Effects and molecular mechanism of exogenous L-carnitine on excessive endoplasmic reticulum stress-mediated hepatic pyroptosis in severely scald rats
-
摘要:
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。 Abstract: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. -
Key words:
- Burns /
- Pyroptosis /
- Endoplasmic reticulum stress /
- Liver /
- L-carnitine
-
参考文献
(34) [1] PorterC, TompkinsRG, FinnertyCC, et al. The metabolic stress response to burn trauma: current understanding and therapies[J]. Lancet, 2016,388(10052):1417-1426. DOI: 10.1016/S0140-6736(16)31469-6. [2] JeschkeMG, MicakRP, FinnertyCC, et al. Changes in liver function and size after a severe thermal injury[J]. Shock, 2007,28(2):172-177. DOI: 10.1097/shk.0b013e318047b9e2. [3] PriceLA, ThombsB, ChenCL, et al. Liver disease in burn injury: evidence from a national sample of 31,338 adult patients[J]. J Burns Wounds, 2007,7:e1. [4] 褚万立, 柴家科, 冯永强, 等. 严重烧伤大鼠肝脏内质网应激相关蛋白的表达及其意义[J].中华医学杂志,2012,92(12):853-856. DOI: 10.3760/cma.j.issn.0376-2491.2012.12.019. [5] StanojcicM, VinaikR, AbdullahiA, et al. NLRP3 knockout enhances immune infiltration and inflammatory responses and improves survival in a burn sepsis model[J]. Immunology, 2022,165(2):195-205. DOI: 10.1111/imm.13427. [6] Al MamunA, WuY, JiaC, et al. Role of pyroptosis in liver diseases[J]. Int Immunopharmacol, 2020,84:106489. DOI: 10.1016/j.intimp.2020.106489. [7] SadigharaM, JoktajiJP, HajhashemiV, et al. Protective effects of coenzyme Q(10) and L-carnitine against statin-induced pancreatic mitochondrial toxicity in rats[J]. Res Pharm Sci, 2017,12(6):434-443. DOI: 10.4103/1735-5362.217424. [8] BusquetsS, SerpeR, ToledoM, et al. L-carnitine: an adequate supplement for a multi-targeted anti-wasting therapy in cancer[J]. Clin Nutr, 2012,31(6):889-895. DOI: 10.1016/j.clnu.2012.03.005. [9] HotaKB, HotaSK, ChaurasiaOP, et al. Acetyl-L-carnitine- mediated neuroprotection during hypoxia is attributed to ERK1/2-Nrf2-regulated mitochondrial biosynthesis[J]. Hippocampus, 2012,22(4):723-736. DOI: 10.1002/hipo.20934. [10] PatelSP, SullivanPG, LyttleTS, et al. Acetyl-L-carnitine ameliorates mitochondrial dysfunction following contusion spinal cord injury[J]. J Neurochem, 2010,114(1):291-301. DOI: 10.1111/j.1471-4159.2010.06764.x. [11] XiaZ, TianX, KongW, et al. Effects of L-carnitine on liver injury in rats and its impact on blood lipids[J]. Int J Clin Exp Med, 2018, 11(9):9768-9773. [12] LiP, XiaZ, KongW, et al. Exogenous L-carnitine ameliorates burn-induced cellular and mitochondrial injury of hepatocytes by restoring CPT1 activity[J]. Nutr Metab (Lond), 2021,18(1):65. DOI: 10.1186/s12986-021-00592-x. [13] 徐晓东, 邓洋洋, 郑洪新. 糖皮质激素诱导肾虚骨质疏松症大鼠骨组织中OPG/RANKLmRAN及蛋白表达的影响[J].中华中医药学刊,2013,31(12):2623-2627. [14] 中华医学会骨质疏松和骨矿盐疾病分会. 