ABSTRACT
As a threat to human health， steroid-induced osteonecrosis of femur head is a common refractory orthopedic disease mainly caused by glucocorticoids， with poor prognosis and unclear pathogenesis. Osteogenesis-associated signaling pathways play an important role in bone formation. Glucocorticoid-induced abnormal activation and transport of these signaling pathways lead to abnormal differentiation of bone marrow mesenchymal stem cells， dysfunction of bone metabolism， and osteogenesis disorders， which may be the main reasons for the occurrence and development of steroid-induced osteonecrosis of femur head. Bone formation and remodeling need the participation of bone marrow mesenchymal stem cells， which are stem cells characterized by continuous self-renewal and differentiation. The key to strengthening bone remodeling is to improve the osteogenic differentiation capacity， which is the key point to inhibit bone resorption and prevent bone marrow mesenchymal stem cells from differentiating into osteoclasts. Traditional Chinese medicine （TCM） has been used in the treatment of osteonecrosis in ancient times. It is recorded in the Treasury of Words on Materia Medica （《本草汇编》） that "The deficiency in the lower energizer cannot be tonified without Eucommiae Cortexz.The soreness in lower legs cannot be alleviated without Eucommiae Cortex...The pain in the waist and knee cannot be relieved without Eucommiae Cortex...Tonifying liver and invigorating kidney， Eucommiae Cortex is an essential medicine." This indicates that ancient physicians have already begun to use the liver-tonifying， kidney-invigorating， and sinew-bone-strengthening effects of Eucommiae Cortex for the treatment of osteonecrosis. As the national support for the development of TCM strengthens， increasing studies have been conducted on the TCM prevention and treatment of steroid-induced osteonecrosis of femur head. Studies have suggested that Chinese medicinal herbs can exert a positive effect on the differentiation of bone marrow mesenchymal stem cells by affecting targeted signaling molecules， and promote osteogenesis and bone defect repair， thus combating the occurrence and development of steroid-induced osteonecrosis of femur head. The regulation of osteogenic signaling pathway by Chinese medicines to prevent steroid-induced osteonecrosis of femoral head has become a hot research topic. This article reviews the studies about the prevention and treatment of steroid-induced osteonecrosis of femur head with the active components in Chinese medicinal herbs by regulating osteogenic signaling pathways. We then explore the mechanism of the active components in promoting the differentiation of bone marrow mesenchymal stem cells into osteoblasts and inhibiting their differentiation into osteoclasts to facilitate bone formation， aiming to provide a reference for the further study of treating steroid-induced osteonecrosis of femoral head with Chinese medicinal herbs.
ABSTRACT
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.
Subject(s)
Rats , Male , Animals , Femur Head/pathology , Plasminogen Activator Inhibitor 1/adverse effects , Vascular Endothelial Growth Factor A , Femur Head Necrosis/pathology , Rats, Sprague-Dawley , Steroids , Pain , CholesterolABSTRACT
Steroid-induced osteonecrosis of the femoral head (SONFH) is a kind of disease caused by hip joint dysfunction. Long-term or high-dose application of hormone drugs leads to impaired blood supply to the femoral head and the death of a variety of bone stromal cells, resulting in the structural change of trabecular bone in the load-bearing area of the femoral head and irreversible collapse of the femoral head. Early manifestations were hip pain and discomfort, and tenderness in the inguinal region, in the middle stage, pain affects activity; in the later stage, the hip joint space become narrowed, the motion of the hip joint is reduced, and the motion is limited. SONFH has greatly affected the quality of life of patients, so modern medicine is dedicated to improve the symptoms of patients through early intervention, enabling SONFH to get effective treatment and reducing the pressure of life and economic burden of patients. Bone marrow mesenchymal stem cells (BMSCs) have become a focus in the early treatment of SONFH due to its osteogenic differentiation. BMSCs are stem cells with multi-directional differentiation potential, with self-proliferation and differentiation characteristics, which can differentiate into bone, provide mechanical support for the necrotic area and secrete a variety of growth factors to support hematopoiesis, slow down the progress of disease, and extend the service life of their own joints. The unreasonable use of hormones mainly inhibits the osteogenic activity of BMSCs, resulting in the occurrence of SONFH. This paper reviews the research on BMSC osteogenic differentiation in recent years, hoping to improve the therapeutic effect of SONFH.
