Epimedium‐Curculigo herb pair enhances bone repair with infected bone defects and regulates osteoblasts through LncRNA MALAT1/miR‐34a‐5p/SMAD2 axis

Abstract Infected bone defects (IBDs) are the common condition in the clinical practice of orthopaedics. Although surgery and anti‐infective medicine are the firstly chosen treatments, in many cases, patients experience a prolonged bone union process after anti‐infective treatment. Epimedium‐Curculigo herb pair (ECP) has been proved to be effective for bone repair. However, the mechanisms of ECP in IBDs are insufficiency. In this study, Effect of ECP in IBDs was verified by micro‐CT and histological examination. Qualitative and quantitative analysis of the main components in ECP containing medicated serum (ECP‐CS) were performed. The network pharmacological approaches were then applied to predict potential pathways for ECP associated with bone repair. In addition, the mechanism of ECP regulating LncRNA MALAT1/miRNA‐34a‐5p/SMAD2 signalling axis was evaluated by molecular biology experiments. In vivo experiments indicated that ECP could significantly promote bone repair. The results of the chemical components analysis and the pathway identification revealed that TGF‐β signalling pathway was related to ECP. The results of in vitro experiments indicated that ECP‐CS could reverse the damage caused by LPS through inhibiting the expressions of LncRNA MALAT1 and SMAD2, and improving the expressions of miR‐34a‐5p, ALP, RUNX2 and Collagen type І in osteoblasts significantly. This research showed that ECP could regulate the TGF‐β/SMADs signalling pathway to promote bone repair. Meanwhile, ECP could alleviate LPS‐induced bone loss by modulating the signalling axis of LncRNA MALAT1/miRNA‐34a‐5p/ SMAD2 in IBDs.


| INTRODUC TI ON
The infected bone defects (IBDs) are known as one of the most difficult intractable diseases in clinical orthopaedics, 1,2 which are accompanied by aberrant bone formation, progressive bone destruction and systemic inflammatory response. 3,4According to the epidemiological studies of IBDs, the prevalence of IBDs induced by bone fracture is 20%-30%, the incidence of postoperative infection bone defects is 24%, and the rate of combination with antibiotics is still 4.5%. 5Surgical intervention and the placement of vancomycincalcium sulphate beads (VCS) are the most commonly used therapies.
While delayed union is still a mystery, some reports suggest that some patients experience a long bone union process, a delayed union or nonunion after anti-infection treatment of local VCS placement. 6nce, effective treatment options for IBDs shall be found quickly.
On basis of the theory of 'Kidney Governing Bones' in TCM, Chinese herbal medicines for tonifying kidney and strengthening bone have been applied in clinical treatment of bone repair, 7 especially Epimedium brevicornum maxim and Curculigo orchioides Gaertn.][10] The main effective substances of Curculigo orchioides, such as Curculigoside, Orcinol glucoside, Lignan, Curculigo words polysaccharide, could significantly promote the proliferation and alkaline phosphatase activities of MC3T3-E1 and boost bone formation. 11,12Clinical trials have found that application of the herb pair of Epimedium brevicornum maxim and Curculigo orchioides Gaertn (ECP) could enhance bone repair in some metabolic bone diseases, by alleviating the oxidative damage of H 2 O 2 on osteoblasts through PI3K/AKT pathway 13 or AKT/Nrf2/ HO-1 pathway. 14However, the mechanisms and effective ingredients of ECP in IBDs are still unclear.
Previous studies have found that TGFβ/SMADs signalling pathway played a pivotal role in osteoblast lineage commitment and differentiation, skeletal development and homeostasis.This pathway involved the TGFβ receptor, a heterodimeric complex consisting of type I and type II receptors on the plasma membrane.Activation of this receptor leaded to the phosphorylation of SMAD2 and SMAD3 transcription factors, which transduce the signal to the nucleus, ultimately triggering gene transcription. 15Moreover, some of noncoding RNAs (ncRNAs) were confirmed to be upstream regulators of TGFβ/SMADs signalling pathway, 16 including miR-34a-5p, which associated with an inflammatory response in osteoblasts.Our group has previously demonstrated that the miR-34a-5p/SMAD2 signalling axis can enhances bone formation in infected bone nonunion models and attenuates lipopolysaccharide-induced osteoblast inhibition. 17searches showed that Long non-coding RNAs could competitively bind to miRNAs, indirectly regulating the expression of miRNA target genes due to their sponge-like effect.Among these, LncRNA Metastasis Associated in Lung Denocarcinoma Transcript 1 (LncRNA MALAT1), one of the few highly conserved nuclear lncRNAs, has been proved to regulate a variety of physiological processes.
LncRNA MALAT1 can not only act as a sponge of miR-34a-5p in cancer cells, 18 but also enhance osteoblasts' activities in osteoporosis mice by mediating the miR-34c/SATB2 axis. 19However, the mechanisms of whether LncRNA MALAT1 regulates osteoblast proliferation and differentiation in infected bone defects and how ECP regulates LncRNA MALAT1 in IBDs are still unknown.
In this research, UPLC-QTRAP-MS/MS approach was employed to analyse the absorbed constituents, possible targets for bone repair after anti-infective therapy were predicted with the network pharmacology approach, and molecular biology experimental techniques were used to confirm the mechanism of ECP in IBDs.Thus, this study elucidated the metabolic patterns of ECP in vivo, the mechanisms in vitro and provided a primordial complementary remedy for bone remodelling in IBDs.

