Baicalin alleviates osteomyelitis by regulating TLR2 in the murine model

Baicalin alleviates osteomyelitis by regulating TLR2 in the murine model Abstract Osteomyelitis is an inflammation of bone caused by invading organisms. TLR2, inflammatory cytokines and mitogen-activated protein kinase (MAPK) signaling pathway are involved in osteomyelitis. Baicalin, the major active constituent of the isolated root of Scutellaria lateriflora Georgi, has been shown to have anti-inflammatory effects. In this study, the potentials of baicalin against osteomyelitis were evaluated. We treated mice and MC3T3-E1 cells with baicalin together with Staphylococcus aureus infection, and then analyzed the mice bone destruction, the expressions of TLR2 and osteogenic marker, the serum levels of proinflammatory factors and activation of MAPK signaling pathway. We also knocked down TLR2 by shRNA in MC3T3-E1 cells and detected the role of TLR2 in baicalin mediated inhibition of osteomyelitis. It was found that baicalin alleviated bone destruction in osteomyelitis. Baicalin decreased TLR2, alkaline phosphatase, osteopontin and collagen type I expressions. Baicalin decreased serum levels of proinflammatory factors IL-1β, IL-6 and CRP. Baicalin inhibited activation of MAPK signaling pathway. The inhibition of osteomyelitis by baicalin depended on TLR2 inhibition. In summary, baicalin is able to alleviate osteomyelitis by regulating TLR2. osteomyelitis, baicalin, TLR2, inflammation, Staphylococcus aureus INTRODUCTION Osteomyelitis is a progressive infection of bone, which results in inflammatory destruction of the bone, bone necrosis, and new bone formation and may progress to a chronic and persistent state (Lew and Waldvogel 2004). Osteomyelitis is characterized by progressive bone destruction and bone neoformation (Lew and Waldvogel 2004). Bone remodeling depends on the balance between bone resorption and formation and is necessary for skeletal growth and the maintenance of bone structure (Martin and Sims 2005). The resorption of bone by osteoclasts is followed by new bone formation by osteoblasts in a complex cascade of events involving several factors. The maturation of osteoblasts is characterized by the expression of alkaline phosphatase (ALP), which is important for bone matrix deposition and mineralization. Osteomyelitis usually occurs as a result of an infection in one part of the body that is transported through the bloodstream to a bone in a distant location. Osteomyelitis is frequently caused by Staphylococcus bacteria. Staphylococcus aureus is the most common organism, accounting for ∼80% of all osteomyelitis cases (Wright and Nair 2010). Staphylococcus aureus is a Gram-positive bacterium that lives in the microflora of the skin and mucous membranes of humans and animals (Foster 2009). The infection of bone tissue leads to osteoblast apoptosis, prevention of new bone formation and alteration of the bone remodeling process regulated by osteoblasts and osteoclasts (Tucker et al.2000). During osteomyelitis, TLR2 is essential for the recognition of microbial components, in particular lipoproteins/lipopeptides, peptidoglycan and lipoteichoic acid from S. aureus (Chen et al.2014) and triggered the activation of signal transduction pathways that control the expression of genes regulating immune response. Staphylococcus aureus infection resulted in mitogen-activated protein kinase (MAPK) activation, increased level of TLR2 (Chen et al.2014) and increased level of inflammatory factors including IL-1β, IL-6 and TNF-α (Yoshii et al.2002). Baicalin is the predominant flavonoid isolated from the roots of Scutellaria lateriflora Georgi (Huang Qin). It has been reported that baicalin exhibits many different pharmacological activities, including that of an antioxidant (Gao, Huang and Xu 2001), anti-inflammatory (Shen et al.2003), anti-tumor agent (Ikezoe et al.2001) and as an anti-viral (Xu et al.2010). In addition, baicalin was shown to inhibit S. aureus-induced apoptosis (Guo et al.2014) and induce the differentiation of cultured osteoblasts (Guo et al.2011). These biological activities of baicalin suggested a potential role of baicalin in osteomyelitis. In this study, we reported that baicalin alleviates cortical bone destruction of osteomyelitis by inducing expression of osteogenic marker ALP, osteopontin (OCN), collagen type I (COLL1), and decreasing serum level of proinflammatory factors in the murine model. Baicalin inhibited S. aureus-induced TLR2 expression and MAPK signaling pathway activation in MC3T3-E1 cells. Knocking down TLR2 also displayed protection against osteomyelitis, while the inhibition effect of baicalin was no longer significant, suggesting that baicalin protected against osteomyelitis through inhibiting TLR2. MATERIALS AND METHODS Bacteria Staphylococcus aureus was purchased from ATCC (ATCC 43300). From an overnight culture of ATCC 43300 in 9 ml BBL Trypticase Soy Broth (TSB; BD Biosciences, Franklin Lakes, NJ, USA), portions of 100 μl were transferred into sterile tubes containing 3 ml of TSB. These tubes were then incubated for 3 h at 37°C to obtain log-phase growth. After incubation, tubes were centrifuged for 10 min at 3000 rpm, the supernatant was decanted, and the remaining pellet was washed three times with phosphate-buffered saline (PBS) and added to PBS until a McFarland standard of 6 was obtained. Colony forming units (CFU) per milliliter were confirmed by the spread plate method. Murine model of osteomyelitis BALB/c adult male mice (12 weeks old; 20– 25 g) were maintained in our animal facility under specific pathogen-free conditions. Murine model of osteomyelitis was established according to the previous publication (Horst et al.2012). The mice were anesthetized with an intraperitoneal injection of 50 mg of pentobarbital/kg of body weight, and the skin on the left knee was shaved and sterilized with povidone iodine. A skin incision was made over the left knee, and the distal femur was exposed through a lateral parapatellar arthrotomy with medial displacement of the quadriceps-patellar complex. The distal end of the femur was perforated using a high-speed drill with a 0.5-mm sharp steel burr. Then, a channel was created using a 23-gauge (external diameter, 0.6 mm) needle, through which the bioluminescent strain of S. aureus (1.0 × 108 CFU) in 1 μl of medium was inoculated into the medullary cavity of the femur using a Hamilton syringe. PBS was administered to the sham group using the same technique. The burr hole was closed with bone wax, the dislocated patella was reduced, and the muscle and skin openings were closed by sutures. The animals were placed on a heating pad and carefully monitored until recovery. In some experiments, mice were intraperitoneally injected with PBS or baicalin (40 mg/kg) every day until sacrifice. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of Shanghai Jiao Tong University Affiliated Sixth People's Hospital. All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. Microcomputed tomography Analysis of cortical bone destruction was determined by microCT imaging using a μCT50 (Scanco Medical) and the manufacturer's analytical software. Baicalin treatment in mice Baicalin (∼99% purity) was purchased from Hubei Prosperity Galaxy Chemical Co., Ltd, and dissolved in DMSO (final concentration low than 0.1%) for a stock solution of 5.0 mg/ml. The mice were treated with baicalin at the dose of 40 mg/kg intraperitoneally every day starting from 1 day after S. aureus infection until they were sacrificed. RT-PCR The left hind tibiae in mice were crushed in an achate mortar under liquid nitrogen, and total RNA was isolated using an RNeasy Mini kit (Qiagen, CA, USA) according to the manufacturer's instructions with an additional DNase I (Fermentas) treatment step to eliminate residual genomic DNA. Reverse transcription was performed using a reverse transcription kit (Applied Biosystem, Waltham, MA, USA). Real-time quantitative PCR reactions were set up in triplicate with SYBR® Green Master Mix (Biorad, CA, USA) and run on a LightCycler 480 (Roche, Penzberg, Upper Bavaria, Germany). The following primers were used in the current study: ALP (Fwd: 5΄- CC AACTCTTTTGTGCCAGAGA-3΄, Rev: 5΄- GGCTACATTGGTGTTGAG CTTT-3΄), OCN (Fwd: 5΄- CTGACCTCACAGATCCCAAGC-3΄, Rev: 5΄- TGGTCTGATAGCT CGTCACAAG-3΄), COLL1 (Fwd: 5΄- TGTGCCAATTTCATCAAGGTCC-3΄, Rev: 5΄- CTCTTCCCACGAC CGTTTTCA-3΄), β-actin (Fwd: 5΄-AATGGGTCAGAAGG ACTCCT-3΄, Rev: 5΄-ACGGTTGG CCTTAGGGTTCAG-3΄) ELISA ELISA kits including as IL-1β, IL-6 and CRP for cytokine profiling were all obtained from R&D systems (Minneapolis, USA). Blood samples were collected from the infected and the control mouse groups by retro-orbital bleeding before surgery (day 1) and on days 3 and 7 after the operation. The plasma was retrieved for ELISA analysis according to the manufacturer's protocol. Lentivirus preparation shRNA targeting TLR2 (targeted sequence 5΄-GATAATCACCTATCTAGTTTA-3΄) was designed and cloned into lentiviral vector pLKO.5-puro (Sigma, MO, USA) following manufacture's protocol. To make lentivirus, HEK293 cells were cultured and next day pLKO.5-puro-TLR2 shRNA, together with package plasmids pCMV-dR8.2 and envelop plasmid pMD.G were transfected. Lentiviruses were harvested and concentrated as described before. MC3T3-E1 cell culture and treatment MC3T3-E1(ATCC) was cultured in standard tissue culture flasks in α-MEM medium (Invitrogen Corporation, Carlsbad, CA, USA) supplemented with 10% FBS (Gibco, Grand Island, NY, USA), 2% penicillin/streptomycin solution (Sigma-Aldrich, USA) and 1% L-glutamine (Sigma-Aldrich, USA). For cell infection, cells were incubated with prepared bacterial suspensions at a multiplicity of infection (MOI) of 100. Infected cells were pre-incubated for 30 min at room temperature to allow sedimentation of bacteria and then shifted to 37°C. Then, baicalin (50 μM) and/or shTLR2 were treated for 12 h, 48 h or 7 days. For lentivirus transduction, MC3T3-E1 cells were transduced with control TLR2 shRNA lentiviral particles using an MOI of 10 in the presence of hexadimethrine bromide (8 μg/ml), as previously described (Linares et al.2009). ALP assay MC3T3-E1 were seeded into 24-well plates (1 × 105 cells/ml) and infected with S. aureus at an MOI of 100 for 7 days and then ALP activity was assayed using the TRACP and ALP double-staining kit (Takara Bio Inc.) according to the manufacturer's instructions. The absorbance of each well at 405 nm was measured with a microplate reader (Immuno-Mini NJ-2300). Western blot Whole cell extracts were prepared as previously described (Linares et al.2012). A total of 20 μg of proteins from cell lysate were loaded onto a 12% SDS polyacrylamide gel under denaturing conditions and were transferred to a PVDF membrane (Bio-Rad, USA). Membranes were blocked with 10% non-fat dry milk dissolved in PBS with 0.1% Tween-20 overnight with rotation at 4°C. The following day, membranes were probed with TLR2, ERK, p-ERK, JNK2, p-JNK2 and β-actin antibodies for 2 h at room temperature. The membranes were washed and probed with appropriate horseradish peroxidase-conjugated secondary antibody and chemiluminescent substrate (Thermo Fisher Scientific, Waltham, MA) was used to detect the bands. All antibodies were purchased from Abcam. The western blot results were quantitated and analyzed using GS-900 Calibrated Densitometer and software Image Lab (Bio-Rad, USA) following the manufacturer's instructions. Statistical analysis One-way ANOVA analysis followed by a Tukey's post hoc test was used to determine the related protein levels. Statistical difference was considered as significant only if P < 0.05. RESULTS Baicalin alleviates cortical bone destruction in the murine model of osteomyelitis To test the potential protective effect of baicalin in osteomyelitis, we used the murine model of osteomyelitis by inoculating S. aureus into femur. After S. aureus infection, mice were intraperitoneally injected with PBS or baicalin every day for continuously 14 days. Then the cortical bone destruction was analyzed. The views of femurs were shown in Fig. 1A. Sham group (no infection/treatment) displayed normal condition. The femurs from infection/PBS-treated group displayed obvious bone destruction. In contrast, baicalin treatment alleviated bone destruction caused by infection as less bone destruction was detected in femurs from baicalin-treated mice group. After microCT imaging analysis, we did detect significantly less cortical bone destruction score in baicalin-treated mice (Fig. 1B). Thus, our data indicated that baicalin exhibited protective effect against bone destruction in murine model of osteomyelitis. Figure 1. View largeDownload slide Baicalin alleviates cortical bone destruction in the murine model of osteomyelitis. (A) The views of femurs were harvested at 14 days post-inoculation and subjected to microCT analysis. (B) MicroCT imaging analysis of cortical bone destruction. Sham group was control group without S. aureus-infected and untreated with baicalin. Inf group means S. aureus-infected and untreated with baicalin. Inf + Bai group means S. aureus-infected and treated with baicalin (40 mg/kg) on the day post-inoculation. N = 6 mice per group. Values are expressed as means ± SEM of three independent experiments. ** P < 0.01, vs Inf group. Figure 1. View largeDownload slide Baicalin alleviates cortical bone destruction in the murine model of osteomyelitis. (A) The views of femurs were harvested at 14 days post-inoculation and subjected to microCT analysis. (B) MicroCT imaging analysis of cortical bone destruction. Sham group was control group without S. aureus-infected and untreated with baicalin. Inf group means S. aureus-infected and untreated with baicalin. Inf + Bai group means S. aureus-infected and treated with baicalin (40 mg/kg) on the day post-inoculation. N = 6 mice per group. Values are expressed as means ± SEM of three independent experiments. ** P < 0.01, vs Inf group. Baicalin increased expression of osteogenic marker genes In osteomyelitis, S. aureus binds to osteoblasts and inhibits de novo bone formation by preventing expression of key markers of osteoblast growth and division such as ALP, COLL1 and OCN (Widaa et al.2012). Baicalin has been reported to induce these markers expression in cultured osteoblastic cells (Guo et al.2011). Thus, we continued to detect the effect of baicalin on expression of these osteoblast markers