The Effective Mechanism Of SZR Decoction And JUA On Myocardial Cell Apoptosis Induced By Simulated Transport Stress In Rats And H9C2Cells

Transport stress seriously affect the health of livestock and poultry, which also causes enormous losses. The present study investigated the effects of Suanzaoren decoction (SZR) and its main active component on rat myocardial tissue and cell injury induced by simulated transport stress. Firstly, a constant temperature (60rpm,35℃,2h/d, off food and water) was used to simulate rats to mimic the highway transportation in the summer. Clinical manifestations, hormone levels, physiological and biochemical, cardiac function index and heat shock proteins (HSP27/70/90) were used for a success model evaluation. Morphological microstructure and ultrastructure damage were observed with Scanning electron microscopy (SEM) and Transmission electron microscope (TEM) after simulated transport stress. Terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) staining was used to detect the cardiac cell apoptosis. The further mechanism of cardiac cell apoptosis induced by simulated transport stress was discussed in gene level with RT-PCR and signal protein expression with western bloting, also the effect of SZR decoction on it was detected. Afterwards, In vitro, one of the stress hormones norepinephrine (NE) was used to stimulate rat myocardial cells (H9C2), which was evaluated by cell vitality detection with MTT, cell apoptosis assay with flow cytometry and cell morphology observaion with SEM and TEM. DNA microarray and microRNA deep sequencing analysis was used to dicuss the potential mechanism of myocardial cell apoptosis induced by NE, then, the exact mechanism of related microRNA and signal pathway and effect of JUA were detected. Results are as follows:1. The simulated transport treatment induced obvious stress responses with significant decreases in body weight, remarkable increases in rectal temperature, serum corticosterone (CORT), blood glucose (GLU), epinephrine (E) and norepinephrine (NE), as well as expression of HSP27Y70\90mRNA in rat. Continuous transport stress especially in S3d group, the level of serum cardiac troponinT (CTnT), creatine kinase (CK) and lactate dehydrogenase (LDH) was elevated than other groups, which indicated an obvious myocardial damage in rat after simulated transport stress.2. The result showed simulated transport stress enhanced phosphorylation of ASK-1, JNK, P38, ERK and AKT in rat myocardial tissue, indicating that the mitogen-activated protein kinase (MAPK) pathway and AKT pathway were notably activated after stress. While the balance of pro-apoptsis signal and anti-apoptosis signal was destroyed by continuous intensity stress, and then the mitochondrial apoptotic pathway was promoted, the cardic cell eventually inclined to apoptosis.3, SZR decoction played an obvious role in decreasing the serum NE, CK, LDH and the content of CTnT elevation induced by transport stress in rats, also droping the ratio of Bax/Bcl-2, then inhibiting myocardial cell apoptosis, reducing myocardial injury.4, NE induced apoptosis by altering microRNA-325-5p, enhancing the Bax-to-Bcl-2ratio, and MAPK signals in H9C2cells. These changes were attenuated by JUA, the anti-apoptotic effect of JUA was due to its inhibitory effect on the activation of p38/c-Jun N-terminal kinase (JNK) and on the mitochondrial apoptotic pathway activated by NE, and was also attributed to promoting the expression of ERK and AKT signal pathway.Conclusion: The simulated transport treatment caused serious stress responce in rats, and induced myocardial cell apoptosis via influencing MAPK and AKT signaling pathway, also promoted mitochondrial apoptosis pathway, SZR played an obvious protective role in reducing myocardial injury induced by simulation transport stress in rats. NE, one of the hermons that stimulated after transport stress, induced H9C2cell apoptosis, the mechanism of which was similar with that in vivo, also microRNA-325-5p could promote H9C2cell apoptosis induced by NE. The anti-apoptotic effect of JUA was due to its inhibitory effect on the activation of p38, JNK and on the mitochondrial apoptotic pathway activated by NE, and was also attributed to promoting the expression of ERK and AKT signal pathway..