Abstract
We tested the hypothesis that changes reported on functions of neutrophils from streptozotocin-induced diabetic rats involve autophagy impairment. Wistar rats were rendered diabetic by streptozotocin injection (65 mg/kg, i.v.), and the measurements were carried out 2 weeks afterward. Neutrophils were collected through intraperitoneal cavity lavage after 4 h of i.p. oyster glycogen type 2 injection. Neutrophils cultured with PMA (20 nM) for 1 h were used for analysis of plasma membrane integrity, DNA fragmentation, and mitochondrial depolarization by flow cytometry; expression of Atg5, Atg14, Beclin1, LC3BII, and Rab9 by RT-PCR; the contents of caspase 3, LC3BII/LC3BI, and pS6 by western blotting; ATP content by fluorescence essay; reactive oxygen species production by chemiluminescence (Luminol), and autophagy by immunofluorescence tracking LC3B cleavage. Herein, neutrophils from diabetic rats had high DNA fragmentation, depolarization of mitochondrial membrane, low content of ATP, and high content of cleaved caspase 3 after PMA stimulation. Neutrophils from diabetic rats also had low expression of LC3B, failed to increase the expression of Rab9 and Atg14 induced by PMA stimulation. Neutrophils from diabetic animals also had low cleavage of LC3BI to LC3BII and do not present punctate structures that label autophagosomal membranes after stimulus. The changes of neutrophil function reported in diabetic rats do involve impaired autophagy. The suppression of autophagy in neutrophils from diabetic rats may be associated with the activation of the mTOR signaling as indicated by the high content of pS6.
Highlights
Changes in neutrophil functions, such as chemotaxis [1], production of inflammatory mediators [2, 3], phagocytosis [4], bactericidal activity [5, 6], production of reactive oxygen species (ROS) [7, 8], and programed cell death [9], contribute to the high incidence of infections in diabetic patients
GFX and diphenylene iodonium (DPI), inhibitors of NADPH oxidase, fully inhibited ROS production in neutrophils from both groups, indicating that this enzyme complex is the main site of ROS production during Phorbol 12-Myristate 13-Acetate (PMA) stimulation
Neutrophils from diabetic rats had lower expression of LC3B before and after PMA stimulus when compared to the control group (Figure 5C)
Summary
Changes in neutrophil functions, such as chemotaxis [1], production of inflammatory mediators [2, 3], phagocytosis [4], bactericidal activity [5, 6], production of reactive oxygen species (ROS) [7, 8], and programed cell death [9], contribute to the high incidence of infections in diabetic patients. Apoptotic neutrophils have increased expression of pro-apoptotic B-cell lymphoma 2 (BCL-2) family members, activation of caspases, reduction of mitochondrial membrane potential, phosphatidylserine externalization on plasma membrane, and DNA fragmentation [15, 16]. Autophagy plays an essential role in regulating cell death. It prevents apoptosis [17] by removing pro-apoptotic proteins, e.g., B-cell lymphoma-associated X and BCL-2 antagonist killer or by generating caspase inhibitors [18, 19]. Autophagy is an evolutionary cellular degradation mechanism involved in cell homeostasis. It is mainly activated by nutrient starvation [20], oxidative stress [21], endoplasmic reticulum stress [22], and energy deficiency [23]
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