| Diabetic retinopathy (DR), the most common type 2 diabetes complications, has become one of the main causes of blindness. L-carnitine (LC) can promote the fatty acid and glucose metabolism. There were a lot of studies on antioxidation and neuroprotective effects of LC in retinal ganglion cells (RGCs). Alginic acid oligosaccharide can scavenge reactive oxygen species (ROS) and excessive free radical, scavenge lipid peroxidation products, increase the activity of antioxidase function. Thus, this study intends to investigate the change of concentration of LC, Acetyl-L-carnitine(ALC) and Propionyl-L-carnitine(PLC) in plasma from patients with diabetes and diatetic complication, especially patients with diabetic retinopathy (DR). Through the establishment of the high glucose induced damage of retinal ganglion cells (retinal ganglion cells, RGCs) model, to observe the apoptosis of RGCs under high glucose condition, explore the protective effect and mechanism of antioxidative stress of LC and different molecular weight mannuronic acid oligosaccharide on high glucose induced apoptosis of RGCs, and the the synergistic effect between Lcand mannuronic acid oligosaccharide, and then investigate the regulation of Nrf2-Keap 1-ARE pathway in the protective effect of LC in the apoptosis of RGCs.(1) In this paper, the non intervention to collect clinical index and plasma samples of normal subjects (76 cases), diabetic patients (59 cases) and diabetes complications (diabetic retinopathy in 74 cases,51 cases of early diabetic nephropathy,57 cases of diabetic peripheral neuropathy, diabetic hypertension in 69 cases,64 cases of diabetic coronary heart disease), to detect the content of LC, ALC, PLC. The results showed that the concentrations of plasma LC in diabetic patients is lower than normal subjects, and plasma concentration in patients with diabetic complications decrease in diabetic patients remarkably. These suggest that LC supplementation may be benefit for patients with diabetes, especially in patients with diabetic complications,.(2) This paper discussed the protective effect and the mechanism of antioxidative stress of L-carnitine on RGCs apoptosis in high glucose conditions. The RGCs model was established by high glucose injury and application of trypsin digestion and primary culture of rat retinal neural cells. According to the different concentrations of glucose on RGCs after different time background to intervention, the survival of RGCs was observed by trypan blue stained experiment, RGCs apoptosis model was established by 30mmol·L-1 glucose concentration of glucose and cultured for 48h finally. The result of MTT show that the RGCs viability is best in 50-200μmol·L-1 of LC, The resuluts of Hoechst33342 show that the cells apoptosis rate of different concentrations of LC group was significantly lower than the protection of apoptotic cells in high glucose group (P<0.01 or P<0.05). The ROS lever of different group of LC decreased significantly than high glucose group (P<0.01), the content of MDA was lower than high glucose injury group (P<0.01), and the SOD, GSH-Px, CAT and T-AOC levels were higher than the model group with the dose dependent manner. The results suggested that L-carnitine preconditioning can inhibit the apoptosis of RGCs induced by high glucose by decreasing the levels of ROS and lipid peroxidation, enhancing cell antioxidant ability.(3) The protect effect of LC on the apoptosis of RGCs under high glucose condition and the regulation of Nrf2-Keap1-ARE pathway showed that Nrf2 protein of LC pretreatment group was significantly enhanced than high glucose group (P<0.01 or P<0.05). The expression of Keapl protein was not significantly changed (P>0.05). The protein expression of HO-1 and γ-GCS were higher than high glucose injury group (P<0.01) with dose dependent manner. The results suggested that LC has a neuroprotective effect for oxidative damage of RGCs in high glucose through activating the Nrf2-Keapl-ARE pathway, and activating the expression of downstream antioxidant enzyme HO-1 and phase Ⅱ detoxification enzymes y-GCS.(4)At the basis of the isolation and identification of four components of mannuronic acid oligosaccharide M segments (MH-1, MH-2, MH-3, MH-4), MTT method to measure the RGCs viability of mannuronic acid oligosaccharide pretreatment of different molecular weight showed that compared with high glucose group, the 10-1000μg·L-1 range of different molecular weight mannuronic acid oligosaccharide could antagonize the effect of high glucose on the activity of RGCs cells. The protective effect of size sorting is MH-1>MH-2>MH-4>MH-3 at the on the concentration of 100μg·L-1.The different molecular weight mannuronic acid oligosaccharide (MH-1, MH-2, MH-3, MH-4) protection group significantly decreased ROS level(P<0.01), decrease the elevation of intracellular MDA(P<0.01), reduced the lipid peroxidation caused by oxidative damage in RGCs cells, increased antioxidant enzymes SOD, GSH-PX and CAT activity and T-AOC, significantly compared to high glucose injury grop (P<0.05). The results suggest that different molecular weight oligomeric mannuronic oligose can protect the apoptosis of RGCs cells under high glucose condition, and its mechanism may be related to reducing intracellular ROS levels and lipid peroxidation damage, enhancing intracellular antioxidant enzymes SOD, GSH-Px and CAT activity, increasing intracellular total anti oxidative capacity (T-AOC) levels.(5) This paper explores the synergetic protective effect of L-carnitine and mannuronic oligosaccharides on high glucose induced apoptosis of RGCs. We select MH-1, which protective effect on apoptosis of RGCs was maximum, with LC to study the interaction of the mixture, Hoechst33342 staining was used to detect cell apoptosis.Results showed that, under the same conditions, the protective effect of mannuronic oligose MH-1 on apoptosis of RGCs is slightly lower than that of levocarnitine.The apoptosis rate comparison in three mixed groups suggested that protective effect of low concentration mixed group on the apoptosis of RGCs was higher than that of LC(L) group, MH-1(L) group and MH-1(M) group(P<0.05). Protective effect of concentration on apoptosis of RGCs in the mixed group was higher than that of MH-1 (M) group (P<0.05), but had no significant difference with LC (M) group. Protective effect of high concentration group on the apoptosis of RGCs is higher than that of MH-1(H) group, and had no significant difference with LC (H) group. The coefficient of drug interaction (CDI) of LC and mannuronic oligose was 0.598,0.971,1.350, respectively. The results suggest that LC and mannuronic oligose mixture effects on high glucose injury RGCs has synergistic effect. |
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