| Establishment of IBDs model
Healthy New Zealand white male rabbits (3 months, 3.0-3.5 kg weight) were punched a 2 mm-diameter hole in the tibia and sealed with sterile bone wax to establish the bone defects model.Then, the holes were injected with 1 × 10 6 CFU/mL Staphylococcus aureus suspension (China General Microbiological Culture Collection Center, CGMCC) to prepare the IBDs models. 20At the end of 4-week infection, it was diagnosed that 90% rabbits suffer from infected bone defects.The inactivated, necrotic tissue and dead bone in the wound were thoroughly scraped away.As well, the vancomycincalcium sulphate (Van-CS) were prepared by mixing calcium sulphate powder, vancomycin and normal saline in a mixing bowl at a weight ratio of 9.5:1:3.

| Effective evaluation of ECP on bone repair
Following an eight-week treatment period with ECP, the rabbits were humanely euthanized by injecting sodium pentobarbital excessively and the tibias were removed for subsequent experiments.
The tibia specimens underwent examination by micro computed tomography (micro-CT, SkyScan-1172, Bruker, Switzerland).For all scans, the tibia specimens were wrapped in cling film to maintain tissue hydration.The femurs were scanned individually, placed securely within a custom holder.Each scan was conducted with a nominal pixel size of 9 μm, utilizing a 0.5 mm Al filter at a voltage of 60 kV and a current of 167 μA, with an entrance exposure time of 590 ms.An increment of 0.4° was used for each angular step, averaging two frames for increased accuracy.Following standardized reconstruction using NRecon software, the datasets were analysed using CTAn software (Bruker micro-CT, Belgium).The indexes of bone mineral density (BMD) and bone volume fraction (BV/TV) were calculated.
Then, embedded with paraffin, the tibia specimens were cut into 5 μm slices.After treating with 3% H 2 O 2 and 5% BSA, slices were added with anti-RUNX2 (1:200, bs-1134R, Bioss) at 4°C overnight individually and used for immunohistochemical analysis according to the routine protocol.The expressions of RUNX2 were observed by microscope (Nikon Eclipse Ti, 531,608).

| Preparation of ECP extract and ECP-CS
Epimedium brevicornum maxim and Curculigo orchioides Gaertn

| Screening of effective chemical components and analysis of the absorbed constituents
The ECP extract was appropriately diluted to a density of 0.28 g/mL, followed by mixing with an equal volume of methanol, 5-min whirling and 20-min centrifugal at 14,000 r/min.All frozen serum samples were thawed and equilibrated at 4°C prior to analysis.To detect the composition of the sample, 500 μL serum was combined with 1500 μL mixed solution (methanol: acetonitrile = 1:1) and vibrated for 2-3 min.
After 20-min vibration at 14,000 r/min, the mixture was then frozen dry.Finally, the re-dissolution of freeze-dried powder was made in 200 μL of methanol, followed by 20-min subsequent centrifugal at 14,000 r/min.The supernatant was gathered for analytical experi-  S1.The qualitative analysis was made by the UPLC-QTOF-MS/ MS (Ultra performance liquid chromatography-quadrupole-time of flight-mass spectrometer) system based on our previous experiment. 19Meanwhile, the method validation was performed according to some related studies. 21

| Network pharmacology approach
The mol2 files for Curculigoside, Baohuoside I, Epimedin A/B, Icariin and Orcinol glucoside were uploaded to PharmaMapper for predicting the relevant targets of bone repair.The keyword 'bone healing' was selected in the GeneCards database (https:// www.genec ards.org), OMIM database (http:// www.omim.org) and the Drugbank database (https:// www.drugb ank.ca) to find out the relevant targets.
The 'VennDiagram' package in R software was employed to intersect the hub genes between the absorbed constituents and bone repair.
Moreover, the biological mechanisms of hub genes were offered by the Kyoto Encyclopedia of Genes and Genomes (KEGG) (https:// www.kegg.jp/ kegg/ rest/ kegga pi.html) and the gene GO annotation in the R package 'org.Hs. eg.db' (version 3.1.0).All these analyses were made with the 'clusterProfiler' package in R software.