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BACKGROUND: The proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) can delay the procession of steroid-induced femoral head necrosis. Besides, microRNA-141 (miR-141) is one of the important regulatory factors to promote cell proliferation. In addition, velvet antler is a traditional Chinese medicine which has significant roles in repairing bone and tissue and improving health. OBJECTIVE: To investigate whether velvet antler serum can regulate the expression of miR-141 to promote the proliferation of BMSCs, and further delay or reverse the progression of steroid-induced femoral head necrosis. METHODS: BMSCs were isolated and cultured from Sprague-Dawley rats. The passage 3 BMSCs were transfected with miR-141 mimic or miR-141 inhibitors, and then real-time PCR and methyl thiazolyl tetrazolium (MTT) assay were performed for detecting miR-141 expression and cell proliferation, respectively. The passage 3 BMSCs were divided into three groups: control group (α-MEM), dexamethasone group (α-MEM+1 μmol/L dexamethasone), and velvet antler serum group (α-MEM+1 μmol/L dexamethasone+15% velvet antler serum). Expression of miR-141 mRNA was detected by real-time PCR at 24 hours after intervention. The proliferation ability of BMSCs was evaluated by MTT assay at 24, 48, and 72 hours after intervention. RESULTS AND CONCLUSION: After transfection with miR-141 mimic, the expression of miR-141 mRNA was upregulated, while the cell proliferation was reduced. After transfection with miR-141 inhibitor, the expression of miR-141 mRNA was downregulated, while the cell proliferation was increased. The expression of miR-141 mRNA was significantly higher in the dexamethasone group than the control group (P < 0.01), while the treatment with velvet antler serum could significantly downregulate the expression of miR-141 mRNA (P < 0.01). The absorbance of BMSCs in the dexamethasone group was significantly lower than that in the control group (P < 0.01), and the absorbance value in the velvet antler serum group was significantly higher than that in the dexamethasone group (P < 0.01). In conclusion, the serum containing velvet antler can downregulate the expression of miR-141 which is upregulated by dexamethasone and then do help to promote the proliferation of BMSCs.
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Objective: To observe the volume and distribution of necrotic tissue of femoral head in steroid-induced osteonecrosis of femoral head (SONFH) patients by three-dimensional reconstruction of CT. Methods: A clinical data of 25 patients with SONFH between September 2016 and December 2018 was analyzed. There were 22 males and 3 females, with an average age of 38.8 years (range, 20-63 years). The necrosis of the femoral head was in stage Ⅱ of Association Research Circulation Osseous (ARCO). The disease duration ranged from 3 to 18 months, with an average of 9.2 months. A three-dimensional reconstruction with CT data of SONFH patients were performed by Mimics Research 21.0 software and the femoral head was segmented into eight regions by 3-matic Research 13.0 software. The volume of necrotic tissue of the femoral head and the volume rate of necrotic tissue to femoral head were calculated and the distribution was also analyzed. Results: The three-dimensional digital model of the femoral head showed that the necrotic tissue of the femoral head was located above the anterior superior medial, and the area of the necrotic tissue was in a dome-like shape. The results showed that the necrotic tissue in the femoral head was mainly concentrated on the anterior superior internal area, the anterior superior outer area, and the posterior superior internal area. The volume of femoral head was (48 399.52±9 408.90) mm 3, and the volume of necrotic tissue was (20 917.08±6 566.94) mm 3, and the volume ratio of necrotic tissue to femoral head was 44.75%±15.72%. The proportion of necrotic volume in different regions was different, and the necrotic tissues were mainly distributed in the anterior superior internal area, the anterior superior outer area, and the posterior superior internal area. Conclusion: The volume and distribution of necrotic tissue in femoral head can be evaluated quickly and intuitively by three-dimensional reconstruction of CT in Mimics software.
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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.
