Abstract
Background. Osteoporosis (OP) is a major problem that increases the mortality and disability rate worldwide. With an increase in the aging population, OP has become a major public threat to human health. Searching for effective and suitable targets for drug treatment in OP has become an urgent need.
Objectives. Osteoporosis is a metabolic bone disease characterized by reduced bone mass and density as well as micro-architectural deterioration. Icariin is a flavonoid extracted from plants of the genus Epimedium and has been shown to exert potential anti-OP activity. The present study was designed to observe the effect of icariin on OP and to clarify the underlying mechanisms in ovariectomized (OVX) rats.
Materials and methods. Hematoxylin and eosin (H&E) staining, von Kossa staining and micro-computed tomography (micro-CT) confirmed significant bone loss in the OVX group. Protein expression level was detected with western blot analysis.
Results. Icariin reversed a trend of increased bone turnover by reducing serum alkaline phosphatase (ALP), procollagen type I N-terminal propeptide (PINP), tartrate-resistant acid phosphatase isoform 5b (TRACP-5b), and C-telopeptide of type I collagen (CTX-I). Furthermore, icariin decreased sequestosome 1 (p62) and increased microtubule-associated protein 1 light chain 3II/microtubule-associated protein 1 light chain 3I (LC3II/LC3I), autophagy-related protein 7 (Atg7), and Beclin 1 in the femur of OVX rats, improving the indicators of impaired autophagy in OP.
Conclusions. Icariin reversed the significant upregulation of the serine/threonine protein kinase (Akt), mammalian target of rapamycin (mTOR) and unc-51-like autophagy activating kinase 1 (ULK1) at Ser757, and the downregulation of p-AMP-activated protein kinase (p-AMPK) and ULK1 phosphorylated at Ser555 in the OVX rats, suggesting that the mechanism of icariin action in OP treatment involves the activation and suppression of the AMPK/ULK1 and AKT/mTOR/ULK1 autophagy pathways, respectively.
Key words: autophagy, icariin, osteoporosis, AMP-activated protein kinase/Unc-51-like autophagy activating kinase 1, serine/threonine protein kinase/mammalian target of rapamycin/Unc-51-like autophagy activating kinase 1
Background
Osteoporosis (OP) is a common degenerative bone disease characterized by a reduction in bone mass and the degradation of micro-architectural structures.1 Epidemiological surveys show that over 200 million women are affected by OP globally, with 8.9 million fractures each year. According to the latest projections, the prevalence of OP will reach 13.6 million by 2030 in women aged ≥50 years.2 Osteoporosis has become the leading cause of disability and death in the elderly population.
Many phytochemicals are good substitutes for chemically synthesized medications for the treatment of various human diseases.3 Icariin, the main flavonoid isolated from the plant of the genus Epimedium, has been identified as a potential drug for treating OP.4, 5, 6 Icariin mainly affects bone metabolism and promotes bone resorption by regulating estrogen, skeletal accretion, as well as apoptosis and its related signaling pathways. The common signaling pathways include PI3K-Akt, Wnt/β-catenin, and RANKL/RANK/OPG. They are essential for restoring the balance between bone resorption and formation in the bone remodeling process.7
Autophagy is critical for maintaining bone homeostasis through the degradation of abnormal proteins and pathogenic microorganisms. Research links low autophagy to OP.8, 9, 10, 11 Osteoblasts (OBs) and osteoclasts (OCs) play a crucial role in maintaining bone homeostasis, and the regulation of this process involves autophagy; when autophagy is deficient, it disrupts bone homeostasis and leads to OP.12, 13 However, the mechanisms mediating OP autophagy remain unclear.
Objectives
In our research, an OP model was established by ovariectomy in rats and icariin was administered by irrigation to study its effects and potential mechanism in autophagy and improvement of OP.
Materials and methods
Animals
The study was approved by the Animal Ethics Committee of the Shaanxi University of Chinese Medicine (approval No. SCXK (Shaanxi) 2019-001). Thirty female adult Sprague–Dawley (SD) rats (200–250 g), 3–4 months of age, purchased from the Experimental Animal Center of the Air Force Medical University (Xi’an, China), had access to clean water and food during the experiment, and were housed at 25°C with 50 ±10% humidity and a 12 h/12 h light-dark cycle.
In the present investigation, animal feed was purchased from Chengdu Dashuo Biological Technology Co., Ltd. (Sichuan, China), and its composition was the same in all experimental rats.
