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The effect of Moringa oleifera polysaccharides on the regulation of glucocorticoid-induced femoral head necrosis: In vitro and in vivo
⁎Corresponding author at: No.555 Youyi East Road, Xi’an 710068, China. yangquanhao.health@yahoo.com (Yangquan Hao)
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Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Abstract
One of the common challenges in using glucocorticoid in the long term is the development of femoral head necrosis. To address this challenge, the use of glucocorticoid suppressors like plant polysaccharides has been considered. In this study, Moringa oleifera polysaccharide was isolated through hot water–ethanol precipitation method and purified by DEAE-Sepharose fast flow column. Then, they were characterized by FT-IR, NMR, methylation, and chromatography assays. The polysaccharide biocompatibility was investigated by MTT assay and its effect on osteoblasts was evaluated by controlling gene expression. Also, the effect of polysaccharide on dexamethasone-induced femoral head necrosis in rats was assessed by hydroxyproline, hexosamine and morphometric parameters. The results show that 2 Da molecular weight polysaccharide is mainly composed of Rha, Ara, Fru, Xyl, Man and Gal in the molar ratio of 1.7:2.1:3.4:5.9:5.8:1.3. Meanwhile, MTT results on osteoblasts cells showed polysaccharide biocompatibility, while significantly reducing the negative effects of glucocorticoid. Likewise, polysaccharide significantly reduced the levels of apoptosis and intracellular ROS of glucocorticoid-induced femoral necrosis. Moreover, the results of gene expression indicated a decrease in the expression of TNF-α and IL-6 genes using polysaccharide, which is very effective in preventing apoptotic activity. Also, Polysaccharide increased bone density, bone volume per tissue volume, trabecular thickness, and the hexosamine to hydroxyproline ratio in the rat serum in the presence of glucocorticoids, which are very effective in the process of femoral head necrosis. Furthermore, polysaccharide significantly increases the OCN, RUNX2 and COL-1 genes expression in cartilage tissue, which is in line with the result of morphometric parameters. Overall, this study suggests that the use of polysaccharide could result in the treatment of femoral head necrosis.
Keywords
Polysaccharide
Osteoblasts
Apoptosis
Femoral necrosis
Biocompatibility
1 Introduction
Moringa oleifera is a perennial and multi-purpose tree that grows in most parts of the world. Despite the remarkable nutritional properties in all organs of this plant, the leaves of this plant are very important due to their high protein, glucosinolate, fat, antioxidant compounds and poly-phenols (Gopalakrishnan et al. 2016). Moringa is affluent in phytosterols such as stigmasterol and cytosterol, which are precursors to the kind of hormone (Zhao et al. 2019). In addition, the seeds, flowers and gums of this plant have good sources of nitrile, flavonoids, thiocarbamate and glycosides which are effective in anti-inflammatory, anti-bacterial, anti-diabetic, antidiabetic, and anti-hypertensive activities (Dhimmar et al. 2015). In addition, the roots of this plant are used in anti-inflammatory, anti-bacterial and anti-fertility activities (Singh et al. 2020).
Various previous clinical and non-clinical studies have shown that corticosteroid-induced femoral head necrosis is one of the most important problems when using drugs such as dexamethasone and other glucocorticoids used to treat inflammatory and auto-immune diseases (Feng et al. 2017; Liu et al. 2017). Repeated use of steroids causes necrosis of the femoral head, which eventually leads to permanent deformity, bone fractures and imbalances in the bone regeneration process (Luo et al. 2018). Efforts have been made to reduce glucocorticoid levels to control bone necrosis (Jin et al. 2020; Liang et al. 2021). In this regard, osteonecrosis of the femur has been observed even with topical ointment for glucocorticoid occlusion (Kubo et al. 2001; McLean et al. 1995). Today, osteonecrosis occurs in 9–40% of patients on long-term treatment and can occur without even osteoporosis with glucocorticoids (Weinstein 2012). In recent years, herbal medicine has shown considerable evidence to modulate apoptosis. For example, Jiang et al. (2014) reported that by regulating PPARγ expression and activating β-catenin signaling compared to surgery, good results were achieved in reducing bone femur necrosis. However, it is unclear which of the chemical compounds in herbal medicines is responsible for controlling and regenerating femoral head tissue. But, polysaccharides have surprisingly attracted attention in various medical and pharmaceutical fields such as anti-cancer (Kholiya et al. 2020), wound healing (Ghlissi et al. 2020), anti-inflammatory (Yan et al. 2020), antiviral, and antibacterial (Kaczmarek 2020).
In this study, an attempt was made to purify the polysaccharide from Moringa oleifera plant and structural analysis by FT-IR, methylation and NMR methods to provide an opportunity for using polysaccharide to control the activity of femoral head necrosis. Therefore, after investigating the biocompatibility of polysaccharide by MTT method on osteoblasts cells, the function of polysaccharide on the expression of hormone-induced tumor necrosis factors (TNF-α) and interleukin-6 (IL-6) genes and its direct effects on femoral bone necrosis were investigated. Then, in vivo experiments were performed by measuring morphometric parameters and changes in the expression of OCN, RUNX2 and COL-1 genes in cartilage tissue.
