BRAZILIN MEDIATES THE HUMAN PERIODONTAL LIGAMENT STEM CELLS FOR PERIODONTITIS TREATMENT THROUGH THE YAP SIGNALING PATHWAY

Objective : To explore the effects of Brazilin on the Human periodontal ligament stem cells (hPDLSCs) mediating periodontal tissue regeneration in periodontitis and the Yes-associated protein (YAP) pathway for periodontitis. Methods : The anti-inflammatory effects and osteogenic effects of Brazilin in the inflammatory microenvironment were mainly evaluated by polymerase chain reaction (PCR), western blot (WB), staining, and the blocking and deletion of the YAP pathway in vitro by verteporfin (VP) and shRNA to evaluated the effect on the YAP pathway. A rat periodontitis model was established and the therapeutic effects were evaluated by Micro-CT, hematoxylin and eosin (HE) staining, Masson staining, and immunohistochemistry. Results : Brazilin effectively down-regulated the expression of inflammatory factors, showed deeper staining, and showed up-regulation of related osteogenic factors, and inhibition of the YAP pathway is followed by inhibition of the osteogenic effects of Brazilin . Brazilin was effective in reducing inflammation and inhibiting alveolar bone loss in the rat periodontitis model. Conclusion : Brazilin can promote periodontal tissue regeneration in periodontitis through the YAP pathway, providing new ideas for the treatment of clinical periodontitis


Introduction
Periodontitis is an inflammatory and destructive disease of periodontal support tissue that ranks first in the causes of loss of teeth in adults (Peres et al., 2019).Moreover, accumulating data links periodontal disease to systemic diseases, including adverse pregnancy outcomes, cardiovascular disease, cancer, and diabetes (Choi andHyun, 2020; Heikkilä et al., 2018).Therefore, preventing or treating periodontal disease is important for improving oral health and averting systemic diseases.
Traditional treatment methods for periodontitis cannot achieve the ideal regeneration of periodontal tissue defects.Human periodontal ligament stem cells (hPDLSCs) possess characteristics of multipotential differentiation and immunomodulation, and hPDLSCs-mediated periodontal tissue regeneration is now regarded as a promising method for periodontitis treatment (Yan et al., 2018).However, previous studies demonstrated that hPDLSCs differentiation and periodontal tissue regeneration ability declined in the inflammatory microenvironment (Zhao et al., 2020).Therefore, how to regulate the inflammatory response and promote periodontal regeneration is an urgent problem to be solved.
The knowledge that periodontitis is a chronic inflammatory disease provides us with the option of using antiinflammatory agents as therapeutic strategies.Preventing periodontitis by using nonsteroidal anti-inflammatory drugs has been indicated to be effective.However, nonsteroidal anti-inflammatory drugs cause adverse effects, including gastrointestinal effects, renal effects, hemostatic effects, and hypersensitivity reactions, as well as other symptoms including headaches, dizziness, and vertigo (Preshaw, 2018).Natural compounds have been used for the pre-European Cells and Materials Vol.48 2024 (pages 32-44) DOI: 10.22203/eCM.v048a03vention and treatment of periodontitis, receiving extensive attention and research.They are considered more suitable for long-term use than chemical synthetic drugs due to their fewer adverse reactions (Elburki et al., 2014).Caesalpinia sappan L (C. sappan), commonly known as Sumu in China, is a famous herbal medicine.Its heartwood has been traditionally used for various diseases, such as trauma and stasis pain, attributed to its effects of, antiinflammation, dissipating stasis, and easing pain (Nirmal et al., 2015).Brazilin is a major homoisoflavone isolated from C. sappan that is used as marker components for the quality control of C. sappan in the current version of the China Pharmacopoeia (14-yaodian).In traditional Chinese medicine, Brazilin is used for the treatment of increased blood circulation, promotes menstruation, and exhibits analgesic and anti-inflammatory potentials (Li et al., 2017), however, whether Brazilin can reverse the adverse effects of inflammation on hPDLSCs is unknown.
Yes-associated protein (YAP) is a transcriptional coactivator that is regulated by the Hippo signaling pathway, which plays an important role in promoting tissue regeneration and maintaining the self-renewal of stem cells (Jia et al., 2019).Pan proved that bone marrow stem cells (BM-SCs) of YAP knockout mice owned lower osteogenic differentiation ability (Pan et al., 2018).More importantly, the YAP pathway is closely related to hPDLSCs and can be involved in proliferation and apoptosis to regulate its biological behavior (Jia et al., 2018).Regulating YAP expression and thus hPDLSCs to promote periodontitis may be an effective approach.A study has shown that flavonoid drugs bind to and inhibit the expression of YAP (Li et al., 2019), suggesting that YAP may be the pathway by which Brazilin regulates hPDLSCs.
Taken together, the aim of this paper is to explore the effects of Brazilin on the osteogenic differentiation of hPDLSCs under inflammatory conditions and to further explore its therapeutic effect in vivo by establishing a rat periodontitis model.Whether YAP plays a role in this process is the focus of this study.

