| Diabetic retinopathy(DR)is a major retinal vascular complication of diabetes mellitus(DM),and is still the main cause of blindness in DM patients of many countries.The pathogenesis of DR is sort of complicated.At present,more and more domestic and foreign studies have found that the activation of p38 mitogen-activated protein kinase(MAPK)-related pathways plays a vital role in the development of DR.At the same time,the high morbidity and disability rate of DR in DM patients make both patients and doctors more aware of the importance and criticality of early prevention of DR.In this study,based on the previous stage,combined with the experience of the tutor Xiaolin Tong in the clinical treatment of diabetic microvascular complications,Yiqi Tongluo Formula was used for early intervention in type 1 diabetes mellitus(T1DM)rats without retinopathy.Combining the overall level,including serum glycolipid metabolism,oxidative stress,and inflammatory status,with micro level,including retinal and pancreatic pathology,β-cell function,blood-retinal barrier(BRB),and p38 MAPK-related pathways,from multi-level,multi-angle,and multi-dimension,to observe the effect of Yiqi Tongluo Formula on delaying the retinopathy of T1DM rats and explore its internal mechanism,and compare the differences between Yiqi Tongluo Formula and Huoxue Tongluo Formula and their combined use of insulin in improving retinopathy in T1DM rats.It provided the scientific basis,scientific connotation for treatment of T1DM and prevention of DR with "Qi deficiency,collaterals and blood stasis" by the method of supplementing Qi,warming meridians and dredging collaterals under the guidance of the thought of "State-Target-Cause-Effect","early treatment of collaterals and whole process dredging of collaterals".Objective:1.To observe the role of Yiqi Tongluo Formula in improving blood glucose,blood lipid,islet tissue damage,regulating β-cell function and vital proteins in T1DM rats;explore the overall effectiveness and safety of Yiqi Tongluo Formula in delaying T1DM microvascular complications from the perspective of regulating glycolipid metabolism disorders,reducing islet tissue damage,then delay the development of DR;and compare the differences between Yiqi Tongluo Formula and Huoxue Tongluo Formula and their combined use of insulin in improving the above indicators2.To observe the role of Yiqi Tongluo Formula in improving the oxidative stress and inflammation related indicators in serum and retina of T1DM rats;explore whether Yiqi Tongluo Formula can delay the development of DR by reducing oxidative stress and inflammation in T1DM rats with the combination of the whole and the local;and compare the differences between Yiqi Tongluo Formula and Huoxue Tongluo Formula and their combined use of insulin in improving the above indicators.3.To observe the role of Yiqi Tongluo Formula in reducing retina Evans blue(EB)leakage,increasing the expression of vital tight junctions,and improving the pathological morphology of retina in T1DM rats;explore whether Yiqi Tongluo Formula can delay the development of DR by improving BRB and retinal tissue damage and compare the differences between Yiqi Tongluo Formula and Huoxue Tongluo Formula and their combined use of insulin in improving the above indicators4.To observe the role of Yiqi Tongluo Formula in reducing the expression of retinal polycomb repressive complex 2(PRC2),p38 MAPK,matrix metallopeptidase-9(MMP-9)and vascular endothelial growth factor receptor(VEGFR);explore whether Yiqi Tongluo Formula can delay the development of DR by regulating PRC2/p38 MAPK/MMP-9/VEGFR pathway,inhibiting the degeneration of retinal capillaries and angiogenesis;and compare the differences between Yiqi Tongluo Formula and Huoxue Tongluo Formula and their combined use of insulin in improving the above indicators.Methods:1.There were 210 healthy male SPF SD rats,and 25 were randomly selected as the normal group.The remaining rats were injected with streptozotocin(STZ)at a dose of 45 mg/kg in the tail vein at one time to induce the T1DM rat model.175 rats after successful modeling were randomly divided into model group,insulin group,insulin+calcium dobesilate group,Yiqi Tongluo group,insulin+Yiqi Tongluo group,Huoxue Tongluo group,insulin+Huoxue Tongluo Group and 25 in each group.Rats were intragastrically administered the next day after