Unveiling the Role of Sik1 in Osteoblast Differentiation: Implications for Osteoarthritis

Abstract

Osteoarthritis (OA) is a chronic degenerative disease characterized by subchondral osteosclerosis, mainly due to osteoblast activity. This research investigates the function of Sik1, a member of the AMP-activated protein kinase family, in OA. Proteomic analysis was conducted on clinical samples from 30 OA patients, revealing a negative correlation between Sik1 expression and OA. In vitro experiments utilized BMSCs to examine the effect of Sik1 on osteogenic differentiation. BMSCs were cultured and induced toward osteogenesis with specific media. Sik1 overexpression was achieved through lentiviral transfection, followed by analysis of osteogenesis-associated proteins using Western blotting, RT-qPCR, and alkaline phosphate staining. In vivo experiments involved destabilizing the medial meniscus in mice to establish an OA model, assessing the therapeutic potential of Sik1. The CT scans and histological staining were used to analyze subchondral bone alterations and cartilage damage. The findings show that Sik1 downregulation correlates with advanced OA and heightened osteogenic differentiation in BMSCs. Sik1 overexpression inhibits osteogenesis-related markers in vitro and reduces cartilage damage and subchondral osteosclerosis in vivo. Mechanistically, Sik1 modulates osteogenesis and subchondral bone changes through Runx2 activity regulation. The research emphasizes Sik1 as a promising target for treating OA, suggesting its involvement in controlling bone formation and changes in the subchondral osteosclerosis.

Introduction

Osteoarthritis is a frequently occurring degenerative condition that mainly impacts people in their middle to older age.¹ Primary clinical symptoms include pain, edema, joint stiffness, and a limited range of motion. Advanced OA can cause severe disability and loss of work, burdening individuals, families, and society. Its cause and pathogenesis are unclear; however, its incidence is rising, possibly due to an increase in the proportion of obese individuals and an aging society.² OA affects joints and the surrounding tissues, such as the articular cartilage, subchondral bone, and synovium.³ Previous research has predominantly emphasized the involvement of cartilage. However, more recently, there has been increasing attention on the role of subchondral bone in OA recently. Certain researchers have demonstrated that alterations in the microstructure of subchondral bone occur prior to the degradation of articular cartilage.^4,5 Subchondral bone changes in OA involve early increased bone resorption via osteoclast activation and late enhanced bone formation due to increased osteoblast activity.⁶ When the entire joint experiences abnormal mechanical stress, cells increase the production of receptor activator for nuclear factor-κB ligand (RANKL) while reducing the secretion of osteoprotegerin (OPG).⁷ RANKL, by binding to the receptor activator for nuclear factor-κB (RANK) on the surface of osteoclast precursors, promotes the maturation of osteoclasts, leading to increased bone resorption.⁸ As OA progresses, osteocytes regulate the Wnt signaling pathway to modulate the mineralization of osteoblasts, increasing the production of Wnt proteins and reducing the secretion of sclerostin (SOST) to manage the increased abnormal mechanical load.⁹ Mature osteoblasts release RANKL, insulin-like growth factor 1 (IGF-1), transforming growth factor β1 (TGF-β1), and vascular endothelial growth factor (VEGF) under the cartilage, mediating the sclerosis of subchondral bone, generation of osteoclasts, and vascular formation.^7,10 Furthermore, TGF-β1 derived from osteocytes enhances osteoblast-mediated bone synthesis metabolism via activation of Smad 2/3 in the later stages of OA within the subchondral bone.^11,12

The salt-inducible kinases (SIKs), part of the AMP-activated protein kinase (AMPK) family, consist of three members (SIK1-3) and are pivotal in numerous physiological functions through their role as protein kinases.¹³ They are considered key regulatory factors in several metabolic disorders such as obesity and diabetes, influencing fatty acid synthesis and degradation, glucose metabolism, and energy balance by modulating the activity of protein kinases and phosphatases.^14–16 Moreover, Sik1 is hypothesized to play a significant role in the nervous system, affecting cognitive function by regulating synaptic plasticity and neuronal connectivity.^17,18 Kim et al.¹⁹ demonstrated that Sik1 regulates bone cell proliferation and differentiation through the CRTC1-CREB-Id1 axis, thereby modulating bone anabolism. This identifies Sik1 as a potential therapeutic target for disorders related to bone synthesis metabolism. A recent bioinformatics analysis and machine learning study identified Sik1 as a novel target with promising diagnostic and therapeutic potential for OA and aging-related conditions.²⁰ In addition, Zhu et al.²¹ identified Sik1 as a diagnostic marker for synovial tissue in OA using a weighted gene co-expression network. This multifaceted nature positions Sik1 as a protein worthy of further in-depth exploration, potentially offering novel insights and opportunities for future therapeutic developments.

During osteogenic differentiation, crucial transcription factors are instrumental in gene expression regulation. One of the most important transcription factors is Runx2, which is often referred to as the “master regulator” of osteogenesis.^22,23 Runx2 regulates gene expression in osteoblasts, either positively or negatively, by interacting with multiple transcription factors, chromatin-modifying enzymes, and cofactors.²⁴ For example, it can facilitate the differentiation and maturation of osteoblasts by enhancing the transcription of osteoblast-specific marker genes through binding to the cis-acting element 2 (OSE2).²⁵ Komori et al.²⁶ found that ALP, OSX, and OCN were not expressed after knocking out Runx2 expression in an in vivo model, and both intramembranous and endochondral ossification in mice were completely blocked. This result validates the crucial role of Runx2 in osteogenic differentiation in vivo, and Ren et al.²⁷ reached the same conclusion using in vitro experiments.

In the present study, proteomic data analysis on clinical samples of subchondral bone was obtained from 30 patients with OA. The findings unveiled a negative regulatory association between Sik1 and OA. The in vivo and in vitro experiments subsequently confirmed that enhancing Sik1 expression suppresses both the osteogenic differentiation of BMSCs and the sclerosis of subchondral bone. Moreover, based on the previous experimental observations derived from intervening with Runx2, it was concluded that Sik1 impacts osteogenic differentiation and subchondral bone sclerosis by modulating the activity of Runx2. This study offers new understanding into the pathophysiology of osteoarthritis (OA) and identifies Sik1 as a promising therapeutic target for the treatment of OA and other bone metabolism disorders.