2 Material and methods
2.1 Extraction and purification of polysaccharide from Moringa oleifera
Fresh Moringa oleifera roots were provided by Xuwenfarm in Guangdong, China. After washing and drying in the oven, the roots were grounded. Water-extractable way was applied to extract polysaccharides from milled roots. 200 g of roots were boiled in 2 L of distilled water for 3 h. The resulting solution separately was boiled again for 3 h followed by evaporation up to 50 mL by the rotary evaporator system. To precipitate the polysaccharide, the volume of ethanol dehydrate was added and incubated at 4 °C for 24 h. The solution was then centrifuged at 6300 rpm for 4 min at 4 °C. In the next phase, the precipitate was deproteinized by the method described by Zhang et al. (2010). The deproteinized sample was quadrupled in volume by ethanol dehydrate, re-precipitated and finally weighed. Then, for fractionation by a DEAE Sepharose Fast Flow column, approximately 15 g of polysaccharide was dissolved in 8 mL of distilled water. Then, the column was rinsed with 0, 0.1, 0.2, and 0.3 mol/l NaCl. After collecting the elution buffer, the concentration of polysaccharide was determined using the method reported by Masuko et al. (2005) based on phenol–sulfuric acid technique. In order to obtain a higher concentration of polysaccharide, 3500D membrane was used for dialysis of the sample containing polysaccharide.
2.2 Molecular weight and chemical composition of polysaccharide
High-performance liquid chromatography (HPLC) with TSK-G4000 PWXL column (7.8 × 300 mm) and RID-10A detector were used to evaluate the molecular weight of polysaccharides. A 0.45 μm membrane was used to purify the polysaccharide solution at a concentration of 1% (w/v). The mobile phase in the molecular weight analysis was deionized water with a flow rate of 0.6 mL/min. The column temperature and the injection rate were set at 25 °C and 20 μL, respectively. The molecular weight of the sample was evaluated using the standard curve obtained from Tseries Dextran. Also, gas chromatography was used to investigate the composition of monosaccharides. In this regard, 5 mg of polysaccharide with 1.5 mL of trifluoroacetic acid (2 mol/L) was hydrolyzed under nitrogen gas and stored for 8 h at 95 °C. 100 μL of methanol was added to the solution to remove trifluoroacetic acid at 60 °C. 5 mg of hydroxylamine hydrochloride and 0.35 mL of pyridine were added to the mixture. The mixture was then incubated for 30 min at 90 °C. Afterwards, 0.5 mL of acetic anhydride was added to the cooled mixture and incubated at 90 °C for 30 min. After removing the top liquid, the residues were dried. Then, the obtained product was mixed with 0.5 mL of chloroform and approximately 0.2 μL of the obtained sample for gas chromatography–mass spectrometry analysis (by increasing the column temperature from 160 to 210 °C with a passage rate of 1.0 mL/min and a flow ratio: 50 to 1) was used. 7 types of standard monosaccharides including rhamnose, arabinose, fructose, xylose, mannose, glucose and galactose were considered.
2.3 FT-IR, methylation investigation and NMR
To investigate the polysaccharide FT-IR spectrum, 1.5 mg of polysaccharide was mixed with 150 mg of ground dry KBr powder. The mixture was then completely compressed in a 1 mm pellet. The FT-IR spectra of samples were determined using a Nicolet 5700 FT-IR spectrometer in a wave number range of 400–4000 cm−1 applying the KBr way.
Furthermore, in order to perform methylation analysis, 1 mg of polysaccharide was dissolved in 1 mL of dimethyl sulfoxide. It was then methylated using the Kalyan and Paul methods (Anumula and Taylor 1992). The OH adsorption removal in the range of 3200 to 3700 cm−1 was used to determine complete methylation. 5 mg of methylated product was hydrolyzed with 1 mol/L trifluoroacetic acid (4 mL) for 2 h at 120 °C. Then, 50 mg of NaBH4 was used to reduce the sample. Subsequently, the sample was acetylated with acetic-pyridine anhydride (1:1) at 100 °C for 1 h. Finally, methylated alditol acetate was evaluated by gas chromatography–mass spectrometry.
As well, for NMR examination, 50 mg of the sample was dried in vacuum for 6 days. Then, the sample was placed in 1 mL of D2O (99.97%). Subsequently, the sample was exchanged 3-time with freeze drying in D2O deuterium. Finally, the sample was dissolved in 0.5 mL of D2O. Next, the 1H and 13C NMR spectra were registered on a Bruker AV-500 spectrometer, respectively. Chemical shifts were expressed applying acetone as the internal standard.
2.4 Cell proliferation
Early osteoblasts resulting from neonatal rats were sequentially digested with collagen II. The second to fifth digested cells were neutralized and collected. Next, the cells were cultured in Dulbecco’s modified Eagle’s medium added with 10% fetal bovine serum and 1% penicillin/streptomycin in a plate, and maintained in an ambient of 95% air and 5% CO2 at 37 °C. After the cells approached 70% of the confluency, they were treated. During the study, the medium changed at three-day intervals.