successful modeling.The doses of Yiqi Tongluo Formula and Huoxue Tongluo Formula were 7,2 g/kg/d,that of calcium dobesilate was 0.25 g/kg/d,and subcutaneous insulin doses were 1.5 IU/d,rats in normal group and model group were given the same amount of distilled water,once a day for 12 weeks.2.The weight and random blood glucose were measured every two weeks,and samples were taken at the 12th week of the disease course to detect relevant indicators Glucose(HbA1c,FBG)was detected in whole blood or serum of rats in each group as well as lipid(CHO,TG,HDL-c,LDL-c,VLDL-c).At the same time,the differences of glycolipid metabolism in each group were observed.The difference of β-cell function and pathological morphology of islet cells in each group were examined with oral glucose tolerance test(OGTT)and HE staining on paraffin sections.Western blotting was used to detect the expression of insulin receptor substrate 1(IRS-1)and glucose transporter 4(Glut-4)in pancreas.Differences in the vital proteins for maintaining glucose homeostasis were observed3.The enzyme linked immunosorbent assay(ELISA)was used in each group to detect serum oxidative stress indicators(ROS,iNOS,NO,MDA,SOD,GSH,CAT)and inflammatory factors.While western blotting was used to detect the expression of NF-kB p65 and TNF-α in the retina,and the differences in oxidative stress and inflammation levels in both serum and retina of rats in each group were observed.4.Fundus photography,FFA and HE/PAS staining on paraffin sections were used to observe the differences of retinal pathological morphology and vascular lesions in each group5.EB leakage was detected in each group,and the expression of ZO-1,Occludin,Claudin 5,and VE-cadherin in the retina of each group was detected by Western blot and RT-PCR.6.PRC2,EZH2,SUZ12,EED,p38 MAPK,MMP-9,and VEGFR expressions in the retina of each group were detected by Western blot and RT-PCR.The difference of decreasing activation of PRC2/p3 8 MAPK/MMP-9/VEGFR pathway in each group was observed.Results:1.Effects on body weight,glucose and lipid metabolism,islet tissue of T1DM rats:(1)Compared with the normal group rats,the general condition of the model group rats was worse,and the body weight significantly decreased,then slowly increased,and the difference was statistically significant.Compared with the model group,each medication group improved the general state of T1DM rats with varying degrees.The insulin group had the best effect,and the combination of either one of the formula+insulin was better than the two formula alone.Compared with the model group,the weight gain amplitude of the rats in the insulin group increased significantly in each time period,and the difference was statistically significant,and there was no difference in the other medication groups.(2)Compared with the normal group rats,the random blood glucose rise amplitude in the model group increased significantly,and the glucose index(HbAlc,FBG)elevated significantly.The difference was statistically significant.Compared with the model group,the insulin+Huoxue Tongluo Formula group had a significant hypoglycemic effect at 2 weeks of administration.Except for the Huoxue Tongluo Formula group,the Yiqi Tongluo Formula group and the insulin+Huoxue Tongluo Formula group,the other medication groups all showed a hypoglycemic effect at 4 weeks;At 12 weeks,except for the insulin+Yiqi Tongluo Formula group and the Huoxue Tongluo Formula group,the other medication groups showed significant hypoglycemic effects,among which,the insulin group and the insulin+Yiqi Tongluo Formula group had the best hypoglycemic effect.and the difference was statistically significant;On the whole,the random blood glucose rise amplitude in the 6 medication groups decreased in a spiral manner.Compared with the model group,HbAlc in each medication group reduced to varying degrees,and HbAlc in the insulin group and the insulin+calcium dobesilate group reduced significantly,and the difference was statistically significant.Compared with the model group,the FBG of each medication group reduced,and the difference was not statistically significant.