ABSTRACT
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.
Subject(s)
Animals , Humans , Rats , Glucocorticoids/pharmacology , Human Umbilical Vein Endothelial Cells , Neovascularization, Pathologic/metabolism , Rats, Sprague-Dawley , Vascular Endothelial Growth Factor A/metabolismABSTRACT
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.
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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.
ABSTRACT
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.
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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.
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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