Cloning and catalytic analysis of Isatis indigotica chalcone isomerase in vitro / 中国中药杂志

Chalcone isomerase is a key rate-limiting enzyme in the biosynthesis of flavonoids in higher plants, which determines the production of flavonoids in plants. In this study, RNA was extracted from different parts of Isatis indigotica and reverse-transcribed into cDNA. Specific primers with enzyme restriction sites were designed, and a chalcone isomerase gene was cloned from I. indigotica, named IiCHI. IiCHI was 756 bp in length, containing a complete open reading frame and encoding 251 amino acids. Homology analysis showed that IiCHI was closely related to CHI protein of Arabidopsis thaliana and had typical active sites of chalcone isomerase. Phylogenetic tree analysis showed that IiCHI was classified into type Ⅰ CHI clade. Recombinant prokaryotic expression vector pET28a-IiCHI was constructed and purified to obtain IiCHI recombinant protein. In vitro enzymatic analysis showed that the IiCHI protein could convert naringenin chalcone into naringenin, but could not catalyze the production of liquiritigenin by isoliquiritigenin. The results of real-time quantitative polymerase chain reaction(qPCR) showed that the expression level of IiCHI in the aboveground parts was higher than that in the underground parts and the expression level was the highest in the flowers of the aboveground parts, followed by leaves and stems, and no expression was observed in the roots and rhizomes of the underground parts. This study has confirmed the function of chalcone isomerase in I. indigotica and provided references for the biosynthesis of flavonoid components.

A new method for evaluating regional cerebral blood flow changes: Laser speckle contrast imaging in a C57BL/6J mouse model of photothrombotic ischemia / 华中科技大学学报（医学）（英德文版）

The present study aimed to improve the processing of data acquired from laser speckle contrast imaging (LSCI) to provide a standardization method to explore changes in regional cerebral blood flow (rCBF) and to determine the correlations among rCBF, cerebral ischemic lesion volume and microvascular density over time in a focal ischemic region. C57BL/6J mice were subjected to focal photothrombotic (PT) ischemia. rCBF was measured using LSCI at different time points before and after PT ischemia through an intact skull. Standardized rCBF (SrCBF), defined as the ratio of rCBF measured in the ipsilateral region of interest (ROI) to that in the corresponding contralateral region, was calculated to evaluate potential changes. In addition, the volume of the ischemic lesion and the microvascular density were determined using Nissl staining and immunofluorescence, respectively. The relationships among the ischemic lesion volume, microvascular density and SrCBF were analyzed over time. The results showed that the cortical rCBF measured using LSCI following PT ischemia in the C57BL/6J mice gradually increased. Changes in the cerebral ischemic lesion volume were negatively correlated with SrCBF in the ischemic region. Changes in the microvascular density were similar to those observed in SrCBF. Our findings indicate that LSCI is a practical technique for observing changes in murine cortical rCBF without skull opening and for analyzing the relationships among the ischemic lesion volume, microvascular density and SrCBF following focal cerebral ischemia. Preliminary results also suggest that the use of LSCI to observe the formation of collateral circulation is feasible.

Effects of continuous positive airway pressure therapy on plasma aldosterone levels in patients with obstructive sleep apnea: A meta-analysis / 华中科技大学学报（医学）（英德文版）

Obstructive sleep apnea (OSA) is an independent risk factor for cardiovascular diseases. Aldosterone was reported to be increased in patients with OSA and correlated with OSA severity. Many studies investigated the effect of continuous positive airway pressure (CPAP) therapy on plasma aldosterone concentrations (PAC) in OSA patients. The results, however, were inconsistent. In the present study, we aimed to evaluate the effects of CPAP therapy on PAC by performing a meta-analysis. Literature search was carried out in electronic databases including PubMed/Medline, Cochrane Library, Embase and Web of Science. Eligible full-text articles were identified, and important data were extracted. Pooled analysis was performed using the STATA12.0 and RevMan 5.2. Standardized mean difference (SMD) was calculated to estimate the treatment effects. A total of eight studies involving 219 patients were included for our final analysis. PAC was found unchanged after CPAP treatment in OSA patients (SMD=-0.36, 95% CI:-0.91 to 0.18, Z=1.32, P=0.19). Meanwhile, CPAP therapy showed no impact on PAC (SMD=-0.21, 95% CI:-0.85 to 0.42, Z=0.66, P=0.51) in a separate meta-analysis including 3 randomized controlled trials. In conclusion, the evidence for the use of CPAP therapy to decrease PAC in OSA patients is low, and further studies are still warranted.

