Yuanbin Zhang, Yuliang Zhang*, Xingzhong Hu, Yong Lang and Qi Sun

Department of Orthopedics, Fuyang Orthopedics and Traumatology Affiliated Hospital of Zhejiang University of Traditional Chinese Medicine, Hangzhou, Zhejiang 311400, China

*Corresponding Author:
Yuliang Zhang
Department of Orthopedics, Fuyang Orthopedics and Traumatology Affiliated Hospital of Zhejiang University of Traditional Chinese Medicine, Hangzhou, Zhejiang 311400, China
E-mail: zyl63313718@163.com
Date of Received 30 April 2022
Date of Revision 04 February 2023
Date of Acceptance 29 September 2023
Indian J Pharm Sci 2023;85(5):1452-1457  

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DOI: 10.36468/pharmaceutical-sciences.1215

Abstract

This study aimed to investigate the impact of the luteolin-microRNA-335-3p pathway on the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells. Cell proliferation was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay and osteogenic markers were assayed by Western blotting. Luteolin treatment demonstrated a dose-dependent promotion of bone marrow mesenchymal stem cells proliferation, accompanied by an increase in osteogenic marker content within the cells. Additionally, luteolin treatment dose-dependently up-regulated microRNA-335-3p levels. The restoration of microRNA-335-3p further augmented bone marrow mesenchymal stem cells proliferation and up-regulated osteogenic differentiation markers. Conversely, knockdown of microRNA-335-3p negated the promoting effects of luteolin on bone marrow mesenchymal stem cells. Luteolin facilitates osteogenic differentiation and proliferation in bone marrow mesenchymal stem cells by elevating microRNA-335-3p. This molecular pathway holds promise as a potential therapeutic target for enhancing bone regeneration and combating bone-related disorders.

Keywords

Luteolin, microRNA-335-3p, bone marrow mesenchymal stem cells, proliferation, osteogenic differentiation

Osteoporosis, featured by declined bone mineral density and increased fracture risk, has become a significant public health concern worldwide. The prevalence of osteoporosis continues to rise with age, leading to considerable morbidity and socioeconomic burden[1]. Therefore, exploring novel therapeutic strategies to enhance bone formation and counteract bone loss has emerged as a critical area of research.

Bone Marrow Mesenchymal Stem Cells (BMSCs) play a pivotal role in maintaining skeletal homeostasis through their ability to differentiate into osteoblasts, the bone-forming cells responsible for bone regeneration and repair[2,3]. Various signaling pathways and regulatory factors tightly govern osteogenic differentiation and proliferation of BMSCs, and understanding these mechanisms holds the key to developing effective therapies for bone-related disorders[4,5].

MicroRNAs (miRNAs) have emerged as essential post-transcriptional regulators, capable of influencing various cellular processes, including osteogenic differentiation and proliferation. Among these, miR-335-3p has recently garnered attention for its regulatory role in bone metabolism. Previous studies have manifested that miR-335-3p negatively modulated osteogenesis by targeting key genes involved in the osteogenic pathway[6-9]. However, the potential modulation of miR-335- 3p to promote osteogenic differentiation and proliferation remains an underexplored aspect of bone biology.

Luteolin, a flavonoid abundantly present in various plant-based sources[10], has exhibited diverse biological properties[11-13]. Recent evidence suggests that luteolin may also have beneficial effects on bone health[14]. However, the molecular mechanisms underlying its osteogenic potential and its interaction with miRNAs in BMSCs remain elusive.

In this context, our research article aims to elucidate the function of luteolin in promoting osteogenic differentiation and proliferation in BMSCs. In addition, we hypothesized that luteolin treatment enhances miR-335-3p content, subsequently influencing the expression of osteogenic genes, thus facilitating osteogenic differentiation and proliferation in BMSCs.

Materials and Methods

Cell culture and treatment:

BM-MSCs were purchased from Saiye (Guangdong, China) and cultured in Dulbecco's Modified Eagle Medium (DMEM) and 10 % Fetal Bovine Serum (FBS), 2 mmol/L glutamine, and 1 % antibiotics in a 5 % Carbon dioxide (CO2) incubator at 37° (all from HyClone, Logan, Utah, United States of America (USA)). Cells passages were collected for functional analysis.

BM-MSCs at logarithmic phase (5.0×105) were treated with increasing doses of luteolin (0, 2, 4 or 8 μmol/l) (Chengdu Master Biotechnology Co. Ltd., Sichuan, China) for 24 h.

