Mechanisms Of ORAI/STIM1 In The Regulation Of Vasoreactivity In Rats With Severe Hemorrhagic Shock

Objective:Shock is a common complication of traumatic injury that endangers life and is one of the diseases with a high mortality rate in China and even in the world.However,hemorrhagic shock is a kind of hypovolemic shock,the death rate of which is second only to craniocerebral injury in the classification of causes of death,accounting for 20%～25%of the total number of deaths.Based on the existed research,the therapeutic effect of hemorrhagic shock has been improved.However,intractable hypotension caused by vascular hyporesponsiveness in late shock is still a difficult problem to overcome in shock treatment.Finding new therapeutic targets to improve vascular hyporesponsiveness is of great significance for the treatment of hemorrhagic shock.In the late stage of shock,mitochondrial structure and functional damage of arterial smooth muscle cells,dysfunction of potassium and calcium channels,cell membrane hyperpolarization,intracellular calcium overload and calcium desensitization are all closely related to vascular hyporesponsiveness.But the related treatment to prevent hyporeactivity in shock still can not achieve satisfying result.The intracellular calcium homeostasis is regulated by both extracellular Ca²⁺influx and intracellular calcium store Ca²⁺release.Calcium release-activated calcium（CRAC）channel,as an important calcium channel in the regulation of intracellular calcium homeostasis,contributes to the component of Store-Operated Ca²⁺Entry（SOCE）and is widely distributed in various cells in vivo.It’s been reported that CRAC plays an important role in vascular smooth muscle cell proliferation,thrombosis,angiogenesis and vascular tension regulation,and CRAC dysfunction is one of the important mechanisms involved in cardiovascular diseases.CRAC is mainly composed of ORAI and Stromal interaction molecule 1（STIM1）.As intracellular Ca²⁺sensor and oxidative stress sensor,STIM1 is spatially coupled with intracellular mitochondria.Previous studies have shown mitochondrial dysfunction after severe hemorrhagic shock,and protection of mitochondria can significantly promote the recovery of vascular reactivity in late shock,but whether ORAI/STIM1-mediated calcium influx can affect mitochondrial function and then regulates vascular reactivity in late shock has not been reported.This article will combine the rat model of severe hemorrhagic shock and the in vitro cell hypoxia and reoxygenation model to investigate the changes of intracellular Ca²⁺signal mediated by ORAI/STIM1 channels,mitochondrial function,and vascular hyporesponsiveness in mesenteric arterial smooth muscle cells of severe hemorrhagic shock rats and its molecular mechanism.Methods:The experimental methods are mainly divided into two parts.First,a Sprague-Dawley（SD）rat model of severe hemorrhagic shock was constructed,and the tension measurement in isolated mesenteric artery was used to investigate the involvement of SOCE in the regulation of vasoresponsiveness in shock rats.q PCR,ELISA,and Western Blotting were used to detect the expression of ORAI and STIM1 in mesenteric artery.Adenovirus-infected rats overexpressing or interfering with STIM1 were used to detect the effects of STIM1 expression on the responsiveness and calcium sensitivity of mesenteric artery vessels in shock.Secondly,the rat mesenteric artery smooth muscle primary cells were cultured and a hypoxia-reoxygenation model was established.The effect of hypoxia-reoxygenation on cell viability was measured using the CCK-8 kit.q PCR,ELISA,and Western Blotting were used to detect the effect of hypoxia and reoxygenation on the expression level of ORAI,STIM1,and Cyclophilin D（CypD）,which is the important structural regulatory proteins of mitochondrial permeability transition pores（MPTPs）.In addition,after infecting smooth muscle cells with overexpressing STIM1adenovirus,flow cytometry or cellular immunofluorescence was used to detect the effect of STIM1 overexpression on MPTPs opening of smooth muscle cells exposed hypoxia and reoxygenation,and Western Blotting was used to detect whether the overexpression level of CypD was regulated by STIM1overexpression in smooth muscle cells following exposure to hypoxia and reoxygenation.The change of intracellular calcium signal induced by SOCE was detected by TILLvis ION micro-fluorescence ion imaging system.Results:（1）Changes of blood pressure,heart rate and multiple organ injury,as well as the survival time（less than 24 h）in rats with severe hemorrhagic shock indicated that the severe hemorrhagic shock model has been successfully constructed.（2）Vascular tension measurement showed that compared with the sham group,after incubated with 2μmol/L TG,the maximum contraction of mesenteric artery in rats with severe hemorrhagic shock caused by 5μmol/L phenylephrine was significantly decreased（P<0.01）.（3）The detection of gene and protein expression showed that compared with the sham group,the expression of ORAI protein in the mesenteric arteries of shock rats was not significantly changed（P>0.05）,while the expression of STIM1 was significantly down-regulated（P<0.05）.（4）After the rats being infected either with STIM1 overexpression adenovirus or adenovirus-sh STIM1,the vascular tension measurement showed that compared with the control group（Ad-NC）,the EC₅₀ of PE induced contraction is significantly decreased in the STIM1 over-expressing group（Ad-STIM1）and the E_max decreased significantly（P<0.05）.Compared with the control group（Adscramble）,the EC₅₀of PE and Ca²⁺significantly increased and the E_max of PE and Ca²⁺significantly decreased in the adenovirus-sh STIM1 group（Adsh STIM1）（P<0.05）.（5）The detection of intracellular calcium showed that compared with the sham group,the SOCE in the mesenteric artery smooth muscle of the rats induced by TG was significantly increased（P<0.01）.（6）Effects of hypoxia and reoxygenation on cell viability.Compared with the control group,the maintaining cells in hypoxia/reoxygenation less than 24 h did not affect cell viability,while hypoxia/reoxygenation time exceeded 24 h significantly reduced the activity of rat mesenteric artery smooth muscle cells（P<0.01）.（7）The detection of gene and protein expression showed that compared with the control group,the expression of STIM1 was significantly decreased in the primary cultured rat mesenteric artery smooth muscle cells exposed to hypoxia for 24 h and followed by reoxygenation for 24 h（P<0.05）.（8）The MPTPs test results showed that compared with the control group,the calcein fluorescence intensity significantly reduced and the expression of CypD significantly increased（P<0.01）in mesenteric artery smooth muscle cells H24-R24 group,suggesting that hypoxia and reoxygenation result in the increased opening of MPTPs significantly,which lead to mitochondrial dysfunction in smooth muscle cells.However,the overexpression of STIM1 in smooth muscle cells could significantly inhibit the opening of MPTPs and increase the expression of CypD in smooth muscle cells exposed to hypoxia and reoxygenation.（9）Intracellular Ca²⁺signal tests showed that compared with control group,the store-operated calcium influx in H24-R24 group was significantly increased（P<0.001）.Conclusion:Down-regulation of STIM1 contribute to the decreased mesenteric artery vascular responsiveness in rats with severe hemorrhagic shock,overexpression of STIM1 can improve vascular reactivity by inhibiting the opening of MPTPs in smooth muscle cells caused by hypoxia and reoxygenation.