Correlation between autophagy and polarization of macrophages in atherosclerosis plaque in arteriosclerosis obliterans amputees / 药学学报

This study was designed to investigate the correlation between autophagy and polarization of macrophages in atherosclerosis (AS) plaque in arteriosclerosis obliterans amputees. Femoral artery specimens from arteriosclerosis obliterans amputees were performed hematoxylin and eosin (HE) staining, oil red O and immunofluorescence staining to observe the morphology of atherosclerotic plaque, phenotype of macrophages and autophagy in plaque; using real-time quantitative RT-PCR technology to detect the mRNA level of M1 and M2 type markers in arterial tissue; to analyze polarized signal pathway and autophagy protein levels in macrophages by Western blotting. Arterial specimens staining showed obvious lipid deposition and obvious infiltration of amount of foam cells and inflammatory cells. Macrophages were mainly expression M1 type in percentage in fibrous plaque. Although both M1 and M2 macrophages were upregulated in atheromatous plaque, the increase was dominant in M2 type in percentage. The level of autophagy was significantly higher in the atheromatous plaque than that of fibrous plaque. The expression of tumor necrosis factor- α (TNF-α), monocyte chemotactic protein-1 (MCP-1), inducible nitric oxide synthase (iNOS), interleukin-6 (IL-6) and interleukin-12 (IL-12) mRNA was significantly higher in fibrous plaque than that of atheromatous plaque (P < 0.01 or 0.05), and arginase-1 (Arg-1), transforming growth factor-β (TGF-β), CD163 and interleukin-10 (IL-10) mRNA was significantly lower than that in atheromatous plaque (P < 0.01). The levels of p-STAT1 and NF-κB were significantly increased in fibrous plaque (P < 0.01), while p-STAT6 expression was significantly increased in atheromatous plaque (P < 0.01). The level of LC3-II was significantly higher in atheromatous plaque than that in fibrous plaque (P < 0.01). Macrophages in early atherosclerotic plaque were induced to M1 type through p-STAT1/NF-κB pathway and expressed moderate levels of autophagy; while macrophages in advanced plaques were induced to polarization of M2 type through p-STAT6 pathway. M2 macrophages expressed a higher level of autophagy than M1 macrophages.

A new method for evaluating regional cerebral blood flow changes: Laser speckle contrast imaging in a C57BL/6J mouse model of photothrombotic ischemia / 华中科技大学学报（医学）（英德文版）

The present study aimed to improve the processing of data acquired from laser speckle contrast imaging (LSCI) to provide a standardization method to explore changes in regional cerebral blood flow (rCBF) and to determine the correlations among rCBF, cerebral ischemic lesion volume and microvascular density over time in a focal ischemic region. C57BL/6J mice were subjected to focal photothrombotic (PT) ischemia. rCBF was measured using LSCI at different time points before and after PT ischemia through an intact skull. Standardized rCBF (SrCBF), defined as the ratio of rCBF measured in the ipsilateral region of interest (ROI) to that in the corresponding contralateral region, was calculated to evaluate potential changes. In addition, the volume of the ischemic lesion and the microvascular density were determined using Nissl staining and immunofluorescence, respectively. The relationships among the ischemic lesion volume, microvascular density and SrCBF were analyzed over time. The results showed that the cortical rCBF measured using LSCI following PT ischemia in the C57BL/6J mice gradually increased. Changes in the cerebral ischemic lesion volume were negatively correlated with SrCBF in the ischemic region. Changes in the microvascular density were similar to those observed in SrCBF. Our findings indicate that LSCI is a practical technique for observing changes in murine cortical rCBF without skull opening and for analyzing the relationships among the ischemic lesion volume, microvascular density and SrCBF following focal cerebral ischemia. Preliminary results also suggest that the use of LSCI to observe the formation of collateral circulation is feasible.

Correlation between autophagy and polarization of macrophages in atherosclerosis plaque in arteriosclerosis obliterans amputees / 药学学报