2.5 Mtt assay
The osteoblasts cells were cultured at a density of 4 × 103 cells/well into 96-well plate and then incubated for 12 h at 37 °C with 5% CO2. After 12 h, the cells were treated and incubated for 24 h with different concentrations of dexamethasone (0.1, 0.5, 1, and 5 μM) as a glucocorticoid. Then, the culture medium was changed to a medium containing determined concentrations of polysaccharide (25, 50, 100, and 150 μg/mL) and incubated for 24 h. To determine the separate effect of glucocorticoid and polysaccharide on the cells, each of them was added separately to the cell culture medium at the concentrations stated above and incubated for 24 h. Finally, all of the mediums were replaced by PBS, and 0.5 mg/mL MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] was diffused in media and incubated for 4 h (50 μL/well). After incubation, the culture medium was removed and the formed crystals were dissolved in 100 μL dimethyl sulfoxide by shaking for 2 min. Then, the absorbance of the cells was measured at λ = 570 nm using the Eq (1). The cell viability was stated as the ratio of optical density.
Where, PSabsorbance is the individual absorbance of the polysaccharide loaded well and Cabsorbance is the mean absorbance of control. Mean standard deviations were considered for each well.
2.6 Apoptosis and reactive oxygen species (ROS) assays
To estimate the apoptosis, flow-cytometry was applied. According to the method described above, the cells (4 × 104 cells/well) were sited into a 6-well plate and treated with polysaccharide in the incubator for 24 h. Next, the cells were collected with 3000 g centrifugation at 4 °C for 3 min, and washed with PBS. Afterwards, the cells were resuspended in 100 μL per tube of containing Annexin V binding buffer (HEPES buffer: 0.1 M, NaCl 1.4 M, CaCl2 25 mM, pH 7.4). Then, 2 μL of Annexin V conjugated Alexa Fluor 488 was added to the cells solution and retained in dark for 15 min at 21 °C. Subsequently, 400 μL of the binding buffer and 5 μL of 50 μg/ml propidium iodide were added to the solution and kept in ice. Finally, samples were studied by FACscan (BD Bioscience, USA).
To determine intracellular ROS in the presence of polysaccharide (15, 25, 50, 100 and 150 µg/mL µg/mL), 4 × 104 cells/well were cultured into 6-well plates for 24 h. Next, the fluorescent intensity based on DCF measurement was assessed.
2.7 Analysis of TNF-α and IL-6 in vitro
Total RNA isolation was carried out according to the manufacturer’s protocols in control and polysaccharide (25, 50, 100, and 150 μg/mL) groups through Trizol reagent. The RNA was then dignified at 260–280 nm using a UV–VIS spectrophotometer (Eppendorf). To omit any contaminant genomic DNA, the RNA was treated with DNAse I and cDNA was generated from 1 µg of RNA applying revert Aid First Strand cDNA Synthesis Kit (Fermentas). The following primers were applied:
TNF-α
forward 5′- CAGGCGGTGCCTATGTCTC-3′,
reverse 5′- CGATCACCCCGAAGTTCAGTAG-3′;
IL-6
forward 5′- GCCAGAGTCCTTCAGAGAGATACA-3′
reverse 5′- CTTGGTCCTTAGCCACTCCTTC-3′;
β-actin
forward 5′-AATTCCATCATGAAGTGTGA-3′
reverse 5′-ACTCCTGCTTGCTGATCCAC-3′.
Quantitative PCR was done by an ABI 7500 real-time PCR system (ABI, USA) using a SYBR Premix Ex Taq Reagent Kit (Takara) based on the manufacturers’ protocols. The cycle threshold (Ct) values of each target gene were deducted from the Ct values of the housekeeping gene (ΔCt). Target gene ΔΔCt was evaluated as the ΔCt of the target gene minus the ΔCt of control. The fold change in mRNA expression of TNF-α, and IL-6 was assessed as 2−ΔΔCt. The β-actin amplification was applied as a housekeeping gene.
2.8 In vivo assays
In order to evaluate the effect of polysaccharide on rats with femoral head necrosis, rats with an average weight of 220 g and 11 weeks of age were selected. Then, rats were divided into three groups including the control (n = 6), the glucocorticoid (n = 6) (injection of dexamethasone at a dose of 0.2 g/kg every four days) and the glucocorticoid plus polysaccharide (n = 6) (daily intake of 6 g/kg (an average of 1.32 g/rat) polysaccharide by oral gavage along with injection of dexamethasone at a dose of 0.2 g/kg every four days). Rats were kept in cages for 50 days under normal conditions with 12 h of light and free access to water and food. No deaths were observed in any of the groups. All stages of animal testing and care were in full compliance with Chinese Ethics under the supervision of the Animal Welfare and Use Committee of Xi'an Jiaotong University.
