| Diabetic Cardiomyopathy(DC),which triggered by metabolic disturbance,is a specific kind of diabetic heart disease,and it can cause the change of myocardial ultra structure,finally leads to left ventricular hypertrophy,dysfunction during diastolic and/or systolic.It is a kind of cardiac dysfunction that different from coronary artery disease,microvascular disease and cardiac neuropathy.With the incidence of Diabetes Mellitus(DM) increased and the course extended year by year,DC along with DM has become a leading cause of death.Therefore,the main research area in the field of diabetic angiocardiopathy is proving up the pathogenesis of DC and seeking for the prophylactic agent of DC.Recently,Traditional Chinese Medicine has attracted more and more attention in the treatment of DM and diabetic angiocardiopathy.Commonly used clinical prescriptions are Gegenqinlian decoction,Dachaihu decoction Huanglianjiedu decoction,Sanhuang decoction, etc,and Berberine,baicalin,matrine is a commonly used clinical prescriptions.Modern pharmacological studies have shown that these three kinds of Chinese medicine and its main chemical constituents in the treatment of diabetes and cardiovascular disease have a certain effect.Berberine(Ber) is the primary alkaloid inside Coptis chinensis Franch,cortex phellodendri\ Radix Berberidis.Large quantity,of studies have proved that Ber has the cardiovascular pharmacological activity of anti-arrhythmic,lowering blood pressure and cardiac,it also have the ability to lowering blood sugar,regulate blood fat,reform IR and restrain glycosylated protein.But there's no research report about whether Ber has the ability to cure DC.Baicalin(Bai) is a chief flavanoid in the Plant of Baicalin,it can generate baicalein and glucuronic acid after hydrolyze.According to the recent research,Bai not only has the pharmacological action of lowering blood pressure,and anti-inflammatory,but also has the ability to anti-lipid peroxidation,calcium antagonists and Catecholamine channel blockers, inhibit aldose reduction enzyme(AR),inhibitory protein non-enzymatic glycation,inhibition of protein kinase C activation,regulation of lipid metabolism and cytokines,the protection of endothelial cells,inhibition of vascular smooth muscle cell proliferation,protect myocardial ischemia / reperfusion injury.Matrine(Mat) is the active constituent of Chinese herbal medicine like Sophora alopecuroides L\Matrine and Subprostrate Sophora Root,and it is the primary matrine alkaloid. Mat has wide cardiovascular pharmacological action,and deeply research has been made in the field of antiarrhythmic,hypolipidemic,positive inotropic effect.In vitro study showed that Mat can inhibit aldosterone and the hyperplasia of cardiac fibroblasts caused by angiotensin-Ⅱ. Therefore,Mat may have the pharmacological action of anti ventricular remodeling.The topic established DC cell model by high sugar and high glucose plus high insulin. Through the in vitro experimental study of Ber,Bai,Mat on myocardial cells and cardiac fibroblasts in DC morphology,cell proliferation and the impact of pathology,this paper would evaluate their preventive and therapeutic effect on DC and discuss the metabolic mechanism of Ber during curing DC.Finally clarify the pathogenesis of DC,the role of drug therapy,as well as aspects of berberine on the prevention and treatment of diabetic cardiomyopathy.1 Cultured and identified of cardiomyocyte cell(MC) and cardiac fibroblast(CFB) in neonatal rat ventricularObjective:Isolation culture cardiomyocyte cell and cardiac fibroblast that-meet the experimental requirements and establish the best period of the experimental.Methods:Cardiac tissue was digested to single cell suspension by mechanical and enzyme.Cardiomyocyte cell and cardiac fibroblast was separated according to different adherence time and inhibition of chemical reagents in order to get high purity,and strong activity of myocardial cells and cardiac fibroblasts.Cell was identified by morphology and immunochemistry.Cytoactive was detected by trypan blue.Detected the cytoactive by MTT colorimetric,and mapped the cell proliferation curves.Results:①Cardiomyocyte was looked like fusiform or irregularity,cell nucleus was obviously bulged,and rhythmic mpulse was seen.Cytoactive＞90%;α-Sarcomeric actin antibody dyeing was positive(rate≥90%),90%,Fibronectin antibody dyeing was negative.After separation it grew in good condition within 144 hours.