from mice at day 1 and day 7 after bacterial infection by RT-PCR. As shown in Fig. 2A, there is no significant difference of ALP expression among control, infection only and infection/baicalin-treated groups at day 1. In contrast, S. aureus infection inhibited ALP expression on day 7. Baicalin treatment on mice infected with S. aureus significantly increased ALP mRNA level. Similarly, baicalin significantly increased both OCN (Fig. 2B) and COLL1 (Fig. 2C) mRNA level on day 7. Thus, our data indicated that baicalin increased key markers of osteoblast growth and division including ALP, COLL1 and OCN, suggesting baicalin promoted osteoblast proliferation and differentiation. Figure 2. View largeDownload slide The effect of baicalin on osteogenic marker genes in the murine model of osteomyelitis. The mRNA level of (A) alkaline phosphatase (ALP), (B) osteopontin (OCN) and (C) collagen type I (COLL1) were tested by qPCR. Sham group was control group without S. aureus infection and untreated with baicalin. Inf group was S. aureus-infected and baicalin untreated group. Inf + Bai group was S. aureus-infected and baicalin-treated group (40 mg/kg) on the day post-inoculation. N = 6 mice per group. Values are expressed as means ± SEM of three independent experiments. ** P < 0.01, vs Inf group. Figure 2. View largeDownload slide The effect of baicalin on osteogenic marker genes in the murine model of osteomyelitis. The mRNA level of (A) alkaline phosphatase (ALP), (B) osteopontin (OCN) and (C) collagen type I (COLL1) were tested by qPCR. Sham group was control group without S. aureus infection and untreated with baicalin. Inf group was S. aureus-infected and baicalin untreated group. Inf + Bai group was S. aureus-infected and baicalin-treated group (40 mg/kg) on the day post-inoculation. N = 6 mice per group. Values are expressed as means ± SEM of three independent experiments. ** P < 0.01, vs Inf group. Baicalin decreased serum level of proinflammatory factors IL-6, IL-1β and CRP in osteomyelitis Elevated level of proinflammatory factors including IL-6, IL-1β and CRP has been reported to be associated with osteomyelitis (Yoshii et al.2002; Michail et al.2013). As baicalin had the anti-inflammatory effect (Chou et al.2003; Lin et al.2010), we next test the effects of baicalin on the serum level of IL-6, IL-1β and CRP in mice with osteomyelitis at day 1, day 3 and day 7 after S. aureus inoculation. As shown in Fig. 3A, the uninfected/no baicalin-treated mice had very low level of serum IL-6 at day 1, day 3 and day 7. IL-6 level at day 1 in infected mice was also low, but reached the climax at day 3. At day 7, the IL-6 level remained at a high level while decreased a little bit when compared with day 3. In contrast, baicalin-treated mice had significantly decreased level of serum IL-6 at day 3 and day 7 when compared to infected group, indicating baicalin treatment inhibited proinflammatory cytokine IL-6 production in osteomyelitis. We got similar results for IL-1β (Fig. 3B) and CRP (Fig. 3C). Although baicalin treatment did not decrease IL-1β or CRP level at day 3, it did decrease IL-1β and CRP level at day 7. Thus, our data indicated that baicalin displayed anti-inflammatory effect in osteomyelitis, which contributed to its protective effect in osteomyelitis. Figure 3. View largeDownload slide The effect of baicalin on serological evaluation in the murine model of osteomyelitis. The mRNA level of (A) IL-6, (B) IL-1β and (C) CRP were tested with ELISA. The blood was harvested at day 1, 3 and 7 post-inoculation. N = 6 mice per group. Values are expressed as means ± SEM of three independent experiments. ** P < 0.01, vs Inf group. Figure 3. View largeDownload slide The effect of baicalin on serological evaluation in the murine model of osteomyelitis. The mRNA level of (A) IL-6, (B) IL-1β and (C) CRP were tested with ELISA. The blood was harvested at day 1, 3 and 7 post-inoculation. N = 6 mice per group. Values are expressed as means ± SEM of three independent experiments. ** P < 0.01, vs Inf group. Baicalin inhibited S. aureus-induced TLR2 expression and MAPK signaling pathway activation in MC3T3-E1 cells Staphylococcus aureus infection was reported to induce TLR2 expression and activate MAPK signaling pathway, resulting in inflammation in osteomyelitis (Chen et al.2014). As baicalin exhibited protection in osteomyelitis, we further detected the effect of baicalin on TLR2 expression and MAPK signaling pathway activation after S. aureus infection in osteoblastic cell line MC3T3-E1. First, we confirmed previous published results that S. aureus induced TLR2 expression and activated MAPK signaling pathway in MC2T3-E1 cells. As shown in Fig. 4A, TLR2 protein levels increased at 12 and 48 h after S. aureus infection. We also detected obvious increased p-ERK and p-JNK2 in S. aureus-infected cells. Once baicalin applied to cells, the TLR2 protein level was decreased and less p-ERK and p-JNK2 were detected. After quantitation, we can see baicalin significantly inhibited TLR2 expression at 12 h and 48 h after infection (Fig. 4B). Baicalin also significantly inhibited ERK (Fig. 4C) and JNK2 (Fig. 4D) activation. Figure 4. View largeDownload slide Baicalin downregulated TLR2 expression and MAPK pathway in MC3T3-E1 cells after S. aureus infection. (A) Western blot analysis of TLR2, ERK and JNK on 0, 12 and 48 h after S. aureus infection with or without baicalin treatment. (B–D) Quantitation of relative protein expression. Values are expressed as means ± SEM of three independent experiments. *P < 0.05, ** P < 0.01, vs Inf group. Figure 4. View largeDownload slide Baicalin downregulated TLR2 expression and MAPK pathway in MC3T3-E1 cells after S. aureus infection. (A) Western blot analysis of TLR2, ERK and JNK on 0, 12 and 48 h after S. aureus infection with or without baicalin treatment. (B–D) Quantitation of relative protein expression. Values are expressed as means ± SEM of three independent experiments. *P < 0.05, ** P < 0.01, vs Inf group. Baicalin protected against osteomyelitis through inhibiting TLR2 Baicalin has been reported to decrease expression of TLR2 during S. aureus infection and other bacterial infection and inhibited the activation of downstream signaling pathway (Hao et al.2012; Guo et al.2014). Our data also showed inhibition of TLR2 by baicalin in osteomyelitis mice model. As TLR2 plays essential role in osteomyelitis, we hypothesize that baicalin protected against osteomyelitis through decreasing/inhibiting TLR2. To test our hypothesis, we knocked down TLR2 in MC3T3-E1 cells using shRNA together with baicalin treatment. Lentivirus encoding TLR2 shRNA decreased endogenous TLR2 level induced by S. aureus infection (Fig. 5A). Baicalin alone also decreased TLR2 level, but with less efficiency when compared to shRNA treatment. TLR2 shRNA/baicalin combined treatment showed similar inhibition effect to TLR2 shRNA treatment, indicating that shRNA displayed the dominant role in decreased TLR2. In addition, knocking down TLR2 by shRNA inhibited both ERK and JNK2 activation (Fig. 5A and B), indicating that S. aureus infection activated TLR2 downstream MAPK signaling pathway. Baicalin alone inhibited both ERK and JNK2 activation and we did