Cell transfection:

30 nm miR-335-3p mimic (miR-335-3p), inhibitor (anti-miR-335-3p) or the control (miR-NC or anti-miR-NC) (Gene Pharma, Shanghai, China) were transfected into BM-MSCs for 24 h. After the validation of transfection efficiency, cells were treated with 8 μmol/l luteolin for further investigation.

3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyl-2HTetrazolium Bromide (MTT) assay:

BM-MSCs were inoculated into a 96-well plate for 48 h and 72 h, then per well was added with 10 μl MTT reagent (Solarbio, Beijing, China) and incubated for 4 h. Following 150 μl dimethyl sulfoxide reaction, the absorbance was tested at 490 nm.

Western blot:

Radioimmunoprecipitation Assay (RIPA) protein lysis solution was used to extract total cell proteins. The solution was boiled in a boiling water bath for 5 min, and then separated by 12 % Sodium Dodecyl- Sulfate Polyacrylamide Gel Electrophoresis (SDSPAGE), followed by shifting to the Polyvinylidene Difluoride (PVDF) membrane. Then primary incubation and secondary incubation with corresponding antibodies were performed. The Enhanced Chemiluminescence (ECL) substrate kit was utilized to detect protein signals and ImageJ software was applied to determine the gray values. All primary antibodies included Runt-Related Transcription Factor 2 (RUNX2) (ab76956, 1:1000), Osteocalcin (OCN) (ab93876, 1:1000), Osteopontin (OPN) (ab214050, 1:1000) and Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH) (ab8245, 1:5000) were provided by Abcam (Cambridge, United Kingdom)

Quantitative ReverseTranscription-Polymerase Chain Reaction (qRT-PCR):

The Trizol reagent was applied to incubate with BM-MSCs to extract total Ribonucleic Acid (RNA), which were then synthesized to complementary Deoxyribonucleic Acid (cDNA). Then amplification reaction using SYBR Green PCR Master Mix was conducted (Invitrogen) with U6 as an internal reference. The 2-ΔΔCt method was applied to assess miR-335-3p content. The primers for qRT-PCR, miR-335-3p: Forward 5ʹ-UUUUUCAUUAUUGCUCCUGACC-3ʹ and reverse 5ʹ-CCAGTCTCAGGGTCCGAGGTATTC- 3ʹ; U6: Forward 5ʹ-CTCGCTTCGGCAGCACA-3ʹ and reverse 5ʹ-AACCGCTTCACGAATTTGCGT- 3ʹ.

Statistical analysis:

All experimental data are manifested as the mean±- standard deviation. The comparison was conducted using Analysis of Variance (ANOVA) in multiple groups or t test in two groups. p<0.05 was considered statistically significant difference.

Results and Discussion

As shown in Table 1, the proliferation of BM-MSCs was dose-dependently increased after treating with 2, 4, or 8 μmol/l luteolin relative to the untreated cells (Negative Control (NC) group). The contents of RUNX2, OCN and OPN proteins in BM-MSCs were dose-dependently elevated with the treatment of increasing doses of luteolin compared with the untreated cells (NC group) as shown in fig. 1 and Table 2. As exhibited in Table 3, the treatment of luteolin increased miR-335-3p expression in BMMSCs at concentrations of 2, 4, or 8 μmol/l luteolin. Compared with the introduction of miR-NC, miR- 335-3p transfection markedly elevated its level in BM-MSCs (Table 4). Thereafter, it was found that miR-335-3p mimic induced proliferation in BMMSCs (Table 4).

luteolin

Fig. 1: The protein bands of RUNX2, OCN and OPN with the increasing doses of luteolin

Luteolin OD (490 nm)
48 h 72 h
NC 0.38±0.05 0.45±0.04
2 μmol/l 0.47±0.05* 0.52±0.05*
4 μmol/l 0.68±0.07* 0.73±0.09*
8 μmol/l 0.92±0.07* 1.14±0.10*
F 140.655 156.216
p 0.000 0.000

Table 1: The Effects of Luteolin on BM-MSCs Proliferation

Luteolin RUNX2 OCN OPN
NC 0.41±0.04 0.35±0.03 0.25±0.04
2 μmol/l 0.53±0.05* 0.44±0.04* 0.37±0.04*
4 μmol/l 0.69±0.04* 0.58±0.05* 0.48±0.05*
8 μmol/l 0.86±0.07* 0.71±0.03* 0.65±0.03*
F 129.821 152.542 157.591
p 0.000 0.000 0.000

