Bone marrow mesenchymal stem cells (BMSCs) are multi-potent, with the ability to differentiate into adipocytes, chondrocytes, or osteoblasts.[1-3] Many diseases, such as osteoporosis or steroid-induced osteonecrosis of the femoral head (SONFH), occur because the balance between osteogenic and adipogenic differentiation is disrupted.[4,5] BMSC apoptosis and proliferation are important prerequisites in their differentiation. Yet, past studies on SONFH have focused on the unbalanced differentiation of BMSCs while ignoring their apoptosis and proliferation.
Dexamethasone (Dex) is one of the common synthetic glucocorticoids, with the effects of immunosuppression and anti-inflammation. The long-term use of hormonal drugs may lead to femoral head necrosis. Indeed, low concentrations of Dex can induce BMSCs to differentiate into bone and fat,[7,8] while high concentrations (10–6 mol/ L) can promote and inhibit, respectively, BMSC apoptosis and proliferation.[9,10] This effect likely contributes to hormonal-drug-induced SONFH. In a prior study, we revealed that certain non-coding RNAs may be related to Dex-induced apoptosis of BMSCs. Here, to explore drug and gene-level treatments, we attempt to elucidate the molecular signaling pathways related with Dex-induced BMSC apoptosis.
Baicalin (BA) is the main active ingredient extracted from Scutellaria baicalensis. Because of its anti-tumor, anti-oxidation, and anti-inflammatory effects,[12-14] it has been broadly applied in traditional Chinese medicine. In orthopedics, BA can be used to treat osteoarthritis due to its anti-inflammatory effects on chondrocytes.[15-17] Further, it can reduce apoptosis by enhancing chondrocyte autophagy protection and ameliorating endoplasmic reticulum stress. BA has been reported to increase mesenchymal stem cell (MSC) survival after transplantation by reducing apoptosis, as well as the survival of cardiomyocytes treated with H2O2. The hedgehog (HH) signaling pathway is highly conserved; it regulates embryonic development and adult tissue homeostasis. Its abnormal activation can promote tumorigenesis and tumor development.[23-26] BA can reduce apoptosis via the HH signaling pathway. As reported in two stud-ies,[28,29] activating the HH signaling pathway reduces the apoptosis of MSCs. Therefore, this study aimed to explore whether BA can block Dex-induced BMSC apoptosis through activating the HH signaling pathway.
Based on prior research, we hypothesize that BA reduces Dex-induced BMSC apoptosis through activating the HH signaling pathway. Therefore, we conducted this experiment to verify this hypothesis.
Human BMSC (hBMSC) primary cultures were obtained from three patients (aged 45 years, 47 years, and 52 years) with fractures of femoral neck and received total hip arthroplasty at the Affiliated Hospital of Qingdao University.
All patients signed informed consent forms. This study was approved by the Ethics Committee of the Affiliated Hospital of Qingdao University (No. QYFYWZLL26792).
Human BMSC isolation, cultivation, and treatment
hBMSCs were extracted from bone marrow tissue as described in previous study. They were cultivated in culture flasks in low glucose Dulbecco's Modified Eagle Medium containing 1% fetal bovine serum. The culture flasks were placed in a humidified incubator (ThermoFish Scientific, Massachusetts, USA). When confluence reached 8% to 9%, the cells were digested with trypsin and passaged into new culture flasks at a ratio of 1:2.
Primary hBMSCs were either treated with 10–6 mol/L Dex alone or with BA (5.0 μmol/L, 10.0 μmol/L, and 50.0 μmol/L, respectively) for 24 hours followed by co-treatment with 5.0 μmol/L, 10.0 μmol/L, or 50.0 μmol/L BA and 10–6mol/L Dex. Depending on the experimental condition, the cells were treated with drugs for a different number of days.
Cell phenotype identification
Each time the primary hBMSCs were extracted, they were subjected to phenotype identification. First, third-generation hBMSCs were re-suspended in 100 μL phosphate buffered saline (PBS) at a concentration of 1 × 10 cells/ mL. The cell suspensions were then incubated with cluster of differentiation 34(CD34)-phycoerythrin (PE), CD45-PE, CD73-fluorescein isothiocyanate (FITC), and CD90-FITC for 45 minutes with the condition of room temperature and in darkness. All antibodies were purchased from BD Biosciences (USA). After washing twice with PBS, those single-stained cells were re-suspended in 100 μL PBS. The negative control was unstained cells. The five groups of cell samples were processed using flow cytometry (Apogee A50-MICRO flow cytometer, Apogee, London, UK).
