[Effect of Jianpi Huogu Formula on function damage of vascular endothelial cells induced by glucocorticoid].

This study aimed to observe the intervention effect of Jianpi Huogu Formula(JPHGF) on the functional damage of vascular endothelial cells caused by glucocorticoid, and explore its action mechanism from the PI3 K/Akt and mitogen activated protein kinase(MAPK) signaling pathways. The extracted thoracic aorta ring of normal SD rats were intervened first with vascularendothelial growth factor(VEGF, 20 µg·L-1) and/or sodium succinate(MPS, 0. 04 g·L-1) in vitro and then with JPHGF(8, 16, and 32 µg·L-1) for five mcontinuous ethylpdays, rednisolofollowed nebythe statistics of the number, length, and area of microvessels budding fromvascular rings. In addition, the human umbilical vein endothelial cells(HUVECs) induced by VEGF(20 µg·L-1) were added with MPS(0. 04 g·L-1) and then with JPHGF(8, 16, and 32 µg·L-1) for observing the migration, invasion, and luminal formation abilities of HUVECs in the migration, invasion and luminal formation experiments. The protein expression levels of PI3 K, p-Akt, p-JN K, and p-ERK in HUVECs were assayed by Western blot. The results showed that JPHGF dose-dependently improved the num-ber,length, and area of microvessels in MPS-induced rat thoracic aortic ring, reversed the migration, invasion and lumen formation abiliti es of HUVECs reduced by MPS, and up-regulated the protein expression levels of PI3 K, p-Akt, and p-JNK in HUVECs. All thesehave suggested that JPHGF exerts the protective effect against hormone-induced damage to the angiogenesis of vascular endothelial cells by activating the PI3 K/Akt and MAPK signaling pathways, which has provided reference for exploring the mechanism of JPHGF in treating s teroid-induced avascular necrosis of femoral head(SANFH) and also the experimental evidence for enriching the scientific connotationof spleen-invigorating and blood-activating therapy.

Impaired osteogenic differentiation associated with connexin43/microRNA-206 in steroid-induced avascular necrosis of the femoral head.

Connexin(Cx)43 and microRNA(miR)-206 play an important role in osteogenesis. However, their role in steroid-induced femoral head osteonecrosis (SANFH) is still ambiguous. The present study aimed to establish a rabbit model and investigate osteogenesis in steroid-induced femoral head osteonecrosis occurring via Cx43/miR-206 and the changes of Wnt/ß-catenin signal pathway-related proteins. A total of 72 adult New Zealand white rabbits were divided randomly into a model group (Group A) and a control group (Group B) of 36 rabbits each. Group A was injected intravenously with lipopolysaccharide (10µg/kg body weight, once per day). After 48h, three injections of methylprednisolone (MPS; 20mg/kg body weight) were administered intramuscularly at 24-hour intervals. Group B were fed and housed under identical conditions but received saline injections. All animals were sacrificed at two, four, and eight weeks from the first MPS injection. Typical early osteonecrosis symptoms were observed in Group A. The expression of miR-206 in Group A was significantly higher than that of Group B. The mRNA and protein levels of Cx43, ß-catenin, runt-related transcription factor 2, and alkaline phosphatase gradually decreased while Dickkopf-1 (Dkk-1) gradually increased in Group A compared with Group B. These findings indicated that Cx43/miR-206 is involved in the pathogenesis of early stage SANFH and may be associate with Wnt/ß-catenin signal pathway.

Dried root of Rehmannia glutinosa extracts prevents steroid-induced avascular necrosis of femoral head by activating the wingless-type (Wnt)/ß-catenin signal pathway.

