| Objective1.To establish a hyperglycemic myocardial model on H9C2 to simulate diabetic cardiomyopathy,and study the effect of Xie Huo Hu Xin Fang on the model.2.To establish the HPLC fingerprints and analyze the effective components of Xie Huo Hu Xin Fang.3.To explore the mechanism of Xie Huo Hu Xin Fang on the intervention of hyperglycemic myocardial model on H9C2 cell in autophagy cardiomyocytes.Methods1.With the HPLC-MS technology,this study establish and analyze the fingerprint of Xie Huo Xin Fang.After contrasting the reference materials,we conjectured the corresponding chemical structure and analyze the effective components of the compound preparation in serum and metabolites of rabbit.2.H9C2 cardiomyocytes were cultured in vitro and intervened with different concentrations of high glucose and serum containing drugs.And the diabetic cardiomyocyte model is studyed by high glucose.3.10 SPF male rabbits were fed by Xie Huo Hu Xin Fang decoction once a day.The same amount of normal saline and trimetazidine were used to feed the blank control and western medicine rabbits group on the same condition.The feeding amount standard is 1.5g/kg.After a continuous perfusion for 1 week,the serum was collected after the administration of 0.5h,1h,1.5h and 2h in seventh days of irrigation.The rabbits in different drug serums,different high glucose DMEM mediums and different markers were the intervention of H9C2 myocardial cells.The real-time live cell imaging technology was used to observe the proliferation,apoptosis and autophagy condition.And the LC3 Ⅱautophagy marker protein content were detected by Western blotting.ResultsThe study on the fingerprint chromatogram identified 22 peaks.With the SHIMADZU MetID database Solution and mass spectrometry analysis,we found 11 peaks for shift components of the blood and metabolites,included C22H17N04,C20H17N04,C19H13N04,C21H21N04,C15H1005,C15H806,C15H1004,C16H1205,C9H80,C20H27N04 and C21H2209.In the high glucose environment of 100mmol/L,100 times TCM serum inhibited the autophagy and promoted the autophagy in normal growth of H9C2 cardiac myocytes,which promoted the proliferation of H9C2 cardiomyocytes.In the high glucose environment of 100mmol/L with 10 times TCM serum and in the high glucose environment of 150mmol/L with 100 times TCM serum can inhibit H9C2 myocardial cell proliferation at the same time,also inhibit autophagy and normal growth of H9C2 myocardial cells.As the autophagy marker protein,LC3 Ⅱ was detected by Western blotting.Compared with the other intervention group,the expression of LC3 Ⅱ in the medium groups was increased(P<0.05).There was no significant difference between the control group and the control group(P>0.05).ConclusionThis experiment used HPLC-MS hplc-tofms to analyze the effective components of Xie Huo Hu Xin Fang.The analysis of the condition can make the sample stable and repeatable.The high similarity of fingerprint showed that this method was accurate and feasible.It provided a basis compound for further research of pharmacodynamic material.The experiment also showed that Xie Huo Hu Xin Fang could inhibit the proliferation of H9C2 cells and inhibit the occurrence of autophagy under the high sugar environment.The increased expression of LC3 Ⅱ of H9C2 myocardial cell autophagy may be induced by high glucose.So high glucose may lead to myocardial autophagy degradation,resulting from abnormal accumulation of degradation products.This study showed that Xie Huo Hu Xin Fang could inhibit cell proliferation and apoptosis,and delay the progression of the disease.That was mainly induced by high glucose through the degradation of H9C2 myocardial cell autophagy inhibition. |
