International Immunopharmacology 129 (2024) 111596

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International Immunopharmacology
journal homepage: www.elsevier.com/locate/intimp

Ozone therapy (O2-O3) alleviates the progression of early intervertebral

disc degeneration via the inhibition of oxidative stress and the interception
of the PI3K/Akt/NF-κB signaling pathway
Najah Elmounedi a, *, Walid Bahloul a, b, Abdelkader Kharrat c, Mabrouk Horchani d,
Hichem Ben Jannet d, Ahmed Racem Guidara a, b, Hassib Keskes a, b
a
Cell Therapy and Experimental Surgery of Musculoskeletal System LR18SP11 Lab, Faculty of Medicine, Sfax, Tunisia
b
Department of Orthopedics and Traumatology, CHU Habib Bourguiba, Sfax, Tunisia
c
Doctor Rhymatologist, Sfax, Tunisia
d
Laboratory of Heterocyclic Chemistry, Natural Products and Reactivity (LR11Es39), Medicinal Chemistry and Natural Products, Faculty of Science of Monastir,
University of Monastir, Avenue of Environment, Monastir 5000, Tunisia

A R T I C L E I N F O A B S T R A C T

Keywords: Intervertebral disc degeneration (IDD) stands for the most frequent cause of low back pain. Finding a cure for this
Intervertebral disc degeneration disease is an important challenge as current conservative treatments and surgical interventions fail to bring a
Percutaneous treatment solution to this disease. Ozone therapy (O2-O3) has yielded outstanding outcomes in intervertebral disc pa­
ROS
thology. The ozone’s efficacy in the treatment of IDD remains unconfirmed. This study aimed to assess the
O2-O3
PI3K/NF-kB pathway
effectiveness of intradiscal ozone injection on IDD induced in a rat. Effects of ozone therapy on the viability of
nucleus pulposus cells were evaluated by CCK-8 assays. Macrophage immunoreactivity was detected by
immunohistochemical, the expression of collagen type II was evaluated by western blot, and measurement of
oxidative stress parameters was realized. Molecular docking studies were carried out in order to predict the
interaction formed between O3 and the target enzymes, on the one hand, O3 with PI3K and, on the other hand, O3
with COX-2. IRM, X-ray, hematoxylin-eosin, and bleu alcian staining were realized to assess the therapeutic
impacts of ozone in the puncture-induced rat model of IDD. In vivo, O3 ameliorated the IDD in the early stage of
this disease. It was also displayed in molecular docking that O3 might bind to PI3K to suppress the PI3K/Akt/NF-
κB signaling pathway. This study’s results show that the O3 should be administered at the low grade of IDD and at
an early stage because it cannot restore the advanced inflammatory alteration of the IVD. Our results corrobo­
rated also that O3 inhibits the progression of IDD via the PI3K/Akt/NF-κB signaling pathway, which supports O3
as an effective therapeutic option for treating IDD.

1. Introduction
Low back pain (LBP) is the 2nd leading cause of medical consultation
in the industrialized countries (behind cardiac disorders) [1]. It is also
the cause of many frequent, repeated or prolonged work stoppages,
often accompanied by psychological and social repercussions [2,3,4,5].
Lumbar pain is recognized as one of the most expensive conditions for
social security [6]. The management of LBP patients is, therefore, a
major socio-economic issue. This type of LBP, called discogenic LBP,
affects about nearly 600 million people worldwide [7]. It is linked in 40
% of cases with intervertebral disc degeneration (IDD) [8].
Despite the pathogenesis of IDD is not entirely understood,
interleukin-1β (IL-1β) has been shown to play a crucial function in the
progression of IDD [9], IL-1β level has been shown to increase as IDD
progresses [10].
The nuclear factor kappa B (NF-κB) signaling pathway plays a crucial
role in the IL-1β-induced inflammatory response, stimulated the extra­
cellular matrix (ECM) degradation, aggravated the oxydative stress, and
induced apoptosis [11,12]. In response to certain stimuli, including IL-
1β, phosphorylated IκBα exhibits nuclear localization signals on the NF-
κB complex, which activates the p65 subunit of nuclear factor (NF-κB)
(p65) to be phosphorylated and translocated to the nucleus to stimulate
the transcription of particular genes [8]. Moreover, during IDD pro­
gression, the NF-κB signaling pathway has also been demonstrated to be

* Corresponding author at: Sfax Faculty of Medicine, Majida Boulila Road, Sfax 3029, Tunisia.
E-mail address: najah.mounedi12@gmail.com (N. Elmounedi).