Results

Quantitative proteomics analysis and validation

A total of 60 patients with primary OA were included in the present study, consisting of 43 women and 17 men. The average age of the patients was 70.95 ± 7.40 (Supplementary material, Figure S2A), with a range of 57 to 86 years. The average BMI was 29.54 ± 2.86 (Supplementary material, Figure S2B). Of these, 45 patients had a BMI ≥ 27.9, aligning with the observation that the majority of OA patients were obese. All subjects were in the end stages of the disease, with K-L grades of 3–4 (Supplementary material, Figure S2C). The Lysholm score averaged 65.02 ± 9.27 (Supplementary material, Figure S2D), the VAS score was 7.28 ± 1.26 (Supplementary material, Figure S2E), and the WOMAC score was 84.8 ± 10.5 (Supplementary material, Figure S2F). The clinical data of all patients corresponded to the epidemiological characteristics of OA. Among the 60 patients, three cases were randomly selected. Their preoperative knee joint AP and LAT (anterior-posterior and lateral), knee double AP, MRI, postoperative knee joint AP and LAT, and intraoperative images are all shown in Figure 1A to C.

Figure 1.

Radiological data and label-free quantitative proteometric analysis of clinical samples. (A–C) Representative preoperative knee joint AP and LAT, knee double AP, MRI, postoperative knee joint AP and LAT, and intraoperative photos of three randomly selected cases. (A) A 72-year-old female patient with left knee OA of K-L grade 4. (B) A 69-year-old female patient with right knee OA of K-L grade 4. (C) A 73-year-old male patient with left knee OA of K-L grade 4. The clinical samples utilized the weight-bearing area of the tibial plateau for the OA group and the nonweight-bearing area for the control group. (D and E) Results of differential proteomic analysis, showing significant downregulation of Sik1 expression in the OA group. (F and G) Functional enrichment analysis results using KEGG and GO, highlighting the functional pathways associated with the differentially expressed proteins. (H) Validation of the transcriptional levels in the subchondral bone of OA, revealed a significant decrease in Sik1 mRNA expression compared to the control group. *P < 0.05. OA, osteoarthritis; AL and LAT, Anterior posterior and lateral; K-L, Kellgren–Lawrence; Sik1, salt-inducible kinase 1; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Proteomic data analysis of subchondral bone in patients with OA identified 296 proteins with distinct expression patterns between the OA and control groups. In particular, Sik1 showed significantly lower expression levels in the OA group (Figure 1D and E). Subsequently, functional enrichment analysis was performed on these proteins. The Gene Ontology (GO)^28,29 results highlighted terms such as osteoblast differentiation (Figure 1F). Kyoto Encyclopedia of Genes and Genomes (KEGG)^30,31 analysis indicated that the differentially expressed proteins were associated with osteoclast differentiation (Figure 1G). Subsequently, the transcription levels of Sik1 in clinical samples of subchondral bone from an additional five patients with OA were assessed. A statistically significant decrease in the mRNA expression levels of Sik1 in the OA group was observed compared with the control group. This result corroborated the earlier proteomic data analysis (Figure 1H). Combined with the results of the functional enrichment analysis, it was hypothesized that Sik1 influenced the progression of OA by affecting the differentiation process of osteoblasts.

Sik1 expression is significantly reduced within the subchondral bone during the late stages of OA

The expression of Sik1 in the subchondral bone knee joints in patients with OA was assessed to validate the role of Sik1 in this disease. Immunohistochemistry (IHC) results revealed that Sik1 expression was higher in the control subchondral bone than in the OA group, indicating a downregulation of Sik1 expression in OA (Figure 2A and F). Moreover, patients with advanced OA exhibited significantly increased expression levels of osteogenesis-related genes including Runx2, ALP, OSX, and OCN in the subchondral bone, suggesting enhanced bone formation in this area (Figure 2B to F). Histopathological changes and subchondral bone content in clinical samples were assessed using several staining techniques, including Safranin O-fast green, hematoxylin and eosin, Masson, and Alizarin red. According to the hematoxylin and eosin staining, the extracellular matrix in OA subchondral bone was noticeably expanded compared to the control group (Figure 2G and K). Masson staining revealed an appreciable increase in fibrillogenesis of subchondral bone in the OA group, and the distinction between cartilage and subchondral bone was less pronounced than in the control group, illustrating the pathology of deteriorated cartilage in advanced OA (Figure 2H). Safranin O‑fast green staining displayed increased bone trabecular area in the OA group and a paler hue of cartilage tissue, attributed to the redistribution of proteoglycans released following cartilage deterioration (Figure 2I). Alizarin red staining highlighted calcium deposition in the trabecular bone post-new bone formation in the subchondral bone, with the positive area in the OA group being notably larger than in the control group (Figure 2J). Overall, in late-stage OA, osteoblasts were abnormally activated, leading to increased bone formation, which resulted in thickening of the subchondral bone plate and trabecula, accompanied by subchondral bone sclerosis. Sik1 expression significantly decreased during this phase, suggesting that Sik1 may play a pivotal role in the onset and progression of OA.

Figure 2.

Histopathological and immunohistochemical analysis of clinical samples showed that Sik1 expression was low in the subchondral bone. (A–E) Immunohistochemical analysis showed that the expression of Sik1 in the subchondral bone of the OA clinical specimens was lower than that of the control group, while the expression of RUNX2, ALP, OSX, and OCN in the subchondral bone of the OA clinical specimens was higher than that of the control group. Red triangles show positive cells, bar, 200 μm. (F) Quantization (A–E). (G–J) Hematoxylin and eosin, Masson, Safranin O, and Alizarin Red S staining showing the subchondral bone trabecular area and the relative quantitative analyses. Scale bar, 500 μm. (K) Quantization (G–J). (L–S) Flow cytometry analysis of the expression of surface markers of BMSCs. BMSCs were negative for CD14, CD34, CD45, HLA-DR, and NC3 and positive for CD73, CD90, and CD105, the latter of which are MSC-specific. Data are presented as the mean ± SD of three repeats. **P < 0.01, ***P < 0.001, ****P < 0.0001. BMSC, bone marrow stem cell; Sik1, salt-inducible kinase 1; ALP, alkaline phosphatase.

