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The Kaohsiung Journal of Medical Sciences logoLink to The Kaohsiung Journal of Medical Sciences
. 2021 Aug 2;37(12):1089–1100. doi: 10.1002/kjm2.12426

Astragalus polysaccharide ameliorates steroid‐induced osteonecrosis of femoral head through miR‐206/HIF‐1α/BNIP3 axis

Shen‐Yao Zhang 1, Fan Wang 1, Xiang‐Jing Zeng 1, Zhen Huang 1, Ke‐Fang Dong 1,✉
PMCID: PMC11896451  PMID: 34338434

Abstract

Declining autophagy and rising apoptosis are the main factors driving the development of steroid‐induced osteonecrosis of the femoral head (SONFH). Here, we showed that astragalus polysaccharide (APS) improved femoral head necrosis via regulation of cell autophagy and apoptosis through microRNA (miR)‐206/hypoxia inducible factor‐1 (HIF‐1α)/BCL2 interacting protein 3 (BNIP3) axis. The expression of miR‐206, HIF‐1α, and BNIP3 in SONFH specimens and cell model were measured using qPCR. SONFH cell model was treated with APS. Cell autophagy was evaluated using LC3‐immunofluorescence assays. Flow cytometry was conducted to assess cell apoptosis. Apoptosis‐related proteins and autophagy‐related proteins were determined using western blot. Besides, dual‐luciferase reporter assay was employed to investigate the relationship between miR‐206 and HIF‐1α. Here we showed that miR‐206 expression was upregulated in SONFH tissues and cell model. APS promoted autophagy and inhibited apoptosis in SONFH cell model via downregulating miR‐206. What is more, HIF‐1α was the target of miR‐206. Knockdown of HIF‐1α reversed the recovery effect of miR‐206 inhibitor on SONFH cell model. Furthermore, BNIP3 was the target of HIF‐1α. HIF‐1α overexpression promoted autophagy and inhibited apoptosis, and knockdown of BNIP3 abolished the recovery effect of HIF‐1α overexpression in SONFH cell model. These results provided evidence that APS reduced miR‐206 expression, and the downregulated miR‐206 increased BNIP3 expression by targeting HIF‐1α to promote autophagy and inhibit bone cell apoptosis. Our research proved that APS effectively improved SONFH by regulating cell autophagy and apoptosis.

Keywords: astragalus polysaccharide, BNIP3, HIF‐1α, miR‐206, steroid‐induced osteonecrosis of the femoral head

Abbreviations

APS

astragalus polysaccharide

SONFH

steroid‐induced osteonecrosis of the femoral head

HIF‐1α

hypoxia inducible factor‐1α

BNIP3

Bcl2 interacting protein 3

miRNA

microRNA

UTR

untranslated region

ONFH

osteonecrosis of the femoral head

IF

immunofluorescence

ANOVA

analysis of variance

qRT‐PCR

quantitative real‐time polymerase chain reaction

SDS‐PAGE

sodium dodecyl sulfate‐polyacrylamide gel electrophoresis

WB

western blot

1. INTRODUCTION

Osteonecrosis of the femoral head (ONFH) is a progressive orthopedic disease that eventually leads to the collapse of femoral head and osteoarthritis. 1 Steroid‐induced osteonecrosis of the femoral head (SONFH) is a common type of ONFH. 2 , 3 In China, about 5,000,000–7,500,000 people suffer from femoral head necrosis, and the rate grows by 100,000–200,000 people every year. 4 However, the femoral head preservation treatment for SONFH is not ideal. 1 Therefore, exploring the mechanism of SONFH is of great significance for the effective clinical treatment of SONFH. Previous study showed that excessive apoptosis of bone cells was the main cause of SONFH. 5 Besides, Luo et al indicated that autophagy participated in the pathological process of SONFH and was closely related to apoptosis. 6 However, there are few studies about autophagy and apoptosis in SONFH. Here, we aimed to explore the regulation mechanism of bone cell apoptosis and autophagy in SONFH.