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Objective: To investigate the effect and mechanism of Mailuoning Compound for treatment of early steroid-induced osteonecrosis of femoral head (SONFH) in rats by obturator nerve block. Method: 24 rats were injected with endotoxin 10 μg·kg-1 through tail vein. After 24 hours, prednisolone acetate 20 mg·kg-1 was given by intraperitoneal injection, once every 24 hours for 3 consecutive days. After successful modeling, the rats were randomly divided into the model group (n=12), the treatment group (n=12) and the normal control group (n=6). In the treatment group, 2 mL·kg-1 of Mailuoning compound was injected into the obturator nerve from the 4th day, 3 times a week for 8 weeks. The arterial blood was collected from rats on the first day of the 9th week after model building to detect the content of blood lipid; the femoral head was taken to prepare the paraffin section, and the pathological changes of femoral head was observed and the changes of empty bone lacuna rate, bone trabecular area and bone lacuna area were quantitatively analyzed; The changes of bone morphogenetic proteins(BMPs),transforming growth factor-β1(TGF-β1),vascular endothelial cell growth factor(VEGF),and Ⅷ factor related antigen(Ⅷ-R Ag) were quantitatively analyzed by immunohistochemical method. Result: In the model group, the bone trabeculae were sparse, thin, disorganized and broken; some of the bone cells were necrotic and the number of empty bone lacunae was increased. In the treatment group, the number of trabeculae was increased; the structure was clear, most of which was normal bone cells, with a few necrotic bone cells, and the number of empty bone lacunae was decreased obviously. The rate of empty bone lacuna and the area of bone lacuna in the treatment group were significantly lower than those in the model group (Pβ1 and the microvessel density of Ⅷ-R Ag in the treatment group were significantly higher than those in the model group (PPConclusion: Mailuoning compound can improve the microcirculation state of femoral head, promote the formation of new bone and blood vessel in femoral head by regulating the expression of VEGF, BMPs, TGF-β1, Ⅷ-R Ag and down-regulating blood lipid content, thus effectively controlling the development of early SONFH. This can provide a theoretical basis for the treatment of early SONFH.
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Recent studies have shown that TCM treatment for bone metabolic diseases, such as steroid-induced avascular necrosis of the femoral head, has definite efficacy and has received extensive attention. However, due to lack of molecular biological basis of pharmacodynamics mechanism, it has not yet reached the standard of scientific treatment. The discovery of OPG/RANK/RANKL system has become a new breakthrough point in the prevention and treatment of bone metabolic diseases. The pathogenesis of deficiency of spleen and kidney - blood stasis - caused by phlegm arthralgia caused by blood stasis is closely related to the system. The effect of the final control of the system from the deficiency theory can be expressed through the axial micro-information. This article discussed the TCM syndrome differentiation of steroid-induced osteonecrosis of the femoral head and the relation of regulation of bone metabolism combined with the OPG/RANK/RANKL system, and provided the basis for prevention and treatment of this disease.
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ABSTRACT:Objective To explore the differential expression of microRNAs (miRNAs)in steroid-induced osteonecrosis (ON).Methods Bone tissues were selected from patients with steroid-induced ON between the necrotic zone of femoral heads and its femoral neck to analyze the miRNA expression profile using the microarray. The most differentially expressed miR-125a-3p and miR-1 7-5p in microarray analysis were further confirmed by real-time quantitative PCR.Results According to the microarray screening,8 miRNAs were upregulated and 3 miRNAs were downregulated in the necrotic zone of femoral heads samples (Fold>2,P <0.05 ).Results of real-time PCR revealed that miR-125a-3p was upregulated and miR-1 7-5p was downregulated in the necrotic zone of femoral heads samples,which were in agreement with the microarray data.Conclusion MiRNA’s differential expression profile of human steroid-induced ON samples was obtained.Among the differentially expressed miRNAs, miR-125a-3p and miR-1 7-5p are the most apparently differentially expressed miRNAs,which may be related to the pathogenesis and development of steroid-induced ON.
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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.
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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.
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[Objective]To investigate the morphological changes that take place in the subchondral cocortical bone in steroid-induced osteonecrosis and analyze the reasons leading to humeral head collapse in juvenile rabbits.[Method]Five-six month-old female rabbits were separated by two groups.A modified version of the methods was used to replicate steroid enhanced osteonecrosis anminal humeral head models with Shwartzman reaction in group A,and group B served as the single control.Each humeral head was obtained 10 weeks after the drugs injection.Subchondral cortical bone was observed,and the number of haversian canals was counted.The microcirculatory changes were also detected with scanning electron microscope.[Result]In group A,the Haversian canals in' subchondral area almost disappeared;the subchondral cortical bone disappeared with its arch,dome and bridge structures.Microcirculatory stasis happened in the subchondral vessels.Some humeral heads collapse were observed.While in group B,subchondral cortical bone is integrity and continuity,forming arch,dome and brige structures with subtrabecular bone.[Conclusion]The disappearance of the subchondral cortical bone is a major reason leading humeral head collapse,and ischemia is the critical reason of it.