Characteristic changes of blood stasis syndrome in rat model of steroid-induced femoral head necrosis based on the combination of disease, syndrome, and symptom / 中国中药杂志

The approach combining disease, syndrome, and symptom was employed to investigate the characteristic changes of blood stasis syndrome in a rat model of steroid-induced osteonecrosis of the femoral head(SONFH) during disease onset and progression. Seventy-two male SD rats were randomized into a healthy control group and a model group. The rat model of SONFH was established by injection of lipopolysaccharide(LPS) in the tail vein at a dose of 20 μg·kg~(-1)·d~(-1) on days 1 and 2 and gluteal intramuscular injection of methylprednisolone sodium succinate(MPS) at a dose of 40 mg·kg~(-1)·d~(-1) on days 3-5, while the healthy control group received an equal volume of saline. The mechanical pain test, tongue color RGB technique, gait detection, open field test, and inclined plane test were employed to assess hip pain, tongue color, limping, joint activity, and lower limb strength, respectively, at different time points within 21 weeks of modeling. At weeks 2, 4, 8, 12, 16, and 21 after modeling, histopathological changes of the femoral head were observed by hematoxylin-eosin(HE) staining and micro-CT scanning; four coagulation items were measured by rotational thromboelastometry; and enzyme-linked immunosorbent assay(ELISA) was employed to determine the levels of six blood lipids, vascular endothelial growth factor(VEGF), endothelin-1(ET-1), nitric oxide(NO), tissue-type plasminogen activator(t-PA), plasminogen activator inhibitor factor-1(PAI-1), bone gla protein(BGP), alkaline phosphatase(ALP), receptor activator of nuclear factor-κB(RANKL), osteoprotegerin(OPG), and tartrate-resistant acid phosphatase 5b(TRAP5b) in the serum, as well as the levels of 6-keto-prostaglandin 1α(6-keto-PGF1α) and thromboxane B2(TXB2) in the plasma. The results demonstrated that the pathological alterations in the SONFH rats were severer over time. The bone trabecular area ratio, adipocyte number, empty lacuna rate, bone mineral density(BMD), bone volume/tissue volume(BV/TV), trabecular thickness(Tb.Th), trabecular number(Tb.N), bone surface area/bone volume(BS/BV), and trabecular separation(Tb.Sp) all significantly increased or decreased over the modeling time after week 4. Compared with the healthy control group, the mechanical pain threshold, gait swing speed, stride, standing time, and walking cycle of SONFH rats changed significantly within 21 weeks after modeling, with the greatest difference observed 12 weeks after modeling. The time spent in the central zone, rearing score, and maximum tilt angle in the open field test of SONFH rats also changed significantly over the modeling time. Compared with the healthy control group, the R, G, and B values of the tongue color of the model rats decreased significantly, with the greatest difference observed 11 weeks after modeling. The levels of total cholesterol(TC), total triglycerides(TG), low-density lipoprotein-cholesterol(LDL-C), and apoprotein B(ApoB) in the SONFH rats changed significantly 4 and 8 weeks after modeling. The levels of VEGF, ET-1, NO, t-PA, PAI-1, 6-keto-PGF1α, TXB2, four coagulation items, and TXB2/6-keto-PGF1α ratio in the serum of SONFH rats changed significantly 4-16 weeks after modeling, with the greatest differences observed 12 weeks after modeling. The levels of BGP, TRAP5b, RANKL, OPG, and RANKL/OPG ratio in the serum of SONFH rats changed significantly 8-21 weeks after modeling. During the entire onset and progression of SONFH in rats, the blood stasis syndrome characteristics such as hyperalgesia, tongue color darkening, gait abnormalities, platelet, vascular, and coagulation dysfunctions were observed, which gradually worsened and then gradually alleviated in the disease course(2-21 weeks), with the most notable differences occurred around 12 weeks after modeling.

Optimization of expression yield in a stable cell line expressing a novel mutated chimeric tissue plasminogen activator (mt-PA)

Abstract The development of stable cell lines producing recombinant proteins is very time-consuming and laborious. One of the practical approaches successfully performed is Fluorescence-Activated Cell Sorting (FACS). A mutated chimeric tissue plasminogen activator (mt-PA) was developed by removing the first three domains of t-PA, insertion of GHRP sequence and mutation toward resistance to plasminogen activator inhibitor-1 (PAI-1). In the current study, a new stable CHO-DG44 cell line producing mt-PA was developed by two sequential clonal selections: FACS and clonal-selection by limiting dilution. Furthermore, the expression was more evaluated using two different expression media. Finally, the high-producing clones were selected based on the dot blot and amidolytic activity test. The transfection efficiency of CHO-DG44 cells was 38% as measured by flow cytometry on green fluorescent protein (GFP). After performing FACS on stable cell pools, the expression yield was increased to fifty-fold. In terms of growth profile, CD-DG44 showed higher viability and cell density results than ProCHO5 medium. The expression of mt-PA was significantly higher in CD-DG44 than in ProCHO5, 765 and 280 IU/mL, respectively. Our data indicated that selection of an appropriate expression medium played a critical role in the development of potent producing stable cells by FACS.

Síndrome metabólica: um estado pró-trombótico / Metabolic syndrome: a prothrombotic state

Dentre os vários componentes considerados na definição da síndrome metabólica encontra-se o estado pró-trombótico. Esse estado pró-trombótico é caracterizado pelo desequilíbrio entre fatores pró-coagulantes e pró-fibrinolíticos. Clinicamente o estado pró-trombótico da síndrome metabólica pode ser caracterizado pela elevação do fibrinogênio e do PAI-1 e pela ativação de vias de coagulação. Além da ativação de vias de coagulação e do aumento de fatores que desencadeiam fenômenostrombóticos no paciente com a síndrome metabólica, o agrupamento de fatores de risco cardiovascular encontrado nesses pacientes está associado também com a disfunção endotelial. O endotélio, como já se sabe, regula funções tais como controle do tônus vascular, metabolismo de lipídios, equilíbrio fibrinólise-trombose, agregação plaquetária, resposta inflamatória (permeabilidade celular) e proliferação celular vascular. De modo que ele assume papel muito importante como elo fisiopatológico desde a patogênese da aterosclerose incipiente até as complicações mais graves. Essa situação é preocupante pelas evidências epidemiológicas, que demonstram risco de eventos cardiovasculares aterotrombóticos aumentado associado coma síndrome metabólica.

Plasminogen activator inhibtior-1 and fibrinogen in ischemic heart disease

Three groups were included in the study. Group I of 40 males with stable angina with mean age of 52.1 +/- 5.43 years. Group II of 40 male patients with acute myocardial infarction [MI] with a mean age of 51.2 +/- 5.7 years. Control group of 10 normal males with mean age of 50.8 +/- 8.04 years. The results showed significant elevation of serum cholesterol in groups I and II over control group [P <0.0001 - <0.05], respectively. Triglyceride levels were elevated nonsignificantly in group I [P >0.05] but showed highly significant elevation in group II over controls [P <0.0001]. Plasma fibrogen levels were significantly elevated in both groups over controls [P <0.0001]. Plasma PAI-1 showed a nonsignificant difference in group I [P >0.05] and a highly significant elevation in group II over control group [P <0.0001]. Patients with acute MI [group II] showed a significant rise of triglycerides and PAI-1 over those with stable angina [group I] [P <0.0001]. The results of this study showed that plasma fibrinogen is a significant risk factor in both stable angina and acute MI. They also support the hypothesis that there is a significant correlation between triglycerides and PAI-1 as important risk factors only in acute MI, while serum cholesterol is rather more important as atherogenic factor