MicroRNAs (miRNAs) are endogenous noncoding RNAs with a length of 20–24 nt. MiRNAs were reported to be closely related to the formation of osteoblasts and osteoclasts. 7 MiRNA(miR)‐206 was also reported to be upregulated in the SONFH animal model. 8 Besides, previous study displayed that miR‐206 could suppress angiogenesis in colorectal cancer via targeting HIF‐1α. 9 HIF‐1α was reported to be downregulated in the SONFH animal model. 2 Moreover, HIF‐1α was proved to be an activator of autophagy: hypoxic preconditioning could upregulate autophagy of SH‐SY5Y cells by increasing the expression of HIF‐1α. 10 What is more, downregulated HIF‐1α promoted excessive apoptosis of osteocytes in SONFH and excessive apoptosis might related to the reduction of autophagy. 2 BNIP3 was reported to act as the target molecule of HIF‐1α. 11 Zhang et al indicated that HIF‐1α activated BNIP3 and subsequently induced autophagy, as well as inhibiting apoptosis during myocardial ischemia–reperfusion injury.

APS is the main active ingredient of the traditional Chinese medicine astragalus, which has a variety of biological activities. 12 Previous study showed that APS could promote osteogenic differentiation of bone marrow mesenchymal stem cells. 12 Besides, APS had a good antiapoptotic effect: it could inhibit the apoptosis of bone marrow mesenchymal stem cells caused by ferric ammonium citrate treatment. 13 Furthermore, APS could regulate apoptosis by regulating the expression of miRNAs in many diseases. 14

Based on the research described above, this study hypothesized that APS downregulated the expression of miR‐206 in SONFH, and low expression of miR‐206 promoted autophagy and inhibited apoptosis of bone cells by targeting HIF‐1α to increase the expression of BNIP3, thereby improving the symptoms of SONFH. Our study proposed a new insight for the pathological mechanisms of SONFH.

2. MATERIALS AND METHODS

2.1. Collection of clinical samples

Ten patients diagnosed with SONFH and 10 patients with femoral neck fracture were recruited from The Second Affiliated Hospital of Hunan University of Chinese Medicine. All participants were between 40 and 60 years of age. All SONFH patients undergone X‐ray diagnosis and had a clear history of steroid application. The exclusion criterion is that patients had severe chronic diseases, such as congenital diseases, cardiovascular diseases, diabetes, and so on. Bone marrow tissue of the proximal femur was obtained from 10 patients with SONFH and 10 patients with femoral neck fractures as the controls. During total hip arthroplasty (THA) surgery, tissues were stored at −80°C for further research. All volunteers signed the informed consents before surgery, and the protocols of this study were approved by The Second Affiliated Hospital of Hunan University of Chinese Medicine. The IRB approval number is 2020‐KY‐114.

2.2. Cell culture and construction of SONFH cell model

Murine long bone osteocyte‐Y4 (MLO‐Y4) cell line was obtained from American type culture collection (ATCC, VA). MLO‐Y4 cells were cultured in type‐I collagen (0.15 mg/ml in 0.02 M acetic acid and PBS)‐coated cell culture flask. All cells were cultured in complete growth medium (Bioind, Haemek, Israel) mixed with a‐MEM, 5% fatal bovine serum (FBS) (Gibco, MD), and 1% penicillin–streptomycin solution (Sangon, Shanghai, China). All cells were cultured at 37°C, in a humidified atmosphere with 5% CO2. MLO‐Y4 cells were treated with 10−5 M dexamethasone for 72 h to construct SONFH cell model. 15 APS was purchased from YuanYe Biotechnology Co., Ltd (Shanghai, China), with a purity of 98%, and was formulated with saline to a concentration of 10 mg/ml. For APS treatment, cells were treated with 10 mg/ml APS for 24 h.

2.3. Plasmid constructs and transfection

The HIF‐1α overexpression plasmid (oe‐HIF‐1α), the short hairpin RNA against BNIP3 (sh‐BNIP3), the short hairpin RNA against HIF‐1α (sh‐HIF‐1α), and inhibitor or mimics of miR‐206 as well as their negative controls (oe‐NC, sh‐NC, inhibitor NC, and mimics NC) were purchased from GenePharma (Shanghai, China). Cells were transfected with oe‐HIF‐1α or sh‐HIF‐1α or sh‐BNIP3 or miR‐206 mimics or miR‐206 inhibitor and their negative controls using Lipofectamine™ 3000 (Invitrogen, CA) for 24 h according to the manufacturer's instructions.

