[Effects and mechanism of diammonium glycyrrhizinate on liver injury in severely scalded rats].

Objective: To investigate the effects and mechanism of diammonium glycyrrhizinate (DG) on liver injury in severely scalded rats. Methods: The experimental research method was used. Fifty-four female Sprague-Dawley rats aged 7-9 weeks were divided into sham injury group with simulated injury on the back, and simple scald group and scald+DG group with scald of 30% total body surface area on the back, with 18 rats in each group. Rats in sham injury group were not specially treated after injury, and rats in simple scald group and scald+DG group were rehydrated for antishock. Besides, rats in scald+DG group were injected intraperitoneally with 50 mg/kg DG at post injury hour (PIH) 1, 25, and 49. Rats in the three groups were collected, the serum content of liver function injury related indexes including aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), total protein, and albumin was measured by automatic biochemical assay analyzer, and serum content of ornithine carbamoyl transferase (OCT) was measured by enzyme-linked immunosorbent assay method at PIH 24, 48, and 72; hepatic histopathological changes at PIH 72 were observed by hematoxylin-eosin staining; the mRNA expressions of B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), glucose regulated protein 78 (GRP78), activating transcription factor 4 (ATF4), and protein kinase R-like endoplasmic reticulum kinase (PERK) in liver tissue were detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction at PIH 24, 48, and 72. The protein expressions of Bcl-2, Bax, GRP78, PERK, and ATF4 in liver tissue were detected by Western blotting at PIH 72 in sham injury group and PIH 24, 48, and 72 in simple scald group and scald+DG group. The number of samples was 6 in each group at each time point. Data were statistically analyzed with analysis of variance for factorial design, one-way analysis of variance, and Bonferroni test. Results: Compared with that in sham injury group, the serum content of AST, ALT, and LDH was significantly increased (P<0.01), and the serum content of total protein and albumin was significantly decreased (P<0.05 or P<0.01) of rats in simple scald group at all post-injury time points. Compared with those in simple scald group, the serum AST content of rats in scald+DG group at PIH 24 was decreased significantly (P<0.05); the serum AST, ALT, and LDH content of rats in scald+DG group at PIH 48 was decreased significantly (P<0.01), and the serum total protein content was increased significantly (P<0.01); the serum AST, ALT, and LDH content of rats in scald+DG group at PIH 72 was decreased significantly (P<0.01), and the serum total protein and albumin content was increased significantly (P<0.01). At PIH 24, 48, and 72, the serum OCT content of rats in simple scald group was (48.5±3.9), (40.8±2.4), and (38.7±2.0) U/L, which was significantly higher than (15.1±2.5), (15.7±2.6), and (16.4±3.7) U/L in sham injury group (P<0.01), and (39.0±4.5), (31.8±2.0), and (22.1±2.6) U/L in scald+DG group (P<0.05 or P<0.01). At PIH 72, the cells in liver tissue of rats in sham injury group had normal morphology and regular arrangement, with no obvious inflammatory cell infiltration; the cells in liver tissue of rats in simple scald group had disordered arrangement, diffuse steatosis, and moderate inflammatory cell infiltration; the cells in liver tissue of rats in scald+DG group arranged regularly, with scattered steatosis and a small amount of inflammatory cell infiltration. Compared with those in sham injury group, the Bcl-2 mRNA (P<0.05 or P<0.01) and protein expressions of liver tissue were significantly decreased, and the mRNA (P<0.01) and protein expressions of Bax were significantly increased in rats in simple scald group at PIH 24, 48, and 72. Compared with those in simple scald group, the mRNA (P<0.05) and protein expressions of Bax in liver tissue of rats in scald+DG group were decreased significantly at PIH 48; the mRNA (P<0.01) and protein expressions of Bax in liver tissue of rats in scald+DG group were significantly decreased, and the mRNA (P<0.01) and protein expressions of Bcl-2 were significantly increased at PIH 72. Compared with those in sham injury group, the mRNA (P<0.05 or P<0.01) and protein expressions of ATF4, GRP78, and PERK in liver tissue were significantly increased in rats in simple scald group at all post-injury time points. Compared with those in simple scald group, the mRNA (P<0.01) and protein expressions of ATF4 in liver tissue of rats in scald+DG group at PIH 48 were significantly decreased, and the mRNA (P<0.05 or P<0.01) and protein expressions of ATF4, GRP78, and PERK were significantly decreased in liver tissue of rats in scald+DG group at PIH 72. Conclusions: DG can effectively reduce the degree of liver injury in rats after severe scald, and the mechanism may involve alleviating endoplasmic reticulum stress and mitigating mitochondrial damage.

[Effects and molecular mechanism of exogenous L-carnitine on excessive endoplasmic reticulum stress-mediated hepatic pyroptosis in severely scald rats].

Objective: To investigate the effects and molecular mechanism of exogenous L-carnitine on hepatic pyroptosis mediated by excessive endoplasmic reticulum stress in severely scald rats. Methods: The 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 1ß(IL-1ß) 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-1ß 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-1ß 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. Results: At 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-1ß 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-1ß 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-1ß 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-1ß 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-1ß 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-1ß in cells were not significantly changed (P>0.05). Conclusions: In 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.

Cytokine gene expression in the livers of ducklings infected with duck hepatitis virus-1 JX strain.

Duck hepatitis virus type 1 (DHV-1) causes a highly contagious disease in ducklings and is often associated with liver necrosis, hemorrhages, and high mortality. In the current study, the expression levels of gene transcripts encoding proinflammatory cytokines and the virus were measured by quantitative reverse-transcription PCR in duck livers after infection with a DHV-1 JX isolate obtained from natural cases in Hubei Province, China. In addition, sera IL-1ß, IL-6, and alanine aminotransferase levels were quantified. Liver histopathology was examined following DHV-1 infection. The ducklings died within 1 to 2 d postinfection (d.p.i.) because of typical liver degeneration, hemorrhage, necrosis, and bile-duct epithelial cell proliferation. Transcripts of the cytokines IFN-α, IL-6, TNF-α, and IL-10 decreased by 0.5 d.p.i. and then gradually increased at 1 d.p.i. Similarly, DHV-1 JX 3D gene levels in the liver sharply increased at 1 d.p.i. and then maintained a high level. In contrast, liver TNF-α and IL-1ß transcripts showed no increased expression of the cytokine gene postinfection and significantly decreased compared with the expression at 0.25 d.p.i., only the expression of IFN-α transcripts increased 128-fold by 1 d.p.i. Changes in the serum IL-6 level remained relatively stable postinfection and not significantly different compared with that of the control (P > 0.05), whereas serum levels of IL-1ß significantly decreased at 0.5 d.p.i. and increased from 1 d.p.i. onwards (P < 0.05). Serum alanine aminotransferase levels significantly increased 2 d.p.i. compared with that of the control group (P < 0.01), which seemed to keep with the number of dead ducks. The cytokines exhibited a biphasic pattern following DHV-1 JX infection. Taken together, the data indicated that duckling liver inflammatory responses were produced following experimental DHV-1 JX infection involving multiple cytokines.