| Objective:1.To observe the levels of GRP78 and GRP78 acetylation in myocardial tissue of traumatized rats in vivo,and to explore the relationship between GRP78 acetylation and myocardial apoptosis.2.To further analyze the effect of acetylation of GRP78 on apoptosis of injured cardiomyocytes in vitro,and to explore the possible mechanisms.3.To investigate the effect of GRP78 acetylation on GRP78 expression in cardiomyocytes after trauma in vitro.Methods:1.In vivo level: Thirty-six male rats aged 6-8 weeks and weighing 180-200 g were equally randomized to a sham-trauma group(Sham group)and 5 trauma groups at 0,3,6,12 and 24 h post-trauma.After the rats were anesthetized intraperitoneally,the trauma rat model was prepared by Noble-Collip trauma instrument,and the sham trauma rat model was prepared by fixing the rats with tape in the trauma instrument.1.1 The expression of GRP78,caspase-3,HDAC6 and P300 protein were detected with western blot in rats myocardial tissue.1.2 The m RNA levels of GRP78,HDAC6,and P300 were determined with real-time PCR in rats myocardial tissue.1.3 The expression levels of GRP78,HDAC6,and P300 were processed with immunohistochemical in rats myocardial tissue.1.4 The level of GRP78 acetylation was detected with immunoprecipitation in rats myocardial tissue.1.5 The level of myocardial apoptosis was detected with TUNEL staining in rats myocardial tissue.2.In vitro level:2.1 H9C2 cardiomyocytes were cultured in Dulbecco’s Modified Eagle Medium(DMEM)containing 10% fetal bovine serum,which was used as sham trauma group.Rat serum collected at 12 h post-trauma was used instead of FBS and cultured for 6,12 and24 h as the trauma groups.Identify the most obvious time point of trauma by that experiment described above.2.1.1 The expression of caspase-3 protein was detected with western blot in H9C2 cardiomyocytes.2.1.2 H9C2 cardiomyocytes apoptosis was determined with flow cytometry.2.2 Cardiomyocytes in trauma groups were cultured with 0.5 μM Trichostatin A and 10 μM ITSA-1 for 24 h(Most obvious trauma).2.2.1 Acetylation level of GRP78 was detected with immunoprecipitation in H9C2 cardiomyocytes.2.2.2 H9C2 cardiomyocytes apoptosis was determined with flow cytometry.2.2.3 The expression of caspase-3 and GRP78 protein were determined with western blot in H9C2 cardiomyocytes.2.2.4 The expression levels of caspase-3 and GRP78 were detected with immunofluorescence in H9C2 cardiomyocytes.2.2.5 The m RNA level of GRP78 was processed with real-time PCR in H9C2 cardiomyocytes.Results:1.Apoptosis of traumatic cardiomyocytes induced by protracted ERS is correlated with the level of GRP78 expression and acetylation.1.1 GRP78 expression in rat myocardial tissue was significantly increased at 6 h after trauma.Western blot results showed that GRP78 protein expression began to increase at 3 h after trauma and reached the highest level at 6 h(P < 0.05),and immunohistochemical staining positive spots also appeared the most at 6 h(P < 0.05),and real-time PCR results showed that GRP78 m RNA was significantly increased at 3 h after trauma(P <0.05)and peaked at 6 h(P < 0.05).1.2 The acetylation level of GRP78 in rat myocardial tissue increased significantly at 6 h and 12 h after trauma.Immunocoprecipitation showed that the acetylation level of GRP78 in myocardial tissue increased significantly at 6 h and 12 h after trauma(P < 0.05).1.3 Myocardial tissue of rats showed significant apoptosis at 6 h and 12 h after trauma.Western blot results showed that caspase-3 protein expression peaked at 12 h after trauma,and TNNEL staining positive spots also appeared most at 12 h(P < 0.05).2.There exists a dynamic balance between HAT and HDAC during the ERS process.2.1 HDAC6 increased at 3 h after trauma and subsequently decreased.Western blot results showed that the protein expression level of HDAC6 in myocardial tissue of traumatized rats showed a peak at 3 h after trauma(P < 0.05),and immunohistochemical staining results showed the same phenomenon(P < 0.05),and real-time PCR results showed that the expression level of HDAC6 m RNA in myocardial tissue began to increase immediately after trauma and peaked at 3 h(P < 0.05).2.2 P300 was significantly increased at 6 h and 12 h after trauma.Western blot and immunohistochemical staining results showed that the protein expression of P300 in myocardial tissue of traumatized rats was significantly increased at6 h and 12 h after trauma(P < 0.05),and P300 m RNA expression peaked at 6 h(P <0.05).2.3 With the lapse of time,the expression of HDAC6 and P300 exhibited an ebb-and-flow trend.Western blot and immunohistochemical staining results showed that HDAC6 expression increased at 3 h and decreased significantly at 6 h and 12 h after trauma(P <0.05);P300 reached the highest peak value at 6 h and 12 h after trauma(P < 0.05).Thus,With the lapse of time,the expression of HDAC6 and P300 exhibited an ebb-and-flow trend.3.Inhibition of HDAC function promotes apoptosis of traumatic cardiomyocytes by increasing the acetylation level of GRP78.3.1 Apoptosis was most evident in H9C2 cardiomyocytes cultured in trauma serum for 24 h.The isolated myocardial trauma model was successfully established by culturing H9C2 cells with traumatized rat serum.Western blot results showed that cardiomyocyte apoptosis was most pronounced after 24 h of culture(P < 0.05),and flow cytometry results showed that early apoptotic cells were significantly increased.3.2 Inhibition of HDAC significantly increased the level of acetylation of GRP78.Immunoprecipitation analysis showed that the acetylation level of GRP78 was significantly increased in the HDAC inhibitor Trichostatin A(TSA)group(P < 0.05),while it was decreased in the HDAC agonist ITSA-1 group(P < 0.05).3.3Inhibition of HDAC significantly increases the apoptosis of H9C2 cardiomyocytes.Flow cytometry analysis revealed that early apoptotic cells accounted for more than half(73%)of the total number of cells in the TSA group,while the level of apoptosis in the ITSA-1 group decreased compared with the trauma group(6.6%).Western blot and immunofluorescence staining showed that caspase-3 expression level was significantly higher in the TSA group than in the control group(P < 0.05).4.Elevated levels of acetylation of GRP78 decrease GRP78 protein expression,but did not affect GRP78 m RNA expression.Western blot results showed that the increase of GRP78 acetylation level resulted in a significant decrease in GRP78 protein expression level(P < 0.05)and a significant decrease in the number of immunofluorescent staining positive cells(P < 0.05)after TSA inhibited HDAC function.Real-time PCR showed that increasing levels of GRP78 acetylationdid not affect GRP78 m RNA expression(P > 0.05)after HDAC function was inhibited by TSA.Conclusion:With time of ERS prolonging after trauma,the increased acetylation level of GRP78 promoted cardiomyocyte apoptosis.And the decrease of GRP78 protein expression level may be related to the increase of its acetylation level,but does not affect its transcription level. |
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