Isolation and Culture of hPDLSCs
The study protocol was approved by the Medical Ethics Committee of the Affiliated Stomatology Hospital of Kunming Medical University, China (KYKQ2021MEC025).All participating patients signed a written informed consent form.Briefly, periodontal tissues were gently separated from the middle third of the molar root and digested in a solution of 4 mg/mL type II dispase (Sigma, St Louis, MO, USA) and 3 mg/mL type I collagenase (Gibco, Grand Island, NE, USA) for 30 min at 37 °C.The obtained cells were cultured in a basic medium (α-MEM supplemented with 10 % FBS, 100 mg/mL streptomycin, and 100 U/mL penicillin) and incubated at 37 °C in 5 % CO 2 .

Identification of hPDLSCs
The characteristics of hPDLSCs were examined by detecting the colony formation efficiency, differentiation ability, and phenotypic molecular markers of mesenchymal stem cells.The cells (500) were cultured, and the aggregation of more than 50 cells was identified as a colony.The osteogenic induction medium consisted of a basic medium containing 50 µg/mL vitamin C (Sigma, St Louis, MO, USA) and 10 mM β-glycerophosphate (Solarbio, Beijing, China).The adipogenic induction medium consisted of a basic medium containing 2 µM dexamethasone, 0.2 mM indomethacin, 0.01 mg/mL insulin, and 0.5 mM 3-isobutyl-1-methylxanthine (Sigma, St Louis, MO, USA).For chondrogenic differentiation, 6 × 10 5 cells were centrifuged and cultured in a chondrogenic differentiation medium (Cyagen, Santa Clara, CA, USA).Mineral nodules, lipid droplets, and chondrogenic cells pellets were detected by Alizarin Red Staining (ARS), Oil Red O staining, and alcian blue, respectively.Mesenchymal markers, including CD29, CD44, CD34, and CD45 (BD Biosciences, Milpitas, CA, USA), were detected by flow cytometry.

Alkaline Phosphatase (ALP) Staining and Alizarin Red Staining (ARS) Assay
Cells were counted and seeded into 6-well plates at a density of 2 × 10 5 per well.When the cells growth fused to 90 %, each group was replaced with an osteogenic induction medium, different concentrations of Brazilin were added to each group for intervention, and a complete change of fluid was carried out every 3 days.The culture was terminated on day 14, BCIP/NBT ALP staining kit (Beyotime, Shanghai, China) and alizarin red staining kit (Cyagen, Santa Clara, CA, USA) was performed according to the instructions.To investigate the osteogenic properties in the inflammatory microenvironment, 10 µg/mL LPS and (or) 5 µM Brazilin were co-cultured in the medium, followed by the staining www.ecmjournal.orgEuropean Cells and Materials Vol.48 2024 (pages 32-44) DOI: 10.22203/eCM.v048a03described above.Observe under an inverted microscope and photographs were taken.