https://doi.org/10.1016/j.intimp.2024.111596
Received 18 December 2023; Received in revised form 19 January 2024; Accepted 23 January 2024
1567-5769/© 2024 Elsevier B.V. All rights reserved.
N. Elmounedi et al.
downstream of the PI3K/Akt signaling pathway [13]. Therefore, the
PI3K/Akt/NF-κB signaling pathway is considered the primary pathway,
and PI3K is viewed as a potential target for the therapy of IDD.
Many factors contributed IDD. Among them, we cite the oxidative
stress which can disturb the anabolic/catabolic balance in the nucleus
pulposus cells (NPCs), therefore accelerating the progression of IDD
[14]. In the treatment of IDD, targeting oxidative stress could be a
promising therapeutic technique [15]. Excessive oxidative stress has
been shown in studies to regulate inflammatory release, which is also a
key factor in IDD [16,17]. Anti-inflammatory and antioxidant therapy
could thus be a very effective treatment for IDD.
Therapeutic planning ought to start with a conservative approach. In
case of failure, surgical intervention is required. However, surgery ought
to be a last resort given the high incidence of complications. To avoid it,
numerous minimally invasive approaches have been developed in recent
decades. Among these techniques; The oxygen-ozone therapy (O2-O3),
from its intervention in the clinical practice over the 1990s [18], it has
found a large application in Asia and Europe on account of its significant
clinical amelioration and very low costs [19]. In the orthopedic area, O2-
O3 can be administered via several ways, including subcutaneous,
intramuscular, intra-articular, intradiscal, intraforaminal, pregangli­
onic, and periarticular [20,21].
O3 has emerged as a feasible and safe therapeutic option for different
diseases in humans including osteomyelitis, hepatitis, chronic obstruc­
tive pulmonary disease, cystitis, and rheumatoid arthritis…etc. In or­
thopedics, ozone therapy has also been proven effective in the treatment
of arthrosis and herniated discs [22].
The therapeutic effectiveness of the medical ozone on IDD is
controversial. Some authors claim that ozone therapy has no effect on
IDD [23], whereas, others hold that it is effective in the treatment of
IDD. Only three investigations have showed the effectiveness of ozone
therapy in the treatment of IDD [24,25,26]. Regrettably, there is no
Fig. 1. The effect of ozone on NP cell (NPC) viability. NPCs were treated with different volumes of ozone for 24 and 48 h. Cell viability was measured using a CCK-8
assay. **p < 0.01, *p < 0.05 versus the control group.
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International Immunopharmacology 129 (2024) 111596
experimental research on the mechanism of action of ozone in the
treatment of IDD. To overcome this deficiency, we performed an
experimental study on rats.
Ozone is delivered in an associated formula with oxygen at very low
concentrations (The volume of the gas mixture should be between 8 and 10
ml) (2 % of O3 with 98 % of O2), it is usually administered from the con­
centration of 20–40 µg/ml, where it can set up anti-inflammatory, immu­
nomodulatory, bactericidal, antifungal, antiviral, and analgesic effects [27].
Recently, to our knowledge, no experimental study has been pub­
lished on the possible relationship between IDD and response to oxygen-
ozone therapy. In our study, we aim to assess the outcomes of the O2-O3
mixture intradiscal administration in the treatment of two grades of IDD
(mild and severe). The radiographic, histological, and, immunohisto­
chemical examinations were utilized to assessed the impact of intra­
discal ozone injection on IDD.
2. Materials and methods
2.1. Extraction and culture of nucleus pulposus cell (NPC)
The NPC tissues were carefully extracted from the coccygeal inter­
vertebral disc (IVD) (Fig. 1A) of 1-month-old male sprague Dawley (SD)
rats, and then cut the specimens into small sections, rinced with
phosphate-buffered saline (PBS), and incubated with 0.25 % trypsin
solution containing 0.2 % mg/ml type II collagenase at 37 ◦ C for 12 h
[28]. NPC were then cultured in DMEM/F12 supplemented with 10 %
fetal bovine serum, 100U/mL penicillin, and 100 μg/mL streptomycin in
an incubator with 5 % CO2 at 37 ◦ C.
2.2. Cell viability assay
The Cell Counting Kit-8 (CCK-8) (Dojindo, Kumamoto, Japan) was
N. Elmounedi et al. International Immunopharmacology 129 (2024) 111596

Fig. 2. A) Schematic illustration of the establishment of the IDD rat model and the experimental design to evaluate the effect of ozone in vivo, B) Medical ozone
production by the use of Alnitec Ozo2futura® generator, C) Intradiscal injection of ozone therapy.

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N. Elmounedi et al.
performed to test the viability of NPC in accordance with the manu­
facturer’s recommendations. NP cells were treated with different vol­
umes of O2-O3: 0.05, 0.1, 0.2, or, 0.4 ml with a fixed concentration of 30
µg/ml of O2-O3 for 24 h and 48 h in order to determine the appropriate
treatment volume. Thereafter, 10 µl of CCK-8 was added to the cells and
incubated at 37 ◦ C for 4 h. A spectrophotometer was then used to
measure the absorbance at 450 nm [29].
2.3. Puncture-induced IDD model
All surgical interventions and treatments procedures were approved
by the Tunisian Ethical Committee for the Care and Use of Laboratory
Animals, in accordance with the internationally recognized principles
and judged commendable by the Committee of Animal Ethics (Ethics
No. 94–1939). Adult healthy male Sprague Dawley (SD) rats (3 months
old and weighing 200–250 g, n = 45) were purchased from the Central
Pharmacy (SIPHAT, Tunisia) were used in this research. All rats were
housed in groups of 5 rats per cage with free access to a commercial
pellet diet (SICO Sfax, Tunisia) and water ad libitum. The rats were kept