| Cell culture
The MC3T3-E1 cell line, derived from mouse calvaria pre-osteoblasts, was adopted as a model of osteoblasts according to previous report 22 (collectively referred to as osteoblasts).All osteoblasts during the experiment were cultured in α-MEM medium (Genomcell bio; China) containing 10% fetal bovine serum.Osteoblasts were grown in a humidified incubator (95% air, 5% CO 2 at 37°C).Fresh medium was utilized and changed every 2-3 days.Upon reaching 80% confluency, the cells were digested, counted and prepared into cell suspensions in different culture vessels for additional studies.

| CCK-8 assay
The cytotoxicity of LPS and the therapeutic effect of ECP-BS/ ECP-CS on osteoblasts was assessed by the CCK-8 assay.Osteoblasts were put in a 96-well plate at a concentration of 5 × 10 4 cells/mL.Subsequently, the treatment of osteoblasts with (0-100 μg/mL) LPS or (0-20%) ECP-BS/ECP-CS was made at 24 h and 48 h, respectively.
Then, osteoblasts were exposed to CCK-8 solution and analysed with a microplate reader at λ = 450 nm (Thermo Fisher Scientific Inc, USA).

| Quantitative real-time PCR (qRT-PCR) assay
The extraction of total RNA was made from osteoblasts by SPARKeasy Biotechnology Co Ltd, China).In each of the samples, the reference control gene utilized was β-actin, while U6 was implemented as the internal control to account for miR-34a-5p.Quantitative real-time PCR was made with the Biosystems 7500 Real-Time PCR system.The 2 −ΔΔCT approach was adopted for calculating the relative gene expression data.Table S3 lists the primers adopted in this research.

| Western blot assay
The extraction and lysis of total proteins were made through the use of RIPA lysis buffer and were subsequently quantified.Subsequently, the transfer of proteins underwent separation via SDS-PAGE, was made onto polyvinylidene difluoride membranes (PVDF) (Millipore, Bedford, MA, USA), next to 1-h blocking with 5% BSA at room temperature.

| Cell differentiation and mineralization assay
The ALP staining was utilized to assess cell differentiation capacity.
Osteoblasts were cultured in six-well plates and treated accordingly.
After treatment for 24 h, pre-cooled PBS was adopted to wash cells for three times before fixation with 4% paraformaldehyde at 37°C for 10 min and, next to staining by the ALP staining kit (20210520; KGI Biotechnology, China).
The Alizarin Red S staining was made to evaluate cell mineralization capability.To induce calcification, osteoblasts were cultured in induction medium containing ascorbic acid (50 μg/L, A171447, Aladdin, China), β-Sodium glycerophosphate (10 mmol/L, BCCD6874, Sigma-Aldrich, USA) and dexamethasone (1 × 10 −8 mmol/L, B1704084, Aladdin, China) for 21 days.The medium was refreshed at 48-h intervals during induction.After successful calcification, cells were fixed by the same method mentioned above and then stained with 0.1% Alizarin Red S solution (130-22-3, Aladdin, China) for 1 h at 37°C.The calcium nodules appeared as orange or red nodules were then discovered under a microscope (Nikon Eclipse Ti, 531,608).

| Dual-luciferase reporter assay
The Starbase (http:// starb ase.sysu.edu.cn/ ) was employed to predict the binding sites of LncRNA MALAT1 and miRNA-34a-5p.For the luciferase assay, HEK 293 cells were plated in 96-well plates and cultured for 24 h.Subsequently, cells were co-transfected with 50 nM of either Negative Control or miRNA-34a-5p mimic, along with the dual-luciferase vectors containing either WT-MALAT1 or MUT-MALAT1, using the LipoHigh transfection reagent (GenePharm Pharmaceutical Technology Co., Ltd.Shanghai, China) for 5 h.After transfection, the cells were refreshed with α-MEM medium containing 10% fetal bovine serum and incubated at 37°C with 5% CO 2 for 24 h.Following the protocols outlined in the dual-luciferase reporter assay kit (Yeasen Biotechnology Co., Ltd., Shanghai, China), the cells were lysed using Passive Lysis Buffer and added to the Firefly luciferase reaction solution for 30 min.The activity of Firefly luciferase (F) was then measured.Subsequently, the Renilla luciferase reaction solution was added, and the mixture was thoroughly mixed using a vibrator to detect the activity of Renilla luciferase (R) within 30 min.Finally, the association between miRNA-34a-5p and LncRNA MALAT1 was quantitatively evaluated by calculating the ratio of Renilla luciferase activity to Firefly luciferase activity.