This study was designed to investigate the correlation between autophagy and polarization of macrophages in atherosclerosis (AS) plaque in arteriosclerosis obliterans amputees. Femoral artery specimens from arteriosclerosis obliterans amputees were performed hematoxylin and eosin (HE) staining, oil red O and immunofluorescence staining to observe the morphology of atherosclerotic plaque, phenotype of macrophages and autophagy in plaque; using real-time quantitative RT-PCR technology to detect the mRNA level of M1 and M2 type markers in arterial tissue; to analyze polarized signal pathway and autophagy protein levels in macrophages by Western blotting. Arterial specimens staining showed obvious lipid deposition and obvious infiltration of amount of foam cells and inflammatory cells. Macrophages were mainly expression M1 type in percentage in fibrous plaque. Although both M1 and M2 macrophages were upregulated in atheromatous plaque, the increase was dominant in M2 type in percentage. The level of autophagy was significantly higher in the atheromatous plaque than that of fibrous plaque. The expression of tumor necrosis factor-α (TNF-α), monocyte chemotactic protein-1 (MCP-1), inducible nitric oxide synthase (iNOS), interleukin-6 (IL-6) and interleukin-12 (IL-12) mRNA was significantly higher in fibrous plaque than that of atheromatous plaque (P β (TGF-β), CD163 and interleukin-10 (IL-10) mRNA was significantly lower than that in atheromatous plaque (P κB were significantly increased in fibrous plaque (P P P κB pathway and expressed moderate levels of autophagy; while macrophages in advanced plaques were induced to polarization of M2 type through p-STAT6 pathway. M2 macrophages expressed a higher level of autophagy than M1 macrophages.

Effect of ASO Blood Stasis Syndrome Serum on Vascular Endothelial Cell Injury and Regulation of Taohong Siwu Decoction on it / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To explore the effect of arteriosclerosis obliterans (ASO) blood stasis syndrome (BSS) serum on vascular endothelial cell injury and to study the regulation of Taohong Siwu Decoction (TSD) on it.</p><p><b>METHODS</b>Umbilical vein endothelial cell culture system was established. The serum endothelial cell injury model with ASO BSS was prepared. Low, medium, and high concentrations TSD containing serums were respectively added. The endothelial cell proliferation activity was observed by MTT method. Ultrastructures of endothelial cells were observed under transmission electron microscope. Changes of intracellular calcium ion concentration and the cytoskeleton were observed under laser confocal microscope. Contents of ET, NO, and transforming growth factor beta1 (TGF-beta1) in endothelial cell culture supernatant were detected by ELISA.</p><p><b>RESULTS</b>In ASO BSS serum group endothelial cell proliferation activities decreased, the cell structure was obviously destroyed, calcium ion concentration increased, contents of ET, NO and TGF-beta1 increased significantly (P < 0.01), and ET/NO ratio was imbalanced. After incubating with TSD drug containing serum, endothelial cell proliferation activities and injured cell structures were obviously improved; ET, NO and TGF-beta1 levels decreased (P < 0.05, P < 0.01), ET/NO ratios approximated to the normal level.</p><p><b>CONCLUSION</b>The main mechanism of TSD for treating ASO ASS lied in improving injured vascular endothelial cells and endocrine disorder.</p>

Taohong Siwu Decoction regulated functions of endothelial cells and treated arteriosclerosis obliterans: an experimental study / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To discuss the effect of Taohong Siwu Decoction (TSD) in regulating functions of endothelial cells and treating arteriosclerosis obliterans (ASO).</p><p><b>METHODS</b>The ASO model was prepared by using high-fat diet plus intimal injury. They were randomly divided into the model group (n = 10), the normal control group (n = 9), the low dose TSD group (group A, n = 12), the middle dose TSD group (group B, n = 10), and the high dose TSD group (group C, n = 9). Eight weeks after modeling, the limb blood perfusion was observed using laser Doppler flowmetry. The arterial morphology was observed using light microscope and transmission electron microscope. The number of circulating endothelial cells (CECs) was determined using Percoll density gradient centrifugation method. Serum levels of TNF-alpha, IL-1, ET-1, and NO were detected using double antibody sandwich assay of enzyme linked immunosorbent assay (ELISA).</p><p><b>RESULTS</b>The ASO rat model was successfully established. Blood lipids levels significantly increased, the blood perfusion of left hind limbs significantly decreased, the number of CECs in the peripheral blood significantly increased, the arterial lumen was irregularly narrowed, the ultra-structure of vessel walls was damaged, serum levels of TNF-alpha, IL-1, and ET-1 significantly increased, and the serum level of NO significantly decreased in the model group, showing statistical difference when compared with the normal control group (P < 0.01). Compared with the model group, significant improvement in the aforesaid indices was shown in group B and C (P < 0.05, P < 0.01).</p><p><b>CONCLUSIONS</b>The injury and abnormal functions of endothelial cells is an important pathological process of ASO. As an effective recipe for treating ASO, TSD could protect vascular endothelial cells and improve the secretion function of vascular endothelial cells.</p>

Effect of gene transfer using nanoparticles as gene vector in different animal models / 中国医学科学院学报

