Study On The Effect And Mechanism Of Hyperbaric Oxygen Therapy Combined With Alginate Oligosaccharide On D-Galactose-Induced Cardiac Senescence In Mice

Objective: In this study,the senescent mice model was established using D-galactose(Dgal)with the aim of investigating the protective effect of hyperbaric oxygen therapy(HBOT)combined with alginate oligosaccharide(AOS)on the senescent heart in mice and exploring the potential mechanisms.Materials and Methods: 45 male Kunming mice were randomly grouped into Control group,D-gal group,D-gal+HBOT group,D-gal+AOS group and D-gal+HBOT+AOS group.Control group was injected subcutaneously with sterilized saline(5 ml/kg·d)for 8weeks,the other four groups were injected subcutaneously with D-gal(200 mg/kg·d)for8 weeks,the D-gal+HBOT group also required HBOT once daily for 2 weeks starting from the first day of the 7th week of D-gal injection,the D-gal+AOS group received subcutaneous abdominal AOS(150 mg/kg·d)for 8 weeks starting from the first week,and the D-gal+HBOT+AOS group took both interventions.The daily behavior of mice was observed daily and body weight data was recorded once a week.After the experiment,the histopathological changes of mice cardiac tissue were visualized by Hematoxylin-eosin staining;the degree of fibrosis in cardiac tissue was visualized by Masson staining and Sirius red staining;total superoxide dismutase(T-SOD),catalase(CAT)activity and malondialdehyde(MDA)content in serum of mice were detected by oxidative stress related kits;the protein expression levels of aging-related markers p53 and p16,cardiac function-related proteins atrial natriuretic peptide(ANP)and brain natriuretic peptide(BNP),antioxidant proteins CAT,superoxide dismutase1(SOD1)and superoxide dismutase2(SOD2),inflammation associated protein tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6),and the activation status of extracellular regulated protein kinascs/nuclear transcription factor-κB(ERK/NF-κB)signaling pathway in heart tissues of mice was measured by Western blot;the levels of IL-6,TNF-α and interleukin-1β(IL-1β)in serum of mice were examined by ELISA kit.Results: 1.Mice in the Control group had good appetite,agile movement and stable weight gain,while mice in the D-gal group showed gradual loss of appetite,slow response,slow weight gain and a significantly lower cardiac index than the Control group(P<0.01).The above characteristics were significantly alleviated by AOS and HBOT intervention(P<0.01),and the combination of the two alleviated them more significantly(P<0.01).2.The results of cardiac tissue staining revealed that myocardial cells in the D-gal group had enlarged gaps,disorganized arrangement and increased fibrosis,and after HBOT and AOS intervention,myocardial cell damage was reduced and the level of fibrosis was decreased,and the tissue morphology in the two combined treated group was close to that of normal mice heart.3.Western blot results demonstrated that the expression of aging-related proteins p53 and p16 and cardiac function-related proteins ANP and BNP in the heart tissue of mice in the D-gal group was markedly higher than that in the Control group(P<0.01),and HBOT and AOS could down-regulate the expression of these proteins(P<0.01),and down-regulation was more significant in the combined group(P<0.01).4.The oxidative stress kit detected that the T-SOD and CAT activities in the serum of mice in the D-gal group decreased(P<0.01)and the MDA content increased(P<0.01)compared with the Control group,and after the intervention of HBOT or AOS,the T-SOD and CAT activities increased(P<0.01)and the MDA content decreased(P<0.01),and the effect after the combination was greater than which in the single treated group(P<0.01～0.05).Western blot results of mice heart tissue revealed that the SOD1,SOD2 and CAT protein expression in D-gal group was reduced compared with the Control group(P<0.01),while HBOT and AOS could upregulate these proteins expression(P<0.01),and the upregulation effect was more significant in combined group(P<0.01～0.05).5.Western blot results showed activation of the ERK/NF-κB pathway in the heart tissue of D-gal treated mice,as evidenced by increased p-ERK/ERK ratio and upregulation of pIκBα and cytosolic NF-κB p65 protein expression,which was statistically significant compared with the Control group(P<0.01),while HBOT and AOS could downregulate pERK/ERK ratio and p-IκBα and cytosolic NF-κB p65 protein expression(P<0.01),and the effect in the combined group was stronger than the single treated group(P<0.01～0.05).6.Western blot results showed that the expression of TNF-α and IL-6 in the heart tissue of mice in the D-gal group was upregulated compared to the Control group(P<0.01),and HBOT and AOS downregulated TNF-α and IL-6 protein expression(P<0.01),and the effect was more pronounced in the combined group(P<0.01).the ELISA kit results demonstrated that,compared to the Control group,the serum levels of TNF-α,IL-6 and IL-1β was dramatically enhanced in mice of the D-gal group(P<0.01),and the levels of pro-inflammatory factors was reduced after HBOT and AOS intervention(P<0.01),and the reduction was more pronounced in the combined group(P< 0.01～0.05).Conclusions: Combined application of HBOT and AOS delayed the cardiac senescence in D-gal induced senescence model in mice,which may be related to attenuation of oxidative stress and inhibition of ERK/NF-κB inflammatory pathway.