Real-Time Polymerase Chain Reaction (RT-PCR)
To examine the effects of Brazilin on the mRNA expression of inflammatory mediators, hPDLSCs were treated with 10 µg/mL LPS for 24 h, followed by treatment with 2.5, 5, and 10 µM Brazilin for 24 h (coincubation).To examine the effects of Brazilin on the mRNA expression of osteogenic markers, an osteogenic induction medium with various Brazilin concentrations (2.5, 5, and 10 µM) was used to culture hPDLSCs for 7 and 14 d.Furthermore, the impacts of 5 µM Brazilin on osteogenic markers in inflammatory microenvironments were investigated.An osteogenic induction medium was used with 5 µM Brazilin and 10 µg/mL LPS to culture hPDLSCs for 7 and 14 d.
The RNA of treated cells was isolated using the Eastep® Super Total RNA Extraction Kit (Promega, Fitchburg, WI, USA) according to the manufacturer's protocol.cDNA was synthesized from 1 µg of total RNA using the PrimeScript RT reagent kit with gDNA Eraser (Takara, Kyoto Prefecture, Japan) at 37 °C for 15 min and inactivated at 85 °C for 5 s.RT-PCR was performed using SYBR qPCR Master mix (Vazyme, Nanjing, China).During PCR, a dissociation curve was constructed in the range of 60 °C to 95 °C, and the cycling parameters of PCR were followed: 1 cycle for 30 s at 95 °C, 40 cycles for 10 s at 95 °C, and 30 s at 60 °C.Gene expression was presented using 2 −∆∆Ct values, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal control.

Western Blot (WB) Analysis
To examine the anti-inflammatory effects of Brazilin in LPS-induced hPDLSCs, WB analysis was performed to quantify the protein expression levels of inflammatory mediators.Cells were lysed using RIPA buffer containing 1 % protease inhibitor (Solarbio, Beijing, China).BCA protein assay kit (Beyotime, Shanghai, China).Protein samples (20 µg/lane) were loaded on a 12 % SDS-polyacrylamide gel, separated, and then transferred to polyvinylidene difluoride (PVDF) membranes (Biosharp, Hefei, China), which were blocked with QuickBlock™ Blocking Buffer (Beyotime, Shanghai, China) for 1 hour at room temperature.Then, the membrane was further incubated overnight at 4 °C with primary antibodies against GAPDH, Interleukin-8 (IL-8), Matrix Metalloproteinases 1 (MMP1), and Matrix Metalloproteinases 3 (MMP3), all were purchased from Proteintech, Wuhan, China.The membrane was then incubated with an HRP-conjugated secondary antibody (Proteintech, Wuhan, China) for 1 h.The proteins were visualized using an imaging system.

Animal Experiments
All experimental procedures involving animals were reviewed and approved by the Ethics Committee of Kun-ming Medical University.A total of 30 6-week-old male Wistar rats (SD rats) with weighing 200 g on average were kept for 1 week under standard laboratory conditions to adapt to the environment.All studies involving animals were strictly adhered to the ARRIVE guidelines and the National Research Council's Guide for the Care and Use of Laboratory Animals.Periodontitis was induced with ligation silk over the second molar of the maxilla for a month.Then, the right maxillaries (N = 5) were scanned by Micro-CT to evaluate the completion of the experimental periodontitis model.The study contains 5 groups: (1) control, (2) periodontitis group (initially ligated rat only), (3) solvent group (initially ligated rat with solvent), ( 4) low Brazilin group (initially ligated rat with 5 µM Brazilin), and ( 5) high Brazilin group (initially ligated mice with 10 µM Brazilin).After a month, the maxillaries were fixed for Micro-CT evaluation, histological staining, Masson's trichrome staining, and immunohistochemical (IHC) staining.

Micro-CT and Analysis
The maxillary bone samples of the SD rats were trimmed and placed into a 4 % paraformaldehyde fixative solution for 24 h.The next day, the samples were collected and prepared for micro-CT scanning with a Skyscan 1176 scanner (Bruker, Karlsruhe, Germany).The scanning parameters were as follows: a rotation angle of 360°, a tube voltage of 70 kV, a tube current of 353 µA, an X-ray exposure time of 404 ms, and a scanning layer thickness of 18 µm.For the assessment of alveolar bone loss, the distance between the cementoenamel junction and the alveolar bone crest (CEJ-ABC) for the distal buccal root of the first molars was measured in 3D images viewed from the buccal side.The measurements were repeated three times per site, and mean distances in millimeters were obtained.

Hematoxylin and Eosin (HE) Staining and Masson's Trichrome Staining
The right maxillaries were collected and fixed in 4 % paraformaldehyde for 48 h and then placed into 10 % EDTA decalcifying solution for 1 month.The samples were dehydrated with a series of graded ethanol solutions and embedded in paraffin wax.The embedded maxillaries were cut in a buccal-lingual direction to acquire transverse sections (5 µm thick).The prepared sections were stained with HE and Masson's trichrome staining.

