 4 (TRPV4) can reduce neuronal injury in temporal lobe epilepsy (TLE) model mice. This study investigated the role of TRPV4 in pyroptosis during TLE. In the hippocampus of pilocarpine-induced status epilepticus (PISE) mice, the ratio of inactive calpain 1 protein level to its total protein level (inactive/total calpain 1) significantly decreased, while the ratio of inactive calpain 2 protein level to its total protein level remained unchanged. The protein levels of NLRP3, cleaved cas-1 (c-cas-1), interleukin (IL)-1β, and N-GSDMD increased, with more GSDMD-immunofluorescence-positive (GSDMD⁺) cells and fewer surviving pyramidal neurons observed in the hippocampus of PISE mice. Calpain inhibition with MDL-28170 reversed these changes, except for the elevated NLRP3 levels. Inhibitors targeting NLRP3 (MCC950) and cas-1 (Ac-YVAD-cmk) blocked the increase in c-cas-1, IL-1β, and N-GSDMD levels in the hippocampus of PISE mice. TRPV4 inhibition via HC-067047 increased the inactive/total calpain 1 ratio, decreased NLRP3, c-cas-1, IL-1β, and N-GSDMD protein levels, reduced GSDMD⁺ cells number, and improved pyramidal neuron survival in the hippocampus of PISE mice. Conversely, TRPV4 activation with GSK1016790A decreased the inactive/total calpain 1 ratio, elevated NLRP3, c-cas-1, IL-1β, and N-GSDMD levels, and increased GSDMD⁺ cells number in the hippocampus. In the hippocampus of GSK1016790A-injected mice, the inactive/total calpain 1 ratio was increased by MDL-28170, and c-cas-1, IL-1β, and N-GSDMD protein levels were markedly attenuated by MDL-28170, MCC950, and Ac-YVAD-cmk, respectively. In conclusion, TRPV4 inhibition mitigates pyroptosis in PISE mice by downregulating the calpain 1-NLRP3/cas-1-GSDMD pathway, ultimately reducing neuronal damage.

Keywords: Calpain; Caspase-1; Gasdermin D; Pyroptosis; Temporal lobe epilepsy; Transient receptor potential vanilloid 4.

Fig. 1

Changes in calpain 1–NLRP3/cas-1–GSDMD pathway in the hippocampus of PISE mice. (A) The inactive/total calpain 1 ratio markedly decreased (independent-samples t test, t = 9.42, P < 0.01) while the inactive/total calpain 2 ratio did not change (independent-samples t test, t = − 0.16, P = 0.87) in the hippocampus of PISE mice. (B) The protein levels of NLRP3 (Mann–Whitney U test, P < 0.01), c-cas-1 (Mann–Whitney U test, P < 0.01), IL-1β (Mann–Whitney U test, P < 0.01), and N-GSDMD (Mann–Whitney U test, P < 0.01) increased markedly in the hippocampus of PISE mice. (C) The number of GSDMD⁺ cells (red arrow) increased markedly in the hippocampus in PISE mice (independent-samples t test, t = − 18.01, P < 0.01). (D) GSDMD⁺/NeuN⁺ (D-i, white arrow), GSDMD⁺/GFAP⁺ (D-ii, white star) and GSDMD⁺/Iba-1⁺ (D-iii, white triangle) cells in the hippocampus of control and PISE mice. Scale = 50 μm. ×4 objective for the top row in D-i, D-ii and D-iii; ×10 objective for C-DAPI, C-GSDMD, C-Merge; ×20 objective for C-enlarge and the down row in D-i, D-ii and D-iii. **P < 0.01 vs. control

