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.

Effect of icariin on early steroid-induced osteonecrosis of the femoral head in rabbits / 中国修复重建外科杂志

Objective: To explore the effect of icariin on early steroid-induced osteonecrosis of the femoral head in rabbits. Methods: Fifty mature New Zealand rabbits (weighing, 2.5-3.0 kg) were randomly divided into control group ( n=10), model group ( n=20), and experimental group ( n=20). The rabbits of model and experimental groups were injected with lipopolysaccharide and methylprednisolone to establish the animal model of early steroid-induced osteonecrosis of the femoral head. The rabbits of experimental group were feeded with icariin solution once a day for 6 weeks since the first injection of methylprednisolone, whereas the rabbits of control and model groups were given normal saline at the same time points. The left femoral heads were removed after 6 weeks and gross morphological features were evaluated. Micro-CT scan was performed to analyze the trabecular microstructure with the following parameters: trabecular bone volume to total volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Tn), and trabecular separation (Tb.Sp). The Micro-CT scan was also converted to three-dimensional reconstruction images for observation. HE staining was applied to observe the trabecular structure and morphological changes of osteocytes and marrow adipocytes. It was also used to determine whether the samples of femoral heads occurred osteonecrosis based on the criteria for pathological diagnosis, and calculate the rate of empty lacunae. Results: Seven rabbits died during the study, and 9, 16, and 18 rabbits in the control, model, and experimental groups, respectively, enrolled the final analysis. Compared with control group, the femoral head collapse and trabecular breaks were more obvious, and the trabeculae were sparse with irregular arrangement in the model group according to the results of gross observation, Micro-CT scan, and three-dimensional reconstruction images. But in the experimental group, the surface of femoral head was slight shrinking without obvious collapse, and the degeneration of trabecular structure was mild. According to bone microstructures analysis, the Tb.N, Tb.Tn, and BV/TV of femoral head in model and experimental groups were lower than those in control group, while the Tb.Sp in the model and experimental groups were significantly higher. The Tb.N, Tb.Tn, and BV/TV of femoral head in experimental group were higher than those in model group, while the Tb.Sp in the experimental group was significantly lower. The differences between groups were all significant ( P<0.05). In the model group, HE staining showed that the number of osteocytes reduced, the number of empty lacunae increased, and the marrow adipocytes piled up in the space between femoral trabeculae, some even mashed together like a cyst. In the experimental group, the trabecular structure was still relatively complete compared with model group, no obvious apoptosis of osteocytes was observed, the size and number of adipocytes were basically normal. None of the animals in control group occurred osteonecrosis of the femoral head based on the criteria for pathological diagnosis, and the incidence of osteonecrosis were 81.3% (13/16) in the model group and 66.7% (12/18) in the experimental group, and the difference was not significant ( P=0.448). The rate of empty lacunae of osteonecrotic femoral heads in the model group was 33.1%±1.4%, which was higher than that in experimental group (18.9%±0.8%) and in control group (12.7%±1.5%), and the differences between groups were significant ( P<0.05). Conclusion: The icariin has a protective effect on the early steroid-induced osteonecrosis of the femoral head in rabbits, which can decrease osteocytes apoptosis, improve the bone microstructure, and delay such disease processes.

Proteomics Research of Chinese Medical Syndromes of Steroid-induced Osteonecrosis of the Femoral Head / 广州中医药大学学报

Objective To screen the differentially expressed proteins of blood stasis blocking tendon and vessel syndrome and liver-kidney deficiency syndrome of the steroid-induced osteonecrosis of the femoral head ( SONFH) by proteomic technology, so as to supply evidence for Chinese medical syndrome classification. Methods The serum was taken separately from 10 patients with blood stasis blocking tendon and vessel syndrome, 10 patients with liver-kidney deficiency syndrome and 10 healthy volunteers. The two dimensional electrophoresis combined with mass spectrometric method was applied to screen and identify the differentially expressed proteins, and then the obtained protein candidates were verified by Western blotting method. Results Seven proteins were identified from differentially expressed protein spots, including hemoglobin subunit delta, actin, complement C4, antithrombin-Ⅲ, apolipoprotein A-IV, leucine-rich alpha-2 glycoprotein and serum amyloid A-2 protein. We found that antithrombin-Ⅲ and serum amyloid A-2 protein had specific expression in the SONFH patients with blood stasis blocking tendon and vessel, and complement C4 and leucine-rich alpha-2-glycoprotein had specific expression in the SONFH patients with liver-kidney deficiency syndrome. The results of Western blot method showed that the expression levels of complement C4 and antithrombin-Ⅲ were down-regulated in SONFH patients ( P<0.05 compared with the healthy volunteers) , the down-regulation of complement C4 was more obvious in SONFH patients with liver-kidney deficiency syndrome (P<0.05) and the down-regulation of antithrombin-Ⅲ was more obvious in the patients with blood stasis blocking tendon and vessel (P<0.05), the results being accorded with those of proteomic detection. Conclusion Antithrombin-Ⅲand serum amyloid A-2 protein may be the specific serum protein markers of SONFH patients with blood stasis blocking tendon and vessel syndrome, and complement C4 and leucine-rich alpha-2-glycoprotein may be the specific serum protein markers of SONFH patients with liver-kidney deficiency syndrome.

Experimental research on the effects of metformin on steroid -induced osteonecrosis of mice femoral head / 实用医学杂志

Objective To explore the effect of prevention and treatment on steroid-induced osteonecrosis of mice femoral head(ONFH) treated with metformin. Methods Thirty-six Kunming mice were randomly divided into three groups (n = 12):A (Control Group), B (Model Group)and C (Prevention Group). For producing ONFH mice models, did the intraperitoneal injection of horse serum (10 mL/kg) to B and C firstly. After two weeks, continuing the intraperitoneal injection of horse serum (5 mL/kg) again with the prednisolone intramuscularly [45 mL/(kg· day), totally for 5 days]. Meanwhile, feeding normal saline 10 mL/(kg·day) to B and feeding metformin hydrochloride [0.2 g/(kg·day)] to C. For A, mice were only given normal saline intramuscularly and intragastrically in equal quantity at the same time. The contents of serum cholesterol (TC), triglycerid (TG), plasma von willebrand factor (VWF) and plasminogen activator inhibitor 1 (PAI-1) were determined at the 2nd, 4th and 6th week after treatment. The micewere sacrificed at 2nd, 4th, and 6th weekafter treatment, and femoral heads were harvested to do histopathology analysis. Results The appearance and shape of the femoral head and the surface of cartilages were normal. The percentage of empty osteocyte lacunae in B was significantly higher than that in C (P 0.05). TC and TG contents in C were significantly lower than that in B in 2th、4th、6th weeek(P<0.05), and higher than that in A(P<0.05). VWF and PAI-1 level in C were significantly lower than that in B at 2nd and 4th week (P<0.05), but there were no statistical significance at 6th week. there were no statistical significance for the comparison between A and C. Conclusion Metformin can prevent steroid-induced ONFH by improving hyperlipemia, hypercoagulability and hypofibrinolysis, then effectively prevent osteonecrosis.