【Abstract】 Objective The present study employed both static and dynamic imaging modal ities to study bothintra- and extravascular events attributing to steroid-associated osteonecrosis (ON) using an experimental protocol with a single low-dose l i ppolysaccharide (LPS) injection and subsequently three injections of high-dose methylprednisolone (MPS). Methods Fourteen 28-week-old male New Zealand white rabbits received one intravenous injection of LPS (10 μg/ kg). After 24 hours, three injections of 20 mg/kg of MPS were given intramuscularly at a time interval of 24 hours. Additional 6 rabbits were used as controls. Dynamic MRI was performed on bilateral femora for local intraosseous perfusion before and after LPS injection. Blood samples were collected for haematological examinations before and after LPS injection. Bilateral femora were dissected and decalcified for microCT-based microangiography. ON lesion, intravascular thrombus and extravascular marrow fat cell size were examined histopathologically. Results Intravascular thrombus was observed in all ON rabbits. Extravascular marrow fat cell size was significantly increased in ON rabbits than that of the controls (P lt; 0.05). Compared to basel ine, a significant decrease in ratio of tissue-type-plasminogen-activator/plasminogen-activator inhibitor 1,activated-partial- thromboplatin-time, and a significant increase in ratio of low-density-l ipoprotein/high-density-l ipoprotein were only found in ON rabbits (P lt; 0.05). Dynamic MRI showed a significant decrease in the perfusion index ‘maximum enhancement’ in the ON rabbits (P lt; 0.05) and microCT-based microangiography showed blocked stem vessels in ON samples.Overall, 93% of the rabbits (13/14) developed ON and no rabbits died throughout the experiment period. Conclusion Bothintra- and extravascular events were found attributing to the steroid- associated ON based on our experimental protocol with a single low-dose LPS injection and subsequent three injections of high-dose MPS. Both high ON incidence and no mortal ity in rabbits treated with this inductive protocol suggested its effectiveness for future studies on evaluation of therapeutic efficacy of interventions developed for prevention of steroid-associated ON.
Objective To investigate the effect of steroid receptor coactivator family in initiation, development, treatment and prognosis of breast cancer. Methods The literatures in recent years which have related to the effect of steroid receptor coactivators in breast cancer are reviewed. Results Steroid receptor coactivators are essential for several kinds of steroid hormones binding to steroid receptors, so they are important accessory factors that induce the initiation, development and recurrence of breast cancer, and predictive factors that estimate the prognosis of breast cancer. Conclusion Inhibition of the expression and signaling pathway of steroid receptor coactivators may be effective for breast cancer prevention and treatment.
Objective To investigate the significance of sensory neuropeptides [calcitonin gene related peptide (CGRP) and substance P (SP)] in steroid-induced avascular necrosis of the femoral head (ANFH) by using a rabbit model. Methods Fifty-five adult female Japanese White rabbits (weighing 3 kg and aging 24 months) were randomly divided into experimental group (n=45) and control group (n=10). The rabbits in experimental group received a single intramuscularinjection of methylprednisolone at a dose of 4 mg/kg and then were sacrificed after 3 days (n=15), 1 week (n=15), and 2 weeks (n=15) of injection. The rabbits in control group were fed without any treatment. The necrosis of the femoral head was observed. And the expressions of the monoclonal antibodies CGRP and SP were observed with immunohistochemical staining. Also, the integrated absorbance (IA) value of the positive area was calculated. Results All rabbits survived to the end of the experiment. There was no necrosis of the bone or bone marrow in experimental group at 3 days; whereas ANFH was observed in 5 rabbits at 1 week (33%) and in 8 rabbits at 2 weeks (53%). There were significant differences in the rate of ANFH between 1 week, 2 weeks and 3 days (P lt; 0.05); but there was no significant difference between 1 week and 2 weeks (P gt; 0.05). The intensity of CGRP immunoreactivity increased and reached the peak at 1 week, and then decreased at 2 weeks in experimental group. The IA value of CGRP in experimental group at 1 week was significantly higher than that of control group and that of experimental group at 3 days (P lt; 0.05). The IA value of CGRP in experimental group at 2 weeks was significantly lower than those at 3 days and 1 week (P lt; 0.05). The intensity of SP immunoreactivity decreased and reached the lowest at 1 week, and then increased. The IA value of SP in experimental group at 1 week was significantly lower than that of control group and that of experimental group at 2 weeks (P lt; 0.05). Conclusion The sensory neuropeptides may be affected by the steroid, which may play a key role in the process of steroid-induced ANFH by imbalance of bone metabol ism, disturbance of the microcirculation of bone, and disorder of the protective pain-transmission.