Sik1 expression is downregulated during BMSC osteogenic differentiation

To elucidate the regulatory relationship between Sik1 and osteogenic differentiation, the expression levels and changes of Sik1 during osteogenic differentiation induction in BMSCs were assessed. Sik1 transcription levels steadily decreased from days 0 to 14 following the initiation of osteogenic differentiation with OIM in BMSCs (Figure 3A). Similarly, Sik1 protein expression levels were also significantly reduced during differentiation (Figure 3B and C), in agreement with the bioinformatics analysis and IHC results. During differentiation, the transcriptional levels and protein expression levels of osteogenic genes, such as Runx2, ALP, OSX, and OCN were assessed. The results indicated a significant increase in the expression of all of these genes (Figure 3A to C). In addition, ALP activity increased in a time-dependent manner (Figure 3D and E). Collectively, these findings demonstrate the successful establishment of the BMSC osteogenic differentiation model.

Figure 3.

During BMSC osteogenic differentiation, Sik1 expression decreased, while overexpression of Sik1 inhibited BMSC osteogenic differentiation. (A and B) The mRNA and protein expression levels of Sik1 decreased in a time-dependent manner during osteogenic differentiation. Concurrently, the osteogenic differentiation index increased with the progression of the osteogenic differentiation process. (C) quantization (B). (D and E) ALP staining was performed on BMSCs on days 7 and 14 of osteogenic induction differentiation. Scale bar, 500 μm. (F) Overexpression of Sik1 resulted in decreased mRNA and protein expression levels of the osteogenic indicators. (H) Quantization (G). (I and J) Overexpression sik1 reduces ALP activity. Scale bar, 500 μm. Data are presented as the mean ± SD of three or five repeats. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. BMSC, bone marrow stem cell; Sik1, salt-inducible kinase 1; ALP, alkaline phosphatase.

Overexpression of Sik1 in BMSCs inhibits osteogenesis in vitro

To further understand the regulatory role of Sik1 in OA in vitro, Sik1 was overexpressed during the induced differentiation of BMSCs. The experimental findings revealed a substantial upregulation of Sik1 at both the transcriptional and translational levels in the overexpression group, thus confirming the efficacy of the overexpression approach (Figure 3F to H). Moreover, the pronounced overexpression of Sik1 yielded a substantial downregulation in the mRNA and protein expression levels of essential osteogenic markers, namely Runx2, OSX, ALP, and OCN (Figure 3F to H). The ALP staining results further corroborated the observations, revealing a marked reduction in ALP activity in the Sik1 overexpression group compared with the control group (Figure 3I and J). These findings collectively suggest the possibility that Sik1 Overexpression exerted a suppressive influence on the in vitro osteogenic differentiation of BMSCs.

Inhibiting the expression of Runx2 suppresses the osteogenic differentiation of BMSCs

To investigate the regulatory role of Runx2 in the development of OA, Runx2 was inhibited during the osteogenic differentiation of BMSCs. Initially, CCK8 experiments were performed which showed that treating BMSCs with a concentration of 200 μm CDAA522 (inhibitor of Runx2) for 24 h achieved optimal results without any adverse effects on cell viability (Supplementary material, Figure S3L and M). Following the inhibition of Runx2 expression, a noticeable reduction in the levels of OSX, ALP, and OCN was observed (Supplementary material, Figure S3A to I), along with a significant decrease in ALP activity (Supplementary material, Figure S3J and K). These findings suggest that inhibiting Runx2 expression effectively impeded the osteogenic differentiation of BMSCs.

Sik1 modulates osteogenic differentiation of BMSCs via Runx2 in vitro

To elucidate the mechanism through which Sik1 regulates osteogenic differentiation via Runx2, Sik1 was overexpressed while concurrently activating Runx2. Initially, CCK8 experiments were performed to determine the optimal concentration of MDP (activator of Runx2) and established that varying treatment durations did not adversely affect BMSCs (Figure 4F and G). Following the simultaneous overexpression of Sik1 and inhibition of Runx2 expression, noteworthy results were observed. Sik1 transcription and translation levels exhibited a significant increase, while the expression of Runx2 displayed the opposite trend. Simultaneously, a substantial reduction in the expression of osteogenic markers and a notable decrease in ALP activity was observed (Figure 4A to E). These findings suggest that overexpression of Sik1 and concurrent Runx2 inhibition effectively hindered the osteogenic differentiation of BMSCs. Subsequently, when Sik1 and Runx2 were concurrently overexpressed, it was evident that the Sik1 expression levels remained unaffected by variations in Runx2 expression. This indirectly suggested that Sik1 likely acted upstream of Runx2.

Figure 4.

Sik1 affects osteogenic differentiation via the regulation of Runx2. (A and B) Following Sik1 overexpression, inhibiting Runx2 using CA, and activating Runx2 using MDP, the alterations in the transcriptional and translational levels of Sik1 and osteogenic markers were assessed. (C) Quantization (B). (D and E) ALP staining was used to assess ALP activity in the induced BMSCs undergoing osteogenic differentiation up to the fourteenth day under various conditions. Scale bar, 500 μm. (F) Effect of varying concentrations of MDP on BMSCs; 10 μM MDP resulted in a pronounced toxic effect on BMSCs. Thus, 10 μM was identified as the optimal concentration of MDP for BMSCs treatment. (G) No apparent cytotoxicity was detected in BMSCs following treatment with the optimal concentration over different durations. Data are presented as the mean ± SD of three or five repeats. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. CA, CADD522; MDP, muramyl dipeptide; ALP, alkaline phosphatase; Sik1, salt-inducible kinase 1.

Next, expression of osteogenic markers and ALP activity were assessed, it was noted that under the conditions of Sik1 overexpression, the overexpression of Runx2 led to an increase in osteogenic marker expression levels and ALP activity compared to Runx2 inhibition. However, significant differences persisted when compared to the unaltered control group (Figure 4A to E). In summary, these results support the notion that Sik1 regulated osteogenic differentiation by modulating the activity of Runx2.

