Role Of Endoplasmic Reticulum Stress In Cardiomyocyte Apoptosis Induced By Mechanical Trauma

Mechanical trauma refers to the destruction of the structural integrity of human tissues and organs or the disorders of physiological functions caused by various mechanical injury factors.It is well known that severe mechanical trauma can directly lead to failure of vital organs such as heart?cardiac rupture and coronary dissection?,brain?cerebral hemorrhage,skull fracture?,etc.It can also cause secondary injury of multiple parts and organs through various injury factors.It is reported that about 6million people in the world suffer from mechanical trauma every year,while young people account for more than 50%.Therefore,it is of great clinical significance to prevent and treat mechanical trauma.It has been reported that some patients with systemic mechanical trauma do not have direct heart damage after 24 hours hospital admission,but have heart disease such as cardiac dysfunction,arrhythmia,heart failure,and even myocardial infarction several days or weeks after discharge.The trauma-induced secondary cardiac injury?TISCI?is occult and easily missed,which seriously threatens the health and safety of patients.It has been concerned by more and more domestic and foreign scholars.So far,the mechanism is not yet clear.Apoptosis of cardiomyocytes is an important cause of TISCI.However,it remains unclear that how mechanical trauma causes myocardial cell apoptosis and subsequent secondary heart damage.It is generally believed that apoptosis is the result of a cascade of caspases,and there are mainly three apoptotic pathways including Caspase-8 mediated death receptor pathway,Caspase-9 mediated mitochondrial apoptosis pathway and Caspase-12 mediated endoplasmic reticulum stress pathway.These three pathways eventually activate Caspase-3,and thus lead to apoptosis.Our previous study found that myocardium Caspase-12 was activated in the early stage of mechanical trauma by detecting Caspase-8,9,12 activities in the myocardium of rats.However,it is not clear whether TISCI activates endoplasmic reticulum and further induces endoplasmic reticulum stress apoptosis pathway.Endoplasmic reticulum?ER?is an important organelle in eukaryotic cells and plays an important role in maintaining Ca²⁺homeostasis,lipid synthesis,protein folding and transport.Disruption of the endoplasmic reticulum homeostasis leads to endoplasmic reticulum stress?ERS?.A certain degree of ERS activates the unfolded protein response?UPR?,which increases the degradation of associated protein,restores normal physiological function of the endoplasmic reticulum and promotes cell survival.In contrast,excessive or sustained ERS initiates ERS apoptotic signaling pathway.Therefore,this study aims to investigate the role of endoplasmic reticulum stress in mechanical trauma-induced cardiomyocyte apoptosis by establishing in vivo and in vitro research models and using drug interventions,which would be helpful in finding new targets for clinical prevention and treatment of TISCI,and providing new directions and ideas for effectively reducing mechanical traumatic mortality.Objective:To observe the role of endoplasmic reticulum stress in cardiomyocyte apoptosis induced by mechanical trauma from both in vivo and in vitro levels.Methods:1.Experimental animals and grouping:Male SD rats of 180-220 g were randomly divided into sham group?Sham?,trauma group?Trauma?and trauma+ERS inhibitor intervention group?Trauma+4-PBA?.Traumatic rats were divided into five groups,including 0,3,6,12 and 24 hours after trauma?n=8/group?.TISCI rat model was established using a Noble-Collip drum.2.Experimental cells and grouping:H9c2 cardiomyocytes were randomly divided into normal serum culture group?Control?,sham serum culture group?Sham?,trauma serum culture group?Trauma?and trauma serum+ERS inhibitor intervention group?Trauma+4-PBA?.In the trauma serum culture group,abdominal aortic blood was taken from the rats of 3 h,6 h,12 h,and 24 h after trauma group.The trauma serum was disinfected with a 0.22?mM ILLEX?GP disposable needle filter and then added to DMEM medium.Myocardial injury model induced by mechanical trauma serum was established by culturing H9c2 cardiomyocytes for 12 hours using trauma serum.3.In vivo experiments methods:ERS markers GRP78,PERK,p-PERK,IRE1?,p-IRE1?,ATF6,and ERS-related apoptosis proteins CHOP,Caspase-12,Caspase-3,Cleaved-Caspase-3 protein expression were detected by immunohistochemistry and Western blot.Caspase-3 activity was detected by Caspase-3 activity kit.4.In vitro experiments method:The viability of H9c2 cardiomyocytes was detected by CCK-8 kit.ERS markers GRP78,PERK,p-PERK,IRE1?,p-IRE1?,ATF6,and ERS-related apoptosis protein CHOP,Caspase-12,Caspase-3,and Cleaved-Caspase-3protein was detected by Western blot.The cardiomyocyte apoptosis was detected by Hoechst 33258 staining and flow cytometry.Results:1.Endoplasmic reticulum stress was an important cause of mechanical trauma inducing myocardial cell apoptosis in rats.1.1 Mechanical trauma increased the expression of ERS-related proteins GRP78,p-PERK,p-IRE1?and ATF6 in heart of ratsThe results of immunohistochemistry showed that the positive staining granules of GRP78,p-PERK,p-IRE1?,and ATF6 were higher than the sham group.Western blot results showed that compared with the trauma group,the expression of GRP78,p-PERK and