detect an enhanced inhibition of ERK, and JNK2 activation by TLR2 shRNA/baicalin combined treatment. These results suggested that S. aureus infection predominantly activatedTLR2-mediated MAPK pathway, while there were other pattern recognition receptors active by S. aureus, which partially activated downstream MAPK pathway. Figure 5. View largeDownload slide Baicalin affected osteogenic differentiation of MC3T3-E1 cells after S. aureus infection by TLR2. (A) Western blot analysis of TLR2, ERK and JNK on 48 h after S. aureus infection treated with different treatment. (B) Quantitation of relative protein expression. (C) ALP activity on day 7 after different treatment. Values are expressed as means ± SEM of three independent experiments. *P < 0.05, ** P < 0.01. Figure 5. View largeDownload slide Baicalin affected osteogenic differentiation of MC3T3-E1 cells after S. aureus infection by TLR2. (A) Western blot analysis of TLR2, ERK and JNK on 48 h after S. aureus infection treated with different treatment. (B) Quantitation of relative protein expression. (C) ALP activity on day 7 after different treatment. Values are expressed as means ± SEM of three independent experiments. *P < 0.05, ** P < 0.01. Osteoblast apoptosis and osteogenic differentiation in response to bacterial invasion were dependent on TLR2 expression and JNK activation (Chen et al.2014). Previously, we found that baicalin increased expression of osteogenic marker ALP. So we continued to detect the effect of knocking down TLR2 on ALP expression. As shown in Fig. 5C, baicalin treatment increased ALP expression. Knocking down TLR2 significantly increased ALP expression too. Interestingly, combination of TLR2 knocking down and baicalin treatment had similar effect on ALP expression when compared to TLR2 knocking down alone, suggesting that the effect that baicalin enhanced ALP expression depended on TLR2. Taken together, our results suggested that baicalin prevented against osteomyelitis through inhibiting TLR2. DISCUSSION Osteomyelitis is a common manifestation of invasive S. aureus infection characterized by widespread bone loss and destruction (Carek, Dickerson and Sack 2001). Bone is a dynamic structure that is in a continuous process of remodeling, which is primarily driven by two cell types: osteoblasts, which deposit bone matrix, and osteoclasts, which remove it (Raggatt and Partridge 2010). The balance of absorption and deposition of bone matrix facilitated by these two cell types determines the structure, density and strength of bone tissue. Bone remodeling is controlled by a wide variety of systemic factors including hormones and steroids, and local factors such as cytokines and growth factors. Particularly, in such bone active cytokines, IL-1β, IL-6 and TNF-α have long been recognized as stimulators of osteoclasts, and thus lead to bone resorption. During S. aureus infection, the levels of TLR2, IL-1β, IL-6, CRP and TNF-α appear to be raised (Yoshii et al.2002; Guo et al.2014). In our mice model, we also detected increased serum level of inflammatory factors IL-1β, IL-6 and CRP (Fig. 2). The TLR pathways connecting infection detection and cellular responses indicated by cytokine expression are channeled through the MyD88 and IRAK4 proteins and then into several independent MAPK shunts, including ERK and JNK (Oda, Kitano 2006). These pathways then proceed to the gene expression level where they affect cellular survival decision making and other cellular response to infection, such as cytokine production (Chen et al.2016). In this study, we found that S. aureus infection in MC3T3-E1 cells increased TLR2 expression and activated both ERK and JNK MAPK signaling pathway (Fig. 4). TLR2 and JNK have been shown to be involved in S. aureus-induced apoptosis in MC3T3-E1. Chen and the colleagues reported that S. aureus induced apoptosis in association with the upregulation of TLR2 through a mechanism dependent on the activity of the pro-apoptotic JNK pathway. Silencing of TLR2 suppressed the effects of S. aureus infection on apoptosis and the calcification of osteoblasts via a mechanism involving the JNK pathway as inhibiting JNK had similar effects (Chen et al.2014). Thus, inhibiting TLR2-mediated downstream signaling pathway activation should be a potential approach to treat osteomyelitis. Baicalin exhibited the anti-inflammatory capability and has been proven to suppress TLR2 expression (Guo et al.2014) and suppress MAPK signaling pathway (Yang et al.2015), which suggested a potential role of baicalin to treat osteomyelitis. As predicted, in our mice model, baicalin prevented the bone destruction induced by S. aureus infection (Fig. 1) and decreased the serum level of proinflammatory cytokines IL-6, IL-1β and CRP, which are the typical markers for osteomyelitis (Fig. 3) (Yoshii et al.2002). Baicalin has also been shown to induce the differentiation of cultured osteoblasts and increase the osteoblasts mineralization and the levels of bone differentiation marker ALP, osteonectin, osteocalcin and COLL1 (Guo et al.2011). And consistent to this, we identified that in the mice model with osteomyelitis, application of baicalin increased expression of osteonectin, osteocalcin and COLL1 (Fig. 2). These results also suggested that baicalin may prevent osteoblasts apoptosis induced by S. aureus infection and promote the osteoblasts proliferation/differentiation, which benefits for bone recovery. In previous study, TLR2 was observed to be activated by S. aureus infection and TLR2 expression was increased following infection (Yang et al.2008; Guo et al.2014). In current study, TLR2 level was increased in MC3T3-E1 cells after S. aureus infection. The results were similar to the previous study. The cell wall components peptidoglycan and lipoteichoic acid from S. aureus have been found to activate TLR2 and its downstream MAPK signaling pathway. Baicalin treatment significantly decreased TLR2 level and MAPK JNK and ERK activation (Fig. 4), indicating that baicalin treatment had the anti-inflammatory effect. We demonstrated that silencing of TLR2 by lentivirus transduction in MC3T3-E1 cells prevented activation of MAPK JNK and EKR signaling pathway after S. aureus infection, indicating an essential role of TLR2 in S. aureus-induced inflammation. Silencing of TLR2 also resulted in an increased expression of ALP, similar to the effect of baicalin treatment. More importantly, the baicalin effect on ALP induction depended on TLR2 as baicalin did not enhance the ALP production induced by TLR2 silencing, suggesting that baicalin functioned through TLR2 inhibition. Thus, TLR2 was an essential factor in osteomyelitis. Effective TLR2 inhibitors may be utilized to treat osteomyelitis as well. CONCLUSION We demonstrated that baicalin alleviated bone destruction in osteomyelitis by inhibiting TLR2 expression and downstream MAPK activation. Baicalin alleviates osteomyelitis by regulating TLR2. FUNDING This work was funded by China Postdoctoral Science Foundation (2017M611573). Conflict of Interest. 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Cytokine  2002; 19: 59– 65. Google Scholar CrossRef Search ADS PubMed  © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Pathogens and Disease Oxford University Press

