ABSTRACT
Objective To investigate the effects of heat shock protein Gp96 on alcoholic liver fibrosis in mice. Methods A total of 220 male healthy C57BL/6 J mice were randomly divided into four groups; normal control group (n = 10), saline+alcohol induced liver fibrosis group (n = 70), the injection of CRISPR expression Gp96-sgRNA3 by tail vein+ alcohol induced liver fibrosis group (n = 70), the intraperitoneal injection of nuclear factor kappa B(NF-κB) inhibitors PDTC+alcohol induced liver fibrosis group (n = 70). The blood was got from eyeballs and the mice were killed after 8 weeks of ethanol induction. We detected the activity of serum aspartate aminotransferase (AST) in mice of different groups. The pathological changes were detected by HE staining, sirius red staining and periodic acid-Schiff (PAS) staining in the liver of mice. The expression of Gp96 and transforming growth factor βl ( TGF-βl ) were detected by Western blotting. Results Compared with the normal control group, the AST enzyme activity and liver fibrosis increased significantly, glycogen decreased significantly in other three groups (P<0.01). Compared with the saline+alcohol group, the AST enzyme activity and liver fibrosis increased more significantly, glycogen decreased more significantly, Gp96 expression decreased significantly and TGF-βl expression increased significantly in Gp96-sgRNA3+ alcohol group and NF-κB inhibitors PDTC+ alcohol group (P<0.01 or P<0.05). Conclusion The injection of CRISPR expression plasmid Gp96-sgRNA3 by tail vein significantly inhibited the Gp96 expression, promoted the degree of alcoholic liver fibrosis in mice, and NF-κB signaling pathway played a certain role in regulating the expression of Gp96.
ABSTRACT
Objective To investigate the role of Toll like receptor 4/nuclear factor-κB (TLR4/NF-κB) signal pathway on myocardial dysfunction after cardiac arrest-cardiopulmonary resuscitation (CA-CPR) in animal model. Methods Twenty-six pigs were randomly divided into sham group (n = 6), CA-CPR 12 hours group (n = 10) and CA-CPR 24 hours group (n = 10). The model of CA-CPR was reproduced by endocardial electrical stimulation for 8 minutes followed by CPR, and the pigs in sham group were only given anesthesia and tracheal intubation. The changes in hemodynamics including mean arterial pressure (MAP) and cardiac output (CO), as well as morphology and ultrastructure of myocardial cells were observed before and after CPR. The levels of serum tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were determined by enzyme linked immunosorbent assay (ELISA), and protein and mRNA expressions of TLR4/NF-κB in the myocardium were determined by Western Blot and real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR), respectively. Results Hemodynamic disturbance and myocardial serious injury were observed in CA-CPR groups. Compared with sham group, the levels of serum TNF-α were markedly increased 0.5 hour after return of spontaneous circulation (ROSC) in CA-CPR 12 hours and 24 hours groups (pg/L: 62.49±6.66, 48.39±2.37 vs. 10.75±0.74, both P < 0.05), and peaked at 2 hours (pg/L: 70.93±5.51, 66.03±2.60 vs. 10.87±0.91, both P < 0.05) followed by a gradual decline. The levels of serum IL-6 at 0.5 hours after ROSC in CA-CPR 12 hours and 24 hours groups were markedly higher than those of sham group (pg/L: 14.42±1.99, 11.23±1.12 vs. 8.75±0.74, both P < 0.05), and peaked at 12 hours (pg/L: 36.50±2.91, 38.15±1.26 vs. 8.88±0.62, both P < 0.05) followed by a gradual decline. The protein expressions of TLR4 and NF-κB in the myocardium were significantly increased in CA-CPR 12 hours and 24 hours groups as compared with sham group [TLR4 protein (gray value): 0.11±0.03, 0.24±0.05 vs. 0.05±0.02; NF-κB protein (gray value): 0.27±0.04, 0.24±0.03 vs. 0.09±0.02, all P < 0.05]. The mRNA levels of TLR4 in CA-CPR 12 hours and 24 hours groups were increased by approximately (9.93±1.07) folds and (9.21±1.27) folds of sham group respectively, and NF-κB mRNA expressions were increased by (4.44±0.96) folds and (6.09±0.81) folds of sham group respectively (all P < 0.01). Conclusion Activation of TLR4/NF-κB signal pathway may be one of the main pathological mechanisms of post resuscitation myocardial injury in a porcine model of CA-CPR.