The Impact of Brazilin on YAP in Vitro
Verteporfin (VP), a YAP pathway inhibitor, was selected to inhibit YAP.The hPDLSCs were cultured in a medium with or without Verteporfin.After seven days of induction, hPDLSCs were harvested and protein expression was analyzed by Western blot analysis.To demonstrate the role of YAP in the osteogenic differentiation of hPDLSCs, YAP was stably silenced by short hairpin RNA (shRNA) duplex oligo targeting YAP (pEF-1aF-GFP-puro) packaged to lentiviruses by Genechem Company (Shanghai, China).After 72 h, the transfection reagents-containing medium was changed to a normal culture medium.

Immunofluorescence Staining
After washing with PBS, the cells were fixed with paraformaldehyde for 10 min and then permeated with 1 % Triton X-100 for 10 min.Then, the samples were blocked with 5 % BSA for 30 min at room temperature.Subsequently, the samples were incubated with primary antibodies (YAP; 1:200; Cell Signaling Technology, Boston, MA, USA) at 4 °C overnight.Alexa Fluor 594-conjugated goat anti-rabbit IgG (1:300; Abcam, Cambridge, UK) was incubated with the samples for 1 h at 37 °C in a dark room.The nuclei were stained with 4 ′ ,6-diamidino-phenylindole (DAPI; 1:500; Solarbio, Beijing, China).Images were taken with an inverted fluorescence microscope (Leica Microsystems, Wetzlar, Germany).

Statistical Analysis
All statistical analyses were calculated using Graph-Pad Prism 8.0.2 (GraphPad Software, Inc., San Diego, CA, USA).The data were analyzed using ANOVA and post hoc tests using the Dunnett test.In addition, the data are presented as the mean ± SD.The results were considered statistically significant with a p value; p < 0.05 was considered statistically significant (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Characterization of hPDLSCs
hPDLSCs exhibited typical long spindle-shaped morphology and were arranged in a vortex, as observed by an inverted microscope (Fig. 1A).Colony formation assays confirmed that hPDLSCs were able to form cells clusters (Fig. 1B).The osteogenic/adipogenic/chondrogenic differentiation assay detected mineralized nodules, lipid droplets, and chondrogenic cells pellets after induction (Fig. 1C-E).Flow cytometry analyses showed that these cells were highly positive for the mesenchymal stem cell-positive markers CD 29 (95.92 %) and CD90 (98.48 %) but were negative for CD 34 (1.02 %) and CD 45 (0.98 %) (Fig. 1F-I).Taken together, these results confirmed that the isolated hPDLSCs were of mesenchymal origin and had powerful multipotency and self-renewal ability.

Effects of Brazilin on Cells Viability
It was observed that hPDLSCs treated with 20 to 80 µM Brazilin exhibited smaller cells numbers from Day 3 compared with the control, Brazilin at 2.5 µM, 5 µM, and 10 µM exerted no significant cytotoxic effects on the viability of hPDLSCs from Days 1 to 7 (Fig. 2).Therefore, Brazilin at these concentrations was used in the subsequent study.

Effects of Brazilin on the Osteogenic Differentiation of hPDLSCs
As shown in Fig. 3A, the ALP staining results showed that the staining areas in the Brazilin group were significantly larger than those in the control group and osteogenic induction medium (OIM) group, treatment of hPDLSCs with Brazilin (2.5 µM, 5 µM, 10 µM) resulted in increased mineral nodules compared to the control group and OIM group according to the ARS.As presented in Fig. 3B, mRNA levels of osteogenic markers in hPDLSCs were enhanced compared to the expression in the control group after 7 and 14 d of osteogenic induction.When treated with 5 µM Brazilin, the mRNA expression of Runt-related transcription Factor 2 (RUNX2), Osterix (OSX ), Osteocalcin (OCN), and Collagen type I (COL1) was enhanced compared to the expression in the OIM group on Day 14.