Steroid-induced avascular necrosis of femoral head (SANFH) is one of the most common complications caused by long-term or excessive clinical use of glucocorticoids. This study aimed to investigate the effects of dried root of Rehmannia glutinosa extracts (DRGE) in SANFH. First, SANFH rat model was established by dexamethasone (Dex). Tissue change and proportion of empty lacunae were detected by hematoxylin and eosin staining. Protein levels were detected by western bloting analysis. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was performed to assess apoptosis of femoral head tissue. Cell viability and apoptosis of MC3T3-E1 cells were assessed by Cell Counting Kit-8 assay and flow cytometry. ALP activity and cell mineralization were detected by ALP staining assay and Alizarin red staining. The findings showed that DRGE improved tissue damage, inhibited apoptosis, and promoted osteogenesis in SANFH rats. In vitro, DRGE increased cell viability, inhibited cell apoptosis, promoted osteoblast differentiation, reduced the levels of p-GSK-3ß/GSK-3ß, but increased the levels of ß-catenin in cells treated with Dex. Furthermore, DKK-1, an inhibitor of the wingless-type (Wnt)/ß-catenin signaling pathway, reversed the effect of DRGE on cell apoptosis and ALP activity in cells treated with Dex. In conclusion, DRGE prevents SANFH by activating the Wnt/ß-catenin signaling pathway, indicating that DRGE may be a hopeful choice drug to prevent and treat patients with SANFH.

Dexamethasone promotes the endoplasmic reticulum stress response of bone marrow mesenchymal stem cells by activating the PERK-Nrf2 signaling pathway.

The pathogenesis of steroid-induced avascular necrosis of femoral head (SANFH) is complex, and there is a lack of effective early prevention method. The aim of the present study was to evaluate the effect of dexamethasone (DEX) on the biological behavior of bone marrow mesenchymal stem cells (BMSCs) and to explore the possibility of DEX in the clinical treatment of SANFH. The effect of DEX on the proliferation of BMSCs was evaluated by Counting Kit-8 assay, western blot assay, and enzyme-linked immunosorbent assay. Flow cytometry and western blot assay were performed to detect the effect of DEX on the apoptosis of BMSCs. Quantitative real-time PCR and western blot assay were performed to detect the effect of DEX on the expression of endoplasmic reticulum stress (ERS)-related genes. Immunoblotting analysis was conducted for detecting the nuclear-cytoplasmic distribution of Nrf2. DEX could significantly inhibit the proliferation of BMSCs and promote apoptosis of BMSCs. DEX could increase the expression of PERK, ATF6, and IRE1a, and induce nuclear translocation of Nrf2. The addition of ML385 could reverse the effect of DEX on BMSCs. DEX could activate the PERK-Nrf2 pathway to promote ERS and finally affect the cell proliferation and apoptosis of BMSCs.

Influence of MicroRNA-141 on Inhibition of the Proliferation of Bone Marrow Mesenchymal Stem Cells in Steroid-Induced Osteonecrosis via SOX11.

OBJECTIVE: To investigate whether miR-141 and the sex determination region of Y chromosome box 11 (SOX11) play roles in steroid-induced avascular necrosis of the femoral head (SANFH), and to explore whether miR-141 could target SOX11 to influence the proliferation of bone marrow mesenchymal stem cells (BMSC). METHODS: Bone marrow mesenchymal stem cells (BMSC) were isolated and cultured from 4-week-old Sprague Dawley rats. A flow cytometry assay was performed to identify BMSC. BMSC were divided into two groups: a control group and a dexamethasone (DEX) group. BMSC were transfected by miR-141 mimic, miR-141 inhibitor, and SOX11. Real-time polymerase chain reaction (PCR) assay was performed to investigate the mRNA expression of miR-141 and SOX11. The results were used to determine the effect of transfection and to verify the expression in each group and the association between miR-141 and SOX11. Luciferase reporter assay revealed the targeted binding site between miR-141 and the 3'-untranslated region of SOX11 mRNA. MTT assays were performed to investigate the proliferation of BMSC in the miR-141 mimic, miR-141 inhibitor, and SOX11 groups. RESULT: The results of the flow cytometry assay suggested that cells were positive for CD29 and CD90 while negative for CD45. This meant that the isolated and cultured cells were not hematopoietic stem cells. In addition, cell transfection was successful based on the expression of miR-141 and SOX11. According to the results of real-time PCR assay, the mRNA expression of miR-141 in SANFH was upregulated (4.117 ± 0.042 vs 1 ± 0.027, P < 0.001), while SOX11 was downregulated (0.611 ± 0.055 vs 1 ± 0.027, P < 0.001) compared with the control group. Based on the results of the luciferase experiment, MiR-141 could directly target the expression of SOX11. Inhibition of miR-141 could upregulate the expression of SOX11 (2.623 ± 0.220 vs 1 ± 0.095, P < 0.001) according to the results of a real-time PCR assay. MiR-141 inhibited the proliferation of BMSC (0.618 ± 0.092 vs 1.004 ± 0.082, P < 0.001), while suppression of miR-141 increased the proliferation of BMSC (0.960 ± 0.095 vs 0.742 ± 0.091, P < 0.001). Furthermore, according to the results of the MTT assay, SOX11 promoted the proliferation of BMSC (1.064 ± 0.093 vs 0.747 ± 0.090, P < 0.001). CONCLUSION: MiR-141 inhibited the proliferation of BMSC in SANFH by targeting SOX11. Inhibition of miR-141 upregulated the expression of SOX11 and promoted the proliferation of BMSC. MiR-141 and SOX11 could be new targets for investigating the mechanism of SANFH.

