| Objective To observe the effect of Shanghan Classical Recipe Didang Decoction and Xiaoxian Xiong Decoction(hereinafter abbreviated to Didang Xianxiong Decoction)and the treating phlegm and blood stasis therapy on the myocardial tissue microvessel,myocardial fibrosis and extracellular mechanism caused by the AGEs-RAGE axis of diabetic cardiomyopathy rats.To investigate the relationship between the intervention mechanism of diabetic myocardial lesions and the AGEs-RAGE axis and its induced myocardial lesions.It was attempted to prove that the effect of Phlegm and Blood Stasis Therapy on diabetic cardiomyopathy is closely related to the activation of AGEs-RAGE axis.It provides the basis ofobjective experimental for the clinical prevention and treatment of diabetic cardiomyopathy,and lays a foundation for the development and ap plication of the prescription.Methods Adaptive feeding for 7 days,120 he ALT-711 hy male SD rats with body mass of220±20g were randomly divided into two groups,one group was 10 and treated as NC group.The other group was 110 for modeling,replicating the diabetic mellitus(DM)model.The 110 SD rats to be modeled were fasting for 24 hours,but the water was not inhibited.The single intraperitoneal injection of streptozotocin(STZ)solution was carried out at 55mg/kgdose.Finally,a total of 120 rats were successfully modeled.Then the DCM rats were randomly divided into: Model group(MC),Removing Phlegm and Blood Stasis group(RPDBS),AGEs specific protein cross-linked inhibitor group(ALT-711),Resolving Phlegm group(RP),Dissolving Blood Stasis group(DBS),18 in each group.Methods of administration: The NC and MC groups were fed with 1.0ml/(100g·d)distilled water.RPDBS group,RP group and DBS group were calculated according to the conversion coefficient of human and mouse,and the corresponding decoction was given to the stomach according to the dosage of 4.05,7.02,8.10g/(kg ? d);ALT-711 group: gavage at 0.3mg /(100g · d)(currently used now).The daily gastric perfusion time was fixed for 8 Weeks.the end of the 8th week,the material was taken after intraperitoneal injection of 3%pentobarbital sodium(30mg/kg)and the material was quickly obtained after anesthesia.1 Effect of Phlegm and Blood Stasis therapy on AGEs-RAGE axis activation:(1)Real-time quantitative PCR and western blot were used to detect the expression of RAGE.(2)Real-time quantitative PCR and western blot were used to detect the expression of PPAR?.2 Effect of treating therapy of Phlegm and Blood Stasis on myocardial microvascular lesions in DCM rats induced by AGEs-RAGE axis:(1)Real-time quantit ative PCR detection of c-jun gene expression.(2)The expression of p-JAK1 and p-STA T1 in cardiac muscle tissue was detected by Western blot.3 Effect of the therapy of Phlegm and Blood Stasis on myocardial extracellular stasis lesions in DCM Rats Induced by AGEs-RAGE axis:(1)Western blot was used to detect the expression of TGF-?1 in myocardium.(2)The expression of ROS in myocard ium was detected by ELISA kits.(3)The expression of type ? and type III collagen in myocardium was determined by immunohistochemistry.Results Twenty-two SD rats were eliminated due to death and other reasons during the experiment.Compared with MC group,the general situation of each group was improved in different degrees,among which the improvement was most obvious in ALT-711 group and RPDBS group.1(1)Real-time quantitative PCR and Western blot were used to detect RAGE expression.Compared with the NC group,the expression of RAGE in the myocardium of MC group was up-regulated,that were statistical significance(p<0.01).The protein and gene expression of RAGE in myocardial tissues of each group were down-regulated and significantly different that compared with the MC group(p<0.01).The RAGE gene and protein expression levels of RPDBS group were significantly lower than those of RP and DBS group(p<0.01).