under controlled conditions: temperature (23 ± 1 ◦ C), with a relative
humidity of 30–60 % and under an artificial light/dark cycle of 12 h.
After anesthesia using a mixture of ketamine (90 mg/kg, Panpharma,
France) and xylazine (10 mg/kg, Unimed, Tunisia) by intramuscular
injection, the rats were placed in a prone position. The coccygeal (Co)
IVD levels Co6/7, Co7/8 and Co8/9 were detected by palpation and
confirmed by an X-ray. The puncture site was disinfected with ethanol
and the needles: 21G or 25G (SOFAP, Tunisia) were used to puncture the
IVDs. In these discs the tip of the needle was prudently inserted into the
center of the NP at a controlled depth of penetration of 5 mm, rotated
360◦ two times, and held for 30 s pre-extraction. All the needles utilized
were marked to prevent exceeding the depth of 5 mm. To reduce pain,
the rats received an intramuscular injection of Tramadol HCl® Ampule
100 mg (Teriak, Zaghouan, Tunisia) as an analgesic agent, in terms of 3
mg/kg for three days after animal model establishment [30].
2.4. Experimental groups
45 rats SD were randomly divided into nine groups of five rats:
Control group: No intervention was performed.
-Group 25G « 3(25G) »: the IVDs underwent a puncture by a 25-
gauge needle inducing a IDD. The rats were sacrificed 3 weeks later.
-Group 25G « 6(25G) »: the IVDs underwent a puncture by a 25G
gauge needle inducing a IDD. The rats were sacrificed 6 weeks later.
-Group 21G « 3(21G) »: the IVDs were punctured with a 21G gauge
needle. The rats were sacrificed 3 weeks after
-Group 21G « 6(21G) »: the IVDs were punctured with a 21G gauge
needle. The rats were sacrificed 6 weeks later.
-Treated group « 3(25-O3) »: 3 weeks after establishing an animal
model of IDD by 25G needle, the rats were treated with 0.1 ml of O2-
O3 (30 µg/ml).
-Treated group « 6(25-O3) »: 6 weeks after establishing an animal
model of IDD by 25G, the rats were treated with 0.1 ml of O2-O3 (30
µg/ml).
-Treated group « 3(21-O3) »: 3 weeks after establishing an animal
model of IDD by 21G needle, the rats were treated with 0.1 ml of O2-
O3 (30 µg/ml).
-Treated group « 6(21-O3)»: 6 weeks after establishing an animal
model of IDD by 21G needle, the rats were treated with 0.1 ml of O2-
O3 (30 µg/ml) (Fig. 2A, C).
The frequency of ozone therapy (one dose) is performed by referring
to previous clinical studies [24,25,26].
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International Immunopharmacology 129 (2024) 111596
2.5. Intradiscal injection of medical ozone (O3) to rats
Three and six weeks after the animal model establishment the rats
received 0.1 ml of a gas mixture O2-O3 with the concentration of 30 µg/
ml (30 µg O3 per 1 ml of O2) by using a 29-gauge needle (Fig. 2C). This
caliber of needle was chosen based on our trial results because it does
not cause injury leading to IDD [31]. The animals were sacrificed one
week after the ozone injection (Fig. 2A). The mean time of injection was
about 10 sec. After all processes, needles were taken out and the punc­
ture area was squeezed for 20 sec.
2.6. Ozone generation
The oxygen-ozone gas mixture was produced just before adminis­
tration, using an Alnitec Ozo2futura® generator (Catania, Italy)
(Fig. 2B). The O2 used for the production came from pure medical ox­
ygen cylinders. O3 was generated in stipulated doses by regulating O2
flow; it was packed in a Terumo1 (SOFAP, Tunisia) latex-free syringe
and held in a vertical position to avoid gas leakage until employed.
2.7. Radiographic analysis of disc height
Using the same method of anesthesia as detailed above pre- and post-
ozone injection at 3, and, 6 weeks. The lateral plain radiographs of the
caudal vertebrae were taken by a Siemens Multix ® (Germany; 35 kV,
2.5 ms, 1 m) and were scanned and digitally loaded with an image
capture application. The IVD height were analyzed by using the public
domain image analysis program by the U.S. National Institutes of
Health: Image J (https://imagej.nih.gov/ij/). The disc height index
(DHI) was computed through the use of the method proposed by Masuda
et al. [32] (Fig. 3A,B).
2.8. Magnetic resonance imaging (MRI) analysis
The rats’ coccyx was evaluated by 1.5 T MRI system (Bruker Bio­
Spin). The signal and structural modifications of the rat tail IVDs were
assessed by sagittal T2-weighted MRI slices at 3 and 6 weeks after ozone
injection. We utilized a T2-weighted sagittal plane with the following
parameters: repetition time/echo time, 2000/70 ms; the field of view,
50 mm; the number of averages, 2. The slide thickness was 1 mm; in­
terval, 0 mm; matrix, 256. A 2-cm-diameter surface receive coil and a 60
mm volume resonator were used. To rate the severity of IDD a five-grade
modified Pfirrmann system was utilized [33]. The interpretation of the
MRIs was realized by three separate researchers. The quantitative data
were exhibited as the average of the three assessments.
2.9. Blood sample collection
Animals were euthanized by excess anesthesia with ketamine, the
blood samples were collected via a cardiac puncture in EDTA tubes and
were instantly used for the determination of the hematological param­
eters such as white blood cells (WBC) count, red blood cells (RBC) count
and platelets (PLT). The quantification of these parameters was realized
by the use of an automatic hematological assay analyzer (KX21 hemo­
gram, department of hematology, CHU Habib Bourguiba, Sfax).
2.10. Macroscopic analysis
After euthanasie, the rat’s caudal vertebras were carefully harvested
and fixed in 4 % paraformaldehyde for 24 h and decalcified using 10 %
EDTA solution for three weeks. The whole IVDs with adjacent verte­
brates bodies were sectioned mid-sagittally. Thompson Grading System
[34] was then used for the macroscopic assessment of IDD.
N. Elmounedi et al. International Immunopharmacology 129 (2024) 111596