2.4. Dual‐luciferase reporter assay

We predicted the target of gene binding using a common online tool, TargetScan (http://www.targetscan.org/). The 3′‐UTR sequence of HIF‐1α, together with mutated sequence within the predicted target sites, was synthesized and inserted into the pmiR‐GLO dual‐luciferase miRNA target expression vector (Promega, WI). Cells were plated onto the 96‐well plates at a density of 5 × 103 cells/well and co‐transfected with wild‐type (WT) or mutant constructs (MUT) and miR‐206 mimics or NC mimics. After a 48 h period of transfection, luciferase activity was measured with a dual‐luciferase reporter assay system (Promega, WI). Firefly luciferase activity was normalized against Renilla luciferase activity.

2.5. Flow cytometry

After transfection, cells were stained with FITC‐Annexin V and PI using the Annexin V‐FITC apoptosis detection kit (Beyotime, Shanghai, China) according to the manufacturer's protocol. The stained cells were analyzed using the FAC Scan flow cytometry (Beckman, CA).

2.6. Immunofluorescence

MLO‐Y4 cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X‐100, and blocked with 1% bovine serum albumin. Next, cells were incubated with primary antibody against LC3 (1:500, Abcam, UK) at 4°C overnight. Finally, cells were washed with PBS and then incubated with fluorescent Cy3‐conjugated secondary goat anti‐rabbit antibodies (dilution 1:100; Abcam, UK) for 1 h at room temperature. Cells were counter‐stained with 4′,6‐diamidino‐2‐phenylindole (DAPI) and fluorescence images were viewed using a FV 1000 Olympus IX‐81 (Olympus Corporation, Tokyo, Japan). The images were analyzed using Image‐Pro Plus 6.0 software (Media Cybernetics).

2.7. Quantitative real‐time polymerase chain reaction

Total RNA was extracted by using TRIzol reagent (Invitrogen, CA), and cDNA was synthesized using HiFiScript cDNA synthesis kit (Life Technologies, CA). The cDNA was used for quantitative real‐time polymerase chain reaction (qRT‐PCR) with specific primers for different target genes. PCR reactions were conducted on an Eppendorf MasterCycler RealPlex4 (Eppendorf, Wesseling‐Berzdorf, Germany) using an Ultra SYBR Mixture kit (Thermo Fisher Scientific, MA). PCR thermal cycling conditions were 95°C for 3 min, followed by 40 cycles at 95°C for 15 s and 60°C for 30 s. Relative expression levels were calculated by the 2−ΔΔCt method. GAPDH was used to normalize target genes, and U6 was used to normalize miRNA expression levels. The primers used in the reaction were provided by GenePharma (Shanghai, China). The primers used in the study were listed as follows:

miR‐206 F: ACAACAAGGACCGGTTGCAGA.

miR‐206 R: GGGCATACATCGGCTAATACA.

HIF‐1α F: AGGAGCCTGATGCTCTCACT.

HIF‐1α R: TGGGACTGTTAGGCTGGGAAA.

BNIP3 F: TCCTGGGTAGAACTGCACTTC.

BNIP3 R: GCTGGGCATCCAACAGTATTT.

GAPDH F: AGGTCGGTGTGAACGGATTTG.

GAPDH R: TGTAGACCATGTAGTTGAGGTCA.

U6 F: CGACAAGACGATCCGGGTAAA.

U6 R: GGTTGAGGAGTGGGTCGAAG.

2.8. Western blotting

The proteins were isolated from cells by using RIPA mixed with 1% protease inhibitor and phosphorylase inhibitor, and the protein concentrations were detected by BCA™ Protein Assay Kit (Beyotime, Shanghai, China). The lysate was mixed with 5 × SDS sample buffer and boiled for 10 min. Lysate samples were separated on sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), and transferred to polyvinylidene fluoride (PVDF) membrane (Millipore, MA). The blots were blocked with 5% milk blocking solution for 1 h at room temperature and then incubated overnight with primary antibodies purchased from Abcam, including HIF‐1α (Abcam, 1:1000, ab179483), BNIP3 (Abcam, 1:1000, ab109362), LC3 (Abcam, 1:1000, ab63817), p62 (Abcam, 1:1000, ab56416), Beclin1 (Abcam, 1:1000, ab210498), Bax (Abcam, 1:1000, ab182733), C‐caspase‐3 (Abcam, 1:1000, ab2302) and C‐caspase‐9 (Abcam, 1:1000, ab2324), and GAPDH (Abcam, 1:1000, ab8245). Anti‐GADPH antibody was served as a loading control. After washed with Tris‐buffered saline with Tween (TBST), membranes were further incubated with an HRP‐labeled secondary antibody (Abcam, 1:10,000, ab7090, ab97035) for 1 h at room temperature. The blots were visualized by ECL WB Detection Reagents (Beyotime, Shanghai, China) and the images were performed by GEL imaging system (Bio‐Rad, CA). The quantification of proteins was analyzed by the software Image J.