(2)Real-time quantitative PCR and Western blot were used to detect the expression of PPAR?.Compared with the NC group,the expression of PPAR? gene and protein in atrial tissue of MC group decreased significantly(p<0.01).Compared with the MC group,the expression level of PPAR? gene and protein in each drug group increased significantly(p<0.05,p<0.01),the expression level from high to low order: ALT-711 group,RPDBSgroup,RPgroup and DBS group.2(1)Western blot was used to detect the expression of p-JAK1 and p-STAT1 in cardiac muscle tissue : The expression levels of p-JAK1 and p-STAT1 in myocardial tissue of MC group were significantly increased that compared with NC group,(p<0.01).compared with MC group,the expression of p-JAK1 and p-STAT1 were diversity conspicuousness decreased in each drug group(p<0.01).Compared with that in RP group and DBS group,the expression level of p-JAK1 and p-STAT1 protein in RPDBS group was significantly lower(p<0.01),but not in ALT-711 group(p<0.01).(2)Real-time quantitative PCR detection of c-jun gene expression.Compared with the NC group,the expression level of c-jun gene in myocardial tissue of MC group was significantly up-regulated(p<0.01).Compared with the MC group,the expression level of c-jun gene was down-regulated in each drug group(p<0.05,p<0.01),that the expression level of c-jun gene in RPDBS group was significantly down-regulated compared with that in RP group and DBS group(p<0.01),that the ALT-711 group outperforms the RPDBS group(p<0.05).3(1)Western blot assay for the expression of TGF-?1 protein in cardiac muscle tissue.The expression level of TGF-?1 in myocardial tissue of MC group was up-regulated that compared with the NC group(p<0.01),Comparison of differentdrug groups and MC group,the expression level of TGF-?1 was down-regulated(p<0.01).The comparison between groups showed that the ALT-711 group was level of significance test lower than the RPDBS group(p<0.01),that the RPDBS group decreased significantly compared to RP group and DBS group(p<0.05).(2)The expression of ROS in myocardial tissue was detected by Enzyme Linked Immunosorbent Assay.The expression of ROS in MC group increased,and the difference was significant that compared with NC group(p<0.01).Compared with MC group,the ROS content of each drug group was significantly reduced(p<0.01),the ROS content from low to high order:ALT-711 group,RPDBSgroup,RPgroup and DBS group.(3)The expression of Type ?and Type III collagen was determined by immunohistochemistry.The proteins of Col?and Col? in NC group were expressed in cytoplasm,and a small amount of brown and yellow substances were observed.Compared with the NC group,the plasma and membrane of rat cardiomyocytes can be seen as brown yellow matter(p<0.01).Compared with the MC group,the brown matter areas of ALT-711 group and RPDBS group were obviously reduced,and the distribution was point-like(p<0.01).The brown matter in the pictures of RP group and DBS group still had the small area band distribution(p<0.01).The improvement of RPDBS group is better than that of RP group and DBS group(p<0.05),which is equivalent to that of ALT-711 group(p>0.05).Conclusion1 The Chinese medicine Didang Xianxiong Decoction with the function of "Treatment of Phlegm and Blood Stasis in Search and Eliminate Collateral Channels" can obviously improve the general condition of DCM model rat(including mental state,hair the condition,tail skin damage degree etc),Which is equivalent to ALT-711 group effect,which shows that the Chinese medicine has the certain advantage in preventing and treating the myocardial lesion of DCM rat.2 The intervention mechanism of Didang Xianxiong Decoction with the function of "Treatment of Phlegm and Blood Stasis in Search and Eliminate Collateral Channels" on DCM microvascular disease,extracellular stasis resistance disease,fibrosis disease and so on is related to the activation and regulation of AGEs-RAGE axis.3 The Chinese medicine composition has the obvious curative effect on DCM rat.It is superior to Xuefu Zhuyu decoction and Xiaoxian Xiong decoction for clearing away heat,eliminating phlegm and dissipating mass. |
PDF Full Text Request