Fig. 2

Effect of calpain inhibitor MDL-28170 on calpain 1–NLRP3/cas-1–GSDMD pathway in the hippocampus of PISE mice. (A) The calpain inhibitor MDL-28170 markedly increased the inactive/total calpain 1 rato in the hippocampus of PISE mice (Mann–Whitney U test, P < 0.01). (B) MDL-28170 did not affect NLRP3 protein levels in the hippocampus of PISE mice (independent-samples t test, t = 0.74, P = 0.47), but decreased c-cas-1 (independent-samples t test, t = 7.81, P < 0.01), IL-1β (independent-samples t test, t = 6.30, P < 0.01), and N-GSDMD protein levels (Mann–Whitney U test, P < 0.01). (C) MDL-28170 increased GSDMD⁺ cells (red arrow) number in the hippocampus of PISE mice (independent-samples t test, t = 8.83, P < 0.01). (D) GSDMD⁺/NeuN⁺ (D-i, white arrow), GSDMD⁺/GFAP⁺ (D-ii, white star) and GSDMD⁺/Iba-1⁺ (D-iii, white triangle) cells in the hippocampus of vehicle- and MDL-28179-treated PISE mice. (E) The numbers of surviving pyramidal neurons reduced in the hippocampal CA1 (independent-samples t test, t = 22.76, P < 0.01) and CA2/3 (independent-samples t test, t = 28.02, P < 0.01) area of PISE mice (E-i). Administration of MDL-28170 to PISE mice increased the numbers of surviving pyramidal cells in the hippocampal CA1 (independent-samples t test, t = 6.88, P < 0.01) and CA2/3 area (independent-samples t test, t = 6.15, P < 0.01) (E-ii). Scale = 50 μm. ×4 objective for the top row in D-i, D-ii and D-iii; ×10 objective for C-DAPI, C-GSDMD, C-Merge, and the middle column in E-i and E-ii; ×20 objective for C-enlarge, the down row in D-i, D-ii, and D-iii; ×40 objective for the left and right columns in E-i and E-ii. ^##P < 0.01 vs. PISE + vehicle (ip.), **P < 0.01 vs. control

Fig. 3

Effects of NLRP3 and cas-1 inhibitors on c-cas-1, IL-1β, and N-GSDMD protein levels in the hippocampus of PISE mice. A and C. NLRP3 inhibitor MCC950 decreased c-cas-1 (A, Mann–Whitney U test, P < 0.01; C, independent-samples t test, t = 10.47, P < 0.01), IL-1β (A, independent-samples t test, t = 5.75, P < 0.01; C, independent-samples t test, t = 8.50, P < 0.01), and N-GSDMD protein levels (A, independent-samples t test, t = 5.98, P < 0.01; C, Mann–Whitney U test, P < 0.01) in the hippocampus of control and PISE mice. B and D. Cas-1 inhibitor Ac-YVAD-cmk decreased c-cas-1 (B, independent-samples t test, t = 5.04, P < 0.01; D, independent-samples t test, t = 9.50, P < 0.01), IL-1β (B, independent-samples t test, t = 5.91, P < 0.01; D, independent-samples t test, t = 11.54, P < 0.01), and N-GSDMD protein levels (B, independent-samples t test, t = 8.14, P < 0.01; D, independent-samples t test, t = 12.08, P < 0.01) in the hippocampus of control and PISE mice. E. Administration of MCC950 to PISE mice increased the numbers of surviving pyramidal neurons in the hippocampal CA1 (independent-samples t test, t = 10.01, P < 0.01) and CA2/3 area (independent-samples t test, t = 14.50, P < 0.01). Scale = 50 μm. ×10 objective for the middle column in E; ×40 objective for the left and right columns in E. ^{^^}P < 0.01 vs. control (ip.), ^<<P < 0.01 vs. control (icv.), ^##P < 0.01 vs. PISE + vehicle (ip.), ^&&P < 0.01 vs. PISE + vehicle (icv.)

