Role Of Stellate Ganglion Block Improving Vascular Reactivity In Rats Following Hemorrhagic Shock And Its Mechanism

Hemorrhagic shock is a common clinical critical illness.Vascular hyporeactivity is a refractory factor of hemorrhagic shock and the main cause of death.Return of post-hemorrhagic shock mesenteric lymph(PHSML)is one of the important mechanisms leading to vascular hyporeactivity after shock.Stellate ganglion block(SGB)can reduce sympathetic excitability and so as to promote local blood circulation.However,it is unclear whether SGB could improve vascular hyporeactivity after shock.Vascular smooth muscle cells(VSMCs),as stromal cells in the vascular wall,participate in all physiological and pathological changes in the vascular wall.In the pathogenesis of some vascular diseases,autophagy of VSMCs cells is activated,and excessive autophagy causes necrosis and apoptosis of VSMCs.Based on these clues,we proposed a series of questions.Whether VSMC excessive autophagy was involved in the pathogenesis of vascular hyporeactivity caused by hemorrhagic shock? Whether SGB improved vascular hyporeactivity during shock throught the inhibition of VSMC autophagy? Was vascular hyporeactivity induced by PHSML return mediated VSMC excessive autophagy? It needs to be confirmed by further study.In this study,we constructed the conscious hemorrhagic shock model,and tested the effects of SGB,PHSML,autophagy inhibitors and agonists on vascular reactivity of mesenteric secondary arterioles after shock in rats.At the cell level,vascular smooth muscle cell viability,cell contractility and autophagy related protein expression were detected after treatments of PHSML,PHSML obtained from the rats underwent hemorrhagic shock plus SGB treatment(PHSML-SGB),autophagy inhibitors and agonists,respectively.Together,the current study will provide new experimental data for the prevention and treatment of vascular hyporeactivity after hemorrhagic shock,and expand the clinical application of SGB.Firstly,the rat model of hemorrhagic shock was established along with SGB or not,for the preparation of PHSML and PHSML-SGB.Forty-two healthy adult male rats were randomly divided into seven groups(n = 6),including Sham,Sham+SGB,Shock,Shock+SGB,Shock+3-MA,Shock+SGB+Rapa and Shock+SGB+PHSML groups.The intestinal fistulas at a fixed position were taken at 3 hours or corresponding time points after fluid resuscitation in each group.The mesenteric secondary arterioles were separated under the posture microscope to prepare the arteriole ring.The tension-contractile reactivity of microvessels under different treatments was detected by four-channel in vitro vascular tension meter.The results showed that there was no significant difference in contractile response of mesenteric arterioles to different concentrations of NE between the Sham and Sham+SGB groups(P>0.05).The reactivity of intestinal arterioles to NE in Shock group was significantly lower than that in Sham group(P<0.05).The reactivity of mesenteric arterioles to NE in Shock+SGB group was significantly higher than that in Shock group,and there was no significant difference in contractile response of mesenteric arterioles to different concentrations of NE between Shock+SGB group and Sham group(P<0.05).Autophagy inhibitor 3-MA significantly increased the reactivity of mesenteric arterioles to gradient NE in rats with hemorrhagic shock(P<0.05).On the contrary,autophagy agonist Rapa decreased the contractile response of blood vessels to gradient NE in Shock+SGB group(P<0.05).Intravenous infusion of PHSML decreased the contractile reactivity of blood vessels to gradient NE in Shock+SGB group(P<0.05).The VSMCs cells were divided into control group(Control),post-hemorrhagic shock mesenteric lymph group(PHSML),post-hemorrhagic shock mesenteric lymph after stellate ganglion block group(PHSML-SGB),post-hemorrhagic shock mesenteric lymph with 3-MA group(PHSML+3-MA),PHSML-SGB combined with PI3 K inhibitor LY294002 treatment group(PHSML-SGB+LY294002),PHSML-SGB combined with Akt inhibitor MK2206 group(PHSML-SGB+MK2206),and PHSML-SGB combined with Rapa group(PHSML-SGB+Rapa).The cell viability of each group was detected by CCK-8 method.The contractile response of cells in each group to NE was observed by the cumulative transmittance of VSMCs to fluorescence-labeled albumin after Transwell double-chamber culture.The results of CCK-8 assay showed that PHSML significantly inhibited the cell viability of VSMCs(P<0.05),while 3-MA could counteract the inhibitory effect of PHSML on VSMCs cell viability(P<0.05).The cell viability of PHSML-SGB group was significantly higher than that of PHSML group(P<0.05).At the same time,LY294002,MK2206 and Rapa could all inhibit the viability of VSMCs cells in PHSML-SGB group(P<0.05).The results of VSMCs contractile reactivity showed that the cumulative transmittance of VSMCs to fluorescence-labeled albumin after PHSML treatment was significantly lower than that of Control group(P<0.05).The cumulative transmittance of VSMCs to fluorescence-labeled albumin in PHSML-SGB and PHSML+3-MA groups was significantly higher than that in PHSML group(P<0.05).However,the cumulative transmittance of VSMCs to fluorescence-labeled albumin in PHSML-SGB+LY294002 group was significantly higher than that in PHSML-SGB group(P<0.05).There was no significant difference in the cumulative transmittance of fluorescence labeled albumin between PHSML-SGB+MK2206,PHSML-SGB+Rapa groups and PHSML-SGB group(P>0.05).Finally,according to the different treatment factors,the normal VSMCs were divided into four groups: Control,PHSML,PHSML-SGB and PHSML+3-MA.The expression and localization of microtubule associated protein light chain 3(LC3-II)were determined by immunofluorescence.The expressions of autophagy marker proteins LC3-II,Beclin1 and p62 were detected by Western blotting.Compared with the normal group,the expression of LC3-II and Beclin1 in PHSML group was significantly increased,while the expression of p62 was significantly decreased(P<0.05).In contrast,PHSML-SGB and 3-MA treatment decreased the LC3-II and Beclin1 expression induced by PHSML(P<0.05),and increased the expression of p62(P<0.05).In summary,the results show that SGB improved vascular reactivity after hemorrhagic shock.VSMCs excessive autophagy is involved in the process of vascular hyporeactivity after hemorrhagic shock.SGB inhibits PHSML-mediated vascular hyporeactivity by reducing VSMCs excessive autophagy.