effect of celastrol on bone structure and mechanics in arthritic rats

Arthritis of the purpose (RA)
Characterized by chronic inflammation that leads to bone and cartilage damage in joints.
Despite recent progress in RA Management, adverse reactions, lack of efficacy and economic barriers to treatment still limit the success of treatment.
Therefore, safer and less costly treatments are needed to control inflammation and bone absorption.
We have previously demonstrated that celastrol is a candidate for RA treatment.
We observed that it suppressed two kinds of il (IL)-
1 Beta and tumor necrosis factor (TNF)
In vitro, it has Resistance
In vivo inflammatory properties and the ability to reduce the presence of slide CD68 macrophages.
Here, our goal is to evaluate the effect of celastrol in local and systemic bone loss.
Methods a dose of 1 μg/g/day was given to female Wistar adjuvant-
Induced arthritis rats
Rats were killed 22 days after disease progression, blood, femoral, tibia and paw samples were collected for bone remodeling marker quantification, 3-
Point bending test, Micro
CT analysis, measurements of nano-depression and Fourier transform infrared spectroscopy, and evaluation of immune tissue chemistry.
Results we observed that celastrol preserved the structure of the joints and reduced the number of single-core cells and bone cells present in the joints of arthritis.
In addition, triptolide reduced its resistance
Pre-collagen type 1 amino-resistant P.
End-front peptide and C-end cross-linked end peptide of type II collagen serum level.
It is important that celastrol prevents bone loss and deterioration of bone microstructure.
Celastrol also preserved the nano-properties and mineral content of the bones.
In addition, the animal bones treated with celastrol are not so fragile, as illustrated by the increase in maximum load and yield displacement.
Conclusion these results suggest that celastrol reduces bone absorption and cartilage degradation and maintains bone structure properties.
Purpose:RA)
Characterized by chronic inflammation that leads to bone and cartilage damage in joints.
Despite recent progress in RA Management, adverse reactions, lack of efficacy and economic barriers to treatment still limit the success of treatment.
Therefore, safer and less costly treatments are needed to control inflammation and bone absorption.
We have previously demonstrated that celastrol is a candidate for RA treatment.
We observed that it suppressed two kinds of il (IL)-
1 Beta and tumor necrosis factor (TNF)
In vitro, it has Resistance
In vivo inflammatory properties and the ability to reduce the presence of slide CD68 macrophages.
Here, our goal is to evaluate the effect of celastrol in local and systemic bone loss.
Methods a dose of 1 μg/g/day was given to female Wistar adjuvant-
Induced arthritis rats
Rats were killed 22 days after disease progression, blood, femoral, tibia and paw samples were collected for bone remodeling marker quantification, 3-
Point bending test, Micro
CT analysis, measurements of nano-depression and Fourier transform infrared spectroscopy, and evaluation of immune tissue chemistry.
Results we observed that celastrol preserved the structure of the joints and reduced the number of single-core cells and bone cells present in the joints of arthritis.
In addition, triptolide reduced its resistance
Pre-collagen type 1 amino-resistant P.
End-front peptide and C-end cross-linked end peptide of type II collagen serum level.
It is important that celastrol prevents bone loss and deterioration of bone microstructure.
Celastrol also preserved the nano-properties and mineral content of the bones.
In addition, the animal bones treated with celastrol are not so fragile, as illustrated by the increase in maximum load and yield displacement.
Conclusion these results suggest that celastrol reduces bone absorption and cartilage degradation and maintains bone structure properties.
What do you already know about this topic?
Triptolide has Resistance
Characteristic of inflammation.
What has been added to this study?
Celastrol reduces bone absorption and cartilage degradation.
Celastrol retains bone structure.
What impact does this have on clinical practice?
According to the data of these animal models, scelastrol may be a useful drug for treating arthritis.
Introduction to arthritis (RA)
It\'s a chronic immune.
It is estimated that the global prevalence of inflammatory diseases is 1% per cent.
RA is characterized by chronic edema and inflammation of the joint slide tissue.
With the development of the disease, cartilage and bones are damaged, resulting in joint damage.
Loss of bone around the joint and throughout the body leads to increased risk of fracture in patients.
Bone loss of 2-5 in RA is due to the imbalance between bone synthesis and degradation of bone fragments, bone absorption dominates bone formation.