Epimedium protects steroid-induced avascular necrosis of femoral head in rats by inhibiting autophagy.

The effect of epimedium extracting solution on bone mineral density (BMD) of steroid-induced avascular necrosis of femoral head (SANFH) in rats was evaluated to further explore its function mechanism. Twenty-four Sprague-Dawley (SD) rats (male/female: 1/1) were randomly divided into three groups: the control (n=8), the glucocorticoid (n=8) and the epimedium (n=8) group. Rats in the glucocorticoid and the epimedium group were injected with prednisolone acetate injection in gluteal muscles with 15 mg/kg/day twice a week. The epimedium group was given 10 ml/kg ephedra extracting solution containing crude drug with the concentration of 1.5 g/ml daily by gavage. After 6 weeks, all the experimental rats were sacrificed and materials were extracted. The expression of autophagy-related proteins were detected by observing the bone of the femoral head. After comparison of the control group with the model group in BMD, it was found that there were significant differences (P<0.05). There were no significant differences between the control and the epimedium group (P>0.05). Neither between the glucocorticoid and the epimedium group (P<0.05). Epimedium extracting solution can significantly enhance the BMD of femoral heads, prevent osteoporosis and lead to collapse, increase the expression of apoptotic and protective proteins and reduce the expression of autophagy-related proteins, thus providing a preliminary theoretical study for the prevention and treatment of SANFH.

[Effect and mechanisms of vitamin E on early steroid-induced avascular necrosis of femoral head in rats].

OBJECTIVE: To investigate the possibility of mitochondria-dependent apoptosis as a mechanism of early steroid-induced avascular necrosis of femoral head (SANFH) in rats and vitamin E as a possible prevention strategy. METHODS: Seventy-two male Sprague Dawley rats were randomly divided into control group, model group, and intervention group, with 24 rats in each group. The rats in control group were not treated as normal control. The rats in model group and intervention group were established early SANFH models by lipopolysaccharide combined with methylprednisolone injection. At the same time, the rats in intervention group were injected with vitamin E (40 mg/kg) every day for 7 days. At 2, 4, and 8 weeks after the final injection, the bilateral femoral heads were harvested and observed by HE staining, TUNEL assay, immunohistochemical staining, and Western blot. The rate of empty lacunae, apoptotic index, and the expressions of Caspase-9, Caspase-3, and cytochrome-c (Cyt-c) proteins were calculated. RESULTS: According to histological staining, there were significant differences in the rate of empty lacunae between intervention group and control group at 8 weeks ( P<0.05) and between intervention group and model group at 4 and 8 weeks ( P<0.05). The apoptotic index of intervention group was significantly lower than that of model group at each time point ( P<0.05). And there was significant difference between the intervention group and the control group at 8 weeks ( P<0.05). According to immunohistochemistry staining and Western blot, the expressions of Cyt-c, Caspase-9, and Caspase-3 all significantly decreased in intervention group than those in model group at each time point ( P<0.05); and the differences were significant between intervention group and control group at 8 weeks ( P<0.05). CONCLUSION: Vitamin E can delay the progression of early SANFH by reducing mitochondrial dependent osteocyte apoptosis.

