This research was designed to investigate the protective effects of TSPN on steroid-induced avascular necrosis of the femoral head (ANFH) and the likely mechanisms of those effects. As an in vivo study, TSPN was shown to be protective against steroid-induced ANFH due to the upregulation of VEGF-A. Furthermore, TSPN attenuated the apoptosis of osteocytes and reduced the expression of Caspase-3 relative to the model group. As an in vitro study, TSPN exerted a concentration-dependent protective effect against apoptosis in MC3T3-E1 cells. Moreover, TSPN (at a dose of 100
Corticosteroid usage is the most common nontraumatic cause of avascular necrosis of the femoral head (ANFH), which can cause interruptions of the blood supply to the bone [
Panax notoginseng is the root of
The aims of the present study were to investigate the protective effect of TSPN on steroid-induced ANFH and to elucidate whether TSPN can protect osteocytes by influencing apoptosis and modulating the expression of Caspase-3 in vitro and in vivo.
Adult male Sprague-Dawley rats (body weight 300–350 g) were raised under controlled temperature (
The rats were randomly divided into the following three groups: a blank control group (
All rats were killed by anaesthesia overdose, and the femoral heads were fixed in paraformaldehyde for 48 h and decalcified with 10% EDTA solution for 2 weeks. Next, the specimens were embedded in paraffin, cut along the coronal plane of the femoral head, and stained with haematoxylin and eosin (H&E).
Total RNA from the femoral head was isolated using Trizol Reagent (Invitrogen) according to the manufacturer’s protocol. The integrity and concentration were quantified using a NanoDrop 1000 spectrophotometer (Thermo Scientific). The cDNA was synthesized from the extracted total RNA (500 ng) by reverse transcription according to the instructions of the kit (Fermentas). Real-time quantitative PCR was performed on a 7500 Real-Time PCR System (ABI). Each 10
The detections of apoptotic cells in the tissues were performed with an Apoptosis Assay Kit (Roche). Briefly, the paraffin slides were dewaxed and rehydrated, proteinase K was added, and the slides were incubated for 30 min at 37°C. Afterwards, the sections were permeabilized for 20 min and incubated with TUNEL reaction mixture for 60 min at 37°C in the dark. Next, the slides were incubated with 0.03% diaminobenzidine (DAB) for 3–5 min and counterstained with haematoxylin. Nuclei exhibiting intense, homogenous dark brown staining were considered to be indicative of TUNEL-positive cells.
Briefly, after deparaffinization and rehydration, the sections were immersed in 3% hydrogen peroxide to block endogenous peroxidase activity followed by incubation with normal rabbit serum for 30 min at 37°C. Next, the primary anti-Caspase-3 and anti-VEGF-A antibodies were added to mark their specific antigens, and the sections were incubated overnight at 4°C. The next morning, the sections were incubated with horseradish peroxidase- (HRP-) labelled secondary antibodies for 50 min at 37°C. The sections were then incubated with 0.03% DAB and counterstained with haematoxylin. The negative control sections underwent identical staining with the exception that the primary antibody was excluded.
MC3T3-E1 cells (subclone 14) were cultured in cell culture dishes containing alpha minimum essential medium (
The MC3T3-E1 cells were seeded onto 96-well culture dishes (5 × 104 cells/well), allowed to attach overnight, and subsequently exposed to dexamethasone (10−6 M) and different concentrations of TSPN (50
The TUNEL assays were performed with a TUNEL kit (Beyotime) to label the 3′-end of the fragmented DNA of the apoptotic cells. Briefly, the cells were exposed to dexamethasone (10−6 M) and TSPN (100
After treatment, the cells were washed thrice with cold PBS and then lysed in a lysis buffer. The samples were centrifuged at 18,000 ×g for 1 h at 4°C. The supernatants were collected as whole cell lysates. The protein concentrations were estimated, and equal amounts of proteins (10
Caspase-3 activity was measured using a colorimetric assay kit (Beyotime) following the manufacturer’s instructions. Briefly, after treatment, the cells were collected and lysed with lysis buffer on ice for 15 min, and the supernatant was then incubated with specific colorimetric peptide substrates (Ac-DEVD-pNA) for 60 min at 37°C. The reaction products were measured at 405 nm with a microplate reader (Eppendorf, Germany). The Caspase-3 activities are expressed as fold increases relative to the baseline observed in the negative controls.