| Statistical analysis
In this study, the results of our analysis were displayed as the mean ± SD and analysed by GraphPad Prism 6.0 (GraphPad Software, San Diego, CA, USA).Variations between multiple groups were decided by one-way ANOVA followed.Statistical significance was considered at p < 0.05 and p < 0.01.

| ECP reversed the inhibition of bone formation caused by IBDs
To explore the bone repair effect of ECP, we analysed bone tissue samples from each group.The results showed that there was obvious bone loss in Model group by comparing with Control group.In the Van-CS group, a modest amount of fresh bone was present at the defect site, but the defect cavity was still visible.Nevertheless, the cortical bone structure of the Van-CS + ECP group had become complete and continuous, and the defect cavity was reverted to its normal state essentially (Figure 1A,B).According to statistical analysis, Model group displayed greatly lower BV/TV and BMD than Control group (Figure 1E).Besides, Van-CS + ECP group displayed higher BV/TV and BMD than Model and Van-CS groups significantly (Figure 1E).These findings indicated that ECP could significantly increase bone repair in IBDs.
According to the HE staining results, a lot of inflammatory cells were found in the bone tissue of the Model group by comparing with Control group (Figure 1C).The inflammatory state of the Van-CS group was mitigated, while the bone defect was aggravated.Interestingly, the bone cortex of Van-CS + ECP group was substantially improved, and the inflammatory cells between the bone tissues were greatly decreased (as shown in Figure 1C).On the other hand, IHC staining results indicated that the expressions of RUNX2, identified as the early osteoblastic marker, in Model group were greatly decreased by comparing with Control group.Moreover, Van-CS and Van-CS + ECP groups showed greatly higher the protein expressions of RUNX2 than Model group.It was worth mentioning that the positive expressions of RUNX2 in the Van-CS + ECP group were higher than that in Van-CS group (Figure 1D,F).These outcomes hinted that ECP could significantly grow the expressions of RUNX2 to facilitate bone repair in IBDs.

| Chemical constituents' characterization in ECP extract and Quantification of 6 components in ECP-CS
In order to analyse the chemical constituents' characterization in ECP extract and ECP-CS, the UPLC-QTOF-MS/MS methods were employed.The results showed that 34 constituents were identified in the ECP extract (Tables S4-S6, Figure S1), and 6 constituents (Curculigoside, Baohuoside I, Epimedin A/B, Icariin and Orcinol glucoside) (Table S7, Figure S2) were verified to absorb into the serum.Furthermore, 6 absorbed constituents in ECP-CS were quantitative analysed by using UPLC-QTRAP-MS/MS methods.First of all, as shown in Table S8 and Figure S3, the selectivity, accuracy, precision, stability, extraction recovery and matrix effect of UPLC-QTRAP-MS/MS approach were met the requirements, except the low-concentration icariin couldn't be detected.It indicated that the UPLC-QTRAP-MS/MS approach was feasible to detect the concentrations of the 6 components.Finally, the results showed that the concentrations of Baohuoside I, Icariin, Epimedin B, Orcinol glucoside, Curculigoside and Epimedin A in serum were 2.99 ng/mL, 0.94 ng/mL, 18.14 ng/mL, 1581.00 ng/mL, 4.52 ng/mL and 3.58 ng/ mL, respectively (as shown in Table 1).

