Two proteins of similar molecular weight (named as ASPR-C-1 and ASPR-C-2) from the crude drug of Angelica sinensis were purified and characterized by 80% ammonium sulfate precipitation, Sephadex G-50 gel filtration chromatography, and DEAE-Sepharose anion exchange chromatography. The molecular weight of ASPR-C-1 and ASPR-C-2 on SDS-PAGE was 17.33 kDa and 17.18 kDa, respectively. They were mainly monomeric in solution, but partially formed dimers and they were glycoproteins with glycosyl content of 2.6% and 8.2%, respectively. Both ASPR-C-1 and ASPR-C-2 were identified to be members of pathogenesis-related 10 family of proteins by matrix-assisted laser desorption ionization time-of-flight mass spectrometry and have ribonuclease activities with the specific activity of 73.60 U/mg and 146.76 U/mg, respectively. The optimum pH of the two isoforms was similar, at about 5.6, while their optimum temperatures were different. The optimum temperature of ASPR-C-1 was 50 ℃, and that of ASPR-C-2 was 60 ℃. Both isoforms presented highest thermal stability at 60 ℃. However, ASPR-C-2 was more thermotolerant than ASPR-C-1. The latter was rapidly inactivated and retained only about 20% residual activity while the former still maintained about 80% of its original activity at a higher treatment temperature (80 to 100 ℃). In addition, Fe²⁺ had an activating effect on the ribonuclease activities of two isoforms while Ca²⁺, Mg²⁺, Zn²⁺, Mn²⁺, Ag⁺, Cu²⁺, EDTA (Elhylene diamine tetraacetic acid), dithiothreitol and sodium dodecylsulphate showed different degrees of inhibition of the enzyme activities. Our findings provide a foundation for further research on the biological function of PR-10 protein from Angelica sinensis.
Angelica sinensis ; Chromatography, Gel ; Chromatography, Ion Exchange ; Electrophoresis, Polyacrylamide Gel ; Enzyme Stability ; Hydrogen-Ion Concentration ; Kinetics ; Molecular Weight ; Protein Isoforms ; Temperature

OBJECTIVE: To assess the association of 5A/6A polymorphism in the promoter region of MMP3 gene with the stability of extracellular matrix of atherosclerotic plaque. METHODS: Clinical data of 776 consecutive patients undergoing percutaneous coronary intervention (PCI) was reviewed. MMP3 gene polymorphisms and serum level of MMP3 for the second admission were collected. The target gene fragment containing MMP3 promoter region was transfected into HepG2 vector cells. The influence of the polymorphism on the expression of the MMP3 gene was determined in vitro. RESULTS: Compared with the first admission data, the proportion of mutant MMP3 genotypes (5A/5A+5A/6A) was significantly higher in patients with acute myocardial infarction (AMI) compared with the control group (37.6% vs. 24.9%, P<0.01). 64.1% of the patients carrying the 5A allele had AMI, whereas only 50.11% of those carrying the 6A allele had AMI (P<0.01). The proportion of wild-type MMP3 genotype (6A/6A) was significantly higher in the stenotic group compared with the non-restenosis group (79.5% vs. 66.5%, P<0.01). Restenosis has occurred in 9.5% of patients harboring the 5A allele compared with 16.2% in those carrying the 6A allele (P<0.01). In addition, serum level of MMP3 in the restenosis group was significantly lower than that of the non-restenosis group (P<0.01). In vitro studies confirmed that the expression of pGL2-Basic/6A was significantly lower than that of pGL2-Basic/5A. CONCLUSION The 5A/6A polymorphism in the promoter region of the MMP3 gene may influence its transcriptional activity and impact on the degradation or push-up of extracellular matrix, resulting in a difference in the stability of atherosclerosis plaques, which in turn may induce different pathological processes in AMI or restenosis after stenting.
Case-Control Studies ; Extracellular Matrix ; Genetic Predisposition to Disease ; Genotype ; Humans ; Matrix Metalloproteinase 3 ; genetics ; Percutaneous Coronary Intervention ; Plaque, Atherosclerotic ; genetics ; Polymorphism, Genetic ; Promoter Regions, Genetic