The data are presented as the means ± the standard deviations (S.D.s) and were analysed with one-way ANOVA using SPSS 16.0 (SPSS Inc., Chicago, IL, USA). A
H&E staining indicated that the samples obtained from the blank control group exhibited a clear normal ultrastructure of the femoral head (Figure
Protective effects of TSPN against steroid-induced ANFH in a rat model. (a) H&E staining of femoral heads of each group (
Immunohistochemical analyses of VEGF-A expression revealed that there were rare VEGF-A-positive areas in the model group, while the TSPN group rats exhibited dramatic increases in the numbers of VEGF-A-positive areas in the femoral head (Figure
As shown in Figure
TSPN attenuated apoptosis and reduced Caspase-3 expression in osteocytes in the model of steroid-induced ANFH. (a) TUNEL staining of femoral heads in each group (
The immunohistochemical results provided supporting evidence that TSPN reduced the expression of Caspase-3. As shown in Figure
As shown in Figure
Protective effects of different doses of TSPN in MC3T3-E1 cells. Hoechst 33258 staining of the cells (
The TUNEL staining results (Figure
TSPN reduced Caspase-3 expression and activity in MC3T3-E1 cells. (a) TUNEL staining of the apoptotic cells (×200). The positive cells are indicated with white arrows. (b) Western blot for Caspase-3 protein. (c) Caspase-3 activity levels:
Animal models have been widely used to investigate the pathological mechanisms of steroid-induced ANFH. The rat model used in the present study successfully induced osteonecrosis as indicated by histological examinations, and this finding is consistent with a previous report [
In the present study, following treatment with TSPN for 6 weeks, the development of dexamethasone-induced ANFH was remarkably prevented. The H&E staining observations revealed that the rats in the TSPN group exhibited nearly normal ultrastructures of the femoral head. Furthermore, we found that TSPN raised VEGF-A expression compared to the model group. These results indicate that TSPN has a potential protective effect on ANFH that is similar to its effects on ischemic diseases.
An early report [
In the present study, the TUNEL procedure was applied to the femoral head, and positive labelling was confirmed along with the presence of typical signs of nuclear condensation and shrinkage. These results indicate that there were plentiful apoptotic osteocytes in the model group and that TSPN obviously attenuated osteocyte apoptosis. Our results indicate that TSPN elicited antiapoptotic effects in osteocytes in this ANFH rat model.
Dexamethasone has been widely reported to play a pivotal role in the proliferation and differentiation of osteoblasts [
Although the mechanism underlying the apoptosis that is involved in steroid-induced ANFH remains unclear, it has been found that Caspase-3 might play an important role in the development of this disease. A recent study demonstrated that the apoptotic process in steroid-induced ANFH develops with the upregulation of Caspase-3 [
Taken together, these results show that TSPN has a positive protective effect on steroid-induced ANFH that is mediated by reductions in the rate of apoptosis in osteocytes due to the inhibition of Caspase-3 activation. TSPN might be a useful protective phytomedicine for patients who are in need of corticosteroid treatments and are at risk of developing ANFH. However, further in vitro and in vivo studies should be performed to support the clinical application of TSPN.
The authors declare that there is no conflict of interests regarding the publication of this paper.
Heng-feng Yuan, Jian-feng Pan, and Shuo Li contributed equally to this work.
This research was financially supported by the National Natural Science Foundation of China (no. 81171671) and the Shanghai Natural Science Foundation (no. 12410710200).