Anti-Oxidant Effect of Lycium Barbarum Polysaccharides on the Retina of Streptozotocin-induced Diabetes Rats

Aim: To investigate the effect of Lycium barbarum polysaccharides (LBP) on the oxidation damage of retina of streptozotocin-induced diabetes rats. Methods: The diabetes rats were induced by injection streptozotocin intraperitoneally. LBP was intragastrically administered to the rats of the LBP group. Body weight and blood glucose were measured every four weeks. In the 24th week, the SOD and MDA of retina were measured, the expression of VEGF mRNA of retina was tested by RT-PCR, the ultrastructure was observed by transmission electron microscopy. Results: The body weight decreased and the blood glucose increased in DM group and LBP group (P<0.01, VS control group). The body weight and blood glucose is the same level between DM group and LBP group (body weight P=0.503, blood glucose P=0.984). The SOD in DM group decreased and the MDA increased (P<0.05, VS control group). The SOD in LBP group increased and the MDA decreased (P<0.05, VS DM group). The expression of VEGF mRNA was highest in DM group and lowest in control group, and decreased in LBP group (P<0.05, VS DM group). The ultrastructure changes occur in almost all retinal nerve tissues in DM group, as represented by the number and morphology of mitochondria. A small amount of mild mitochondrial changes could be observed in the endochylema of bipolar and Müller cells in LBP group. Conclusion: LBP can reduce the oxidative damage, alleviate the pathological changes in mitochondria, prevent the apoptosis of nerve cells and block the progression of diseases to the vascular tissue.


Establishment of Streptozotocin-Induced Diabetic Rat Model
The [3] SD rats were randomly divided into the DM group (15 rats) and the control group (5 rats). They were raised adaptively for 1 week, and fasted for 10 hours before modeling. Streptozotocin solution was injected into rats in the DM group via the lower left abdominal cavity(65mg·kg -1 ). Citric acid-citrate buffer solution of the same quantity was injected into 5 rats in the control group. 72 hours after, blood was taken from the caudal vein to measure blood glucose. When the rats showed a blood glucose level ≥ 16.7mmol·L -1 , the measurement of blood glucose level was repeated after 1 week. The rats which blood glucose level ≥ 16.7 mmol·L -1 were picked out for the following experiment.

Experimental Grouping and Observation
Thirteen rats, which passed the modeling, were randomly divided into two groups: the diabetic model group (DM group) and the LBP treatment group (LBP group). Every morning, LBP was intragastrically administered to the rats of the LBP group, whereas physiological saline was intragastrically administrated to the DM group and the control group. Body weight and blood glucose were measured every four weeks.

Observation of SOD and MDA Level of Retina
The rats were weighed and blood glucose were measured after drug administration for 24 weeks. The rats were intraperitoneally injected with napental(60mg·Kg -1 ) and the eyeballs were removed.
The retina was peeled under a ophthalmologic microscope. The retina tissue was added into the pre-cooled physiological saline at 4°C and then cleaned twice. The physiological saline at 4°C was added at a ratio of 1:9. The solution was then homogenized and centrifugated (2000rpm, 10min). The supernatant fluid was retrieved for measurements with a reagent kit, according to the manufacturer's instructions.

Expression of VEGF mRNA in the Rat Retina Tested by RT-PCR
The eyeballs of the rats in each group were taken to peel the retina under a microscope for ophthalmologic operation. The TRIzol method was used to extract total RNA and prepare cDNA by reverse transcription. The cDNA was PCR-amplified, and then agarose gel electrophoresis was performed. A gel imaging system was used for gray scale scanning. The absorbance value was recorded and the VEGF/β -Actin specific value was determined.

Ultrastructure Observation by Transmission Electron Microscopy
Glutaraldehyde myocardial perfusion was performed and the eye ball was rapidly removed. Retina tissue was taken and cut into small rectangular pieces, which were then fixed in glutaraldehydeparaformaldehyde solution overnight. After fixing in osmic acidpotassium ferrocyanide for cleaning, alcoholized uranyl acetate dye solution was used for En Bloc staining. Alcohol-acetone was used for dehydration in the gradient slope. Epoxide resin embedding medium was used for embedding. The specimens were sliced into ultrathin sections. Uranyl acetate and lead citrate were used for staining; they were observed under a Philips transmission electron microscope.

Statistical Analysis
Data were represented as mean±SD. Statistical analysis was performed by SPSS13.0 statistical software. Statistical differences between groups were analyzed by one-way analysis of variance (ANOVA). P<0.05 was considered statistically significant.

The difference of Body Weight During Observation
The body weight of the rats increased in control group whereas decreased in DM group and LBP group (P<0.01 VS control group).
The change of body weight is the same level between DM group and LBP group (P=0.984) (Table 1, Figure 1).

The Variation of Blood Glucose During Treatment
The blood glucose of the rats in DM group and LBP group is higher than the glucose in control group (P<0.01 VS control group).
The blood glucose is the same level between DM group and LBP group (P=0.503) (Table 2, Figure 2, 3).

Expression of VEGF mRNA by RT-PCR
The expression of VEGF mRNA in the rat retina was highest in DM group and lowest in control group. The level of VEGF mRNA decreased in LBP group (P<0.05, VS DM group) (Table 4, Figure 6).

Ultrastructure Observation by Transmission Electron Microscopy
The  activating NADPH oxidase combined with epicyte [8,9]. The study of diabetic renal disease indicated that the inhibition of NADPH oxidase can prevent AGE-induced renal damage in diabetic patients [10]. Under normal conditions, the antioxidant system in organisms can eliminate the oxidative substances produced by metabolism, and maintains the dynamic balance of the internal environment.
For example, superoxide dismutase (SOD) can eliminate all kinds of oxygen radicals. When an organism produces more oxygen radicals beyond its antioxidant ability, ROS damages its tissues and organs.
These attack the mitochondrial membrane, resulting in apoptosis in the mitochondrial pathways [11]. Excessive ROS also activates the action pathways of all known diabetic complications, including the polyol pathway, PKC, AGES precursors and amidohexose pathway, thereby inducing diabetic complications in multiple organs [12]. found that LBP can remove redundant free radicals in the body [13], improve the activity of antioxidant enzymes [14,15] and increase the survival rate and promote the growth of rat retinal ganglion cells [16]. In our study, no significant difference in blood glucose level was found between the LBP group and the DM group before treatment, showing that the two groups were comparable.
For blood glucose and body weight after treatment, no significant difference was also found between the two groups. These findings show that LBP could not reduce the blood glucose of animals. This result differs from that derived by Liu Ping [17]. The method they adopted was low dosage combined with high amounts of fat and sugar. The blood glucose of the modeled animal was 13.43 ± 1.36 mmol·L -1 . Our study adopted the high-dosage streptozocin simple modeling method. The blood glucose of the diabetic model rats were more than 20 mmol·L -1 . Therefore, our research can rule out the possibility of LBP indirectly curing diabetic retinopathy by reducing blood glucose. Significant pathological changes were not