Effects Of ?3-polyunsaturated Fatty Acids On Myocardial Systolic Dysfunction Induced By Hemorrhagic Shock And Its Mechanism

Myocardial systolic function plays a main role in the maintenance of hemodynamics homeostasis and tissue perfusion.In the process of hemorrhagic shock,insufficiency of effective circulatory volume leads to systemic hypotension directly and ischemic hypoxia of gastrointestinal tract,therefore,the return of bacteria,endotoxin and other injurious factors contained in mesenteric lymph into the systemic circulation damages myocardial cells,and eventually causes cardiac dysfunction.Therefore,the key link of shock therapy is to ameliorate systolic dysfunction of myocardial cells and restore the pump activity of the heart.From this view,further exploration of the mechanisms by which shock causing systolic dysfunction of myocardial cells has important clinical significance for the protection of myocardial cells,other organs dysfunction induced by severe shock.Omega-3 polyunsaturated fatty acids(?-3PUFAs),as one of nutrients and cell membrane ingredients,involves in the synthesis of lipid mediators,the production and release of inflammatory factor,and then regulation of inflammatory response.Moreover,?-3PUFAs has protective effect for the intestinal barrier function.Cell autophagy is responsible for many physiological processes and involves in the development of divers diseases.Meanwhile,cell autophagy plays a certain role in the pathogenesis of organ injuries after hemorrhagic shock.However,whether ?-3PUFAs can alleviate the systolic dysfunction of myocardial cells following hemorrhagic shock and,if so,its mechanism is associated with cell autophagy has not been reported.Based on an observation of ?-3PUFAs treatment on the survival time and survival rate of rats subjected to hemorrhagic shock,the study further investigated the effects of ?-3PUFAs and autophagy signaling tool reagents on the damage,systolic function,and autophagy of myocardial cells in shocked rats,and observed the related mechanism aimed to the intestinal injury.To achieve it,the study may provide a certain experimental proof for the prevention and treatment of myocardial systolic function caused by hemorrhagic shock.Firstly,rat model of hemorrhagic shock was prepared.Briefly,anesthesia of healthy male rats was induced with inhalation of 3% isoflurane in a box filled with oxygen and maintained by mask inhalation of 1.5% isoflurane in the process of surgery.Bilateral femoral arteries and unilateral femoral vein were cannulated.After equilibrium of 30 minutes,conscious rats were bled with 40% of total blood volume within 10 minutes and then fluid resuscitation was performed after maintaining hypotension for 1 hour.Twelve healthy male rats were divided into shock or shock plus ?-3PUFAs groups to observe the effect of ?-3PUFAs on the survival time and survival rate of rats subjected to hemorrhagic shock.Results showed that all of 6 rats in shock group died within 30 h and the survival time of 3 rats in shock plus ?-3PUFAs group was more than 50 h,and the remaining 3 animals in shock plus ?-3PUFAs group died within 30 h.Results suggested that ?-3PUFAs has significant intervention effects on hemorrhagic shock.In order to further validate the effect of ?-3PUFAs on myocardial systolic function of rats subjected to hemorrhagic shock,the next set of experiment was carried out.Twelve healthy male rats were subjected to hemorrhagic shock procedure by routine method and after fluid resuscitation completed,mesenteric lymph duct was cannulated to collect shock mesenteric lymph(PHSML)for the further study.Forty-eight rats were randomly divided into sham(sham),sham plus ?-3PUFAs(sham+ ?-3PUFAs),shock,shock plus ?-3PUFAs(shock+?-3PUFAs),shock plus PHSML plus ?-3PUFAs(shock+PHSML+3PUFAs),shock plus 3-methyladenine(3-MA)(shock+3-MA),shock plus ?-3PUFAs plus rapamycin(RAPA)(shock+?-3PUFAs+RAPA),shock plus ?-3PUFAs plus PHSML plus 3-MA(shock+3PUFAs+PHSML+3-MA)groups with each group 6 rats.Rats in shock,shock+?-3PUFAs,shock+ PHSML+ ?-3PUFAs,shock+3-MA,shock+?-3PUFAs+RAPA and shock+?-3PUFAs+3-MA+PHSML groups were conducted by conscious animal disease model of hemorrhagic shock using above mentioned means and maintained hypotension for 60 min before the fluid resuscitation.The fluid resuscitation was performed by infusion of the shed blood plus equal volumes of compound sodium chloride solution in shock group,infusion of the shed blood plus equal volumes of compound sodium chloride solution containing ?-3PUFAs(0.2 g/kg)in shock+?-3PUFAs group,infusion of the shed blood plus equal volumes of compound sodium chloride solution containing ?-3PUFAs(0.2 g/kg)and PHSML(1 ml/kg)in shock+ PHSML+3PUFAs group,infusion of the shed blood plus equal volumes of compound sodium chloride solution containing ?-3PUFAs(0.2 g/kg)combined with intraperitoneal injection of RAPA(10 mg/kg)in shock+?-3PUFAs+RAPA group,infusion of the shed blood plus equal volumes of compound sodium chloride solution containing ?