Effect of Epimedii Folium Total Flavonoids on Angiogenesis of Ischemic Myocardium in Rats After Acute Myocardial Infarction / 中国实验方剂学杂志

Objective:To observe the effect of total flavonoids from Epimedii Folium （TEF） on the angiogenesis of ischemic myocardium in rats after acute myocardial infarction （AMI） and discuss its molecular biological mechanism of attenuating myocardial ischemia and improving cardiac function. Method:AMI in rats was induced through the ligation of left anterior descending coronary artery. All male SD rats were randomized into sham-operated group， model group， diltiazem group （10 mg·kg^-1·d^-1）， and TEF low-dose and high-dose groups （100 and 200 mg·kg^-1·d^-1）， with 8 rats in each group. After modeling， rats in the diltiazem group and TEF groups were given corresponding doses of diltiazem and TEF， respectively， and those in the model group and sham-operated group received normal saline of equivalent volume， once a day for 7 days. After the administration， VisualSonics Vevo2100 imaging system was used to detect the cardiac structure and function and hematoxylin-eosin （HE） staining to observe the histomorphological changes in myocardial ischemic area. Immunohistochemistry was employed to analyze the expression of CD31 and <italic>α</italic>-smooth muscle actin （<italic>α</italic>-SMA） in ischemic myocardium and Western blot to detect the expression of vascular endothelial growth factor-receptor 2 （VEGF-R2） and phosphorylation of protein kinase B （Akt） in ischemic myocardium. Real-time PCR was applied to quantify the mRNA levels of vascular endothelial growth factor （VEGF） and basic fibroblast growth factor （bFGF）. Result:Compared with the sham-operated group， the model group demonstrated significant increase in left ventricular systolic diameter （LVIDs）， left ventricular internal diameter at end-diastole （LVIDd）， left ventricular end-systolic volume （LVEVs）， and left ventricular end-diastolic volume （LVEVd）， significant decrease in End-systolic thickness of left ventricular anterior wall （LVAWs）， end-diastolic thickness of left ventricle anterior wall （LVAWd）， end systolic thickness of left ventricular posterior wall （LVPWs）， stroke volume （SV）， ejection fraction （EF）， fractional shortening （FS）， and cardiac output （CO）， obvious pathological changes in the ischemic myocardium， and plummet of the expression of CD31 and <italic>α</italic>-SMA （<italic>P</italic><0.01）， Akt phosphorylation level， protein level of VEGF-R2， and mRNA levels of VEGF and bFGF （<italic>P</italic><0.05， <italic>P</italic><0.01）. High-dose TEF significantly alleviated the pathological changes of ischemic myocardium as compared with the model group. Moreover， TEF high-dose group showed significantly lower levels of LVIDs， LVIDd， LVEVs， and LVEVd， significantly higher levels of LVAWs， LVAWd， LVPWs， SV， EF， FS， and CO， higher expression of CD31 and <italic>α</italic>-SMA （<italic>P</italic><0.05， <italic>P</italic><0.01）， and higher levels of VEGF-R2 protein， phosphorylated Akt， and VEGF and bFGF mRNA than the model group （<italic>P</italic><0.05， <italic>P</italic><0.01）. Conclusion:TEF can effectively improve myocardial perfusion in peri-myocardial infarction area and attenuate ventricular remodeling and heart failure after AMI by up-regulating the expression of bFGF， VEGF， and VEGF-R2 in ischemic myocardium following AMI and activating phosphatidylinositol 3-kinases （PI3K）/Akt/VEGF signaling transduction pathway which can promote angiogenesis in ischemic myocardium.

