Polycytosine RNA-binding protein 1 regulates osteoblast function via a ferroptosis pathway in type 2 diabetic osteoporosis

Abstract

BACKGROUND

Recently, type 2 diabetic osteoporosis (T2DOP) has become a research hotspot for the complications of diabetes, but the specific mechanism of its occurrence and development remains unknown. Ferroptosis caused by iron overload is con-sidered an important cause of T2DOP. Polycytosine RNA-binding protein 1 (PCBP1), an iron ion chaperone, is considered a protector of ferroptosis.

AIM

To investigate the existence of ferroptosis and specific role of PCBP1 in the development of type 2 diabetes.

METHODS

A cell counting kit-8 assay was used to detect changes in osteoblast viability under high glucose (HG) and/or ferroptosis inhibitors at different concentrations and times. Transmission electron microscopy was used to examine the morphological changes in the mitochondria of osteoblasts under HG, and western blotting was used to detect the expression levels of PCBP1, ferritin, and the ferroptosis-related protein glutathione peroxidase 4 (GPX4). A lentivirus silenced and overexpressed PCBP1. Western blotting was used to detect the expression levels of the osteoblast functional proteins osteoprotegerin (OPG) and osteocalcin (OCN), whereas flow cytometry was used to detect changes in reactive oxygen species (ROS) levels in each group.

RESULTS

Under HG, the viability of osteoblasts was considerably decreased, the number of mitochondria undergoing atrophy was considerably increased, PCBP1 and ferritin expression levels were increased, and GPX4 expression was decreased. Western blotting results demonstrated that infection with lentivirus overexpressing PCBP1, increased the expression levels of ferritin, GPX4, OPG, and OCN, compared with the HG group. Flow cytometry results showed a reduction in ROS, and an opposite result was obtained after silencing PCBP1.

CONCLUSION

PCBP1 may protect osteoblasts and reduce the harm caused by ferroptosis by promoting ferritin expression under a HG environment. Moreover, PCBP1 may be a potential therapeutic target for T2DOP.

Key Words: Polycytosine RNA-binding protein 1; Ferroptosis; Reactive oxygen species; Ferritin; Osteoblast; Type 2 diabetic osteoporosis

Core Tip: Type 2 diabetic osteoporosis (T2DOP) is one of the most common complications of type 2 diabetes and its incidence has increased annually to seriously affect human health. T2DOP has a complicated environment and pathogenesis, including high glucose (HG) toxicity, inflammatory response, and iron metabolism disorders. However, the molecular mechanisms underlying T2DOP remain unclear. This study demonstrated that polycytosine RNA-binding protein 1 (PCBP1) protects osteoblasts and reduces the damage caused by ferroptosis by promoting ferritin expression in a HG environment in T2DOP, suggesting the promising therapeutic potential of PCBP1 in T2DOP.

INTRODUCTION

Recently, the incidence of diabetes, especially type 2 diabetes, has increased annually, posing a serious threat to human health[1]. There are > 100 complications of diabetes, including diabetic osteoporosis, diabetic nephropathy, diabetic foot, diabetic heart disease, diabetic vascular disease, and diabetic peripheral neuropathy[2]. Type 2 diabetic osteoporosis (T2DOP) is particularly common, especially in the elderly population. As individuals age, they have a considerably higher risk of fractures, which seriously affects their quality of life and imposes a heavy burden on society[3]. Several types of osteoporosis exist. Compared with type 1 diabetic osteoporosis and other types of osteoporosis, T2DOP has a more complicated environment and pathogenesis, including high glucose (HG) toxicity, inflammatory response, advanced glycation end products aggregation, and iron ion metabolism disorders[4]. However, the exact cause of T2DOP has not been fully elucidated.

Iron is a crucial trace element that sustains the life of organisms. The metabolic process of iron has a complete control system in advanced animals, including humans, to ensure that the body maintains a steady state. Moreover, iron homeostasis is essential for cells to perform physiological functions[5]. Ferritin, the main intracellular iron storage protein, is widely expressed in animals, plants, and microorganisms. Ferritin can protect cells from the toxic effects of excessive iron and release iron ions to meet the functional requirements of cells when iron is deficient[6]. Thus, it is essential for the maintenance of iron homeostasis.

