| Background:Diabetic heart disease is one of the most important complications which affects the quality of life of diabetes mellitus(DM)patients and endanger their lives.It has a huge burden on national health and social economy.Left ventricular diastolic dysfunction(LVDD)is one of the early clinical manifestations of cardiac function damage in patients with type 2 diabetes(T2DM).Due to its hidden symptoms and signs,it is easy to be ignored in diagnosis and treatment.At present,the prevention and treatment methods for T2DM with LVDD are very limited,so it is urgent to find a more safety and effective treatment.Traditional Chinese medicine(TCM)has advantages in treating diabetic heart disease.Yi-Xiao Formula(YXF),a TCM herbal compound based on the "heart collateral micro-zhengjia" theory,is created for the treatment of diabetic heart disease.Previous research has found that YXF had a certain protective effect on early period of diabetic heart disease patients,and could reduce the degree of myocardial fibrosis in spontaneously type 2 diabetic KKAy mice,but its mechanism needs to be further research and validated in clinical trials.This study further explored the possible mechanism and target of YXF in the treatment of myocardial fibrosis in T2DM through animal experiments and clinical trials,in order to provide a theoretical basis for clinical practice and new drug development.Objective:1 To observe the effect of YXF intervention on the improvement of glucose metabolism,myocardial pathological damage,cardiac structure and function in spontaneously type 2 diabetic KKAy mice,and to observe the effect of YXF intervention on transforming growth factor-β/Sma mothers against decapentaplegic(TGF-β/Smads)signaling pathway in cardiac tissue.In order to find the intervention target of YXF in treating diabetic heart disease,and provide an important basis for new drug development.2 To observe the effect of YXF on the glucose metabolism,echocardiography,TCM symptoms and signs,and serum myocardial fibrosis indicators in T2DM with LVDD(syndrome of qi-yin deficiency with blood stasis)patients.In order to verify the clinical efficacy and mechanism of YXF in treating T2DM with LVDD patients,and to provide an important method for early diagnosing and treating diabetic heart disease.Methods:1 Animal Experiment:Seventy-two male KKAy mice and twenty-four male C57BL/6J mice were studied at 12 weeks of age.After 2 weeks adaptive feeding,KKAy mice with fasting blood glucose≥13.9 mmol/L were diabetic mice and were included in the follow-up experiments.KKAy mice were randomly divided into model group,YXF group and losartan group,with twenty-four mice in each group.Twenty-four C57BL/6J mice with normal blood glucose were selected as the control group.The YXF group mice was gavaged with YXF 7.7 g/(kg·d),while the losartan group mice was gavaged with losartan 10 mg/(kg·d).The model group and control group mice were gavaged with equal doses of distilled water.The gavage intervention lasted 12 weeks once a day.At the 0th,4th,8th,and 12th weeks of intervention,six mice were randomly selected from each group for anesthesia and sample collection,before which the fasting blood glucose and echocardiography were detected.The cardiac tissue of mice was collected after sacrifice for HE staining,transmission electron microscopy,Masson staining,and pathological analysis.The expression of collagen Ⅰ(COL-1)and collagen Ⅲ(COL-3)in cardiac tissue were detected by Immunohistochemistry(ICH).The relative mRNA levels of TGF-β1 and Smad7 in myocardium were analyzed by real-time quantitative polymerase chain reaction(RT-qPCR).The relative protein levels of TGF-β1,Smad2,p-Smad2,Smad3,p-Smad3 and Smad7 in myocardium were detected by western blot(WB).Statistical analysis was carried out with SPSS 26.0.2 Clinical Study:Using the method of randomized,controlled and prospective study,cases of T2DM with LVDD(syndrome of qi-yin deficiency with blood stasis)were collected from the inpatient department in Dongzhimen Hospital,Beijing University of Chinese Medicine from September 2021 to January 2023,and divided into T2DM with LVDD group and T2DM with LVDD+YXF group according to the randomization principle grouping.The T2DM with LVDD group was cured with basic therapy,while the T2DM with LVDD+YXF group was cured with YXF on the basis of basic therapy for 8 weeks.Fasting blood glucose(FBG),postprandial blood glucose of 2 hours(2hPG),glycated albumin(GA),glycated hemoglobin(HbA1c),echocardiogram,TCM syndrome scores and serum level of TGF-β1,C-terminal propeptides of collagen type Ⅰ(PⅠCP)and N-terminal propeptides of collagen type Ⅲ(PⅢNP)of all the patients were detected and statistically analyzed at the 0th and 8th weeks by using SPSS 26.0Results:1 The results of experiments showed that in terms of glucose metabolism,the FBG level in the YXF group was significantly lower than that in the model group from the 8th week of intervention(P<0.01).In the pathology of cardiac HE staining and transmission electron microscopy,the myocardial pathological damage in the model group was more severe,after the intervention of YXF and losartan,the above pathological lesions could be alleviated to varying degrees.In terms of cardiac structure,the levels of left ventricular anterior wall thickness(LVAWT)and left ventricular posterior wall thickness(LVPWT)in the YXF and losartan group were significantly lower than those in the model group from the 8th week of intervention(P<0.01),while the level of left ventricular end diastolic diameter(LVEDD)was