Fig. 3. A) Evaluation of coccygeal disc height by radiographic imaging representative X-ray images of tail discs of the four groups at 3 and 6 weeks post punction (B)
Schematic representation of measurement of disc height index (DHI). The DHI was measured from radiographs using Image J. Changes in the DHI of punctured discs
are expressed as percentages (%DHI = post-punctured DHI/pre-punctured DHI x100%). C) Changes in the percent of disc height index (%DHI) in the four groups at
the two time points (3 and 6 weeks). At 3 Weeks the mean of the %DHI of the discs decreased significantly compared to that the 21G and 25G groups (*p < 0.05). No
significant (ns) differences were detected at 6 weeks (P˃0.05). The data were expressed as mean ± SEM.

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2.11. Histological analysis
After macroscopic analysis, the samples were dehydrated in graded
ethanol, cleared in xylene twice, embedded in paraffin blocks, sectioned
into sagittal sections of 5-μm and stained with hematoxylin and eosin
(H&E). Sections were then visualized and evaluated under a light mi­
croscope. The H&E staining was graded according to the scoring system
of Lai et al [35] histopathological assessments were realized by two
experienced pathologists. The scores ranging from 0 point for a normal
IVD to 16 points for a severely degenerated IVD (two in each category).
We used the alcian blue staining to localize the glycosaminoglycan
(GAG) tenor as detailed by Stapleton et al [36].
2.12. Immunohistochemical (IHC) staining of CD-68
Immunohistochemistry was performed for CD-68. Briefly, the sec­
tions were deparaffinized with xylene and rehydrated by graded
ethanol. The endogenous peroxidase activity was blocked with 3 % H2O2
for 10 min at 37 ◦ C. The sections were rinsed two times in PBS solution
for 5 min and boiled in 0.01 M citric acid buffer for antigen retrieval at
95 ◦ C for 15–20 min, followed by incubation with primary antibodie
anti-CD-68 clone ED1 (AbD Serotec, Raleigh, NC). All sections were then
incubated overnight at 4 ◦ C. The signal was treated with DAB substrate
(DAKO, Cambridge, UK). The slides were counterstained with H&E and
mounted. Next, the slides were dehydrated, washed, mounted, and
imaged through the use of a DXM 1200C (Nikon, Japan) CCD camera.
2.13. Oxidant assays
The oxidative stress parameters were detected in the manipulated
discs 3 weeks post O2-O3 injection and reported by the protein quantity
(/mg of protein). The quantification was realized according to the
technique proposed by Lowry et al [37]. Malondialdehyde (MDA) level
was determined according to the method of Draper and Hadley [38].
Advanced oxidation of protein products (AOPP) levels were estimated
according to the technique of Kayali et al [39].
2.14. Antioxidant assays
Measurement of superoxide dismutase (SOD) activity was assessed in
supernatant fractions depending on the method described by Beau­
champ and Fridovich [40] based on the capacity to inhibit the photo­
reduction of nitroblue tetrazolium (NBT) at 25 ◦ C. One unit of SOD
activity was fixed as the amount of enzyme that inhibited 50 % of the
NBT decreased. At 560 nm, the reaction product was measured, and the
values were represented as Units/mg of protein.
Catalase activity (CAT) was analyzed spectrophotometrically
assessed at 240 nm based on Aebi’s method [41] by measuring the
diminution of H2O2 at 240 nm for 60 s. One unit of catalase activity
catalyzed the defacement of 1 µmol of H2O2/min. The enzyme activity
was measured as µmoles H2O2 used/min/mg of protein.
2.15. Western blot
Each NPCs samples were rinced with PBS for 3 times. Then, the total
protein of NPCs was lysed by RIPA buffer. The protein concentration was
measured by the BCA kit, according to the manufacturing protocols. 20 μg
of total protein was loaded on to SDS-PAGE electrophoresis and trans­
ferred onto 2.2 µm polyvinylidene difluoride membrane, then blocked
with 5 % non-fat milk solution (Sigma-Aldrich) for 2 h at room tempera­
ture. Next, the membrane was incubated overnight at 4 ◦ C with primary
antibodies against GADPH (1:5000; Abcam), Collagen type II (1:500;
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International Immunopharmacology 129 (2024) 111596
Abcam) diluted in blocking buffer; the corresponding horseradish perox­
idas (HRP)-conjugated secondary antibody was utilized to incubate with
membranes at 37 ◦ C at room temperature for 1 h immediately after dis­
carding the primary antibody by TBST solution. Finally, the target protein
expression was investigated through the use of the ECL solution and the
findings were visualized via an Imaging System (Bio-Rad).
2.16. Molecular modeling
Molecular docking simulations were realized by using Auto Dock 4.2
program package [42]. The structure of ozone (O3) was drawn by
ChemDraw and minimized its energy using ACD (3D viewer) software
(https://www.filefacts.com/acd3d-viewer-freeware-info). The crystal
structure of PI3K (PDB code: 3LJ3) and COX-2 (PDB code: 3LN1) were
obtained from the RCSB PDB database (https://www.rcsb.org/). The
docking assay was performed using AutoDock 4.2 (https://vina.scripps.
edu/) to predict potential interactions between ligand (O3) and both
receptors: PI3K and COX-2. The three-dimensional (3D) images of the
protein–ligand interaction were presented using PyMoL.
2.17. Statistical analysis
For the comparison of the %DHI of discs pre-and post-ozone
administration, data were analyzed by the use of a paired-samples t-test.
Mann–Whitney U tests were utilized to analyze the nonparametric data.
All Statistical analyses were realized using GraphPad Prism 8 (USA).
Data were presented as the means ± standard error of the mean (mean
± SEM). A p-value < 0.05 was considered a statistically significant
difference.
3. Results
3.1. Effect of O2-O3 on rat NP cellular viability
To determine the appropriate volume for the subsequent experi­
ments, we utilized the CCK-8 assay to determine the effects of O2-O3 on
cell viability. As shown in Fig. 1B, NP were treated with different vol­
umes of O2-O3 for 24 h and 48 h. A concentration higher than 30 µg/ml
showed obvious cell toxicity. Thus, we chose a concentration of 30 µg/
ml for use in the subsequent experiments.
3.2. Radiographic analysis
In week 3, the 25-O3 group presented significant disc space nar­
rowing compared to the 25G group (P < 0.05). Additionally, there was a
significant difference between the 21-O3 group and the 21G group (P <
0.05). In Week 6, there were no significant differences among the four
groups in %DHI (Fig. 3C).
3.3. MRI assessment
MRI is another valuable technique to evaluate the structure of IVDs
and water content (Fig. 4A). The 21G, 25G, and 21-O3 groups demon­
strated significantly reduced intensity (P < 0.01) compared to the con­
trol group at 3 weeks. However, no significant MRI changes were found
between the control and the 25-O3 groups at 3 weeks. Moreover, sig­
nificant T2 intensity change was found in the four groups (21G, 25G, 21-
O3, and 25-O3) compared to the control group at 6 weeks (Fig. 4B).
3.4. Hematological parameters
Our results showed that disc punctured with 21G or 25G produced
N. Elmounedi et al. International Immunopharmacology 129 (2024) 111596

Fig. 4. A) MRI images of rat spines, B) Quantification of the Pfirrmann grade.

significant changes in hematological parameters (Table 1). In compari­ group, a clear differentiation between NP and AF was detected. Also, the
son with the control group, significant increases in WBC counts and limit between these two disc compositions (NP and annulus fibrosus
platelets were observed in the 21G and 25G groups. However, the level (AF)) was visible, while for a degenerated IVD, The NP lost its homo­
of RBC was reduced in these groups. O2-O3 administered to the 25G geneity and became fibrous. Furthermore, the NP-AF distinction became
group repaired those parameters to near normalcy. difficult to observe according to the grade of IDD (Fig. 5). Based on the
Thompson Grading System, at week 3, the differences between the
control group and the 25G group, between the control group and the
3.5. O3-O2 ameliorates IDD process in a puncture-induced rat model
21G group and between the control group and 21-O3 group were sig­
nificant (P < 0.01). However, there is no significant difference between
3.5.1. Macroscopic evaluation
the control group and the 25-O3 group (P˃0.05). At week 6, there were
According to the macroscopic sections, in the IVD of the control
significant differences between the control group and the other four
groups (P < 0.01) (25G, 21G, 25-O3 and 21-O3 groups) (Fig. 5J).
Table 1
Hematological parameters in the different groups:
3.5.2. Histologic assessment
Parameters WBC (103/µl) RBC (106/µl) PLT (103/mm3) As shown in Fig. 6, In the control group, we observed normal IVD
Control 12.96 ± 0.41 8.21 ± 0.66 483.2 ± 7.81 appearance, NP cells were evenly distributed, and no AF rupture was
3(21G) 19.58 ± 0.12 α*** 7.7 ± 0.31 α*** 920 ± 5.12 α*** detected. (Fig. 6A, B). The boundary between NP and AF has been well
3(25G) 18.23 ± 0.65 α*** 7.50 ± 0.03 α*** 935 ± 5.33 α***
defined (Fig. 6A1). 3 weeks after IVD puncture with a 25G needle (25G
3(21-O3) 16.65 ± 0.88 α* β**£** 7.8 ± 0.62 α* β**£** 695 ± 6.27 α**β**£**
3(25-O3) 13.32 ± 0.62***£** 8.15 ± 0.05***£** 488 ± 7.21***£** group) we noticed NP atrophy and a reduction in the number of cells
(Fig. 8D), and AF deformation (Fig. 8E). In addition, endplate and
Values are expressed as mean ± SEM. Symboles (*, ∕= et + ). * p < 0,05; ** p <
vertebral body niches were filled with inflammatory cells (Fig. 8F). In
0,01 et *** p < 0,001 α: compared to control group; β: compared to 21G group
group 21G the NP disappeared and was replaced by fibrous tissue
and; £: compared to 25G group. WBC; white blood cells count, RBC; red blood
(Fig. 6C), thus, the ordered arrangement of AF was destroyed (Fig. 6E).
cells count, and PLT; platelets.