2.9. Data analysis

All experiments were repeated at least three times. Data were expressed as mean ± SD. Statistical analysis was performed using GraphPad Prism 6 software. Differences were analyzed by Student’s t‐tests between two groups or one‐way analysis of variance (ANOVA) for more than two groups. The P values less than 0.05 were considered significant.

3. RESULTS

3.1. Differential expression of miR‐206, HIF‐1α, and BNIP3 in SONFH

Firstly, we detected the expression of miR‐206, HIF‐1α, and BNIP3 in the bone tissues of SONFH patients and in the SONFH cell model established by dexamethasone. The qRT‐PCR showed that miR‐206 in SONFH bone tissues was markedly elevated compared with normal bone tissue, while HIF‐1α and BNIP3 were evidently reduced (Figure 1(A‐C)). What is more, Pearson correlation analysis showed that miR‐206 expression was negatively correlated to HIF‐1α expression in SONFH bone tissues (Figure 1(D)). And correlation analysis displayed a significant positive correlation between HIF‐1α expression and BNIP3 expression in SONFH bone tissues (Figure 1(E)). We observed similar trends of miR‐206, HIF‐1α, and BNIP3 expression in SONFH cell model compared with normal MLO‐Y4 cells (Figure 1(F‐H)). The above results indicated that miR‐206, HIF‐1α, and BNIP3 regulated the progression of SONFH.

FIGURE 1.

FIGURE 1

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Differential expression of miR‐206, HIF‐1α, and BNIP3 in SONFH. (A‐C) MiR‐206, HIF‐1α, and BNIP3 expression in SONFH bone tissues and normal bone tissues were determined by qRT‐PCR. (D) Correlation analysis of miR‐206 expression and HIF‐1α expression in necrotic SONFH tissues. (E) Correlation analysis of HIF‐1α expression and BNIP3 expression in necrotic SONFH tissues. (F‐H) MLO‐Y4 cells were treated with 10−5 M dexamethasone for 72 h to construct SONFH cell model. MiR‐206, HIF‐1α, and BNIP3 expressions in MLO‐Y4 cells were detected by qRT‐PCR. *P < 0.05, **P < 0.01, ***P < 0.001

3.2. APS promoted autophagy and inhibited apoptosis in SONFH cell model

Further research was performed to explore the regulatory effect of APS on autophagy and apoptosis in SONFH cell model. Firstly, the results showed miR‐206 expression in SONFH cell model was significantly downregulated after treatment of APS (Figure 2(A)). IF showed that the fluorescence intensity of autophagy marker LC3 was significantly upregulated in SONFH cell model after treatment of APS, representing a significant increase in the number of cells undergoing autophagy (Figure 2(B)). Western blot analysis showed that LC3II/LC3I ratio and Beclin1 were higher while p62 was lower in SONFH cell model after treatment of APS, confirming the promotion of autophagy (Figure 2(C)). Cell apoptosis rate of SONFH cell model was decreased after treatment of APS, as detected by flow cytometry (Figure 2(D)). The apoptosis‐related protein markers—Bax, C‐caspase‐3 and C‐caspase‐9—were significantly reduced and Bcl‐2 was increased in SONFH cell model after treatment of APS, also showing the inhibition of cell apoptosis (Figure 2(E)). Overall, these results suggested that APS promoted autophagy and inhibited apoptosis in SONFH cell model.

FIGURE 2.