Osteogenic and adipogenic differentiation of hBMSCs
Third-generation hBMSCs were inoculated into six-well plates. To induce osteogenic differentiation, when cell confluence reached 60%, the complete medium would be replaced with an osteogenic medium (Fuyuanbio, Shanghai, China) and cultured for 14 days. To induce adipogenic differentiation, when cell confluence reached 90%, the complete medium would be replaced with an adipogenic induction medium (Fuyuanbio) and cultured for 21 days. The cells were washed with PBS twice, followed by fixation with formalin (10%). Afterward, the cells were stained with alkaline phosphatase (Hat Biotechnology Co. Ltd., Xi’an, China) or Oil Red O (Solarbio, Beijing, China) for 30 minutes.
Cell viability analysis
For 96-well plates, 5 × 103 cells per well were inoculated, and further processed after 24 hours according to their experimental groups. Cell viability was determined by Cell Counting Kit-8 (CCK-8) assay kit (Solarbio). Complete medium (100 μL) as well as CCK-8 reagent (10 μL) were both added to each well. With the incubation of 1 to 4 hours in a cell incubator, the absorbance was measured at 450 nm by a microplate reader (Tecan, Austria). Notably, the experiment was repeated three times.
Assessment of apoptotic morphology in Dex-treated hBMSCs
For 24-well plates, a total of 5 × 104 cells per well were inoculated for 24 hours, followed by further procession based on their experimental group. Hoechst 33342/ propidium iodide (PI) Kit (Solarbio) was adopted for the assessment of apoptotic cells’ morphology following the manufacturer's instructions. Apoptotic cells that displayed morphological characteristics under a fluorescence microscope, such as chromosome aggregation, nuclear division, and nuclear fragmentation, were identified and counted. Red and blue hyper-fluorescence could be observed in necrotic cells. The experiment was repeated three times.
Flow cytometry analysis of apoptotic cells
Third-generation cells were cultured in six-well plates until the confluence reached approximately 80%. The cells were subsequently treated with different drugs according to their assigned group. After staining with an AnnexinV-FITC/PI apoptosis detection kit (Absin Biotechnology Co. Ltd., Shanghai, China), an Apogee A50-MICRO flow cytometer (Apogee) was used to quantify the percentage of apoptotic cells. The experiment was repeated three times.
Western blotting analysis
To extract total protein, pre-chilled RIPA lysis buffer (Solarbio) having 1% protease inhibitor (MedchemEx-press) was applied to lyse the treated hBMSCs. The bicinchoninic acid protein detection kit (Meilunbio, Dalian, China) was used to determine protein concentration. Protein loading buffer (EpiZyme, Shanghai, China) was added to the extracted total protein solution at a ratio of 1:4, which was then heated to 100°C for 10 minutes. Depending on the molecular weight of the target protein, 6.0% or 12.5% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (EpiZyme) was used to separate the protein samples (30 μg). Next, the proteins were electro-transferred onto polyvinylidene fluoride (PVDF) membranes (MilliporeSigma, Shanghai, China). Subsequently, those membranes were placed in tris-buffered saline with Tween 20 (TBST) (Solarbio) containing 5% skimmed milk powder for 1.5 hours. After washed thrice with TBST, PVDF membranes were first incubated with the primary antibody at the temperature of 4°C overnight and then the corresponding secondary antibodies for 1.5 hours. The PVDF membranes were washed three times with TBST. The target bands were visualized with ECL-PLUS reagent (MilliporeSigma), and further scanned by the BioSpectrum imaging system (ThermoFisher Scientific, Massachusetts, USA). ImageJ software (version 1.52u) was used for quantifying gray values of the target bands; the results were normalized to glyceraldehyde 3-phosphate dehydro-genase (GAPDH). The experiment was repeated three times.
The primary antibodies included: rabbit anti-human sonic hedgehog (SHH, 2207T), suppressor of fused (SUFU; 2522S), zinc finger protein GLI-1 (GLI-1; 3538T), apoptosis regulator BAX (Bax; 5023T), B-cell lymphoma 2 (Bcl-2; 4223T), caspase-3 (14220T), and cleaved caspase-3 (14220T). They were all purchased from Cell Signaling Technologies (Danvers, USA). The GAPDH primary antibody as well as all secondary antibodies were provided by Elabscience (Shanghai, China). The antibody dilution solution (Boster Biological Technology, Shanghai, China) was used to dilute all antibodies according to the manufacturer's instructions.
