Amala Reddy, Sujatha Sundaresan
Animal Cell Culture Laboratory, Department of Biotechnology, SRMIST, Kattankulathur, Tamilnadu, India.
Bioimpacts. 2021;11(1):15-22. doi: 10.34172/bi.2021.03. Epub 2019 Dec 25.
Inflammation is the primary response caused due to harmful stimuli which are followed by the increased draining of plasma and immune cells from the body into the site of the injured tissue. A signaling cascade of growth factors and cytokines propagates and eventually matures in the inflammatory site involving the blood vessels and immune markers within the injured tissue in order to promote the renewal of the degenerated tissue. During a chronic disorder like diabetic foot ulcer, there is an obstinate inflammation which may act as a prime factor for limb amputation and upon persistent prevalence may even lead to death. This study focuses on the mode of action of ALK-F (alkaloid fraction) isolated from in attenuating the nitric oxide production which was estimated by Griess assay, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) expression was analyzed by ELISA and expression of COX-2 and iNOS by RT-PCR and western blotting in LPS stimulated RAW 264.7 macrophages. Total intracellular ROS was analyzed by DCFH-DA probing and the presence of quinazoline alkaloid (vasicine) in the ALK-F was evidenced by high performance liquid chromatography (HPLC). The ALK-F of exhibited a significant inhibitory effect on LPS elicited nitrite production (13.2 ± 1.06 µM), iNOS, and COX-2 (2.6 and 3.3 fold) in a dose-dependent manner. There was a significant decrease in the generation of these pro-inflammatory cytokines TNF-α (1102 ± 1.02 pg/mL) and IL-6 (18 ± 0.87 ng/mL) and total intracellular ROS in the highest tested concentrations (1 µg and 10 µg) of ALK-F of A. vasica. HPLC analysis by the gradient elution method revealed the presence of 12% of quinazoline alkaloid vasicine in the crude alkaloid fraction. Thus this study communally suggests that attenuation of nitric oxide and the dysregulation of genes responsible for inflammation which deliberates to conflict against inflammation and provide remedial benefits in diabetic wound care.
炎症是由有害刺激引起的主要反应，随后血浆和免疫细胞从身体向受伤组织部位的引流增加。生长因子和细胞因子的信号级联反应在炎症部位传播并最终成熟，涉及受伤组织内的血管和免疫标志物，以促进退化组织的更新。在糖尿病足溃疡等慢性疾病中，存在顽固的炎症，这可能是肢体截肢的主要因素，持续存在甚至可能导致死亡。本研究重点关注从[植物名称未给出]中分离出的ALK - F（生物碱部分）的作用模式，该模式通过格里斯测定法评估其对一氧化氮产生的影响，通过酶联免疫吸附测定法分析肿瘤坏死因子 -α（TNF -α）和白细胞介素 -6（IL -6）的表达，并通过逆转录 - 聚合酶链反应（RT - PCR）和蛋白质印迹法分析脂多糖（LPS）刺激的RAW 264.7巨噬细胞中环氧合酶 -2（COX -2）和诱导型一氧化氮合酶（iNOS）的表达。通过2′,7′ - 二氯二氢荧光素二乙酸酯（DCFH - DA）探针分析细胞内总活性氧（ROS），并通过高效液相色谱法（HPLC）证明ALK - F中喹唑啉生物碱（鸭嘴花碱）的存在。[植物名称未给出]的ALK - F对LPS诱导的亚硝酸盐产生（13.2±1.06 μM）、iNOS和COX -2（分别为2.6倍和3.3倍）具有显著的剂量依赖性抑制作用。在最高测试浓度（1 μg和10 μg）的[植物名称未给出]的ALK - F中，这些促炎细胞因子TNF -α（1102±1.02 pg/mL）和IL -6（18±0.87 ng/mL）的产生以及细胞内总ROS显著降低。通过梯度洗脱法的HPLC分析显示，粗生物碱部分中存在12%的喹唑啉生物碱鸭嘴花碱。因此，本研究共同表明，一氧化氮的减少以及负责炎症的基因失调有助于对抗炎症，并在糖尿病伤口护理中提供治疗益处。