Ferroptosis is a non-apoptotic programmed death pathway that is dependent on iron ions and reactive oxygen species (ROS). When iron ions are excessively accumulated in cells, the large amount of ROS produced by the Fenton reaction attacks biological macromolecules, resulting in an imbalance in iron homeostasis in the cell, ultimately leading to cell death[7]. Numerous studies have investigated ferroptosis; however, there have been few studies on bone metabolism. For example, Lu et al[8] reported that ferroptosis may occur during the pathogenesis of osteoporosis, as detected using a mouse model of osteoporosis and bioinformatics methods. Seibt et al[9] found that glutathione peroxidase 4 (GXP4) is a key regular of ferroptosis. Our previous studies revealed that iron overload caused by HG is an important factor causing osteoblast injury, and ROS levels are considerably increased during this period[10]. These findings suggest that ferroptosis is closely associated with the pathogenesis of diabetic osteoporosis; however, the specific occurrence process and whether ferritin is involved in this process require further examination.

Polycytosine RNA-binding protein 1 (PCBP1) is an iron ion chaperone protein widely present in cells that can bind iron ions and transfer iron ions to ferritin, thereby increasing the load of iron ions in ferritin[11]. Among the PCBP family, PCBP1 is one of the most studied and has a wide range of functions, including regulating gene transcription, maintaining mRNA stability, promoting translation, and acting as an iron ion partner[12-14]. Ryu et al[15] reported that mice lacking PCBP1 exhibited small cell iron deficiency anemia, which was compensated by regulating erythropoietin. Protchenko et al[16] found that unchaperoned iron in PCBP1-delated mouse hepatocytes produced ROS, indicating that PCBP1 may be a key factor in preventing ferroptosis. To the best of our knowledge, no study has confirmed whether PCBP1 is involved in the occurrence and development of T2DOP, and the relationship between changes in PCBP1 levels, ferritin, and ferroptosis in osteoblasts remains unknown.

Therefore, this study aimed to determine the existence of ferroptosis and the role of PCBP1 in the pathogenesis of T2DOP. We hypothesized that the compensatory increase of PCBP1 in T2DOP may lead to increased ferritin levels in osteoblasts, as well as reduce the excessive accumulation of iron ions and slow the occurrence and development of ferroptosis. This study first detected the expression levels of PCBP1 and ferritin in a HG environment, and then tested the relevant indicators, such as ferritin, after lentiviral silencing and overexpression of PCBP1. The current findings will help elucidate the pathogenesis of osteoporosis in type 2 diabetes and provide a theoretical basis for the treatment of metabolic diseases that target PCBP1 to regulate ferroptosis.

MATERIALS AND METHODS

Cell culture and related reagents

The human osteoblast-like cell line, hFOB1.19, was purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences, cultured in DMEM/F12 medium (glucose concentration 17.5 mmol/L; Hyclone; Cytiva) supplemented with 10% FBS (Hyclone; Cytiva) and 1% penicillin (Invitrogen; Thermo Fisher Scientific, Inc.), and cultured at 34 ˚C in a 5% CO₂ incubator. The solution was changed every two days. When the cells were 80% confluent, they were subcultured with trypsin-EDTA (Sigma, America).

PCBP1 Lentiviral reagents were purchased from GeneChem Co., Ltd (Shanghai, China). PCBP1 (EPR11055, 1:500), ferritin (EPR3005Y, 1:500), and GPX4 (EPNCIR144, 1:500) antibodies were purchased from Abcam. Osteoprotegerin (OPG, sc-390518, 1:500) and osteocalcin (OCN, sc-390877, 1:500) antibodies were purchased from Santa Cruz Biotechnology, Inc. The human bone mineralization induction culture base was purchased from Cyagen Biosciences, Inc. β-actin (bsm-33036M, 1:1000) and all secondary antibodies (goat anti-rabbit IgG, bs-40295G-HRP, 1:5000 and goat anti-mouse IgG, bs-40296G-HRP, 1:5000) were purchased from BIOSS.

Cell viability analysis

Cell viability was detected using a cell counting kit (CCK)-8 (Abcam) assay, according to the manufacturer’s instructions. Cells from each group treated with different glucose concentrations (17.5, 26.25, 35, 43.75, and 52.5 mmol/L, Sigma, America) and/or ferroptosis inhibitor (Ferrostatin-1, Fer-1, Sigma, America) were seeded in 96-well plates at a density of 6 × 10³ cells/well, and cultured with 10% serum-containing medium. When the percentage of cell growth reached 70%–90%, the serum concentration of the medium was reduced to 1%, and the culture was continued to maintain the cells in the same division state as much as possible. After 24, 48, and 72 h, 10 µL CCK-8 solution was added to each well, and the reaction was continued in the incubator for 4 h. The optical density (OD) value of the cells in each well was measured using a microplate reader at a wavelength of 490 nm. Relative cell activity (%) = (OD value of wells in treatment group-OD value of blank group)/(OD value of control group-OD value of blank group).