Objective To explore the significance of osteocyte apoptosis in steroidinduced osteonecrosis of the femoral head. Methods SixtyNew Zealand rabbits were divided into experimental group and control group(n=30). The experimental group was given 10 ml/kg of horse serum intravenously 2 times at 2 weeks intervals and an intraperitoneal injection of 45 ml/kg·d of methylprednisolone acetate for 3 days；the control group was given equal isotonic Na chloride. Osteocyteapoptosis was observe by means of TUNEL. Results The number of apoptosis in the experimental group(112.33‰±26.12‰) was significantly higher than that in the control(47.01‰±22.95‰) (Plt;0.01)in the 4th week. With time, osteocytes apoptosis progressively increased. In the 6thand 8th weeks, the percentage of empty osteocyte lacunae in the experimental group (17.23%±3.44%, 28.56%±3.45%) was significantly higher than that in the control group (11.29%±2.89%,11.26%±2.75%，Ｐlt;0.05). The transmission electron microscope showed that the characteristics of osteocyte apoptosisincluded intact nuclear membrane,comdensed chromatin and increased electron dense. Conclusion Osteocytes apoptosis may play a key role in the process of steroidinduced early osteonecrosis of the femoral head.
ObjectiveTo screen for the differentially expressed genes in steroid-induced osteonecrosis of the femoral head (ONFH) by gene microarray.
MethodsThe femoral head tissue of ONFH was harvested from 3 patients with steroid-induced ONFH, aged 25, 31, and 38 years, respectively. Normal tissue was harvested from a 26-year-old male remains contributor. HE staining of the specimens was performed for observing the histology manifestation; the total RNA was extracted for measuring the purity; cDNA probe was synthesized by reverse transcription, and then were hybridized as the cDNA microarray for scanning of fluorescent signals and differentially expressed genes in the tissues.
ResultsHE staining of normal tissue showed complete unit composed of lamellar bone, continuous and complete lamellar bone with a concentric arrangement around blood vessels, and normal bone cells in the trabecular bone lacuna. In ONFH tissue, adipose tissue increased in the medullary cavity, with increased fat cells filling in the medullary cavity and extruding capillary, and with decreased bone cells in the bone trabecula, which had deeply-stained nuclear chromatin, pyknotic or cracking nucleus, and even bone cells disappeared in the part of the bone lacuna, and trabecular bone became thin, sparse, interrupt, reduced area in visual field/unit. Total RNA extraction electrophoretogram displayed clear bands of 28S and 18S, and the brightness ratio of the 28S:18S was 2:1, indicating good total RNA quality. And 44 genes were differentially expressed, and there were 28 up-regulated genes and 16 down-regulated genes, including cell/organism defense genes, cell structure/motility genes, cell division genes, cell signaling/cell communication genes, cell metabolism genes, gene/protein expression genes, and unclassified genes.
ConclusionThe analysis of the gene expression profile of steroid-induced ONFH can provide evidence for the pathogenesis of ONFH.
Objective To investigate the postoperative biochemical changes and the strategy of steroid treatment for autoimmune pancreatitis （AIP）. Methods Six patients with AIP from January 2007 to October 2011 in this hospital were included in this study. Four patients received pancreaticoduodenectomy or distal pancreatectomy for being preoperatively misdiagnosed as malignant tumors. Two patients avoided the unnecessary resection and received the internal drainage for obstructive jaundice and postoperative steroid medication because of a confirmation of AIP by intraoperative frozen biopsy. All the patients underwent routine examinations， including liver function， CA19-9 level， and imaging data. Measurement of serum IgG or IgG4 was performed for fewer patients after operation. Results CA19-9 levels were normal or slightly elevated in six AIP patients. For five patients with AIP in the head of pancreas， preoperative serum γ-GT levels were elevated. For these patients， whether pancreaticoduodenectomy or the internal drainage were performed， TB/DB levels were transiently elevated on day 1 or 4 after operation but dropped to below preoperative levelsor normal levels on day 7 after operation， and serum γ-GT levels among them presented a downward. Serum γ-globulin levels exhibited a downward trend among four AIP patients after resection， while a upward trend was found in another two AIP patients receiving internal drainage. From the limited data， elevated serum level of IgG in the AIP patients were found. As to IgG4， the only case was tested on month 7 after operation， the serum IgG4 level was elevated. CT showed that the swollen status of pancreas obviously reduced on month 3 after operation as compared with before operation.Conclusions Postoperative steroid therapy of AIP should be based on the serum γ-globulin level and operation method. CT or MRI imaging is used to evaluate the response to steroid treatment for the AIP patients.
