Materials and methods
In the present study, exposure of erythrocytes to HC plasma induced a significant increase of cholesterol inclusion into erythrocytes membrane compared to control ones (P⩽0.01). On the other hand, treatment of HC group with CS or CS plus l-arginine significantly decreased cholesterol loading into cell membranes compared to untreated HC group. Fig. 1 displays these results. In respect of SOD, GPx, and CAT, these results indicated that, treatment of control erythrocytes with CS, l-NAME and l-arginine preserves activity of these glycyrrhetinic acid and their ratios (SOD/CAT and SOD/GPx) at values near that of control one. However, a significant (P⩽0.05) decrease in SOD, GPx, and CAT activities was observed in erythrocytes incubated with HC compared to control. Conversely, treatment of HC group with CS, or CS plus l-arginine preserves activity of measured enzymes and their ratios compared to HC group. Table 1 represents these results.
In the current work, HC exposure induced marked decrease of GSH/GSSG ratio; however, MDA and PCC were significantly increased compared to control erythrocytes (P⩽0.05). On the other side, treatment of HC with CS or CS plus l-arginine keeps GSH/GSSG, MDA and PCC at values similar to control group, see Table 2. Erythrocytes SA contents were significantly decreased by incubation of cell to HC plasma. However, treatment l-Arginine, CS or CS plus l-arginine prevent HC induces SA loss. Fig. 2 represents these results. Plasma TAC significantly decreased in HC group compared to control. However, no significant difference was noted between control and control treated with CS or CS+l-arginine. Treatment of HC group with CS or Cs+l-arginine prevents HC-induced TAC depletion. Fig. 3 annotates these results.
In respect of NO variables, the exposure of erythrocytes to l-NAME caused a significant decrease in NOS activity and NO level compared to control group. A similar result was observed in HC treated group. On the other side, l-arginine treatment induced a significant increase in NOS activity compared to control; however, CS induces a marked but non-significant increase of NOS compared to control group. l-arginine treatment alone fails to prevent HC induced NOS inactivation. However, treatment of HC with l-arginine+CS prevented HC-induced decrease in NOS activity. By contrast, arginase activity was increased by HC exposure; however, CS or CS plus l-arginine treatment prevents HC induced arginase activation. Similar results were observed for arginase/NOS ratio. Table 3 explains these data.
Previous studies reported that an increase in arginase activity plays a crucial role in the cardiovascular pathobiology, so that the inhibition of arginase may protect against vascular diseases (Rabelo et al., 2015). Therefore, this study was conducted to explore the protective role of CS against HC induced erythrocyte’s arginase activation. HC disrupts erythrocytes cholesterol homeostasis, triggers ROS production and depletes antioxidant capability of cells (Devrim et al., 2008). However, cholesterol-lowering therapy prevents HC induced erythrocytes dysfunction (Franiak-Pietryga et al., 2009; Uydu et al., 2012). Herein, incubation of erythrocytes with cholesterol enriches plasma increased cholesterol inclusion into erythrocytes. Similar results were documented in several previous studies (Uydu et al., 2012; Harisa and Badran, 2015). In contrary, CS prevents cholesterol deposition into erythrocyte membranes; similarly, it has been reported that cholesterol lowering therapy decreases erythrocytes membrane cholesterol (Uydu et al., 2012). The attraction between CS and cholesterol decreases cholesterol inclusion into erythrocytes membrane.
In the current study, SA was assessed to confirm the safety of CS addition to erythrocytes SA as well as SA elicits antioxidant action. It has been reported that, ROS induced desialylation by depletion of SA content from cell surfaces (Pawluczyk et al., 2014; Harisa, 2015). Therefore, significant decrease in SA content was noticed in cells incubated with high cholesterol. The decreases of erythrocytes SA content increase their friction among themselves and endothelial cells; this may trigger vascular dysfunction (Fan et al., 2012). By contrast, treatment with CS keeps SA levels at values near that of control. This is in agreement with many studies which demonstrated that CS preserves antioxidants capacity (Anandan et al., 2013). In the present study, plasma TAC levels were significantly decreased by HC incubation. Likewise, several studies reported that oxidative stress exposure induced decline of TAC levels. The decrease in plasma TAC was demonstrated in HC induced oxidative stress (Devrim et al., 2008). Oxidative stress environment causes a plethora of changes in erythrocytes including loss of antioxidant power and enhances proteins and lipids oxidation.
Materials and methods