Experimental agents
Icariin (20200719) was purchased from Xi’an Yuhui Biotechnology Co. Ltd (Xi’an, China). Alkaline phosphatase (ALP) (JL26470), procollagen type I N-terminal propeptide (PINP) (JL49448), tartrate-resistant acid phosphatase isoform 5b (TRACP-5b) (JL12318), and C-telopeptide of type I collagen (CTX-I) (JL20748) enzyme-linked immunosorbent assay (ELISA) kits were purchased from Jianglai (Shanghai, China). Autophagy-related protein 7 (Atg7) (8558), Akt (9272), p-Akt (4058), Beclin1 (3495), mammalian target of rapamycin (mTOR) (2972), p-unc-51-like autophagy activating kinase 1 (ULK1) (Ser555) (5869), p-mTOR (2971), ULK1 (8054), p-ULK1 (Ser757) (14202), and AMP-activated protein kinase (AMPK) (5832) were purchased from Cell Signaling Technology (Danvers, USA). Microtubule-associated protein 1 light chain 3 (LC3) (GTX127375) was purchased from Genetex (Louis Park, USA), sequestosome 1 (p62) (ab109012) from Abcam (Cambridge, UK), β-actin (bs-0061R) from Bioss (Woburn, USA) and p-AMPK (13S4010) from Bioword (Louis Park, USA). Bicinchoninic acid (BCA) kits were purchased from Thermo Fisher Scientific (Waltham, USA) and dithiothreitol (DTT; 1 mol/L) was obtained from Sigma-Aldrich (St. Louis, USA).
Instruments
Mini-PROTEAN Tetra electrophoresis and blots, a protein semi-dry transfer device, and a gel imaging system were used (Bio-Rad, Hercules, USA). A full wavelength microplate reader (Tecan, Zurich, Switzerland), an automatic high-speed refrigerated centrifuge (Sigma-Aldrich), a SCIENTZ-48 Tissue Grinder (Xinzhi, Hunan, China), an X-Ray Micro-CT SkyScan 1276 (Bruker, Billerica, USA), and a VS200 digital slide scanner (Olympus, Tokyo, Japan) were utilized for tissue analysis.
Groups and animal treatments
Thirty female adult SD rats were randomly divided into control (sham-operated+saline, n = 10), model (ovariectomy+saline, n = 10) and icariin (ovariectomy+icariin, n = 10) groups. The model and icariin groups were subjected to bilateral ovariectomy, and the same amount of fat tissue near the ovary was removed in the control group. One week after the operation, intragastric administration of saline (0.5 mL/100 g) was carried out for 12 consecutive weeks in the model group, to which icariin (120 mg/kg) was added in the icariin group. Body weight of the animals was measured 14 times per week. Rats were intraperitoneally anesthetized with 10% chloral hydrate (300 mg/kg), and, under anesthesia, blood was collected from the abdominal aorta until the blood flow was exhausted. Death was confirmed by cardiac and respiratory arrest. Bones were frozen to −80°C for testing. None of the rats had peritonitis or its associated symptoms after the administration of 10% chloral hydrate. Rats weighed 340–403 g, and less than 10 mL of blood was collected from each rat at the time of sacrifice.
Measurement of serum metabolism indexes
Serum PINP, ALP, CTX-I, and TRACP-5b are indicators for evaluating bone formation and resorption ability. The levels of ALP (JL26470), PINP (JL49448), TRACP-5b (JL12318), and CTX-I (JL20748) were detected with ELISA. Blood samples were stored at room temperature for 2 h, centrifuged at 4°C for 5 min (3000 rpm), and the serum was separated. The samples were prepared according to the index instruction, a standard curve based on the standard solutions was created, and the sample (50 µL) and horseradish peroxidase (HRP; 100 µL) were added to each well. The well was sealed and placed in a 37°C incubator, and after 60 min, the liquid was poured off, and the sample dried on the absorbent paper. The washing solution (350 µL) was added to each well and maintained for 1 min at room temperature; after repeating 5 times, reaction substrate solutions A (50 µL) and B (50 µL) were added to each well, and they were incubated for 15 min in a 37°C incubator. The termination solution (50 µL) was added to each well for 15 min. A regression equation was formulated according to the standard curve, and the sample optical density (OD) value (λ = 450 nm) was taken into the equation to calculate the sample concentration. The sample size was 7.
