| Diabetes mellitus(DM)is a serious threat to human health,and its complications,Diabetic retinopathy(DR),are one of the leading causes of blindness in humans.Therefore,how to prevent and cure DR has become an important subject in the field of treatment of chronic complications of diabetes.DR is not only a retinal microvascular lesion,but also a neurodegenerative disease of the retina.It is found that apoptosis is an important process of DR nerve tissue injury.Protein kinase B(PKB/Akt)signal transduction pathway by Akt,Bcl-2-associated death protein(Bad)and cysteine aspartic proteinase-9(Caspase-9)is composed of a series of related factors in promoting cell growth,proliferation,apoptosis,differentiation and metabolism so,through the activation of Akt,the phosphorylation and phosphorylation of the downstream of a series of target protein,which play an important role,especially its effect on cell apoptosis.Sericin is a kind of silkworm cocoon extract,which is composed of 18 kinds of amino acids with high nutritional value.There has been a lot of research results show that sericin can effectively reduce type diabetes 2 mellitus(T2DM)rats blood glucose,and the target organs of diabetic rats including protective effect on diabetic kidney and central nervous system,but the protective effect of DR research is not many.In this study,SPF rats were selected as the research object,and the T2 DM rat model was established to investigate the effects of sericin on the PKB/Akt signal transduction pathway in the process of apoptosis of retinal nerve cells in T2 DM rats,so as to provide a new method for the treatment of DR in rats.Objective:Through the observation of type 2 diabetes sericin protein kinase during the apoptosis of retinal nerve cells in rats with PKB/Akt signal transduction pathway on related factors Akt,Bad and Caspase-9 expression,to investigatethe protective effects of sericin on DR and its possible mechanism.Methods:To clean grade healthy male Sprague-Dawley(SD)rats as the experimental object,a total of 36.The rats were randomly divided into groups according to the random number table method.The normal control group(NC group)do not need any treatment,given common forage;diabetic model group(DM group),sericin treatment group(ST group)with high caloric diet,and intraperitoneal injection of STZ(25mg/kg/d),continuous injection of 3D,the establishment of rat model of type 2 diabetes,fasting blood glucose(FBG)value is more than 16.7mmol/L as a model of standard model;after the completion of ST group(2.4g/kg/d,35d)of sericin by gavage,NC group and DM group were given equal physiological saline solution gavage,and the time was 35 d.2 The FBG of rats in each group was measured by ONE TOUCH blood glucose meter.3 Pathological changes of retina were observed by HE.4 The apoptosis of retinal neurons in each group was detected by TUNEL method.5 The expression of p-Akt,Bad and Caspase-9 protein in retina were detected by Western blot.6 RT-PCR was used to detect the expression of p-Akt,Bad,Caspase-9,and m RNA in rat retina.Results:1 FBG of rats in each group: DM group FBG was significantly higher than the NC group(P<0.01),ST group FBG was significantly lower than the DM group(P<0.01).2 Pathological changes of retina in rats: NC group rats showed the whole retinal layers of the retina layer structure is clear,smooth surface without internal limiting membrane bulge,all cells of normal morphology,uniform size,neatly arranged;the retina of rats in the DM group the overall structure less clear,obvious swelling in the surface membrane,even some parts offracture of retinal ganglion cells(RGCs)vacuole shape changes,pyknosis,cell number reduced,arranged in disorder;retinal internal limiting membrane of rats in the ST group showed mild swelling,mild reduction,the number of cells arranged in mild disorder.3 Apoptosis was detected by TUNEL assay.The apoptotic cells were mainly brown and granular,and each section was visible.Compared with NC group,the apoptotic cells in DM group and ST group were widely distributed,and the results showed that the apoptosis products were mainly concentrated in the RGCs layer and the inner nuclear layer.AI results: compared with NC group,the AI of retinal nerve cells in DM group was significantly higher(P<0.01).Compared with DM group,the AI of retinal neurons in ST group was significantly lower(P<0.01),the difference was statistically significant.4 The expression of p-Akt in retina of rats in each group: the results of Western blot showed that the p-Akt protein band was visible at 56 KD of protein Marker,and the band of β-actin protein in was visible at 43 KD of protein Marker.RT-PCR results showed that the p-Akt m RNA band was visible at 271 bp DNA Marker,and the band of β-actin m RNA was visible at the level of DNA Marker of 452 bp.The expression of p-Akt protein and m RNA in the retina of DM group was significantly lower than that of NC group(P<0.01).Rats in ST group were significantly higher than those in DM group(P<0.01).5 The expression of Bad in rat retina: Western blot showed that the Bad protein band was visible at 18 KD of protein Marker,and the band of β-actin protein was visible at the protein Marker of 43 KD.The RT-PCR results show that the Bad m RNA band is visible at the 109 bp level of DNA Marker,and the band of β-actin m RNA can be seen at the level of DNA Marker of 452 bp.The expression of Bad protein and m RNA in retina of rats in DM group was significantly higher than that in NC group(P<0.01).Rats in ST group were significantly lower than those in DM group(P<0.01).6 The expression of Caspase-9 in rat retina: Western blot showed that the Caspase-9 protein band was visible at 48 KD of protein Marker,and the bandof β-actin protein was visible at the protein Marker of 43 KD.The RT-PCR results show that the Caspase-9 m RNA band is visible at the 247 bp level of DNA Marker,and the band of β-actin m RNA can be seen at the level of DNA Marker of 452 bp.The expression of Caspase-9 protein and m RNA in retina of rats in DM group was significantly higher than that in NC group(P<0.01).Rats in ST group were significantly lower than those in DM group(P<0.01).Conclusion:Sericin may play an important role in the protection of DR by regulating the expression of related factors in PKB/Akt signal transduction pathway,that is up regulation of p-Akt and down regulation of Bad and Caspase-9. |
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