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N. Elmounedi et al. International Immunopharmacology 129 (2024) 111596

Fig. 5. (A-I) Macroscopic appearance of intervertebral discs in the control and the 3 experimental groups. Macroscopic observations revealed a decrease in disc
height and disappearance of nucleus pulposus structure in group 21G at 6 weeks. (J) Thompson grading scores at different groups and time points. **P < 0.01
comparing the 3 experimental groups with the control group.

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N. Elmounedi et al. International Immunopharmacology 129 (2024) 111596

Fig. 6. Hematoxylin and eosin stained sections of all studied groups. (A) Control disc, (B, F) IVDs three weeks after puncture by 21G, (C, G) disc punctured by 25G or
21G and received medical ozone. (D) Histological score of the four groups at 3 and 6 weeks post surgery. ns: not significant, ***P < 0.01.

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N. Elmounedi et al.
Fig. 7. Hematoxylin and eosin (H&E) stained sections of the two groups 21-O3 (A) and 21G (D) before and after ozone injection. (B, C, E and F) are high-
magnification AF images.
The disc punctured with 25G and treated with ozone after 3 weeks (25-
O3 group) showed repair of the structure of the IVD and disappearance
of the signs of degeneration. It was clearly shown that a large part of the
NP was restored. The AF in this group showed a regular annular struc­
ture (Fig. 8B). The inflammatory cells that have been aggregated in the
niches of the vertebral bodies have almost disappeared. A limited
number of these cells remained in the endplate niches (Fig. 8I).
At 6-week time point, H&E staining revealed that the effects of ozone
on the morphological preservation of NP and ECM were much weaker
than at the 3-week time point. No improvement was detected, suggest­
ing that ozone improves IDD phenomena during short-term treatment
(3 weeks).
Alcian blue (AB) staining gives clearer identification of the proteo­
glycan (PG) in the tissue. Fig. 8 shows the structural alterations of each
group in AB staining as well as H&E staining.
In the 21-O3 group, we noticed the persistence of signs of degener­
ation, therefore, no improvement was observed in the ozone-treated disc
at 3 and 6 weeks post-puncture (Fig. 6G, F). This allowed us to conclude
that ozone is unable to restore the advanced alteration of IVD.
The result obtained allows us to conclude that ozone therapy should
be administered in mild to moderate stages of IDD, since a sufficient
number of NP cells is necessary for this therapy to be effective. On the
other hand, at an advanced stage of IDD, we noticed a total disappear­
ance of NP cells, which makes the intradiscal injection of O2-O3
ineffective.
At high magnification of the two IVDs (group 21G and 21-O3) we
observed a slight improvement in the structure of the AF (decrease in the
number of lamellar fractures) (Fig. 7) by comparison between the disc
treated and not treated with ozone therapy.
As for the histological score. At 3 weeks, significant differences were
detected between the control group and the groups: 25G, 21G, and 21-
O3 (P < 0.001) and between the 25G and 25-O3 groups (P < 0.001).
Also, no significant difference was observed between the control group
and the 25-O3 group (Fig. 6I).
At 6 weeks, no significant difference was observed between the 25G
and 25-O3 groups or between the 21G and 21-O3 groups (P˃0.05)
(Fig. 6I). On the other hand, significant differences were recorded be­
tween the control group and the four other groups (P < 0.001). In
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International Immunopharmacology 129 (2024) 111596
summary, these findings showed that ozone has a role in the protection
of IVD against IDD at an early stage and at low grade of this desease.
For the study of macrophage immunoreactivity, the results demon­
strate an increased positivity 3 weeks after punction in the group of 25G,
which diminishes after the ozone administration (in the group of 25-O3)
(Fig. 9). This may explain that the O3 causes macrophage activation and
increased phagocytosis.
3.5.3. Quantification of collagen type II (Coll II) by western blot
The expression levels of coll II-associated proteins of the ECM in the
three groups (control, 25G, and 25-O3) were examined (Fig. 10A).
Coll II is a major component of the ECM of NP that is responsible for
fluid retention in tissues and preservation of NP resiliency and volume.
The abnormal decrease in synthesis or degradation of coll II can lead to
the failure of the NP structure and the development of IDD. Following
the ozone treatment, the NPCs expressed additional levels of coll II
(Fig. 10A). Expression of coll II was significantly decreased in the 25G
group (P < 0.01). In contrast, after ozone treatment, coll II expression
was significantly up-regulated (P < 0.01) (Fig. 10B). This result suggests
that ozone could protect NP by regulating the expression of one of the
components of the ECM.
3.5.4. Effect of repeated administration of ozone therapy
In order to adopt a new therapeutic strategy allowing better
improvement of IDD, we thought of increasing the number of injections
of O2-O3 (one injection per week for 5 weeks) in the 25-O3 group. Three
weeks after the establishment of the IDD model by the 25G needle, the
rats received 0.1 ml of an O2-O3 gas mixture at concentrations of 30 µg/
ml (30 µg of ozone per 1 ml of oxygen) once a week for five weeks.
Fig. 11 shows histological sections taken from the IVD of control rats’
treated with repeated injections of ozone (0.1 mg/week for 5 weeks).
The control group showed normal IVD appearance, AF morphology
included well-organized collagen lamellae with no ruptured or serpen­
tine fibers. The tissues between the AF and the NP were intact without
interruption. For the group treated several times with ozone, we noticed
a strong disorganization of the AF lamellae. NP cells were small and
irregularly shaped; residual cells were clustered and separated by dense
areas of PGs. These data prove that ozone causes damage to IVDs if
N. Elmounedi et al. International Immunopharmacology 129 (2024) 111596

Fig. 8. (A-I) Hematoxylin-eosin (H&E)-stained sections. (J-O) Alcian blue-stained sections of NP, AF, and CEP in the groups: Negative control, 25G and 25-O3.
Abbreviations: AF: Annulus Fibrosis, NP: Nucleus Pulposus, CEP: Cartilage Endplate, AB: Alcian Blue.

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N. Elmounedi et al. International Immunopharmacology 129 (2024) 111596

Fig. 9. Immunohistochemical staining of NP and AF with the CD-68 antibody three weeks after the establishment of an animal model of IDD (25G).

