Mekala Naveen Kumar, Sasikumar Shyama, Akula Kranthi Kiran, Parekh Yash, Rao Ch Mohan, Bokara Kiran Kumar
College of Medicine, Central Michigan University Mt Pleasant, MI 48858, USA.
Department of Biomedical Engineering, Indian Institute of Technology Hyderabad Kandi-502285, Sangareddy, Telangana, India.
Am J Stem Cells. 2020 Dec 25;9(5):68-77. eCollection 2020.
HspB5 (αB-crystallin) is known to be involved in a variety of cellular functions, including, protection of cells from oxidative damage and inhibiting apoptosis. Neural stem/progenitor cells (NSPCs) have significant therapeutic value, especially in the NSC/NPC transplantation therapy. However, the viability of the transplanted NSPCs remains low because of various factors, including oxidative stress.
The current investigation explored the possible role of HspB5 in the protection of mouse NSPCs (NSPCs) against paraquat-induced toxicity.
The recombinant human HspB5 was expressed in and was purified using gel filtration and Ion-exchange chromatography. The biophysical characterization of HspB5 was carried out using DLS, CD, and Analytical Ultracentrifugation (SV); the chaperone activity of HspB5 was determined by alcohol dehydrogenase aggregation assay. We have subjected the mNSPCs to paraquat-induced oxidative stress and monitored the protective ability of HspB5 by MTT assay and Hoechst-PI staining. Furthermore, increase in the expression of the anti-apoptotic protein, procaspase-3 was monitored using western blotting.
The recombinant HspB5 was purified to its homogeneity and was characterized using various biophysical techniques. The externally added FITC-labeled HspB5 was found to be localized within the cytoplasm of NSPCs. Our Immunocytochemistry results showed that the externally added FITC-labeled HspB5 not only entered the cells but also conferred cytoprotection against paraquat-induced toxicity. The protective events were monitored by a decrease in the PI-positive cells and an increase in the procaspase-3 expression through Immunocytochemistry and Western blotting respectively.
Our results clearly demonstrate that exogenously added recombinant human HspB5 enters the NSPCs and confers protection against paraquat toxicity.
已知热休克蛋白B5（αB-晶状体蛋白）参与多种细胞功能，包括保护细胞免受氧化损伤和抑制细胞凋亡。神经干细胞/祖细胞（NSPCs）具有显著的治疗价值，尤其是在NSC/NPC移植治疗中。然而，由于包括氧化应激在内的各种因素，移植的NSPCs的存活率仍然很低。
本研究探讨热休克蛋白B5在保护小鼠神经干细胞（NSPCs）免受百草枯诱导的毒性方面的可能作用。
重组人热休克蛋白B5在大肠杆菌中表达，并用凝胶过滤和离子交换色谱法纯化。通过动态光散射（DLS）、圆二色光谱（CD）和分析超速离心（SV）对热休克蛋白B5进行生物物理表征；通过乙醇脱氢酶聚集试验测定热休克蛋白B5的伴侣活性。我们使小鼠神经干细胞遭受百草枯诱导的氧化应激，并通过MTT试验和Hoechst-PI染色监测热休克蛋白B5的保护能力。此外，使用蛋白质印迹法监测抗凋亡蛋白原半胱天冬酶-3表达的增加。
重组热休克蛋白B5被纯化至同质，并使用各种生物物理技术进行表征。发现外部添加的异硫氰酸荧光素（FITC）标记的热休克蛋白B5定位于神经干细胞的细胞质内。我们的免疫细胞化学结果表明，外部添加的FITC标记的热休克蛋白B5不仅进入细胞，而且赋予细胞对百草枯诱导的毒性的保护作用。通过免疫细胞化学和蛋白质印迹法分别监测PI阳性细胞的减少和原半胱天冬酶-3表达的增加来观察保护作用。
我们的结果清楚地表明，外源添加的重组人热休克蛋白B5进入神经干细胞并赋予其对百草枯毒性的保护作用。
