Redhwan Moqbel Ali Moqbel, M G Hariprasad, Samaddar Suman, Bafail Duaa Abdullah, Hard Sumaia Abdulbari Ahmed Ali, Guha Sourav
Department of Pharmacology, KLE College of Pharmacy, Bengaluru, Karnataka, India; Basic Science Research Center (Off-Campus), KLE College of Pharmacy, Bengaluru, Karnataka, India.
Department of Pharmacology, KLE College of Pharmacy, Bengaluru, Karnataka, India; Basic Science Research Center (Off-Campus), KLE College of Pharmacy, Bengaluru, Karnataka, India.
Int J Biol Macromol. 2024 Dec;282(Pt 2):136964. doi: 10.1016/j.ijbiomac.2024.136964. Epub 2024 Oct 28.
Hyperglycemia induces an excessive production of superoxide by the mitochondria's electron-transport chain triggers several pathways of injury contributing to the development of diabetic complications. This increase in oxidative and nitrosative stress triggers the activation of PARP-1, a nuclear enzyme, through mechanisms such as DNA damage. siRNA-chitosan nanoparticles were formed based on electrostatic interaction, their particle size, zeta potential, STEM, and cellular uptake were characterized. Neuro2a cells were treated with low glucose (LG) and high glucose (HG) for 24 and 48 h. Neuro2a cells were pre-treated with negative siRNA, naked siRNA, siRNA-Lipofectamine™3000, and ChNPs-5. qRT-PCR was used to analyze the expression of regulatory, inflammatory, and apoptotic biomarkers. The siRNA-chitosan complex at the weight ratio 1:3000 were approximately uniform spheres with particle size 150.5 nm and a positive zeta potential of about +41.5 mV. The uptake of FITC-labeled siRNA nanoparticles into Neuro2a cells was significantly higher compared to naked siRNA, as visualized using fluorescence microscopy, with no notable cytotoxicity compared to normal cells. High glucose stimulation of Neuro2a cells increased PARP1 expression, and with siRNA-ChNP (1:3000) treatment, significant inhibition of PARP1 expression is observed that consequently reversed the expression of regulatory genes like SIRT1, FOXO1, FOXO3, and p53. PARP-1 inhibition reduced HG-induced inflammatory response, including NF-kB, IL6, IL1β, TNFα, iNOS, and TGF-β expression, and HG-induced apoptosis response, such as Cas-3, Cas-9, BAK, BAX, and AIF expression. This study highlights the crucial role of siRNA delivery via ChNPs and PARP-1 inhibition in hyperglycemia-induced oxidative stress in Neuro2a cells and PARP-1 inhibition may be a feasible strategy for the treatment of hyperglycemia-induced oxidative stress.
高血糖通过线粒体电子传递链诱导超氧化物过度产生，触发多种损伤途径，导致糖尿病并发症的发生。氧化应激和亚硝化应激的增加通过DNA损伤等机制触发核酶PARP-1的激活。基于静电相互作用形成了siRNA-壳聚糖纳米颗粒，并对其粒径、zeta电位、扫描透射电子显微镜（STEM）和细胞摄取进行了表征。将Neuro2a细胞分别用低糖（LG）和高糖（HG）处理24小时和48小时。Neuro2a细胞分别用阴性siRNA、裸siRNA、siRNA-脂质体转染试剂™3000和壳聚糖纳米颗粒（ChNPs-5）进行预处理。采用定量逆转录聚合酶链反应（qRT-PCR）分析调节、炎症和凋亡生物标志物的表达。重量比为1:3000的siRNA-壳聚糖复合物为近似均匀的球体，粒径为150.5nm，正zeta电位约为+41.5mV。通过荧光显微镜观察，与裸siRNA相比，FITC标记的siRNA纳米颗粒进入Neuro2a细胞的摄取量显著更高，与正常细胞相比无明显细胞毒性。高糖刺激Neuro2a细胞增加了PARP1的表达，而用siRNA-壳聚糖纳米颗粒（1:3000）处理后，观察到PARP1表达受到显著抑制，从而逆转了SIRT1、FOXO1、FOXO3和p53等调节基因的表达。PARP-1抑制降低了高糖诱导的炎症反应，包括核因子κB（NF-κB）、白细胞介素6（IL6）、白细胞介素1β（IL1β）、肿瘤坏死因子α（TNFα）、诱导型一氧化氮合酶（iNOS）和转化生长因子β（TGF-β）的表达，以及高糖诱导的凋亡反应，如半胱天冬酶3（Cas-3）、半胱天冬酶9（Cas-9）、促凋亡蛋白BAK、促凋亡蛋白BAX和凋亡诱导因子（AIF）的表达。本研究强调了通过壳聚糖纳米颗粒递送siRNA和抑制PARP-1在高血糖诱导的Neuro2a细胞氧化应激中的关键作用，抑制PARP-1可能是治疗高血糖诱导的氧化应激的一种可行策略。