Effects of Brazilin on Inflammatory Mediator Production in Pg-LPS-Treated hPDLSCs
As Fig. 4 shows inflammatory mediators were significantly upregulated by 10 µg/mL Pg-LPS stimulation compared with the control group.Treatment with Brazilin at different concentrations caused a great decrease in IL-8 pro-duction compared with that of the LPS group.Brazilin inhibited IL-8 release in a dose-dependent manner with 10 µM Brazilian having the best inhibitory effect.Brazilin also had inhibitory effects on MMP1 and MMP3.The production of MMP1 was significantly inhibited by 5 µM Brazilin, and the expression of MMP3 was significantly inhibited by 2.5 µM, 5 µM, and 10 µM Brazilian with 5 µM Brazilin having the best inhibitory effect.

Effect of Brazilin in Promoting Osteogenic Differentiation in Inflammatory States
Following osteogenic induction for 14 d or 21 d, the hPDLSCs were positively stained by ALP or ARS (Fig. 5A).However, the ability of the cells to undergo osteogenic differentiation was largely impaired by the addition of LPS, whereas Brazilin was able to rescue the inflammationinduced reduction in osteogenesis.Similarly, the protein levels of OSX, and Osteopontin (OPN) in hPDLSCs and the mRNA levels of osteogenic genes (including ALP, COLI, OSX, and OCN) were significantly decreased compared with those under normal conditions at Days 7 (Fig. 5B,C).However, Brazilin cotreatment increased the expression of osteogenic genes that were damaged by LPS.

Micro-CT Analysis
As shown in Fig. 6A, the Micro-CT images showed that significant loss of alveolar bone occurred in the periodontitis group compared to the black group, the solvent group exhibited a similar degree of loss, and there was a reduction in the degree of alveolar bone loss in the Brazilin group.As illustrated in Fig. 6B, the CEJ-ABC

Histological Analysis
Hematoxylin and eosin (HE) staining was performed to further histologically evaluate periodontium status in all groups (Fig. 6A).There was an apparent increase in the numbers of inflammatory cells in the periodontitis group and solvent group, indicating the status of persistent inflammation.In contrast, the treatments with a low Brazilin group and high Brazilin group decreased the number of infiltrated inflammatory cells, mitigated the inflammation, and improved the alveolar bone morphology to various degrees in the periodontitis.Newly formed bone was observed with Masson's trichrome staining in the low Brazilin group and high Brazilin group (Fig. 6A).Moreover, we confirmed that mineralized alveolar bone had formed in- side that was treated with Brazilin.Immunohistochemical staining was conducted to further evaluate the inflammation condition and restoration of the alveolar bone condition.The staining sections showed that periodontitis induced the highest level of MMP1-positive cells in rats and the reduced amount of MMP1 was observed in the low Brazilin group and high Brazilin group, among which the low Brazilin group accomplished the most favorable outcome, the osteogenic marker OCN was increased in low and high Brazilin group (Fig. 6C).

Blocking YAP Attenuated the Brazilin-Induced Promotion of Osteogenesis of hPDLSCs
To elucidate the role of the YAP pathway in Brazilininduced osteogenic promotion of hPDLSCs under inflammatory conditions, the specific inhibitor, VP, was used to block the YAP pathway.As the results were shown in Fig. 7A, we found that the LPS+VP+Brazilin groups repressed the Brazilin-enhanced ALP staining of hPDLSCs.ARS staining also revealed that the LPS+VP+Brazilin groups inhibited the enhanced in vitro mineralization by Brazilin.Similarly, verteporfin also partially blocks the osteogenic promotion of Brazilin in the inflammatory environment.As shown in Fig. 7B, Brazilin upregulated the protein lev-els of RUNX2, OPN, and OCN in hPDLSCs under inflammatory conditions.However, the positive effect of Brazilin on LPS-induced inflammation diminished in the YAP knockdown groups, indicating that Brazilin promotes osteogenic differentiation of hPDLSCs via YAP under inflammatory conditions.In the immunofluorescence staining results (Fig. 7C), compared with the LPS+Brazilin group, the fluorescent presence of YAP was still observed in the LPS+VP+Brazilin group after the addition of VP.