Drug intervention has no effect on the overall function of mice

To evaluate the impact of PA, CA, and MDP on mice in general, changes in their body weight during their growth were measured, and tissue samples of their livers, kidneys, spleens, and serum were collected for analysis. The research results indicated that over time, the body weight of mice in all groups increased, with no significant differences observed among the subgroups (Supplementary material, Figure S4A). Hematoxylin and eosin staining results showed that there were no notable structural changes in the liver (Supplementary material, Figure 5B), kidneys (Supplementary material, Figure 5C), and spleen (Supplementary material, Figure 5D) of the mice in each group when compared to the sham group. Functional changes were assessed through serum indicators, and there were no significant differences in liver, kidney, spleen, heart, and glucose metabolism functions between the sham group and the other groups (Supplementary material, Figure S5A to D), indicating that the interventions used in the present study did not have any adverse effects on the mice. However, there were significant differences in antioxidant indicators. When compared to the sham group, the DMM surgery group exhibited a significant increase in MDA levels, along with significant decreases in SOD, GSH-px, CAT, GSH, and T-AOC levels (Figure S5E). These differences were more pronounced at 8 weeks compared to 4 weeks. Importantly, the overexpression of Sik1 reversed this trend, suggesting that Sik1 overexpression can enhance the antioxidant capacity of mice in the OA model, thereby slowing down the progression of OA. During the osteogenic differentiation of BMSCs, overexpression of Sik1 elevates the levels of reactive oxygen species (ROS) (Figure S6). This phenomenon has been validated through in vitro experiments, further confirming the role of Sik1 in enhancing the antioxidant capacity of BMSCs.

Figure 5.

Effects of the interventions on the growth and development of mice and on the structures of the liver, spleen, and kidney. (A) Schematic representation outlining the design of the animal experiment. Intraperitoneal injections were administered by group initiation on the fourth day after DMM. All treatments were delivered twice a week for 8 weeks, wherein both the sham and DMM groups received an equivalent volume of normal saline. Histological examination using hematoxylin and eosin staining of the (B) liver, (C) kidney, and (D) spleen of the mice revealed no significant alterations in the basic structural characteristics when compared to the Sham group. Scale bar, 50 and 200 μm. DMM, destabilizing the medial meniscus.

Sik1 alleviates subchondral osteosclerosis via Runx2 in vivo

To validate the mechanism of action of Sik1 in OA, in vivo experiments were used to alter the expression levels of Sik1 and Runx2 and then evaluated the impact of Sik1 on OA using histological staining and μ-CT scanning. Safranin O-fast green staining results revealed that compared to the sham group, the DMM surgery group showed a significant increase in OARSI scores (Figure 6A and B, and S), indicating worsened cartilage damage. Hematoxylin and eosin staining showed that the HC became thinner, while the CC became thicker, and the cartilage surface exhibited widespread damage, appearing rougher (Figure 6G and H, and T–U). The results of IHC analysis indicate a decrease in the expression levels of Aggrecan and Col-II in the extracellular matrix (ECM) of cartilage (Supplementary material, Figure S7A and B, D and E). μ-CT analysis demonstrated that 8 weeks postsurgery, the DMM surgery group exhibited evident subchondral osteosclerosis, with significant increases in BV/TV, Tb. N, and BMD, and a significant decrease in Tb.Sp (Figure 7A and B, G and H, M and N, and S to V). However, these changes were alleviated following Sik1 overexpression. Compared to the DMM group, DMM mice overexpressing Sik1 exhibited thicker HC and thinner CC (Figure 6H and I). Furthermore, the elevated OARSI scores (Figure 6B and C), the decrease in Aggrecan and Col-II (Figure S7C), and the subchondral osteosclerosis (Figure 7B and C, H and I, and N and O) observed in the DMM group were reversed by Sik1 overexpression. These findings indicate that Sik1 overexpression alleviated cartilage damage and subchondral osteosclerosis caused by OA.

Figure 6.

Overexpression of Sik1 reversed cartilage destruction in the DMM mouse model. (A–F) Safranin O-fast green staining results in mice. (G–L) Hematoxylin and eosin staining of mice; black and white lines denote the thickness of HC (hyaline cartilage) and CC (calcified cartilage), respectively. (M–R) Results of Masson staining in mice. (S) OARSI score reflects structural damage of the cartilage in mice. (T–U) Thickness of the HC and CC. Scale bars, 50 and 100 μm. Data are presented as the mean ± SD of measurements taken by five different examiners. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. HC hyaline cartilage; CC, calcified cartilage; DMM, destabilizing the medial meniscus; OARSI, Osteoarthritis Research Society International; Sik1, salt-inducible kinase 1.

Figure 7.

Overexpression of Sik1 reversed subchondral bone sclerosis in the DMM mouse model. (A–F) 2D μ-CT scans depicting the tibial subchondral bone. (G–L) 3D reconstructions derived from the μ-CT images. (M–R) 2D μ-CT reconstructions showcasing tibial subchondral bone. Quantification of the bone morphological parameters: (S) BV/TV, (T) Tb.N, (U) BMD, and (V) Tb.Sp. Data are presented as the mean ± SD of eight mice. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Sik1, salt-inducible kinase 1; DMM, destabilizing the medial meniscus; CT, computed tomography; BV/TV, bone volume fraction; Tb.N, trabecular number; BMD, bone mineral density.

Subsequently, the role of Runx2 in OA was validated, and the inhibition of Runx2 expression led to improvements in cartilage damage (Figure 6B to D, and H to J) and subchondral osteosclerosis (Figure 7B to D, H to J, and N-P) in DMM mice. Combining these results, Sik1 was overexpressed while simultaneously inhibiting or activating Runx2 expression. In comparison to the classic cartilage damage induced by DMM, Sik1-overexpressing DMM mice, irrespective of whether Runx2 was inhibited or overexpressed, exhibited significant improvements in pathological staining after 8 weeks: OARSI scores decreased (Figure 6B, E and F), CC thickness decreased, HC thickness increased (Figure 6H, K and L) and overall cartilage damage improved. μ-CT results also demonstrated that compared to the DMM group, BV/TV, Tb. N and BMD decreased, while Tb. Sp increased in the intervention groups, indicating a reversal of subchondral osteosclerosis (Figure 7B, E, F, H, K and L, N, and Q and R). While both interventions resulted in improvements, mice overexpressing both Runx2 and Sik1 exhibited a more significant reduction in cartilage damage (Figure 6S to V) and subchondral osteosclerosis (Figure 7S–V) compared to those with Runx2 inhibition. These combined results strongly support the role of Sik1 in mitigating subchondral osteosclerosis in OA through its modulation of Runx2, thereby inhibiting the progression of OA.

