Silibinin Promotes Bone Regeneration Through HIF-1α/VEGF and Notch Signaling Pathway in Ovariectomized Rats

Objective The purpose was to observe whether systemic administration with silibinin(SIL) have an positive effect on bone defect regeneration through HIF-1α/VEGF and Notch signaling pathway in an ovariectomized(OVX) rat model. The MC3T3-E1 cells were co-cultured with lower SIL and higher SIL and induced to osteogenesis, and the cell viability, osteogenic activity were observed by Cell Count Kit-8(CCK-8), Alkaline phosphatase (ALP) staining, Alizarin Red(RES) staining and Western blotting(WB). After the drilling defect model was established, the OVX rats were treated with SIL for 12 weeks. Micro-CT, histology and Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis were used to observe the therapeutic effect and explore the possible mechanism. CCK-8, ALP and ARS staining results show that the cell mineralization and osteogenic activity of LSIL and HSIL group is signicantly higher than the Con group. Protein expressions show that related regulatory proteins such as ALP, OPN, RUNX-2, OC, VEGFA, HIF-1α, Notch 1, JAG 1, HEY 1 and HES 1 of LSIL and HSIL group are signicantly higher than Con group. Micro-CT and Histological analysis evaluation show that group SIL + OVX presented the stronger effect on bone regeneration, bone mineralization, higher expression of VEGFA and HIF-1α, when compared with OVX group. RT-qPCR analysis shows that SIL + OVX group showed increased Notch 1, HES1, HEY1 and JAG1 than the OVX group(p < 0.05). with SIL a scheme for rapid condylar and these effects may activating HIF-1α/VEGF and Notch signaling pathway. bone defects is lacking and limited. Based on these previous studies, hypothesized that systemic administration with silibinin may have a positive effect on bone defect regeneration through Notch signaling pathway and HIF-1α/VEGF signaling pathway in an OVX rat model. The aim of the present study was to investigate the effect of systemic administration with silibinin on bone defect in an OVX rat model, and preliminary exploration of possible mechanisms by activating the Notch signaling pathway and HIF-1α/VEGF signaling pathway.


Introduction
Postmenopausal osteoporosis is characterized by reduced bone mineral density (BMD) and deterioration of bone microarchitecture resulting in increased bone fragility and an increased risk of developing fractures, which commonly happened in postmenopausal women [1,2]. Most osteoporotic fractures and bone defects are commonly seen and happen after low-energy trauma [3,4]. The bone is a kind of mineralized connective tissue which has the ability to repair minor injuries by remodeling. When the bone defect reaches a certain limit, it is di cult to complete the defect healing by its own repair ability. The autologous bone graft has good biocompatibility and bone-inducing ability, demonstrating the gold standard for bone defect healing [5]. However, autologous bone grating is limited by donor site morbidity, which will inevitably lead to extension of surgical time, increase of surgical sites and infection chance and aggravation of patient pain [6,7]. Especially for the elderly with many basic diseases and they are not allowed to tolerate long-term surgery. Therefore, it poses new challenges for our clinicians.
Silibinin(SIL), a avonoid obtained from the dried fruits of Silybum marianum L. [8], is mainly composed of four avonoids and can exert multiple biological effects [9]. Previous studies also have reported that SIL supplementation is not only able to prevent oxidative stress-induced cell cytotoxicity in bone marrow mesenchymal stromal cells (MSCs), but also e ciently inhibited oxidative stress and protects their osteoblastic differentiation [10][11][12][13]. Currently, SIL is also reported to be potential in regulating osteoblastic cell growth and functions, and maintaining bone mass [14,15]. Vascular endothelial factor (VEGF) can not only promote survival, proliferation and migration of endothelial cells, but also stimulate the growth of new blood vessels into damaged sites and regulate osteogenic growth factors to stimulate bone repair and bone regeneration. [16]. Hypoxia inducible factor 1(HIF-1), a transcription factor, expressed ubiquitously in almost all mammalian cells, which becomes activated under hypoxia [17].
Moreover, VEGF is a major target gene of HIF-1α. Activation of VEGF signaling activates the HIF-1α signaling pathway to promote angiogenesis and bone growth [18]. Recently, accumulating evidence proves that notch signaling regulates mesenchymal stem cells proliferation and differentiation, skeletal tissue development and regeneration [19].
Although current studies have con rmed that silibinin play an important role in the process of bone remodeling, systemic administration of silibinin in the treatment of osteoporotic bone defects is lacking and limited. Based on these previous studies, hypothesized that systemic administration with silibinin may have a positive effect on bone defect regeneration through Notch signaling pathway and HIF-1α/VEGF signaling pathway in an OVX rat model. The aim of the present study was to investigate the effect of systemic administration with silibinin on bone defect in an OVX rat model, and preliminary exploration of possible mechanisms by activating the Notch signaling pathway and HIF-1α/VEGF signaling pathway.