| Network pharmacology predicted the therapeutic targets of the absorbed constituents in the process of inflammatory and bone repair
Based on the results of GeneCards and OMIM databases, there were 356 genes related to bone repair, and 80 genes were related to 6 absorbed constituents (Curculigoside, Baohuoside I, Epimedin A/B, Icariin and Orcinol glucoside).Then, 11 hub genes were sought out by intersecting bone repair related genes and 6 components target genes using the 'VennDiagram' package in R software (Figure 2A,B).The main hub targets genes were MMP13, BMP7, MAPK14, BMP2, TGFBR2, KDR, PPARG, ESR1, MAPK1, ESR2 and CYP19A1.Meanwhile, the GO annotation and KEGG enrichment analysis were adopted for exploring the biological function of these hub targets.The Network pharmacology prediction results showed that 64 biological process (BP) and 10 signalling pathways were related with bone repair and inflammatory, such as osteogenic TGFβ signalling pathway, Hippo signalling pathway, angiogenesis related VEGF signalling pathway, MAPK and IL-17 signalling pathway (Figure 2C,D).Most importantly, the 6 components in the ECP have been proved to be an effective treatment for bone repair through intervening osteogenesis, angiogenesis or anti-inflammatory function.Above all, according to the preliminary experiments and literature searches, TGFβ/SMADs signalling pathway was selected for further research.

| LncRNA MALAT1 regulated genes involved in the differentiation and mineralization of osteoblasts
According to the TargetScan (http:// www.targe tscan.org/ vert_ 80/ ) prediction, miR-34a-5p had LncRNA MALAT1 binding places in its 3′UTR.In order to investigate whether LncRNA MALAT1 directly targets miR-34a-5p, we constructed luciferase reporter genes with   Based on the experimental results, it was evident that knockdown of lncRNA MALAT1 significantly suppressed the expression of osteoblasts' signature genes, thereby inhibiting the differentiation and mineralization abilities.Additionally, LncRNA MALAT1 could directly regulate the expression of miR-34a-5p and impacted the expression of SMAD2.Thus, the above outcomes suggested that LncRNA MALAT1 could regulate osteoblastic differentiation and mineralization by mediating miR-34a-5p.

| MiR-34a-5p regulated genes involved in the propagation and mineralization of osteoblasts
For the investigation into the function of miR-34a-5p, we constructed miR-34a-5p mimics plasmid and inhibitor plasmids.According to the qRT-PCR results, overexpression of miR-34a-5p could greatly reduce the gene expression of SMAD2, while increase the gene expressions of ALP, RUNX2 and Collagen type І.The miR-34a-5p inhibitor had exactly the opposite effects on osteoblasts (Figure 3G).Western Blot outcomes also demonstrated that miR-34a-5p inhibitor drove the protein expression of SMAD2, but inhibited the protein expressions of ALP, RUNX2 and Collagen type І (Figure 3E,F).Similarly, as shown in Alizarin Red S staining and ALP staining results, miR-34a-5p mimics drove the abilities of propagation and mineralization in osteoblasts, compared with NC group (Figure 3J,K).
Our findings demonstrated that upregulating the expression of miR-34a-5p could enhance the expression of osteoblasts' genes, which associated with differentiation and mineralization, thereby promoting the osteogenic differentiation and mineralization process.Besides, SMAD2 was downregulated as well.If miR-34a-5p was inhibited, the opposite would be true.Furthermore, our previous experiments have validated the targeted binding between miRNA and SMAD2.Collectively, our results indicated that miR-34a-5p might drive the propagation and differentiation of osteoblasts by mediating the expression level of SMAD2.

| SMAD2 regulated genes involved in the propagation and mineralization of osteoblasts
In order to determine the bioactive function of SMAD2, the siS-MAD2 plasmid was constructed.The gene expressions of ALP, RUNX2 and Collagen type І were significantly increased (Figure 4A), and the corresponding protein expressions were remarkably raised (Figure 4B,I) when siSMAD2 was transferred into osteoblasts.
Moreover, siSMAD2 dominated in promoting the propagation and mineralization of osteoblasts compared with NC group, as confirmed by Alizarin Red S staining and ALP staining (Figure 4C,D).
These results indicated that Low-expression SMAD2 could directly stimulate the expression levels of osteoblasts' signature genes, related to osteoblast proliferation and mineralization capacity, consequently impacting osteoblasts' biological functions.These outcomes strongly showed that the knockout of SMAD2 promoted the differentiation and differentiation of osteoblasts.

