| Objective:1.To explore the correlation between different stages of diabetic retinopathy and different stages of diabetic nephropathy from the point of view of ophthalmology2.To explore the mechanism of nephropathy in diabetic retinopathy rats by establishing a diabetic rat model.3.To observe the intervention effect of traditional Chinese medicine on retinopathy and renal histopathology in rats with diabetic retinopathy based on AMPK,and to explore its mechanismMethods:1.Clinical study methodsA study was conducted on patients with type 2 diabetes mellitus with diabetic retinopathy who were hospitalized in the Department of Diabetes Ophthalmology,Ophthalmology Hospital,Chinese Academy of traditional Chinese Medicine from January 2017 to December 2018.A total of 230 patients with complete clinical data,laboratory indicators and auxiliary examination were included,including 129 males and 101 females.According to the(DR)diagnostic criteria of diabetic retinopathy,the patients were divided into Ⅰ-Ⅵ stage,Ⅰ-Ⅲ stage was non-proliferative diabetic retinopathy group(BDR group)and Ⅳ-Ⅵ stage was proliferative diabetic retinopathy group(PDR group).According to the clinical diagnostic criteria of diabetic nephropathy((DKD)),the patients were divided into two groups:non-diabetic nephropathy group(NK group)and diabetic nephropathy group(DKD group)According to the clinical stage of CKD,the DKD group was divided into two groups:1Mel stage 2(K1 group)and 3Mel stage 5(K2 group).To study the correlation between DR stage and nephrotic stage,clinical general data,blood glucose and blood lipid.2.Experimental research methods6-week-old SPF male SD rats were selected,10 rats were randomly selected as normal group,and the other 40 rats were induced diabetic rats by intraperitoneal injection of 2%streptozotocin(STZ)55mg/kg.The rats after successful modeling were randomly divided into control group and experimental group(high,middle and low dose groups).The rats were given intragastric administration of Chaigui decoction for 24 weeks.During this period,the general state of rats in each group was dynamically observed,and the body weight and random blood glucose of rats were monitored every 4 weeks.After 24 weeks of intragastric administration,the rats were killed,and the optic omentum and kidney tissues were removed.Paraffin sections were made to observe the pathomorphological changes and autophagy of retina and kidney by HE staining and immunohistochemical staining.RT-PCR was used to determine the expression of AMPK,Beclin-1 and LC3 in retina and kidneyResults:1.Clinical research result1)BDR group(n=104)and PDR group(n=126).The male/female ratio of BDR group was 1.12 and that of PDR group was 1.42.Age,age of DM onset,creatinine,urinary microalbumin,urinary immunoglobulin G,α 1-microglobulin and urinary transferrin had significant differences between the two groups(P<0.01).There was significant difference in the course of hypertension between the two groups(P<0.05).It is suggested that there is a correlation between age,especially the age of onset of DM and DR stage.The earlier and younger the age of onset of DM is,the more serious the degree of DR is.The course of hypertension was shorter in PDR group.The related indexes of renal function were correlated with DR stage,and the renal function was worse in PDR group2)Among the 230diabetic retinopathy patients,99(95.2%)were in BDR group(Ⅰ-Ⅲ),4(3.8%)in K1 group,1(84.9%)in K2 group(1.0%);PDR(Ⅳ-Ⅵ)),14(11.1%)in K1 group and 5(4.0%)in K2 group.Whether there is diabetic nephropathy between BDR group and PDR group has statistical significance.The correlation coefficient of Spearman between DR stage(I-VI stage)and DKD group was 0.168,and the correlation coefficient of Pendall analysis was 0.94,0.000<0.01.The results showed that the correlation coefficient between Spearman stage and Kendall analysis was 0.168 and 0.000<0.01,respectively.Therefore,it can be considered that there is a correlation between DR and DKD stages.It is suggested that there is a positive correlation between DR stage and DKD stage,and the degree of renal lesion is significantly aggravated in PDR group.However,people with diabetic retinopathy are not necessarily complicated with diabetic nephropathy3)DR stage was negatively correlated with age,age of onset of DM and course of hypertension,and the correlation coefficients were-0.279,-0.253 and-0.133,respectively,in which age,age of onset of DM and course of hypertension were P<0.01 and P<0.05,respectively.It is suggested that the patients with younger age,younger age of DM onset and shorter course of hypertension have higher DR stages.4)There was no correlation between DR stage and blood β2-MG and urea nitrogen,but 2DR stage was positively correlated with urinary protein,microalbuminuria,urinary immunoglobulin G,α 1 microglobulin,urinary transferrin and creatinine,and the correlation coefficients were 0.215,0.295,0.262,0.206,0.2700.204,respectively.It shows that with the development of DR staging,urinary protein,urinary micro-albumin and creatinine are also changing,showing an increasing trend2.Experimental research result1)There were significant differences in body weight among different time points(4w,8w,12w,16w,20w,24w)(P<0.01).It was found that there was a significant difference in body weight between the normal group and the model group after 4 weeks(P<0.01).At the 24th week,the advantage appeared in the middle dose group,and there was significant difference between the middle dose group and the model group(P<0.01).Compared with the normal group,the blood glucose of rats in the model group increased significantly.Compared with the blood glucose control of rats in the intragastric administration group,it can be seen that the middle dose group has the best blood glucose control effect in 4w-12w.2)Compared with the normal group,the glomerular basement membrane of the model group slightly thickened,the diffuse Mesangial matrix widened,the fibrosis was serious,the capillary wall thickened obviously,the renal capsule