| Background: Schisandrae Chinensis Fructus (SCF) or Schisandrae SphenantheraeFructus (SSF), the fruit of Schisandra chinensis (Turcz.) Baill. or Schisandra sphenantheraRehd.et Wils has been used as a tonic and astringent in traditional Chinese medicine (TCM)for centuries. Arecent study demonstrated that SCF or SSF was the anti-oxidant componentherb in a Chinese medicinal which is commonly used for the treatment of coronary heartdisease. However, very little information is available expliciting the effects activeconstituents in SCF and SSF. Therefore, we developed an analytical method for screeningthe active ingredients from SCF and SSF by combining cardiac muscle/cell membranechromatography (CM/CMC) with liquid chromatography–tandem mass spectrometry(LC/MS), and explored the protective role of active ingredients in myocardialischemia–reperfusion (I/R) injury.Methodology: First of all, CM/CMC-Offline-LC/MS was used to scan and analyz theeffective anti-myocardial ischemia component from extracts of SCF and SSF. Cardiacmuscle cells isolated from rat thoracic aortas were used for preparation of the stationaryphase of the CM/CMC column. The suitability and reliability of theCM/CMC-offline-LC/MS method was assessed using nitrendipine and nifedipine aspositive controls, glimepiride and gliclazide as negative control, and this method was thenapplied to screen bioactive components from the extracts of FSC and FSS. Retentionfractions from the CM/CMC column were collected and then analyzed by LC/MS under theoptimized conditions offline.Later, the deoxyschizandrin (DSD) and schisantherin A (STA) intracenous injectionemulsions were prepared by nanomizer system after ultrasonding, and the oily phase wassoybean oil and soya lecithin was used as emulsifier. High performance liquidchromatography method was established for determining the content of DSD and STA inthe intracenous injection emulsions. Last but not least, the key of this project is we explored the protective role of DSD andSTA in myocardial ischemia–reperfusion injury. In berif, anesthetized male rats weretreated once with DSD and STAthrough the tail vein after45min of ischemia, followed by2-h reperfusion. Cardiac function, infarct size, biochemical markers, histopathology andapoptosis were measured during experiments. Protein expression of caspase-3and mRNAexpression of gp91phoxin myocardial tissue were assessed respectively. Neonatal ratcardiomyocytes were pretreated with DSD and STA and then damaged by H2O2. Apoptosisof cardiomyocytes was tested by a flow cytometric assay.Principal Findings：Our CM/CMC-LC/MS method can be applied for screeninganti-myocardial ischemia component from extracts collected from SCF and SSF. And wefound that DSD and STA could bind to rat cardiomyocyte membranes. This findingsuggested their potential role in protecting myocardial cells. To overcome the problem ofpoor solubility of DSD and STA, we made them into intracenous injection emulsions. TheDSD and STAintracenous injection emulsions have stable preparation.Compared with the I/R group:(i) DSD and STA could significantly reduce theabnormalities of LVSP, LVEDP,±dp/dtmaxand arrhythmias, thereby showing theirprotective roles in cardiac function;(ii) DSD and STA could significantly attenuate theinfarct size and MDA release while increasing SOD activity, suggesting a role in reducingmyocardial injury;(iii) tissue morphology and myocardial textual analysis revealed thatDSD and STA mitigated changes in myocardial histopathology;(iv) DSD and STAdecreased apoptosis (33.56±2.58%to10.28±2.80%and10.98±1.99%, respectively) andcaspase-3activity in the myocardium (0.62±0.02OD/mg to0.38±0.02OD/mg and0.32±0.02OD/mg, respectively), showing their protective effects upon cardiomyocytes; and (v)DSD and STA had similar protective effects on I/R injury as those seen with the positivecontrol metoprolol.(vi) In vitro, DSD and STAcould significantly decrease the apoptosis ofneonatal cardiomyocytes.Conclusions: These data suggest that DSD and STA can protect against myocardialI/R injury. The underlining mechanism may be related to their role in inhibiting cardiomyocyte apoptosis. Our study suggests DSD and STA have potential clinicalapplication value in treating ischemic heart disease. |
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