6. we have reported an increase in il levels before (IL)-
1β is in the fluid of the recently started arthritis and the already identified RA patient.
7 This may be explained by activating the half enzyme
1. responsible for the processing of pro-IL-
Since the early RA, we have also observed an increase in 1β.
8 in vitro drug screening by using THP-1 macrophage-
Like cell lines, we have identified the reduction of IL-
1 Beta and tumor necrosis factor (TNF)
Another central pro-inflammatory factor in RA pathology.
Among them, celastrol is considered as a promising treatment candidate for arthritis because it is able to reduce the level of IL-
1β and tumor necrosis factor, by inhibiting the activation of both
1. nuclear factor kappa B (NF-kB).
The red moon is apentacyclic-
A plant used from traditional Chinese medicine-Tripterygium wilfordii F (TwHF). The anti-
The inflammatory properties of Celastrol are mainly attributed to the production of cytokines and the regulation of the expression of inflammatory mediators, the regulation of the function of inflammatory cells in 10-14, 15-18 and regulation of autophagy in vitro.
19 20 we have recently described in vivo that celastrol has significant resistance
Inflammation and anti-proliferation properties, with the decrease in the number of inflammatory cells in the whole slide, the most important thing is to inhibit the number of CD68 macrophages, which is a biomarker of the efficacy of RA drugs.
In this study, we hypothesize that celastrol can control inflammation as well as local and systemic bone absorption that occurs in arthritis.
Our aim is to evaluate celastrol\'s ability to prevent inflammatory-induced disorders of bone structure and strength
Induced arthritis (AIA)rat model.
Materials and methodsweek-
Aged female Wistar rats weighing 230-
Purchased 250g from Charles River international laboratory (Wilmington, United States).
Charles River Laboratory used Freund to completely supplement the branch bacteria and inject them on the right foot pad to optimize induction of auxiliary diseases (
Solution: 1 ml of 40 mg/wax oil of M; 0.
1 ml solution per animal).
Maintain AIA rats in specific pathogens
Free facilities, random check-in in each group under standard laboratory conditions (
At 20 °C-22 °C, at 10-hour light/14-hour dark)
And get food for free (
Wensm RM3, SDS diet, UK)and water (Ultrapure).
In addition, in order to minimize the discomfort of animals, shaved flowers were used as bedding in the double-layer GR1800 cage (
Tecniplast, Kettering, United Kingdom)
There are five animals in each cage.
According to Directive 2010/63/EU, all animal procedures are approved by the agency\'s Animal Welfare Agency (
Pelo berm-
Animal, ORBEA-iMM)
With the permission of the competent Portuguese authorities (
Director veterinentée veterinária, license number: 0421/000/2016). Humane end-
When the maximum inflammation score is presented, the score is determined and the animal is sacrificed (0–3)
More than two claws or when weight drops by more than 20%.
Triptolide treatmentThe dose of triptolide (1u2009µg/g/day)
What was used in this study was based on what was used in our previous study and in other studies.
23 In addition, we have reported that this dose has effectively suppressed fasciitis in the AIA rat model, and there is no evidence that the drug
Induced toxicity. 21 Celastrol (
Sigma in Sintra, Portugal)
Reserve solution of 100 ml in px (DMSO)
Since the fourth day of disease induction, in the early treatment group of AIA rats, it was dissolved in normal saline solution and was injected into the abdominal cavity (n=15)
In the advanced treatment group since 11 days of disease induction (n=15)
It lasted until 22 days.
Due to the plateau effect of inflammatory manifestations, studies using an acute AIA model are usually completed at this point in time.
A healthy group of non
Untreated arthritis and arthritis in women
Matched rats were executed 22 days after disease induction (
Chronic clinical stage)
Used as a control in all experiments.
Calculate the sample size in each group using the free sample size calculated for G * Power V. 3. 1. 9. 2 software (
Types of power analysis: a priori; alpha(α)
Error Probability: 0. 05; power (1-beta(β)
Error probability): 0. 95;
Effect size d: 1. 526112;
Actual power: 0. 9576654).
In the pre-clinical stage of AIA progression, there is still a lack of evidence of inflammation or bone erosion in the lateral hind paw and front paw.