Objective To investigate the clinical features of patients with heart failure and the safety and efficacy of noninvasive ventilator in patients with heart failure.Methods Sequentially enrolled 65 patients who were diagnosed with decompensated heart failure in Tianjin Chest Hospital Heart Center from October 2016 to October 2017 and who had acute heart failure during hospitalization requiring noninvasive ventilator,were divided into the HF-PEF group (n=19) and HF-REF group (n=46).The clinical data of the two groups and the observation indexes before and after the application of the non-invasive ventilator were compared.Results Comparing the admission data of the two groups,the proportion of patients with hypertension (57.9％ vs 21.7％,P=0.005) and LVEF(％) (53.00±4.85 vs 33.07±7.24,P＜0.01)were significantly higher in the HF-PEF group than those in the HF-REF group;LVEDD (mm) in the HFPEF group was significantly lower than that in the HF-REF group (50.00±5.23 vs 63.82±8.95,P＜0.01).In the two groups of patients with acute left heart failure,blood lactate levels (mmol/L) in the HF-PEF group (4.20±1.06 vs 2.02±0.88,P＜0.05) and systolic blood pressure (mmHg) (151.32±43.40 vs 117.90± 19.55,P＜0.05) were significantly higher than those in the HF-REF group.After the application of non-invasive ventilator,systolic blood pressure (mmHg) (34.38±9.36 vs 16.94±5.19,P=0.038) and PaCO2 (mmHg)(2.49±0.98 vs-0.06±0.00,P=0.025),and lactic acid (mmol/L) (2.06±0.67 vs 0.04±0.01,P=0.001) were significantly lower in the HF-PEF group than those in the HF-REF group.While the NT-proBNP level (ng/L) (13 064.90±1 963.83 vs 11 687.13±1 028.03,P=0.848) did not decrease significantly,and the time of non-invasive ventilator application (h)was significantly longer than that in the HF-REF group (152.74±10.61 vs 71.03±10.41,P=0.013).Conclusions Hypertension is the main cause of HF-PEF group.The systolic blood pressure and blood lactate level in HF-PEF patients with acute left heart failure are significantly higher than HF-REF patients.Non-invasive ventilator is also safe and effective for the treatment of acute left heart failure in HF-PEF patients,but HF-PEF patients with acute left heart failure have a longer clinical remission time.

OBJECTIVETo observe the impacts of thermosensitive moxibustion (TSM) on the expressions of nitric oxide (NO), typeⅠdisintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4), typeⅡcollagen and proteoglycan (PG) in the rabbit models of knee osteoarthritis (KOA) and explore the mechanism of TSM on KOA.

METHODSA total of 42 Japanese long-eared male rabbits were divided into a blank group (6 rabbits), a model group (6 rabbits), a moxibustion group (24 rabbits) and a sham-operation group (6 rabbits) according to the random number table. In the blank group, the rabbits were fed normally. In the model and moxibustion groups, the papain injection was given to establish KOA models. The rabbits in the sham-operation group were treated with the intracavity injection of 0.9% NaCl solution. The rabbits were forced to move for 30 min every day, continuously for 15 days during modeling. At the end of modeling, in the moxibustion group, moxibusiton was applied at "Dubi" (ST 35), once a day, 40 min each time, for 14 days totally. According to the temperature changes during moxibustion, the rabbits were divided into a TSM group and a non-TSM group. 6 rabbits were collected randomly from the two groups. The usual feeding was given in the blank group, the model group and the sham-operation group, without any intervention. The body mass and behavioristics changes were observed in each group. At the end of treatment, the nitrate reduction method was adopted to determine NO expression in the serum. The real-time PCR was adopted to determine the expressions of ADAMTS-4, typeⅡcollagen and PG in the cartilage.

RESULTS① After modeling, compared with the blank group, the body mass was all reduced and the Lequesne MG score was increased in the model group, TSM group, non-TSM group and sham-operation group (<0.05, <0.01). After intervention, compared with the blank group, the body mass was decreased and the Lequesne MG score was increased in the model and sham-operation groups (<0.05, <0.01). Compared with the model group, the body mass was increased and the lequesne MG score was decreased in the TSM, non-TSM, and sham-operation groups (<0.05, <0.01). Compared with the non-TSM group, the body mass in the TSM group was increased remarkably (<0.05), but the difference in Lequesne MG score was not statistically significant (>0.05). ② After intervention, compared with the blank group, the expressions of NO and ADAMTS-4 were all increased and the expressions of typeⅡcollagen and PG were decreased in the model group, TSM group, non-TSM group and sham-operation group (<0.05, <0.01). Compared with the model group, the expressions of NO and ADAMTS-4 were all remarkably lower and the expressions of typeⅡcollagen and PG were increased in the TSM group, non-TSM group and sham-operation group (<0.05, <0.01). Compared with the non-TSM group, the expressions of NO and ADAMTS-4 were all remarkably lower and the expressions of typeⅡcollagen and PG were increased in the TSM group after intervention (all <0.05).

CONCLUSIONThe thermosensitive moxibustion alleviates the inflammatory reactions and protects the joint cartilage through inhibiting the expressions of NO and ADAMTS-4 to achieve the effects in the treatment of KOA.