(3)Compared with the normal group rats,the lipid index(CHOL,TG,HDL-c,LDL-c,VLDL-c)in the model group increased significantly,and the difference was statistically significant.Compared with the model group,CHOL,HDL-c,LDL-c,and VLDL-c in the insulin group decreased significantly,CHOL、LDL-c、VLDL-c in the insulin+calcium dobesilate group decreased significantly,CHOL、LDL-c、VLDL-c in the insulin+Yiqi Tongluo Formula group decreased significantly,CHOL、LDL-c、VLDL-c in the insulin+Huoxue Tongluo group decreased significantly,and the differences were all statistically significant.Among them,the combination of either one of the formula+insulin was better than the two formula alone,and the insulin+Yiqi Tongluo group had the best lipid-lowering effect.(4)Compared with the normal group rats,the area under curve(AUC)of blood glucose of the model group rats increased,and the difference was statistically significant.Compared with the model group,except for the insulin group,the AUC of each medication group reduced,and the difference was not statistically significant.(5)HE staining:Compared with the normal group rats,the sample from the model group rats showed islet atrophy,the islet cell randomly distributed,the cells proliferating on one side,and the nuclei densely arranged.Except for the insulin group,the damaged islet cells were improved to a different extent,atrophy reduced,and the islet cell distribution became more regular in each of the medication groups.(6)Compared with the normal group rats,the expression of IRS-1 and Glut-4 in the model group rats decreased significantly,and the difference was statistically significant.Compared with the model group,the expressions of IRS-1 and Glut-4 in each medication group increased significantly,and the difference was statistically significant.Among them,the combination of either one of the formula+insulin was significantly worse than the two formula alone,and the insulin+calcium dobesilate group had the best effect,followed by the insulin+Yiqi Tongluo Formula group.2.Effects on oxidative stress and inflammation in T1DM rats:(1)Compared with the normal group rats,serum ROS,iNOS,NO,and MDA in the model group rats all increased significantly,and the difference was statistically significant.Compared with the model group,except for MDA in the Yiqi Tongluo group,ROS,iNOS,NO,and MDA in each medication group decreased significantly,and the differences were statistically significant.Overall,the combination of either one of the formula+insulin was significantly better than the two formula alone.The insulin+Huoxue Tongluo group had the best effect,followed by the insulin+calcium dobesilate group.(2)Compared with the normal group rats,the SOD and CAT in the serum of the model group rats reduced significantly,and the difference was statistically significant.Compared with the model group,the levels of SOD,GSH and CAT increased in each medication group.(3)Compared with the normal group rats,the serum levels of TNF-a and IL-1β in the model group rats increased significantly,and the difference was statistically significant.Compared with the model group,the two indexes levels of each medication group reduced significantly,and the difference was statistically significant Among them,the combination of either one of the formula+insulin was significantly worse than the two formula alone,and effect of the insulin was the best.(4)Compared with the normal group rats,the expression of TNF-α in the retina of the model group rats increased significantly,and the difference was statistically significant,and the NF-kB p65 increased slightly.Compared with the model group,the expression of TNF-α in the insulin group and the Yiqi Tongluo group reduced significantly,and the difference was statistically significant3.Effect on pathological morphology of retina in T1DM rats:(1)Compared with the normal group rats,the model group rats showed lens opacity,blurred fundus periphery,widened retinal vein diameter and smaller arteriovenous ratio.The lens opacity,the widening of the retinal vein diameter of the rats in each medication group were alleviated to varying degrees.(2)HE staining:Compared with the normal group rats,the sample from the model group rats showed thinner retina,cells arranged loosely and irregularly in each layer,swollen and thickened inner limiting membrane,and proliferation in the pigment epithelial cells.In each medication group,the pathological changes in the retina were reduced to varying degrees,the cells in each layer arranged more regularly,the inner limiting membrane becoming thinner,swelling reduced and pigment epithelial cell proliferation reduced.