2.8.1 Morphometric parameters and hydroxyproline to hexosamine ratio
To evaluate bone mass density in rats, first, after anesthesia with chloral hydrate (10%), the femoral head of the left lower limb was isolated from the rat. After separating the muscles and fascia, to evaluate the bone parameters, the head of the right femur of mice was scanned with a micro-CT scan (Skyscan 1176; Bruker MicroCT, Kontich, Belgium) with a resolution of 9 µm and their two-dimensional images were analyzed using CTAn software (v.1.13; Bruker MicroCT) for bone mineral density, bone volume per tissue volume and trabecular thickness. Also, in order to determine the hexosamine and hydroxyproline concentrations in the blood serum of rats, blood samples of each rat were prepared on days 25 and 50 and their plasma was separated by centrifugation. Then, based on the methods of Blumenkrantz and Asboe-Hansen (1976) and Neuman and Logan (1950) hexosamine and hydroxyproline concentrations were measured, respectively.
2.8.2 Analysis of OCN, RUNX2, and COL-1 in vivo
After separating the right femoral head from the mice, the cartilage samples were prepared from the articular cartilage of the femoral head using a sharp knife and frozen with liquid nitrogen. Total RNA was extracted by applying Trizol reagent following the manufacturer's protocol. Then, the PCR process was performed on cells derived from rat tissue based on section 2.7. The primers include of:
OCN
forward 5′- CCCCCTCTAGCCTAGGACC-3′
reverse 5′- ACCAGGTAATGCCAGTTTGC-3′;
RUNX2
forward 5′- CCGAGACCAACCGAGTCATTTA-3′
reverse 5′- AAGAGGCTGTTTGACGCCAT-3′;
COL-1
forward 5′- GACATCCCACCAATCACCTG-3′
reverse 5′-CGTCATCGCACAACACCTT-3′;
β-actin
forward 5′-AATTCCATCATGAAGTGTGA-3′
reverse 5′-ACTCCTGCTTGCTGATCCAC-3′.
2.9 Statistical analysis
Data statistical results were analysed using Statistical Product and Service Solutions (SPSS) 13.0 software (SPSS, Inc., Chicago, IL, USA). The differences between the groups were analysed with analysis of variance. Statistical differences were measured at levels of *P < 0.05, and **P < 0.01.
3 Results and discussion
3.1 Isolation, molecular weight and composition of polysaccharide
The extraction efficiency of polysaccharide through water extraction, ethanol precipitation and deproteinization was approximately 6.17%. The outputs of Fig. 1A shows the highest concentrations of polysaccharide at the peak of 0.891, 0.553 and 0.325 mg/mL. The molecular weight of polysaccharide was assessed from a calibration curve supplied with standard dextran as 1.93 × 105 Da. In addition, the results of Fig. 1B and 1C show that the monosaccharide composition in the sample is mainly from Rha, Ara, Fru, Xyl, Man and Gal in the molar ratio of 1.7:2.1:2.6:5.5:4.2:1.3 with the glucose trace. The results show that Xyl (∼30.3%) and Man (∼26.9%) are two abundant monosaccharides.
(A) The elution profile of the crude polysaccharide isolated from Moringa oleifera root on a cellulose DEAE sepharose fast flow chromatography eluted with stepwise gradient of NaCl aqueous solutions (0, 0.1, 0.2 and 0.3 M) at a flow rate of 1.0 mL/min. GC–MS analysis of standard (B) monosaccharide and (C) Moringa oleifera root polysaccharide. D: Infrared Spectrum of Moringa oleifera root polysaccharide. E: 1H NMR spectrum of Moringa oleifera root polysaccharide. F: 13C NMR spectrum of Moringa oleifera root polysaccharide.
3.2 Characterization of polysaccharide
As shown in Fig. 1D, the highest tensile peak is in the range of 3400–3420 cm−1. Adsorptions in region 2940 cm−1 indicates carbon-hydrogen group, 2160 cm−1 as carbon–carbon group, 1740 cm−1, 1610 cm−1, 1415 cm−1 and 1325 cm−1 as carbon-hydrogen or carbon–oxygen groups, and also 1220–1020 cm−1 indicates pyran ring structure. In addition, high adsorption in regions 820–960 cm−1 to β-glycosidic bonds and two adsorption peaks in regions 760 cm−1 and 535 cm−1 also indicate α-glycosidic bonds and the carbon–carbon–oxygen group, which are common bonds in the structure of polysaccharides.
The results of methylation, to determine the lateral and main structure of polysaccharide based on retention time and ions fragments characteristics, illustrate the presence of D-galactitol; L-arabinitol; L-rhamnitol and D-galactitol in the structure of polysaccharide. Also, the results show that there is non-reduced L-rhamnopyranosyl, D-galactopyranosyl, L-arabinopyranosyl and D-galactopyranosyl. Also, the result of methylated carboxyl in the polysaccharide and the presence of alditol acetates together with the new and high peaks D-galactitol show that D-GalpA It is also present in polysaccharide.
On the other hand, NMR results, which confirm the results of FT-IR, show five peaks in the 1H spectrum chromatogram that refer to chemical components and chemical bandage (Fig. 1E). Among the signals provided, the signal 5.12 to 5.20 ppm in the 1H spectrum shows the α-Araf residue. While signals 4.82 to 4.93 ppm are also related to β-Galp/α-Galp residues. Signals 1.17 to 1.23 ppm also indicate the bonding of C to H1, H2, H3. In addition, the 13C NMR spectrum outputs revealed that signals adjacent to 5.17 ppm represent the α-Araf residue and signals 99.54 to 100.43 ppm also indicate the β-Galp residue (Fig. 1F). Specifically, the chemical shifts of anomeric protons and carbon resonance respectively below 4.77 ppm and above 100.45 ppm indicate β-linked-anomeric of the Galp residues, which is consistent with the methylation finding.
