The Protective Effect Of High Dose Of Hydrogen Breathing On Endometriosis In Rats And Its Mechanism

Background: Endometriosis(EMT)is an estrogen-dependent benign disease characterized by implantation and growth of endometrial tissue outside the uterine cavity,and causes chronic pelvic pain,dysmenorrhea and infertility,affects 5%~10% of women in their reproductive ages.Most of the current treatment modalities aim estrogen reduction as goal of treatment,but the outcomes of the treatments are menopause like side effects and symptom return upon discontinuation of the treatment.Therefore,it is still necessary to explore novel therapeutic strategies for reversing the clinical symptoms of EMT patients and improving their quality of life.Despite the lack of complete understanding of the etiology of EMT,it is well-established that an EMT-related imbalance between reactive oxygen species(ROS)and antioxidants exists.The elevated oxidative stress in endometriosis may either be a cause or a consequence of the pathophysiology of endometriosis.Recent promising evidence also indicates that the effectiveness of targeting oxidative reduction as the goal of treatment for the disease.It seems that the endometriosis treatment by targeting a reduction in oxidative stress appears to be a promising strategy.Hydrogen is used to be thought as an inert gas and has been applied to prevent decompression sickness in deep divers for safety profiles.In 2007,it was reported that an inhalation of H2 has antioxidant properties that can protect the brain ischemia reperfusion injury by selectively reducing oxidative stress.This report aroused considerable interest worldwide,and many researchers have demonstrated the therapeutic effects of molecular hydrogen on various diseases through its antioxidative capability.Compared with other antioxidants,hydrogen has several advantages: safety,efficient and cheap.Female Sprague-Dawley rats were chosen to establish a stable EMT model and to evaluate the protective effect of hydrogen and its mechanism on endometriosis in rats.Material / Methods 1.Model of EMT: The endometrium tissues of female SD rats were autotransplanted into the ovary,the uterosacral ligament and the abdominal wall.2.Experimental animal groups and treatment:(1)Model group(EMT):animals subjected to the EMT model only;(2)Hydrogen treatment group(EMT+P1H2): rats inhaled the 67%H2+33%O2 mixture for 1 hour per day 4 weeks after the EMT surgery,and the course lasted 4 weeks;(3)Hydrogen prevention group(EMT+P2H2): animals inhaled the 67%H2+33%O2 mixture for 1 hour per day immediately after the EMT surgery,and the course lasted 4 weeks;(4)Nitrogen treatment group(EMT+P1N2): animals inhaled the 67%N2+33%O2 mixture for 1 hour per day 4 weeks after the EMT surgery,and the course lasted 4 weeks;(5)Nitrogen Prevention group(EMT+P2N2): animals inhaled the 67%N2+33%O2 mixture for 1 hour per day immediately after the EMT surgery,and the course lasted 4 weeks.3.Test Index: The total weight of ectopic endometrium was measured 8 weeks after the EMT operation,and MDA,SOD,GSH,CAT,COX-2,MMP-9 etc.in ectopic endometrium tissue and serum.Results: 1.Autotransplantation method can establish a stable endometriosis animal model: Autotransplantation of endometriosis in the ovary,the uterosacral ligament,and the abdominal wall of rats can all establish the stable endometriosis model.The successful modeling rats of the three site had no significant difference.2.67% hydrogen breathing reduces the severity of the endometriosis: The total weight of transplants and of EMT+P1H2 and EMT+P2H2 group had a significant difference with EMT group,respectively(P<0.05).However,animals administrated with 67% nitrogen breathing failed to show the significant protection.In addition,compared with the rats in the EMT group,the number of PCNA-positive cells of the lesions was decreased significantly in the EMT+P1H2 and EMT+P1H2 group respectively(P<0.05),but the EMT+P1N2 and EMT+P2N2 group did not show the significant change.3.67% hydrogen breathing reduces the oxidative stress index in endometrial tissues: The EMT+P1H2 and EMT+P2H2 group show significant increases in activities of SOD,GSH and CAT in serum and tissue of the rats compared with the EMT group respectively(P<0.05),but both the EMT+P1N2 and the EMT+P2N2 group did not show the significant change.The EMT+P1H2 and EMT+P2H2 group also presents the significant decrease in the serum and tissue activity of MDA compared with the EMT group(P<0.05),and the protection of 67% nitrogen inhalation still can not be found.4.67% hydrogen breathing reduces proteins and genes expression of MMP-9 and COX-2: In the EMT+P1H2 and the EMT+P2H2 group,the number of MMP-9+ cells in endometrial tissue was significantly decreased compared with the EMT group(P<0.05),and similar condition can also be found in the number of COX-2+ cells(P<0.05).However,in the EMT+P1N2 and EMT+P1N2 group,the proteins expression of MMP-9 and COX-2 was not changed according to the statistics.In addition,the expression of gene MMP-9-m RNA was statistically decreased in the EMT+P1H2 and the EMT+P2H2 group compared with the EMT group respectively(P<0.05),and the expression of gene COX-2-m RNA presented similar significant results in the EMT+P1H2 and the EMT+P2H2 group(P<0.05).The two genes were not statistically changed in the EMT+P1N2 and EMT+P2N2 group compared with the EMT group respectively.Conclusion: These results suggest that autotransplantation method can establish a stable endometriosis animal model and that 67%H2+33%O2 breathing has a beneficial effect on EMT model rats,and inhalation of a high dose of hydrogen could be a potential method applied in clinical practice.