The Research On The Mechanism Of Compatible Chinese Herbal Medicine Effective Components To Improve Diabetic Cardiomyopathy By P38MAPK Pathway

Diabetic cardiomyopathy (DCM) is an independent of the myocardial disease.Every year about 2-3% of diabetic patients complicated with cardiovascular disease,it was a study as long as 8 years showed that 4.9% male and 3.2% female diabetic patients died of cardiomyopathy at the age of 65-74 years each year.In contrast,the non diabetes population was 1.9% and0.9%,both of which has significant difference. Along with the prolonging duration of DCM,the prognosis is getting worse,and the incidence of heart failure and reinfarction is higher. The high prevalence of disability and fatality rate in patients with the danger of overall health,11.6% of health care expenditures was spent on prevention and treatment of diabetes in 2010 years, the World Health Organization estimated diabetes and vascular disease caused economic losses amounted to 557.7 billion dollar from 2005 to 2015 years in China.Pathogenesis of DCM is complex, involving advanced glycation end products, oxideti--ve stress, lipid metabolic disorders, inflammatory reaction, myocardial cell metabolism, myocardial microvascular disease, autonomic neuropathy multiple.There is no clear diagnostic criteria for it currently.Therefore, it can make a comprehensive judgement on the basis of clinical symptoms,laboratory examination,imaging examination, myocardial biopsy methods and the exclusion of coronary heart disease, hypertension, valvular heart disease and other cardiomyopathies. Early symptoms include cardiac insufficiency and congestive heart failure. Terminal symptoms include diastolic dysfunction, arrhythmia, widely focal myocardial necrosis,cardiogenic shock,and even sudden death in critically patients.Pathological manifestations contain myocardial hypertrophy,diffuse myocardial microangiopathy,reduced capillary density,endothelial and subendothelial fibrous hyper--plasia and basement membrane thickening.In view of the great harm caused by DCM for patients and society,and explore its pathogenesis and find effective treatment methods of scientific research become an urgent problem to be solved. Western medicine treatment is mainly to correct metabolic disorder and symptomatic treatment,not only to delay the development of cardiomyo--pathy heart failure, but also prevent the occurence of other cardiovascular complications. First is controlling blood sugar,second is controlling the lipid metabolism,Third is the use of RASS antagonists,fourth is the application of protein non enzymatic glycosylation inhibitors,fifth is to treat arrhythmia,heart failure, cardiac insufficiency,other symptoms and non drug therapy. According to the theory of Traditional Chinese medicine (TCM), Qi and yin deficiency and blood stasis as the important pathogenesis. The clinicians use syndrome differentiation combined with disease differentiation,prominent main treatment and differentiation throughout.Recently further clinical and basic researches have appear--ed, such as TCM prescriptions,compound preparation,single herb and its extract,natural medicine research and development, multiple target effect,overall adjustment,individual--ized treatment,and gradually improve the symptoms of patients and improve the quality of life, the comprehensive treatment is still the basic principles. Improvement of compatibility of TCM prescription optimization, dosage form, new drug research and development is imperative, but also the research group of drugs between the synergistic relationship,and different clinical stages of optimal treatment and medication dose, which provides new ideas for the treatment of DCM.[Objective] To observe the compatibility of TCM components(40% Ophiopogon japoni--cus polysaccharide,30% coptis alkaloid,30% Panax Notoginseng Saponins) on safety factors of DCM rabbit models:ALT, AST, Scr, BUN; the relevant indicators of blood glucose:GlU, FBG, GHB, GSP, INS, IRI, FRA, AR; TC, TG, blood lipids HDL-C,LDL-C; oxidative stress related indicators:AGEs, NO,MDA, SOD, GSH-PX;cytokine related indicators:hs-CRP, IL-6, TNF-a, TGF-β1, bFGF, PDGF, ICAM-1, VCAM-1,ET,PAI-1; apoptosis index:AI, Caspase-3, bcl-2; the signaling pathway protein expression of TGF-β1-p38MAPK-CREB.In order to explore compatible Chinese herbal medicine effective components to improve myocardial cell proliferation, differentiation, apoptosis by p38MAPK pathway and provide the basis for the possible mechanism and treatment of diabetic cardiomyopathy and new target.