p-IRE1?in the myocardium began to increase 3 h after mechanical trauma,and reached the maximum at 6 h?P<0.01?.While the expression of ATF6 was significantly increased at 6 h after mechanical trauma?P<0.05?,and reached its peak at 12 h?P<0.01?.1.2 Mechanical trauma elevated the expression of ERS-associated apoptosis protein CHOP,Caspase-12,and Caspase-3 in rat heartsThe results of immunohistochemistry showed that the positive staining granules of CHOP,Caspase-12 and Caspase-3 in the trauma group were higher than those in the sham group.Western blot results showed that compared with the sham group,the expression of myocardial CHOP and Caspase-3 protein began to increase at 6 h after trauma?P<0.05?,and the expression of Caspase-12 protein reached the highest at this time point?P<0.01?.1.3 ERS inhibitor 4-PBA reduced the expression of myocardial CHOP and Caspase-12 in rats with mechanical traumaIn this experiment,6h after trauma was selected as the observation time point.Rats were injected intraperitoneally with 100 mg/kg 4-PBA.And the expression of CHOP and Caspase-12 was detected by immunohistochemistry and Western blot.CHOP and Caspase-12 positive staining particles were reduced after intervention with 4-PBA inhibitor.The protein expression levels of CHOP and Caspase-12 were also decreased in myocardial tissue?P<0.05?.1.4 ERS Inhibitor 4-PBA reduced cardiomyocyte apoptosis in rats with mechanical traumaAfter treatment with ERS inhibitor 4-PBA,cardiomyocyte apoptosis was detected by immunohistochemistry,Western blot and Caspase-3 activity kits.The results showed that the positive staining granules of Caspase-3 in the trauma group were significantly increased compared with the sham group,and the protein expression?P<0.05?and activity?P<0.05?of Caspase-3 were also elevated.After the inhibitor 4-PBA intervention,compared with the trauma group,the positive staining of Caspase-3 was significantly reduced,and its protein expression and activity level also significantly decreased?P<0.05?.2.Endoplasmic reticulum stress is an important cause of mechanical trauma serum inducing cardiomyocyte apoptosis2.1 Establishment of cardiomyocyte injury model with traumatic serumThe H9c2 cardiomyocytes were cultured with fetal bovine serum,and the serum of the sham rats and 3 h,6 h after trauma rats were respectively added to DMEM medium.After cultured for 1 h,3 h,6 h,12 h,24 h,and 48 h,the cell viability was measured by CCK-8 method.The results showed that compared with the sham serum culture group,the viability of H9c2 cells significantly decreased at 12 h?P<0.01?after cultured with 3and 6 h traumatic serum.The survival rate of cells at 24 h and 48 h was significantly decreased,but there was no significant difference compared with 12 h?P>0.05?.The above results suggest that H9c2 cardiomyocytes were cultured at 3 h and 6 h after trauma at 1 h,3 h,6 h,12 h,24 h and 48 h,and they were found to have cell survival at 12 h of culture time.Therefore,H9c2 cardiomyocytes was cultured for 12 hours in the following study.2.2 The expressions of ERS-related proteins GRP78,p-PERK,p-IRE1?,and ATF6in H9c2 cells increased after cultured with traumatic serum at different time pointsWestern blot analysis showed that compared with the trauma serum culture group,the expression of GRP78 protein,p-PERK and p-IRE1?was highest in the H9c2cardiomyocytes after cultured with serum at 6 h after trauma?P<0.01?.The level of ATF6protein was highest in H9c2 cardiomyocytes after cultured with serum at 12 h after trauma?P<0.01?.2.3 The expressions of ERS-associated apoptotic proteins CHOP,Caspase-12,and Caspase-3 in H9c2 cells increased after cultured with traumatic serum at different time pointsWestern blot analysis showed that compared with the sham serum culture group,the expression of CHOP and Caspase-3 protein in H9c2 cardiomyocytes reached a peak cultured with serum at 12 h after trauma?P<0.01?.The expression of Caspase-12 protein was highest in H9c2 cardiomyocytes cultured with serum at 6 h after trauma?P<0.01?.2.4 After pretreatment with inhibitor 4-PBA,the expression of CHOP and Caspase-12 reduced in H9c2 cardiomyocytes cultured with traumatic serumAccording to in vivo study results,after pretreatment with 4-PBA?5 mM?,H9c2cardiomyocytes were cultured with serum at 6 h after trauma for 12 h and detected by Western blot.The results showed that compared with the sham serum culture group,the expression of CHOP and Caspase-12 protein increased in the traumatic serum culture group?P<0.05?.After the intervention with the inhibitor 4-PBA,the expression of CHOP and Caspase-12 protein decreased?P<0.05?.2.5 After pretreatment with inhibitor 4-PBA,cardiomyocyte apoptosis decreased in H9c2 cardiomyocytes cultured with traumatic serumAfter administration of 4-PBA in vitro,cardiomyocyte apoptosis was detected by Western blot,Hoechst 33258 staining and flow cytometry.The results showed that compared with the sham serum culture group,cardiomyocyte apoptosis significantly increased?P<0.05?.in the traumatic serum culture group.After the intervention with the inhibitor 4-PBA,cardiomyocyte apoptosis obviously decreased?P<0.05?.Conclusion:1.Mechanical trauma can promote the occurrence of myocardial ERS,and activate the ERS apoptotic pathway to induce cardiomyocyte apoptosis.2.ERS plays an important role in the process of myocardial cell apoptosis induced by mechanical trauma.