Discussion

OA is a common chronic degenerative disease with a multifaceted pathophysiology influenced by numerous factors. The prevalence of osteoarthritis (OA) has been on the rise in recent years. Its advanced stages are characterized primarily by the deterioration of articular cartilage, inflammation of the synovial membrane, and hardening of the subchondral bone³². Although considerable research has long focused on the degeneration of articular cartilage, more recently there has been a gradual increase in research on the role of the subchondral bone in recent years. Sik1, as a protein kinase, plays a role in various physiological processes, including metabolic regulation, cell proliferation, inflammation, and neural transmission.^33,34 It achieves these functions by regulating intracellular signaling pathways and is associated with the progression of several chronic diseases.^16,17 Sik1 is considered a key regulator in bone metabolism, exerting a negative influence on both in vivo and in vitro bone formation through the activation of the CRTC1-CREB pathway.¹⁹ This further validates the accuracy of the experimental findings of the present study. Following the suppression of Sik1/2 and CRTC2, a significant reduction in parathyroid hormone-related protein (PTHrP) stimulation on Wnt 7b and Wnt 11 transcription levels was observed. This suggests the potential involvement of Sik1 in phosphorylating co-activators bound to p-Smads in the nucleus, thereby modulating the transcription of Wnt 7b and Wnt 11 in osteoblasts.³⁵ This also supports the notion that silencing Sik1 can enhance Wnt/β-catenin activation.³⁶ Another study related to PTH found that several key biological effects of PTH 1R in bone development and remodeling are achieved through the inhibition of Sik1.³⁷ Sik1 also plays a distinctive role in osteoclastogenesis; inhibiting Sik1 significantly reduces bone pit formation in RANKL-stimulated RAW 264.7 cells and primary BMM, thereby significantly impacting osteoclast generation.³⁸ Current therapeutic approaches for bone metabolic disorders, such as osteoporosis and OA, predominantly target osteoclasts (bisphosphonates or cathepsin K inhibitors) or osteoblasts (synthetic analogs of parathyroid hormone) to address bone remodeling³⁸. Sik1 may represent a potential novel therapeutic target for bone metabolic disorders. Our research specifically focuses on the role of Sik1 in osteoblasts, aiming to elucidate the underlying relationships in this context. In this study, proteomic analysis of subchondral bone was conducted, revealing a marked reduction in Sik1 expression in the subchondral bone of OA patients. A significant negative correlation between Sik1 and OA was found. Combining the functional enrichment analysis of differentially expressed genes, it was hypothesized that Sik1 may influence the progression of OA by regulating osteogenic differentiation. In the subchondral bone samples of patients with OA, the expression of Sik1 is significantly reduced, consistent with the results of bioinformatics analysis. Furthermore, during the induction of osteogenic differentiation of BMSCs, the expression of Sik1 decreased in a time-dependent manner. These findings confirmed our hypothesis.

OA is a disease that affects the entire joint and has an impact on all the structures surrounding the joint. Subchondral bone and cartilage together constitute the bone-cartilage unit, a complex structure integral to the pathophysiology of OA.^39,40 In the development of OA, early pathological changes in subchondral bone include an abnormal increase in bone turnover, with abnormally activated osteoclasts causing bone resorption to exceed bone formation, leading to early transient osteoporosis. As OA progresses to its later stages, there is a shift in the role of osteoblasts. This change effectively reverses the earlier bone metabolism process, leading to increased bone formation, which in turn results in an increase in bone mass and the hardening of the subchondral bone.^12,41 Unfortunately, these positive changes in the bone are offset by the gradual weakening of the cartilage’s protective function, which ultimately accelerates the progression of OA.⁴² DMM is currently one of the most commonly used surgical models for the study of chronic OA in mice.⁴³ It changes the mechanical stability of the knee joint by severing MMLT, thereby inducing cartilage injury, subchondral osteosclerosis, and osteophyte formation.^44,45 This process closely resembles the pathological development of OA in humans.^43,44,46 DMM surgery is characterized by its low cost, high reproducibility, and reliability.⁴⁶ Yan et al.⁴⁷ conducted a study using the DMM-induced OA model and observed late-stage pathological changes in OA ∼8 weeks after the surgical procedure. In the present study, late-stage changes of OA were also observed, including cartilage degradation and subchondral bone sclerosis, at 8 weeks postsurgery. Notably, the overexpression of Sik1 was found to have a mitigating effect on these changes, effectively slowing down the progression of OA. Furthermore, research has identified Runx2 as a major regulatory factor in osteoblast differentiation and bone formation. Knocking down the expression of Runx2 can inhibit osteoblast differentiation and other regulatory factors related to bone formation.^22,48 OSX plays a pivotal role in the regulation of osteoblast differentiation and bone formation, functioning downstream of Runx2.^49,50 Binding sites exist between the two, and they form a Runx2-OSX complex through the interaction of protein-protein interactions and binding to homologous DNA sequences.^51,52 This complex acts synergistically to activate the genetic program of osteoblasts, resulting in the production of bone-specific matrix,⁵¹ the results of the present study further substantiate this fact. Inhibiting the expression of Runx2 can be observed to suppress the osteogenic differentiation of BMSCs. Similarly, when Runx2 expression is inhibited in vivo, it can be observed that subchondral bone sclerosis in DMM mice is reversed, thereby slowing the progression of OA. These findings underscore the pivotal roles played by Sik1 and Runx2 in the development of OA.

Sik1 primarily regulates gene expression by interacting with transcription factors.³³ Runx2, a major transcription factor associated with the Runt family, may be inhibited by Sik1, thus slowing down the promotion of OA. In the present study, both in vivo and in vitro experiments demonstrated that intervention targeting Runx2 expression did not affect the transcription and translation levels of Sik1, whereas changes in Sik1 levels affected the expression of Runx2, thereby indicating that Sik1 acts on targets upstream of Runx2. In in vitro experiments, Runx2 was inhibited or overexpressed while simultaneously overexpressing Sik1. It was observed that both treatments could promote osteogenic differentiation of BMSCs. Compared with the overexpression of Sik1 and inhibition of Runx2, the promoting effect of simultaneously overexpressing Sik1 and Runx2 was more significant, but it still did not reach the level of the simple induction differentiation group. Runx2 overexpression did not fully reverse the reduced osteogenic differentiation induced by Sik1 overexpression to the pre-intervention level. During in vivo experiments, it was observed that both inhibition or activation of Runx2 expression following Sik1 overexpression improved subchondral osteosclerosis in DMM mice, but inhibition of Runx2 expression resulted in a more significant improvement than that in mice with Runx2 overexpression. This suggests that activation of Runx2 expression attenuates the remission effect of subchondral osteosclerosis induced by Sik1 overexpression. It was confirmed that Sik1 affected osteogenic differentiation and subchondral bone sclerosis by regulating the activity of Runx2, thereby influencing the progression of OA. Several potential mechanisms are proposed here. First, Sik1 may modulate the expression or activity of Runx2 by regulating specific signaling pathways. For example, there is an interaction between Sik1 and the AMPK signaling pathway,³³ and AMPK has been shown to affect the activity of Runx2.^53,54 Second, Sik1 may directly interact with Runx2 at the transcriptional level, thereby regulating osteoblast differentiation and bone matrix synthesis. In addition, it is speculated that Sik1 may also influence Runx2 through other pathways yet to be discovered, such as by modulating signaling pathways such as TGF-β, NF-κB, MAPK, or PI3K/Akt. Further research is required to validate these potential mechanisms. Based on the findings of the present study as well as the literature review, an explanation for the role of Sik1 is hypothesized; when Sik1 is activated in the cell nucleus, it impedes the expression of Runx2 or the Runx2-OSX complex, thereby suppressing the expression of genes related to bone formation. In the context of OA, PKA phosphorylates Sik1 which then binds to 14-3-3, resulting in its localization and inactivation in the cytoplasm.⁵⁵ In the cell nucleus, the absence of the inhibitory effect of Sik1 promotes the expression of genes associated with bone formation by Runx2, consequently accelerating the progression of OA (Figure 8).