In the acute clinical stage, swelling, inflammation, and joint erosion of the hind paw progressed steadily, reaching the plateau stage in the chronic stage.
24 rats were killed by CO2 anesthesia and blood, femoral, tibia and paw samples were collected.
Immune tissue chemical staining of tissue protein K and bone calcium positive cells in the posterior pawsLeft claw sample from the ankle joint, the materials collected at the time of sacrifice were immediately fixed in a 10% neutral buffered formaldehyde solution, then dehydrate in 10% of the acetate.
The sample is then dehydrated and embedded in paraffin, continuously sliced using a slicer with a thickness of 5 m and mounted on a microscope slide.
The immunolocalisation in broken bone and bone cells is in the lower bone/foothills of the tibia cartilage by staining with protease K (
Broken cell markers;
Mature broken cell enzyme
Biorbyt, Cambridge, UK)
Bone calcium protein (
Bone mark;
Index of bone activity;
Abcam, Cambridge, UK)
One resistance and then EnVision (
Dako, Glostrup, Denmark).
Color is developed in solution containing diamino-group
IV hydrochloric acid (
Sigma in Sintra, Portugal), 0.
5% hydrogen peroxide in phosphate
Buffer salt water buffer (pH 7. 6).
The slides are dyed and installed with Su Mu essence.
A semi-quantitative score of 0-3 was used to visually evaluate the joints of rats (
0: 0%-25% staining;
1: 25%-50% staining;
2: 50%-75% staining;
3: dyeing more than 75%)
Positive stained cells for each total cell count in tissue sections.
Analysis of 9 21 25 slides using Leica DM2500 microscope (
Wetzlar Leica Microsystems, Germany).
Serum biochemical measurements of bone and cartilage turnover were analyzed by quantifying the level of TRACP-5b (tartrate-
Acid-resistant phosphate 5b), P1NP (
Pre-amino collagen type 1-
Terminal peptide)and CTX-II (
C-end cross-linked end peptide of type II collagen)
Determination of serum in rats by ELISA (
The immune diagnosis system of British bolldon).
All commercial analyses are carried out according to the manufacturer\'s instructions to generate a standard curve using the provided reference concentration.
Sample measurement using tablet reader Infinite M200 (
Tecan, Mannedorf, Switzerland). Micro-
CT analysis of the performance of the tibia trabecular and cortex
Micro resolutionCT system (
Bruker micro-SkyScan 1272
CT of Kontich, Belgium).
Wet bones are wrapped in parafilm and covered with dental wax to prevent drying and movement during scanning. X-
The voltage of the detonator is set to 50kV and the beam is filtered with 0.
5mm aluminum filter.
The sample location and camera settings are adjusted to provide 3.
0 m homophobic pixel size and projected images are collected every 0 times. 2°.
Density calibration is performed for ha dies with a density of 250 mg/cm 2 and 750 mg/cm 2.
Image reconstruction was completed with NRecon software (V. 1. 6. 9. 8; Bruker micro-CT)
Appropriate corrections were made to reduce beam hardening and ring artifacts.
Manually divide the trabecular bone from the cortical bone, more than 1400 pieces analyze the parameters of the trabecular bone, and start with 200 pieces from the distal end of the long plate.
More than 300 pieces were analyzed for cortical bone parameters and 1800 pieces were started from the distal end of the long plate.
Analysis is consistent with guidelines for assessing the microstructure of rodent bones using trace elementsCT.
The morphology of 26 trabecular bone was analyzed by applying global threshold and freckle removal, which was three-dimensional(3D)analyses.
Cortical bone areas of interest (ROI)
Through ROI-
Shrink packing surgery also provides the shape of the cortical bone for both
Analysis of size and shape.
The blood vessels are then divided using an adaptive threshold. Blood vessels (porosity)
Analysis using 3D morphological analysis.
Using CSM-perform nanoindentation measurements
Nano hardness tester system (Indentation V. 3. 83;
CSM instruments, Switzerland)
Equipped with a Berkovich-
Based on Pyramid diamonds. After micro-CT, 0.
Cut the top of the Shin 5mm and embed the lower part in a low viscosity epoxy resin (
HELSINKI, Finland, kenolin AB, buerle, ebosin). Slow-
Remove 10% bone length using a speed diamond saw.