Inhibitory mechanism of icariin against oxidative stress-induced calcification in aortic vascular smooth muscle cells / 中国中药杂志

This study aimed to observe the inhibitory effect of icariin against oxidative stress-induced calcification in aortic vascular smooth muscle cells(VSMCs) and elucidate the molecular mechanism of icariin in inhibiting endoplasmic reticulum stress(ERS)-mediated atherosclerotic calcification, so as to provide new ideas for exploring the anti-atherosclerotic mechanism of Epimedii Folium. The VSMCs in rat thoracic aorta were subjected to adherent culture and then treated with the complete calcification DMEM containing high glucose and hydrogen peroxide(H_2O_2) for three weeks. The resulting calcified VSMCs were divided into different treatment groups. Icariin was added one week after calcification induction for protecting the VSMCs, whose viability was then detected using cell counting kit-8(CCK-8). Alizarin red-S staining was conducted to observe the calcification degree. The activity of alkaline phosphatase(ALP) in VSMCs was measured using the disodium phenyl phosphate substrate and the calcium content was measured by arsenazo Ⅲ method. The mRNA expression levels of ossification-related factors including osteocalcin(OC), osteopontin(OPN), Runt-related transcription factor 2(Runx2), and type Ⅰ collagen(Col Ⅰa) were detected by real-time PCR. Western blot was carried out to determine the protein expression levels of α-smooth muscle actin(α-SMA), Runx2, activating transcription factor 4(ATF4), and eukaryotic translation initiation factor(eIF)-2α. The results showed that H_2O_2 significantly induced the calcification of VSMCs, increased the ALP activity and calcium content in VSMCs, promoted OC, OPN, Runx2, and Col Ⅰa mRNA expression and Runx2 protein expression, and reduced α-SMA protein expression. The ATF4 protein expression and eIF2α phosphorylation were also elevated significantly. Icariin reversed the calcification of VSMCs induced by H_2O_2, inhibited ALP activity and calcium content in VSMCs, down-regulated the mRNA expression levels of OC, OPN, Runx2 and Col Ⅰa and Runx2 protein expression, and relatively up-regulated the expression of α-SMA. The expression of ATF4 and phosphorylation of eIF2α also declined significantly. All these have demonstrated that icariin inhibited VSMCs calcification by down-regulating the ossification-related factors and lowering ALP activity and calcium content in VSMCs. Besides, the down-regulation of Runx2 expression and the inhibition of ATF4 and eIF2α-mediated cellular calcification pathway in ERS might also be involved in such calcification-suppressing process.

Centrifugal force stretcher a new of in vitro mechanical cell stimulator / 生理学报

A number of mechanical cell stimulators have been used to study the effect of mechanical stimulation on cells in vitro. But the efficiency of these devices is not fully desirable. We recently developed a new device for mechanical cell stimulation, the centrifugal force stretcher, and compared its efficacy with that of the traditional Flexercell Strain Unit. When the mechanical stretcher circumrotates with certain speed, cardiac myocytes attached on the plate are stretched and elongated by centrifugal force. Neonatal rat cardiac myocytes were isolated by enzymatic dissociation from the hearts of 3~5 d old Sprague Dawley rats, and were mechanically stimulated by traditional 20% stretch and 180 r/min centrifugal force for 12 and 24 h. The effects of mechanical stimulation on the hypertrophic response of neonatal rat cardiac myocytes and production of angiotensin II (Ang II) were examined. Compared with the non-stretch group, the radioactivity of (3)H-leucine incorporated into the stretch-stimulated cardiac myocytes in the centrifugal force stretch group was significantly higher [(1295.17+/-51.19) vs (1122.67+/-51.63) in 12 h; (1447.5+/-35.96) vs (1210.67+/-90.92) in 24 h, P<0.05]. Ang II was also dramatically increased by 128% in 12 h (P<0.05) and 139% in 24 h (P<0.01). After the myocytes was stretched for 24 h, the LDH level in the medium in the Flexercell Strain Unit group was significantly higher than that in the centrifugal force group [(14.5+/-8.7) U/L vs (7.8+/-4.3) U/L, P<0.05]. The centrifugal force stretcher is a new and improved mechanical cell stimulator with the same effects on the protein synthesis and Ang II secretion of the cardiac myocytes, and the damage to the cells bronght by this stimulator is relatively slighter in comparison with the Flexercell Strain Unit.