Figure 8.

Mechanistic diagram. The potential mechanisms for the role of Sik1 in OA. Sik1 inhibits the expression of the Runx2 or Runx2-osx complex in the cell nucleus, thereby regulating the promoting effect of OA. In OA, Sik1 is phosphorylated by PKA, and binds with 14-3-3, leading to its translocation and inactivation in the cytoplasm, and Runx2 promotes the production of bone-forming genes, accelerating the progression of OA without the inhibitory effect of Sik1 in the nucleus. Sik1, salt-inducible kinase 1; OA, osteoarthritis; PKA, protein kinase A.

In future studies, validating the interaction between Sik1 and Runx2, as well as their roles in the development of OA should be addressed. Co-immunoprecipitation techniques will be used to detect their direct interaction, as well as employing analytical methods at both the transcriptional and protein levels to assess the regulatory effects of Sik1 on the expression and activity of Runx2. These experiments will enhance our understanding of the Sik1-Runx2 interaction and lay the groundwork for novel OA therapeutic strategies.

Materials and Methods

Clinical sample collection and analysis

Tibial plateau samples were obtained from 60 primary knee osteoarthritis patients undergoing total knee replacement surgery at the Third Department of Orthopedics, General Hospital of Ningxia Medical University, between June 2022 and June 2023. The weight-bearing tibial plateau was designated as the OA group, while the contralateral tibial plateau served as the control group. Cartilage and subchondral bone were separated. Fresh tissue samples were sectioned and sequentially rinsed three times with cold PBS followed by saline. They were subsequently stored at –80 °C for future analysis. We conducted statistical analyses on the patient’s age, BMI, preoperative visual analog scale (VAS) pain score, Lysholm score, Western Ontario and McMaster Universities (WOMAC) Osteoarthritis Index, and Kellgren–Lawrence (K-L) grade. The diagnosis of OA was confirmed following the American College of Rheumatology’s diagnostic criteria. Patients presenting with secondary OA, including those with underlying conditions like connective tissue diseases or trauma, were excluded from the study. Clinical data for all patients were sourced from the hospital’s information system.

Prior to enrollment in this study, informed consent was obtained from all patients. The study received approval from the Ethics Committee of the General Hospital of Ningxia Medical University, under approval number KYLL-2021-269. Patient information was securely stored to ensure confidentiality.

Tandem mass spectrometry using liquid chromatography and label-free measurement of tissue proteomes

After random selection, tissue samples from 30 patients were used for subsequent experiments. These include 30 tissue samples from the OA group and 30 tissue samples from the control group. Each group of 10 samples was mixed to form one sample, ultimately creating three OA group samples and three control group samples. Subsequently, label-free quantification and tandem liquid chromatography/mass spectrometry on tissue proteomic samples were conducted. Trypsin was used to digest the protease into short peptides, which were then separated using a high pH reversed-phase peptide process. The data collection process utilized liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The analysis of results was conducted with the LIMMA package (version 3.54.0)⁵⁶ in R (version 4.2.2) to identify differentially expressed proteins between the disease group and the control group. The criteria for determining differentially expressed proteins were a |logFC| >1 and P values < 0.05. The pheatmap and ggplot2 packages in R were utilized to create visualizations of differentially expressed proteins. Specifically, heatmaps were generated using pheatmap, while volcano plots were produced with ggplot2.

Cell culture

BMSCs were extracted from the femur and tibia of 6-week-old C57/BL6J mice using the standard whole bone marrow direct adherence method. The bone marrow was briefly rinsed with DMEM-low glucose (Gibco; Thermo Fisher Scientific, Inc.), then cultured in DMEM supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.), 100 units/mL penicillin, and 100 µg/mL streptomycin. Following a 3-day incubation period, nonadherent cells were discarded, and the culture medium was replenished with fresh media. The cells were cultured in a humidified incubator set at 37 °C with 5% CO₂. Flow cytometry was utilized to isolate BMSCs, identifying them based on the presence of CD73, CD90, and CD105 and the lack of CD14, CD34, CD45, HLA-DR, and NC3 (Figure 2L to S). BMSCs from passages 3–5 were induced to differentiate. For osteoblast differentiation induction, 10 mM β-glycerophosphate (MilliporeSigma), 100 nM dexamethasone (MilliporeSigma), 50 µM ascorbic acid (MilliporeSigma), and supplemented DMEM were used as the osteogenesis induction media (OIM). BMSCs were initially seeded into six-well plates at a density of 5 × 10⁵ cells per well. When the cells reached 80% confluence, the complete medium was replaced with osteogenic induction medium (OIM), which was refreshed every 48 h.