ADAMTS4 Protein ; metabolism ; Animals ; Cartilage ; metabolism ; Collagen Type III ; metabolism ; Male ; Moxibustion ; Nitric Oxide ; blood ; Osteoarthritis, Knee ; therapy ; Proteoglycans ; metabolism ; Rabbits ; Random Allocation

OBJECTIVETo observe the effects of heat-sensitive moxibustion on corticotropin releasing hormone (CRH), adrenocorticotrophic hormone (ACTH) and corticosterone (CORT) in rats with irritable bowel syndrome (IBS), and to explore the possible mechanism of heat-sensitive moxibustion on IBS.

METHODSAccording to random number table, 56 SD male rats were randomly divided into a blank group (=8), a model group (=8), a moxibustion group (=32), and a mifepristone group (RU-486 group,=8). The rats in the blank group were treated with normal feeding; the rats in the model group, RU-486 group and moxibustion group were treated with chronic non-predictable stimulation for 21 days to establish IBS model. After model establishment, the rats in the moxibustion group were treated with moxibustion at "Mingmen" (GV 4) for 40 min, once a day for 14 days; the tail temperature was recorded every 5 min; according to the change of tail temperature, the rats were divided into a heat-sensitive moxibustion group and a non-heat-sensitive moxibustion group, and 8 rats were randomly selected in the two groups. The rats in the RU-486 group were treated with gastric administration of RU-486 for 14 days, while the rats in the blank group, model group and moxibustion groups were treated with identical volume of 0.9% NaCl. The rat general condition, body mass, behavioristics, intestinal propulsive rate and visceral sensitivity were observed in each group; ELISA method was used to detect serum CRH, ACTH and CORT; optical microscope was applied to observe the morphological changes of colon.

RESULTS(1) After model establishment, rats were in rest state, fatigued, with withered hair and dim ear; the stool was dry or watery; the body mass were slowly increased; the number of crossed grid and standing frequency were significantly reduced; visceral sensitivity was increased and intestinal propulsion rate was decreased; no obvious inflammatory cell infiltration was observed under microscope. (2) After intervention, compared with the blank group, the body mass and visceral sensitivity in the RU-486 group were not significantly different (both>0.05), but the intestinal propulsion rate was decreased significantly (<0.01). Compared with the blank group, the body mass of heat-sensitive moxibustion group and non-heat-sensitive moxibustion group was lower (both<0.01), but the visceral sensitivity and intestinal propulsion rate were similar (both>0.05). Compared with the model group, the body mass and visceral sensitivity were improved in the RU-486 group (<0.05,<0.01), but the intestinal propulsion rate was similar (>0.05). The body mass, visceral sensitivity and intestinal propulsion rate of the heat-sensitive moxibustion group and the non-heat-sensitive moxibustion group were superior to those of the model group (<0.05,<0.01), and the body mass and intestinal propulsion rate of heat-sensitive moxibustion group were superior to those of non-heat-sensitive moxibustion group (both<0.05). (3) After intervention, compared with the blank group, the contents of CRH, ACTH and CORT in the model group were significantly increased (<0.05,<0.01). Compared with the model group, the contents of CRH, ACTH and CORT of the heat-sensitive moxibustion group were statistically reduced (<0.05,<0.01), and the contents of CRH and ACTH in the non-heat-sensitive moxibustion group were statistically reduced (<0.05,<0.01); the content of CRH in the RU-486 group was reduced (<0.05), but the contents of ACTH and CORT were increased (<0.05,<0.01). Compared with the non-heat-sensitive moxibustion group, the heat-sensitive moxibustion group was better in the improvement of CRH (<0.05), but there was no significant difference of ACTH and CORT between the two groups (both>0.05).

CONCLUSIONHeat-sensitive moxibustion could reduce the contents of CRH, ACTH and CORT through the HPA axis, and improve the function of gastrointestinal motility to treat IBS.

The fusion of cell permeable peptide TAT and bifunctional antioxidant enzymes, GST (Glutathione sulfur transferase)-TAT-SOD1 (Cu, Zn superoxide dismutase), is an intracellular superoxide scavenger. Compared with SOD1-TAT, GST-TAT-SOD1 has better protective effect on oxidative damage but less transduction efficiency. A novel cell permeable bifunctional antioxidant enzymes with the fusion of GST, SOD1 and polyarginine R9 was constructed for higher transduction efficiency. The full nucleotide sequence of SOD1-R9 was synthesized and inserted into the prokaryotic expression vector pGEX-4T-1 with the GST tag. After the successful construction of the prokaryotic expression vectors of GST-SOD1-R9, the recombinant vector was then transformed into Escherichia coli BL21 (DE3) and the GST-SOD1-R9 fusion protein was produced with the induction of IPTG. The soluble expression of GST-SOD1-R9 fusion protein was combining with the induction temperature and time. The best soluble expression was obtained with the induction temperature of 25 ℃ and the induction time of 11 h. The fusion protein was purified through the combination of 80% ammonium sulfate precipitation and affinity chromatography using glutathione agarose, and verified by SDS-PAGE and special enzymatic activity. The thermal and pH stability of GST-SOD1-R9 fusion protein were analyzed and the SOD and GST activity of fusion protein were proved to be well maintained under physiological conditions. Finally, the transduction efficiency of GST-SOD1-R9 fusion protein was proved to be better than GST-TAT-SOD1 fusion protein (P<0.05). These works establish a foundation for further study of the protective effect of GST-SOD1-R9 fusion protein against oxidative damage.