FIGURE 2

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APS promoted autophagy and inhibited apoptosis in SONFH cell model. (A) MiR‐206 expressions in MLO‐Y4 cells were assessed using qRT‐PCR. (B) LC3 ‐immunofluorescence assays in MLO‐Y4 cells, the number of cells undergoing autophagy were quantitatively counted. (C) Protein levels of LC3II/LC3I, Beclin1, and p62 in MLO‐Y4 cells were determined by Western blot. (D) Flow cytometry was performed to evaluate cell apoptosis of MLO‐Y4 cells, the number of apoptotic cells were quantitatively counted. (E) Protein levels of Bcl‐2, Bax, C‐caspase‐3, and C‐caspase‐9 in MLO‐Y4 cells were determined using Western blot. *P < 0.05, **P < 0.01, *** P < 0.001

3.3. APS promoted autophagy and inhibited apoptosis in SONFH cell model by inhibiting the expression of miR‐206

To investigate whether APS promoted autophagy and inhibited apoptosis in SONFH cell model by regulating miR‐206, overexpression of miR‐206 was performed. The qRT‐PCR result displayed miR‐206 in SONFH cell model was significantly decreased after treatment of APS, while it was enhanced after miR‐206 overexpression (Figure 3(A)). IF subsequently displayed that cell autophagy of SONFH cell model was increased by treatment of APS, which was abolished by miR‐206 mimics (Figure 3(B)). The LC3II/LC3I ratio and Beclin1 were upregulated in SONFH cell model after treatment of APS, and p62 was downregulated, while miR‐206 mimics eliminated the effect of APS (Figure 3(C)). Flow cytometry demonstrated cell apoptosis of SONFH cell model was significantly decreased following the treatment of APS, which was eliminated by miR‐206 overexpression (Figure 3(D)). Moreover, the protein levels of Bax, C‐caspase‐3, and C‐caspase‐9 were downregulated in SONFH cell model after treatment of APS, and the protein level of Bcl‐2 was upregulated, while miR‐206 mimics reversed the effect of APS (Figure 3(E)). In summary, APS promoted autophagy and inhibited apoptosis in SONFH cell model by inhibiting the expression of miR‐206.

FIGURE 3.

FIGURE 3

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APS promoted autophagy and inhibited apoptosis in SONFH cell model by inhibiting the expression of miR‐206. (A) Expression of miR‐206 in SONFH cell model after treated with miR‐206 mimics and APS was determined using qRT‐PCR. (B) Expression of LC3 in SONFH cell model after treated with miR‐206 mimics and APS was assessed using IF, and the number of autophagic cells were quantitatively counted. (C) Western blot was performed to assess the protein levels of LC3II/LC3I, Beclin1, and p62 in SONFH cell model after treated with miR‐206 mimics and APS. (D) Cell apoptosis of SONFH cell model after treated with miR‐206 mimics and APS was determined using flow cytometry, and the numbers of apoptotic cells were quantitatively counted. (E) Protein levels of Bcl‐2, Bax, C‐caspase‐3, and C‐caspase‐9 in SONFH cell model after treated with miR‐206 mimics and APS were detected using Western blot. *P < 0.05, **P < 0.01, ***P < 0.001

3.4. Knockdown of miR‐206 targeted HIF‐1α to promote autophagy and inhibit apoptosis in SONFH cell model

The binding site of miR‐206 to HIF‐1α was predicted by software TargetScan (http://www.targetscan.org/vert_72/) (Figure 4(A)). Dual‐luciferase reporter assay subsequently demonstrated that luciferase activities of HIF‐1α‐WT group cells were significantly downregulated after treated with miR‐206 mimics, while the luciferase activities of HIF‐1α‐MUT group remained unchanged, validating HIF‐1α was the target of miR‐206 (Figure 4(B)). The qRT‐PCR and western blot results demonstrated that HIF‐1α was significantly downregulated after overexpression of miR‐206, while HIF‐1α expression was significantly elevated after silencing miR‐206 (Figure 4(C,D)). Then, we aim to investigate whether miR‐206 targeted HIF‐1α to promote autophagy and inhibited apoptosis in SONFH cell model. The HIF‐1α mRNA expression was significantly upregulated after knockdown of miR‐206, while the alteration was significantly inhibited after inhibition of HIF‐1α (Figure 4(E)). What is more, cell autophagy of SONFH cell model was increased by miR‐206 silence, which was abolished by sh‐HIF‐1α transfection (Figure 5(A)). The LC3II/LC3I ratio and Beclin1 were upregulated in SONFH cell model after knockdown of miR‐206, and the protein level of p62 was downregulated, while transfection with sh‐HIF‐1α reversed the effect of miR‐206 inhibitor (Figure 5(B)). Flow cytometry subsequently showed that cell apoptosis of SONFH cell model was diminished by miR‐206 inhibitor treatment, while it was enhanced by HIF‐1α knockdown (Figure 5(C)). Co‐transfection with miR‐206 inhibitor and sh‐HIF‐1α resulted in the increased protein levels of Bax, C‐caspase‐3, and C‐caspase‐9 and the decreased protein level of Bcl‐2 in SONFH cell model compared with the miR‐206 inhibitor group (Figure 5(D)). Taken together, these results showed that downregulated miR‐206 promoted autophagy and inhibit apoptosis by targeting HIF‐1α in SONFH cell model.