②Cardiac fibroblasts was looked like fusiform or irregularity triangle,Cell body was larger and kytoplasm was tenuity.Fibronectin antibody dyeing was positive,(rate≥95%),α-Sarcomeric actin antibody dyeing was negative. After the separation it reached a exponentially growth within 120 hours.Conclusion:Myocardial cells and cardiac fibroblasts purity and activity in line with the experimental requirements,the best experimental time period is 48 hours to 120 hours after separation.2 Effect of Bet,Bai and Mat on normal MC and CFBObjective:Observing how the Ber,Bai,Mat impact ordinary MC and CFB with different density.Methods:Measure the cell activity via MTT colormetric and Measure intracellular protein content through Coomassie brilliant blue method.Observe Ber with density 0.47μmol/L～60μmol/L,Bai with the density 0.47μmol/L～120μmol/L and Mat with the density 62.5μmol/L～2000μmol/L,find the effectiveness on ordinary MC and CFB after 48 hours.Results:①Ber with the density 7.5μmol/L～30μmol/L,Bai with the density 0.47μmol/L～30μmol/L,Mat with the density 125μmol/L～500μmol/L,Rosiglitazone with the density 1.6μmol/L～2μmol/L,gliclazide with the density 96μmol/L～120μmol/L and captopril with the density 32μmol/L～40μmol/L have no effect on MC cytoactive and protein.②Ber with the density 7.5μmol/L～60μmol/L,Bai with the density 0.94μmol/L～120μmol/L,Mat with the density 125μmol/L～500μmol/L,Rosiglitazone with the density 1.6μmol/L～2μmol/L,gliclazide with the density 96μmol/L and captopril with the density 32μmol/L～40μmol/L can inhibit ordinary CFB cytoactive and protein.Conclusion:Ber with the density 7.5μmol/L～30μmol/L,Bai with the density 0.94μmol/L～30μmol/L,Mat with the density 125μmol/L～500μmol/L,Rosiglitazone with the density 1.6μmol/L,2μmol/L,gliclazide with the density 96μmol/L and captopril with the density 32μmol/L～40μmol/L have no effect on ordinary MC cytoactive,but they can inhibit CFB proliferate.3 Effect of Bet,Bai and Mat on MC and CFB models induced by high glucoseObjective:Construct a model of MC and CFB induced by high glucose,then observe the effectiveness of Ber,Bai and Mat on MC induced by high glucose and CFB.Methods:Construct a model of MC and CFB induced by high glucose model with glucose with density 25mmol/L.Observe the effectiveness of Ber,Bai,Mat,rosiglitazone, gliclazide and captopril on MC and CFB induced by high glucose through testing cytoactive, area(image analysis method),protein content and collagen protein content that secreted by CFB(hydroxyproline-alkaline hydrolysis method)Results:①Glucose with density 25mmol/L can significantly increase the intracellular protein content of MC and CFB and stimulate CFB proliferation and collagen protein content that secreted by CFB.②Bet with density 3.75μmol/L～30μmol/L can effectively inhibit collagen protein content,area of MC induced by high glucose.It also inhibit cytoactive,area and collagen protein content of CFB,but has no effect on cytoactive of MC.③Bai with density 3.75μmot/L～30μmol/L can significantly inhibit collagen protein content,area of MC and CFB induced by high glucose.Bai with density 30μmol/L can effectively inhibit proliferation,collagen protein content Of CFB induced by high glucose.④Mat with density 62.5μmol/L can stimulate MC proliferation.Mat with density 500～μmol/L can inhibit protein content,area of MC induced by high glucose,it also inhibit proliferation and protein content of CFB.All Mat testing group can inhibit the area of CFB induced by high glucose,but they have no effect on collagen protein content increasing that induced by high glucose.