Polish the surface of the sample using sic sandpaper with reduced mesh size (
800, 1200, 2400 and 4800)
Complete with a cloth containing 0. 05u2009µm γ-
Alumina particles.
From the previous work, the indentation scheme was adopted, 27, and 8 compliant indentation were made on the cortical bone and the trabecular bone (
The so-called \"advanced \")
Load protocol.
All dents are performed under an optical microscope to achieve the precise location of the dents at the center of the target area in the tissue.
28 in the advanced protocol, a ladder loading waveform with a loading/unloading rate of 20mN/min is applied and an intermediate load is applied-hold-
At a maximum load of 10mn, the phase for a duration of 30 m/s remains the same. The hardness (HIT)
, Indentation modulus (EIT)
, Indentation creep (CIT)
Elastic part of indentation work (ηIT)
Measurement by advanced protocol using Oliver and Pharr29 methods.
In the nano-indentation technique, a CSM instrument microscope equipped with color cameras was used to obtain histological images of rat tibiae from the backbone cortex region.
The plate structure of bone tissue was evaluated by histological score.
This assessment was conducted blindly using a semi-quantitative score to analyze the structure of the plate bone (
1: mainly parallellamellae;
2: concentric parallel-
Thin sheets of the same proportion;
3: mainly concentric flakes).
At a magnification of × 200, the ratio of the area/Total tissue area of the bone cell cavity was also evaluated to analyze the percentage of the bone cell cavity gap as a percentage of the total tissue area.
The collection and analysis methods used are also applicable to the evaluation of bone volume/tissue volume in tissue morphology measurement techniques.
30 all variables are expressed and calculated on the recommendation of the American Institute of Bone and Mineral Research, 31 using morphological measurement procedures (ImageJ 1.
46 r with plugin BoneJ).
Ftirquantification samples for nano-indentation are also used for Fourier transform infrared spectroscopy (FTIR).
The chemical composition was measured from the bone surface by HYPERION 3000 infrared microscope (
Bierica, Brooke optics, Massachusetts, United States of America)
Total reflection using attenuation (ATR)objective.
Compress the ATR Crystal onto the bone at constant load and record the spectral image with a focal plane array detector.
The spatial and spectral resolutions are set to 1 m and 2 cmcm-1, respectively.
The average spectrum between 840and3300-cm-1 is 32 times, and the two spectra (32×32 spectra)
Collected from trabecular bone and cortical bone respectively.
Analyze data using custom scripts in a matlab environment (
MathWorks, Natick, United States of America).
For each spectrum, by integrating the spectrum between 1595-1720 cmcm-1, 900-1185 cmcm-1, and 850-895 cmcm-1, respectively
Blood vessels and other porous structures were removed by excluding spectra with a maximum phosphate peak height of less than 0.
5 absorption units.
Average content and-
Established parameters of bone composition (
Calcium carbonate: amide I, minerals: matrix and calcium carbonate: Phosphate)
Finally, the threshold spectrum is calculated. 323-
In order to study the bone strength after celastrol treatment, 3-
Point bending test in universal testing machine (
Instron 3366, Instron, Norwood, USA)with a load-cell of 500N.
The femur is placed horizontally, with the front side up, on the stent with a span of 5mm.
The load is applied at a constant speed of 0.
005 mm/s until a fault occurs.
First, the stiffness is analyzed by fitting.
A polynomial function of the linear part of the load deformation data recorded.
The breakpoint at the time of reaching the maximum is defined, and the corresponding deformation and absorption energy are analyzed.
Statistical analysis the main result of this study is to prevent bone structure and mechanical damage caused by inflammation, and the second result is the structural and mechanical deterioration of bone in the chronic stage of arthritis development in the AIA rat model.
Some of the above features are analyzed using descriptive statistics (
The median of the minimum and maximum intervals of continuous variables)and box-plots.
Differences between the three independent groups (
Early and late treatment groups and untreated arthritis rats)
About these featuresParameter Mann-
The Whitney test was performed with Bonferroni correction.
Therefore, with Bonferroni correction, we divide the global significance level into 0.
05 according to the number of independent tests (3)
To obtain a Bonferroni critical value, the test must have a p-value