The effect of hyperbaric oxygen therapy in the inflammatory response in a mouse model of endometriosis: An experimental study

Abstract Background Endometriosis pathogenesis is related to the inflammation shown by the secretion of pro-inflammatory mediators. This hypoxia condition can stimulate this condition. Objective To investigate the effect of hyperbaric oxygen therapy (HBOT) on the inflammation reaction of endometriosis-induced mice. Materials and Methods The animals were designated into 3 groups: I) the pre-test group, II) the post-test group receiving the HBOT, and III) the post-test group without HBOT. All groups were subjected to induction of endometriosis by xenotransplantation for 15 days. HBOT was given 30 min 3 times a day for 10 days. The evaluation of the HBOT effect was conducted by examining the endometrial tissue. The inflammation level was evaluated using the Klopfleisch semiquantitative scoring system (index remmele scale), whilst the expression of nuclear factor kappa (NF κ B) beta was measured by immunohistochemical staining. Results The results showed that group I demonstrated the highest level of inflammation degree (9.41 ± 1.99) compared to the post-test groups (group II: 1.60 ± 0.53; group III: 2.42 ± 0.53). The HBOT-groups was found to have the lowest inflammation level compared to the non-HBOT group (p = 0.020). The results demonstrated that HBOT lowered the peritoneal inflammation degree caused by the endometrial lesion in mice. NF κ B expression on the post-test groups was significantly decreased, compared to the pre-test group (p ≤ 0.001), with a strong correlation between the NF κ B expression and the peritoneal inflammation level (p ≤ 0.001, r = 0.670). Conclusion HBOT significantly reduced the inflammation level on the endometrial lesion in mice, involving the NF κ B pathway.


Introduction
Inflammation theory is one of the molecular Hyperbaric oxygen therapy (HBOT) is a medical tested to treat hypoxia-related medical conditions, including inflammatory diseases (6). A previous study demonstrated the positive effect of HBOT in reducing the pro-inflammatory cytokine HIF-1α in endometriosis lesions, possibly due to the clearance of HIF-1α molecules in an oxygen-rich environment (7).

Chemical products
All chemical substances used in this study were purchased from Sigma-Aldrich (St. Louise, MO, USA).

Experimental animals
24 healthy adult female swiss albino mice (average age of 4 wk), with a weight range of 25-30 gr, were obtained from the Veteriner Farma Centre, Surabaya, Indonesia. Before starting the experiment, the animals were acclimatized for 1 wk for the adaptation process. The animal with more than 10% weight loss was excluded from the study.

Experimental design
Using a randomized controlled study design, the animals were designated into groups I, II, and III

Preparation of endometrial cells
The endometrial cells were collected during the surgery from uterine adenomyosis women. The

Induction of endometriosis
The procedures for the xenotransplantation of endometrial cells on the mice were performed according to the previously published protocol (8).
After the adaptation, each mouse was injected with

HBOT
One day after the endometriosis induction, the mice in group I was placed inside the hyperbaric chamber to receive the HBOT. The oxygen dose in the chamber was 100% O 2, with a flow rate of 8-10 L/min. The hyperbaric oxygen treatment was given as 2.4 atm pressure for 3 × 30 min with a 5-min air break. The treatment was given for 10 days in a row.

Histological evaluation
After the experiment, each group was

Immunostaining for NF B
The tissue slides for immunohistochemistry staining were prepared following protocol described previously (10). The antibody and staining kits were purchased from Santa Cruz Biotechnology, CA, and the staining was  (Table   II).

NF B expression
The expression of NF B on the peritoneal tissue using immunohistochemical staining is shown in arrows ( Figure 3)

The correlation between NF B expression and the degree of inflammation
The

Discussion
Our HBOT has been used in treating various medical conditions and has been proved to reduce the inflammatory mediator level (5,19). In many studies on HBO treatment, the therapeutic effects were dose-and timedependent (19,20). Administration of HBO treatment on human blood-derived monocytemacrophages for 3 hr reduced the Interleukin 1β synthesis, while prolonged HBOT for 12 hr increased the cytokine production (22). In indomethacin-induced enteropathy in rats, HBOT reduced the TNF-expression after 12 hr and 24 hr treatment, but the effect was null after 48 hr. The same study demonstrated that HBOT showed no effect on the IL-1b production after 12 hr treatment. However, the positive effect was observed after 24 hr treatment, which showed that HBOT significantly lowered the IL-1β secretion (23). With regards to the HBOT effect on the inflammatory mediators in endometriosis, it has been reported that HBOT showed an observable effect on lowering the TNF-levels after 6 wk of treatment (20). Therefore, all this evidence may explain the level of NF B shown from our result, which is likely related to the dose and duration of the HBOT used in this study.

Conclusion
Our study showed that HBO therapy reduced the inflammatory state in endometrial lesions, possibly via the alteration of the NF B pathway.
Further investigation in the duration and dose of HBOT is needed to elucidate the molecular mechanism of HBOT in endometriosis. Our results have shown that HBOT significantly reduced the inflammation level on the endometrial lesions, with a low level of NF B expression.