ROS detection

Cells in the logarithmic growth phase were collected and inoculated into 6-well plates at a density of 5 × 10⁶ cells/mL and cultured in an incubator at 37.5 ˚C for 72 h. We used 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA, Sigma, America) to evaluate the ROS level. Briefly, DCFH-DA was diluted (1:1,000) with serum-free medium to a final concentration of 10 μmol/L. After the cells were collected, they were suspended in diluted DCFH-DA, incubated in a cell incubator for 20 min, inverted, and mixed every 3–5 min. After washing the cells thrice with serum-free cell culture solution, the ROS level in the cells in each group was measured using a flow cytometer at an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

Western blotting

After the cells were digested with trypsin-EDTA, a lysis buffer (Abcam) containing protease and phosphatase inhibitor cocktail was added, placed on ice for lysis for 30 min, and then centrifuged at 12000 × g at 4 ˚C for 30 min, after which the supernatant containing total protein was collected. After the total protein was extracted, the protein concentration was determined using the BCA method. The protein sample (50 μg protein) was separated using 12% sodium lauryl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane (MerckMillipore, America) at 60 V for 2 h. The membrane was blocked with a blocking buffer containing 5% skim milk for 2 h before being incubated overnight at 4 ˚C with primary antibodies (diluted at 1:100 to 1:1000). Subsequently, the membrane was incubated with a HRP-conjugated secondary antibody (anti-mouse or anti-rabbit; IgG was diluted at 1:6000 or 1:10000) for 2 h at room temperature. The bands were visualized with an EC3 imaging system (Analytik Jena AG), and the ratio of the OD of each band to β-actin, the internal reference protein, was measured using ImageJ 1.48V software (National Institutes of Health).

Lentiviral infection

The cells were seeded at a density of 4 × 10⁴ cells/mL in a 96-well culture plate, with a volume of 90 μL per well, and cultured in a 37.5 ˚C incubator for 24 h. An appropriate amount of the virus stock solution was diluted with enhanced infection solution to a titer of 1 × 10⁸ TU/mL, which was added to the osteoblast-like cells for virus infection, and then the culture plate was returned to the incubator for incubation. After cells were infected for 3-4 d, the fluorescence expression was observed using a fluorescence microscope. Cell clones with positive expression were selected and expanded to obtain cell with stable expression. At a later stage of infection, the cells were exchanged and passaged according to their growth, to obtain stable expressing cell lines.

Transmission electron microscope

After the cells were digested with trypsin-EDTA, the suspended cells were collected via centrifugation and washed three times with pre-chilled phosphate buffer solution (Gibco, America). The cells were then fixed with 5% glutaraldehyde (Sigma, America) fixing solution for 1 h. After washing the cells again, they were routinely dehydrated, embedded, sectioned, and stained. Morphological changes in mitochondria were observed using a transmission electron microscope.

Induction of mineralization

Cells were seeded at a density of 2 × 10⁴ cells/cm² in a 6-well plate coated with 0.1% gelatin, and 2 mL of medium was added to each well, which was then cultured in a 37.5 ˚C, 5% CO₂ incubator. When the degree of cell fusion reached 60%–70%, the medium in the 6-well plate was replaced with complete osteogenic induction differentiation medium (Cyagen Biosciences, Inc.), which was replaced with fresh osteogenic induction differentiation medium every three days. After 2–4 wk of induction, the cells were stained with Alizarin Red (Shanghai Beyotime Co., Ltd), and the cell mor-phology was observed under an inverted fluorescence microscope.

Statistical analysis

Each experiment was repeated ≥ 3 times, and the data are presented as mean ± SD. Data were analyzed using the SPSS software (version 22.0; IBM Corp.). An unpaired t-test was used to compare two groups, whereas a one-way ANOVA was used to compare multiple groups. P < 0.05 was considered statistically significant.