Lentiviral transduction

GeneCopoeia’s lentiviral vectors (pEZ-Lv195) were employed to overexpress Sik1, utilizing the OmicsLink^™ Expression Clone (EX-Mm30183-Lv195; GeneCopoeia, Inc., Rockville, MD) and the Negative Clone (EX-NEG-Lv195; GeneCopoeia, Inc., Rockville, MD). Utilizing the calcium phosphate technique, 293 T cells (CL-0005; Pricella) were cotransfected with 10 µg of lentiviral vector, packaging plasmid psPAX2, and envelope plasmid pMD2.G. Transfection was carried out with Lipofectamine 3000 (catalog no. L3000001; Thermo Fisher Scientific Inc.). The cells were initially incubated at 37 °C for 6–8 h. Following this, the medium was replaced, and the incubation was extended for an additional 48 h. Subsequently, the supernatant was collected and passed through a 0.45 µm filter to obtain the viral particles. The viral supernatant was mixed with polybrene to achieve a final concentration of 10 µg/mL, following which it was applied to BMSCs and incubated overnight at 37 °C. After 24 h, the medium was refreshed, and the cells were maintained in continuous culture. Puromycin at a concentration of 2 µg/mL was used to select cells overexpressing Sik1. Continuous selection and passaging were performed for three generations to ensure stable expression. These procedures resulted in BMSCs that stably overexpressed the Sik1 gene, which were subsequently utilized for further experiments.

Western blotting

The cells were lysed on ice using the Mammalian Protein Extraction Reagent (Thermo Fisher Scientific, Inc.). Protein concentration was measured with a BCA assay kit (Nanjing KeyGen Biotech Co., Ltd) according to the manufacturer’s instructions. The samples were then denatured by heating at 100 °C for 20 min with loading buffer (Takara Bio, Inc.) to prepare protein extracts. Identical protein quantities (25 µg per sample) were subjected to separation using a 10% SDS-PAGE gel. Subsequent to separation, the proteins were transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were then blocked with 5% nonfat dry milk for 60 min at room temperature. Following this, they were incubated overnight at 4 °C with primary antibodies against Sik1 (1:1,000, Abcam, Cat: ab217809), Runx2 (1:1,000, Abcam, Cat: ab236639), OSX (1:1,000, Abcam, Cat: ab209484), OCN (1:1,000, Abcam, Cat: ab133612), ALP (1:1,000, Abcam, Cat: ab67228), and either β-Actin or GAPDH (1:5,000, Abcam, Cat: ab8226, Cat: ab181602) as loading controls. The membranes were washed five times with TBST-20 and subsequently incubated with an HRP-conjugated secondary antibody (1:10,000, Abcam, Cat: ab175733) for 1 h at room temperature. Following additional washes, the blots were visualized using an enhanced chemiluminescence kit (ABclonal) and analyzed with ImageJ software (version 1.48, National Institutes of Health).

Extraction of RNA and reverse transcription-quantitative PCR (RT-qPCR)

Total cellular RNA was extracted using TRIzol^® reagent (Thermo Fisher Scientific). Subsequently, 1 µg of RNA from each sample was reverse transcribed into cDNA with the PrimeScript^™ RT reagent kit (Takara Bio) incorporating random hexamer primers, adhering to the manufacturer’s protocol. qPCR analysis was conducted using the TB Green^® Premix Ex Taq^™ II (Takara Bio) on an iQ5 real-time PCR system (Bio-Rad Laboratories). The prepared reaction mixtures, with a total volume of 20 µL, comprised the following components: 10 µL of 2× TB Green Premix Ex Taq II, 0.4 µL of forward primer (10 µM), 0.4 µL of reverse primer (10 µM), 1 µL of cDNA template (equivalent to 50 ng), and 8.2 µL of nuclease-free water. The thermal cycling protocol utilized an initial denaturation at 95 °C for 2 min succeeded by 40 cycles. Each cycle included a denaturation step at 95 °C for 15 s and an annealing/extension step at 60 °C for 30 s. β-Actin was used as the quantitative internal control gene, and data were analyzed using the 2^-ΔΔCt method. All reactions were executed in quintuplicate, and the specific primer sequences used for qPCR are listed below: Sik1 (NM_010831) forward 5′-CAAGACACGGTTAGATTCTAGCAAT-3′ and reverse 5′-CTGTGACAATGTAGAGCATATCCTT-3′; Runx2 (NM_009820) forward 5′-AGCAGCACTCCATATCTCTACTAT-3′ and reverse 5′-TCAGCGTCAACACCATCATTC-3′; ALP (NM_007431) forward 5′-GAATCGGAACAACCTGACTGAC-3′ and reverse 5′-GGTCCATCTCCACTGCTTCA-3′; OSX (NM_130458) forward 5′-TCACCTGCCTGCTCTGTTC-3′ and reverse 5′-GCGGCTGATTGGCTTCTTC-3′; OCN (NM_007541) forward 5′-CCTGAGTCTGACAAAGCCTTC-3′ and reverse 5′-GCGGTCTTCAAGCCATACTG-3′; β-Actin (NM_007393) forward 5′-GCTACGTGGCCCTCGACTTC-3′ and reverse 5′-CTCGTGGATGCCGCAGGATT-3′.

Histopathological analysis and immunohistochemical analysis

Samples were decalcified for 21 days in an EDTA-buffered saline solution (pH 7.4, 0.25 mol/L) following fixing overnight in 4% paraformaldehyde. The longitudinal tissue sections, each 4 µm thick, were analyzed using a range of stains, including Safranin O-fast green, hematoxylin and eosin, Masson’s trichrome, and Alizarin red, to assess histological alterations. The articular cartilage was graded using the Osteoarthritis Research Society International (OARSI) grading system. Measurements were taken of both hyaline cartilage (HC) and calcified cartilage (CC) thickness. Observations were made with an Olympus DP74 microscope (Olympus Corporation), and ImageJ software was used for image analysis. For immunohistochemistry (IHC) analysis, tissue sections were incubated overnight at 4 °C with primary antibodies targeting Sik1, Runx2, OCN, ALP, or OSX (all at 1:200 dilution, Abcam). The following day, sections were incubated at room temperature for 1 h with HRP-conjugated secondary antibodies (1:2,000, Abcam).

ALP staining

BMSCs seeded at 5 × 10⁵ cells/well in six-well plates were induced with OIM after 24 h. ALP staining assessed their osteogenic capacity on days 3, 7, and 14 post-induction. Cells were rinsed thrice with PBS, fixed with 4% paraformaldehyde for 20 min, and stained with 5-bromo-4-chloro-3-indolyl phosphate (BCIP)/nitroblue tetrazolium (Beyotime Institute of Biotechnology, C3206) for 30 min, as per the manufacturer’s instructions. The reaction was halted by adding distilled water. The images were captured using an Olympus DP74 light microscope. Following the manufacturer’s guidelines, ALP activity was assessed with an ALP Activity Assay Kit (Beyotime Institute of Biotechnology, P0321S).