FIGURE 4.

FIGURE 4

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Silence of miR‐206 targeted HIF‐1α to promote autophagy and inhibit apoptosis in SONFH cell model. (A) Software prediction of the base sequence of the binding site of miR‐206 and HIF‐1α. (B) Detection of luciferase activity using dual‐luciferase reporter assay. (C) Expressions of miR‐206 and HIF‐1α in MLO‐Y4 cells after treated with miR‐206 mimics and miR‐206 inhibitor were assessed using qRT‐PCR. (D) Protein levels of HIF‐1α in MLO‐Y4 cells after treated with miR‐206 mimics and miR‐206 inhibitor were evaluated by western blot. (E) MiR‐206 and HIF‐1α expression in SONFH cell model after treated with sh‐HIF‐1α and miR‐206 inhibitor were evaluated using qRT‐PCR. *P < 0.05, **P < 0.01, ***P < 0.001

FIGURE 5.

FIGURE 5

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Silence of miR‐206 targeted HIF‐1α to promote autophagy and inhibit apoptosis in SONFH cell model. (A) Expression of LC3 in SONFH cell model after treated with sh‐HIF‐1α and miR‐206 inhibitor were evaluated using IF, and the number of cells undergoing autophagy were quantitatively counted. (B) Protein levels of LC3II/LC3I, Beclin1, and p62 in SONFH cell model after treated with sh‐HIF‐1α and miR‐206 inhibitor were assessed by Western blot. (C) Cell apoptosis of SONFH cell model after treated with sh‐HIF‐1α and miR‐206 inhibitor was evaluated using flow cytometry, and the numbers of apoptotic cells were quantitatively counted. (D) Western blot was performed to assess protein levels of Bcl‐2, Bax, C‐caspase‐3, and C‐caspase‐9 in SONFH cell model following treatment of sh‐HIF‐1α and miR‐206 inhibitor. *P < 0.05, **P < 0.01, ***P < 0.001

3.5. Overexpression of HIF‐1α mediated BNIP3 to promote autophagy and inhibit apoptosis in SONFH cell model

Then, we wanted to explore the downstream target of HIF‐1α in regulating autophagy and apoptosis in SONFH cell model. BNIP3 was reported to be able to regulate cell autophagy in response to hypoxia. 16 In the present study, we found that HIF‐1α overexpression markedly upregulated the expression of BNIP3 evaluated by both qRT‐PCR and western blot, while BNIP3 expression was significantly decreased following silence of HIF‐1α (Figure 6(A,B)). For further examining the effects of BNIP3 on HIF‐1α mediated biological functions in vitro, we both knocked down BNIP3 and overexpressed HIF‐1α in SONFH cell model. BNIP3 mRNA expression was significantly upregulated after overexpression of HIF‐1α, while knockdown of BNIP3 reversed the effect of oe‐HIF‐1α (Figure 6(C)). IF subsequently displayed that cell autophagy was increased in SONFH cell model after treatment of oe‐HIF‐1α, which was abolished by sh‐BNIP3 (Figure 6(D)). What is more, the protein levels of LC3II/LC3I and Beclin1 were elevated in SONFH cell model after transfection with oe‐HIF‐1α, and the protein level of p62 was declined, while inhibition of BNIP3 reversed the effect of oe‐HIF‐1α (Figure 6(E)). Furthermore, the results of flow cytometry demonstrated that cell apoptosis of SONFH cell model was obviously downregulated following oe‐HIF‐1α transfection, while was abolished by sh‐BNIP3 (Figure 6(F)). The decreased protein levels of Bax, C‐caspase‐3, and C‐caspase‐9 and the increased protein level of Bcl‐2 in SONFH cell model resulted from transfection of oe‐HIF‐1α were abolished by the silence of BNIP3 (Figure 6(G)). In summary, overexpression of HIF‐1α could promote autophagy and inhibit apoptosis by mediating BNIP3 in SONFH cell model.

