Gastrodin prevents steroid-induced osteonecrosis of the femoral head
DOI: 10.3760/cma.j.issn.0366-6999.20141371
Department of Orthopedics, Renmin Hospital of Wuhan University, Wuhan, Hubei 430060, China (Zheng HF, Yang EP, Peng H, Li JP, Chen S, Zhou JL, Fang HS, Qiu B and Wang Z)
Correspondence to: Dr. Peng Hao, Department of Orthopedics, Renmin Hospital of Wuhan University, Wuhan, Hubei 430060, China (Tel/Fax: 86-27-88041911 ext. 82031. Email: penghao8668@https://www.sodocs.net/doc/8916365729.html,)
This work was supported by a grant from the National Natural Science Foundation of China (No. 81301592).
Original article
Gastrodin prevents steroid-induced osteonecrosis of the femoral head in rats by anti-apoptosis
Zheng Huifeng, Yang Erping, Peng Hao, Li Jianping, Chen Sen, Zhou Jianlin, Fang Hongsong, Qiu Bo and Wang Zhe Keywords: glucocorticoids; gastrodin; femoral head osteonecrosis; prevention; apoptosis
Background Gastrodin, as one of the major components extracted from the Chinese herb Gastrodia elata Bl., has many biologic effects, one of which is anti-apoptosis. Apoptosis is considered to be one of the pathogenetic mechanisms in steroid-induced osteonecrosis of the femoral head (ONFH). Therefore, we performed this study to investigate whether gastrodin has the potential to prevent steroid-induced ONFH.
Methods All 18 male adult Wistar rats were divided equally into three groups: the steroid group, the gastrodin+steroid group, and the control group. Osteonecrosis was induced by low-dose lipopolysaccharide and subsequent high-dose methylprednisolone. Histomorphometric method was used to determine the incidence of osteonecrosis. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay was performed to detect apoptotic index of osteocytes and osteoblasts. Real-time PCR and Western blotting were performed to detect mRNA and protein expression of Bax, Bcl-2, and Caspase-3. Fisher’s exact probability test and one-way analysis of variance (ANOVA) with Turkey’s post hoc test were used to examine significant differences between groups.
Results The incidence of osteonecrosis in the gastrodin+steroid group (16.7%) was significantly lower than that in the steroid group (83.3%). According to TUNEL assay, the apoptotic indices in the steroid group, the gastrodin+steroid group, and the control group were 91.1%, 27.1%, and 5.4%, respectively, and the differences were significant between groups. Compared with the control group and the gastrodin+steroid group, the mRNA and protein expression levels of Bax and Caspase-3 were significantly higher in the steroid group, but the Bcl-2 mRNA and protein expression levels were significantly lower.
Conclusion Gastrodin could prevent steroid-induced ONFH by anti-apoptosis.
Chin Med J 2014;127 (22): 3926-3931
G
lucocorticoids have been widely used for many years for the treatment of many diseases; for instance, systemic lupus erythematosus.1 However, glucocorticoids use also could cause many complications, for example, the osteonecrosis of the femoral head (ONFH).2,3 Hong and Du 4 reported that 23.9% of patients (16/67) who had undergone glucocorticoid treatment for severe acute respiratory syndrome suffered from ONFH. Since steroid-induced ONFH tends to occur in relatively young patients and has disabling effect, its prevention becomes very important.So far, the proposed pathogenesis of steroid-induced ONFH includes increased vasoconstriction,5 apoptosis,6 oxidative stress,7 lipid metabolism disturbance,8 and disturbances of the coagulation-fibrinolysis system.9 Although the definite pathogenesis of steroid-induced ONFH has not been clarified, apoptosis has been demonstrated as one pathogeny of steroid-induced ONFH by many researches.10,11
Gastrodin is one of the major and bioactive components extracted from the Chinese herb Gastrodia elata Bl. Many researches have shown that gastrodin has the anti-apoptotic action.12-14 Therefore, we suppose gastrodin may have the potential to prevent the steroid-induced ONFH.