Materials And Methods
Experimental animals 30 mature female Sprague Dawley rats(Purchased at Zhejiang Laboratory Animal Center, Zhejiang, China), aged 3 months with a body weight of 230±26 g, were included in this study. Every four animals were acclimatized in a standard cage under a 12-hour light-dark cycle and allowed free access to water and rodent diet. All the animal experiments were conducted in accordance with international standards on animal welfare as well as being compliant with the Animal Research Committee of the university(approval number:LLSC-2020-082).

Animal experiments
All osteoporosis models of estrogen de ciency induced by bilateral ovariectomy were established as described previously [20,21]. 12 weeks after sham operation(Sham) and bilateral ovariectomy(OVX), all rats were randomly divided into 3 groups: Sham group (Sham), OVX rats group (OVX) and OVX rats + silybin treatment group (SIL+OVX). Next, the rats of three groups established an intramedullary defect in the distal femur (1.5 mm in diameter×4mm in depth) using a 1.5 mm diameter clinical drill bits(Stryker Corporation, US) as described previously [22,23]. The rats were classi ed to SIL+OVX were treated with silibinin intraperitoneally with a dose of 50mg/kg (Sigma-Aldrich) [24]. Two intraperitoneal injections of calcein(20 mg/kg) were injected on the 3rd and 10th day before the rats were sacri ced. After 12 weeks of treatment, the rats undergoing bone defect surgery were sacri ced using an overdose of chloral hydrate. Serum and femur samples were harvested. Femurs were xed at 4°C with 4% paraformaldehyde.
Whole blood was frozen at − 80°C for later use.

Cell culture and cell viability Measurement
As an osteoblast precursor cell line, MC3TE-E1 was obtained from the Institute of Biochemistry and Cell Biology, CAS (Shanghai, China). MC3TE-E1 were cultured in 24-well plates at 1×10 4 cells per well with growth culture medium. After culturing for 24 h, cells were treated with phosphate-buffered saline (PBS), silibinin(20 μmol/l, LSIL) and silibinin(50 μmol/l, HSIL). The Cell Count Kit-8 (YEASEN, Shanghai, China) was used to evaluate the effects of silibinin on cell viability.

Microcomputed tomography (Micro-CT) scanning
The distal femur was analyzed with anisotropic voxel size of 10μm through the Micro-CT (Bruker Skyscan 1272 system, Kontich, Belgium). The parameter is set to 55 kV and 114 m A with a thickness of 0.048 mm per slice in medium-resolution mode, 1024 reconstruction matrix, and 200 ms integration time. These images and parameters of trabecular bone parameters with a distance of 1 mm proximal from the end of the growth plate in femoral metaphysis were compared between the Sham group and OVX group to con rm the osteoporosis rat model. For evaluation of bone formation in the defect area, the central 1.5mm-diameter region of the defect was de ned by drawing circular contour as a consistent volume of interest (VOI). After 3D reconstruction, bone mineral density(BMD), bone mineral content(BMC), bone volume fraction(BV/TV), trabecular number(Tb.N), trabecular thickness(Tb.Th), trabecular separation(Tb.Sp) were automatically determined for identi cation of osteoporosis model while BMD, BV/TV, Tb. N, Tb. Th, Tb. Sp, the mean connective density (Conn.D) in VOI regions were used to evaluate new bone formation, using a protocol provided by the manufacturer of the Micro-CT scanner as previously described [25,26].
Histomorphometric analysis and immuno uorescence staining Part of the femora was decalci ed in 10% EDTA (pH 7.4) for 4 weeks and then embedded in para n. Four-micrometer-thick longitudinally oriented along the defect sections were used for staining. HE staining was performed to observe the trabecular bone and bone formation in the defect area. VEGF and HIF-1α staining were used to quantify the expression of angiogenic factors in the defect area. In brief, fresh bone sections were stained with individual primary antibodies to rats VEGFA(Abcam, ab206887, 1:100) and HIF-1α(Abcam, ab180880, 1:100), overnight at 4°C. Subsequently, the secondary antibodies conjugated with uorescence (Jackson Immuno Research, 415-605-166, 1:500; 315-605-003, 1:250) were used at room temperature for 1h while avoiding light and observed under a confocal microscope (FLUOVIEW FV300, Olympus). Calcein double labelling in undecalci ed bone slices were observed under a uorescence microscope(FLUOVIEW FV300, Olympus) to quantify bone mineralization in the defect area.