All data were analyzed using SPSS 26.0 software (IBM, New York, USA). One-way analysis of variance (ANOVA) was carried out for the comparison among groups, while the independent Student's t test was used to compare between two groups. The data were described as the mean ± standard deviation. P values < 0.05 were considered to be statistically significant. Figures were created by GraphPad Prism 8 software (GraphPad, Inc. CA, USA).
After three generations of culturing, the bone-marrow extracted cells displayed a spindle shape [Figure 1A]. Following the induction of osteogenesis or adipogenesis, respectively, alkaline phosphatase or Oil Red O staining was used to determine the cells’ ability to differentiate into osteoblasts or adipocytes [Figure 1B and C]. The flow cytometry detection of cell surface markers revealed that most cells expressed typical BMSC markers: CD73 (95.50%) and CD90 (92.00%); few cells expressed atypical markers: CD34 (0.22%) and CD45 (0.24%) [Figure 1D].
Role of BA in cell viability
BA's molecular formula is given in Figure 2A. A complete medium containing BA at 2.5 μmol/L, 5.0 μmol/L, 10.0 μmol/L, 50.0 μmol/L, or 100.0 μmol/L was used to cultivate hBMSCs for 6 days. BA at concentrations of 2.5 μmol/L, 5.0 μmol/L, 10.0 μmol/L, 50.0 μmol/L had almost no effect on cell viability (P > 0.05), while BA at 100.0 μmol/L significantly inhibited hBMSC viability (P < 0.05). Concurrently, BA was unable to promote hBMSC proliferation [Figure 2B]. Based on these results, BA at concentrations of 5.0 μmol/L, 10.0 μmol/L, or 50.0 μmol/L were used in the following experiments.
BA reverses Dex-induced inhibition of hBMSC proliferation
CCK-8 analysis was used to evaluate hBMSC proliferation under different treatment conditions. When compared to the control group, hBMSCs in the other groups exhibited a decrease in proliferation (P < 0.05). However, when compared to the Dex group, cells treated with BA demonstrated a concentration-dependent increase in proliferation, whereby proliferation increased with increasing BA concentrations (P < 0.05; Figure 2C and D). These results indicate that BA can attenuate the inhibitory effects of high doses of Dex (10–6 mol/L) on hBMSC proliferation.
BA blocks Dex-induced hBMSC apoptosis
High concentrations of Dex (10–6 mol/L) can cause hBMSC apoptosis. Apoptotic cells display chromosome aggregation, nuclear division, and nuclear fragmentation.
After staining with Hoechst 33342/PI, cells were observed under a fluorescence microscope, where it was found that BA reduced the proportion of apoptotic cells caused by Dex (P < 0.05; Figure 3A and B).
Flow cytometry was carried out for the evaluation of hBMSC apoptosis under the different treatments. When compared to the control group, hBMSCs in the other groups exhibited an increase in apoptosis (P < 0.05). However, in contrast with the Dex group, hBMSC apoptosis in BA-treated cells was decreased in a concentration-dependent manner; that is, it decreased with increasing BA concentrations [Figure 3C and D].
The degree of apoptosis was further examined at the molecular level. Western blotting was used to quantify the expression of apoptosis-related proteins such as Bax, Bcl-2, caspase-3, and cleaved caspase-3 in hBMSCs. High doses of Dex (10–6 mol/L) increased the expression level of apoptosis related proteins, Bax, Bcl-2, cleaved caspase-3, compared to Dex group (P < 0.05; Figure 4A and B). The changes in the expression of these apoptosis-related molecules depended on BA concentration [Figure 4B]. Altogether, these data indicate that BA can inhibit Dex-induced apoptosis.
BA reverses Dex-induced apoptosis through the activation of the HH signaling pathway
In addition to detecting apoptosis-related molecules, we used western blotting to assay several proteins of the HH signaling pathway. In hBMSCs treated with Dex (10–6mol/L), SHH and GLI-1 protein levels were significantly reduced while SUFU expression was increased (P < 0.05, P< 0.05, P< 0.05). These results imply that Dex inhibits the HH signaling pathway. When we treated the cells with BA, SHH and GLI-1 protein expression increased, while SUFU expression decreased (P < 0.05, P< 0.05, P< 0.05; Figure 4A and C).