In this work, we studied the ability of gastrodin in preventing the occurrence of steroid-induced ONFH using
an animal model. Moreover, we investigated the effect of gastrodin on steroid-induced osteocyte and osteoblast apoptosis and expression of apoptosis-related genes and proteins.
METHODS
Animals
Eighteen male adult Wistar rats (12 weeks) were obtained from the Hubei Province Center for Disease Control and Prevention. All rats were housed individually in custom-designed Plexiglas cages (55 cm × 35 cm × 26 cm) under standard laboratory conditions (12/12-hour light/dark cycle, 24–25°C, and humidity 50%–55%) and allowed free access to food and water during the study. All experiment protocols adhered to the Guidelines for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication, revised 1996) and approved
by the Ethics Committee for Animal Research, Wuhan University, China.
Grouping and treatment
All rats were divided into three groups by randomized block design according to weight. (1) The gastrodin+steroid g r o u p(n=6):a l l r a t s w e r e g i v e n50m g/k g gastrodin (Kunming Pharma Corp., Kunming, China) intraperitoneally for 28 days. The dose and processing time of gastrodin were determined by preliminary experiment. On days 1 and 2, the animals were given 1.8 mg/kg lipopolysaccharide (LPS, Escherichia coli serotype 055: B5; Sigma, St. Louis, MO, USA) intravenously 2 hours after intraperitoneal injection of gastrodin. On days 3, 4, 5, 6, and 7, the animals were given 25 mg/kg methylprednisolone (Pfizer Pharmaceuticals, China) intramuscularly 2 hours after intraperitoneal injection of gastrodin to promote the development of femoral head necrosis as described by Okazaki et al.15,16 (2) The steroid group (n=6): all rats were given 0.9% saline in the same mode for 28 days. Moreover, LPS and methylprednisolone were given as the gastrodin+steroid group. (3) The control group (n=6): all rats were given 0.9% saline in the same mode at the same time points to replace gastrodin, LPS, and methylprednisolone. The animals were killed 3 weeks after the last methylprednisolone injection. The bilateral femurs were harvested from each rat; the right femur was fixed in 4% paraformaldehyde–0.1 mol/L phosphate buffer (pH 7.4), and the left femur was put in liquid nitrogen immediately and then stored at –80°C. Histopathology
After fixation in 4% paraformaldehyde–0.1 mol/L phosphate buffer (pH 7.4) for 24 hours, the femurs were decalcified with 10% ethylenediaminetetraacetic acid–0.1 mol/L phosphate buffer (pH 7.4) for 5 weeks. After decalcification, the tissues were dehydrated in graded ethanol, embedded in paraffin, cut into 4-μm thick sections along the coronal plane (four sections for each femoral head), and processed for routine hematoxylin and eosin staining for the evaluation of osteonecrosis. All sections were assessed blindly by two independent authors, and the diagnosis of osteonecrosis was established based on the diffuse presence of empty lacunae or pyknotic osteocyte nuclei in the bone trabeculae, with or without surrounding bone marrow cell necrosis. If the diagnoses differed between the two examiners, a consensus was reached by discussing the histologic findings without knowledge of the group from which the sample was obtained. The rats with at least one osteonecrotic lesion in the area examined were considered to have developed osteonecrosis.
TUNEL assay
Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay was used to detect apoptotic cells, with a TUNEL Assay Kit (KeyGEN BioTECH, Nanjing, China), according to the manufacturer’s instructions. Cells with brown nuclei were assessed as positive. The percentage of apoptotic cells (apoptotic index) was evaluated by five randomly selected high-power fields (×200) in each slide from three groups.
Real-time quantitative PCR
Total RNA was extracted from the femoral heads of rats with Trizol reagent according to the manufacturer’s instructions. The concentration of RNA was quantified by measuring the absorbance at 260 nm (A260). The purity of RNA was assessed by determining the A260/A280 ratio. The extracted total RNA (0.84 μg) was reverse transcribed with PrimeScript RT reagent Kit (TaKaRa Company, Dalian, China) on a PCR apparatus (Eastwin Life Sciences Inc., Beijing, China). The cDNA was kept at ?20°C prior to PCR amplification. RT-PCR reactions were performed in 48-well optical PCR plates using SYBR Green/Flourescein qPCR Master Mix (2X) (Fermentas China Co., Ltd, Shenzheng, China) on an Eco Real-Time PCR System (Illumina China, Shanghai, China). A 2?ΔΔCT was used for analyzing the data. Primer sequences are listed in Table 1.
