ObjectiveTo investigate the effects and molecular mechanism of exogenous L-carnitine on hepatic pyroptosis mediated by excessive endoplasmic reticulum stress in severely scald rats.
MethodsThe experimental research method was adopted. According to the random number table (the same group method below), fifteen female Sprague Dawley rats aged 6-8 weeks were divided into sham-injury group, scald alone group, and scald+carnitine group (with 5 rats in each group), and full-thickness scald of 30% total body surface area were made on the back of rats in scald alone group and scald+carnitine group, and rats in scald+carnitine group were additionally given intraperitoneal injection of L-carnitine. At post injury hour (PIH) 72, The levels of aspartate aminotransferase (AST) and alanine dehydrogenase (ALT) of biochemical indicators of liver injury were detected by automatic biochemical analyzer with the sample number of 5. At PIH 72, liver tissue damage was detected by hematoxylin-eosin staining. At PIH 72, The mRNA levels of nucleotide-binding oligomerization domain-containing protein-like receptor family pyrin domain containing 3 (NLRP3), cysteine aspartic acid specific protease 1 (caspase-1), gasderminD (GSDMD), and interleukin 1β(IL-1β) in liver tissue as pyroptosis-related markers and glucose regulatory protein 78 (GRP78) and CCAAT/enhancer binding protein homologous protein (CHOP) in liver tissue as endoplasmic reticulum stress-related markers were detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction (RT-qPCR). Protein expression levels of GRP78, CHOP, NLRP3, caspase-1, caspase-1/p20, GSDMD-N, and cleaved IL-1β in liver tissue were detected by Western blotting, and the sample numbers were all 5. HepG2 cells as human liver cancer cells were divided into dimethyl sulfoxide (DMSO) group, 0.1 μmol/L tunicamycin (TM) group, 0.2 μmol/L TM group, 0.4 μmol/L TM group, and 0.8 μmol/L TM group and were treated accordingly. After 24 h of culture, cell viability was detected by cell counting kit 8, and the intervention concentration of TM was screened, and the sample number was 5. HepG2 cells were divided into DMSO group, TM alone group, and TM+carnitine group, and treated accordingly. After 24 h of culture, the protein expression levels of GRP78, CHOP, NLRP3, caspase-1, caspase-1/p20, GSDMD-N, and cleaved IL-1β in cells were detected by Western blotting, and the sample numbers were all 3. Data were statistically analyzed with one-way analysis of variance and least significant difference-t test.
ResultsAt PIH 72, the AST and ALT levels of serum in scald alone group were (640±22) and (157±8) U/L, which were significantly higher than (106±13) and (42±6) U/L in sham-injury group, respectively, with t values of -46.78 and -25.98, respectively, P<0.01. The AST and ALT levels of serum in scald+carnitine group were (519±50) and (121±10) U/L, which were significantly lower than those in scald alone group, respectively, with t values of 4.93 and 6.06, respectively, P<0.01. At PIH 72, the morphology of liver tissue of rats in sham-injury group were basically normal with no obvious inflammatory cell infiltration; compared with those in sham-injury group, the liver tissue of rats in scald alone group showed a large number of inflammatory cell infiltration and disturbed cell arrangement; compared with that in scald alone group, the liver tissue of rats in scald+carnitine group showed a small amount of inflammatory cell infiltration. At PIH 72, the mRNA expression on levels of NLRP3, caspase-1, GSDMD, and IL-1β in liver tissue of rats in scald alone group were significantly higher than those in sham-injury group (with t values of 34.42, 41.93, 30.17, and 15.68, respectively, P<0.01); the mRNA levels of NLRP3, caspase-1, GSDMD, and IL-1β in liver tissue of rats in scald+carnitine group were significantly lower than those in scald alone group (with t values of 34.40, 37.20, 19.95, and 7.88, respectively, P<0.01). At PIH 72, the protein expression levels of NLRP3, caspase-1, caspase-1/p20, GSDMD-N, and cleaved IL-1β in liver tissue of rats in scald alone group were significantly higher than those in sham-injury group (with t values of 12.28, 26.92, 5.20, 10.02, and 24.78, respectively, P<0.01); compared with those in scald alone group, the protein expression levels of NLRP3, caspase-1, caspase-1/p20, GSDMD-N, and cleaved IL-1β in liver tissue of rats in scald+carnitine group were significantly decreased (with t values of 10.99, 27.96, 12.69, 8.96, and 12.27, respectively, P<0.01). At PIH 72, the mRNA levels of GRP78 and CHOP in liver tissue of rats in scald alone group were significantly higher than those in sham-injury group (with t values of 21.00 and 16.52, respectively, P<0.01), and the mRNA levels of GRP78 and CHOP in liver tissue of rats in scald+carnitine group were significantly lower than those in scald alone group (with t values of 8.92 and 8.21, respectively, P<0.01); the protein expression levels of GRP78 and CHOP in liver tissue of rats in scald alone group were significantly higher than those in sham-injury group (with t values of 22.50 and 14.29, respectively, P<0.01), and the protein expression levels of GRP78 and CHOP in liver tissue of rats in scald+carnitine group were significantly lower than those in scald alone group (with t values of 14.29 and 5.33 respectively, P<0.01). After 24 h of culture, the cell survival rates of 0.1 μmol/L TM group, 0.2 μmol/L TM group, 0.4 μmol/L TM group, and 0.8 μmol/L TM group were significantly decreased than that in DMSO group (with t values of 4.90, 9.35, 18.64, and 25.09, respectively, P<0.01). Then 0.8 μmol/L was selected as the intervention concentration of TM. After 24 h of culture, compared with that in DMSO group, the protein expression levels of GRP78 and CHOP in cells in TM alone group were significantly increased (with t values of 10.48 and 17.67, respectively, P<0.01), and the protein expression levels of GRP78 and CHOP in TM+carnitine group were significantly lower than those in TM alone group (with t values of 8.08 and 13.23, respectively, P<0.05 or P<0.01). After 24 h of culture, compared with those in DMSO group, the protein expression levels of NLRP3 and GSDMD-N in cells in TM alone group were significantly increased (with t values of 13.44 and 27.51, respectively, P<0.01), but the protein expression levels of caspase-1, caspase-1/p20, and cleaved IL-1β in cells were not significantly changed (P>0.05); compared with that in TM alone group, the protein expression levels of NLRP3 and GSDMD-N in cells in TM+carnitine group were significantly decreased (with t values of 20.49 and 21.95, respectively, P<0.01), but the protein expression levels of caspase-1, caspase-1/p20, and cleaved IL-1β in cells were not significantly changed (P>0.05).
ConclusionsIn severely scald rats, exogenous L-carnitine may play a protective role against liver injury by inhibiting the pathways related to excessive endoplasmic reticulum stress-mediated pyroptosis.