<p><b>OBJECTIVE</b>To evaluate the effect of antisense monocyte chemotactic protein-1 (A-MCP-1) nanoparticles (NPs) as gene carrier on gene transfer in two kinds of animal models.</p><p><b>METHODS</b>Poly (lactic acid-co-glycolic acid) (PLGA) was used to make the NPs loaded with A-MCP-1 through a double-emulsion/solvent evaporation technique. NPs size was assessed by dynamic laser defractometer. The particle morphology was observed by scanning electron microscopy. DNA content in the NPs was measured by dissolving known amounts of NPs in chloroform and extracting DNA with water. In vitro release was performed in tris-EDTA buffer at 37 degrees C using double-chamber diffusion cells. The receiver buffer was replaced daily. The A-MCP-1 NPs was transfected into the cultured smooth muscle cells. PCR was used to evaluate the transfection of A-MCP-1. Cationic lipid (Lipofectamine) was used to transfect A-MCP-1 as control. After 48 hours incubation, cells were digested and examined by polymerase chain reaction. Twenty New Zealand white rabbits under jugular vein to artery bypass grafting procedure were divided into four groups: the first group received grafts treated with A-MCP-1 NPs, the second group received grafts treated with cationic liposome (dioleoyl trimethyl ammonium propane)-A-MCP-1, the third group received grafts treated with plasmid DNA, and the fourth group received grafts without transfection as control. Fourteen days after surgery the grafts were harvested. The expression of A-MCP-1 and its effect on MCP-1 in vein grafts were detected by dot blotting. The morphology of the grafts was investigated. To establish abdominal aortic aneurysms rats model, rats were randomly divided into three groups: A-MCP-1 NPs injection group, shame NPs injection group and control groups (without injection). Two weeks after surgery, diameter of abdominal aorta was measured and aortic tissue was obtained for PCR analysis to evaluate the A-MCP-1 expression. Western blot were applied to detect the inhibitory effect to the expression of MCP-1 mRNA and CD68 protein by A-MCP-1 NPs.</p><p><b>RESULTS</b>NPs size ranged 198nm to 205nm with average around 201.4 nm. DNA content in the NPs was 4.14%. NPs showed steady release rate in vitro in Tris-EDTA solution. It released faster in the first week then maintained a slowly sustained release up to 16 days. In cell culture A-MCP-1 gene successfully transfected into smooth muscle cells by NPs vector. In vein grafting animal model, A-MCP-1 expression was detected in the vascular walls of NPs and cationic lipid treated groups. The degree of vascular hyperplasia in the gene NPs treated group was significantly lower than that in control group. There was no significant difference in the inhibition of intimal hyperplasia between NPs and cationic lipid treated groups. Two weeks after transfection in abdominal aortic aneurysm rats models, the abdominal aortic diameter of A-MCP-1 NPs injection group was (1.79 +/- 0.12) mm, significantly smaller than that of control groups [shame NPs group was (2.58 +/- 0.21) mm, and saline group was (2.63 +/- 0.29) mm] (P < 0.01). The expressions of MCP-1 mRNA and CD68 protein in A-MCP-1 NPs injection group were 12.5 +/- 1.5 and 17.6 +/- 2.1, which were much lower than those in control group [in shame NPs group, which were 35.7 +/- 4.5, 42.3 +/- 5.7 (P < 0.01), and saline group which is 32.4 +/- 3.9, 39.8 +/- 4.8 (P < 0.01)]. Specific band of A-MCP-1 was detected only in the A-MCP-1 NPs injection group by PCR.</p><p><b>CONCLUSION</b>A-MCP-1 gene NPs can be successfully used in rabbit vein grafting model and abdominal aortic aneurysm rats models, and may be potentially applied in clinical practice.</p>

Vascular smooth muscle cell proliferation and apoptosis of the aortic wall during the development of abdominal aortic aneurysm / 中国普通外科杂志

Objective To explore vascular smooth muscle cell(SMC) proliferation and cell apoptosis during the development of abdominal aortic aneurysm(AAA). Methods The animal model of AAA was established in Wistar rats and the specimens were harvested at the 3rd day,and 1、2、3 and 4 week after the model initiation. In situ end-labeling of DNA fragments (TUNEL) was used to detect SMC apoptosis and immunohistochemical staining was applied to investigate the expression of SMC apoptosis markers(bcl-2,bax),proliferating cell nuclear antigen (PCNA) and ?-actin. Results TUNEL-positive and PCNA-positive SMC reached the maximum at 2～3 week and 1 week respectively;The count of TUNEL-positive SMC was less than PCNA-positive SMC during the period of day 3 to 1 week and that was vice versa from 2nd to 4th week with SMC amount significantly decreased;Bcl-2 and bax protein was strongly expressed at 1 week and 3 week after operation(all P