The Role And Mechanism Of Mast Cell Activation Induced By Microbubbles In Decompression Sickness Skin Lesion

Decompression sickness is a systemic disease caused by unsafe desaturation of dissolved physiologic inert gas in the body and due to improper decompression of the external environment,resulting in the formation of bubbles in and out of blood vessels and tissues.Its symptoms and signs mainly involve the skin,skeletal muscle,nervous system,cardiopulmonary system and so on.Skin injury caused by decompression sickness is often clinically manifested as purplish papules and marble-like markings,accompanied by skin itching,burning sensation and ant walking sensation.Among them,skin injury can be the first symptom of decompression sickness,and is an early assessment indicator of mild or type I decompression sickness.The skin lesions tend to occur in the parts of the body where the fat layer is thick,and have the characteristics of short duration and quick fading.Although skin injury is not fatal,its progression and extent of injury are often closely related to the severity of systemic symptoms.The cause of skin lesions caused by decompression sickness is still controversial,and there are three hypotheses: in-situ bubble,cutaneous arterial bubble embolism,and cerebral arterial bubble embolism.Our research have found that the formation of skin in-situ bubbles may be an important factor leading to cutaneous decompression sickness,and there were obvious microvascular dilatation and increased vascular permeability and other inflammatory reactions in the skin lesions.These inflammatory effects were consistent with the in situ bubble hypothesis that the bubbles in the local skin lesions blocked microvessels and induced local inflammation.In addition,there are not a few cases of decompression sickness combined with skin damage,except for a few cases reported that the skin manifestations of decompression sickness are often combined with right-left pathway abnormalities such as patent foramina ovale,which will detect bubbles in the arterial system,and no effective evidence of cutaneous artery embolism and cerebral artery embolism has been found.Therefore,we propose the scientific hypothesis that in-situ bubbles are the cause of skin decompression sickness.Considering that the previous research successfully constructed an animal model of decompression sickness in Bama pigs with significant clinical manifestations of skin symptoms,we explored the rationality and scientificity of the in-situ bubble hypothesis on this basis.The previous research team has confirmed that microbubbles are the pathophysiological basis of vascular endothelial damage caused by decompression sickness.When the environmental pressure is rapidly and greatly reduced,bubbles are formed in the blood vessels of the body,causing vascular embolism and tissue compression through physical effects and a series of biochemical effects.As the fundamental cause of decompression sickness,decompression bubbles also act as an important strigger in the process of cutaneous decompression sickness.The microbubbles formed in the skin tissue can act as the effector cells of the inflammatory process through mechanical stress,shear force,extrusion,etc.,resulting in a variety of inflammatory cascade effects.Mast cells play a critical role in the pathogenesis of a series of inflammatory and allergic skin disorders,which can be activated by various physical and chemical stimuli in vivo and in vitro,and the pre-synthesized mediators in the cytoplasm can be rapidly released through the process of degranulation to play a pro-inflammatory response.Some cytokines and chemokines can also be synthesized and released to directly and indirectly affect other immune cells in the tissue,causing the amplification of inflammatory effects.Based on the previous findings of the research group,this project intends to carry out researches at the cellular and animal levels.Using mouse primary mast cells and rat mast cell line RBL-2H3 as research objects,the role and mechanism of microbubble touch on mast cells were investigated from the aspects of cell function and structure.In vivo experiments,Bama pigs were studied to confirm the involvement and mechanism of mast cell activation in skin damage caused by decompression sickness,and to provide theoretical basis for the hypothesis of in situ bubble of skin damage caused by decompression sickness.Part Ⅰ: Functional Changes and Mechanism of Mast Cell Activation induced by microbubbleObjective: To explore the effects of microbubble contact on mast cell activation and degranulation,intracellular calcium mobilization,and the involvement of TRP channels.Methods: In this part,murine bone marrow-derived mast cells and rat mast cell lines were used.Toluidine blue staining and flow cytometry detecting the double positive expressions of CD117 and FcεRⅠα on the surface of mast cells were utilized to identify mouse bone marrow-derived