Upregulation of microRNA-320 decreases the risk of developing steroid-induced avascular necrosis of femoral head by inhibiting CYP1A2 both in vivo and in vitro.

Steroid-induced avascular necrosis of femoral head (SANFH) occurs frequently in patients receiving high-dose steroid treatment for these underlying diseases. The target of this study is to investigate the effect of microRNA-320 (miR-320) on SANFH by targeting CYP1A2. CYP1A2 expression was detected using immunohistochemistry. Specimens were collected from patients with SANFH and femoral neck fracture. Seventy rats were assigned into seven groups. The targeting relationship between miR-320 and CYP1A2 was verified by bioinformatics website and dual luciferase reporter gene assay. RT-qPCR and Western blot analysis were used to detect miR-320 and CYP1A2 expressions. The enzymatic activity of CYP1A2 was detected by fluorescence spectrophotometry. Hemorheology and microcirculation were measured in rats. MiR-320 expression decreased and CYP1A2 expression and enzymatic activity increased in SANFH patients compared to those with femoral neck fracture. CYP1A2 was the target gene of miR-320. Hemorheology and microcirculation results showed that up-regulated expression of CYP1A2 promoted the development of SANFH while increased expression of miR-320 inhibited the development of SANFH. Compared with the SANFH group, the SANFH + miR-320 mimic group showed increased miRNA-320 expression, and decreased CYP1A2 expression and enzymatic activity. Opposite results were found in the SANFH + miR-320 inhibitor group. The SANFH + miR-320 inhibitor + pCR-CYP1A2_KO group showed decreased miRNA-320 expression and the SANFH + pCR-CYP1A2_KO group showed decreased CYP1A2 expression and enzymatic activity. Our findings provide evidences that miR-320 might inhibit the development of SANFH by targeting CYP1A2.

Sildenafil improves blood perfusion in steroid-induced avascular necrosis of femoral head in rabbits via a protein kinase G-dependent mechanism.

OBJECTIVE: The aim of the study were to evaluate the effect of sildenafil against avascular necrosis of femoral head (ANFH) in a rabbit model, and to study the role of protein kinase G (PKG) pathway and vascular endothelial growth factor (VEGF) in ANFH. METHODS: Three weeks after inducing ANFH with methylprednisolone injection, 45 female adult New Zealand white rabbits were divided into three groups and treated as follows: group SI received daily intraperitoneal sildenafil with a dose of 10 mg/kg per day; group SD received daily sildenafil identically to group SI plus auricular vein injection DT3 (a specific PKG inhibitor); group NS received only normal saline. The blood perfusion function in the femoral head was measured by perfusion MRI and ink artery infusion. Bilateral femora heads were examined histopathologically for the presence of osteonecrosis; VEGF of tissue was examined by Western blot analysis; cGMP level and PKG activity were also measured. RESULTS: The incidence of ANFH in SI group was significantly lower than that observed in NS and SD groups (p < 0.05). VEGF in SI group was increased compared to NS group. cGMP level and PKG activity were also significantly different between NS and SI group (p < 0.05). However, these effects of sildenafil in SD group were all markedly inhibited by the administration of DT3 compared to SI group. CONCLUSION: Sildenafil appear to increase the perfusion of femoral head by up-regulating VEGF through PKG pathway. The increased perfusion of femoral head could prevent ANFH.