Western blotting
The femoral heads of rats were powdered, followed by homogenization in ice-cold radio immunoprecipitation assay (RIPA) lysis buffer (Beyotime Institute of Biotechnology, China) containing phenylmethylsulfonyl fluoride (Beyotime Institute of Biotechnology). The samples were centrifuged once at 12 000 r/min at 4°C for 5 minutes to remove cell debris, nuclei, and large particulates. The supernatant containing the cytosolic protein fraction was then collected. A quarter volume of 5 × loading buffer was added and boiled at 100°C for 10 minutes, then stored at –20°C until electrophoresis. Proteins were separated by 12% sodium dodecyl sulfate polyacrylamide gel and transferred to polyvinylidene difluoride membranes (Millipore Corporation, Bedford, MA, USA). After being blocked with 5% defatted milk powder for 2 hours, the membrane was incubated at 4°C overnight with rabbit anti-Bax antibody (1:500, Bioworld, Nanjing, China), rabbit anti-Bcl-2 antibody (1:500, Bioworld), rabbit anti-Caspase-3 antibody (1:1 000, Proteintech Group Inc., Wuhan, China), and mice anti-β-actin antibody (Wuhan Boster Biological Technology Ltd, Wuhan, China) as primary antibodies, respectively, followed by exposure to horseradish peroxidase-conjugated secondary antibodies (1:50 000, Wuhan Boster Biological Technology Ltd) at 25°C for 2 hours. The proteins on the membrane were visualized using SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific Inc., MA, USA), exposed to Kodak X-ray film, then photographed by the Geliance 200 Imaging System. The optical density
Table 1. Real-time quantitative PCR primer sequences Genes Forward Reverse
β-Actin5′-CACGATGGAGGGGC
CGGACTCATC-3′
5′-TAAAGACCTCTATGC
CAACACAGT-3′
Bax5′-GGCGATGAACTGGA
CAACAA-3′
5′-CAAAGTAGAAAAGGGCAACC-3′Bcl-25′-GGTGAACTGGGGGA
GGATTG-3′
5′-GCATGCTGGGGCCATATAGT-3′Caspase-35′-GGACCTGTGGACCT
GAAAAA-3′
5′-GCATGCCATATCATCGTCAG-3′
of the bands was analyzed using Bandscan software. The expression of Bax, Bcl-2, and Caspase-3 were then normalized against β-actin.
Statistical analysis
Categorical data, that is, incidence of osteonecrosis, were analyzed using Fisher’s exact probability test. Numerical data in each group were expressed as mean ± standard deviation (SD). Statistical analysis was performed using SPSS 19.0 software (SPSS Inc., Chicago, IL, USA). One-way analysis of variance (ANOV A) with Turkey’s post hoc test was used to examine significant differences between groups. Statistical significance was set at P <0.05.
RESULTS
Incidence of osteonecrosis
All rats survived during the experimental period. Figure 1 shows the histopathological appearance of the femoral head in the three groups after hematoxylin and eosin staining at week 3 after the last methylprednisolone injection. The incidence of osteonecrosis was 83.3% (5/6) in the steroid
group and 16.7% (1/6) in the gastrodin+steroid group, while no osteonecrosis was observed in the control group (0). The incidence of osteonecrosis in the gastrodin+steroid group was significantly lower than that in the steroid group (P <0.05).
