| Hemorrhagic shock is one of the most common clinical diseases,which is often caused by a large number of blood loss.Then,the body shrink the surrounding blood vessels and increase vascular resistance and heart rate through neuro-humoral secretion regulation mechanism,resulting in dysfunction and disease of tissue and organ and multiple organ dysfunction syndrome(MODS)finally.The key treatments of hemorrhagic shock are replenishment of blood volume in time,treatment of its etiology and blocking its continued blood loss.But the liquid resuscitation will produce a large number of cytotoxic substances such as hydrogen peroxide and hydroxyl free radicals,which will increase cell damage caused by hemorrhagic shock.Thus,when blood products are unavailable and the control of direct bleeding delays,the type of fluid for resuscitation plays a vital role.But from isotonic crystals to colloids,hypertonic solutions,artificial oxygen carriers,blood substitutes,pharmaceuticals,no one can not only restore the organization of oxygen supply and perfusion,but also improve the cell damage and inflammatory response effectively caused by hemorrhagic shock and reperfusion.Lung,liver,kidney,brain,heart,gastrointestinal and other internal organs damage often occur after hemorrhagic shock and resuscitation.Liver injury after hemorrhagic shock and resuscitation is a common clinical emergency,which is often secondary to the lung and gastrointestinal injury.If the patient suffers from a serious liver injury,the mortality rate almost reach 100% because of the lack of direct and effective prevention and treatment measures.Hemorrhagic shock and resuscitation leads to systemic inflammatory response syndrome(SIRS).The main pathophysiological characteristics of liver injury induced by hemorrhagic shock and resuscitation manifest the damage of liver and capillary endothelial cell damage,increased permeability,diffuse liver edema,intractable hypoxemia.The pathogenesis of liver injury after hemorrhagic shock and resuscitation is very complex,the underlying reason include liver energy metabolism disorders,removal of dysfunction and intractable hypoxemia and uncontrolled inflammation reaction,oxidative stress reaction caused by reperfusion injury.Therefore,it is of great clinical significance to control the occurrence,development and prognosis of systemic uncontrolled inflammatory response,improve intractable hypoxia effectively,and reduce the mortality of liver injury after hemorrhagic shock resuscitation.Since 2007,a large number of studies have found that molecular hydrogen can selectively remove strong free radicals,and control of free radicals timely,play the role of reducing the inflammatory response,inhibiting oxidative stress,and apoptosis.In recent years,the use of hyperoxygenated solution(HOS)through non-airway assisted oxygen supply has been passed a large sample of clinical validation,a large number of studies have proved that HOS is currently the most effective way to alleviate severe hypoxia induced by hemorrhagic shock.In this study,we first pioneered the preparation of dissolved hydrogen content ≥0.50mmol/L,oxygen content≥20mg/L hyperoxygenated hydrogen rich solution(HHOS).We established the model of severe hemorrhagic shock according to the method described by wiggers,the mean arterial blood pressure(MAP)was reduced to 30 ~ 40 mm Hg by exsanguinating blood form femoral artery,and MAP 30~40mm Hg was maintained for 60 min by bloodletting or autologous blood transfusion.Then,we resuscitate with Ringer’s solution(LRS),HOS,Hydrogen-rich solution(HHS),HHOS through femoral vein respectively.At the time of 2h and 6h after resuscitation,the blood samples and liver tissue samples were obtained for the protein,lipid,deoxyribonucleic acid(DNA),free radical,antioxidant capacity and inflammatory factor were used for detection to explore the effect of HHOS on liver injury after hemorrhagic shock and resuscitation in rats and its possible mechanism.The experiments show that compared with groups treated with LRS,the serum ALT,AST concentrations and the concentration of TNF-α and IL-6 activity and MDA content in liver tissues were significantly decreased,the T-SOD activity and IL-10 positive cells in liver tissues were significantly increased,the histopathological and subcellular structure changes were significantly attenuated,the number of caspase-3 and TUNEL positive cells was reduced in the group treated with HHS,HOS,HHOS at 2h and 6h after resuscitation,and the above the protective effect of HHOS is stronger than HOS and HHS group.These results suggest that HHOS can provide effective oxygen supply and reduce reperfusion injury for the body at the same time,and provide a new strategy of treatment for