3.3 Cell viability, apoptosis and ROS analysis
As shown in Fig. 2A, increasing the glucocorticoid concentration from 0.1 to 5 μM decreases the viability of cells. On the other hand, based on Fig. 2B, compared to controls of 25, 50, and 100 μg/mL polysaccharides, its improved osteoblast cell viability and even a relative increase in cell growth up to 3.9%, 7.7%, and 12.8 %و respectively. However, a concentration of 150 μg/mL significantly reduces the viability of osteoblast cells. While the presence of polysaccharide significantly controls the effect of glucocorticoid on cell viability (Fig. 2C). According to the results, the use of 100 μg/mL polysaccharide is recommended to control the cell viability in the presence of glucocorticoid. Meanwhile, the result of apoptosis in Fig. 2D and 2E shows that the use of glucocorticoid significantly increases the percentage of apoptosis in early apoptosis (20.3%) and late apoptosis (14.3%) quadrants compared to the control. The use of polysaccharides reduces the percentage of apoptosis in early apoptosis (10.6%) and late apoptosis (10.8%) quadrants up to ∼ 49 and ∼ 25%, respectively (Fig. 2F). As expected, based on the results of apoptosis, the level of intracellular ROS is significantly reduced from 319 to 224 by the use of polysaccharide in the presence of glucocorticoid (Fig. 2G). However, compared to the control group, the use of polysaccharides to reduce the negative effects of glucocorticoids has not been able to completely stop the level of intracellular ROS. Therefore, the use of polysaccharides has an inhibitory effect on intracellular ROS production, and probability modulates the progressive activity of femoral necrosis.
(A) Effects of glucocorticoid on cell viability in vitro. (B) Effects of Moringa oleifera root polysaccharide on cell viability in vitro. (C) Effect of Moringa oleifera root polysaccharide on cell viability pre-treated with glucocorticoid in vitro. Two-dimensional contour density plots of osteoblasts cells determined by flow cytometry assay. (D) control, (E) glucocorticoid, (F) polysaccharide + glucocorticoid. Cell necrosis and apoptosis measured using propidium iodide (PI) and Annexin V-FITC staining, and (G) the effects of glucocorticoid and polysaccharide + glucocorticoid on the ROS production.a,b,c,dLeast square means with different letters in superscripts are different at *P < 0.05.
3.4 Effects of polysaccharide on TNF-α and IL-6 mRNA expression in vitro
Studies of gene expression levels in Fig. 3A and 3B show that the expression of TNF-α and IL-6 genes increase significantly in the presence of glucocorticoid. While the use of polysaccharide significantly reduces the expression of TNF-α and IL-6 genes. Although different concentrations of polysaccharide in the presence of the glucocorticoid reduce the expression of TNF-α gene, but the greatest decrease in the expression of TNF-α gene to 150 μg/mL (2.3 times glucocorticoid + polysaccharide group) and 100 μg/mL (2.12 times glucocorticoid + polysaccharide group) of polysaccharide. However, only concentrations of 100 and 150 μg/mL of polysaccharide reduced IL-6 gene expression. Therefore, the inhibitory effect of polysaccharide on TNF-α gene is greater than IL-6 gene and the crossroads of the reduction of both genes is 100 μg/mL of polysaccharide.
Effects of Moringa oleifera root polysaccharide on TNF-α (A) and IL-6 (B) expression in osteoblasts cells induced by glucocorticoid. (C) Effect of polysaccharide on bone mineral density. (D) Effect of polysaccharide on bone volume per tissue volume. (E) Effect of polysaccharide on trabecular thickness. (F) Effects of Moringa oleifera root polysaccharide on OCN, RUNX2 and COL-1 expression in cartilage tissue triggered by glucocorticoid. a,b,c,d,eLeast square means with different letters in superscripts are different at *P < 0.05.
3.5 Effect of polysaccharide on morphometric parameters and hydroxyproline to hexosamine ratio
The results of Fig. 3C showed that injection of dexamethasone for 50 days caused a loss of mineral density in the femur compared to the control group. While the use of Moringa oleifera polysaccharide at a concentration of 6 g/kg significantly enhances bone density. This result revealed that long-term use of Moringa oleifera polysaccharide can prevent the negative effects of glucocorticoids and improve bone density. The bone volume per tissue volume of the mice in the dexamethasone group was 35.1%, that was drastically lower than that in the control (86.2%). Whereas supplementation with Moringa oleifera polysaccharide meaningfully raised the bone volume per tissue volume up to 62.4% (Fig. 3D). Moreover, a similar effect was identified regarding the trabecular thickness among groups (Fig. 3E). In the following, the outcomes of Table 1 revealed that dexamethasone significantly increased hydroxyproline concentration. While it reduces the concentration of hexosamine in rat serum and also decreases the hexosamine to hydroxyproline ratio. Using Moringa oleifera polysaccharide at a concentration of 6 g/kg, the process of decreasing the hexosamine concentration and hexosamine to hydroxyproline ratio was stopped and increased significantly compared to the Glucocorticoid group.