| Elimination of LncRNA MALAT1 or SMAD2 counteracted the inhibitory role of miR-34a-5p inhibitor in propagation and mineralization of osteoblasts
In order to verify the feasibility of the LncRNA MALAT1/miR-34a-5p/SMAD2 signalling pathway, miR-34a-5p inhibiting agent was selected to verify whether siMALAT1 and siSMAD2 could reverse its activities.As shown in Figure 4F, the gene expressions of SMAD2, ALP, RUNX2 and Collagen type І in miR-34a-5p Inhibitor + siMALAT1 group and miR-34a-5p Inhibitor + siSMAD2 group were markedly higher than those in miR-34a-5p inhibitor group.
Meanwhile, the protein expressions of SMAD2, ALP, RUNX2 and Collagen type І were dramatically elevated compared with miR-34a-5p inhibitor group as well (Figure 4E,J).The outcomes of Alizarin Red S staining and ALP staining displayed that miR-34a-5p Inhibitor + siMALAT1 group and miR-34a-5p Inhibitor + siSMAD2 group could increase the number of osteoblasts and mineralized nodules, which reverse the damage caused by miR-34a-5p inhibitor (Figure 4G,H).
Interestingly, we found that we could partially rescue the inhibitory effect of miR-34a-5p on osteoblasts by inhibiting the expression of LncRNA MALAT1, which was the upstream factor regulating miR-34a-5p.However, if we specifically targeted and suppressed the downstream target of miR-34a-5p-SMAD2, it would be feasi-

| LPS induced the proliferation and mineralization of osteoblasts
To assess the cytotoxicity of LPS, osteoblasts were incubated with 0, 10, 20, 40, 60, 80 or 100 μg/mL LPS for 24 h and 48 h, and the cell survival rates were measured by a microplate reader.As shown in After that, simultaneously transferring into siMALAT1 and miR-34a-5p mimics, the gene expressions of miR-34a-5p, SMAD2, ALP, RUNX2 and Collagen type І were grown and the gene expression of SMAD2 was declined memorably (Figure 6A).Meanwhile, the protein expressions of ALP, RUNX2 and Collagen type І were more improved, the protein expression of SMAD2 was further reduced (Figure 6B,E).Subsequently, it could have a better effect of the gene

| DISCUSS ION
It is noteworthy that bone repair is a troublesome problem in IBDs.
According to the TCM theory, tonifying kidney and strengthening bone medicine are the principal treatment methods in numerous bone related diseases. 23Both Epimedium breviconu Maxim (Epimedium) and Curculigo orchioides Gaertn (Curculigo orchioides), have the effects of nourishing kidney yang, strengthening bones and muscles and eliminating wind. 24,25Pharmacological research has displayed that the flavonoids in Epimedium 9,26 or polysaccharides in Curculigo orchioides 27,28 could mediate osteoblasts' activities and then promote bone repair.
Staphylococcus aureus is the most studied bacteria in bone infection, which have been reported to stimulate bone resorption by secreting immune modulating proteins. 29Meanwhile, vancomycin has been considered as the 'last resort' treatment of infections caused by Staphylococcus aureus over the decades. 30Thus, in the present study, the tibias of rabbits were infected with Staphylococcus aureus for 4 weeks and then implanted Van-CS to establish the IBDs model. 20The results showed that there was obvious bone loss in Model group and a defect cavity in Van-CS + ECP group, which indicated that the IBDs model had been successfully constructed.
Transforming growth factor-beta (TGFβ)/bone morphogenic protein (BMP) signalling have widely recognized roles in bone formation and have two pathways: canonical SMAD-dependent pathways 31 and non-canonical SMAD-independent signalling pathway (such as MAPK). 32Researchers found that both of them converge on the RUNX2 gene to control cell differentiation. 33The coordinated activity of RUNX2 and TGFβ/BMP-activated SMADs is essential for skeleton formation.Our results showed that BV/TV, BMD and the protein expressions of BMP-2 and RUNX2 in Van-CS + ECP group were higher than those in Model and Van-CS groups, which were consistent with the regulatory machinery of RUNX2 for skeletal gene expression 32 and the antagonism of BMP2 and TGFβ signalling during osteogenesis. 18These results suggested that ECP exert protective effects on bone repair in IBDs.These outcomes hinted that ECP could significantly increase the protein expressions of BMP-2 and RUNX2 to facilitate bone repair in IBDs.Above all, the results suggested that ECP exert protective effects on bone repair in IBDs.
However, there were still significant works to be done in investigating the absorbed constituents and molecular mechanisms.
It is well-known that the active ingredients of Chinese Medicine exerted pharmacological effects through entering the bloodstream and transporting into various target organs and cells. 34nce, UPLC-QTRAP-MS/MS method was employed to analysis the absorbed constituents of ECP.Six components were detected in ECP-CS, including Curculigoside, Baohuoside I, Epimedin A/B, Icariin and Orcinol glucoside.6][37] Meanwhile, Baohuoside I is one of the metabolites of Icariin in vivo and also one of the main active flavonoid components of the Epimedium brevicornum maxim. 38Previous studies have shown that Curculigoside, the effective substance of Curculigo orchioides, could attenuate wear particle-induced periprosthetic osteolysis by promoting osteoblastic MC3T3-E1 cell differentiation and inhibiting osteoclast BMSC formation. 39It is important to note that there is currently a lack of literature on the correlation between Orcinol glucoside and bone.Our study revealed that Orcinol glucoside was the highest concentration of the absorbed constituents.As a result, we intend to pursue further research on Orcinol glucoside with an aim to explore its potential as a novel therapeutic agent for bone regeneration.
To explore the mechanisms of ECP in IBDs, the network pharmacology approaches were performed.The results showed that these six absorbed constituents were concentrated in TGFβ/SMADs signalling pathway 40 for bone repair and inflammatory, which were the key to the treatment of IBDs. 41Research has proven that TGFβ/ SMADs signalling pathway could affect the activities of osteoblasts, which exert a key effect on the control of bone remodelling. 42mbining with previous literature searches and extensive literature, it was ultimately predicted that these six components in ECP extract might promote osteoblasts' activities to stimulate the bone repair in infected bone defects through the TGFβ/SMADs signalling pathway.
4][45] Indeed, the reported role for LncRNA MALAT1 in mediating osteoblast differentiation has been linked to the sponging of several miRNAs including miR-204, 46 miR-30 44 and miR-143. 47In our study, the luciferase reporter assay IBDs models were divided into Model group, Van-CS group and Van-CS + ECP group.The Control group were healthy New Zealand white male rabbits.There were 10 animals in each group.The rabbits in the Van-CS and Van-CS + ECP groups received the implantation of Van-CS in the bone defects.After 4-week antiinfection, rabbits in Van-CS + ECP group received ECP intragastrically at 3.34 g/kg/day for 8 weeks.Housed in individual cages, all of the animals were fed in the same way.This study was carried out in the Animal Experiment Center of Zhejiang Academy of TCM (Hangzhou, China, SCXK 2019-0010).

