| ObjectiveThe aim of this study is to explore the mechanism of Tangluoning in the prevention and treatment of DPN by targeting SIRT1/P53 pathway in animal experiments.The role of SIRT1/P53 pathway in DPN pathogenesis was verified through sciatic nerve histopathologic observation and SIRT1/P53 pathology-related protein expression.MethodsTen 8-week-old SPF C57BL/6J male mice were fed ordinary diet and set as normal group.Thirty-five SPF grade male KK-Ay mice were fed high fat diet for four weeks,and random blood sugar of mice was measured to be≥16.7mmol/l,which was regarded as successful modeling of diabetes.After successful modeling,they were still fed high fat diet to maintain high blood sugar state.Five mice died from various causes during the modeling process were removed,and the remaining KK-Ay mice successfully modeled were randomly divided into model group(n=10),high-dose Tangluoning group(n=10),low-dose Tangluoning group(n=10),and the low-dose Tangluoning group was gavage with Tangluoning crude drug 10 g/(kg·d).For 8 weeks,the high-dose Tangluoning group was given 20 g/(kg·d)Tangluoning crude drug,and the normal group and model group were given equal volume of distilled water.The behavior of mice was observed every day,and the changes of blood glucose and body weight of mice at 0w and 2w,4w,6w and 8w after administration were detected.At the 8th week,LFB myelin sheath staining,HE staining,TUNEL staining and transmission electron microscopy were used to observe the pathological morphological changes of sciatic nerve.The expressions of SIRT1,P53,Bax,Bcl-2,cleaved caspase-3 in sciatic nerve tissue were detected by Western Blot.Results1.Behavioral observation results of mice in each group:The model group of mice had yellow and lustrous hair color,listless spirit,slow action,polydipsia,polyuria,and heavy odor;the low-dose Tangluoning group and the high-dose Tangluoning group also showed the above symptoms,but less severe than the model group.2.Comparison results of body weight of mice in each group:comparison between groups:compared with normal group,body weight of model group,the low-dose Tangluoning group and the high-dose Tangluoning group increased significantly in the same period(P<0.0001);Compared with the model group,the body weight of the high sugar group decreased significantly at 6 weeks(P<0.0001),while the body weight of the low sugar group had no significant difference(P>0.05).Compared with the model group,the body weight of the lowdose Tangluoning group and the high-dose Tangluoning group decreased significantly at 8 weeks(P<0.0001),which was statistically significant.Intra-group comparison:body weight of normal group at 2,4,6 and 8 weeks was significantly higher than that at 0 weeks(P<0.05);The body weight of the model group decreased significantly from 0 to 8 weeks(P<0.05).There was no significant difference in body weight between the low-dose Tangluoning group and the high-dose Tangluoning group(P>0.05).3.Comparison results of blood sugar in each group:blood sugar in model group,the lowdose Tangluoning group and the high-dose Tangluoning group was significantly higher than that in normal group during the same period(P<0.0001);Compared with model group,blood glucose levels in the low-dose Tangluoning group and the high-dose Tangluoning group were significantly decreased at 4,6 and 8 weeks(P<0.05).There was no difference in blood glucose level between the low-dose Tangluoning group and the high-dose Tangluoning group(P>0.05).4.The results of LFB myelin staining of sciatic nerve in each group:The myelin sheath of sciatic nerve tissue in the model group was broken and demyelinated.The myelin sheath of sciatic nerve tissue in the treatment group was broken,but it was significantly improved compared with the model group.5.HE staining results of sciatic nerve in each group:The nerve fibers in the model group were sparsely distributed,unevenly arranged,disorderly and scattered,with decreased number,unclear structure and irregular shape.Some nerves were swollen,and the gap between nerve fibers was increased.The myelin sheath of some nerve fibers was swollen and the density was uneven.Demyelination changes occurred,axonal atrophy became thinner,and Schwann cells were significantly reduced.Compared with the model group,the damaged nerve fibers in the high-dose Tangluoning group were partially restored,the myelin sheath morphology tended to be regular,the thickness was uniform,the axon was located in the central axis of the nerve fibers,and the degree of lesion was lighter.Compared with the model group,the nerve fibers in the low-dose Tangluoning group were partially restored,the myelin sheath was broken,and some myelin sheaths were vacuolated.6.TUNEL staining results of sciatic nerve of mice in each group:Compared with normal group,the percentage of positive sciatic nerve cells in model group,the high-dose Tangluoning group and the low-dose Tangluoning group was significantly different,with statistical significance(P<0.05).Compared with model group,there was no difference in the percentage of positive cells between the high glucose group and the low-dose Tangluoning group(P>0.05).7.Electron microscope observation results of sciatic nerve of mice in each group:In the model group,the myelin sheath layer structure of the mice was loose and shrunk in a large area,plaque-like demyelination and segmental demyelination were observed,vacuolar degeneration was observed,and multiple apoptotic bodies were observed.The distribution of nerve microfilaments and blood vessels was rich;the mice the low-dose Tangluoning group showed myelin degeneration of the sciatic nerve,vacuolar degeneration in the cytoplasm,but the myelin sheath layers were arranged neatly;the structure of sciatic nerve myelin sheath in the highdose Tangluoning group was relatively complete,the myelin sheath was swollen,and the morphology tended to be regular.8.Western Blot results:Western Blot results showed that compared with the normal group,the relative expression of SIRT1 protein in the model group was decreased(P<0.0001),and the protein expressions of acetyl-p53,Cleaved Caspase-3 and Bax/Bcl-2 ratio were increased(P<0.0001).Compared with the model group,the relative expression of SIRT1 protein in the high-dose Tangluoning group and the low-dose Tangluoning group was increased(P<0.0001),and the protein expression of acetyl-p53 and Cleaved Caspase-3 and the ratio of Bax/Bcl-2 were decreased(P<0.0001).Conclusions1.Tangluoning improves the general state and blood sugar of diabetic mice;2.Tangluoning can up-regulate the expression of SIRT1 protein and down-regulate the expression of P53,Bax/Bcl-2 and cleaved caspase-3 protein by activating SIRT1/P53 pathway,thereby improving the morphology of sciatic nerve tissue in diabetic mice,reducing apoptosis and slowing down the development of DPN. |
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