Baicalin alleviates osteomyelitis by regulating TLR2 in the murine model

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Abstract

Abstract Osteomyelitis is an inflammation of bone caused by invading organisms. TLR2, inflammatory cytokines and mitogen-activated protein kinase (MAPK) signaling pathway are involved in osteomyelitis. Baicalin, the major active constituent of the isolated root of Scutellaria lateriflora Georgi, has been shown to have anti-inflammatory effects. In this study, the potentials of baicalin against osteomyelitis were evaluated. We treated mice and MC3T3-E1 cells with baicalin together with Staphylococcus aureus infection, and then analyzed the mice bone destruction, the expressions of TLR2 and osteogenic marker, the serum levels of proinflammatory factors and activation of MAPK signaling pathway. We also knocked down TLR2 by shRNA in MC3T3-E1 cells and detected the role of TLR2 in baicalin mediated inhibition of osteomyelitis. It was found that baicalin alleviated bone destruction in osteomyelitis. Baicalin decreased TLR2, alkaline phosphatase, osteopontin and collagen type I expressions. Baicalin decreased serum levels of proinflammatory factors IL-1β, IL-6 and CRP. Baicalin inhibited activation of MAPK signaling pathway. The inhibition of osteomyelitis by baicalin depended on TLR2 inhibition. In summary, baicalin is able to alleviate osteomyelitis by regulating TLR2. osteomyelitis, baicalin, TLR2, inflammation, Staphylococcus aureus INTRODUCTION Osteomyelitis is a progressive infection of bone, which results in inflammatory destruction of the bone, bone necrosis, and new bone formation and may progress to a chronic and persistent state (Lew and Waldvogel 2004). Osteomyelitis is characterized by progressive bone destruction and bone neoformation (Lew and Waldvogel 2004). Bone remodeling depends on the balance between bone resorption and formation and is necessary for skeletal growth and the maintenance of bone structure (Martin and Sims 2005). The resorption of bone by osteoclasts is followed by new bone formation by osteoblasts in a complex cascade of events involving several factors. The maturation of osteoblasts is characterized by the expression of alkaline phosphatase (ALP), which is important for bone matrix deposition and mineralization. Osteomyelitis usually occurs as a result of an infection in one part of the body that is transported through the bloodstream to a bone in a distant location. Osteomyelitis is frequently caused by Staphylococcus bacteria. Staphylococcus aureus is the most common organism, accounting for ∼80% of all osteomyelitis cases (Wright and Nair 2010). Staphylococcus aureus is a Gram-positive bacterium that lives in the microflora of the skin and mucous membranes of humans and animals (Foster 2009). The infection of bone tissue leads to osteoblast apoptosis, prevention of new bone formation and alteration of the bone remodeling process regulated by osteoblasts and osteoclasts (Tucker et al.2000). During osteomyelitis, TLR2 is essential for the recognition of microbial components, in particular lipoproteins/lipopeptides, peptidoglycan and lipoteichoic acid from S. aureus (Chen et al.2014) and triggered the activation of signal transduction pathways that control the expression of genes regulating immune response. Staphylococcus aureus infection resulted in mitogen-activated protein kinase (MAPK) activation, increased level of TLR2 (Chen et al.2014) and increased level of inflammatory factors including IL-1β, IL-6 and TNF-α (Yoshii et al.2002). Baicalin is the predominant flavonoid isolated from the roots of Scutellaria lateriflora Georgi (Huang Qin). It has been reported that baicalin exhibits many different pharmacological activities, including that of an antioxidant (Gao, Huang and Xu 2001), anti-inflammatory (Shen et al.2003), anti-tumor agent (Ikezoe et al.2001) and as an anti-viral (Xu et al.2010). In addition, baicalin was shown to inhibit S. aureus-induced apoptosis (Guo et al.2014) and induce the differentiation of cultured osteoblasts (Guo et al.2011). These biological activities of baicalin suggested a potential role of baicalin in osteomyelitis. In this study, we reported that baicalin alleviates cortical bone destruction of osteomyelitis by inducing expression of osteogenic marker ALP, osteopontin (OCN), collagen type I (COLL1), and decreasing serum level of proinflammatory factors in the murine model. Baicalin inhibited S. aureus-induced TLR2 expression and MAPK signaling pathway activation in MC3T3-E1 cells. Knocking down TLR2 also displayed protection against osteomyelitis, while the inhibition effect of baicalin was no longer significant, suggesting that baicalin protected against osteomyelitis through inhibiting TLR2. MATERIALS AND METHODS Bacteria Staphylococcus aureus was purchased from ATCC (ATCC 43300). From an overnight culture of ATCC 43300 in 9 ml BBL Trypticase Soy Broth (TSB; BD Biosciences, Franklin Lakes, NJ, USA), portions of 100 μl were transferred into sterile tubes containing 3 ml of TSB. These tubes were then incubated for 3 h at 37°C to obtain log-phase growth. After incubation, tubes were centrifuged for 10 min at 3000 rpm, the supernatant was decanted, and the remaining pellet was washed three times with phosphate-buffered saline (PBS) and added to PBS until a McFarland standard of 6 was obtained. Colony forming units (CFU) per milliliter were confirmed by the spread plate method. Murine model of osteomyelitis BALB/c adult male mice (12 weeks old; 20– 25 g) were maintained in our animal facility under specific pathogen-free conditions. Murine model of osteomyelitis was established according to the previous publication (Horst et al.2012). The mice were anesthetized with an intraperitoneal injection of 50 mg of pentobarbital/kg of body weight, and the skin on the left knee was shaved and sterilized with povidone iodine. A skin incision was made over the left knee, and the distal femur was exposed through a lateral parapatellar arthrotomy with medial displacement of the quadriceps-patellar complex. The distal end of the femur was perforated using a high-speed drill with a 0.5-mm sharp steel burr. Then, a channel was created using a 23-gauge (external diameter, 0.6 mm) needle, through which the bioluminescent strain of S. aureus (1.0 × 108 CFU) in 1 μl of medium was inoculated into the medullary cavity of the femur using a Hamilton syringe. PBS was administered to the sham group using the same technique. The burr hole was closed with bone wax, the dislocated patella was reduced, and the muscle and skin openings were closed by sutures. The animals were placed on a heating pad and carefully monitored until recovery. In some experiments, mice were intraperitoneally injected with PBS or baicalin (40 mg/kg) every day until sacrifice. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of Shanghai Jiao Tong University Affiliated Sixth People's Hospital. All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. Microcomputed tomography Analysis of cortical bone destruction was determined by microCT imaging using a μCT50 (Scanco Medical) and the manufacturer's analytical software. Baicalin treatment in mice Baicalin (∼99% purity) was purchased from Hubei Prosperity Galaxy Chemical Co., Ltd, and dissolved in DMSO (final concentration low than 0.1%) for a stock solution of 5.0 mg/ml. The mice were treated with baicalin at the dose of 40 mg/kg intraperitoneally every day starting from 1 day after S. aureus infection until they were sacrificed. RT-PCR The left hind tibiae in mice were crushed in an achate mortar under liquid nitrogen, and total RNA was isolated using an RNeasy Mini kit (Qiagen, CA, USA) according to the manufacturer's instructions with an additional DNase I (Fermentas) treatment step to eliminate residual genomic DNA. Reverse transcription was performed using a reverse transcription kit (Applied Biosystem, Waltham, MA, USA). Real-time quantitative PCR reactions were set up in triplicate with SYBR® Green Master Mix (Biorad, CA, USA) and run on a LightCycler 480 (Roche, Penzberg, Upper Bavaria, Germany). The following primers were used in the current study: ALP (Fwd: 5΄- CC AACTCTTTTGTGCCAGAGA-3΄, Rev: 5΄- GGCTACATTGGTGTTGAG CTTT-3΄), OCN (Fwd: 5΄- CTGACCTCACAGATCCCAAGC-3΄, Rev: 5΄- TGGTCTGATAGCT CGTCACAAG-3΄), COLL1 (Fwd: 5΄- TGTGCCAATTTCATCAAGGTCC-3΄, Rev: 5΄- CTCTTCCCACGAC CGTTTTCA-3΄), β-actin (Fwd: 5΄-AATGGGTCAGAAGG ACTCCT-3΄, Rev: 5΄-ACGGTTGG CCTTAGGGTTCAG-3΄) ELISA ELISA kits including as IL-1β, IL-6 and CRP for cytokine profiling were all obtained from R&D systems (Minneapolis, USA). Blood samples were collected from the infected and the control mouse groups by retro-orbital bleeding before surgery (day 1) and on days 3 and 7 after the operation. The plasma was retrieved for ELISA analysis according to the manufacturer's protocol. Lentivirus preparation shRNA targeting TLR2 (targeted sequence 5΄-GATAATCACCTATCTAGTTTA-3΄) was designed and cloned into lentiviral vector pLKO.5-puro (Sigma, MO, USA) following manufacture's protocol. To make lentivirus, HEK293 cells were cultured and next day pLKO.5-puro-TLR2 shRNA, together with package plasmids pCMV-dR8.2 and envelop plasmid pMD.G were transfected. Lentiviruses were harvested and concentrated as described before. MC3T3-E1 cell culture and treatment MC3T3-E1(ATCC) was cultured in standard tissue culture flasks in α-MEM medium (Invitrogen Corporation, Carlsbad, CA, USA) supplemented with 10% FBS (Gibco, Grand Island, NY, USA), 2% penicillin/streptomycin solution (Sigma-Aldrich, USA) and 1% L-glutamine (Sigma-Aldrich, USA). For cell infection, cells were incubated with prepared bacterial suspensions at a multiplicity of infection (MOI) of 100. Infected cells were pre-incubated for 30 min at room temperature to allow sedimentation of bacteria and then shifted to 37°C. Then, baicalin (50 μM) and/or shTLR2 were treated for 12 h, 48 h or 7 days. For lentivirus transduction, MC3T3-E1 cells were transduced with control TLR2 shRNA lentiviral particles using an MOI of 10 in the presence of hexadimethrine bromide (8 μg/ml), as previously described (Linares et al.2009). ALP assay MC3T3-E1 were seeded into 24-well plates (1 × 105 cells/ml) and infected with S. aureus at an MOI of 100 for 7 days and then ALP activity was assayed using the TRACP and ALP double-staining kit (Takara Bio Inc.) according to the manufacturer's instructions. The absorbance of each well at 405 nm was measured with a microplate reader (Immuno-Mini NJ-2300). Western blot Whole cell extracts were prepared as previously described (Linares et al.2012). A total of 20 μg of proteins from cell lysate were loaded onto a 12% SDS polyacrylamide gel under denaturing conditions and were transferred to a PVDF membrane (Bio-Rad, USA). Membranes were blocked with 10% non-fat dry milk dissolved in PBS with 0.1% Tween-20 overnight with rotation at 4°C. The following day, membranes were probed with TLR2, ERK, p-ERK, JNK2, p-JNK2 and β-actin antibodies for 2 h at room temperature. The membranes were washed and probed with appropriate horseradish peroxidase-conjugated secondary antibody and chemiluminescent substrate (Thermo Fisher Scientific, Waltham, MA) was used to detect the bands. All antibodies were purchased from Abcam. The western blot results were quantitated and analyzed using GS-900 Calibrated Densitometer and software Image Lab (Bio-Rad, USA) following the manufacturer's instructions. Statistical analysis One-way ANOVA analysis followed by a Tukey's post hoc test was used to determine the related protein levels. Statistical difference was considered as significant only if P < 0.05. RESULTS Baicalin alleviates cortical bone destruction in the murine model of osteomyelitis To test the potential protective effect of baicalin in osteomyelitis, we used the murine model of osteomyelitis by inoculating S. aureus into femur. After S. aureus infection, mice were intraperitoneally injected with PBS or baicalin every day for continuously 14 days. Then the cortical bone destruction was analyzed. The views of femurs were shown in Fig. 1A. Sham group (no infection/treatment) displayed normal condition. The femurs from infection/PBS-treated group displayed obvious bone destruction. In contrast, baicalin treatment alleviated bone destruction caused by infection as less bone destruction was detected in femurs from baicalin-treated mice group. After microCT imaging analysis, we did detect significantly less cortical bone destruction score in baicalin-treated mice (Fig. 1B). Thus, our data indicated that baicalin exhibited protective effect against bone destruction in murine model of osteomyelitis. Figure 1. View largeDownload slide Baicalin alleviates cortical bone destruction in the murine model of osteomyelitis. (A) The views of femurs were harvested at 14 days post-inoculation and subjected to microCT analysis. (B) MicroCT imaging analysis of cortical bone destruction. Sham group was control group without S. aureus-infected and untreated with baicalin. Inf group means S. aureus-infected and untreated with baicalin. Inf + Bai group means S. aureus-infected and treated with baicalin (40 mg/kg) on the day post-inoculation. N = 6 mice per group. Values are expressed as means ± SEM of three independent experiments. ** P < 0.01, vs Inf group. Figure 1. View largeDownload slide Baicalin alleviates cortical bone destruction in the murine model of osteomyelitis. (A) The views of femurs were harvested at 14 days post-inoculation and subjected to microCT analysis. (B) MicroCT imaging analysis of cortical bone destruction. Sham group was control group without S. aureus-infected and untreated with baicalin. Inf group means S. aureus-infected and untreated with baicalin. Inf + Bai group means S. aureus-infected and treated with baicalin (40 mg/kg) on the day post-inoculation. N = 6 mice per group. Values are expressed as means ± SEM of three independent experiments. ** P < 0.01, vs Inf group. Baicalin increased expression of osteogenic marker genes In osteomyelitis, S. aureus binds to osteoblasts and inhibits de novo bone formation by preventing expression of key markers of osteoblast growth and division such as ALP, COLL1 and OCN (Widaa et al.2012). Baicalin has been reported to induce these markers expression in cultured osteoblastic cells (Guo et al.2011). Thus, we continued to detect the effect of baicalin on expression of these