FIGURE 6.

FIGURE 6

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Overexpression of HIF‐1α mediated BNIP3 to promote autophagy and inhibit apoptosis in SONFH cell model. (A) The mRNA levels of BNIP3 and HIF‐1α in MLO‐Y4 cells following treatment with OE‐HIF‐1α and sh‐HIF‐1α were determined using qRT‐PCR. (B) Western blot was conducted to evaluate protein levels of BNIP3 and HIF‐1α in MLO‐Y4 cells after treated with OE‐HIF‐1α and sh‐HIF‐1α. (C) The mRNA levels of BNIP3 and HIF‐1α in SONFH cell model after treated with OE‐HIF‐1α and sh‐BNIP3 were evaluated by qRT‐PCR. (D) Expression of LC3 in SONFH cell model after treated with OE‐HIF‐1α and sh‐BNIP3 were detected by IF, and the number of cells undergoing autophagy was quantitatively counted. (E) Western blot was performed to assess the protein levels of LC3II/LC3I, Beclin1, and p62 in SONFH cell model after treated with OE‐HIF‐1α and sh‐BNIP3. (F) Cell apoptosis of SONFH cell model after treated with OE‐HIF‐1α and sh‐BNIP3 was detected by flow cytometry, and the numbers of apoptotic cells were quantitatively counted. (G) Western blot was performed to assess the protein levels of Bcl‐2, Bax, C‐caspase‐3, and C‐caspase‐9 in SONFH cell model after treated with OE‐HIF‐1α and sh‐BNIP3. *P < 0.05, **P < 0.01, ***P < 0.001

4. DISCUSSION

SONFH is a progressive disease caused by excessive use of glucocorticoids and eventually leads to the collapse of the femoral head and hip dysfunction. 2 Unfortunately, the explicit mechanisms of SONFH pathogenesis and progression are still unclear. Previous studies displayed that long‐term exposure to a high dose of Dex (10−6 mol/L) could induce apoptosis and inhibit the autophagy of BMSCs, which might lead to the pathogenesis of SONFH. 17 , 18 Autophagy involves degradation of dysfunctional cellular components through the actions of lysosomes. Autophagy can maintain cell homeostasis and ensures cell survival under stressful conditions. Multifunctional roles of autophagy exert its potential for both adaptive and harmful outcomes. The malfunction of autophagy plays a pathogenic part in human diseases including SONFH. 6 There are considerable cross‐talks between the autophagy and cell apoptosis. 19 Apoptosis is the process of programmed cell death involving a series of characteristic cell changes. At present, people's understanding of the relationship between autophagy and apoptosis can be roughly divided into three types:

  1. Both autophagy and apoptosis can cause cell death: This type of view includes (a) autophagy and apoptosis have no interaction, and autophagy causes dysfunction cell death, but apoptosis causes living cell death 20 ; (b) autophagy is at the upstream of apoptosis, and it can also independently trigger cell death while triggering apoptosis 21 ; (c) apoptosis can inhibit the occurrence of autophagy. When apoptosis is inhibited, autophagy is induced to play the role of killing cells. 22

  2. Autophagy inhibits apoptosis: Autophagy inhibits apoptosis by degrading damaged proteins and reducing DNA damage. 23

  3. Autophagy promotes apoptosis: Autophagy itself does not cause cell death, but it provides energy for the formation and phagocytosis of apoptotic bodies, thereby promoting the occurrence of apoptosis. 24

Bone cell apoptosis is considered to be a prominent pathological feature in SONFH. 25 Autophagy contributes to bulk degradation of cytoplasm and mitochondrion. 26 Autophagy influences mitochondrial recycle and can thus modulate hepatic apoptosis via caspase‐dependent cell apoptosis. 19 More importantly, evidence showed that apoptosis caused by defects in autophagy was the main pathogenesis of SONFH. 6 Therefore, enhancing autophagy, which in turn suppresses apoptosis is considered an effective treatment strategy for SONFH. Here, we proposed that APS could inhibit bone cell apoptosis by enhancing autophagy via regulating miR‐206/HIF‐1α/BNIP3 functional axis, thereby delaying the process of femoral head necrosis and improving SONFH.