(
Zhejiang Chinese Medical University Chinese Medicine Slices Co., Ltd, Lot 220,701) were mixed in a weight proportion of 1:1, based on clinical experience.The mixed materials were subjected to an overnight soaking in 10 × distilled water (V/W) overnight, followed by two rounds of boiling at 100°C 1 hour each time and concentrated to 7.4 g/mL.The ECP extract was stored at −20°C before using.Sprague-Dawley rats (male, body weight 200 ± 20 g) were randomly fallen into Control group and ECP group with 15 rats in each group.The rats in ECP group were given ECP extract at dose of 7.4 g/ kg/day.Following the last gavage, blood sample was extracted from the abdominal aorta.Finally, the ECP containing medicated serum (ECP-CS) was filtered through 0.2 μm microporous membrane and stored at −80°C.

| 7 of 18 MIAO
et al.LncRNA MALAT1, including wild-type (WT) or mutant (MUT).The findings of dual-luciferase reporter assay unveiled that upregulation of miRNA-34a-5p greatly impeded the luciferase reporter activity of the vector with the WT LncRNA MALAT1 3′UTR (Figure 3A).In summary, LncRNA MALAT1 could directly regulate the expression of miR-34a-5p.To dig the biological role of LncRNA MALAT1 in osteoblasts, low-expression plasmid siMALAT1 was transferred into osteoblasts.The qRT-PCR outcomes displayed that low-expression of LncRNA MALAT1 could sensibly increase the gene expression of miR-34a-5p, while obstruct the gene expression of SMAD2.Meanwhile, the gene expressions of ALP, RUNX2 and Collagen type І, markers of differentiation and mineralization in osteoblasts, were significantly increased as well (Figure 3B).As shown in Figure 3C,D, the results of Western Blot were consistent with that of qRT-PCR.The protein expression of SMAD2 was decreased, while the protein expressions of ALP, RUNX2 and Collagen type І were grown.Meanwhile, the differentiation and mineralization of osteoblasts were observed macroscopically by F I G U R E 1 The bone repair effect of ECP in IBDs.(A) The appearance observation of tibia specimens in each group.(B) The micro-CT scanning images of lesion sites in each group.(C) The HE staining images of tibia specimens (10×, scale bar = 1 mm, 40×, scale bar = 200 μm).(D) The IHC staining images of BMP-2 in each group (200×, scale bar = 50 μm).(E) The BV/TV and BMD indexes (F) The protein degrees of RUNX2 in bone tissue.(*p < 0.05, **p < 0.01, vs.Control group; # p < 0.05, ## p < 0.01, vs. Model group; n = 3).