Measurement of bone morphology
Hematoxylin and eosin (H&E) staining was used to evaluate the morphological features of the femur. Right distal femurs (1-cm specimens) were fixed for 48 h with 4% paraformaldehyde, decalcified with 10% ethylenediaminetetraacetic acid (EDTA), embedded in paraffin, and sectioned (5-μm thickness). Sections were stained with H&E prior to digital slide scanning. The H&E staining was performed according to the reference literature.14
Detection of calcium nodules
Calcification nodules can reflect the level of the OB mineralization. Calcium nodules were observed using Von Kossa staining. Right distal femurs (1 cm specimens) were fixed for 48 h with 4% paraformaldehyde, embedded with paraffin, sectioned, and washed subsequently with xylene, gradient alcohol and distilled water. In the next step, 3% silver nitrate was added for 1 h and washed with water for 5 min, counterstain with toluidine blue for 5 min, then washed with distilled water repeatedly, dried, and sealed with neutral glue. The sample size was 3.
Micro-computed tomography
A micro-computed tomography (micro-CT) system was employed for trabecular bone mass measurement. At the time of execution, the left femur, tibia and vertebrae (L4) were separated and fixed for 24 h in 4% paraformaldehyde before flushing with 70% ethanol. Distal femoral and proximal tibia metaphysis were scanned at a resolution of 8 μm (80-kV voltage, 80-μA current). Micview V2.1.2 software (GE Healthcare, Chicago, USA) was used to analyze the data. Regions of interest segmented with a fixed threshold were reconstructed into 3-dimensional images. Trabecular bone was quantified using bone marrow density (BMD), bone volume over total volume (BV/TV), bone surface over bone volume (Bs/Bv), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular spacing (Tb.Sp). The sample size was 4.
Western blot analysis
Femur (500 mg) at −80°C was cut into fragments with bone shears, ground into powder in liquid nitrogen, and collected in a centrifugal tube. Samples were lysed using 700-μL radioimmunoprecipitation assay (RIPA) buffer and ground with a high-throughput grinder. The homogenized tissues were centrifuged (14,000 rpm, 20 min) at 4°C. The supernatant was transferred to a new EP tube, and 10 μL was separated for quantitative analysis using a BCA protein assay kit. The remaining part was treated with RIPA, ×2 loading buffer, and 1 M DTT by volume, heated for 8 min at 100°C, cooled, mixed and frozen at −80°C. Proteins (80 μg/well) were separated on a 10–15% sodium dodecyl sulfate polyacrylamide gel(SDS-PAGE). Separated proteins were subjected to electroblotting on polyvinylidene fluoride (PVDF) membranes. The membranes were then blocked and incubated with designated primary antibodies (rabbit anti-rat) overnight at 4°C (all antibodies were diluted). Using β-actin (1:5000) as internal control, the autophagy proteins Atg7 (1:2000), p62 (1:2000), LC3 (1:2000), Beclin1 (1:2000), Akt (1:1000), p-Akt (1:1000), mTOR (1:1000), p-mTOR (1:1000), p-AMPK (1:1000), ULK1 (1:1000), p-ULK1 (Ser555) (1:1000), and p-ULK1 (Ser757) (1:1000) were detected. On the following day, samples were washed 3 times (5 min/time) with Tris-buffered saline with Tween (TBST) and were incubated with goat anti-rabbit IgG conjugated to HRP for 2 h, and then the PVDF membranes were washed 4 times (5 min/time) with TBST. Finally, the membranes were exposed using a Molecular Imager® ChemiDoc™ XRS System, and the western blot data were analyzed using ImageJ software (National Institutes of Health (NIH), Bethesda, USA). The sample size was 3 for the western blot.
Statistical analyses
The analysis was carried out using IBM SPSS Statistics for Windows, v. 26.0 (IBM Corp., Armonk, USA). Data are presented as mean ± standard deviation (M ±SD). A normality test and homogeneity of variance test were first used for multiple group data; one-way analysis of variance (ANOVA) was used if the data were in accordance with normality and homogeneity of variance; otherwise, the Kruskal–Wallis H test was conducted. A normality test, homogeneity of variance test, sphericity test, and repeated measures analysis of variance test were used for repeated measure data. Fisher’s least significant difference (LSD) or Dunn’s post hoc test were performed if differences were significant. Differences were considered of statistical significance with p < 0.05.
Results
Body weight and condition of the OP rats were ameliorated by icariin
Body weight values increased in all groups after 12 weeks, with a significant uptrend in the model group compared to the control and icariin groups from the 2nd week. Icariin appeared to control body weight but not at a statistically different level when compared to the model group. The fur of the rats in the model group was drier, yellower and duller than that of the control group; reflexes were also slower. Compared to the model group, the icariin group rats exhibited more sleek and white fur, faster reaction times, and reduced body weight (Figure 1 and Table 1).