enlarged and the renal glomeruli dilated with epithelial vacuole-like changes;the renal interstitium was obviously fibrous with inflammatory cell infiltration.The retina became thinner,the arrangement of cells was disordered,the number and volume of ganglion cells decreased obviously,the capillaries dilated,the lumen thickened and vacuoles formed,the capillaries of nerve fiber layer and inner plexiform layer dilated,and there were red blood cells in the cavity.There was edema in the inner boundary membrane and nerve fiber layer,the reticular structure became loose,the arrangement of photoreceptor cell layer was loose,the capillary lumen in the pigment epithelium was obviously dilated,there were red blood cells in the lumen,and some red blood cells leaked into the tissue outside the blood vessels.The pathological changes of renal tissue and retina in DM rats were confirmed3)Immunohisto chemical resultsRenal tissue:compared with the normal group,the protein expression of AMPK,Beclin-1 and LC3-Ⅱ in the model group was inhibited,and the positive expression cells were decreased,and the levels of AMPK,Beclin-1 and LC3-Ⅱ in the high,middle and low dose groups were significantly higher than those in the model group.Compared with the middle dose group,the renal tissue AMPK in the model group,high dose group and low dose group decreased significantly,and the protein expression of Beclin-1 and LC3-Ⅲ in the model group was significantly lower than that in the middle dose group It is suggested that the expression of AMPK,Beclin-1 and LC3-Ⅱ in renal tissue of rats is inhibited in high glucose state.Chaigui Tang Jiajian recipe may increase the expression of autophagy by activating AMPK,in renal tissue and improve the pathological changes of renal tissue in rats with high glucose.Retina:compared with the normal group,the activities of AMPK,Beclin-1 and LC3-Ⅱ in the model group were inhibited and the positive surface cells decreased,and the levels of AMPK,Beclin-1 and LC3-Ⅱ in the retina in the high,middle and low dose groups were significantly higher than those in the model group.Compared with the middle dose group,the levels of AMPK,Beclin-1 and LC3-Ⅱ in the retina of the model group were significantly higher than those of the middle dose group.Compared with the middle dose group,the level of retinal LC3-Ⅱ in the low dose group was significantly lower than that in the middle dose group(P<0.05).It is suggested that Chaigui decoction may increase the expression of autophagy in gastric perfusion group by activating retinal AMPK,thus improving the course of retinopathy in rats with high glucose.Correlation:there was a significant correlation between the AO values of AMPK,Beclin-1 and LC3-Ⅱ in retina and renal tissue,and the correlation coefficients were 0.782,0.872 and 0.782,respectively.It is suggested that there is a positive correlation between the expression of AMPK,Beclin-1 and LC3-Ⅱ in retina and renal tissue.4)PCR resultRenal tissue:compared with the normal group,the mRNA expression of AMPK,Beclin-1 and LC3-Ⅱ in the model group was significantly lower than that in the model group.2Compared with the model group,the mRNA expression of AMPK,Beclin-1 and LC3-Ⅱ in the gastric perfusion group was significantly higher than that in the model group.3Compared with the middle dose group,the mRNA expression of AMPK and LC3-Ⅱ in the model group and low dose group decreased significantly,and there was significant difference between the model group and the middle dose group.(4)compared with the middle dose group,the mRNA expression of Beclin-1 in the model group decreased significantly.It is suggested that the relative expression of AMPK,Beclin-1 and LC3-Ⅱ mRNA in renal tissue of model rats is inhibited,and Chaigui decoction may increase the expression of autophagy by increasing the relative expression of AMPK mRNA in renal tissue,thus alleviating the pathological changes of renal tissue in diabetic rats.Retina:compared with the normal group,the mRNA expression of AMPK,Beclin-1 and LC3-Ⅱ in the model group was lower,and the difference was statistically significant.2 compared with the model group,the mRNA expression of AMPK,Beclin-1 and LC3-Ⅱ in the gastric perfusion group was significantly higher than that in the model group.3Compared with the middle dose group,the mRNA expression of AMPK and LC3-Ⅱdecreased in the model group and low dose group,and the mRNA expression of Beclin-1 decreased in the model group,high dose group and low dose group compared with the middle dose group,and there was significant difference between the model group,the high dose group and the low dose group.It is suggested that the relative expression of AMPK,Beclin-1 and LC3-II mRNA in the retina of rats with high glucose is inhibited compared with that of normal rats,and Chaigui decoction can increase the relative expression of AMPK mRNA,thus improving the degree of retina lesion in rats with high glucose,and the possible mechanism is autophagy.Conclusions:1.There was a positive correlation between the stage of diabetic retinopathy and the stage of diabetic nephropathy.The proportion of diabetic nephropathy in patients with proliferative diabetic retinopathy is significantly increased,and the degree of diabetic nephropathy is more serious.However,not all patients with diabetic retinopathy are complicated with diabetic nephropathy.2.The stage of diabetic retinopathy was positively correlated with urinary protein,microalbuminuria,urinary immunoglobulin G,α1 microglobulin,urinary transferrin and creatinine,and negatively correlated with age and the age of onset of DM.Therefore,the earlier the age of onset of diabetes,the more serious the patients with retinopathy,the more serious the indexes of renal function.3.The expression of AMPK,Beclin-1 and LC3-Ⅱ protein decreased in retina and kidney of model group.Chaigui decoction could regulate autophagy by increasing the expression of AMPK in kidney and retina,thus improving the pathological structure of retina and kidney of DM rats. |
PDF Full Text Request