⑤All testing group can inhibit the cytoactive,protein content,area,collagen protein content of MC and CFB.Conclusion:Ber,Bai,Mat can inhibit the area,protein content of MC and CFB induced by high-glucose.Ber and Bai can also inhibit cytoactive,area,protein content and collagen protein content of CFB,but Mat have no effect on it. 4 Effect of Bet,Bai and Mat on MC and CFB models induced by high glucose and high InsulinObjective:Observe separately the effectiveness of Ber,Bai,Mat on MC and CFB induced by high glucose plus high insulin.Methods:Use glucose with density 25mmol/L plus insulin with density 10-7mol/L to construct a model of MC and CFB induced by high glucose plus high insulin,and then observe the effectiveness of Ber,Bai and Mat on MC and CFB induced by high glucose plus high insulin.Results:①High glucose plus high insulin can significantly increase the cytoactive, protein content,area and collagen protein content of MC and CFB.②Ber with density 1.5μmol/L～30μmol/L can inhibit protein,area of MC and CFB induced by high glucose plus high insulin.Ber with density 30μmol/L can inhibit cytoactive of MC.③Bai with density 15μmol/L～30μmol/L can significantly inhibit,the area of MC and CFB induced by high glucose plus high insulin.Bai with density 30μmol/L can inhibit cytoactive,protein content and collagen protein content of MC and CFB.④Mat with density 250μmol/L～500μmol/L can inhibit cytoactive of MC and CFB induced by high glucose plus high insulin.Mat with density 500μmol/L can inhibit protein content,area of MC induced by high glucose plus high insulin,it also can inhibit protein content of CFB induced by high glucose plus high insulin. All testing group of Mat can inhibit the area of CFB,but they have no significant effect on collagen protein content.⑤All testing group can inhibit the cytoactive,area of MC induced by high glucose plus high insulin,they can also inhibit cytoactive,area,protein content and collagen protein content of CFB induced by high glucose plus high insulin.But they all have no significant effect on protein content of MC induced by high glucose plus high insulin.Conclusion:Ber,Bai and Mat can inhibit MC and CFB cell proliferation and hyperplasia induced by high glucose plus high insulin.Ber and Bai can also inhibit collagen protein content of CFB,but Mat have no effect on it.5 The mechanism of Berberine anti-diabetic cardiomyopathyObjective:Observe the effectiveness of Ber on MC induced by high glucose plus high insulin secreting TNF-αand CFB secreting TGF-β1.Methods:Using Glucose with density 25mmol/L plus insulin with density 10-4mmol/L to construct MC and CFB model.Observe the effectiveness of Ber,Bai and Mat on MC and CFB induced by high glucose plus high insulin through testing the content of TNF-αinside MC culture solution(ELISA-method),testing the content of NO inside CFB culture solution (Nitrate reduction method) and testing the content of TGF-β1(Nitrate reduction method).Results:All testing group of Ber can inhibit the content of TNF-αinside MC induced by high glucose plus high insulin and inhibit the content of TGF-β1 inside CFB,they can also inhibit the NO.content decreasing inside CFB.The positive drug also have the inhibition.Conclusion:The effectiveness of Ber on protecting myocardial cell,anti myocardial fibrosis and assisting cardiac ventricular remodeling may have relative with decreasing the content of TNF-α,TGF-β1,and increasing the content of NO.As a consequence of the above,High glucose and high insulin can active myocardial cell and cardiac fibroblasts,area,protein content and CFB secretion collagen.Ber,Bai and Mat can inhibit hyperplasia and myocardial fibrosis through inhibiting myocardial cell cytoactive, area,protein content and inhibiting CFB proliferation,area,protein,content and collagen protein content.The effectiveness of Ber on protecting myocardial cell,anti myocardial fibrosis and assisting cardiac ventricular remodeling may have relative with decreasing the content of TNF-α,TGF-β1,and increasing the content of NO,but need to do a future study on it. |
