Impact of Heat Shock Protein A 12B Overexpression on Spinal Astrocyte Survival Against Oxygen-Glucose-Serum Deprivation/Restoration in Primary Cultured Astrocytes

Abstract

Heat shock protein A 12B (HSPA12B) is a newly discovered member of the heat shock protein 70 family. Preclinical evidence indicates that HSPA12B helps protect the brain from ischemic injury, although its specific function remains unclear. The aim of this study is to investigate whether HSPA12B overexpression can protect astrocytes from oxygen-glucose-serum deprivation/restoration (OGD/R) injury. We analyzed the effects of HSPA12B overexpression on spinal cord ischemia-reperfusion injury and spinal astrocyte survival. After ischemia-reperfusion injury, we found that HSPA12B overexpression decreased spinal cord water content and infarct volume. MTT assay showed that HSPA12B overexpression increased astrocyte survival after OGD/R treatment. Flow cytometry results showed a marked inhibition of OGD/R-induced astrocyte apoptosis. Western blot assay showed that HSPA12B overexpression significantly increased regulatory protein B-cell lymphocyte 2 (Bcl-2) levels, whereas it decreased expression of the Bax protein, which forms a heterodimer with Bcl-2. Measurements of the level of activation of caspase-3 by Caspase-Glo®3/7 Assay kit showed that HSPA12B overexpression markedly inhibited caspase-3 activation. Notably, we demonstrated that the effects of HSPA12B on spinal astrocyte survival depended on activation of the PI3K/Akt signal pathway. These findings indicate that HSPA12B protects against spinal cord ischemia-reperfusion injury and may represent a potential treatment target.

Inhibition of heat shock protein family A member 8 attenuates spinal cord ischemia–reperfusion injury via astrocyte NF-κB/NLRP3 inflammasome pathway

“…Although HSPA8 shares some of the structural and functional similarities with HSP70, there are some different properties between HSPA8 and the HSP70 family members [ 28 ]. Previous studies have shown that overexpression of members of the HSP70 family, such as HSP70 and HSPA12B, exerts a neuroprotective effect under ischemia/hypoxia [ 25 , 29 ]. HSP70 overexpression inhibits OGD/R-induced apoptosis of bEnd.3 cells [ 29 ], and overexpression of HSPA12B protects spinal astrocytes from ischemic injury [ 25 ].…”

“…Primary rat spinal cord astrocytes were isolated from SD rats and cultured according to the Xia et al method [ 25 ]. Briefly, the spinal cord tissues were digested with 0.25% trypsin for 6 min, and the supernatant was further digested in DMEM/F12 (PM150310; Procell, Wuhan, China) medium containing 10% fetal bovine serum (FBS) at 37 °C with 5% CO 2 .…”

“…To archive the adequate HSPA8-overexpression spinal astrocytes, the cells were infected with LV-sh-HSPA8 or its negative control (LV-sh-NC) for 72 h in the 6-well plate (4 × 10 5 cells per well) in a humidified incubator (HF-100, Healforce, Shanghai, China) with 5% CO 2 at 37 °C. Subsequently, these cells were subjected to an OGD/R procedure that was performed based on a previous study [ 25 , 26 ]. Briefly, in the oxygen and glucose deprivation phase, the medium was washed with glucose-free Hanks balanced salt solution and changed to glucose-free DMEM/F12.…”

The Importance of Natural Antioxidants in the Treatment of Spinal Cord Injury in Animal Models: An Overview

“…As expected, this intentional insult which mimics hypoxic/ischemic conditions in vivo caused considerable oxidative stress: an increase in ROS and MDA content, as well as a decreased SOD activity. Also, it increased the expression of proapoptotic Bax and caspase-3 proteins, as well as the reduction of the antiapoptotic protein Bcl-2 [ 158 ]. The results of TET as a pretreatment to oxygen-glucose-serum deprivation/reoxygenation injury showed a dose-dependent suppression of Akt phosphorylation and NF- κ B activity and inhibition of the elevated caspase-3 activity.…”

Regional differences in the inflammatory and heat shock response in glia: implications for ALS

“…Glial cells also confer protection to neurons through the protective intracellular heat shock response (HSR) (Gleixner et al 2016; Xia et al 2016). The HSR is activated under conditions of cellular stress and leads to an increase in the synthesis of several stress-inducible chaperones called heat shock proteins (Hsps) in the cytosol and in subcellular organelles such as the ER and mitochondria (Kalmar and Greensmith 2017).…”