osteoblast markers from mice at day 1 and day 7 after bacterial infection by RT-PCR. As shown in Fig. 2A, there is no significant difference of ALP expression among control, infection only and infection/baicalin-treated groups at day 1. In contrast, S. aureus infection inhibited ALP expression on day 7. Baicalin treatment on mice infected with S. aureus significantly increased ALP mRNA level. Similarly, baicalin significantly increased both OCN (Fig. 2B) and COLL1 (Fig. 2C) mRNA level on day 7. Thus, our data indicated that baicalin increased key markers of osteoblast growth and division including ALP, COLL1 and OCN, suggesting baicalin promoted osteoblast proliferation and differentiation. Figure 2. View largeDownload slide The effect of baicalin on osteogenic marker genes in the murine model of osteomyelitis. The mRNA level of (A) alkaline phosphatase (ALP), (B) osteopontin (OCN) and (C) collagen type I (COLL1) were tested by qPCR. Sham group was control group without S. aureus infection and untreated with baicalin. Inf group was S. aureus-infected and baicalin untreated group. Inf + Bai group was S. aureus-infected and baicalin-treated group (40 mg/kg) on the day post-inoculation. N = 6 mice per group. Values are expressed as means ± SEM of three independent experiments. ** P < 0.01, vs Inf group. Figure 2. View largeDownload slide The effect of baicalin on osteogenic marker genes in the murine model of osteomyelitis. The mRNA level of (A) alkaline phosphatase (ALP), (B) osteopontin (OCN) and (C) collagen type I (COLL1) were tested by qPCR. Sham group was control group without S. aureus infection and untreated with baicalin. Inf group was S. aureus-infected and baicalin untreated group. Inf + Bai group was S. aureus-infected and baicalin-treated group (40 mg/kg) on the day post-inoculation. N = 6 mice per group. Values are expressed as means ± SEM of three independent experiments. ** P < 0.01, vs Inf group. Baicalin decreased serum level of proinflammatory factors IL-6, IL-1β and CRP in osteomyelitis Elevated level of proinflammatory factors including IL-6, IL-1β and CRP has been reported to be associated with osteomyelitis (Yoshii et al.2002; Michail et al.2013). As baicalin had the anti-inflammatory effect (Chou et al.2003; Lin et al.2010), we next test the effects of baicalin on the serum level of IL-6, IL-1β and CRP in mice with osteomyelitis at day 1, day 3 and day 7 after S. aureus inoculation. As shown in Fig. 3A, the uninfected/no baicalin-treated mice had very low level of serum IL-6 at day 1, day 3 and day 7. IL-6 level at day 1 in infected mice was also low, but reached the climax at day 3. At day 7, the IL-6 level remained at a high level while decreased a little bit when compared with day 3. In contrast, baicalin-treated mice had significantly decreased level of serum IL-6 at day 3 and day 7 when compared to infected group, indicating baicalin treatment inhibited proinflammatory cytokine IL-6 production in osteomyelitis. We got similar results for IL-1β (Fig. 3B) and CRP (Fig. 3C). Although baicalin treatment did not decrease IL-1β or CRP level at day 3, it did decrease IL-1β and CRP level at day 7. Thus, our data indicated that baicalin displayed anti-inflammatory effect in osteomyelitis, which contributed to its protective effect in osteomyelitis. Figure 3. View largeDownload slide The effect of baicalin on serological evaluation in the murine model of osteomyelitis. The mRNA level of (A) IL-6, (B) IL-1β and (C) CRP were tested with ELISA. The blood was harvested at day 1, 3 and 7 post-inoculation. N = 6 mice per group. Values are expressed as means ± SEM of three independent experiments. ** P < 0.01, vs Inf group. Figure 3. View largeDownload slide The effect of baicalin on serological evaluation in the murine model of osteomyelitis. The mRNA level of (A) IL-6, (B) IL-1β and (C) CRP were tested with ELISA. The blood was harvested at day 1, 3 and 7 post-inoculation. N = 6 mice per group. Values are expressed as means ± SEM of three independent experiments. ** P < 0.01, vs Inf group. Baicalin inhibited S. aureus-induced TLR2 expression and MAPK signaling pathway activation in MC3T3-E1 cells Staphylococcus aureus infection was reported to induce TLR2 expression and activate MAPK signaling pathway, resulting in inflammation in osteomyelitis (Chen et al.2014). As baicalin exhibited protection in osteomyelitis, we further detected the effect of baicalin on TLR2 expression and MAPK signaling pathway activation after S. aureus infection in osteoblastic cell line MC3T3-E1. First, we confirmed previous published results that S. aureus induced TLR2 expression and activated MAPK signaling pathway in MC2T3-E1 cells. As shown in Fig. 4A, TLR2 protein levels increased at 12 and 48 h after S. aureus infection. We also detected obvious increased p-ERK and p-JNK2 in S. aureus-infected cells. Once baicalin applied to cells, the TLR2 protein level was decreased and less p-ERK and p-JNK2 were detected. After quantitation, we can see baicalin significantly inhibited TLR2 expression at 12 h and 48 h after infection (Fig. 4B). Baicalin also significantly inhibited ERK (Fig. 4C) and JNK2 (Fig. 4D) activation. Figure 4. View largeDownload slide Baicalin downregulated TLR2 expression and MAPK pathway in MC3T3-E1 cells after S. aureus infection. (A) Western blot analysis of TLR2, ERK and JNK on 0, 12 and 48 h after S. aureus infection with or without baicalin treatment. (B–D) Quantitation of relative protein expression. Values are expressed as means ± SEM of three independent experiments. *P < 0.05, ** P < 0.01, vs Inf group. Figure 4. View largeDownload slide Baicalin downregulated TLR2 expression and MAPK pathway in MC3T3-E1 cells after S. aureus infection. (A) Western blot analysis of TLR2, ERK and JNK on 0, 12 and 48 h after S. aureus infection with or without baicalin treatment. (B–D) Quantitation of relative protein expression. Values are expressed as means ± SEM of three independent experiments. *P < 0.05, ** P < 0.01, vs Inf group. Baicalin protected against osteomyelitis through inhibiting TLR2 Baicalin has been reported to decrease expression of TLR2 during S. aureus infection and other bacterial infection and inhibited the activation of downstream signaling pathway (Hao et al.2012; Guo et al.2014). Our data also showed inhibition of TLR2 by baicalin in osteomyelitis mice model. As TLR2 plays essential role in osteomyelitis, we hypothesize that baicalin protected against osteomyelitis through decreasing/inhibiting TLR2. To test our hypothesis, we knocked down TLR2 in MC3T3-E1 cells using shRNA together with baicalin treatment. Lentivirus encoding TLR2 shRNA decreased endogenous TLR2 level induced by S. aureus infection (Fig. 5A). Baicalin alone also decreased TLR2 level, but with less efficiency when compared to shRNA treatment. TLR2 shRNA/baicalin combined treatment showed similar inhibition effect to TLR2 shRNA treatment, indicating that shRNA displayed the dominant role in decreased TLR2. In addition, knocking down TLR2 by shRNA inhibited both ERK and JNK2 activation (Fig. 5A and B), indicating that S. aureus infection activated TLR2 downstream MAPK signaling pathway. Baicalin alone inhibited both ERK and JNK2 