Fig. 4

Effect of TRPV4 antagonist on calpain 1–NLRP3/cas-1–GSDMD pathway in the hippocampus of PISE mice. A and C. Administration of TRPV4 antagonist HC-067047 increased inactive/total calpain 1 ratio in the hippocampus of control (A, Mann–Whitney U test, P < 0.01) and PISE mice (C, independent-samples t test, t = − 17.09, P < 0.01). B and D. Administration of HC-067047 decreased NLRP3 (B, HC-067047: independent-samples t test, t = 6.49, P < 0.01; D, PISE + HC-067047: Mann–Whitney U test, P < 0.01), c-cas-1 (B, HC-067047: independent-samples t test, t = 6.53, P < 0.01; D, PISE + HC-067047: independent-samples t test, t = 9.38, P < 0.01), IL-1β (B, HC-067047: independent-samples t test, t = 4.23, P < 0.01; D, PISE + HC-067047: independent-samples t test, t = 12.31, P < 0.01), and N-GSDMD protein levels (B, HC-067047: independent-samples t test, t = 5.28, P < 0.01; D, PISE + HC-067047: Mann–Whitney U test, P < 0.01). E and F. Administration of HC-067047 decreased GSDMD⁺ cells (red arrow) numbers in the hippocampus of control (E-i, HC-067047: independent-samples t test, t = 4.34, P < 0.01) and PISE mice (F-i, PISE + HC-067047: Mann–Whitney U test, P < 0.01; white star). GSDMD⁺/NeuN⁺ (white arrow), GSDMD⁺/GFAP⁺ (white star) and GSDMD⁺/Iba-1⁺ (white triangle) cells in the hippocampus in control (E-ii, E-iii and E-iv) and PISE mice (F-ii, F-iii and F-iv) treated with either vehicle or HC-067047. G. HC-067047 treatment increased the numbers of surviving neurons in the hippocampal CA1 (G-i, independent-samples t test, t = 5.14, P < 0.01; G-ii, independent-samples t test, t = 10.12, P < 0.01) and CA2/3 (G-i, independent-samples t test, t = 2.35, P = 0.03; G-ii, independent-samples t test, t = 14.20, P < 0.01) area of control (G-i) and PISE mice (G-ii). Scale = 50 μm. ×4 objective for the top row in E-ii, E-iii, E-vi, F-ii, F-iii, F-vi; ×10 objective for E-i-DAPI, E-i-GSDMD, E-i-Merge, F-i-DAPI, F-i-GSDMD, F-i-Merge, and the middle columns in G-i and G-ii; ×20 objective for E-i-enlarge, F-i-enlarge, the down row in E-ii, E-iii, E-vi, F-ii, F-iii and F-vi; ×40 objective for the left and right columns in G-i, and G-ii. ^<P < 0.05, ^<<P < 0.01 vs. control (icv.), ^&&P < 0.01 vs. PISE + vehicle (icv.)

Fig. 5

Effect of TRPV4 agonist on calpain 1-NLRP3/cas-1-GSDMD pathway in the hippocampus. A. The TRPV4 agonist GSK1016790A decreased inactive/total calpain 1 ratio (independent-samples t test, t = 12.72, P < 0.01) but did not change inactive/total calpain 2 ratio (independent-samples t test, t = 0.19, P = 0.86) in the hippocampus. B and C. GSK1016790A increased NLRP3 (B, Mann–Whitney U test, P < 0.01), c-cas-1 (B, Mann–Whitney U test, P < 0.01), IL-1β (B, Mann–Whitney U test, P < 0.01), and N-GSDMD (B, Mann–Whitney U test, P < 0.01) protein levels, and GSDMD⁺ cells (red arrow) number (C, independent-samples t test, t = − 20.96, P < 0.01) in the hippocampus. D. GSDMD⁺/NeuN⁺ (D-i, white arrow), GSDMD⁺/GFAP⁺ (D-ii, white star) and GSDMD⁺/Iba-1⁺ (D-iii, white triangle) cells in the hippocampus of control and GSK1016790A-injected mice. E and F. The calpain inhibitor MDL-28170 increased inactive/total calpain 1 ratio (E, Mann–Whitney U test, P < 0.01) and decreased c-cas-1 (F, independent-samples t test, t = 8.73, P < 0.01), IL-1β (F, independent-samples t test, t = 9.35, P < 0.01), and N-GSDMD protein levels (F, independent-samples t test, t = 13.52, P < 0.01) but did not affect NLRP3 protein levels (F, independent-samples t test, t = − 0.20, P = 0.85) in the hippocampus of GSK1016790A-injected mice. G and H. The NLRP3 inhibitor MCC950 (G) and cas-1 inhibitor Ac-YVAD-cmk (H) decreased c-cas-1 (G, independent-samples t test, t = 8.17, P < 0.01; H, Mann–Whitney U test, P < 0.01), IL-1β (G, independent-samples t test, t = 11.62, P < 0.01; H, Mann–Whitney U test, P < 0.01) and N-GSDMD protein levels (G, Mann–Whitney U test, P < 0.01; H, independent-samples t test, t = 13.38, P < 0.01) in the hippocampus in GSK1016790A-injected mice. Scale = 50 μm. ×4 objective for the top row in D-i, D-ii and D-iii; ×10 objective for C-DAPI, C-GSDMD and C-Merge; ×20 objective for C-enlarge, the down row in D-i, D-ii and D-iii. ^<<P < 0.01 vs. Control (icv.), ^$$P < 0.01 vs. GSK1016790A + vehicle, ^%%P < 0.01 vs. GSK1016790A + vehicle