Hydroxyproline (HOP) (μg/mg)
Hexosamine (HOM) (μg/mg)
HOM/HOP
(25-day)HOM/HOP
(50-day)
Days
25
50
25
50
Control
57.92c
59.01c
11.69a
12.22a
0.201
0.207
Glucocorticoid
72.61a
80.74a
6.85b
5.96c
0.094
0.073
Polysaccharide + Glucocorticoid
65.87b
72.05b
7.59b
8.93b
0.115
0.123
3.6 Effects of polysaccharide on OCN, RUNX2 and COL-1 mRNA expression in vivo
The results of RT-qPCR analysis on cartilage tissue of the right femoral head of rats in Fig. 3F showed that the use of dexamethasone drastically reduced the levels of RUNX2, COL-I and OCN mRNA after the treatment period compared to the control. Application of Moringa oleifera polysaccharide in combination with glucocorticoid during the treatment period increased all expression concentrations of RUNX2, COL-I and OCN mRNA genes associated with osteogenesis to varying degrees compared to the glucocorticoid group. Nevertheless, the expression level of the above genes is still low compared to the control group. In addition, the results indicate a greater increase in OCN gene expression compared to RUNX2. Overall, the results show that the presence of Moringa oleifera polysaccharides effectively prevents glucocorticoid suppressive activities on bone-forming genes.
4 Discussion
Because femoral head necrosis is seen as a multifactorial disorder in patients who have been taking corticosteroids for a long time, the use of glucocorticoid suppressants seems to be necessary to prevent the disease. Depending on the complexity of the disorder, there are a variety of treatments for the disease. One of the methods to prevent and treat this anomaly is to use plant polysaccharide (Huang et al. 2019). Despite the variety of compounds in the roots of Moringa oleifera, in this study, part of the polysaccharide was isolated and investigated by FT-IR, methylation and NMR methods. Similar to the results of Roy et al. (2007) this study showed that the extracted polysaccharide has the carbohydrates typical structure. However, there is a challenge to more accurately identify the status and purity of the polysaccharide based on the results of Faizi et al. (2014) and Chhikara et al. (2020) who exhibited that the Moringa oleifera has at least one hundred active compounds.
Because an imbalance between osteoblasts and osteoclasts causes osteoporosis, apoptosis of osteoblasts in the presence of glucocorticoid compounds can impair bone regeneration and repair (Canalis et al. 2007). The results of this study using osteoblast cells, in line with the results of Cui et al. (2019b), show that the use of plant polysaccharide such as Moringa oleifera root polysaccharide is very effective in reducing the level of apoptosis and cell death. Thus, the concentration of 25 to 100 µg/mL of polysaccharides not only had a positive effect on the survival of osteoblast cells, but also reversed the process of cell death in glucocorticoid-induced osteoblasts after 24 h. This process of reducing death is in line with the reduction of intracellular ROS, which can prevent further inflammation based on ROS reduction (Guon and Chung 2017; Oguntibeju et al. 2020). Increased intracellular ROS can cause tissue damage that plays an important role in the destruction of femoral head necrosis (Deng et al. 2018). The presence of galactan compounds in the extracted polysaccharide based on methylation and NMR results can be effective in reducing inflammation caused by intracellular ROS based on the description of Barbosa et al. (2020). For example, Cui et al. (2019a) demonstrated that the use of polysaccharide containing galactan on the LPS-induced macrophage cell reduces the expression of TNF-α and IL-6 genes and reduces the level of inflammation. However, the direct effect of galactan on reducing inflammation needs to be investigated more specifically. In the following, Huang et al. (2019) and Cui et al. (2020), respectively, were able to reduces femoral head necrosis in an animal model by suppressing the apoptotic cell death pathway through the polysaccharides extracted from Agrimonia pilosa and safflower. This study, similar to the above study, shows that the use of polysaccharides prevents the development of femoral head necrosis by suppressing the expression pathway of TNF-α and IL-6 genes in addition to reducing the level of inflammation. Meanwhile, using reversal of other femoral head necrosis signaling pathways through the Dipsacus asper polysaccharide by Sun et al. (2019) explained in an animal model.