Superoxide dismutase (SOD) family is necessary to protect cells from the toxicity of reactive oxygen species produced during normal metabolism. Among SODs, manganese-containing superoxide dismutase (Mn-SOD, SOD2) is the most important one. The DNA fragment containing the full nucleotide of full-length human SOD2 was synthesized and inserted into the prokaryotic expression vector pGEX-4T-1 with tag GST. DNA construct was then transformed into Escherichia coli BL21 (DE3) and expression was induced with IPTG at 25 ℃. The recombinant fusion protein GST-SOD2 (46 kDa) was purified from the bacterial lysate by GST resin column affinity chromatography. GST tag was cleaved with thrombin, and a crude SOD2 recombinant protein (25 kDa) was obtained and further purified by heparin affinity chromatography. Activities of the two SOD2 proteins were 1 788 and 2 000 U/mg, respectively. Both SOD2 proteins were stable under physiological condition and cell-penetrating (P<0.05). Our findings open the possibility to study the structure and effects of two full-length recombinant SOD2 proteins.

Objective To explore the correlation of B-type natriuretic peptide (BNP) with blood lipids and renal function in elderly patients with coronary heart disease (CHD).Methods According to the four quantiles of BNP level,325 subjects aged 65 years and over were divided into four groups:group A (≤32.4 ng/L),group B (32.5 ng/L),group C (62.4 ng/L),group D (＞ 162.5 ng/L).The left ventricular ejection fraction (LVEF),left ventricular end-diastole dimension (LVEDD),levels of blood lipids and renal function were compared among the groups.Results With the increase of BNP level,the LVEF was gradually decreased [(63.3±8.2) ％,(59.6±7.4) ％,(57.9±9.7)％,(55.2±10.6)％,respectively,F=11.54,P＜0.01] and the LVEDD was gradually increased (F=6.76,P＜0.01),the level of triglyceride (TG) was gradually decreased (F=2.73,P ＜0.05) in group A,B,C and D.Creatinine clearance was gradually decreased [(1.24±0.31) ml ·s-1 · 1.73 m-2,(1.21±0.31) ml· s-1 · 1.73 m-2,(1.24±0.29) ml · s-1 · 1.73 m 2,(1.09± 0.33) ml · s-1 · 1.73 m-2,respectively,F=3.62,P＜0.05],and blood urea nitrogen was gradually increased (F=4.43,P＜0.05) in group A,B,C and D.Multi-linear regression analysis showed that levels of total cholesterol,triglyceride,low density lipoprotein-cholesterol,high density lipoproteincholesterol,very low density lipoprotein-cholesterol,creatinine clearance and blood urea nitrogen were influencing factors for BNP (all P＜0.05).Conclusions BNP level can be used as a sensitive indicator for the diagnosis of heart failure and the assessment of its severity.The levels of blood lipids and renal function are associated with BNP.

Objective To study the relationship between coronary artery disease (CHD ) and interleukin-6 (IL-6), TNF-α and IL-10. Methods Patients with CHD were included in CHD group (n=628) and patients without CHD con?firmed by coronary angiography were selected in control group (n=540) . The recorded data included age, body mass index (BMI) and serum levels of glucose (GLU), total cholesterol (TC), triglyceride (TG), high density lipoprotein-cholesterol (HDL-C) and low density lipoprotein-cholesterol (LDL-C). Meanwhile serum levels of IL-6, TNF-α, IL-10 were determined by ELISA. Results Serum levels of BMI, LDL-C, TG were higher in CHD group than those in control group(P<0.01). The serum level of HDL-C was significantly lower in CHD group than that in control group(P<0.01). Serum levels of IL-6(P<0.05)and TNF-α(P<0.01)were higher in CHD group than those in control group. The serum level of IL-10 was significant?ly lower in CHD group than that in control group(P<0.01). Conclusion IL-6 and TNF-αare involved in the develop?ment of CHD. IL-10 can inhibit inflammation and protect vessel integrity.