Fig. 10. The protein expression of Coll II was quantified by Western blot in the NPCs of the three groups (control, 3(25G) and 3(25-O3) groups); The thickness of the
bands (which gives us an idea of the expression rate) was measured using the Image J software. The values presented are the means ± SEM (n = 3), **p < 0.01.

injected repeatedly. So a single dose is enough to improve the IDD.
3.5.5. Effect of O2-O3 on oxidative stress
It has been established that the increase in the level of MDA and
AOPP suggests an increase in the production of free radicals in rats [43].
In our present study, we showed that IVD puncture increased MDA and
AOPP levels in both 21G and 25G groups compared to the control group
(P < 0.01) (Table 2). No significant difference was detected in the MDA
and AOPP contents between the 21G and 21-O3 groups (P˃0.05). On the
other hand, significant differences were observed between the 25G and
25-O3 groups (P < 0.01). However, the administration of O3 to the 25G
group significantly decreased the level of these markers. In the low
grade of IDD, O3 can decrease the MDA and AOPP content in the IVD but
this content cannot be reduced when the grade of IDD advanced.
Therefore, free radicals could not be effectively scavenged.
The antioxidant activities of SOD, CAT and GPx were measured.
Compared with the control group, the levels of SOD, CAT and GPx
decrease significantly in the two groups 21G and 25G (P < 0.01)
(Table 2). The SOD, CAT and GPx contents in the 25-O3 group were
significantly higher than in the 25G group (p < 0.001). In the 25-O3
group we observed a restoration of antioxidant enzyme activities in
ozone-treated rats, suggesting the protective effect of O3 against
puncture-induced oxidative damage. On the contrary, no significant
difference was observed between the 21G and 21-O3 groups (P˃ 0.05)
(Table 2).
This shows that in low grade IDD, treatment with O2-O3 could in­
crease SOD, CAT and GPx contents in the IVD and promote the capacity
to scavenge free radicals and minimize oxidative cell damage which can
prevent the destruction of the IVD. On the contrary in the severe degree
of IDD, the rats could not effectively scavenge free radicals.
3.5.6. Molecular docking findings: Effect of O3 on the PI3K/Akt/NF-κB
signaling pathway
To examine whether ozone has a direct affinity with a protein linked
to the PI3K/Akt/NF-κB signaling pathway, we realized a molecular
docking analysis of this compound (O3) with the corresponding protein

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N. Elmounedi et al. International Immunopharmacology 129 (2024) 111596

Fig. 11. Histological sections (H&E and Alcian blue staining) of the rat IVD after administration of repeated doses of O2-O3.

involved in this pathway (PI3K). Prior to molecular docking analysis, the

Table 2 structure of ozone was established by Chemdrew software (Fig. 12A),
Levels of oxidative (MDA and AOPP) and antioxidant (SOD, CAT and GPx)
and the PI3K protein was downloaded from the PDB (Fig. 12D). After
enzyme activities in the IVDs of: control rats, rats with IDD induced by 21G
that, molecular docking analysis was used to assess the affinity between
(21G), rats with IDD induced by 25G needle (25G), ozone-treated 21G rats (21-
O3) and ozone-treated 25G rats (25-O3) (after 3 weeks). O3 and PI3K.
According to space-filling model, the ozone was fully integrated into
Control 3(21G) 3(25G) 3(21- 3(25-O3)
the inhibitory pocket of the crystal structure of PI3K (Fig. 12B). Like­
O3)
wase, the local interaction between ozone and target protein residues
Oxydant Parameters
was revealed by the image of the ribbon pattern (Fig. 12E). Intermo­
MDA (nmoles/ 5.42 ± 6.85 ± 6.21 ± 6.13 ± 5.35 ±
mg proteine) 0.36 0.82** 0.55** 0.49** 0.51∕
=∕=∕
=+++ lecular hydrogen bonds being the most significant interactions for the
AOPP (nmol/ 0.18 ± 0.26 ± 0.21 ± 0.22 ± 0.19 ± inhibition of this enzyme, ozone against PI3K acquired important results
mg proteine) 0.022 0.07** 0.04** 0.08** 0.01∕
=∕=∕
=+++
by showing three hydrogen bonds (3H-bonds) (Fig. 12E), which means
that this molecule (ozone) has a high affinity with PI3K compared to the
Anti-oxydant Parameters reference ligand “pyrrolopyridine-benzofuran” which presented only
SOD (Units/mg 4.45 ± 1.39 ± 1.54 ± 1.51 ± 4.37 ± one hydrogen bond (Fig. 12C). Therefore, ozone treatment intensely
proteine) 0.912 0.18** 0.77** 0.22** 0.82∕
inhibits PI3K and Akt phosphorylation stimulated by IL-1β (Fig. 12B1).
=∕=∕
=+++

CAT (µmoles/ 7.57 ± 2.68 ± 2.61 ± 3.41 ± 7.40 ±

min/mg 0.701 0.41** 0.08** 0.02** 0.23∕
=∕=∕
=+++ In summary, these results demonstrated that ozone treatment inhibits
proteine) the activation of the PI3K/Akt axis.
GPx (nmol de 14.77 ± 7.33 ± 7.11 ± 8.24 ± 13.57 ± In our research, we study the effects of ozone on IDD and the un­
GSH/min/mg 0.077 0.02** 0.16** 0.07** 0.09∕
=∕=∕
=+++
derlying mechanism. We have shown that ozone can inhibit the in­
proteine)
flammatory response induced by IL-1β and promote ECM homeostasis;
Values are expressed as mean ± SEM. Symboles (*, ∕
= et + ). * p < 0,05; ** p < our results demonstrate that O3 limits or even ameliorates the worsening
0,01 and *** p < 0,001 compared to control group; ∕
=p < 0,05; ∕ =p < 0,01 et
=∕ of IDD through the PI3K/Akt/NF-κB signaling pathway, which supports
=∕
∕ = p < 0,001 compared to 21G group and, +<0,05; ++p < 0,01 and +++p <
=∕ O3 as a promising therapeutic agent for the treatment of IDD (Fig. 12B1).
0,001 compared to 25G group. MDA: Malonadialdehyde; AOPP: Advanced Inhibition of NF-κB activation by O3 can therefore lead to a decrease in
oxidation protein product; SOD: Superoxyde Dismutase; CAT: Catalase; et GPx:
inflammation and apoptotic cell death (Fig. 12B1).
Glutathione Peroxydase.

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N. Elmounedi et al. International Immunopharmacology 129 (2024) 111596

Fig. 12. A1) Effect of O3 on the PI3K/Akt signaling pathway. (A) The structure of O3 (B) The space-filling model between O3 and PI3K, (C) the reference ligand
(Pyrrolopyridine-benzofuran) (D) Ribbon model (macrography): the reaction between O3 and PI3K. (E) Ribbon model (local reaction). (): hydrogen bonds. B1)
Schematic representation of the potential protective effects of O3 in the progression of DD. O3 enhances inflammatory responses and ECM degradation via the PI3K/
Akt/NF-κB signaling pathway.