Furthermore, the results of this research in the rat model illustrated that the use of Moringa oleifera polysaccharide can have positive effects on bone volume per tissue volume, trabecular thickness, bone density and hexosamine concentration. The results of micro-CT scans show that polysaccharide can maintain trabecular thickness and bone volume despite the challenge of dexamethasone, which reduces bone volume and trabecular thickness. Increased expression of OCN and RUNX2 genes, which can be effective in maintaining bone volume per tissue volume and thickness of trabeculae (Bai et al. 2021; Ho et al. 2015), confirms the positive effects of Moringa oleifera polysaccharide in the above parameters. Because hydroxyproline (a non-essential amino acid in collagen, the increase of which in the blood indicates the breakdown of collagen) and hexosamine (a component of mucopolysaccharide which reduces osteoblastic formation and bone reabsorption reduces its presence in serum) in blood is considered an indicator of femoral bone necrosis (Hong et al. 2001; Qi and Cao 2001); the positive effect of Moringa oleifera polysaccharides on their control can indicate the favorable effect of polysaccharides on femoral head necrosis. Improving the hexosamine to hydroxyproline ratio using polysaccharides can indicate improved collagen and mucopolysaccharide synthesis. The outcomes of COL-1 gene expression in Fig. 3F confirms the above finding. In this experiment, it was found that the expression level of the COL-1 gene is meaningfully raised by consuming polysaccharide, which is very effective in repairing damaged tissue. Similarly, Cui et al. (2019b) using Safflower polysaccharide were able to improve bone mineral density and hexosamine concentration in the blood serum of rats. In the following, these findings indicate that the therapeutic effect of polysaccharides is effective on femoral bone necrosis.
Overall, the present finding suggests that the polysaccharide from Moringa oleifera root may reduce the apoptosis of osteoblasts, which is effective in the prevention and treatment of femoral head and neck necrosis.
5 Conclusions
The findings of this study emphasize that the use of Moringa oleifera polysaccharide is effective in controlling and regenerating osteoblasts cells, and preventing necrosis of the femoral head. But it still faces significant challenges, such as the lack of complete identification of the Moringa oleifera polysaccharide, and the separate effect of each component on the repair process or the prevention of femoral head necrosis. However, this study reveals that polysaccharide extracted by the hot water–ethanol precipitation method with conventional carbohydrates, high biocompatibility and glucocorticoid suppression can provide a viable pathway for the treatment of femoral head necrosis in patients taking long-term steroidal compounds. In this study, it was found that Moringa oleifera polysaccharide reduces the expression of TNF-α and IL-6 genes, which is very effective in the development of femoral head necrosis. On the other hand, in vivo results show that Moringa oleifera polysaccharide improves femoral head tissue by increasing the hexosamine and decreasing the hydroxyproline along with increasing bone mass density. In general, the oral use of Moringa oleifera polysaccharide in the treatment of femoral head necrosis due to long-term use of glucocorticoids can be considered not only because of the very low side effects of polysaccharide, but also but also it can effectively prevent the progression of femoral necrosis due to its ability to integrate with glucocorticoids that patients need.
Funding
This study was supported by Shaanxi Provincial Administration of traditional Chinese medicine Fund (2020ZXY-010).
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
References
- A comprehensive procedure for preparation of partially methylated alditol acetates from glycoprotein carbohydrates. Analytical biochemistry. 1992;203(1):101-108.
- [Google Scholar]
- Conditional knockout of the PDK-1 gene in osteoblasts affects osteoblast differentiation and bone formation. Journal of Cellular Physiology. 2021;236(7):5432-5445.
- [Google Scholar]
- Immunostimulatory Effect of Sulfated Galactans from the Green Seaweed Caulerpa cupressoides var. flabellata. Marine drugs. 2020;18(5):234.
- [CrossRef] [Google Scholar]
- An assay for total hexosamine and a differential assay for glucosamine and galactosamine. Clinical biochemistry. 1976;9:269-274.
- [Google Scholar]
- Glucocorticoid-induced osteoporosis: pathophysiology and therapy. Osteoporosis International. 2007;18(10):1319-1328.
- [Google Scholar]
- Bioactive compounds, associated health benefits and safety considerations of Moringa oleifera L.: An updated review. Nutrition & Food Science.. 2020;51(2):255-277.
- [Google Scholar]
- Characterization of Moringa oleifera roots polysaccharide MRP-1 with anti-inflammatory effect. International journal of biological macromolecules. 2019;132:844-851.
- [Google Scholar]
- Beneficial contribution of a safflower (Carthamus tinctorius L.) polysaccharide on steroid-induced avascular necrosis of the femoral head in rats. International journal of biological macromolecules. 2019;123:581-586.
- [Google Scholar]
- Structural characterization of a safflower polysaccharide and its promotion effect on steroid-induced osteonecrosis in vivo. Carbohydrate polymers. 2020;233:115856.
- [CrossRef] [Google Scholar]
- Sesamin protects the femoral head from osteonecrosis by inhibiting ROS-induced osteoblast apoptosis in rat model. Frontiers in Physiology. 2018;9:1787.
- [Google Scholar]
- Pharmacological activities of Moringa oleifera: an overview. Res J Pharm Tech. 2015;10:476-480.
- [Google Scholar]
- GC/GCMS analysis of the petroleum ether and dichloromethane extracts of M oringa oleifera roots. Asian Pacific journal of tropical biomedicine. 2014;4(8):650-654.
- [Google Scholar]
- Fludarabine inhibits STAT1-mediated up-regulation of caspase-3 expression in dexamethasone-induced osteoblasts apoptosis and slows the progression of steroid-induced avascular necrosis of the femoral head in rats. Apoptosis. 2017;22(8):1001-1012.
