| Background:Type 2 diabetes mellitus(T2DM)is a metabolic disease caused by defective insulin secretion and/or impaired biological function.It is characterized by chronic hyperglycemia and metabolic disorders.The most serious complication of diabetes mellitus is diabetic vascular disease,which can be divided into two subtypes: microvascular disease and macrovascular disease.Diabetic cardiomyopathy(DCM)is one of the main vascular diseases affecting the heart(for example,ischemic heart disease and congestive heart failure).The role of immune inflammatory response in the pathogenesis of DCM has attracted more and more attention.Currently,there are no effective drugs that can stop or reverse the heart damage caused by diabetes.4-O-Methylhonokiol(MH)are one of the bioactive components extracted from magnolia stem bark.It has been shown that MH can significantly reduce the alginate induced increase of malondialdehyde content in mouse brain cells and Reactive oxygen species(ROS)reactive oxygen species(ROS)in cells,showing significant antioxidant and neuroprotective effects.On the other hand,MH also has anti-inflammatory activity by inhibiting the production of Tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β)through inhibiting Nuclear factor-kappa B(NF-κB).Our previous studies have demonstrated that MH protects against high-fat diet(HFD)induced heart disease in mice by activating nuclear factor 2(Nrf2)and protein kinase B(Akt/PKB,Akt)signaling.Objective:In this study,we aimed to clarify whether treatment with MH improves progression of diabetic heart disease in a T2 D mouse model and,if so,whether the protective effect of MH is related to inhibition of NF-κBmediated immunoinflammatory responses.Research methods:C57BL/6J male mice aged 7 weeks were fed Normal fat diet(ND)or High fat diet(HFD)for 3 months to induce insulin resistance,and then peritoneal injection of streptomycin(STZ,100mg/kg)was used to establish a type 2 diabetes mouse model.Then the model was developed into DCM mouse model,and Blood glucose(GLU)level was measured to evaluate the success of type 2 diabetes mouse model.Male C57BL/6J mice of the same age were injected intrabitoneally with streptothromycin solventsodium citrate(p H 4.5,0.1mol/L)solution as the control group.HFD/STZ mice with hyperglycemia(fasting 3-hour blood glucose ≥220 mg/d L)were treated as T2 D 7 days after STZ injection.The 40 mice were divided into the following 4 groups and given different administration treatments:(1)Control group: control mice were given dimethyl sulfoxide(DMSO)by gavage;(2)Ctrl+MH group: Control mice were given MH solution(1.0mg/kg 5 days a week for 3 months)by gavage;(3)T2D group: Type 2diabetic mice were given DMSO by gavage;(4)T2D+MH group: Type 2diabetic mice were given MH solution(1.0 mg/kg 5 days a week for 3months)by gavage.At the same time,the mice continued to feed ND or HFD.After 3 months of MH treatment,5 mice in each group were sacrificed as a 3-month time point.The remaining mice were fed ND or HFD for 3 months,respectively,and the remaining mice in each group were sacrificed as a 6-month time point.At 3-and 6-month time points,the experimental mice were anesthetized and killed,and the heart tissues of the mice were taken,from which proteins,m RNA or tissue sections were extracted.Cardiac function and structure were evaluated by noninvasive echocardiography in mice.Sirius red staining was used to observe and detect the changes of fibrosis in the heart,and oil red staining was used to observe and detect the changes of lipid aggregation in the kidney.Western blot assay was used to detect the levels of fibrosis(FN,Laminin),proinflammatory cytokines(TNF-α,IL-1β,ICAM),oxidative stress(3-NT,4HNE,SOD2,HO-1),lipid metabolism(CPT1B,CD36)and other signal transduction pathways To further detect the activation levels of NF-κB/PAI1 and GSK-3β in the immunoinflammatory pathway.Through data comparison and analysis,the conclusion is drawn.Research results:(1)MH treatment did not affect the blood glucose level and body weight of diabetic mice or control mice.Blood sugar levels in the T2 D mice were consistently higher than those in the Ctrl group over a 6-month period.After MH treatment,the blood glucose level of mice did not change significantly.Compared with Ctrl group,the weight of T2 D mice was higher than that of Ctrl group,and MH treatment did not significantly change the weight of mice.Secondly,there was no significant difference in the mean tibial length among all groups during the 6-month period.MH treatment did not result in changes in HW/TL in mice.