Objective To evaluate the safety and efficacy of steroid withdrawal in modern triple immunosuppressant (Cycloproine/Tacrolimus, Mycophenolate Mofetil and Steroid) on renal transplantation recipients. Methods We searched MEDLINE (1966-Sep. 2005), OVID (1966-2004), EMBASE (1984-2004), The Cochrane Library (Issue 4, 2005), CBMdisc (1994-2005), and handsearched 7 Chinese Journals. Randomized controlled trials (RCTs) adopting modern triple immunosuppressant, and comparing steroid withdrawal (SW), group and steroid continuing group (SC) were selected. The quality of included studies was evaluated and graded according to Cochrane Reviewer’s Handbook 4.2.5, and meta-analysis was performed by using RevMan 4.2.7 software. Results Nine RCTs including 1 681 patients (845 in SW and 836 in SC) were identified. The average follow-up time was 6-12 months. No significant difference was found in using CsA or Tac in modern triple immunosuppressant. The results of our meta-analysis showed: ① the risk of acute rejection was two times higher in SW than SC (RR 2.05, 95% CI 1.54 to 2.72, P lt;0.000 01), mainly Banff grade I (mild) (RR 1.92, 95% CI 1.16 to 3.17, P =0.01); but no significant differences were found on Banff grade II and III between the two groups. ② the rate of graft and patient survival and chronic rejection were the same between two groups. ③ Steroid withdrawal decreased the incidence of opportunistic infection (mainly caused by simplex herpes virus and Candida) and urinary tract infection. While the incidence of CMV and sepsis infection has no significant difference between two groups. Conclusion Steroid withdrawal within 3 months in modern immunosuppressive regimen ① increases the risk of Banff Grade I rejection reaction, but the moderate and severe rejection are similar between the two groups; ② doesn’t affect the rate of graft, patient survival, and chronic rejection; ③ decreases the incidence of opportunistic and urinary tract infection, but doesn’t improve the CMV infection and sepsis. To prophylaxis serious infection, steroid withdrawal is worth considering under sufficient immunosuppressive regimen. The key point is to balance the benefit and harm for individual recipients.
ObjectiveTo explore the effect of icariin on early steroid-induced osteonecrosis of the femoral head in rabbits.MethodsFifty 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.ResultsSeven 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).ConclusionThe 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.
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】 Objective To investigate both incidence and mechanism attributing to steroid-associated osteonecrosisof femoral head(ONFH) using an experimental protocol with a single low-dose l i popolysaccharide (LPS) injection andsubsequently three injections of high-dose methylprednisolone (MPS). Methods Twenty-five New Zealand white rabbits with body weight of (3.0 ± 0.3) kg were divided randomly into 2 groups. In treatment group, 19 rabbits received one intravenous injection of LPS (10 μg/kg); 24 hours later, three injections of 20 mg/kg of MPS were given intramuscularly at an interval of 24 hours. Additional 6 rabbits which received normal sal ine injection at the same time point were used as controls(control group). The blood samples were collected for hematological examinations before and after LPS injection, MRI was performed on bilateral hip six weeks after last MPS injection, meanwhile, bone marrow was aspirated from femoral head region to evaluate stem cell’s activity. Bilateral femoral heads were harvested to make histopathology examination. Results All animals survived throughout the experiment period except one death on the second day after LPS injection. In the histopathological examinationfor the femoral head, ONFH+ was observed in 16 rabbits (88.9%), and the lesions were mainly in the metaphysis. In ONFH+ rabbits, micro vessels fibrous thrombosis and extravascular marrow fat cell size increasing were found around necrotic bone; The femoral heads of control group had no changes. MRI accurate ratio was 93.8% (15/16). Compared to basel ine, a significant decrease in ratio of tissue plasminogen activator/plasminogen activator inhibitor 1 and activated partial thromboplatin time, and a significant increase in ratio of low-density l ipoprotein/high-density l ipoprotein were only found in ONFH+ rabbits (P lt; 0.05). Meanwhile there was a significant decrease in the number of CFU-F (8.50 ± 9.63) compared with the control (70.17 ± 7.78, P lt; 0.05). Conclusion A single low-dose LPS injection and subsequent three injections of high-dose MPS is effective on building steroid-associated ONFH model, coagulation and l ipometabol ism abnormal ity, activity degeneration of stem cell may be the key factors of ONFH.