- [Google Scholar]
- Polysaccharide from Pimpinella anisum seeds: Structural characterization, anti-inflammatory and laser burn wound healing in mice. International journal of biological macromolecules. 2020;156:1530-1538.
- [Google Scholar]
- Moringa oleifera: A review on nutritive importance and its medicinal application. Food Science and Human Wellness. 2016;5(2):49-56.
- [Google Scholar]
- Moringa oleifera fruit induce apoptosis via reactive oxygen species-dependent activation of mitogen-activated protein kinases in human melanoma A2058 cells. Oncology letters. 2017;14(2):1703-1710.
- [Google Scholar]
- Chitosan nanofiber scaffold improves bone healing via stimulating trabecular bone production due to upregulation of the Runx2/osteocalcin/alkaline phosphatase signaling pathway. International Journal of Nanomedicine. 2015;10:5941.
- [Google Scholar]
- The experimental research of alteration of bone metabolism in steroid-induced necrosis of femoral head. The Journal of Bone and Joint Injury. 2001;16:365-368.
- [Google Scholar]
- A polysaccharide from dried aerial parts of Agrimonia pilosa: Structural characterization and its potential therapeutic activity for steroid-induced necrosis of the femoral head (SANFH) Carbohydrate polymers. 2019;214:71-79.
- [Google Scholar]
- Pravastatin prevents steroid-induced osteonecrosis in rats by suppressing PPARγ expression and activating Wnt signaling pathway. Experimental biology and medicine. 2014;239(3):347-355.
- [Google Scholar]
- Curcumin prevents osteocyte apoptosis by inhibiting M1-type macrophage polarization in mice model of glucocorticoid-associated osteonecrosis of the femoral head. Journal of Orthopaedic Research®. 2020;38(9):2020-2030.
- [Google Scholar]
- Tannic acid with antiviral and antibacterial activity as a promising component of biomaterials—A minireview. Materials. 2020;13(14):3224.
- [Google Scholar]
- Seaweed polysaccharide derived bioaldehyde nanocomposite: Potential application in anticancer therapeutics. Carbohydrate Polymers. 2020;240:116282.
- [CrossRef] [Google Scholar]
- Osteonecrosis of the femoral head that developed after long-term topical steroid application. Journal of orthopaedic science : official journal of the Japanese Orthopaedic Association. 2001;6(1):92-94.
- [Google Scholar]
- Tumor microenvironments self-activated nanoscale metal organic frameworks for ferroptosis based cancer chemodynamic/photothermal/chemo therapy. Acta Pharmaceutica Sinica B 2021
- [CrossRef] [Google Scholar]
- Corticosteroid-induced osteonecrosis of the femoral head: detection, diagnosis, and treatment in earlier stages. Chinese medical journal. 2017;130(21):2601.
- [Google Scholar]
- The role of autophagy in steroid necrosis of the femoral head: a comprehensive research review. International orthopaedics. 2018;42(7):1747-1753.
- [Google Scholar]
- Carbohydrate analysis by a phenol–sulfuric acid method in microplate format. Analytical Biochemistry. 2005;339(1):69-72.
- [Google Scholar]
- Cataracts, glaucoma, and femoral avascular necrosis caused by topical corticosteroid ointment. Lancet (London, England). 1995;345(8945):330.
- [CrossRef] [Google Scholar]
- The determination of hydroxyproline. Journal of Biological Chemistry. 1950;184(1):299-306.
- [Google Scholar]
- Effects of Moringa oleifera on oxidative stress, apoptotic and inflammatory biomarkers in streptozotocin-induced diabetic animal model. South African Journal of Botany. 2020;129:354-365.
- [Google Scholar]
- Study on animal models of hormonal avascular necrosis of the femoral head. Journal of Fujian College of TCM. 2001;11:20-22.
- [Google Scholar]
- Structural investigation of a heteropolysaccharide isolated from the pods (fruits) of Moringa oleifera (Sajina) Carbohydrate research. 2007;342(16):2380-2389.
- [Google Scholar]
- Phytochemical, nutraceutical and pharmacological attributes of a functional crop Moringa oleifera Lam: An overview. South African Journal of Botany. 2020;129:209-220.
- [Google Scholar]
- Protective effects of Dipsacus asper polysaccharide on osteoporosis in vivo by regulating RANKL/RANK/OPG/VEGF and PI3K/Akt/eNOS pathway. International Journal of Biological Macromolecules. 2019;129:579-587.
- [Google Scholar]
- Glucocorticoid-induced osteoporosis and osteonecrosis. Endocrinology and metabolism clinics of North America. 2012;41(3):595-611.
- [Google Scholar]
- Antidepressant effects of a polysaccharide from okra (Abelmoschus esculentus (L) Moench) by anti-inflammation and rebalancing the gut microbiota. International journal of biological macromolecules. 2020;144:427-440.
- [Google Scholar]
- Structure analysis of water-soluble polysaccharide CPPS3 isolated from Codonopsis pilosula. Fitoterapia. 2010;81(3):157-161.
- [Google Scholar]
- Characterization of the chemical composition of Chinese Moringa oleifera seed oil. Journal of the American Oil Chemists' Society. 2019;96(5):523-533.
- [Google Scholar]