3.5.7. Effect of O3 on the expression of cyclooxygenase 2 (COX-2)
Cyclooxygenase 2 (COX-2) is an enzyme that catalyzes the trans­
formation of arachidonic acid into prostaglandins (main mediator of
inflammation, pain and fever). These metabolites are at the origin of
inflammatory processes. We performed a molecular docking analysis of
ozone with the COX-2 protein. The COX-2 protein structure was
downloaded from the PDB (Fig. 13B). Afterwords, molecular docking
analysis was invested to assess the affinity between ozone (ligand) and
COX-2 (receptor). A space-filling model showed that ozone was perfectly
embedded in the inhibitory pocket of the crystal structure of COX-2
(Fig. 13A). Likewase, the local interaction between ozone and related
protein residues was revealed by the image of the ribbon pattern
(Fig. 13B). Ozone against the COX-2 protein is involved in three
hydrogen bonds (3H-bonds) (Fig. 13C), while the reference drug
“Celecoxib” formed only two (Fig. 13D) which means that ozone has a
high affinity with COX-2 compared to the reference ligand.
4. Discussion
The results obtained suggest that the intradiscal injection of ozone
can have a preventive effect on the IDD induced by IVD puncture in rats.
Our experimental study is the first to assess the effect of intradiscal
administration of ozone therapy on the progression of induced IDD in
rats. We believe that the effect of O3 could be due to the anti-
inflammatory and antioxidant effects of O3.
To evaluate the protective impacts, we identified safe volumes of O2-
O3 in NPCs, and this volume was invested as the in vivo experimental
volume.

