| Background: erythropoietin (EPO) can increase myocardial blood vessel formationand inhibit myocardial fibrosis, which improves cardiac function in diabetic rats.Carbamylated erythropoietin(CEPO) is a kind of derivatives of EPO not affectinghematocrit, which is similar to the EPO heart protection. Whether CEPO hasmyocardial protection in diabetic rats is unclearObjective to investigate the protection and the possible mechanism of CEPO againstcardiomyopathy in experimental diabetes mellitus (DM) rat.Methods DM rat model is established by High fat and sμ gar feed add intraperitonealinjection streptozotocin. After confirmed DM, the press to dose and treatment courseof different points in two parts. One is different dosage experiment about CEPO:control group(A group, normal rats, n=10) DM group(B group, n=9) withouttreatment, DM+CEPO treatment at low dosage group (C group,500IU/kg, n=9),DM+CEPO treatment at dosage group (D group,1000IU/kg, n=9), DM+CEPOtreatment at high dosage group(E group,2000IU/kg, n=9), and DM+rhEPOtreatment group (F group,1000IU/kg, n=10),2times a week subcutaneouslyinjecting drμg intervention for4weeks.⑵Different treatments experimental aboutCEPO: Use the previously mentioned control group (A group,4W, normal rats, n=10), DM group (B group,4W, n=9) without treatment, DM+CEPO treatment atdosage group (D group,4W,1000IU/kg, n=9), DM+rhEPO treatment group (F group,4W,1000IU/kg, n=10) for a four-week short intervention. Choose another controlgroup (A ’ group,8W, normal rats, n=10); DM group (B’ group,8W, n=10); DM+CEPO treatment at dosage group (C ’ group,8W,1000IU/Kg, n=9); DM+rhEPOtreatment group (D ’ group,8W,1000IU/Kg, total n=10) for an eight-week long intervention protocol. Healthy rats were used as normal control groups. body weightand water intake were observed. Detect rat blood, Venous blood samples were drawnfor hematology routine examinations, serum glucose, triglycerides and totalcholesterol. Then heart tissues were collected and use HE staining, Masson stainingand electron microscopy myocardial morphological changes. The expression ofmyocardial transforming growth factor β1, connective tissue growth factor, Caspase-3and Bcl-xL protein were detected by Immunohistochemical. The expression of PI3K(p85α)/Akt1mRNA were detected by In situ hybridization. The expression ofp-PI3K (85), p-Akt (ser/thr), and p-ERK (44/42) MAPK protein were detected byWestern blotResults1. CEPO synthesis rate can be achieved (89.72±4.70)%, long-term use it cannotaffecting hematocrit in DM rats.2. In DM rats, serum glucose, triglycerides, total cholesterol were significantlyhigher (p＜0.05). A CEPO effect on blood glucose was no significant difference(p>0.05). CEPO significantly reduced elevated serum triglycerides, and theeffect increases with the dose more pronounced (p <0.05), compared with thesame dose of rhEPO CEPO significantly different (p <0.05).Meanwhile, CEPOreduce serum cholesterol levels more significantly (p <0.05). Compared with4Wgroups, CEPO treatment can significantly reduce cholesterol levels in8W groups(p <0.05).3. In HE staining CEPO and rhEPO group, each group abnormal situationimproved myocardial cells. Masson staining: CEPO can make diabetes increasingmyocardial interstitial collagen decreases, and the greater the dose is moreobvious. rhEPO can also reduce the number of diabetic myocardial tissuecollagen fibers.. TEM: CEPO group and rhEPO group indicates that they can besignificantly reduce diabetes on myocardial ultra structure damage.4. In TUNEL: diabetic myocardial apoptosis increased (21.557±1.915)/mm2, wassignificantly higher (p <0.05). CEPO myocardial apoptosis increased by less than DCM group (p <0.05), is (13.083±2.371)/mm2, but still higher than the controlgroup(p <0.05). rhEPO and CEPO groups were similar, the number of apoptoticcardiomyocytes was (14.476±2.804)/mm2.5. Immunohistochemistry: compared with the control group, diabetic ratmyocardium caspse-3, TGF-β1and CTGF protein expression, Bcl-xL expressiondecreased (p <0.05). Compared with model group, CEPO can reduce elevatedprotein Caspase-3, TGF-β1and CTGF, the anti-apoptotic protein Bcl-xL levels,CEPO in a dose-dependent manner (p <0.05), the same dose of rhEPO groupCEPO group with similar effect (p>0.05). Treatment compared with4W,8Wtreatment CEPO more significantly reduced Caspase-3, TGF-β1and CTGFprotein expression (p <0.05), while Bcl-xL protein levels, but no significantdifference (p>0.05).6. In situ hybridization: compared with the control group, diabetic model ratscardiomyocytes PI3K (p85α) mRNA and Akt1mRNA expression were increased(p <0.05). Compared with model group, CEPO can increase the myocardial tissueof diabetic rats PI3K (p85α) and the mRNA expression of Akt1(p <0.05). As thedose increased, CEPO on diabetic rat heart cells PI3K (p85α) and Akt1mRNA ofthe more obvious effects (p <0.05). High dose CEPO more obvious than rhEPO(p <0.05).4weeks compared with CEPO8weeks, PI3K (p85α) and Akt1ofmRNA were increased, but the difference was statistically not significant(p>0.05).7. Western blot: compared with normal rats, diabetic rat cardiomyocytes p-PI3K(85), p-Akt (ser/thr) and p-ERK (44/42) MAPK protein expression increased(p<0.05).; CEPO can make diabetic rats myocardial p-PI3K (85), p-Akt (ser/thr),and p-ERK (44/42) MAPK protein expression(p <0.05).4weeks and8weeksgroup compared, the difference is not obvious(p>0.05).Conclusions CEPO does not have the function of promote erythropoiesis as EPO andhas no effect on DM rats’ hematocrit for long-term administration. High fat and sμgarfeed long-term add STZ can successfully establish animal model of DM. CEPO can reduce the myocardium damage with lipotoxicity by lowering serum triglyceride andcholesterol in DM rats. It also can protect diabetic rats myocardial tissue structurefrom sμgar or fat causing damage; CEPO has anti-myocardial fibrosis and anti--apoptosis, which mechanisms involved in cell signaling pathways PI3K/Akt andERK (44/42) MAPK cell signaling pathway.Innovative:1. CEPO as a derivative of the EPO does not express the HCT even long-timeand large dose treatment in rats with diabetic cardiomyopathy.2. CEPO can inhibit the diabetic myocardial fibrosis and myocardial apoptosis, itmaybe provide new options for the treatment of diabetic cardiomyopathy and otherheart disease... |
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