Protective effect of Tat fused HPCA protein on neuronal cell death caused by ischemic injury

Abstract

Background: Bain ischemia is a disease that occurs for various reasons, induces reactive oxygen species (ROS), and causes fatal damage to the nervous system. Protective effect of HPCA on ischemic injury has not been extensively studied despite its significance in regulating calcium homeostasis and promoting neuronal survival in CA1 region of the brain.

Objective: We investigate the role of HPCA in ischemic injury using a cell-permeable Tat peptide fused HPCA protein (Tat-HPCA).

Methods: Western blot analysis determined the penetration of Tat-HPCA into HT-22 cells and apoptotic signaling pathways. 5-CFDA, AM, DCF-DA, and TUNEL staining confirmed intracellular ROS production and DNA damage. The intracellular Ca²⁺ was measured in primary cultured neurons treated with H₂O₂. Protective effects were examined using immunohistochemistry and cognitive function tests by passive avoidance test and 8-arm radial maze test.

Results: Tat-HPCA effectively penetrated into HT-22 cells and inhibited H₂O₂-induced apoptosis, oxidative stress, and DNA fragmentation. It also effectively inhibited phosphorylation of JNK and regulated the activation of Caspase, Bax, Bcl-2, and PARP, leading to inhibition of apoptosis. Moreover, Ca²⁺ concentration decreased in cells treated with Tat-HPCA in primary cultured neurons. In an animal model of ischemia, Tat-HPCA effectively penetrated the hippocampus, inhibited cell death, and regulated activities of astrocytes and microglia. Additionally, Cognitive function tests show that Tat-HPCA improves neurobehavioral outcomes after cerebral ischemic injury.

Conclusion: These results suggest that Tat-HPCA might have potential as a therapeutic agent for treating oxidative stress-related diseases induced by ischemic injury, including ischemia.

Keywords: Brain ischemia; Oxidative stress; Protein therapy; Protein transduction domain; Tat-HPCA.

Graphical abstract

Fig. 1

Purification and Transduction of Tat-HPCA protein into HT-22 cells. The constructed map of Tat-HPCA based on the pET15b vector that contains 6 histidine residues (A). Expressed and purified Tat-HPCA proteins were separated by 15 % SDS–PAGE and the protein was confirmed by Western blot analysis with an anti-rabbit polyhistidine antibody (B). Cells were treated with Tat-HPCA proteins (0.5–3 μM) for 1 h (C), or cells were treated with Tat-HPCA proteins (3 μM) for 15–60 min (D). Intracellular stability of transduced Tat-HPCA protein. After Tat-HPCA proteins (3 μM) transduced into the HT-22 cells, the cells were incubated for 1–60 h (E). Transduced Tat-HPCA protein levels were analyzed by Western blotting and the intensity of the bands was measured by densitometer. The distribution of transduced Tat-HPCA protein was examined by confocal fluorescence microscopy (F). Scale bar = 20 μm.

Fig. 2

Effect of Tat-HPCA protein on H₂O₂-induced cellular toxicity. HT-22 cells were pretreated with Tat-HPCA (0.5–3 μM) for 1 h and exposed to H₂O₂ (100 μM) 2 h. Cell viabilities were assessed by WST-1 assay (A) and 5-CFDA, AM (B). Intracellular ROS levels were measured using DCF-DA staining (C). DNA fragmentation was detected by TUNEL staining (D). The fluorescence intensity was measured by an ELISA plate reader. ∗P < 0.01, compared with H₂O₂-treated cells.

Fig. 3

Inhibitory effects of Tat HPCA protein against H₂O₂-induced cellular signaling pathways in HT-22 cells. Tat-HPCA (3 μM) for 1 h before being treated with H₂O₂ (100 μM). H₂O₂-induced JNK activation was examined using Western blot analysis (A). The expression levels of caspase-3, caspase-9, PARP, Bax and Bcl-2 were determined by Western blot analysis (B). The level of intracellular Ca²⁺ was measured in cell culture medium (C). Effects of H₂O₂ and Tat-HPCA on calcium concentration (mM). Band intensity was measured by densitometer. The bars in the figure represent the mean ± SEM obtained from 3 independent experiments. ∗P < 0.01, compared with H₂O₂-treated cells.

Fig. 4

Protective effect of Tat-HPCA protein in ischemic injury animal model. Gerbils were treated with a single injection Tat-HPCA protein (2 mg/kg) before ischemia-reperfusion and killed after 7 days. Transduced Tat-HPCA protein was analyzed by immunostaining using anti-His antibody (A). The hippocampus was stained with CV, Iba-1 and GFAP in sham-, vehicle-, HPCA and Tat-HPCA-treated animals 7 days after I/R (B). Scale bar = 400 and 50 μm. Latency time by passive avoidance (PAT) and mean numbers of errors in 8-arm radial maze test (8-ARMT) in ischemic injury animal model. In the Tat-HPCA-treated group, a significantly delayed latency time in PAT is recorded compared with that in the vehicle groups (C). In the 8-ARMT, the Tat-HPCA-treated group showed a significantly improved number of correct and error choices compared to the vehicle group (D). ∗P < 0.05, compared to the vehicle group. (each group, n = 7).