Effect of Jianpi Huogu Formula on function damage of vascular endothelial cells induced by glucocorticoid / 中国中药杂志

This study aimed to observe the intervention effect of Jianpi Huogu Formula(JPHGF) on the functional damage of vascular endothelial cells caused by glucocorticoid, and explore its action mechanism from the PI3 K/Akt and mitogen activated protein kinase(MAPK) signaling pathways. The extracted thoracic aorta ring of normal SD rats were intervened first with vascularendothelial growth factor(VEGF, 20 μg·L-1) and/or sodium succinate(MPS, 0. 04 g·L-1) in vitro and then with JPHGF(8, 16, and 32 μg·L-1) for five mcontinuous ethylpdays, rednisolofollowed nebythe statistics of the number, length, and area of microvessels budding fromvascular rings. In addition, the human umbilical vein endothelial cells(HUVECs) induced by VEGF(20 μg·L-1) were added with MPS(0. 04 g·L-1) and then with JPHGF(8, 16, and 32 μg·L-1) for observing the migration, invasion, and luminal formation abilities of HUVECs in the migration, invasion and luminal formation experiments. The protein expression levels of PI3 K, p-Akt, p-JN K, and p-ERK in HUVECs were assayed by Western blot. The results showed that JPHGF dose-dependently improved the num-ber,length, and area of microvessels in MPS-induced rat thoracic aortic ring, reversed the migration, invasion and lumen formation abiliti es of HUVECs reduced by MPS, and up-regulated the protein expression levels of PI3 K, p-Akt, and p-JNK in HUVECs. All thesehave suggested that JPHGF exerts the protective effect against hormone-induced damage to the angiogenesis of vascular endothelial cells by activating the PI3 K/Akt and MAPK signaling pathways, which has provided reference for exploring the mechanism of JPHGF in treating s teroid-induced avascular necrosis of femoral head(SANFH) and also the experimental evidence for enriching the scientific connotationof spleen-invigorating and blood-activating therapy.

MicroRNA-34a alleviates steroid-induced avascular necrosis of femoral head by targeting Tgif2 through OPG/RANK/RANKL signaling pathway.

The study aims to investigate the effect of microRNA-34a (miR-34a) targeting Tgif2 on steroid-induced avascular necrosis of femoral head (SANFH) by regulating OPG/RANK/RANKL signaling pathway. SD rats were divided into normal control and model (RNAKL rat models) groups. The model group was further assigned into model control, negative control, miR-34a mimics and miR-34a inhibitors groups. QRT-PCR was applied to detect miR-34a, Tgif2, OPG, RANK and RNAKL mRNA expressions. Femoral head tissues were collected for Micro-CT scanning and HE staining. QRT-PCR and Western blotting were used to detect expressions of miR-34a, Tgif2, OPG, RANK, RANKL and Runx2, OPN and OC in bone tissues. Dual-luciferase reporter gene assay was used to testify the target relationship between miR-34a and Tgif2. Compared with the normal control group, the model group showed increased Tgif2, RANK and RANKL mRNA expressions, but decreased miR-34a and OPG mRNA expressions. Tgif2 mRNA expression was negatively correlated with miR-34a and OPG mRNA expressions. Micro-CT showed cystic degeneration of femoral head, with decreased bone volume/total volume (BV/TV), bone surface area/bone volume and trabecular number in the model control group compared with the normal control group. Compared with the model control group, the miR-34a mimics group showed increased BV/TV and trabecular thickness and Runx2, OPN and OC expressions, while the parameters decreased in the miR-34a inhibitors group. Compared with the normal control group, the other groups showed increased Tgif2, RANK and RANKL expressions but decreased miR-34a and OPG expressions. Compared with the model control group, Tgif2, RANK and RANKL expressions decreased and miR-34a and OPG expressions increased in the miR-34a mimics group, while the miR-34a inhibitors group had a reverse trend in contrast to the miR-34a mimics group. Tgif2 is a target gene of miR-34a. In conclusion, miR-34a can alleviate SANFH through targeting Tgif2 and further regulating OPG/RANK/RANKL signaling pathway. Impact statement miR-34a can alleviate SANFH through targeting Tgif2 and further regulating OPG/RANK/RANKL signaling pathway, which can be used as a new theoretical basis for SANFH treatment.