Apoptosis in the femoral head
As shown in Figure 2A, very few TUNEL-positive cells were observed in the control group, whereas a large number of TUNEL-positive cells were found in the steroid group, including osteocytes, osteoblasts, and bone marrow cells; compared with the steroid group, the number of TUNEL-positive cells was significantly lower in the gastrodin+steroid group. The apoptotic indices in the steroid group, the gastrodin+steroid group, and the control group were 91.1%, 27.1%, and 5.4%, respectively, and the differences were significant between groups (Figure 2B).mRNA expression of Bax, Bcl-2, and Caspase-3
Compared with the control group and the gastrodin+steroid group, the mRNA expression levels of Bax and Caspase-3
were significantly higher in the steroid group, but the Bcl-2
Figure 1. Histological appearance of the femoral head in the three groups. A: The control group, B : the gastrodin+steroid group, and C: the steroid group. The arrows represent empty osteocytic lacunae in the bone trabeculae. It was obvious that there were more empty osteocytic
lacunae in the steroid group (HE staining, original magnification ×200).
Figure 2. Comparison of apoptotic cells. A: Photomicrograph of TUNEL reaction demonstrating the presence of apoptotic cells in the femoral head (Original magnification ×200). Cells with brown nuclei are TUNEL-positive cells (black arrows). It was obvious that there were more TUNEL-positive cells in the steroid group. B: Apoptotic index in the three groups. The apoptotic index in the steroid group was higher than those in the control group and the gastrodin+steroid group. Data values are expressed as mean±SD. *P <0.05 vs. the gastrodin+steroid group. ?P <0.05, ?P <0.05 vs. the control group.
mRNA expression level was significantly lower. Moreover, compared with the control group, the mRNA expression levels of Bax and Caspase-3 in the gastrodin+steroid group were significantly higher, but the Bcl-2 mRNA expression level was significantly lower (Figure 3A). The Bax/Bcl-2 mRNA ratio in the steroid group was significantly higher than those in the control group and the gastrodin+steroid group. Meanwhile, compared with the control group, the Bax/Bcl-2 mRNA ratio in the gastrodin+steroid group was significantly higher (Figure 3B).
Protein expression of Bax, Bcl-2, and Caspase-3
As shown in Figure 4, the protein expression of Bax, Bcl-2, and Caspase-3 had the similar trend to mRNA expression of them in the three groups (Figure 4A and 4B), and the change in Bax/Bcl-2 protein ratio in the three groups was also similar to that of Bax/Bcl-2 mRNA ratio (Figure 4C).
DISCUSSION
Although the exact pathomechanism of the steroid-induced ONFH remains uncertain, apoptosis has been demonstrated as one reason by many researches. Calder et al 10 found evidence of bone cell apoptosis in sections of the femoral head removed from patients with steroid-induced ONFH. They thought glucocorticoids have a direct cytotoxic effect on cancellous bone of the femoral head leading to apoptosis rather than purely necrosis. The animal experiment conducted by Weinstein et al 11 also demonstrated glucocorticoids could promote apoptosis of osteoblasts and osteocytes. They thought accumulation of apoptotic osteocytes may contribute to osteonecrosis. Obviously, drugs that could inhibit apoptosis of osteocytes
may have the ability to prevent the occurrence of steroid-induced ONFH.
As one main active component extracted from the Chinese herb Gastrodia elata Bl., gastrodin has been extensively used in China by Chinese doctors for the treatment of many diseases; for instance, hypertension.17 The previous researches on gastrodin have found that gastrodin has an anti-apoptotic action. Kumar et al 12 reported that gastrodin could prevent neuronal apoptosis in a mouse model of Parkinson’s disease induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Therefore, we suppose that gastrodin could prevent the occurrence of steroid-induced ONFH.In the present study, the steroid-induced ONFH model was successfully established in rats. According to the histopathological results, we found obvious osteonecrosis in the epiphysis of the femoral head characterized by extensive empty osteocytic lacunae in the bone trabeculae in the steroid group (Figure 2C). However, we did not find obvious marrow necrosis, which is probably because it was still in the early lesion. Spencer et al 18 reported that the initial lesion may occur in the bone itself rather than in the marrow space, probably involving a direct cytotoxic
Figure 3. Comparison of mRNA expression. A: mRNA expression of Bax, Bcl-2, and Caspase-3 in the femoral head in the three groups. B: Bax/Bcl-2 mRNA ratio in the three groups. Data values are expressed as mean ± SD. *P <0.05 vs. the gastrodin+steroid group. ?P <0.05, ?P
<0.05 vs. the control group.