Deleting YAP Attenuated the Brazilin-Induced Promotion of Osteogenesis of hPDLSCs
To further elucidate the role of the YAP pathway in Brazilin-induced osteogenic promotion of hPDLSCs.YAP shRNA lentivirus was used to delete YAP expression in hPDLSCs; after 3 days of puromycin selection (1 µg/mL), the knock-down efficiency was verified by qRT-PCR and Western blot.As figure showed in Fig. 8A, both mRNA and protein expression of YAP decreased by YAP shRNA lentivirus compared to the NC group.As shown in Fig. 8B, the YAP knockdown groups repressed the Brazilinenhanced ALP activity of hPDLSCs.Alizarin red staining also revealed that the YAP knockdown groups inhibited the enhanced in vitro mineralization by Brazilin.When YAP was knocked down, shYAP+Brazilin group showed weaker ALP staining and formed a less mineralized matrix (Fig. 8B) than the Brazilin group.The expression of RUNX2, OPN, and OCN in shYAP+Brazilin group decreased as compared with Brazilin group (Fig. 8C).After lentivirus transfection, observation with fluorescence microscope indicated that the transfection efficiency was over 90 %, but its nuclear translocation was partly blocked by YAP shRNA (Fig. 8D).Above results proved knocking down YAP inhibited the Brazilin-induced osteogenic differentiation of hPDLSCs.

Discussion
Inflammation-induced bone loss has recently become a research hotspot due to the profound effects of inflammatory responses on local and systematic bone metabolism.Periodontitis is considered to be one of the diseases with inflammatory responses and destruction of periodontal tissues, including evident alveolar bone loss, which can have varying effects on oral health and general health (Liljestrand et al., 2017; Yoshihara et al., 2004).hPDLSCs play a key role in periodontal healing and have become important seed cells for periodontal tissue regeneration (Zhu and Liang, 2015).However, recent results have shown that it is difficult to attain the desired tissue regeneration.The main problem is that the number of hPDLSCs in patients with periodontitis is decreased, and their functions are impaired, which impedes the regeneration of periodontal tissues.Inflammation control and periodontal tissue regeneration are the key issues in periodontitis treatment of hPDLSCs.
Nowadays, researchers are more interested in finding bioactive compounds from natural origin as drugs, owing to either the high cost of synthetic drugs or the side effects of the synthetic molecules.A mountain of studies has demonstrated that Brazilin plays an important role in eliminating inflammation and protecting against inflammatory bone loss ( Nirmal et al., 2015).Mueller et al. (2016) evaluated Brazilin in RAW 264.7 macrophages and SW1353 chondrocytes and determined the secretion of the proinflammatory cytokines IL-6 and TNF-α and the anti-inflammatory IL-10.Interestingly, Jia et al. (2016) reported Brazilin can suppress the inflammatory response by inhibiting the activation of the Nuclear Factor-κB (NF-κB) signaling pathway.It is reported that Brazilin markedly attenuated mouse collagen-induced arthritis (CIA) and reduced the serum levels of inflammatory cytokines including TNF-α, IL-1β, and IL-6.And Brazilin prevented joint destruction, surface erosion, and enhanced bone formation as revealed by microstructural examinations (Jung et al., 2015).In this study, we explored the effect of Brazilin on anti-inflammatory and LPS from P. gingivalis is considered one of the main pathogenic factors for periodontal inflammation.In this study, hPDLSCs were treated with Pg-LPS (10 µg/mL) to stimulate the inflammatory microenvironment in vitro.The results showed that 10 µg/mL Pg-LPS initiates the production of inflammatory mediators such as interleukin-8 (IL-8), matrix metallopeptidase 1 (MMP1), and matrix metallopeptidase 3 (MMP3).As a major chemoattractant, IL-8 can induce the synthesis of matrix metallopeptidase, which can destroy normal periodontal tissue (Xiao et al., 2016).Therefore, suppressing these inflammatory mediators may help to restrict the progression of periodontal disease.In the present study, three concentrations of Brazilin significantly decreased the mRNA and protein levels of IL-8. 5 µM Brazilin showed the best inhibitory effect on MMP1 and MMP3.This result suggested the anti-inflammatory effects of Brazilin, which help to prevent periodontal destruction.Interestingly, we found that the drug cannot show more efficient inhibitory effects on MMP1 and MMP3 production when the concentration is too high or too low.The occurrence of this phenomenon may be related to the saturation of the receptors on the cells surface.Further increasing the concentration of the drug will no longer enhance its effect (Singh et al., 2016).
The development of clinical therapeutics for use in patients requires effective methods to rescue inflammationmediated changes of hPDLSCs.Therefore, it is of great importance to investigate how to enhance the defective osteogenic differentiation capability of hPDLSCs in the inflammatory microenvironment.Runt-related transcription Factor 2 (RUNX2) was considered to be the central gene involved in osteoblast phenotype induction (Wei et al., 2015).Collagen type I (COL I) is an extracellular matrix protein that stimulates the adhesion and differentiation of osteoblasts (Biasutto and Zoratti, 2014).Osterix (OSX) is a novel osteoblast-specific transcription factor that is essential for osteoblast proliferation, differentiation, and bone formation (Hutchings et al., 2020).Osteocalcin (OCN) is a late marker of osteoblast maturation, playing a key role during the process of bone formation and mineralization (An et al., 2016).The results showed that 10 µg/mL Pg-LPS may hurt the osteogenesis capacity of hPDLSCs.However, Brazilin can significantly reverse the inhibition effect.In addition, through qPCR detection, the expression of the RUNX2, COLI, OSX, and OCN genes was significantly increased after 5 µM Brazilin treatment for 14 days in a noninflammatory environment.This result suggested that consecutive 5 µM Brazilin treatment with osteogenic induction could promote osteogenesis-related gene expression, including both early-stage genes and advanced-stage genes.Of note, compared with the noninflammatory envi- The Hippo pathway plays an important role in many key processes, including oral and maxillofacial development and bone remodeling, whereas the YAP pathway plays an important role in the maintenance of bone homeostasis (Xiang et al., 2018).Several studies have shown that YAP is involved in stem cells and osteoblasts to promote osteogenic differentiation, especially in periodontal tissue remodeling, and regulates periodontal stem cells proliferation and differentiation (Sun et al., 2018; Wu et al., 2021; Zhang et al., 2016).
More importantly, in the inflammatory microenvironment, YAP not only reduces the inflammatory response but also inhibits inflammatory bone resorption (Lv et al., 2018; Yang et al., 2020).Following the use of verteporfin, a YAP inhibitor, RUNX2, OPN, and OCN expressions related to osteogenesis decreased as well as a reduced ALP staining and formation of mineralization nodules, however, in the group with the addition of Brazilin, there was a significant improvement in bone formation.Then we silent YAP caused by shRNA to get a similar result, which suggests that Brazilin may protect bone from inflammation via the YAP pathway and exert a promoting effect on osteogenesis.However, the mechanism of how Brazilin regulates the YAP pathway to promote osteogenesis in the inflammatory microenvironment needs to be investigated next.Taken together, we concluded that Brazilin can not only promote osteogenesis but can also reverse the adverse effects of inflammation on osteogenesis by exerting anti-inflammatory effects.