-3PUFAs(0.2 g/kg),PHSML(1 ml/kg)and 3-MA(30 mg/kg)in shock+?-PUFAs+PHSML+3-MA group,infusion of the shed blood plus equal volumes of compound sodium chloride solution containing 3-MA(30 mg/kg)in shock+3-MA group.Rats in sham and sham+?-3PUFAs groups were anesthetized and their femoral arteries and jugular vein were cannulated,but no blood was withdrawn or infused.In order to maintain the body fluid homeostasis,a small amount of compound sodium chloride solution was given to rats in sham group during the whole experiment period.A small amount of compound sodium chloride solution containing ?-3PUFAs(0.2 g/kg)was given to rats in sham+3PUFAs group.Three hours after the end of fluid resuscitation or corresponding time point,the blood samples of rats were drawn from abdominal aorta for the assay of serum biochemical indexes,heart was removed for the measurement of cardiac systolic function ex vivo and the end of ex vivo experiment ventricular fraction of heart was fixed in 4% paraformaldehyde solution for the next observation of morphology,the remaining fraction of heart was frozen and stored at-80 ?.A 2-cm segment of ileum was dissected 10 cm proximal to the cecum for the determination of water content in tissue and another 2-cm segment of ileum was excised and immersed in 4% paraformaldehyde solution for the next observation of morphology and the expression of microtubule-associated protein light chain 3(LC3 II)was detected by immunohistochemical staining.The results indicated that compared with that of sham and sham+?-3PUFAs groups the activity of plasma CK-MB and LDH-L in rats of shock group significantly increased,the therapy of ?-3PUFAs decreased the activity of plasma CK-MB and LDH-L of shocked rats significantly,the 3-MA had no therapeutic action on the activity of plasma CK-MB and LDH-L of shocked rats,venous injection of shock mesenteric lymph abolished the effect of ?-3PUFAs suppressing the activity of plasma CK-MB and LDH-L of shocked rats,RAPA blunted the effects of ?-3PUFAs suppressing the activity of CK-MB and LDH-L in shocked rats,the 3-MA treatment further exacerbated the negative effect of shock lymph on CK-MB activity.In addition,hemorrhagic shock caused cell rupture and edema,and up-regulation of LC3-II in myocardium;both of ?-3 PUFAs and 3-MA treatments alleviated myocardial damage and LC3-II up-regulation induced by shock;both of intravenous infusion of shock mesenteric lymph and RAPA administration blunted the protective effects of ?-3PUFAs,the 3-MA treatment relieved a certain degree of negative effect of PHSML.Results from isolated heart experiments indicated that compared with those of sham and sham+?-3PUFAs groups the ±dP/dt max and LVSP in rats of shock group significantly decreased,the HR and LVEDP had no statistical difference,?-3PUFAs elevated the ±dP/dt max and LVSP of shocked rats significantly,the venous injection of shock mesenteric lymph abolished the positive effect of ?-3PUFAs on the isolated shocked heart and manifested by significantly reduced ±dP/dt max and LVSP,specific inhibitor of autophagy 3-MA enhanced the ±dP/dt max and LVSP of isolated shocked heart significantly and had no action on the HR and LVEDP,autophagy agonist RAPA had no significant action on the ±dP/dt max,LVSP,HR and LVEDP of shocked rats treated by ?-3PUFAs,the treatment of 3-MA further blunted the negative effect of shock mesenteric lymph on isolated heart and manifested by significantly increased of ±dP/dt and LVSP as well as decreased of LVEDP.Results from intestinal injury indexes indicated that compared with that of sham and sham+?-PUFAs groups the dry/wet ratio of intestine in rats of shock group significantly increased,both of ?-PUFAs and 3-MA decreased the dry/wet ratio of intestine of shocked rats,the venous injection of shock mesenteric lymph abolished the effect of ?-PUFAs reducing the dry/wet ratio of shocked intestine,the 3-MA treatment blunted the negative effect of shock mesenteric lymph on the dry/wet ratio of intestine.Meanwhile,the levels of plasma D-LA and I-FABP in shock group significantly increased when compared to those of sham and sham+?-PUFAs groups,both of ?-PUFAs and 3-MA decreased the levels of plasma D-LA and I-FABP of rats in shock group,the venous infusion of shock lymph abolished the effect of ?-3PUFAs on plasma D-LA in shocked animals and had no significant effect on the levels of I-FABP,the 3-MA treatment blunted the negative effect of shock mesenteric lymph on the levels of plasma D-LA and I-FABP.In summary,the study indicates that the mechanisms of ?-3PUFAs prolonging the survival time and increasing myocardial systolic function of rats subjected to hemorrhagic shock are associated with suppressing cell autophagy and reducing return of PHSML to systemic circulation.Meanwhile,alleviation of intestinal injury may be another mechanism of ?-3PUFAs reducing myocardial injury and preserving cardiac function in shock status.The results suggest that ?-3PUFAs might be one of new medicines to treat myocardial injury induced by severe hemorrhagic shock.