RESULTS

Osteoblast activity decreases in a high glucose environment, which may be associated with ferroptosis

After the cells were treated for 24 h, groups with glucose concentrations of 35 mmol/L or higher showed an osteoblast promotion effect, but there was no major difference between the four groups. After 48 h of treatment, the cell viability of each group was slightly reduced compared with that of the control group, but there was no major difference between all groups. After 72 h of treatment, the viability of the three groups of cells with glucose concentrations greater than 35.00 mmol/L was considerably reduced, but there was no statistical difference between the three groups (Figure 1A). Compared with the control group, the HG group had lower osteoblast viability, and after the addition of the ferroptosis inhibitor, osteoblast viability increased compared with the HG group (Figure 1B). Electron microscopy revealed that the mitochondria in the HG group were fused and shrunken, and the staining intensity was deeper (Figure 2). These results suggest that osteoblast activity decreases in HG environments and may be associated with ferroptosis.

Figure 1 Detection of osteoblast activity after the addition of different glucose concentrations at different time points. A: Osteoblasts considerably reduced in activity after 72 h in glucose concentrations ≥ 35 mmol/L; B: Osteoblasts treated with high glucose (HG) combined with Fer-1 showed increased activity compared with those in the HG group. Data are presented as mean ± SD. ^aP < 0.05 vs control; ^cP < 0.05 vs HG. HG: High glucose.

Figure 2 Observation of mitochondrial changes in osteoblasts in a high glucose environment using electron microscopy. A: In the control group, the mitochondria in the osteoblasts were normal and the ridge lines were clear; B: In the high glucose group, the mitochondria in the osteoblasts were fused and shrunken, and the staining was deeper. HG: High glucose.

PCBP1 expression in osteoblasts increases in a high glucose environment

This study determined the expression level of PCBP1 in osteoblasts in a HG environment using western blotting (Figure 3). PCBP1 expression was higher in osteoblasts under HG environment compared with that in the control group. This indicates that a HG environment can promote the expression level of PCBP1 in osteoblasts.

Figure 3 Detection of polycytosine RNA-binding protein 1 expression in osteoblasts under high glucose. Compared with the control group, polycytosine RNA-binding protein 1 expression was higher in the high glucose group. Data are presented as the mean ± SD. ^aP < 0.05 vs control. HG: High glucose; PCBP1: Polycytosine RNA-binding protein 1.

Lentiviral infection of PCBP1

To confirm the role of PCBP1 in diabetic osteoporosis, the expression level of PCBP1 in hFOB1.19 cells was altered. In total, > 80% of the cells expressed green fluorescent protein (Figure 4A-C). Subsequently, western blotting was used to detect the expression level of PCBP1 after PCBP1-encoded lentivirus infection (Figure 4D), and the results confirmed that the cells were successfully transfected with PCBP1 lentivirus.

Figure 4 hFOB l. 19 cells were infected with polycytosine RNA-binding protein 1-encoded lentivirus, and the expression level of polycytosine RNA-binding protein 1 was detected via western blotting. A: Expression level of green fluorescently labeled PCBP1 in hFOB l. 19 cells was detected using an inverted fluorescence microscope; B: hFOB l. 19 cells were examined using a light microscope; C: Merging of A and B; D: Western blotting results of the protein expression level of polycytosine RNA-binding protein 1 in hFOB l. 19 cells. Data are presented as mean ± SD. ^aP < 0.05 vs control; ^cP < 0.05 vs empty vector virus control. Scale bar = 300 μm. PCBP1: Polycytosine RNA-binding protein 1.

PCBP1 reduces the occurrence of ferroptosis by increasing the expression level of intracellular ferritin in osteoblasts under a high glucose environment

Ferritin, the ferroptosis marker protein GPX4, the osteogenic marker proteins OPG and OCN (Figure 5A), cellular ROS levels (Figure 5B), and alizarin red-stained calcium nodules (Figure 5C) were detected in osteoblasts under HG conditions alone or in combination with the infection of PCBP1 lentivirus via western blotting, flow cytometry, and induction of mineralization.

Figure 5 Polycytosine RNA-binding protein 1 regulation of osteoblastic ferroptosis in a high glucose environment. A: Western blotting results showing the expression levels of ferritin, glutathione peroxidase 4, osteoprotegerin, and osteocalcin after polycytosine RNA-binding protein 1 (PCBP1) lentivirus transfection in a high glucose (HG) environment; B: Flow cytometry results showing the levels of reactive oxygen species after PCBP1 lentivirus transfection in a HG environment; C: Alizarin red calcium nodule staining identified the number of calcium nodules in osteoblasts after PCBP1 lentivirus transfection in a HG environment. Data are presented as the mean ± SD. ^aP < 0.05 vs empty vector virus control; ^cP < 0.05 vs empty vector virus HG. PCBP1: Polycytosine RNA-binding protein 1; HG: High glucose; GPX4: Glutathione peroxidase 4; OPG: Osteoprotegerin; OCN: Osteocalcin.