activation and we did detect an enhanced inhibition of ERK, and JNK2 activation by TLR2 shRNA/baicalin combined treatment. These results suggested that S. aureus infection predominantly activatedTLR2-mediated MAPK pathway, while there were other pattern recognition receptors active by S. aureus, which partially activated downstream MAPK pathway. Figure 5. View largeDownload slide Baicalin affected osteogenic differentiation of MC3T3-E1 cells after S. aureus infection by TLR2. (A) Western blot analysis of TLR2, ERK and JNK on 48 h after S. aureus infection treated with different treatment. (B) Quantitation of relative protein expression. (C) ALP activity on day 7 after different treatment. Values are expressed as means ± SEM of three independent experiments. *P < 0.05, ** P < 0.01. Figure 5. View largeDownload slide Baicalin affected osteogenic differentiation of MC3T3-E1 cells after S. aureus infection by TLR2. (A) Western blot analysis of TLR2, ERK and JNK on 48 h after S. aureus infection treated with different treatment. (B) Quantitation of relative protein expression. (C) ALP activity on day 7 after different treatment. Values are expressed as means ± SEM of three independent experiments. *P < 0.05, ** P < 0.01. Osteoblast apoptosis and osteogenic differentiation in response to bacterial invasion were dependent on TLR2 expression and JNK activation (Chen et al.2014). Previously, we found that baicalin increased expression of osteogenic marker ALP. So we continued to detect the effect of knocking down TLR2 on ALP expression. As shown in Fig. 5C, baicalin treatment increased ALP expression. Knocking down TLR2 significantly increased ALP expression too. Interestingly, combination of TLR2 knocking down and baicalin treatment had similar effect on ALP expression when compared to TLR2 knocking down alone, suggesting that the effect that baicalin enhanced ALP expression depended on TLR2. Taken together, our results suggested that baicalin prevented against osteomyelitis through inhibiting TLR2. DISCUSSION Osteomyelitis is a common manifestation of invasive S. aureus infection characterized by widespread bone loss and destruction (Carek, Dickerson and Sack 2001). Bone is a dynamic structure that is in a continuous process of remodeling, which is primarily driven by two cell types: osteoblasts, which deposit bone matrix, and osteoclasts, which remove it (Raggatt and Partridge 2010). The balance of absorption and deposition of bone matrix facilitated by these two cell types determines the structure, density and strength of bone tissue. Bone remodeling is controlled by a wide variety of systemic factors including hormones and steroids, and local factors such as cytokines and growth factors. Particularly, in such bone active cytokines, IL-1β, IL-6 and TNF-α have long been recognized as stimulators of osteoclasts, and thus lead to bone resorption. During S. aureus infection, the levels of TLR2, IL-1β, IL-6, CRP and TNF-α appear to be raised (Yoshii et al.2002; Guo et al.2014). In our mice model, we also detected increased serum level of inflammatory factors IL-1β, IL-6 and CRP (Fig. 2). The TLR pathways connecting infection detection and cellular responses indicated by cytokine expression are channeled through the MyD88 and IRAK4 proteins and then into several independent MAPK shunts, including ERK and JNK (Oda, Kitano 2006). These pathways then proceed to the gene expression level where they affect cellular survival decision making and other cellular response to infection, such as cytokine production (Chen et al.2016). In this study, we found that S. aureus infection in MC3T3-E1 cells increased TLR2 expression and activated both ERK and JNK MAPK signaling pathway (Fig. 4). TLR2 and JNK have been shown to be involved in S. aureus-induced apoptosis in MC3T3-E1. Chen and the colleagues reported that S. aureus induced apoptosis in association with the upregulation of TLR2 through a mechanism dependent on the activity of the pro-apoptotic JNK pathway. Silencing of TLR2 suppressed the effects of S. aureus infection on apoptosis and the calcification of osteoblasts via a mechanism involving the JNK pathway as inhibiting JNK had similar effects (Chen et al.2014). Thus, inhibiting TLR2-mediated downstream signaling pathway activation should be a potential approach to treat osteomyelitis. Baicalin exhibited the anti-inflammatory capability and has been proven to suppress TLR2 expression (Guo et al.2014) and suppress MAPK signaling pathway (Yang et al.2015), which suggested a potential role of baicalin to treat osteomyelitis. As predicted, in our mice model, baicalin prevented the bone destruction induced by S. aureus infection (Fig. 1) and decreased the serum level of proinflammatory cytokines IL-6, IL-1β and CRP, which are the typical markers for osteomyelitis (Fig. 3) (Yoshii et al.2002). Baicalin has also been shown to induce the differentiation of cultured osteoblasts and increase the osteoblasts mineralization and the levels of bone differentiation marker ALP, osteonectin, osteocalcin and COLL1 (Guo et al.2011). And consistent to this, we identified that in the mice model with osteomyelitis, application of baicalin increased expression of osteonectin, osteocalcin and COLL1 (Fig. 2). These results also suggested that baicalin may prevent osteoblasts apoptosis induced by S. aureus infection and promote the osteoblasts proliferation/differentiation, which benefits for bone recovery. In previous study, TLR2 was observed to be activated by S. aureus infection and TLR2 expression was increased following infection (Yang et al.2008; Guo et al.2014). In current study, TLR2 level was increased in MC3T3-E1 cells after S. aureus infection. The results were similar to the previous study. The cell wall components peptidoglycan and lipoteichoic acid from S. aureus have been found to activate TLR2 and its downstream MAPK signaling pathway. Baicalin treatment significantly decreased TLR2 level and MAPK JNK and ERK activation (Fig. 4), indicating that baicalin treatment had the anti-inflammatory effect. We demonstrated that silencing of TLR2 by lentivirus transduction in MC3T3-E1 cells prevented activation of MAPK JNK and EKR signaling pathway after S. aureus infection, indicating an essential role of TLR2 in S. aureus-induced inflammation. Silencing of TLR2 also resulted in an increased expression of ALP, similar to the effect of baicalin treatment. More importantly, the baicalin effect on ALP induction depended on TLR2 as baicalin did not enhance the ALP production induced by TLR2 silencing, suggesting that baicalin functioned through TLR2 inhibition. Thus, TLR2 was an essential factor in osteomyelitis. Effective TLR2 inhibitors may be utilized to treat osteomyelitis as well. CONCLUSION We demonstrated that baicalin alleviated bone destruction in osteomyelitis by inhibiting TLR2 expression and downstream MAPK activation. Baicalin alleviates osteomyelitis by regulating TLR2. FUNDING This work was funded by China Postdoctoral Science Foundation (2017M611573). Conflict of Interest. 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Pathogens and DiseaseOxford University Press

Published: Mar 1, 2018

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