The Protective Effect And Mechanism Of TFMP And Its Monomers On Diabetic Cardiomyopathy

Cardiovascular complications, including diabetic cardiomyopathy (DCM), havebecome one of the leading causes of the rising morbidity and mortality in both type1and type2diabetes. In addition, DCM, as an independent diabetic cardiaccomplication, is characterized by the myocardial dysfunction in the absence ofhypertension, coronary artery or valvular heart disease which increases the risk ofheart failure. The mainly clinical feature of DCM is left ventricular hypertrophy anddiastolic dysfunction and will occur systolic dysfunction with the progression.TFMP was extract from the leaves of Murraya paniculata (L.) Jack (TFMP). Thecontent of flavonoid was more than92%. The study showed that TFMP wascomposed of a series of small polar methoxy flavonoids, and proved that the highestcontent of TFMP was JA (40.96%) and JB (31.05%). Our previous study showed thatTFMP had hypoglycemic and protective effect of diabetic nephropathy. However, thestudy about the effect of TFMP、JA and JB on the progression of DCM has not beenreported.In the thesis, we have used high fat diet and single low does STZ to inducediabetes to evaluate the protective effect of TFMP on diabetic cardiomypathy, and wehave established the model of oxidative injury and apoptosis in H9c2cells by highconcentration glucose to study the effects of TFMP、JA and JB on H9c2cellsoxidative injury and apoptosis.Flavonoids is one of the major compounds in the leaves of Murraya paniculata.Our previous study showed that TFMP had hypoglycemic effects without harmfulside effects. Therefore, we hypothesized that TFMP may have beneficial influence onthe progression of DCM. In the present study, the effects of TFMP on DCM in highfat diet and STZ-induced diabetic rats were evaluated.1. The protective effects of TFMP on type2diabetic rats(1) Experimental method The type2diabetic rats, induced by high fat diet and low dose STZ, were used toevaluate the effects of TFMP70、35mg/kg. The changes of TC, TG, HDL-c, LDL-cand IL-6in the serum of rats; the activity of SOD, GSH-Px and the content of MDAin myocardial tissue of rats; the expression of TGF-β1and CTGF in myocardial tissuewere detected. The myocardial histopathology and ultrastructure changes wereobserved.(2) Experimental results①Compared with the Normal group, the TC, TG, LDL-c level was increasedsignificantly and the level of HDL-c was decreased significantly in the Model group;compared with the Model group, the TC, TG, LDL-c level was decreased significantlyand the level of HDL-c was increased significantly in the TFMP70、35mg/kg group(P<0.05or P<0.01);②Diabetic rats showed a significant reduction of both SOD andGSH-Px activities while increased the level of MDA in the myocardial tissuecompared with normal rats; the avtivities of SOD, GSH-Px were elevated and thecontent of MDA was reduced significantly in the myocardial tissue of TFMP70、35mg/kg group (P<0.05or P<0.01);③The body weight was decreased and theHW/BW was increased significantly in Model group compared with Normal group;compared with Model group, the body weight was increased and the HW/BW wasdecreased significantly in the TFMP70、35mg/kg group(P<0.05or P<0.01);④In theModel group rats, the level of IL-6and the myocardial tissue staining for TGF-β1and CTGF was significantly higher than that in Normal group; However, comparedwith Model group, the level of IL-6and immunohistochemical staining for TGF-β1and CTGF in myocardial tissue was markedly less in TFMP70、35mg/kg group.⑤The lesion of heart in the TFMP70、35mg/kg were significantly improved comparedwith Model group.2. The effects of different concentration of glucose on H9c2cells.(1) Experimental methodTo investigate the apoptosis-related signaling pathyways that are avtivated inH9c2cells treated with (20、30、50、100mmol/L) glucose. The percentage of survival cells, LDH, SOD, GSH-Px, MDA, the activity of caspase-3，AO/EB,Hoechst33342, Annexin V-FITC/PI and the protein expression of caspase-3andp47phox in H9c2cells were observed.(2) Experimental results①MTT assay was performed to evaluate whether high concentrations of glucoseinhibit the growth of H9c2cells. We found that high concentration of glucose (30、50、100mmol/L) had cytotoxic effect on H9c2cells;②Compared with Normal H9c2cells, the activities of SOD, GSH-Px was decreased significantly, the content of MDAwas increased significantly in high concentration of glucose (30、50、100mmol/L)induced H9c2cells (P<0.05or P<0.01);③High concentration of glucose (20、30、50、100mmol/L) could increased the activities of caspase-3in H9c2cells (P<0.05orP<0.01);④Immunofluorescence staining showed that: the high concentration ofglucose (20、30、50、100mmol/L) induced H9c2cells displayed different levels ofapoptosis;⑤Western Blot to examine the protein expression of caspase-3andp47phox in cells exposed to high concentration of glucose (20、30、50、100mmol/L).The results showed that the protein level of caspase-3and p47phox were elevatedafter high concentration of glucose treatment.3. Effect of TFMP, JA and JB in H9c2cells exposed to high glucose and itsmechanism(1)Experimental methodH9c2cells treated with high concentration of glucose (30mmol/L) was used asModel group. TFMP, JA and JB added into high concentration of glucose (30mmol/L)respectively were used as medicine group intervention. The H9c2cells treated withnormal glucose (5.5mmol/L) was used as Normal group. The H9c2cells of eachgroup were collected and the following measures were carried out: The activities ofSOD, GSH-Px, the content of MDA and the activity of caspase-3were assayed byavailable kits. The apoptosis was observed by staining of AO/EB and Hoechst33342.The rate of apoptosis was assayed by Annexin V-FITC kit. The expression of p47phox,p38MAPK, p-p38MAPK, JNK, p-JNK, p-NF-κB, Bcl-2, Bax, caspase-3protein weredetermined by Western Blot analysis. (2) Experimental results①The activities of SOD, GSH-Px were increased and the content of MDA wasdecreased significantly in TFMP, JA and JB group compared with Model group(P<0.05or P<0.01);②The activity of caspase-3was decreased in TFMP, JA and JBgroup compared with Model group (P<0.05or P<0.01);③The result of AnnexinV-FITC/PI showed that: TFMP, JA and JB can inhibited high glucose induced earlyapoptosis in H9c2cells;④TFMP, JA and JB can decreased the expression ofp47phox, caspase-3. TFMP, JA, JB can up-regulate Bcl-2protein level, and reducedBax protein level. TFMP, JA and JB can reduce the phosphorylation of p38MAPK,JNK and NF-κB. The results showed that high glucose induced oxidative stressthrough the activation of NADPH oxidase, thus activating the p38MAPK, JNK,NF-κB signaling pathway, leading to myocardial apoptosis. The mechanism of TFMP,JA and JB protective effects probably through downregulation of NADPHoxidase-ROS-p38MAPK-JNK-NF-κB signaling pathway.In summary, TFMP has protective effect on the high fat diet and low dose STZinduced diabetic cardiomyopathy. TFMP, JA and JB can extenuate oxidative injury ofH9c2cells induced by high glucose. TFMP, JA and JB can inhibit H9c2cellsapoptosis induced by high glucose. This study preliminary clarified TFMP, JA and JBhave the protective effect of diabetic cardiomyopathy, and confirmed the monomericflavonoids JA and JB is the main active ingredient of TFMP.