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 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. 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-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). 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.
目的： 探讨外源性左旋肉碱对过度内质网应激介导的严重烫伤大鼠肝细胞焦亡的影响及其分子机制。 方法： 采用实验研究方法。将15只6~8周龄雌性SD大鼠按随机数字表法（分组方法下同）分为假伤组、单纯烫伤组、烫伤+肉碱组，每组5只，单纯烫伤组和烫伤+肉碱组大鼠背部制作30%体表总面积的Ⅲ度烫伤，其中烫伤+肉碱组大鼠另外给予腹腔注射左旋肉碱。伤后72 h，采用全自动生化仪检测肝损伤生物化学指标天冬氨酸转氨酶（AST）和丙氨酸转氨酶（ALT）水平，样本数为5。伤后72 h，采用苏木精-伊红染色观察肝组织损伤情况。伤后72 h，采用实时荧光定量反转录PCR（RT-qPCR）法检测肝组织中细胞焦亡相关标志物核苷酸结合寡聚化结构域蛋白样受体热蛋白结构域相关蛋白3（NLRP3）、胱天蛋白酶1（caspase-1）、消皮素D和白细胞介素1β（IL-1β）以及内质网应激相关标志物葡萄糖调节蛋白78（GRP78）、CCAAT/增强子结合蛋白同源蛋白（CHOP）的mRNA水平；采用蛋白质印迹法检测肝组织中GRP78、CHOP、NLRP3、caspase-1、caspase-1/p20、消皮素D-N、剪切型IL-1β蛋白表达水平，样本数均为5。取人肝癌细胞HepG2，分为二甲基亚砜（DMSO）组、0.1 μmol/L衣霉素组、0.2 μmol/L衣霉素组、0.4 μmol/L衣霉素组、0.8 μmol/L衣霉素组，分别作相应处理。培养24 h后，采用细胞计数试剂盒8法检测细胞活力并筛选衣霉素干预浓度（样本数为5）。取HepG2人肝癌细胞，分为DMSO组、单纯衣霉素组和衣霉素+肉碱组，分别作相应处理。培养24 h后，采用蛋白质印迹法检测细胞中GRP78、CHOP、NLRP3、caspase-1、caspase-1/p20、消皮素D-N、剪切型IL-1β蛋白表达水平，样本数均为3。对数据行单因素方差分析和LSD-t检验。 结果： 伤后72 h，单纯烫伤组大鼠血清中AST和ALT水平分别为（640±22）、（157±8）U/L，均分别明显高于假伤组的（106±13）、（42±6）U/L（t值分别为-46.78、-25.98，P<0.01）；烫伤+肉碱组大鼠血清中AST和ALT水平分别为（519±50）、（121±10）U/L，均明显低于单纯烫伤组（t值分别为4.93、6.06，P<0.01）。伤后72 h，假伤组大鼠肝组织形态基本正常，未见明显的炎症细胞浸润；与假伤组相比，单纯烫伤组大鼠肝组织可见大量的炎症细胞浸润，细胞排列紊乱；与单纯烫伤组相比，烫伤+肉碱组大鼠肝组织可见少量的炎症细胞浸润。伤后72 h，单纯烫伤组大鼠肝组织中NLRP3、caspase-1、消皮素D和IL-1β mRNA表达水平均明显高于假伤组（t值分别为34.42、41.93、30.17、15.68，P<0.01）；烫伤+肉碱组大鼠肝组织中NLRP3、caspase-1、消皮素D和IL-1β mRNA表达水平均明显低于单纯烫伤组（t值分别为34.40、37.20、19.95、7.88，P<0.01）。伤后72 h，与假伤组相比，单纯烫伤组大鼠肝组织中NLRP3、caspase-1、caspase-1/p20、消皮素D-N、剪切型IL-1β蛋白表达水平均明显升高（t值分别为12.28、26.92、5.20、10.02、24.78，P<0.01）；与单纯烫伤组相比，烫伤+肉碱组大鼠肝组织中NLRP3、caspase-1、caspase-1/p20、消皮素D-N、剪切型IL-1β蛋白表达水平均明显下降（t值分别为10.99、27.96、12.69、8.96、12.27，P<0.01）。伤后72 h，单纯烫伤组大鼠肝组织中GRP78、CHOP的mRNA水平均明显高于假伤组（t值分别为21.00、16.52，P<0.01），烫伤+肉碱组大鼠肝组织中GRP78、CHOP的mRNA水平均明显低于单纯烫伤组（t值分别为8.92、8.21，P<0.01）；单纯烫伤组大鼠肝组织中GRP78、CHOP的蛋白表达水平均明显高于假伤组（t值分别为22.50、14.29，P<0.01），烫伤+肉碱组大鼠肝组织中GRP78、CHOP的蛋白表达水平均明显低于单纯烫伤组（t值分别为14.29、5.33，P<0.01）。培养24 h后，与DMSO组相比，0.1 μmol/L衣霉素组、0.2 μmol/L衣霉素组、0.4 μmol/L衣霉素组、0.8 μmol/L衣霉素组细胞存活率均明显下降（t值分别为4.90、9.35、18.64、25.09，P<0.01）；选择0.8 μmol/L作为后续衣霉素的干预浓度。培养24 h后，与DMSO组相比，单纯衣霉素组细胞GRP78、CHOP的蛋白表达水平均明显升高（t值分别为10.48、17.67，P<0.01）；与单纯衣霉素组相比，衣霉素+肉碱组细胞GRP78、CHOP的蛋白表达水平均明显下降（t值分别为8.08、13.23，P<0.05或P<0.01）。培养24 h后，与DMSO组相比，单纯衣霉素组细胞NLRP3、消皮素D-N蛋白表达水平均明显升高（t值分别为13.44、27.51，P<0.01），caspase-1、caspase-1/p20、剪切型IL-1β蛋白表达水平均无明显变化（P>0.05）；与单纯衣霉素组相比，衣霉素+肉碱组细胞NLRP3、消皮素D-N蛋白表达水平均明显下降（t值分别为20.49、21.95，P<0.01），caspase-1、caspase-1/p20、剪切型IL-1β蛋白表达水平均无明显变化（P>0.05）。 结论： 在严重烫伤大鼠中，外源性左旋肉碱可能通过抑制过度内质网应激介导的细胞焦亡相关通路发挥对肝损伤的保护作用。.