(2)MH treatment increased left ventricular ejection fraction and alleviated the damage to cardiac function caused by type 2 diabetesEchocardiography showed that the EF and FS values in the left ventricle of T2 D mice decreased significantly at 3 and 6 months,and endsystolic(LV vol;s)significantly increased left ventricular volume.Left ventricular posterior end-diastolic wall thickness(LVPW;d)Significantly X increased at the 6-month time point,suggesting left ventricular hypertrophy.Left ventricular posterior wall end-systolic wall thickness(LVPW;s)significantly decreased at the 6-month time point.After MH treatment,at the 3-month time point,MH treatment significantly reduced the increase of left ventricular end-systolic volume and inhibited the decline of EF and FS in T2 D mice.At the 6-month time point,MH treatment significantly inhibited the decline of EF and FS and the increase of end-diastolic diameter of left ventricular posterior wall thickness.(3)After MH treatment,the myocardial fibrosis changes of DCM were inhibited by inhibiting FN and other fibrotic proteins.Using Sirius red staining,we observed a significant increase in collagen deposition in T2 D mice compared to Ctrl mice at 3 and 6 months,and a significant decrease in myocardial fibrosis in T2 D mice treated with MH compared to T2 D mice.At the 6-month time point,the expressions of fibrosis markers FN and Laminin were significantly increased,and MH treatment significantly decreased the expressions of fibronectin and Laminin(4)MH treatment inhibited the expression of FN and other proteins,and decreased the myocardial inflammatory response induced by type 2diabetesWestern blot was used to detect the protein expressions of tumor necrosis factor TNF-α,interleukin IL-1β and adhesion molecule ICAM-1in experimental mice(Figure 4.6A.B.C).These factors were found to be significantly higher in T2 D mice and significantly inhibited in MH treated T2 D mice at the 6-month time point compared with Ctrl mice.(5)MH treatment inhibited myocardial oxidative stress and regulated lipid metabolism disorder in DCMWestern Blot was used to detect the myocardial oxidative stress level of mice.At the 6-month time point,compared with Ctrl mice,the concentration of 4HNE in the myocardium of T2 D mice was increased,and the content of above lipid peroxides in the myocardium of MH mice was decreased after treatment.MDA was used to detect lipid peroxidation.At the 6-month time point,lipid peroxidation level of T2 D mice was significantly increased,while that of MH mice was significantly decreased.Oil red O staining was used to detect the level of lipid accumulation in the heart.Compared with the Ctrl group,the myocardial lipid accumulation in the T2 D group was significantly increased,while MH significantly reduced the lipid accumulation in the heart.At a 3-month time point,MH treatment significantly inhibited the effect of T2 D on CPT1 B expression in T2 D mice.(6)MH treatment inhibited the activation of NF-κB/PAI1 pathway and suppressed the immunoinflammatory response of DCMThe expression of NF-κB was detected by Western Blot.At the 6-month time point,compared with the Ctrl group,the activity of NF-κB protein in the myocardial cells of T2 D mice was significantly increased,while the activity of NF-κB protein in the myocardial cells of T2D+MH mice was significantly inhibited.At the 3-month time point,compared with the Ctrl group,the expression of PAI1 protein in the myocardial cells of T2 D mice was significantly increased,while the expression of PAI1 protein in the myocardial cells of T2D+MH mice was significantly inhibited compared with the T2 D group.The expressions of GSK-3β and Sirt1 were also detected.At the 3-month time point,T2-induced phosphorylation of GSK-3β in mouse diabetic cardiomyocytes decreased,while MH treatment increased phosphorylation of GSK-3β in T2 D mice.At the 3-month and 6-month time points,Sirt1 expression in T2 D mice was significantly decreased,and at the 6-month time point,MH increased Sirt1 expression in cardiomyocytes of T2 D mice.Conclusion:(1)MH reduced the left ventricular end-systolic volume and increased EF and FS in T2 D mice,thus alleviating the degree of cardiac dysfunction in T2 D mice.(2)MH alleviates myocardial fibrosis in T2 D mice,alleviates heart inflammation and oxidative stress induced by diabetes,regulates lipid metabolism disorder in T2 D mice,and has a protective effect on the heart of T2 D mice.(3)The protective mechanism of MH on the heart of T2 D mice may be related to the inhibition of NF-κB/PAI1 activation level by MH. |
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