Unfractionated heparin ameliorates lipopolysaccharide induced expressions of nitric oxide and reactive oxygen species in renal microvascular endothelial cells / 中华危重病急救医学

ABSTRACT

Objective To observe the effect of heparin on the cellular morphology and the expressions of nitric oxide (NO) and reactive oxygen species (ROS) in renal microvascular endothelial cells (RMVECs) stimulated by lipopolysaccharide (LPS). Methods The three step gradient screen method was used to primarily culture rat RMVECs, and the 3rd and 4th generation cells with excellent growth were collected. The cells were divided into blank control group, 10 mg/L LPS treatment group and 2.5, 5, 10 kU/L heparin pretreatment groups (the corresponding dose of heparin was given 0.5 hour before LPS stimulation). The morphology of the cells at 24 hours after LPS stimulation was observed by transmission electron microscope, the expression of ROS in RMVECs was determined by immunofluorescence at 5, 15, 30, 45 minutes after LPS stimulation, and the expression of NO in RMVECs was determined by nitrate reductase method. Results ① In blank control group, the RMVECs membrane was intact, and the mitochondria and endoplasmic reticulum in cells were clearly visible. The nuclear membrane was complete, and nucleolus was obvious. Cell bubble deformation was obvious at 24 hours after LPS stimulation, especially in the mitochondria and cell membrane. After 10 kU/L heparin pretreatment, the vacuolar degeneration of organelles was significantly reduced, and the cell membrane morphology was stable. ② The increases in ROS and NO in RMVECs could be detected at 5 minutes after LPS stimulation, showed an increase tendency with time prolongation, ROS expression peaked at 30 minutes, NO expression peaked at 45 minutes, which showed significant differences as compared with those of blank control group [30-minute ROS (mean density) 76.2±5.8 vs. 1.5±0.1, 45-minute NO (μmol/L) 70.3±8.6 vs. 1.8±0.1, both P < 0.01]. The expression of ROS and NO production in RMVECs were significantly reduced by heparin, showed a decrease tendency with heparin dose elevation, and the most obvious effect was 10 kU/L of heparin, with significant difference as compared with those of LPS treatment group [30-minute ROS (mean density) 16.8±1.7 vs. 76.2±5.8, 45-minute NO (μmol/L) 11.8±8.6 vs. 70.3±8.6, both P < 0.01]. Conclusions Unfractionated heparin ameliorates LPS induced expressions of NO and ROS in RMVECs and protects the cell morphology. The effect of 10 kU/L heparin is most obvious.