Bone metabolism indexes of OP rats were mediated by icariin
The PINP, ALP, CTX-I, and TRACP-5b are indicators of bone metabolism, reflecting bone formation and resorption ability. In the model group, the expression of PINP, ALP, CTX-I, and TRACP-5b was increased. The 4 indexes were reduced in the icariin group compared with the model group (Figure 2 and Table 2).
Femoral morphological changes and calcium nodules in OP rats were restored by icariin
Morphological features of the femur were evaluated using H&E staining. Compared with the control group, the model group had more pathological changes, such as less femoral trabecular bone, uneven thickness, greater distance, and increased breakpoints. However, compared with the model group, the icariin group displayed multitudinous bone trabecula, and the bone microstructure was generally clear (Figure 3A).
The number of femoral calcium nodules was determined with von Kossa staining (Figure 3B). There was a reduction in the number of calcium nodules in the model group. Compared with the model group, the icariin group displayed an increased trend in calcium nodules, but they were not significantly different (Figure 3C, Table 3).
The BMD and trabecula state in OP rats was altered by icariin
The trabecular bone of the femur, tibia and lumbar spine (L4) were scanned with micro-CT (Figure 4), revealing bone loss and atrophy of the bone trabecula in the model group and an improvement of the trabecular morphology and volume in the icariin group. Bone marrow density in the model group was lower than in the control group, while icariin increased the BMD compared with the model group.
The Bv/Tv, Tb.Th, Tb.N, Bs/Bv, and Tb.Sp parameters reflect the morphological trabecula structure. The Bv/Tv, Tb.Th and Tb.N in the model group were found to be lower than in the control group, while Bs/Bv and Tb.Sp were higher. Icariin increased Bv/Tv, Tb.Th and Tb.N and decreased Bs/Bv and Tb.Sp (Table 4).
Autophagy of the OP rats activated by icariin
Autophagy-associated proteins were evaluated using western blotting. As shown in Figure 5, LC3II/LC3I, Atg7 and Beclin1 were decreased, while p62 was increased in the model group compared to the control group. Icariin increased the levels of LC3II/LC3I, Atg7 and Beclin 1, and decreased the expression of p62 compared to the model group (Table 5).
A bidirectional switch effect of icariin on autophagy pathways
The ULK1 is a pivotal modulator of autophagy initiation.15 The Ser555 and Ser757 phosphorylation sites of ULK1 have distinct effects on autophagy. Phosphorylation at the Ser555 site induces autophagy, while phosphorylation at the Ser757 site inhibits autophagy.16, 17 For the model group, Figure 6E,F show that ULK1 phosphorylated at Ser555 decreased but ULK1 phosphorylated at Ser757 increased. However, icariin increased Ser555-phosphorylated ULK1 and decreased Ser757-phosphorylated ULK1.
The Akt and mTOR, as downstream targets, act on the Ser757 phosphorylation site of ULK1 and are negatively associated with autophagy regulation.18 As shown in Figure 6A–C, phosphorylation of Akt and mTOR resulted in significant activation in the model group. However, icariin suppressed the change.
Another upstream kinase, AMPK, acts on the kinase at the Ser555 phosphorylation site of ULK1. As shown in Figure 6A,D, AMPK levels were significantly suppressed in the model group. Icariin elevated AMPK phosphorylation levels.
The results show that OP modelling in rats suppressed the AMPK/ULK1 pathway and activated the Akt/mTOR/ULK1 pathway, while icariin induced autophagy in a bidirectional switch manner (Table 6).
Discussion
With an aging population, the incidence of OP is a global concern.19 Serious clinical symptoms such as movement restriction, body deformity, chronic pain, and disability seriously reduce the quality of patients’ life, and therefore, an efficient prevention and treatment strategy is urgently required.20 Osteoporosis is treated clinically with synthetic drugs despite their numerous side effects.21 Icariin is a natural herbal extract component that exhibits important pharmacological activities for metabolic bone diseases.22 Autophagy regulates bone balance through bone formation and bone resorption.9 The present study investigated the potential of icariin in OP treatment and elucidated its possible mechanism of action in autophagy.
The OP rat model was established by ovariectomy, simulating bone mass loss and structural changes due to estrogen deficiency. Icariin intervention counteracted the increased weight, graying of hair, and slowing of reflexes that were observed in the model group, possibly by restoring the endocrine imbalance and slowing lipid metabolism caused by OP. Bones maintain a dynamic balance through osteoblastic formation and osteoclastic resorption. The ALP and PINP in serum are markers of osteoblastic formation, and CTX-I and TRACP-5b are specific markers of bone resorption, which increases in high turnover bone disease.23, 24, 25, 26 The blood serum analysis demonstrated that icariin reduced the indicators of bone formation and resorption, and enhanced bone turnover in rats in vivo. Micro-CT is an innovative method for assessing microstructural changes in bone tissue, which is simple, non-invasive and non-destructive to the specimen. Micro-CT scans revealed that icariin increased BMD and improved the trabecula parameters in the OVX rat model. Calcium nodules are mineralized products of the OBs, which can reflect the OB properties. The von Kossa staining revealed an apparent increase in the number of calcium nodules after icariin treatment compared with the OVX group. Therefore, icariin can promote OB formation and differentiation.
