| Objective:The effects of hesperetin on Ang-2 in serum and retina of streptozotocin induced diabetic rats were observed,the effects of angiopoietin-2,integrin ?1 and their related signal pathway expression were studied,and the mechanism of protective effect of hesperidin on diabetic rat retina was discussed,which provided experimental basis for its application in clinical prevention and treatment of diabetic retinopathy.Methods:After one week of adaptive feeding,36 healthy male Sprague-dawley(SD)rats were randomly divided into three groups: the normal(CON)group,the diabetes(DM)group and the hesperetin(hesperetin)group,with 12 rats in each group.DM and Hesperetin groups of rats made diabetes diabetes model: after fasting and drinking water overnight for 12 h,the two groups of rats were injected with 1% streptozotocin(STZ)at 60mg?kg weight,then blood glucose was measured by caudal vein after 72 h.blood glucose was higher than 16.7 and persistent symptoms of polydrink polyuria were used as the model of diabetic rats.CON group of rats were injected with the same amount of 0.1M citrate buffer solution in abdominal cavity.All the three groups of rats were free to eat standard rat food and water.the Hesperetin group received 150 mg/kg of Hesperetin(dissolved in 1% tween-80),1/d,continuous gavage for 12 weeks.the CON group and the DM group received an equal amount of 1% tween-80 gavage of the same amount as the Hesperetin group,1/d,continuous gavage for 12 weeks.Blood glucose and body weight were measured by blood glucose meter and electronic balance.The highest value of blood glucose meter is 33.3 mmol/L,and the value of H1 is the highest value when it is larger than 33.3 mmol/L.After 10% chloral hydrate anesthesia,the supernatant was collected from the heart blood to detect the content of Ang-2,Integrin ?1 and IL-1? in the serum of rats by enzyme-linked immunosorbent assay(ELISA);the rats were killed by deep anesthesia,two rats(four eyes)in each group were randomly selected to remove the eyeballs,and paraffin sections were made by hematoxylin eosin staining(HE)was used to observe the histopathological changes of the retina of rats.The remaining ten rats(twenty eyes)retinas were taken out and the Ang-2,Integrin ?1,PI3 K,Akt,Bcl-2,Bax,Caspase-3 in the retina of rats were detected by RT-PCR and Western blot respectively m RNA and protein expression level.Results:1.Results of blood glucose and body weight of rats in each group:The blood glucose value of DM group was 28.03±3.56 mmol/L and CON group was 5.72±0.74 mmol/L,which was significantly higher than that of CON group,the difference of blood glucose between the two groups is very significant(P<0.01).The blood glucose value of Hesperetin group was 25.76±3.89 mmol/L,which was significantly higher than that of CON group(P<0.01),but there was no significant difference between Hesperetin group and DM group(P>0.05).The body weight of DM group was 235.27±25.76 g and CON group was 478.78±40.66 g,which was significantly higher than that of CON group,the difference of body weight between the two groups was very significant(P<0.01).The body weight of Hesperetin group was 263.76±34.21 g,which was significantly higher than that of CON group(P<0.01),but there was no significant difference between Hesperetin group and DM group(P>0.05);2.Histopathology change of rat retinal sections in each group:The results of HE staining showed that the layers of retina in the CON group were clear,intact and uniform in size.In the DM group,the cell space of retinal ganglion cell layer increased.The retinal structure of the Hesperetin group was relatively complete,the levels between the layers were clear,the ganglion cell layer and the inner and outer nuclear layer were arranged more regularly,the cell space was slightly increased obviously,and the swelling of the inner and outer plexiform layers was significantly improved compared with the DM group;3.Results of serum Ang-2,Integrin ?1 and IL-1? concentrations in rats of each group:Compared with CON group,the concentrations of Ang-2,Integrin ?1 and IL-1? in DM group were significantly higher,the difference was very significant(P<0.01),while the concentrations of Ang-2,Integrin ?1 and IL-1? in Hesperetin group were significantly higher(P<0.05),but they were significantly lower than DM group,the difference was very significant(P<0.01);4.The results of Ang-2,Integrin?1,PI3 K,Akt,Bcl-2,Bax,Caspase-3 RT-PCR in retina of rats in each group:Compared with CON group,the expression levels of Ang-2,Integrin ?1,Bcl-2,Bax and Caspase-3 m RNA in the retina of DM group were significantly higher,the difference was very significant(P<0.01),while the expression levels of PI3 K and Akt m RNA were significantly lower,the difference is statistically significant(P<0.05);Compared with CON group,the expression levels of Ang-2,Integrin ?1,PI3 K,Akt,Bcl-2,Bax and Caspase-3 m RNA in retina of Hesperetin group were significantly higher,the difference is statistically significant(P<0.05?P<0.01);Compared with DM group,the expression levels of Ang-2,Integrin ?1,Bax and Caspase-3 m RNA in the retina of Hesperetin group were significantly lower,the difference is statistically significant(P<0.05?P<0.01),while the expression levels of PI3 K,Akt and Bcl-2 m RNA were significantly higher,the difference is statistically significant(P<0.05?P<0.01);5.The results of Ang-2,Integrin ?1,PI3 K,Akt,Bcl-2,Bax,Caspase-3 Western blot in retina of rats in each group:Compared with CON group,the expression levels of Ang-2,Integrin ?1,Bax protein in the retina of DM group were significantly higher,the difference was very significant(P<0.01),the expression levels of PI3 K,Bcl-2 protein were significantly lower,the difference was very significant(P<0.01),compared with CON group,the expression level of Akt,Caspase-3 protein in DM group had no significant change,the difference was not statistically significant(P>0.05);Compared with the CON group,the expression levels of Ang-2,Akt,Bcl-2 and Bax protein in the retina of Hesperetin group were significantly higher,the difference was very significant(P<0.01),while the expression levels of PI3 K and Caspase-3 protein were significantly lower,the difference was very significant(P<0.01),Compared with CON group,the expression level of Integrin ?1 protein in retina of Hesperetin group had no significant difference(P>0.05);Compared with DM group,the expression levels of Ang-2,Integrin ?1,Bax and Caspase-3 protein in retina of Hesperetin group were significantly lower,the difference was very significant(P<0.01),while the expression levels of PI3 K,Akt and Bcl-2 protein were significantly higher,the difference was very significant(P<0.01).Conclusion:1.The whole body application of Hesperidin had no significant effect on blood glucose and body weight of diabetic rats,and its effect on the structure of diabetic retina was not caused by lowering blood glucose.2.Hesperidin can inhibit the expression of Ang-2 and Integrin ?1 in serum and retina of diabetic rats,reduce the concentration of IL-1? in serum and reduce the inflammatory response,and reduce the early damage of retina in diabetic rats.3.The mechanism of action of Hesperidin on reducing retinopathy in early diabetic rats may be related to activation of PI3K/Akt signaling pathway,up regulation of Bcl-2 expression,and inhibition of Bax and Caspase-3 expression. |
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