[EFFECT OF MACROPHAGE MIGRATION INHIBITORY FACTOR ON VASCULAR REPAIR OF STEROID-INDUCED AVASCULAR NECROSIS OF FEMORAL HEAD IN VITRO].

OBJECTIVE: To interpret the mechanisms of vascular repair disorders in steroid-induced avascular necrosis of the femoral head (SANFH) via detection of the changes of proliferation, migration, and macrophage migration inhibitory factor (MIF)/vascular endothelial growth factor (VEGF) expressions of endothelial cells (ECs) under hypoxia/glucocorticoid. METHODS: According to culture conditions, human umbilical vein ECs (HUVECs) at passage 3 were divided into group A (normal), group B (1.0×10-6 mol/L dexamethasone), group C (hypoxia), and group D (hypoxia+1.0×10-6 mol/L dexamethasone). The cell activity was detected by AlamarBlue; the number of viable cells was detected in live/dead cell staining; the cell morphology was observed after cytoskeleton staining; cell migration ability was compared by scratch test; and the levels of MIF and VEGF expressions were detected by ELISA. RESULTS: At 24 hours after culture, the cell activity and the number of living cells in group C were significantly higher than those in the other 3 groups, showing significant difference between groups (P<0.05), and group D had the worst cell activity and least living cells. Cytoskeleton staining showed that cells had normal morphology in groups A and B; cells had rich cytoskeleton and secretion granules in group C; cytoskeleton form disorder and nucleus pyknosis were observed in group D. Scratch test showed that the cell migration ability of group C was strongest while cell migration ability of group D was weakest. Accumulated concentration of MIF and VEGF in 4 groups significantly increased with time extending. Accumulated concentration of MIF in group C were significantly higher than that in other 3 groups at each time point (P<0.05). Within 24 hours after intervention, stage concentration of MIF during 1-8 hours was significantly lower than that during 0-1 hour and 8-24 hours in every group (P<0.05). Stage concentration of MIF in group C was significantly higher than other groups during 0-1 hour and 8-24 hours (P<0.05). Within 2 hours after intervention, stage concentration of MIF in 4 groups during 0.5-1 hour was significantly higher than that during other stages (P<0.05). Accumulated concentration of VEGF in group C was significantly higher than that in other groups at 8 and 24 hours (P<0.05). The stage concentration of VEGF in groups C and D during 8-24 hours was significantly higher than that during 0-1 hour and 1-8 hours (P<0.05). There was no significant difference in the stage concentration of VEGF within and among group A, B, C, and D at every stage within 2 hours after intervention (P>0.05). CONCLUSIONS: In hypoxia environment, the proliferation and migration of ECs is enhanced, and the secretion of VEGF and MIF is increased. High concentration of dexamethasone will suppress the process above, which induces vascular repair disorders and aggravating SANFH.

[EFFECT OF Wnt/ß-catenin SIGNAL PATHWAY ON APOPTOSIS IN STEROID-INDUCED AVASCULAR NECROSIS OF FEMORAL HEAD IN RATS].