[Methods] 70 rabbits were used in experiment,10 rabbits were selected as the blank control group according to the random number table and were feeding normal diet.Then we injected the ear edge vein of rabbits by 0.9% Sodium Chloride 5ml in the 4th weekend,and 8 weeks normal diet after injection. The remaining 60 rabbits were fed with high fat diet containing 2% cholesterol,0.5% cholic acid and 5% lard(30g/kg·d).In the end of the 4th weekend, they were injected 5% alloxan(100mg/kgBW)by ear vein after fasted 8 hours. We selected the rabbits whose FBG was in the range of 16.7mmol/L-23.5mmol/L three times at random and removed the ones were not in the scope in the 8th week, and 8 weeks high fat diet after modeling.At the 12th weekend,1 health rabbit and 2 model rabbits were selected randomly after anesthesia drawn.The model rabbits myocardial tissue showed that myocardial cell swelling, degeneration,edema and cytoplasm with numerous vacuoles formation, myocardial fiber and interstitial fibrous structure disorder and intercellular boundaries were not clear, the gap widened, interstitial edema, increased perivascular matrix, mesenchyme inflammatory cell infiltration, nuclear fragmentation, nuclear condensation and nuclear loss phenomenon. All of them suggested that diabetic cardiomyopathy model has been established.50 rabbits were successful modeling and grouped according to the balance principle of fasting blood glucose levels and body weight:①model control group(n=10)②TCM components high dose group(n=10)③TCM components medium dose group(n=10)④TCM components low dose group(n=10) ⑤simvastatin control group(n=10).Experimental groups started to fill stomach in the 13th week, the specific dosage for TCM components groups divided into high dose group(450mg/kg·d),medium dose group(150mg/kg·d),low dose group (50mg/kg·d).The simvastatin group was treated with simvastatin(0.8mg/kg·d),blank control group and model control group with(10ml/kg·d) saline gavage.They were filling twice one day for 8 weeks.After fasting 12 hours and anesthesia by 5% sodium 3ml/kg in the 20th weekend, The 3-5ml blood was from the abdominal aorta,and the serum was separated by Fresco, 3000r/m,15min. The heart was Removal and taken part in left ventricular tissue and fixed with 10% neutral formalin solution, dehydrated ethanol, embedded in paraffin, conventional slice thickness 4um.The residual left ventricular tissue was flushing by physiological saline and wrapped with aluminum foil into liquid nitrogen for 3 minutes for frozen, then put it in the -70℃ refrigerator. Application of Colorimetric method for the determination of GlU, FBG, GHB, GSP, NO, NOS, MDA, SOD, GSH-Px, TC, TG, HDL-C, LDL-C, FRA in the serum.Determination of INS content by radioimmunoassay. Application of ElISA determination for AR,AGEs, ET, hs-CRP, IL-6, TNF-a, TGF-β1,bFGF, PDGF,PAI-1, ICAM-1, VCAM-1 in the serum and bcl-2,Caspase-3 in the Myocardial tissue. Western blotting method determination of protein expression from p38MAPK signal transduction pathway related channel and transcription factor p-p38MAPK, TGF-β1, CREB.HE staining of observation of myocardial tissue morphological and pathological changes by light microscope.TUNEL method of myocardial apoptosis and gelimage.[Results](1) The rabbits model were induced by injection of alloxan and high fat diet,the FPG could add up to average 18.0mmol/L in the 8th week,which significantly higher than the blank control group, the difference was significant(P<0.01),and indicated that the diabetic model had been established.In the 12th week,the myocardial tissue pathology showed that cytoplasm with massive vacuolization, cell swelling, degeneration,infiltratio-n of inflammatory cells, nuclear fragmentation and loss of nuclear, myocardial fiber and interstitial fibrous structure disorder.All of them indicated that diabetic cardiomyopathy model had been established.(2)TCM components effected on glycemic indices:After making models, FBG was increased significantly in 8th week, compared with blank control group, there were evident different (P<0.01). high dose TCM components could decrease the content of FBG in the 16th week,high dose, medium dose and low dose TCM components could decrease the content of FBG in the 20th week, compared with model control group, there were evident different (P<0.01).However, simvastatin could not decrease the content of FBG in the 20th week, compared with model control group, there was not evident different(P>0.05).high dose, medium dose group could reduce the content of GHB, GSP, AR, FRA,IRI and INS in the serum,compared with the model group, the difference was significant(P<0.05).High, middle dose groups were benefit for filling the stomach glucose load when 30mim,60min,120min and 180min, low dose group was benefit for glucose load after 30mim,the blood sugar were reduced, there were significant difference(P<0.05).(3)TCM components effected on blood lipid indices:After making models, TC, TG, LDL-C were rised, HDL-C was decreased, compared with the blank control group, the difference had statistical significance(P<0.05).high, middle dose group and simvastatin group of TC, TG, LDL-C were decreased and HDL-C rised by intragastric administration in 20th week,low dose group can decrease TC and LDL-C, compared with the model group, the difference was evident(P<0.05).