Western blotting results demonstrated that, compared with the normal group, ferritin and PCBP1 expression levels were increased in the HG group, while the expression levels of the ferroptosis marker protein GPX4 and the functional indicator proteins OPG and OCN were decreased. Under the same conditions of HG concentration, the expression levels of ferritin, GPX4, OPG, and OCN in the PCBP1 overexpression group were considerably increased compared with those in the HG alone group, while these expression levels were decreased in the knockdown group.

The flow cytometry results indicated that the ROS production was higher in the HG group compared with that in the normal group. Moreover, compared with the HG group, ROS production in the PCBP1 overexpression group was considerably reduced, whereas the knockdown group demonstrated the opposite result.

The osteogenic mineralization induction results showed that calcium nodule deposition was reduced in the HG group compared with that in the normal group. Compared with the HG group, calcium nodule deposition in the PCBP1 overexpression group was considerably increased, while the opposite result was found in the knockdown group. These results indicate that PCBP1 could reduce the toxic effect of ferroptosis on osteoblasts by promoting ferritin expression in a HG environment.

DISCUSSION

This study demonstrated that ferroptosis occurred in osteoblasts after HG treatment, and that PCBP1 and ferritin expression levels were higher than those in normal cells.

Ferroptosis, an iron-dependent lipid peroxide aggregation process, is a recently discovered mechanism of cell death[7]. Ferroptosis has become a research hotspot in several fields. For example, epidemiology and animal studies have reported that iron affects cell proliferation[17,18]. Moreover, abnormal iron metabolism and high intracellular iron content are characteristics of most cancer cells[19]. It has been suggested that oxidative stress may be the main cause of cancer, and that ferroptosis is involved in the pathogenesis of various cancer types[20]. Ferroptosis can lead to numerous diseases, such as liver and kidney damage, iron overload cardiomyopathy, and neurodegenerative diseases[21-23]. Studies have shown that ferroptosis may be a potential mechanism of endothelial cell damage caused by particulate matter 2.5[24]. Moreover, ferroptosis, a novel death mechanism different from that of apoptosis and necrosis, has a wide range of effects on diseases and cell damage. For example, Zhang et al[25] revealed that the upregulation of GPX4 reduces the incidence of ferroptosis and helps decrease secondary brain injury after cerebral hemorrhage. Nguyen et al[26] summarized the basic information of ferroptosis discovery and further explained the role of ferroptosis in human cancer, whereas Zhu et al[27] performed a literature search and analysis to compare the anticancer activity of artemisinin and its derivatives, and reported that ferroptosis was a new mechanism for treating cancer. These studies indicate that ferroptosis is widely studied in the fields of tumors and nerve injury than it is in the field of bone metabolism. These findings suggested that ferroptosis may be involved in the pathogenesis of T2DOP.

Ferritin is a natural reservoir of iron ions in the body, and plays a crucial role in iron storage and the control of intracellular iron distribution[28]. Bu et al[29] reported that ferritin can reduce free iron in cells, thereby inhibiting lipid peroxidation, oxygen free radical damage, and pro-inflammatory cascades that are induced when cells are stressed. Furthermore, Liu et al[30] examined the detoxification and utilization mechanisms of high-concentration heavy metals in the body using blind shrimp from a deep-sea hydrothermal area and found that the heavy chain subunit of ferritin had iron oxidase activity, which could convert divalent iron into non-toxic trivalent iron and store the iron. Santiago González et al[31] studied the iron metabolism of Schwann cells in myelinated nerve fibers, and found that doublesex and mab-3 related transcription factor 1, ferritin, and transferrin receptor 1 were key proteins for early iron uptake and storage in Schwann cells, as well as the normal myelination of the peripheral nervous system. Thus, based on the aforementioned findings, it is suggested that ferritin exerts a detoxification effect on iron overload and a protective effect on cells.

A previous study found that intracellular ferritin heavy peptide 1 and transferrin receptor 1 were upregulated in mice fed a high iron diet[32]. Furthermore, Li et al[33] showed that ferroportin1 protein is localized in the cytoplasm of bone cells of rats and ferroportin1 may be involved in the pathological process of iron overload-induced bone lesions. Additionally, Zhou et al[34] revealed that at the same time as the bone iron content was decreased in iron responsive element binding protein 2-knockout mice, the gene transcription of ferritin was correspondingly reduced. Thomson et al[35] pointed out that when the concentration of iron ions in the cell is low, iron-regulatory proteins /iron-responsive elements (IRP/IRE) prevents the assembly of ribosomes, thereby inhibiting the translation of ferritin. When the concentration of iron ions increases, IRP/IRE decreases, thereby increasing the translation of ferritin to store excess iron. These studies indicate that changes in intracellular iron ions are closely associated with ferritin, which is an important protector against iron overload and ferroptosis in human body.