APS is a macromolecular substance with multiple biological activities. 27 , 28 Numerous evidences showed that APS could activate autophagy 29 and inhibit apoptosis. 30 However, whether APS could promote autophagy and inhibit apoptosis in SONFH was not reported. Here, we showed that APS promoted autophagy and inhibited cell apoptosis, thereby improving femoral head necrosis. MiRNA participates in many biological processes through posttranscriptional regulation of genes. 8 MiR‐206 is closely related to bone diseases: impaired osteoblast differentiation was observed in transgenic mice by overexpressing miR‐206. Our results showed that the expression of miR‐206 was significantly upregulated in both SONFH bone tissues and SONFH cell model. Overexpression of miR‐206 reversed the recovery effect of APS on SONFH cell model, suggesting APS promoted autophagy and inhibited cell apoptosis by downregulating the expression of miR‐206. There were few studies about miR‐206 involved in apoptosis in SONFH, and our research innovatively found that APS participated in the regulation of apoptosis and autophagy in SONFH by regulating miR‐206.

MiRNAs participate in all biological processes through binding to the 3′‐untranslated region (UTR) of the target gene mRNA to regulate expression of gene. 31 Here our results showed that miR‐206 had a binding site to HIF‐1α. HIF‐1α is essential for cell autophagy. 32 , 33 For example, oxygen–glucose deprivation/reperfusion could activate autophagy by upregulating the expression of HIF‐1α and its downstream molecules in SH‐SY5Y cells. 10 When the cell is in a harsh environment, the activation of autophagy can make the cell escape from apoptosis. 34 , 35 Previous study showed that HIF‐1α‐dependent autophagy protected HeLa cells from apoptosis. 36 Our results showed that the expression of HIF‐1α was significantly downregulated in both SONFH bone tissues and SONFH cell model. Knockdown of HIF‐1α reversed the recovery effect of knockdown of miR‐206 on SONFH cell model, suggesting miR‐206 targeted HIF‐1α to promote autophagy and inhibit apoptosis in SONFH cell model.

Previous evidence showed that BNIP3 was the target molecule of HIF‐1α. 37 BNIP3 is reported to be essential for maintaining autophagy. 38 HIF‐1α could activate autophagy by activating BNIP3 to maintain cell survival. 39 Our results showed that expression of BNIP3 was significantly upregulated after overexpression of HIF‐1α, accompanying with increased cell autophagy and decreased cell apoptosis. Furthermore, BNIP3 silence abolished the recovery effect of overexpression of HIF‐1α on SONFH cell model, proving for the first time that HIF‐1α/BNIP pathway was involved in the regulation of SONFH pathogenesis.

In summary, our research proved that APS activated HIF‐1α/BNIP3 axis by downregulating miR‐206, thereby promoting bone cell autophagy and inhibiting bone cell apoptosis, which in turn ameliorate SONFH (Figure 7). Our research clarified the regulatory mechanism of apoptosis and autophagy of SONFH, which was of great significance for the clinical treatment.

FIGURE 7.

FIGURE 7

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Proposed model of the role of APS promoted autophagy and inhibited apoptosis in SONFH via miR‐206/HIF‐1α/BNIP3 axis. APS inhibited the expression of miR‐206, directly targeting HIF‐1α/BNIP3 axis, and thereby promoting bone cell autophagy and inhibiting bone cell apoptosis, which in turn ameliorate SONFH

CONFLICT OF INTEREST

The authors declare no conflicts of interest.

ACKNOWLEDGMENTS

The IRB approval number is 2020‐KY‐114.

Zhang S‐Y, Wang F, Zeng X‐J, Huang Z, Dong K‐F. Astragalus polysaccharide ameliorates steroid‐induced osteonecrosis of femoral head through miR‐206/HIF‐1α/BNIP3 axis. Kaohsiung J Med Sci. 2021;37:1089–1100. 10.1002/kjm2.12426

Funding information The Chinese Medicine Research Project in Hunan Province, Grant/Award Number: 201978

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