liver injury after hemorrhagic shock and resuscitation.In summary,the study proved that HHOS can restore the oxygen supply and perfusion for liver tissue after hemorrhagic shock,and purify the strong free radicals and inflammatory mediators after reperfusion,and it is expected to play the role of the coordination of molecular hydrogen and molecular oxygen to become the ideal liquid for the recovery of hemorrhagic shock,and provide a theoretical basis and experimental basis for the development and utilization of HHOS.Part 1 Study on liver function of rats suffered from hemorrhagic shock andresuscitation with hyperoxygenated hydrogen rich solution Objective 1.To clarify the effect of HHOS on liver function in rats with hemorrhagic shock resuscitation 2.To investigate the effect of HHOS on liver histopathology and ultrastructural changes in rats with hemorrhagic shock resuscitation.Methods 1.SPF grade healthy SD rats,6 to 8 weeks of age,weight 280 ~ 320 g were divided into 5 groups randomly,6 rats in each group,and SHAM group were treated with sham operation.LRS,HOS,HHS and HHOS group were resuscitated with LRS,HOS,HHS and HHOS after 1h of shock in rats,respectively,and obtain blood and tissue specimens at 2h and 6h after resuscitation.2.The serum ALT and AST concentrations were measured by automated analyzer.3.Histopathological changes of liver tissue were observed by H&E staining and electron microscopy.Results 1.The serum ALT and AST results at 2h and 6h after resuscitation of hemorrhagic shock: compared with SHAM group,AST and ALT levels in serum were significantly increased in groups treated with LRS,HOS and HHS at 2h and 6h(P<0.05),and AST and ALT levels in serum were significantly increased in groups treated with HHOS at 6h(P<0.05).Compared with LRS and HOS group,AST and ALT levels in serum were significantly decreased in groups treated with HHOS at 2h and 6h(P<0.05).Compared with HHS group,ALT levels in serum were significantly decreased in groups treated with HHOS at 2h(P<0.05),and AST levels in serum were significantly decreased in groups treated with HHOS at 6h(P<0.05).2.The H&E staining of liver tissue at 2h and 6h after resuscitation of hemorrhagic shock: compared with SHAM group,the other four groups of liver histopathology were changed in different degrees,specifically in: liver tissue structure was significantly disordered,significant swelling steatosis,vacuolar degeneration,point necrosis of liver cells,hepaticlobular vein and hepatic sinus congestion significantly,a large number of inflammatory cell infiltrated around central vein and portal area.In the HHOS group,these histopathological changes were significantly lighter than those in the LRS group,HOS group and HHS group,especially the vacuolar changes were significantly reduced.3.The results of transmission electron microscopy at 2h and 6h after resuscitation of hemorrhagic shock: compared with SHAM group,the ultrastructural changes of liver tissue in the other four groups were changed in different degrees,specifically in: the hepatocyterats of rats arranged disorder,and a large number of autophagosomes,small nuclear pyknosis or heterochromatin aggregated,many mitochondria highly swollened,deformed,ridge reduced,arranged disorder,and even collapsed,vacuolization and endoplasmic reticulum was significantly expanded to vacuolated structure.Capillary bile duct reducted and so on.These ultrastructure changes were significantly lighter than those in the LRS group,HOS group and HHS group,especially the vacuolar changes.Conclusions In this part,It was confirmed that HHOS could relieve liver injury in a rat model of hemorrhagic shock and resuscitation.It was also confirmed that HHOS could increase liver function and reduce histopathology and ultrastructure change.This part of the results provide experimental basis and theoretical basis for the clinical application of HHOS as the ideal liquid for the recovery for hemorrhagic shock,and provides a new way of thinking solution for the study of mechanism of HHOS.Part 2 The effect of hyperoxygenated hydrogen rich solution on liver inflammation in rats with hemorrhagic shock resuscitation Objective 1.To investigate the effect of HHOS on proinflammatory cytokines in rats with hemorrhagic shock resuscitation.2.To investigate the effect of HHOS on the anti-inflammatory factors of liver tissue in rats with hemorrhagic shock resuscitation.Methods1.The concentration of TNF-α and IL-6 was detected using corroding kits according to the instructions.2.The expression of IL-10 in liver tissue of rats was detected by immunohistochemical staining.Results 1.The liver tissue IL-6 and TNF-α results at 2h and 6h after resuscitation of hemorrhagic shock: compared with SHAM group,IL-6 