OBJECTIVE: To investigate the effect of Wnt/ß-catenin signal pathway on the apoptosis in steroid-induced avascular necrosis of femoral head (SANFH) in rats. METHODS: Seventy-two male Sprague Dawley rats (weighing, 200-230 g) were randomly divided into the control group (group A, n=24), the model group (group B, n=24), and the intervening group (group C, n=24). The rats in groups B and C were injected with lipopolysaccharide and methylprednisolone (MPS) to establish the SANFH model. The rats in group C were injected intramuscularly with human recombinant secreted frizzled related protein 1 (SFRP1) [1 µg/(kg·d)] at the first time of MPS administration for 30 days. The rats in group A received saline injection at the same injection time of group B. The general condition of rats in groups B and C was observed during modeling and after modeling. At 2, 4, and 8 weeks after last injection of MPS, 8?rats were sacrificed to harvest the femoral head. Histological staining was performed to evaluate osteonecrosis. Apoptosis was detected via TUNEL staining. The expressions of Wnt/ß-cate nin pathway signaling molecules (activated ß-catenin and c-Myc) were detected by immunohistochemistry and Western blot. RESULTS: Six rats were added in groups B and C because of 6 deaths. The other rats survived to the end of experiment. Normal bone structure was observed in group A; osteonecrosis of bone structure disturbance and disruption of the trabecula were found with time in groups B and C. Group C had the highest empty lacuna rate and apoptosis rate, followed by groups B and A, showing significant difference between groups (P < 0.05). The expression levels of activated ß-catenin and c-Myc were significantly lower in group C than groups A and B (P < 0.05), and in group B than group A (P < 0.05). CONCLUSIONS: Wnt/ß-catenin signal pathway is involved in the pathogenesis in early SANFH model and its?possible mechanism?is to affect the cell cycle and cell apoptosis by the regulation of c-Myc expression.

Efficacy of Wenyangbushen Formula on mRNA Expression of Osteoprotegerin, Receptor Activator of Nuclear Factor-kappa B and Its Ligand in Rabbits with Steroid-induced Avascular Necrosis of Femoral Head / 中国康复理论与实践

Objective:To observe the effect of Wenyangbushen formula on mRNA expression of osteoprotegerin (OPG), receptor activator of nuclear factor- kappa B (RANK) and receptor activator of nuclear factor- kappa B ligand (RANKL) in rabbits with steroid-induced avascular necrosis of femoral head (SANFH). Methods:A total of 46 healthy conventional New Zealand white rabbits were randomly divided into normal group (n = 10) and model building group (n = 36). The modified method of horse serum plus methylprednisolone was used to establish the SANFH model. Two rabbits from the normal group and four from the model building group were used for HE staining. Then the other models were randomly divided into model group, and low-dose, medium-dose and high-dose treatment groups, with eight rabbits in each group. The normal group was given normal saline 10 ml/d, and the treatment groups were given Wenyangbushen formula 6.44 g/(kg·d), 9.66 g/(kg·d) and 12.88 g/(kg·d), respectively, for eight weeks. The mRNA expression of OPG, RANK and RANKL was detected by reverse transcription- polymerase chain reaction. Results:The empty lacuna rate was significantly higher in the model group than in the normal group (t = 17.085, P < 0.001). Compared with the model group, the mRNA expression of OPG increased (P < 0.01), and the mRNA expression of RANK and RANKL decreased (P < 0.01), in the treatment groups. Compared with the low-dose treatment group, the mRNA expression of OPG increased (P < 0.01), and the mRNA expression of RANK and RANKL decreased (P < 0.01), in the medium-dose and high-dose treatment groups. There was no significant difference in the mRNA expression of OPG, RANK and RANKL between the low-dose treatment group and the high-dose treatment group (P > 0.05). Conclusion:Wenyangbushen formula could increase the mRNA expression of OPG and inhibit the mRNA expression of RANK and RANKL in the femoral head tissue of the rabbits with SANFH.

Effect and mechanisms of vitamin E on early steroid-induced avascular necrosis of femoral head in rats / 中国修复重建外科杂志