14
N. Elmounedi et al.
Fig. 13. Effect of O3 on cyclooxygenase 2 (COX-2). (A) The space-filling model between O3 and COX-2, (B) The ribbon model (macrography): the reaction between
O3 and COX-2. (C) The ribbon model (local reaction), (D) the reference ligand (Celecoxib). (): hydrogen bonds.
In this study, we utilized a rat tail IDD model established via
acupuncture as detailed in our previous studies [31]. This animal model
is easy to create, The IVDs in the tail are not difficult to locate and easily
accessible to interventions. It may imitate human IDD in some aspects of
degenerative changes and successfully create radiographic and histo­
logic signs of degeneration [31].
For the hematological parameters, our results are consistent with
previous studies demonstrating that O3 ameliorates blood flow and O2
delivery to ischemic tissues [20]. Another study also provided evidence
that O3 can increase the differentiation of erythroblasts (cells specialized
in the synthesis of hemoglobin and giving rise to RBC), this leads to a
gradual increase in erythrocytes [21]. It was also demonstrated that O3
increases levels of prostacyclin, a known vasodilator [44].
Moreover, the histological findings showed the improvement of the
early IDD treated with ozone therapy. These obtained results are in
agreement with those of Borrelli et al who demonstrated that ozone is at
the origin of the induction of chondrocyte proliferation (which is
responsible for the synthesis of PGs, GAGs and collagen, which is
beneficial for the repair of ECM) and fibroblasts [45]. Ozone also causes
the release of immunosuppressive cytokines like transforming growth
factor β1 (TGF-β1) and interleukin 10 (IL-10) which modulate the syn­
thesis of integrins and promote the synthesis of ECM proteins including
collagen and PGs which are important in tissue remodeling [45,46].
A previous study showed that O3 can reduce the release of matrix
metalloprotease (MMP) (proteolytic enzymes), such as collagenase, and
therefore reduce the destruction of ECM [47]. In addition, similar results
were obtained in a study carried out on rats which emphasized that the
expression of coll II in the MIA (Monosodium Iodoacetate) + O3 group
was upregulated compared to that of the MIA group (animal model of
osteoarthritis (OA)), indicating that O3 inhibited MMP-13 expression
and collagen type II degradation in OA [47].
In the same way, previous histological and biochemical analyzes of
IVDs treated with ozone therapy (from herniated rabbit discs in vivo)
showed that ozone reduced IVD volume by dehydrating the ECM of the
NP through interactions with abundant PGs [48]. Our findings were
consistent with those of previous studies, we also noticed the decrease in
IVD height after the injection of ozone.
The aim of repeated injection of ozone is to create resistance against
oxidative stress by stimulating the antioxidant system [49]. The ob­
tained findings imply that repeated injections of ozone can harm IVDs.
Therefore, a single dose is sufficient to improve early IDD.
In the treatment of herniated disc, it has been shown that O2-O3
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International Immunopharmacology 129 (2024) 111596
dissolves in the intra-discal water and reacts with the macromolecular
components (PGs and GAGs) which is at the origin of the oxidation of the
substrates (glycine, galactose, glucuronic acid…etc.) and the breaking of
inter and intra-molecular chains leading to the disintegration of the 3D
structure. Its collapse releases retained water, which, after reabsorption,
allows a reduction in intradiscal pressure and, finally, the extinction of
pain due to decreased pressure on the nerve root [50]. The final thera­
peutic impact is due to the association of biochemical and vascular-
mediated impacts (correction of local acidosis, improvement of
oxygenation, and disappearance of venous and lymphatic stasis) [51].
Inoue H et al, believed that biological treatments should be given in
the early stages of IDD, because sufficient numbers of NP cells are
needed for these treatments to be effective [52]. Similarly, in a study by
Perri et al on 257 patients [24], a significant improvement was detected
in patients (68.4 %) who presented with Pfirrman grade I, II, and, III of
IDD. This difference is probably associated with the greatly reduced
quantity or even the absence of NP cells in advanced stage of IDD (grade
IV and V of IDD), limiting the expected therapeutic response. These
findings are in agreement with our work.
Our findings agree with those of Bocci [53], who showed that ozone
can cause the decrease of antioxidant enzymes including superoxide
dismutase (SOD), GSH-peroxidases (GSH -Px), GSH-reductase (GSH-Rd)
and catalase (CAT), if the stage of the disease is not too advanced.
O3 reacts with all macromolecules of cell membranes, involving
lipids, proteins, carbohydrates and DNA [54]. More than that, ozone
modifies intracellular activity, increasing the production of ribosomal
and mitochondrial proteins, which explains the high potential for tissue
and organ regeneration [55]. Due to neoangiogenesis, O3 allows
vascularization caused by hyper-oxygenation of tissues. Consequently,
the inhibitory capacity of inflammatory metabolites is enhanced as well
as local tissue tropism (renewal) [52].
Ozone therapy mediates its actions through oxidative products of
lipoperoxidation (LOP) and reactive oxygen species (ROS), which likely
act in two steps in IVD. The first step involves inhibiting inflammation
and decreasing prostaglandin-E2 (PGE2) and pro-inflammatory cytokine
concentrations by depleting phospholipase A2, COX-I and II, kallikreins
and bradykinin. Ozone therapy can also reduce the release of serotonin
and metalloproteinases such as aggrecanase, collagenase and gelatinase,
consequently, preventing the breakdown of ECM [47,53,56]. In the
second step, O3 mediates increased concentrations of inhibitory cyto­
kines (IL-4 and IL-10), antioxidant enzymes, oxidative shock proteins
(hemooxygenase-1) and growth factors (TGF-β), it stimulates the
N. Elmounedi et al.
synthesis of nitric oxide (NO), neoangiogenesis and the release of en­
dorphins, adrenocorticotropic hormone and cortisol. This overview of
activities mediates the restore process via the stimulation of fibroblasts,
chondrocytes and stem cells which synthesize GAGs, PG and collagens
[53].
As proven in a study conducted on rats [57], low concentrations of
O3 stimulate the secretion of macrophages and leukocytes while inhib­
iting the production of prostaglandins and the release of bradykinin. O3
also has an effect on other immune mediators, including vascular
endothelial growth factor (VEGF), platelet-derived growth factor
(PDGF), and TGF-β, ultimately inducing their action [58].
The ozone elevate the concentration of oxygen in tissues suffering
from hypoxemia, due to the restricted microcapillary blood flow
determined by the mechanical disc compression and stimulates fibro­
blastic cells activity to restore the degenerated disc by deposition of
collagen [59].
Intradiscal O3 therapy has been recommended due to its positive
benefits, which include enhanced oxygenation after neoangiogenesis,
anti-inflammatory characteristics, and analgesic effects given by anti­
nociceptive system activation. Intradiscal medicinal O3 suppresses pro-
inflammatory prostaglandin production, inhibits bradykinin release,
and increases the release of proinflammatory cytokine antagonists, all of
which are anti-inflammatory actions [56].
O3 presents a dose/effect ratio, as a result, it is important to inject the
optimal dose of O3 whereas avoiding its toxicity. On the other hand, the
reported side effects are complications due to application errors and the
reported adverse effects of O3 treatment are almost nonexistent [60]. In
addition, early pregnancy conditions involving glucose-6-phosphate
dehydrogenase deficiency, use of angiotensin-converting enzyme
(ACE) inhibitors, hyperthyroidism, thrombocytopenia, severe cardio­
vascular instability, and allergy to O3 can make this a risky treatment
option [61]. Aside from these conditions, O3 is a very safe therapeutic
option, with no official contraindications as stated in various studies
[62].
NF-κB is a most regulator of inflammation in mammalian cells [63].
Consistent with previous studies, NF-κB pathways have been demon­
strated to play an important role in IL-1β induced inflammation [64].
O3 therapy can block the activation of NF-κB and thus inhibit the
release of pro-inflammatory cytokines including IL-1β, IL-6, TNF-α and
COX-2 (These cytokines inhibit the synthesis of aggrecan and collagen
type II, which are the major components of ECM). Which is at the origin
of the inhibition of the degradation of the ECM and the initiation of the
apoptotic pathway, thus begetting the survival and the growth of the
cells [46].
O3 can also promote the production of adenosine triphosphate (ATP)
by the enzymatic pathway of glycolysis and upregulate the oxygenation
of injured cells and therefore avoid cell death [65].
Ozone therapy was not effective for severely degenerated discs. For
early to moderately degenerated discs, this treatment can attenuate IDD.
We anticipate that the results of this study will be useful in treating
people.
Ozone therapy has certain disadvantages. Hemolysis elevated pro­
gressively in high ozone concentrations. In addition, blood ozonation led
to the peroxidation of plasma membrane phospholipids. Ozone becomes
toxic at high concentrations. It is an inconvenient treatment, it is un­
stable and cannot be stored in any form [56,66].
The ozone has side impacts and can irritate the respiratory system in
humans and animals [67]. The eyes and lungs are very sensitive to ozone
so, ozone contact with these organes should be avoided [53]. Inhalation
of O2-O3 may induce acute arterial vasoconstriction in humans [68]. The
low concentrations can achieve a therapeutic effect but the inhalation of
this concentration of O3 may provoke coughing and irritation of the
throat [69]. Inhalation of higher concentrations damages the bronchial
mucosa and pneumocytes and may lead to pulmonary edema [53]. In
our study, O3 doses were used at low concentrations such as 0.1 ml of 30
µg/ml.
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International Immunopharmacology 129 (2024) 111596
The main limitation of our research is that we could only perform
radiological and histopathological examinations and we could not use
other studies such as micro-scanner which could have given more in-
depth information. As we used a reduced number of rats. Although
short, the duration of our study was able to inform us about a significant
slowing down of the natural evolution of IDD. However, a longer
duration in later studies would have told us more about the effectiveness
of our treatment in the long term.
Molecular docking is a very widely used evaluation technique,
requiring confirmation by assay of key proteins (PI3K and COX-2) by
western blot or PCR techniques. We were unable to do these tests due to
lack of funding and the kits are very expensive.
5. Conclusion
In summary, intradiscal ozone injection is a cost-effective procedure.
It seems that O2-O3 should be administered at a low degree of IDD and at
an early stage because ozone cannot repair the advanced inflammatory
alteration of IVD. Our study demonstrated that ozone attenuated the IL-
1β-induced inflammatory reaction and catabolism by intercepting the
PI3K/Akt/NF-κB signaling pathway and COX-2 expression. These find­
ings can support the potential use of ozone as a promising therapeutic
agent in treating IDD. As this was only preliminary investigation, further
studies are required to substantiate these findings and elucidate the
mechanisms underlying O3′s advantageous effects on IVD.
CRediT authorship contribution statement
Najah Elmounedi: Writing – original draft, Software, Resources,
Project administration, Methodology, Investigation, Formal analysis,
Data curation, Conceptualization. Walid Bahloul: Writing – review &
editing, Validation, Supervision. Abdelkader Kharrat: Software, Re­
sources, Methodology. Mabrouk Horchani: Methodology, Investiga­
tion, Data curation, Conceptualization. Hichem Ben Jannet:
Validation. Ahmed Racem Guidara: Visualization, Validation. Hassib
Keskes: Writing – review & editing, Supervision, Funding acquisition.
Declaration of competing interest
There was no external funding in the preparation of this manuscript.
Data availability
Data will be made available on request.
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