mast cells.The degranulation level of mast cells was detected by ELISA,and the expression level of CD63 on mast cells was measured by flow cytometry.The effect of time length of microbubble contact on the degranulation and activation of mast cell were analyzed.The changes of Ca2+ concentration in mast cells labeled by Fluo-4AM fluorescent probes were detected by fluorescence microscopy and quantitatively analysed by flow cytometry.In addition,mast cells were pretreated with different TRP channel inhibitors to examine their inhibitory effects on microbubble-induced degranulation and Ca2+ concentration.Results: The appropriate time point that the degranulation level of mast cells increased significantly of microbubble contact on mast cells was 1 h..Microbubble contact can effectively increase the degranulation of β-Hex and histamine released in two different types of mast cells,and up-regulate the expression of activation marker CD63 on cell surface,accompanied by an increased intracellular Ca2+ concentration.TRPV1 and TRPV4 inhibitors can effectively inhibit mast cell degranulation and calcium inflow induced by microbubbles.Conclusion: Microbubble contact induced mast cell degranulation and calcium mobilization,in which TRPV4 and TRPV1 were potential targets of microbubble contact affecting mast cell function.Part Ⅱ: Cytoskeletal Changes and Mechanism of Mast Cell Activation Induced by MicrobubbleObjective: The effects of mast cell activation induced by microbubble contact on the changes of cell ultrastructure,skeleton structure and cell membrane as well as the related signal pathways involved were preliminarily investigated.Methods: In this part,mast cells and mast cell lines derived from primary mouse bone marrow were used as research objects.The degranulation process of mast cells was detected by cellular immunofluorescence staining,the changes of ultrastructure of mast cells were observed by transmission electron microscopy,and the changes of F-actin cytoskeleton of mast cells were detected by phalloidin staining.Meanwhile,flow cytometry was used to detect the PS externalization marked by Annexin V-FITC on mast cell membrane,and Western Blot was used to detect the expression of cytoskeleton-related signaling pathway proteins.Results: Microbubbles contact inducd mast cells to activate and release tryptase particles.Transmission electron microscopy(TEM)observed the formation of vacuolar vesicles in the cytoplasm.Flow cytometry detected the increased cell membrane PS externalization of activated mast cells and Phalloidin staining indicated the rearrangement of F-actin in microbubble-stimulated mast cells.The relative protein expression levels of Rho A,ROCK1 and Rac1/Cdc42 were increased by Western Blot analysis.Conclusion: Microbubble contact induced mast cell activation and structural changes,and Rho A/ROCK1 signaling pathway was involved in microbubble –induce mast cell structural changes.Part Ⅲ: Effect and Mechanism of Mast Cell Activation in Swine Decompression Sickness Skin LesionsObjective: To explore the effect and mechanism of mast cell activation on swine skin injury caused by decompression sickness in vivoMethods: The symptoms of decompression sickness were successfully induced in Bama pigs,and samples were collected from normal skin,lesion margin and lesion areas of Bama pigs.The inflammatory reaction and degranulation rate of mast cells were observed by pathological section.The skin β-Hex and histamine level in skin tissue were detected by tissue homogenate.The expression of activated mast cells and TRPV channels in skin lesions was marked by tissue immunochemistry and tissue immunofluorescence.The expression of TRPV channels in skin tissues was detected by RT-q PCR.Results: Pathological sections of normal skin,lesion margin,and skin lesion were taken,and HE staining indicated obvious inflammatory reactions in the skin tissues,including epidermal thickening and edema,dermal capillary dilatation and congestion,mixed inflammatory cells infiltration.Toluidine blue and Giemsa staining showed a significant increase in degranulation of mast cells.The contents of β-Hex and histamine in skin lesions were higher than those in normal skin tissues,and were positively correlated with mast cell degranulation rate.Immunohistochemical results showed that the expression of TRPV1/TRPV4 was up-regulated in the epidermis.Tissue immunofluorescence indicated that activated mast cells increased and TRPV1/TRPV4 expression was up-regulated in the lesion tissue.RT-q PCR detected increased m RNA expression of TRPV4 and TRPV1 in the skin lesion.Conclusion: The inflammation of swine skin lesion in decompression sickness was obvious,the degranulation rate of mast cells in dermis was significant,and the content of inflammatory mediators released by mast cells in tissues increased.Activation of TRPV4 and TRPV1 channels mediated mast cell activation to participate in decompression disease skin injury.