Objective: To investigate the possibility of mitochondria-dependent apoptosis as a mechanism of early steroid-induced avascular necrosis of femoral head (SANFH) in rats and vitamin E as a possible prevention strategy. Methods: Seventy-two male Sprague Dawley rats were randomly divided into control group, model group, and intervention group, with 24 rats in each group. The rats in control group were not treated as normal control. The rats in model group and intervention group were established early SANFH models by lipopolysaccharide combined with methylprednisolone injection. At the same time, the rats in intervention group were injected with vitamin E (40 mg/kg) every day for 7 days. At 2, 4, and 8 weeks after the final injection, the bilateral femoral heads were harvested and observed by HE staining, TUNEL assay, immunohistochemical staining, and Western blot. The rate of empty lacunae, apoptotic index, and the expressions of Caspase-9, Caspase-3, and cytochrome-c (Cyt-c) proteins were calculated. Results: According to histological staining, there were significant differences in the rate of empty lacunae between intervention group and control group at 8 weeks ( P<0.05) and between intervention group and model group at 4 and 8 weeks ( P<0.05). The apoptotic index of intervention group was significantly lower than that of model group at each time point ( P<0.05). And there was significant difference between the intervention group and the control group at 8 weeks ( P<0.05). According to immunohistochemistry staining and Western blot, the expressions of Cyt-c, Caspase-9, and Caspase-3 all significantly decreased in intervention group than those in model group at each time point ( P<0.05); and the differences were significant between intervention group and control group at 8 weeks ( P<0.05). Conclusion: Vitamin E can delay the progression of early SANFH by reducing mitochondrial dependent osteocyte apoptosis.

Research Progress in OPG/RANK/RANKL Signal-regulated Mechanism of Steroid Induced Avascular Necrosis of Femoral Head from Phlegm Theory / 中国中医药信息杂志

In recent years, due to the irregular and abused use of glucocorticoid in clinical treatment, the incidence of steroid induced avascular necrosis of femoral head (SANFH) is gradually increasing. TCM for the prevention and treatment of SANFH has received much attention from many scholars. However, due to the lack of the scientific explanation of molecular biology level for its pharmacodynamics mechanism, it is difficult to achieve the purpose of standardized treatment. The discovery of OPG/RANK/RANKL signaling pathway has opened up new shortcuts for the prevention and treatment of bone metabolic diseases. OPG/RANK/RANKL signaling pathway is associated with the pathogenesis of spleen deficiency and phlegm - blood stasis caused by phlegm-blood stasis due to paralysis of SANFH. The treatment efficacy based on the phlegm theory can eventually axial control of the system through the micro-information to express. This article discussed the relevance between the phlegm in the treatment of SANFH and molecular biology mechanism of OPG/RANK/RANKL signal regulation mechanism, and combined the system of bone metabolism regulation mechanism to discuss TCM differentiation of SANFH, with a purpose to provide references for clinical and further study.

EFFECT OF MACROPHAGE MIGRATION INHIBITORY FACTOR ON VASCULAR REPAIR OF STEROID-INDUCED AVASCULAR NECROSIS OF FEMORAL HEAD IN VITRO / 中国修复重建外科杂志

CONCLUSIONS: In hypoxia environment, the proliferation and migration of ECs is enhanced, and the secretion of VEGF and MIF is increased. High concentration of dexamethasone will suppress the process above, which induces vascular repair disorders and aggravating SANFH.

Effect of Xianhua Huogudan Granules on hemorrheology and blood lipid of steroid-induced avascular necrosis of femoral head / 中成药

AIM: Study on the effects of Xianhuahuogudan Granule (XHHGDG) (Radix et Rhizowa Salviae uiltiorrhizae, Rhizoma Drynariae,etc) in treatment of steroid-induced avascular necrosis of femoral head in order to provide basis for clinical use. METHODS: 40 full-grown well New Zealand rabbits were divided randomly into four groups:normal group,model group,MaShGuPian group and XHHGDG group.Except the normal group, The other three groups were administered intramuscularly with steroid.From the 9 th week after production of models,the normal group and the model group were treated with normal saline,XHHGDG group with XHHGDG aqueous solution,and MaShGuPian group with MaShGuPian aqueous solution.All of the rabbits were killed 14 weeks after baseline for the research material. After making pathomorphology of bone,blood viscosity and content of blood lipid will be acquired. RESULTS: The blood viscosity and content of blood lipid were lower significantly in the XHHGDG group than those in the model group.Compared with model group,XHHGDG group significantly showed followings:the small bone trabeculas were fuller;most of bone cells were normal.Although of adipose cell existed in the margin of marrow,the cells for producing blood were enriched;numbers of empty bone lacuna were 16.4%(P