Table 2: The Effects of Luteolin on BM-MSCs Osteogenic Differentiation

Luteolin miR-335-3p
NC 1.00±0.10
2 μmol/l 1.59±0.15*
4 μmol/l 1.84±0.19*
8 μmol/l 2.45±0.22*
F 111.036
p 0.000

Table 3: The Effects of Luteolin On miR-335-3p Expression in BM-MSCs

Group miR-335-3p OD (490 nm)
48 h 72 h
miR-NC 0.99±0.08 0.32±0.05 0.41±0.04
miR-335-3p 2.88±0.32* 0.68±0.04* 0.92±0.07*
t 17.19 16.687 18.977
p 0.000 0.000 0.000

Table 4: The Effects of miR-335-3p on BM-MSCs Proliferation

miR-335-3p mimic introduction in BM-MSCs relative to miR-NC markedly elevated the contents of RUNX2, OCN and OPN proteins as shown in fig.2 and Table 5. Luteolin treatment in BMMSCs increased the proliferation and contents of RUNX2, OCN and OPN proteins, while these effects were abolished after the inhibition of miR- 335-3p as shown in fig. 3 and Table 6.

Group RUNX2 OCN OPN
miR-NC 0.37±0.02 0.32±0.03 0.22±0.04
miR-335-3p 0.54±0.05* 0.49±0.07* 0.45±0.05*
t 9.47 6.697 10.776
p 0.000 0.000 0.000

Table 5: miR-335-3p Effects on Osteogenic Differentiation in BM-MSCs

Group miR-335-3p OD (490 nm) RUNX2 OCN OPN
48 h 72 h
Luteolin+anti-miR-NC 1.02±0.09 0.89±0.06 1.16±0.12 0.84±0.07 0.73±0.06 0.64±0.07
Luteolin+anti-miR-335-3p 0.32±0.05* 0.62±0.04* 0.93±0.05* 0.62±0.05* 0.58±0.04* 0.47±0.03*
t 20.397 11.233 5.308 7.672 6.24 6.697
P 0.000 0.000 0.000 0.000 0.000 0.000

Table 6: miR-335-3p Deficiency Abolishes the Promoting Effects of Luteolin on BM-MSCs

osteogenic

Fig. 2: Up-regulation of miR-335-3p promotes osteogenic differentiation in BM-MSCs

abolishes

Fig. 3: Inhibition of miR-335-3p abolishes the promoting effects of luteolin on BM-MSCs

Osteoporosis, in severe cases, it can cause fractures in patients, which can have adverse effects on their daily lives[15]. BM-MSCs are crucial for maintaining bone resorption and bone formation balance, and are closely related to the pathogenesis of osteoporosis[16]. Luteolin is widely used in medicine[17,18]. Luteolin has been manifested that can impact osteogenic differentiation[19]. Luteolin was able to protect against high glucose-evoked oxidative injury in osteoblasts[20]. According to the findings of Nash et al., the luteolin isolated from tea up-regulated the content of the mineral in human osteoblasts[21]. All the data suggested the possible suppressing effects of luteolin on osteoporosis. In our work, we found that the proliferation of BM-MSCs was dose-dependently increased after treating with 2, 4, or 8 μmol/l luteolin. Moreover, the contents of osteogenic markers RUNX2, OCN and OPN were dose-dependently elevated with the treatment of increasing doses of luteolin in BMMSCs. RUNX2 is essential for bone development and osteodifferentiation, and is involved in encouraging the expression of osteoblast secretion proteins OPN and OCN[22-24]. Therefore, we confirmed that luteolin promoted osteogenic differentiation in BM-MSCs.

In the present work, we also found luteolin increased miR-335-3p levels in BM-MSCs. miRNAs are widely discovered in eukaryotic cells, and can regulate signal transduction pathways and gene expression, thereby involving in development of various cells including BM-MSCs[25-27]. A study showed forced expression of miR-335- 5p promoted osteogenic differentiation in BMMSCs[ 28], which was consistent with our findings with the increased RUNX2, OPN and OCN protein levels after miR-335-5p restoration. Besides, miR- 335-5p restoration triggered proliferation in BMMSCs. In the meanwhile, we also found that miR- 335-5p deficiency abolished the action of luteolin on BM-MSCs.

In all, luteolin accelerates the osteogenic differentiation and proliferation in BM-MSCs through elevation miR-335-5p, bringing to light the possible mechanisms implicated in the function of luteolin in osteoporosis treatment.

Funding:

This work was supported by Zhejiang Traditional Chinese Medicine science and technology plan project(NO.2020ZQ047).

Conflict of interests:

The authors declared no conflict of interests.

References