骨代谢生化标志物临床应用指南[J].中华骨质疏松和骨矿盐疾病杂志,2015,8(4):283-293. DOI: 10.3969/j.issn.1674-2591.2015.04.001. [15] 苏延林, 宁斌. 骨质疏松的骨转换生物标志物及其临床应用[J/CD].转化医学电子杂志,2017,4(6):64-68. DOI: 10.3969/j.issn.2095-6894.2017.06.016. [16] NagyV, PenningerJM. The RANKL-RANK story[J]. Gerontology, 2015,61(6):534-542. DOI: 10.1159/000371845. [17] JeschkeMG, FinnertyCC, HerndonDN, et al. Severe injury is associated with insulin resistance, endoplasmic reticulum stress response, and unfolded protein response[J]. Ann Surg, 2012,255(2):370-378. DOI: 10.1097/SLA.0b013e31823e76e7. [18] RochaJ, Eduardo-FigueiraM, BarateiroA, et al. Erythropoietin reduces acute lung injury and multiple organ failure/dysfunction associated to a scald-burn inflammatory injury in the rat[J]. Inflammation, 2015,38(1):312-326. DOI: 10.1007/s10753-014-0035-7. [19] YangX, BaiH, CaiW, et al. Inhibition of Na+/H+ exchanger 1 by cariporide alleviates burn-induced multiple organ injury[J]. J Surg Res, 2013,185(2):797-804. DOI: 10.1016/j.jss.2013.06.049. [20] 孔维昌, 夏正国, 田曦茜, 等. 早期补充外源性肉碱对严重烫伤大鼠肝脏线粒体损伤的影响及病理机制[J].中华烧伤杂志,2018,34(6):374-379. DOI: 10.3760/cma.j.issn.1009-2587.2018.06.012. [21] BortolottiP, FaureE, KipnisE. Inflammasomes in tissue damages and immune disorders after trauma[J]. Front Immunol, 2018,9:1900. DOI: 10.3389/fimmu.2018.01900. [22] WangZ, ZhangS, XiaoY, et al. NLRP3 inflammasome and inflammatory diseases[J]. Oxid Med Cell Longev, 2020,2020:4063562. DOI: 10.1155/2020/4063562. [23] Fernandes-AlnemriT, WuJ, YuJW, et al. The pyroptosome: a supramolecular assembly of ASC dimers mediating inflammatory cell death via caspase-1 activation[J]. Cell Death Differ, 2007,14(9):1590-1604. DOI: 10.1038/sj.cdd.4402194. [24] ShiJ, ZhaoY, WangK, et al. Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death[J]. Nature, 2015,526(7575):660-665. DOI: 10.1038/nature15514. [25] D'SouzaCA, HeitmanJ. Dismantling the cryptococcus coat[J]. Trends Microbiol, 2001,9(3):112-113.DOI: 10.1016/s0966-842x(00)01945-4. [26] HagarJA, PowellDA, AachouiY, et al. Cytoplasmic LPS activates caspase-11: implications in TLR4-independent endotoxic shock[J]. Science, 2013,341(6151):1250-1253. DOI: 10.1126/science.1240988. [27] KayagakiN, WongMT, StoweIB, et al. Noncanonical inflammasome activation by intracellular LPS independent of TLR4[J]. Science, 2013,341(6151):1246-1249. DOI: 10.1126/science.1240248. [28] ShiJ, ZhaoY, WangY, et al. Inflammatory caspases are innate immune receptors for intracellular LPS[J]. Nature, 2014,514(7521):187-192. DOI: 10.1038/nature13683. [29] SudsawardS, KhunchaiS, ThepmaleeC, et al. Endoplasmic reticulum stress, unfolded protein response and autophagy contribute to resistance to glucocorticoid treatment in human acute lymphoblastic leukaemia cells[J]. Int J