PCBP1, an iron ion partner, can transfer iron ions to ferritin. Halon-Golabek et al[36] reported that, in the skeletal muscle of rats with amyotrophic lateral sclerosis, an increase in PCBP1 protein expression was accompanied by an increase in ferritin light and heavy chains. Moreover, Ryu et al[37] revealed the role of PCBP1 in iron flux via ferritin in developing erythroid cells, and showed that mice lacking PCBP1 could develop small cell anemia. Therefore, PCBP1 is important for ferritin formation and function. However, the effect of PCBP1 on ferritin in osteoblasts remains unknown.

Studies by Zarjou et al[38] showed that ferritin regulates osteoblast activity and osteogenesis. However, to the best of our knowledge, no studies have yet elucidated the regulatory mechanism of ferritin in T2DOP or its effect on T2DOP. Our previous study showed that osteoblasts from T2DOP bone tissues are overloaded with iron, causing the cells to produce an oxidative stress response, which ultimately affects the osteogenic function of the osteoblasts[4,39]. However, the specific processes and mechanisms of iron overload are yet to be fully elucidated.

This study demonstrated that the mitochondria exhibited typical morphological changes[7], and that the expression levels of PCBP1 and ferritin were considerably increased after HG treatment in osteoblasts. After lentiviral silencing and overexpression of PCBP1, ferritin was downregulated and upregulated, respectively. This study identified a key role of PCBP1 in the regulation of ferritin in T2DOP. Ferritin is the main storage protein of intracellular iron and an important component of intracellular iron homeostasis[28]. Thus, it was suggested that the increase in ferritin in the HG environment was due to the regulation of PCBP1, which compensated for the intracellular iron overload.

Ferritin regulated by PCBP1 affects osteoblast function via ferroptosis. Park et al[40] reported that ferroptosis is regulated by ferritin and transferrin receptors. Moreover, Mumbauer et al[41] showed that ferritin can protect cells from harmful effects caused by active oxygen accumulation and ferroptosis. Our previous study revealed that under HG conditions, ROS levels are considerably increased in osteoblasts, which could cause osteoblast function to decline. This study suggests that PCBP1 can reduce the occurrence of ferroptosis by promoting the formation of ferritin, which may be a potential target for the treatment of T2DOP. Future studies will attempt to prepare targeted drugs containing PCBP1, which can target the patient’s osteoblasts via oral administration, intravenous drip, or local injection, to inhibit the occurrence and development of osteoporosis, thereby improving or even reversing osteoporosis.

CONCLUSION

In conclusion, our findings revealed novel regulatory mechanisms involving PCBP1-associated ferroptosis as a promising therapeutic target for clinical therapy of T2DOP.

ARTICLE HIGHLIGHTS

Research background

Type 2 diabetic osteoporosis (T2DOP) has become the most common complication of type 2 diabetes and its incidence has increased annually to seriously threaten human health. Exploring the molecular mechanisms underlying T2DOP is critical for its treatment.

Research motivation

The molecular mechanisms underlying T2DOP are poorly understood.

Research objectives

This study aimed to determine the involvement of ferroptosis and the specific role of polycytosine RNA-binding protein 1 (PCBP1) in the development of T2DOP.

Research methods

A high glucose (HG)-induced in vitro model was established and treated with PCBP1 overexpression lentivirus vectors. The levels of the osteoblast functional proteins osteoprotegerin and osteocalcin, and the ferroptosis-related markers reactive oxygen species and glutathione peroxidase 4 (GPX4) were evaluated.

Research results

HG environment considerably decreased the viability of osteoblasts and enhanced mitochondria atrophy, along with increased PCBP1 and decreased GPX4 expression, whereas treatment with PCBP1 repressed ferroptosis and protected osteoblast function.

Research conclusions

PCBP1 protects osteoblasts from ferroptosis by promoting ferritin expression under the HG stimulation in T2DOP, suggesting the promising therapeutic potential of PCBP1 in T2DOP.

Research perspectives

Our findings revealed a novel regulatory mechanism involved in T2DOP and provided a promising therapeutic approach for clinical use.