CCK-8 assay

A CCK-8 assay (Dojindo Molecular Technologies, Inc.) was used to investigate the effects of CADD522 (CA) and Muramyl dipeptide (MDP) on BMSC viability at various concentrations and treatment durations. CA is an inhibitor of Runx2 (MedChemExpress),⁵⁷ while MDP is an agonist of Runx2 (MedChemExpress).⁵⁸ Cells were seeded in 96-well plates at a density of 5 × 10⁵ and treated with 100 μL of WST-8. After incubating at 37 °C for 1 h, absorbance was measured at 450 nm using an Infinite^® 200 PRO microplate reader.

Construction of the DMM model and animal experiments

A total of 96 female C57BL/6 mice were used in the present study (Provided by Ningxia Medical University, Laboratory Animal Center). All 8-week-old mice (weighing 19–20 g) were kept in a controlled environment at 25 °C with 48% humidity, under a 12-h light/dark cycle, and had unlimited access to food and water. Changes in body weight were documented. The Ningxia Medical University Laboratory Animal Center provided the setting. The mice were divided into six groups (n = 16 per group): Control (sham group), OA (DMM group), DMM + Phanginin A (PA) group, DMM + CA group, DMM + PA + CA group, and DMM + PA + MDP group. Following intraperitoneal injection of 0.2% pentobarbital sodium (40 mg/kg) to induce anesthesia, the OA model was established via the DMM procedure on the right knee joint. After anesthesia, the surgical area was shaved, and the mouse was placed in the supine position with the right hind limb flexed at 90°. The surgical field was thoroughly disinfected and the patellar ligament was exposed (Supplementary material, Figure S1A). The skin along the medial edge of the patellar ligament was excised, the joint capsule was bluntly dissected, and, upon removal of adipose tissue within the joint, the medial meniscus connected to the tibial plateau was observed through the medial meniscotibial ligament (MMTL) (Supplementary material, Figure S1B). After transecting the MMTL (Supplementary material, Figure S1C), the incision was rinsed and the joint cavity was sutured with 7-0 surgical thread, followed by closure of the skin incision. The sham surgery group involved only the incision and layer-by-layer suturing of the joint capsule. The skin incision healed within 4 days post-DMM, and drugs were administered intraperitoneally based on their respective groups. All treatments were given twice a week for 8 weeks, with both the sham and DMM groups receiving an equivalent volume of normal saline. PA (MedChemExpress) is an effective activator of Sik1 expression.^59,60 A total of 4 or 8 weeks postoperation, 8 mice from each group were euthanized by cervical dislocation, and the blood, liver, kidneys, spleen, and knee joint tissues were collected. Serum samples were derived from blood by centrifuging at 3,000 g for 15 min and stored at –80 °C for later analysis. Similarly, liver, kidney, spleen, and knee tissues were appropriately preserved for follow-up research. Figure 5A shows the experimental design.

All animal experiments were conducted with the approval of the Institutional Animal Care and Use Committee (IACUC) at Ningxia Medical University, in compliance with the Institutional Animal Testing Center’s guidelines (approval number IACUC-NYLAC-2023-099).

μ-computed tomography (CT) analysis

The mouse knee joint was scanned using a μ-CT scanner (SkyScan 1276; Bruker Belgium S.A./N.V.), and reconstructed μ-CT images were generated using NRecon version 1.6. Data analysis was performed using the CTAn version 1.9 software (Bruker Belgium S.A./N.V.), and 3D models were visualized using CTVox version 3.3.1 software (Bruker Belgium S.A./N.V.). Quantitative morphometric indices were derived from the microtomographic data based on 3D measurements and visual assessment of the images. The regions of interest for bone parameter analysis were identified as the knee cartilage and subchondral bone. Several metrics were evaluated, including bone volume fraction (BV/TV), bone mineral density (BMD), trabecular number (Tb. N), and trabecular separation (Tb. Sp).

Statistical analysis

The data analysis was performed using GraphPad Prism version 9.0 (GraphPad Software, Inc.) and R. Results are reported as the mean ± standard deviation. A Student’s t test was applied to compare differences between two groups. For comparisons among three or more groups, a variance homogeneity test was first conducted, followed by one-way ANOVA and Tukey’s post hoc multiple comparison test. Statistical significance was set at P < 0.05.

Conclusion

In summary, the present study delved into the role of Sik1 in OA and highlighted its pivotal role in regulating bone metabolism. The findings demonstrated a significant decrease in Sik1 expression levels in patients with OA, and further experiments confirmed the influence of Sik1 on osteogenic differentiation and subchondral bone sclerosis by modulating the activity of Runx2, thereby exerting a crucial impact on OA progression. These findings not only deepen our understanding of the pathological processes underlying OA but also provide novel avenues for developing more precise and effective treatment strategies. It is hoped that these advancements will improve the quality of life for patients with OA and enhance the level of disease management.

Funding Statement

This study was supported by National Natural Science Foundation of China (No. U22A20285); National Natural Science Foundation of China (No. 82160433); Key R&D Project of Autonomous Region (No. 2023BEG02018); Autonomous Region Major Scientific and Technological Achievements Transformation Project (No. 2023CJE09037);Ningxia Medical University General Hospital "Medical Engineering Special" (No. NYZYYG-001).

Author Contributions

Qunhua Jin and Di Xue initiated and designed the study; Kuanmin Tian, Xiaoxin He, Xue Lin, Xiaolei Chen, Yajing Su, Long Ma, Zhibin Lan and Gangning Fen performed animal and molecular biological experiments; Zhidong Lu, Zhirong Chen, Xin Zhao, Liang Zhang and Peng Li analyzed data; Qinqin Hai contributed to data interpretation; and Kuanmin Tian drafted the manuscript. Kuanmin Tian and Xiaoxin He contributed equally to this work. All authors approved the submitted version of the manuscript.

Data Availability Statement

The data sets produced in this study are available in the following databases: ProteomeXchange Consortium: PXD052989 (https://proteomecentral.proteomexchange.org/cgi/GetDataset?ID=PXD052989).

Disclosure Statement

No potential conflict of interest was reported by the authors.

Ethics Statement

Ethics approval for this study was obtained from the ethics committee of the Ningxia Medical University General Hospital (KYLL-2021-269). All participants provided informed consent. All animal studies were conducted in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of Ningxia Medical University (approval no. IACUC-NYLAC-2023-099).