At present, the mechanism and targets for OP are unclear, so there are no particularly effective drugs on the market. In this study, autophagy marker proteins, namely Atg7, LC3II/LC3I, p62, and Beclin 1, were measured. The Atg7, an autophagy-related gene, is essential for autophagy, and the Atg system can promote LC3II binding and assist autophagosome elongation and closure. Beclin 1 is a protein involved in vesicle trafficking and autophagosome maturation, and increased expression of p62 can cause protein-specific autophagy degradation.27 Our study showed that the autophagy activation factors LC3II/LCI, Atg7 and Beclin 1 were reduced while inhibitory factor p62 was increased in OP rats. This situation was reversed after the intervention with icariin. These results confirm other literature precedents indicating that OP is associated with decreased levels of autophagy.28
Our research focused on explaining the bidirectional effects of the autophagy AMPK/ULK1 and AKT/mTOR/ULK1 pathways on OP. The ULK1 is the first initiation complex of autophagy.29 The Ser555 and Ser757 phosphorylation sites of ULK1 have distinct effects on autophagy. The AMPK stimulates the phosphorylation of the ULK1, while AKT and mTOR are inhibitors.30, 31 This paper reveals that, in OP rats, ULK1 protein phosphorylated at the Ser555 phosphorylation decreased, but ULK1 phosphorylated at Ser757 increased. Therefore, impaired autophagy can induce OP, and appropriate activation of autophagy can have a protective effect against OP. The AMPK acted on the kinase at the ULK1 Ser555 phosphorylating site, and the AMPK/ULK1 (Ser555) pathway was suppressed in OP rats in vivo. The mTOR and AKT acted on the ULK1 Ser757 phosphorylating site and the mTOR/AKT/Ulk1 (Ser757) autophagy pathway was promoted. However, the activities of AMPK, AKT, mTOR, ULK1 (Ser757), and ULK1 (Ser555) were modified by the intervention with icariin. The overexpression of AKT, mTOR and ULK1 (Ser757) was suppressed, and low AMPK and ULK1 (Ser555) were improved by icariin. In summary, icariin induced autophagy by activating the AMPK/ULK1 (Ser555) pathway and suppressing the AKT/mTOR/ULK1 pathway.
Icariin affects bone metabolism in various ways. For example, for ULK1, icariin stimulates the osteoblastic differentiation of bone marrow-derived mesenchymal stem cells, adjusting the bone metabolism balance.32 This is a separate mechanism. In this study, only 2 signaling pathways, AMPK/ULK1 and Akt/mTOR/ULK1 in the autophagy pathway, were selected for study. In our subsequent experiments, we will consider using mesenchymal stem cells from bone marrow to induce osteogenic differentiation and confirm the in vivo experiments.
Limitations
Our research explained the bidirectional effects of the autophagy AMPK/ULK1 and AKT/mTOR/ULK1 pathways on OP. Meanwhile, the mechanisms of the icariin treatment in OP by its mediation of autophagy were explained. However, our study still has some limitations. This research is too basic for further analysis, and deeper investigations are needed. Particularly, this study should be repeated with the addition of another group (ovariectomy+icariin+autophagy inhibitor) to prove the participation of autophagy in the mechanism. Thus, we were not able to fully confirm the in vivo results. We will further examine the mechanism in vitro.
Conclusions
In conclusion, daily oral administration of icariin for 12 weeks restored femur microstructure and increased bone turnover, effectively impeding bone loss caused by OP in rats. The molecular mechanism, at least partially, involves the regulation of the AMPK/ULK1 and AKT/mTOR/ULK1 autophagy pathways. The results provide an experimental basis for the further development of icariin in the field of OP treatment.
Supplementary data
The Supplementary materials are available at https://doi.org/10.5281/zenodo.8415761). The package consists of the following files:
Supplementary Table 1. Results of normality and variance homogeneity test of body weights at the 12th week (X±S).
Supplementary Table 2. Results of normality and variance homogeneity test of indexes of bone formation and resorption in serum (X ±S).
Supplementary Table 3. Results of normality and variance homogeneity test of BMD and trabecular parameters in femur.