(4)TCM components effected on oxidative stress indices:After making models,MDA, NO,NOS, AGEs were increased and SOD, GSH-Px decreased, compared with the blank control group, the difference had statistical significance(P<0.05).high, middle dose group and simvastatin groups can decrease MDA,NO,NOS and increase SOD, GSH-Px,high and middle dose groups can decrease AGEs,compared with the model group, the differ--ence were significant(P<0.01).Low dose group can decrease NOS, AGEs and increase GSH-Px, compared with the model group, the difference had statistical significance (P<0.05).(5)TCM components effected on inflammatory reaction indices:After making models, hs-CRP,IL-6,ET,PDGF,ICAM-1,VCAM-1,PAI-1 were increased and bFGF decreased, compared with the blank control group, the difference had statistical significance(P<0.05) high,middle dose and simvastatin groups of hs-CRP,IL-6,ET,PDGF,ICAM-1,VCAM-1, PAI-1 were dreased and bFGF increased;high dose group of TGF-β1 and TNF-α were decreased and simvastatin group of TNF-a was decreased; low dose group of hs-CRP, VCAM-1,PAI-1 were decreased and bFGF was increased;compared with the model group,the difference had significant difference(P<0.05).(6) TCM components effected on p38MAPK signal transduction pathway in channel and transcription factors:The expression of TGF-β1, p-p38MAPK and p-CREB in model group was higher than that in control group, with significant difference (P<0.01).The protein expression in high, medium dose group and simvastatin group of TGF-β1, p-p38MAPK were inhibited,compared with the model group,there were significant difference(P<0.01),the p-CREB protein expression was inhibited significantly, compared with the blank group,there was no significant statistical difference after treat--ment (P>0.05). Low dose group of p-p38MAPK, p-CREB protein expression were inhibited, compared with the model group,there were significant difference(P<0.01), the p-CREB expression suppression was more obvious, compared with the blank group,there was no significant statistical difference after treatment (P>0.05)(7) TCM components effected on myocardial pathology:high dose, middle dose and simvastatin groups of HW,LVW,H/W,LVWI have decreased, compared with the model group, the difference has statistical significance(P<0.01). high dose middle dose.Simvas--tatin groups had inhibited Caspase-3,AI and invoked bcl-2,low dose group has only invoked bcl-2,compared with the model group, the difference has statistical significance (P<0.01).(8) TCM components effected on body weight:After making models, the body weight of rabbits model group, high, medium and low dose group and simvastatin group weight decreased significantly in the 12th week,compared with blank control group, there were obvisious different (P<0.01).The high dose group rabbits body weight increased in 16th week,and high,medium dose group simvastatin group rabbits body weight significantly in 20th week,compared with model group,the difference were significant(P<0.01).(9) TCM components effected on liver and kidney function indicators:After making models, ALT, AST,BUN and Scr were increased significantly, compared with blank control group, there were evident different (P<0.01).ALT, AST,BUN,Scr in high and middle dose group were decreased and BUN,Scr in simvastatin group were reduced by intragastric administration in 20th week,lower than those of model group significant (P<0.01).[Conclusion] (1) Alloxan injection combined with high fat feeding induced rabbits model ,the average fasting blood glucose was 18.0mmol/L in the 8th week,which showed that the diabetes models were established.the DCM model was established by high fat diet in 12th week.(2) TCM components could reduce FBG, GHB, GSP, AR, FRA,IRI and INS,so it may improve glucose tolerance abnormal and insulin resistance.(3) TCM components could decrease TC, TG, LDL-C and increase HDL-C,in order to regulate the lipid disorder.(4) TCM components could decrease AGEs、MDA、NO、NOS and increase SOD、 GSH-Px, which improved oxidative stress injury.(5) TCM components could reduce hs-CRP、IL-6、TNF-α、ETF-β1、PDGF、ICAM-1、 VCAM-1、PAI-1 and rise bFGF,which alleviate the inflammatory reaction.(6) TCM components could regulate p38MAPK/ Caspase-3, bcl-2 pathway and inhibit the apoptosis of myocardial cells,and could regulate TGF-β1-p38MAPK-CREB signal transduction pathways to inhibit myocardial tissue abnormal, cell proliferation, differentiation and other biological effects,which alleviated the process of myocardial hypertrophy,interstitial fibrosis and other diseases.(7)TCM components could reducethe weight of the whole heart,the left ventricle,the weightindex of the left ventricle and the ratio of the weight of the body.(8) TCM components could increase the body weight of the model rabbits.(9)TCM components could decrease ALT, AST, BUN, Scr,and had a safe and reliable protective effect on liver and kidney function.