Figure 4. Comparison of protein expression. A: Western blotting bands of Bax, Bcl-2, and Caspase-3 in the three groups. B: Densitometric analysis of Western blotting bands. C: Bax/Bcl-2 protein ratio in the three groups. Data values are expressed as mean ± SD. *P <0.05 vs. the gastrodin+steroid group. ?P <0.05, ?
P <0.05 vs. the control group.
effect on the osteocytes. The incidence of osteonecrosis in the gastrodin+steroid group (16.7%, 1/6) was significantly lower than that in the steroid group (83.3%, 5/6). Obviously, gastrodin could significantly prevent the occurrence of steroid-induced ONFH.
In addition, the TUNEL assay in this work found that the number of apoptotic cells in the gastrodin+steroid group was significantly lower than that in the steroid group, which demonstrates that the underlying mechanism of gastrodin against osteonecrosis is anti-apoptosis.
In order to explore the further mechanism, we investigated the effect of gastrodin on steroid-induced change of mRNA and protein expression of Bax, Bcl-2, and Caspase-3 in the femoral head by real-time PCR and Western blotting, respectively.
Bcl-2 and Bax all belong to the Bcl-2 family which controls a critical step in commitment to apoptosis by regulating permeabilization of the mitochondrial outer membrane (MOM).19 Briefly speaking, Bcl-2 is an antiapoptotic protein; however, Bax is a proapoptotic protein which can lead to release of components of the intermembrane space, for example, cytochrome c that can activate the final effector of apoptosis. Bcl-2 can bind selectively to the active conformation of Bax to prevent it from inserting into the MOM and prevent the release of mitochondrial proapoptotic factors. Usually, the Bax/Bcl-2 ratio was used to evaluate the apoptotic potential of cells.20 In this study, compared with the control group and the gastrodin+steroid group, the Bax mRNA and protein expression levels were significantly higher in the steriod group, but the Bcl-2 mRNA and protein expression levels were significantly lower. Accordingly, the Bax/Bcl-2 mRNA and protein ratios in the steroid group were significantly higher than those in the control group and the gastrodin+steroid group. This result shows that glucocorticoids could induce osteocyte apoptosis by elevating Bax/Bcl-2 ratio; however, gastrodin could reduce the proapoptotic activity of glucocorticoids by inhibiting elevation of Bax/Bcl-2 ratio, although it cannot reduce the Bax/Bcl-2 ratio to that level in the control group. Caspase-3 is another important apoptosis-related protein, which belongs to the caspase family that plays vital roles in the induction, transduction, and amplification of intracellular apoptotic signals.21,22 Caspase-3 is an apoptosis executioner, which can subsequently cleave distinct cellular proteins such as poly(ADP-ribose)polymerase, lamin, fodrin, and caspase-activated deoxyribonuclease, leading to condensation of chromatins, decomposition of the nuclear membrane, DNA degradation, and apoptotic body formation. In the present study, the Caspase-3 mRNA and protein expression levels were markedly higher in the steroid group than those in the control group and the gastrodin+steroid group. This implies that osteocyte apoptosis induced by glucocorticoids is executed by Caspase-3, and inhibition of Caspase-3 expression may be another mechanism of gastrodin against osteocyte apoptosis.In conclusion, the present work demonstrates that gastrodin can prevent steroid-induced ONFH in rats by anti-apoptosis, and the underlying mechanisms involve reducing Bax/Bcl-2 mRNA and protein ratios and inhibiting Caspase-3 mRNA and protein expression. Moreover, our results provide an experimental basis for clinical application of gastrodin in the prevention of steroid-induced ONFH. However, further studies are required to elucidate more detailed mechanism of gastrodin in preventing steroid-induced ONFH, which will potently support gastrodin as a potential drug for the prevention of steroid-induced ONFH.
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(Received June 12, 2014)
Edited by Ji Yuanyuan