Conclusions
The results of Brazilin on hPDLSCs presented in this study will expand the knowledge of their role in antiinflammatory and osteogenic differentiation.In particular, Brazilin has a positive effect on periodontal tissue regeneration and alleviated local inflammation.Notably, in a rat periodontitis model, Brazilin significantly inhibited alveolar bone loss by reducing inflammation and attenuating alveolar bone loss.We then preliminarily explored the effect of Brazilin on the YAP pathway in vitro, suggesting that Brazilin may be acting through the YAP pathway.This study provides a novel idea for new drug research and development, as well as clinical applications for treating periodontitis.
Fig. 6.The analysis of Brazilin ameliorated bone formation in rat periodontitis models.(A) Micro-CT image, HE staining images, and Masson's trichrome staining images of the periodontium after the treatment in decalcified sections.Black arrows point to the alveolar bone.PDL, periodontal ligament.(B) The distance of cemento-enamel-junction-alveolar bone crest (CEJ-ABC) was plotted.(C) The immunohistochemical analysis of MMP-1 and OCN positive cells (Red arrows indicate positive cells).***p < 0.001, ****p < 0.0001, ns, non significance.

Fig. 8 .
Fig. 8. Effects of shRNA on Brazilin-induced osteogenic promotion of hPDLSCs.(A) The expression levels of YAP mRNA and protein were deleted by shRNA.(B) Representative images of ALP (14 d, scale bar, 100 µm) and ARS staining.(C) Western blotting analysis of protein expression levels of the osteogenesis-associated genes after YAP expression was deleted by shRNA lentivirus.(D) Immunofluorescence staining of YAP.Data are expressed as the mean ± SEM from three independent experiments performed in triplicate.NC, normal control; *p < 0.05, **p < 0.01, ns, non significance.