Reverse transcription and real time polymerase chain reaction (RT-PCR) analysis
According to the manufacturer's instructions, Total messenger RNA (mRNA) was extracted using the total RNA extraction kit (Takara, Kusatsu, Japan). Complementary DNA (cDNA) was obtained from total RNA using rst Strand cDNA Synthesis Kit (Toyobo, Ōsaka, Japan). Then synthetic cDNAs and speci c primers were used for qRT PCR with the TB Green TM Premix Ex Taq II (Tli RNaseH Plus) kit (Takara, Kusatsu, Japan) on the CFX Connect TM Real-Time System (Bio-Rad, Singapore). GAPDH was used as an internal control. Sequences of primers for the reference gene (GAPDH) and interested genes are listed in table 1. Table 1 Nucleotide sequences for real-time RT-PCR primers.

Genes
Forward (

Micro-CT evaluation
The 2D scan images (Figure 1. A-C) and 3D reconstruction images (Figure1. a-c) of Micro-CT clearly shows us the bone remodeling of the defect area after 12 weeks of treatment with different intervention methods. As we expected, the defect area of the Sham group was almost lled with bone tissue, while large amounts of bone tissue was found in the SIL+OVX group, but it was di cult to nd the bone tissue in the OVX group. The quantitative results were expressed as BMD, BV/TV, Tb. Th, Tb. N, Conn. D and Tb. Sp (Figure 1 D). Therapy with SIL showed positive effects on all micro-CT parameters. Compared to groups OVX, SIL treatment shows the better bone microscopic parameters including the higher BMD, BV/TV, Tb. N, Conn.D, Tb. Th, and the lowest Tb. Sp (P<0.05).

Histological and Fluorescent analysis
Histological and uorescent images showing bone repair in defect for different treatments, as shown in Figure 2. In 12 weeks, a large amount of bone tissue lls the defect area in the Sham group. In the OVX group, only a very small amount of new bone tissue can be observed; and large defect areas still exist, while the defect area of SIL+OVX group was signi cantly reduced in rats. In uorescent analysis, treatment with SIL showed the larger calcein green-marked defect area (p<0.05), and exhibited the higher values of relative bone mineralization(green/green marked defect area) (p<0.05), compared to that of the OVX group.
The angiogenesis regulator of bone defect measured by immuno uorescence and WB clearly show us the expression of HIF-1α and VEGFA of the defect area after 12 weeks of treatment with different intervention methods( Figure 2). As we expected, the defect area of the Sham group was almost lled with immuno uorescence for HIF-1α and VEGFA, while large amounts of immuno uorescence was found in the SIL+OVX group, but it was di cult to nd immuno uorescence in the OVX group. The quantitative results measured by WB were expressed as HIF-1α and VEGFA. Therapy with SIL showed positive effects on HIF-1α and VEGFA expressions. Compared to groups OVX, SIL treatment shows the higher protein expression with HIF-1α and VEGFA (P<0.05).

RT-qPCR analysis
Gene expression of defect area bone tissue after different treatment, as shown in Figure 3. In 12 weeks, SIL+OVX group showed increased Notch 1, HES1, HEY1 and JAG1 than the OVX group(p<0.05). These results indicate that the Notch pathway of SIL treatment is activated, and the expression of Notch 1, HES1, HEY1 and JAG1 is up-regulated.

Cell viability and function and related protein expression
In order to determine the effect of silibinin on MC3T3-E1 cells viability and function and related protein expression, this study further conducted Cell Count Kit-8, ALP staining, RES staining and WB analysis. As shown in Figure 4 A, the cell viability of HSIL and LSIL group were signi cantly higher than that of Con group(P<0.05). ALP staining, RES staining with quanti cation of area in osteogenic differentiation of MC3T3-E1 cells is shown in Figure 4D. The mineralized nodules (number per well), mineralized area (%), ALP activity and ALP gray value of HSIL and LSIL group were signi cantly higher than that of Con group(P<0.05). The protein expressions including ALP, OPN, RUNX-2, OC, VEGFA, HIF-1α, Notch 1, JAG 1, HEY 1 and HES 1 of HSIL and LSIL group were signi cantly higher than that of Con group(P<0.05). These results indicate that the treatment with silibinin can signi cantly increase MC3T3-E1 cells viability and function and related protein expression by activating Notch signaling pathway and HIF-1α/VEGF signaling pathway.