significantly higher than those in the model group from the 12th week of intervention(P<0.05).In terms of cardiac function,the levels of mitral orifice blood flow doppler early diastolic E peak/late diastolic A peak(E/A)and mitral annulus tissue doppler early diastolic e’peak/late diastolic a’ peak(e’/a’)of in the YXF and losartan group were significantly higher than those in the model group from the 8th week of intervention(P<0.01),and the level of E/e’ was significantly lower than those in the model group since the 12th week of intervention(P<0.05),There was no significant difference in the levels of left ventricular ejection fraction(LVEF)and left ventricular fractional shortening(LVFS)in each stage of treatment in the YXF group compared with the model group.In terms of Masson staining,the myocardial mesenchyme and peripheral vessel fibrotic area in the YXF and losartan group were significantly lower than those in the model group from the 8th week of intervention(P<0.01).In terms of ICH,the positive expression areas of COL-1 and COL-3 in the myocardium were significantly lower in the YXF and losartan group than those in the model group from the 4th week of intervention(P<0.01).In terms of mRNA levels in cardiac tissue,the mRNA level of TGF-β1 in the YXF and losartan group was significantly lower than the model group(P<0.01),while the mRNA level of Smad7 in the YXF and losartan group was significantly higher than those in the model group(P<0.05).In terms of protein expression levels in cardiac tissue,the levels of TGF-β1,p-Smad2/Smad2 and p-Smad3/Smad3 in the YXF group were significantly lower than the model group(P<0.01),while the level of Smad7 in the YXF group was significantly higher than those in the model group(P<0.01).2 The clinical study results showed that a total of 50 cases were collected in this study,among which 5 cases were dropped off.45 cases were actually completed,including 23 cases in the T2DM with LVDD group and 22 cases in the T2DM with LVDD+YXF group.The two groups of patients had comparability at baseline(P>0.05).After 8 weeks of treatment,in terms of glucose metabolism,2hPG of patients in the T2DM with LVDD+YXF group was significantly lower than that in the T2DM with LVDD group(P<0.05).2hPG,FBG and GA were decreased after treatment(P<0.001 or P<0.05).There was no significant differences in HbAlc between two groups(P>0.05).In terms of cardiac structure and function,the left atrial superior-inferior diameter(LASID)and tricuspid regurgitation velocity(TRV)of patients in the T2DM with LVDD+YXF group were significantly lower than those in the T2DM with LVDD group(P<0.01 or P<0.05).The lateral e’ was increased after treatment while the E/e’(Avg)was decreased after treatment in the T2DM with LVDD+YXF group(P<0.05 or P<0.01).The LVEF and LVFS in both groups were within the normal range.In terms of serum myocardial fibrosis indicators,the serum level of TGF-β1 in the T2DM with LVDD+YXF group was significantly lower than that in the T2DM with LVDD group(P<0.05),and the serum levels of TGF-β1,PⅠCP and PⅢNP were decreased after treatment in the T2DM with LVDD+YXF group(P<0.05 or P<0.01).In terms of the TCM symptom scores,the T2DM with LVDD+YXF group showed a significant improvement in symptoms such as chest tightness,short of breath and reluctant to speak,fatigue,and constipation compared to the T2DM with LVDD group(P<0.05 or P<0.01).Moreover,the TCM symptom scores of the T2DM with LVDD+YXF group had significantly decreased compared to the T2DM with LVDD group from the 4th week(P<0.05).After 8 weeks of treatment,the T2DM with LVDD+YXF group showed a more significant decrease in TCM syndrome score(P<0.001),and the effective rate of TCM syndrome and disease were significantly higher than those of the T2DM with LVDD group(P<0.01 or P<0.05).During the treatment and visit,no adverse reactions or adverse events occurred in both groups.Conclusions:1 Spontaneously type 2 diabetic KKAy mice are relatively stable diabetic heart disease model.After the high-fat diet,the blood glucose of KKAy mice continued to rise,and the thickness of left ventricular wall increased,accompanied by persistent myocardial fibrosis and cardiac diastolic dysfunction.It was consistent with the early clinical characteristics of human diabetic heart disease.2 YXF could significantly reduce the FBG of KKAy mice,relieve diabetic cardiomyopathy,reduce lipid vacuoles deposition,improve left ventricular wall hypertrophy and left ventricular diastolic function,and delay the progress of diabetic heart disease.YXF could down regulate the expression of TGF-β1 and up regulate the expression of Smad7,inhibit the phosphorylation of Smad2 and Smad3,reduce the deposition of COL-1 and COL-3,and inhibit the degree of myocardial fibrosis in diabetes.It is suggested that TGF-β/Smads signaling pathway may be one of the key pathways for YXF to improve myocardial fibrosis and left ventricular diastolic dysfunction in diabetes.3 The combination of YXF and conventional medicine could significantly improve the glucose metabolism,left ventricular diastolic function,and the TCM syndrome score of T2DM with LVDD(syndrome of qi-yin deficiency with blood stasis)patients.At the same time,YXF could reduce the serum level of TGF-β1 PⅠCP and PⅢNP,which indirectly suggested that YXF could alleviate the degree of myocardial fibrosis in patients and it was proven clinical benefits and safety.It provided important clinical evidence for the use of YXF in T2DM with LVDD patients. |
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