and TNF-α levels in liver tissue were significantly increased in groups treated with LRS and HOS at 2h(P<0.05),and IL-6 and TNF-α levels in liver tissue were significantly increased in groups treated with LRS,HOS and HHS at 6h(P<0.05).Compared with LRS and HOS group,IL-6 and TNF-α levels in liver tissue were significantly decreased in groups treated with HHOS at 2h(P<0.05).Compared with LRS,HOS and HHS group,IL-6 and TNF-α levels in liver tissue were significantly decreased in groups treated with HHOS at 6h(P<0.05).2.Immunohistochemical staining showed that compared with SHAM group,IL-10 positive cell in liver tissue were significantly decreased in groups treated with LRS,HOS,HHS and HHOS at 2h and 6h after resuscitation of hemorrhagic shock.Compared with LRS,HOS and HHS group,IL-10 positive cell in liver tissue were significantly increased in groups treated with HHOS at 2h and 6h after resuscitation of hemorrhagic shock.Conclusions In this part,it was clear that HHOS could decrease the proinflammatory cytokines of liver tissue and increase the anti-inflammatory cytokines of rat liver after resuscitation of hemorrhagic shock.The results suggest that the protection of HHOS for liver in hemorrhagic shock and resuscitation may be associated with its inhibition of inflammatory response,which provides a new target for the mechanism of HHOS and lay the theoretical basis and experimental basis for the clinical application of HHOS.Part 3 Effect of the hyperoxygenated hydrogen rich solution on liver oxidative stress in rats with hemorrhagic shock resuscitation Objective1.To investigate the effect of HHOS on hepatic oxidative stress in rats with hemorrhagic shock resuscitation Methods 1.The concentrations of T-SOD and MDA were detected using corroding kits according to the instructions.Results 1.The liver tissue T-SOD and MDA results at 2h and 6h after resuscitation of hemorrhagic shock: compared with SHAM group,the MDA levels in liver tissue were significantly increased,and the T-SOD levels in liver tissue were significantly decreased in groups treated with LRS,HOS and HHS at 2h and 6h after resuscitation of hemorrhagic shock(P<0.05),and the T-SOD levels in liver tissue were significantly decreased in groups treated with HHOS at 2h and 6h after resuscitation of hemorrhagic shock(P<0.05).Compared with LRS,HOS and HHS group,the T-SOD levels in liver tissue were significantly increased,and the MDA levels in liver tissue were significantly decreased in groups treated with HHOS at 2h and 6h after resuscitation of hemorrhagic shock(P<0.05).Conclusions This part of the experiment clear the HHOS can reduce the hepatic oxidative stress.This part results suggest that the protective effect of HHOS on hemorrhagic shock resuscitation may be related with the decrease of oxidative stress of HHOS.It reveals the new mechanism of the protection function of HHOS in a model of hemorrhagic shock and resuscitation in rat,and also provides a new way for the treatment of hemorrhagic shock.Part 4 Effect of the hyperoxygenated hydrogen rich solution on liver apoptosis in rats with hemorrhagic shock resuscitation Objective 1.To clarify the effect of HHOS on liver apoptosis in rats with hemorrhagic shock resuscitation Methods 1.The expression of caspase-3 in liver tissue was detected by immunohistochemicalstaining.2.TUNEL method was used to detect the apoptosis of liver tissue in each group.Results 1.Immunohistochemical staining showed that compared with SHAM group,the caspase-3 positive cell in liver tissue were significantly increased in groups treated with LRS,HOS and HHS at 2h and 6h after resuscitation of hemorrhagic shock(P<0.05).Compared with LRS,HOS and HHS group,the caspase-3 positive cell in liver tissue were significantly decreased in groups treated with HHOS at 2h and 6h after resuscitation of hemorrhagic shock(P<0.05).2.TUNEL staining showed that compared with SHAM group,the TUNEL positive cell in liver tissue were significantly increased in groups treated with LRS,HOS and HHS at 2h and 6h after resuscitation of hemorrhagic shock.Compared with LRS,HOS and HHS group,the TUNEL positive cell in liver tissue were significantly decreased in groups treated with HHOS at 2h and 6h after resuscitation of hemorrhagic shock.Conclusions In this part,it was clear that HHOS could relieve the apoptosis of liver tissue in rats with hemorrhagic shock resuscitation.The results suggested that HHOS could exert the protective effect of liver after the hemorrhagic shock and resuscitation,which lays a foundation for the further study of mechanism of HHOS and also provided a new theoretical basis and experimental basis for the clinical application of HHOS. |
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