Discussion
So far, studies have con rmed that osteoporotic bones undergo a prolonged and impaired healing process in clinical postmenopausal osteoporotic women and experimental estrogen de ciency animal model [27,23]. Despite substantial advances in the eld of bone repair, strengthening the ability of bone formation remains a major challenge that has held back the clinical use of arti cial bone biomaterials. In the present study, we report that systemic administration with silibinin could promote bone regeneration in OVX rats. Our results show that the successful regeneration of bone defect was achieved based on evaluation of Micro-CT, and also con rmed with histological evaluation.
In this study, we compared the changes of bone mineral density and bone mass between Sham-operated rats and OVX rats, and successfully established an osteoporotic rat model through detection and evaluation of bone mineral density. In this model, a standard perforation 1.5 mm in diameter was generated at femoral condyle. Histological evidence and Micro-CT 3D reconstruction image demonstrated that very little new bone tissue was observed within the defected zone following drill-hole surgery in OVX group. The bone defect model used in this study is a mature and easily repeatable model originally used to intramem-branous bone formation, which is better than previously reported using an internal or external xation model [28]. Micro-CT reconstruction and histological examination consistently demonstrated less new bone formation in defect region in OVX rats, suggesting that bone repair ability was impaired during bone healing in the OVX-induced osteoporotic bone. In the study, more new bone tissue in the defect area was observed in the SIL + OVX group at 12 weeks than in the OVX group, suggesting that systemic administration with silibinin could accelerate the healing response. Accordingly, the results of this experiment suggest that systemic administration with silibinin may hold promise for improving bone defect repair under osteoporosis.
In order to further investigate the potential mechanisms, we also performed bone tissue immuno uorescence, qRT-PCR and western blotting experiments to analyze the mRNA expression of related gene, regulatory factor and protein contents. Previous studies indicated that signaling pathway also plays vital roles in mineralization of bone tissue via a direct regulation effect on osteoblastic activity [29,30]. Therefore, RT-PCR was conducted to detect the expression levels of several Notch target genes for bone tissue in the defect area of rats in each group. The results showed that the ovariectomy induces estrogen de ciency decreased the expression of target genes in bone tissues including Notch 1, HES1, HEY1 and JAG1. What's more, the decreased signaling pathway by ovariectomy was restored by SIL treatment. Recently, a large body and animal of emerging evidence has proved that angiogenesis plays a key role in bone repair [31,32]. Previous reports reached consensus that the tightly coupled angiogenesis and osteogenesis play an important role in bone regeneration [33]. Similarly, angiogenesis is regulated by a variety of growth factors, such as VEGFA and HIF-1α [34][35][36]. Among them, VEGFA and HIF-1α are the two most important factors that seem to play coordinated roles in vascular development to support bone regeneration. Osteoblast experiments found that treatment with silibinin can signi cantly increase MC3T3-E1 cells viability and function and related protein expression by activating Notch signaling pathway and HIF-1α/VEGF signaling pathway. Combining the above results, what we can explain is: systemic administration with Sil can markedly promote osteogenesis and angiogenesis, which results in a systemic environment that is more conducive to bone repair and ultimately accelerates bone formation.
As far as we know, this is the rst study of the effect of systemic administration with SIL on the regeneration of bone defect under osteoporotic conditions. Nevertheless, this study had several de ciencies. Firstly, the mechanisms underlying the effects of SIL on osteogenic differentiation of MSCs and osteoclasts should be elucidated. In vitro experiments of osteoblasts lack pathway agonists and inhibitors. Moreover, local delivery systems that allow the sustained release of SIL should be developed for effective bone regeneration in vivo. In addition, the optimal dosage of SIL should be determined for bone regeneration by using animal studies. Besides, there is no positive control group for animal experiments.

Conclusion
In summary, our study suggests that treatment with SIL may be a useful method to improve the initial bone regeneration of defects by increasing bone formation and angiogenesis in osteoporotic rats. Besides, this bene t effect may be mediated by HIF-1α/VEGF and Notch signaling pathway.  Bone regeneration of defected area by histological(A, magni cation, ×10) and uorescent analysis (B, magni cation, ×200). C. Total uorescently marked defect area(%) and Relative bone mineralization (green/green marked defect area) after SIL treatment. Bone tissue protein expression of HIF-1α and VEGFA in defect area measured by immuno uorescence(D) and WB(F); The quantitative results were expressed as uorescently marked defect area(E) and relative protein expression(G). *Vs. Sham group, p<0.05, #Vs. OVX, p<0.05.