(3)PAS staining:Compared with the normal group rats,the inner limiting membrane of the model group rats was swollen and thickened,the capillaries dilated and congested,the vascular endothelial cells significantly proliferated,and the pericytes decreased.In each medication group,the inner limiting membrane of the retina was thinner,the dilation and congestion of capillaries reduced,and the vascular endothelial cells decreased.4.Effect on BRB in T1DM rats:(1)Compared with the normal group,the EB leakage in the model group was significantly increased,with statistically significant differences.Compared with the model group,the EB leakage decreased in each treatment group.Among them,the effect of the combination of either one of the formula+insulin was similar to the two formula alone.(2)Compared with the normal group,the expression of ZO-1,Occludin and VE-cadherin proteins in the model group were significantly decreased,with statistically significant differences.Compared with the model group,the protein expressions of Occludin,Claudin 5 and VE-cadherin in each treated group were significantly increased,with statistically significant differences Insulin+calcium dobesilate group,and insulin+Yiqi Tongluo group effects were the best.(3)Compared with the normal group,the expression of Occludin and Claudin 5 genes in the model group was significantly decreased,and the difference was statistically significant.Compared with the model group,except for the significantly decreased expression of Claudin 5 gene in the Huoxue Tongluo group,the expression of Occludin and Claudin 5 gene in each drug group was significantly increased,with statistically significant differences.And,insulin alone has the best effect5.Effect on p38 MAPK-related pathway in T1DM rats:(1)Compared with normal groups,EZH2,SUZ12,EED,p38 MAPK,MMP-9,and VEGFR were all increased in the model group,with statistically significant differences.Compared with the model group,EZH2,SUZ12,EED,p38 MAPK,MMP-9 and VEGFR were all decreased in each group.On the whole,the group of insulin+Yiqi Tongluo had the best effect,followed by the group of Yiqi Tongluo,and the group of insulin had the worst effect.(2)Compared with the normal group,the expressions of EZH2,SUZ12,EED,p38 MAPK,MMP-9 and VEGFR genes in the retina of the model group were significantly increased,with statistically significant differences.Compared with the model group,except the insulin group,the gene expression level of each drug group was decreased to different degrees.On the whole,the combined use of insulin was significantly worse than the combined use of insulin.The effect of Huoxue Tongluo group was the best,while the insulin group was the worstConclusions:1.At 12 weeks of course,pathological changes occurred in the retinal tissue of T1DM rats with BRB destruction,and early retinopathy was found.2.T1DM rats were successfully modeled and treated with different intervention methods of Chinese and western medicine to prevent the development of DR at an early stage.Insulin combined with Yiqi Tongluo Formula had the best effect on improving BRB damage,enhancing retinal tight junctions,and inhibiting p38 MAPK-related pathways(PRC2/p38 MAPK/MMP-9/VEGFR),which was the intrinsic mechanism for early prevention of T1DM retinopathy in rats3.In the improvement of T1DM retinopathy in rats,insulin combined with Yiqi Tongluo Formula was superior to Yiqi Tongluo Formula alone or Huoxue Tongluo Formula or Huoxue Tongluo Formula combination with insulin.It provided experimental basis for the integrated syndrome differentiation and treatment of T1DM patients and early intervention to prevent DR by combining traditional Chinese medicine with Yiqi Huoxue Tongluo method on the basis of hypoglycemic lowering with western medicine4.Insulin combined with Yiqi Tongluo Formula can regulate glucose and lipid metabolism in T1DM rats,improve pancreatic damage,and inhibit inflammation and oxidative stress,which may be a reason to prevent the development of DR5.Although insulin alone can significantly lower glucose and improve inflammation and oxidative stress,it was significantly inferior to insulin combined with Yiqi Tongluo Formula in improving BRB damage,enhancing retinal tight junctions and inhibiting p38 MAPK-related pathways,proving that insulin alone cannot delay the occurrence and deterioration of DR. |
PDF Full Text Request