F I G U R E 2 | 9 of 18 MIAO
Network pharmacology-based prediction of the six absorbed constituents.(A) Intersection of the six absorbed constituents' targets with osteogenesis-related genes involved in treating bone repair by Venn diagram.(B) Network diagram of osteogenic-core targeted of the 6 absorbed constituents.(C) A circle diagram showed an analysis of the biological processes involved in the core targets of the six absorbed constituents by GO. (D) Analysis of the pathways involved in the core targets of the six absorbed constituents by the KEGG and bubble diagrams.et al.Alizarin Red S staining and ALP staining.Compared with NC group, siMALAT1 could dramatically increase the number of osteoblasts and the degree of mineralized nodules (as shown in Figure 3H,I).

3. 10 |
ECP reversed the inhibition of osteoblasts brought by LPSAs shown in Figure7A, CCK8 assay revealed that the 1% ECP-CS could markedly increase the cell viability of osteoblasts induced by LPS, comparing with LPS and ECP-BS groups.The qRT-PCR outcomes displayed that the gene expressions of LncRNA MALAT1 and SMAD2 were greatly decreased, while the gene expressions of miR-34a-5p, ALP, RUNX2 and Collagen type І were grown (Figure7B).Meanwhile, the protein expression of SMAD2 was downregulated, and the protein expressions of ALP, RUNX2 and Collagen type І were upregulated by 1% ECP-CS (Figure7C,F).According to the outcomes of Alizarin Red S staining and ALP staining, the number of osteoblasts and the size and degree of mineralized nodules were memorably improved when treated with 1% ECP-CS compared with LPS group or 1% ECP-BS (Figure7D,E).These results indicated that ECP-CS substantially attenuated the inhibitory role of LPS in differentiation and mineralization.

F I G U R E 9
substantiated that LncRNA MALAT1 modulate SMAD2 expression via being a miR-34a-5p sponge.According to the outcomes of qRT-PCR and Western Blot, when osteoblasts were transferred into si-MALAT1 or siSMAD2, it could significantly promote differentiation and mineralization of osteoblasts during miR-34a-5p inhibitor coculture.Our results are in line with previous studies showing that LncRNA MALAT1 participates in osteogenic processes by binding miRNAs.Inflammatory bone diseases, including osteomyelitis and periodontitis, were characterized by osteoblast dysfunction, which contributes significantly to inflammation-induced bone degradation 48 Lipopolysaccharide (LPS), a critical inducer of bone erosion associated with gram-negative bacteria, hinders the differentiation and mineralization of osteoblasts.Here in our study, we also validated that LPS regulated the differentiation and mineralization of osteoblasts by mediating the LncRNA MALAT1 /miR-34a-5p/ SMAD2 signalling pathway.When osteoblasts were transferred into siMALAT1, siSMAD2 or miR-34a-5p mimics, it could also substantially reverse the inhibition of normal osteoblasts activities during LPS culture.These results dropped a hint that LPS mediates the differentiation and mineralization of osteoblasts through the LncRNA MALAT1/miR-34a-5p/SMAD2 signalling axis.Furthermore, research indicates that the expression of LncRNA MALAT1 is decreased during the process of osteoclast differentiation, leading to bone resorption, ultimately contributing to low bone mineral density and the development of osteoporosis. 49It is hypothesized that LncRNA MALAT1 plays a significant role in bone repair by promoting osteoblast function and inhibiting osteoclast activity, thereby serving as a potential therapeutic target for the prevention and treatment of osteoporosis and bone defects.Finally, we validated the efficacy of ECP-CS on osteoblasts.The expressions of LncRNA MALAT1 and SMAD2 were blocked, while the expressions of miR-34a-5p, ALP, RUNX2 and Collagen type І were elevated dramatically, when ECP-CS intervened in LPSinduced osteoblasts.Meanwhile, knocking down LncRNA MALAT1 and SMAD2, or enhancing miR-34a-5p in osteoblasts could significantly promote the osteoblasts' differentiation and mineralization abilities in ECP-CS-LPS-cultured osteoblasts.In summary, ECP could alleviate the damage caused by LPS through the LncRNA MALAT1/ miR-34a-5p/SMAD2 axis in osteoblasts (as shown in Figure 9).The completion of this study provided a new strategy and some novel therapeutic targets for the clinical treatment of IBDs.The mechanism of Epimedium-Curculigo herb pair on bone repair after IBDs.