Notably, when cyclopamine (CP), a HH signaling pathway inhibitor (MCE, Shanghai, China), and BA were used concurrently to treat Dex-induced apoptosis of hBMSCs, the flow cytometry analysis revealed that the apoptosis rate of the Dex + BA + CP group was significantly higher than the Dex + BA group (P < 0.05; Figure 5A and B). Moreover, the expression of HH-signaling-pathway-related proteins indicated that CP can inhibit the HH signaling pathway [Figure 6A and C] and significantly weaken the anti-apoptotic effects of BA. We found that in hBMSCs stimulated by Dex, the effects of BA on the expression levels of Bax, Bcl-2, and cleaved caspase-3 were all antagonized by CP (P < 0.05, P< 0.05, P< 0.05; Figure 6A and B). Altogether, these findings indicate that BA exerts its inhibitory effects on Dex-induced apoptosis via the HH signaling pathway.
The value of BMSCs lies in their self-renewing and multidirectional differentiation abilities. The understanding of the mechanisms underlying their abnormal function may help improve the treatment of related orthopedic dis-eases.[4,31] Glucocorticoids are widely used in clinical treatment, but their long-term use can cause SONFH. SONFH is a common orthopedic disease; however, its only treatment is surgery as no drugs are available. Based on previous studies, we inferred that high concentration of glucocorticoids may cause BMSC apoptosis, which may be a reason for the occurrence of SONFH. Dex, a common glucocorticoid, at high concentrations (10–6 mol/L) can promote the apoptosis and inhibit the proliferation of BMSCs.[9,10,32] Therefore, in this study, 10–6 mol/LDex was used to induce hBMSC apoptosis to simulate the microenvironment of cells in SONFH. Exploring a compound that can counteract the negative effects of glucocorticoids may be an effective way to prevent or treat SONFH.
Traditional Chinese medicine extracts are an important topic in medical research. BA has been confirmed to affect the cells’ proliferation as well as apoptosis in various tumors.[33-36] Notably, it has been found to have anti-apoptotic effects on bone cells[18,19] such as chondrocytes and osteoblasts. However, no studies have explored the influence of BA on BMSCs.
The HH signaling pathway is important in MSC proliferation and apoptosis.[28,29] SUFU and GLI-1 are downstream effectors of SHH in the HH signal pathway. More specifically, SUFU releases the bound GLI-1 into the nuclei of cells. In the nuclei, it controls the target genes’ transcription by binding to their promoters. A growing number of studies have reported that many effector molecules that regulate cell proliferation and apoptosis, such as cyclin-D, cyclin-E, Bcl-2, Bax, and caspase-3, are downstream molecules of the HH signaling pathway.[38-40] We detected molecules associated with the HH signaling pathway in hBMSCs cultured with a high concentration of Dex. Reductions in SHH and GLI-1 expression and increases in SUFU expression indicate inhibition of the HH signaling pathway. Decreases in GLI-1 expression leads to the activation of the mitochondrial apoptotic pathway, which manifests in the increase and decrease, respectively, of Bax and Bcl-2. Increases in cleaved caspase-3 levels results in cell apoptosis. Our findings are consistent with those from previous papers. When we added BA to a cell culture medium containing a high concentration of Dex, the HH signaling pathway was activated, which decreased cell apoptosis. Notably, CP, an inhibitor of the HH signaling pathway, partially weakened the anti-apoptotic effects of BA. Altogether, based on these results, we can reasonably infer that BA ameliorates Dex-induced hBMSC apoptosis by activating the HH signaling pathway.
During the experimental process, we set a concentration gradient for BA. At the cellular level, the proportion of apoptotic cells decreased with increasing BA concentrations. At the molecular level, HH-signaling-pathway-related and apoptosis-related molecules varied with BA concentration. The optimal concentration of BA is worthy of further exploration.
There were some limitations in our study. First, hBMSC apoptosis induced by a high concentration of Dex cannot fully encapsulate the pathological changes in the actual disease process. Second, the metabolism of drugs in the body is a complex process that requires further experimental verification in animals.
In conclusion, this study confirms that BA can block the effect of Dex on hBMSC apoptosis by activating the HH signaling pathway, and this effect is concentration-dependent.
The present study was supported by grants from the National Natural Science Foundation of China (Grant No. 81802151); Shandong Province Natural Science Foundation (Grant No. ZR2019MH012); China Postdoctoral Science Foundation (Grant No. 2018M642616); Qingdao Applied Foundational Research Youth Project (Grant No. 19-6-2-55-cg); and Qingdao Traditional Chinese Medicine Science and Technology Project (Grant No. 2021-zyym28).
Conflicts of interest
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