Oncol, 2020,57(3):835-844. DOI: 10.3892/ijo.2020.5089. [30] NybergWA, EspinosaA. Imiquimod induces ER stress and Ca2+ influx independently of TLR7 and TLR8[J]. Biochem Biophys Res Commun, 2016,473(4):789-794. DOI: 10.1016/j.bbrc.2016.03.080. [31] JägerR, BertrandMJ, GormanAM, et al. The unfolded protein response at the crossroads of cellular life and death during endoplasmic reticulum stress[J]. Biol Cell, 2012,104(5):259-270. DOI: 10.1111/boc.201100055. [32] LeeAS. The ER chaperone and signaling regulator GRP78/BiP as a monitor of endoplasmic reticulum stress[J]. Methods, 2005,35(4):373-381. DOI: 10.1016/j.ymeth.2004.10.010. [33] QiZ, ChenL. Endoplasmic reticulum stress and autophagy[J]. Adv Exp Med Biol, 2019, 1206:167-177. DOI: 10.1007/978-981-15-0602-4_8. [34] NovoaI, ZengH, HardingHP, et al. Feedback inhibition of the unfolded protein response by GADD34-mediated dephosphorylation of eIF2alpha[J]. J Cell Biol, 2001, 153: 1011-1022.DOI: 10.1083/jcb.153.5.1011. -
1 假伤组与2个严重烫伤组大鼠伤后72 h肝组织损伤情况 苏木精-伊红×400，图中标尺为50 μm。1A.假伤组大鼠肝组织中的细胞排列规则，未见明显的炎症细胞浸润；1B.单纯烫伤组大鼠肝组织中可见大量的炎症细胞浸润，细胞排列紊乱，并可见弥漫的脂肪病变；1C.烫伤+肉碱组大鼠肝组织中可见少量的炎症细胞浸润以及散在的脂肪病变
注：图中箭头所指部位为病变部位
2 假伤组与2个严重烫伤组大鼠伤后72 h肝组织中细胞焦亡相关标志物表达水平。2A. 实时荧光定量反转录PCR条图（样本数均为5，
注：NLRP3为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，caspase-1为胱天蛋白酶1，IL-1β为白细胞介素1β，GAPDH为3-磷酸甘油醛脱氢酶；条图横坐标下和条带图上方1、2、3分别为假伤组、单纯烫伤组、烫伤+肉碱组；图2A中3组间NLRP3、caspase-1、消皮素D、IL-1β总体比较，F值分别为1 141.59、1 652.32、644.00、144.40，P值均<0.001；图2A中与假伤组比较，aP<0.01；与单纯烫伤组比较，bP<0.01；图2C中3组间NLRP3、caspase-1、caspase-1/p20、消皮素D-N、剪切型IL-1β蛋白总体比较，F值分别为146.94、373.25、77.72、91.26、220.51，P值均<0.001；图2C中与假伤组比较，aP<0.01，与单纯烫伤组比较，bP<0.01
3 假伤组与2个严重烫伤组大鼠伤后72 h肝组织中内质网应激相关标志物表达水平。3A. 实时荧光定量反转录PCR条图（样本数均为5，
注：GRP78为葡萄糖调节蛋白78，CHOP为CCAAT/增强子结合蛋白同源蛋白，GAPDH为3-磷酸甘油醛脱氢酶；条图横坐标下和条带图上方的1、2、3分别为假伤组、单纯烫伤组、烫伤+肉碱组；图3A中3组间GRP78、CHOP总体比较，F值分别为268.17、160.76，P值均<0.001；图3A中与假伤组比较，aP<0.01，与单纯烫伤组比较，bP<0.01；图3C中3组间GRP78、CHOP总体比较，F值分别为164.63、30.88，P值均<0.001；图3C中与假伤组比较，aP<0.01，与单纯烫伤组比较，bP<0.01
4 培养24 h后5组HepG2细胞活力比较（样本数均为5，
注：DMSO为二甲基亚砜；图中横坐标下方1、2、3、4、5分别为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；5组间总体比较F=107.08，P<0.001；与DMSO组比较，aP<0.01
5 3组HepG2细胞培养24 h后内质网应激相关标志物蛋白表达水平比较。5A、5B.分别为条带图及其对应的条图（样本数均为3，
注：GRP78为葡萄糖调节蛋白78，CHOP为CCAAT/增强子结合蛋白同源蛋白，GAPDH为3-磷酸甘油醛脱氢酶；图5A条带上方及图5B横坐标下方的1、2、3分别为二甲基亚砜组、单纯衣霉素组、衣霉素+肉碱组；图5B中3组间GRP78、CHOP总体比较，F值分别为69.72、318.45，P值均<0.001；图5B中与DMSO组比较，aP<0.01；与单纯衣霉素组比较，bP<0.05，cP<0.01
6 3组HepG2细胞培养24 h后细胞焦亡相关标志物蛋白表达水平比较。6A、6B.分别为条带图及其对应条图（样本数均为3，
注：NLRP3为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，caspase-1为胱天蛋白酶1，IL-1β为白细胞介素1β，GAPDH为3-磷酸甘油醛脱氢酶；图6A条带上方及图6B横坐标下方的1、2、3分别指二甲基亚砜（DMSO）组、单纯衣霉素组、衣霉素+肉碱组；图6B中3组间NLRP3、caspase-1、caspase-1/p20、消皮素D-N和剪切型IL-1β总体比较，F值分别为306.32、0.44、5.28、464.71、4.90，P值分别为<0.001、0.664、0.048、<0.001、0.055；图6B中与DMSO组比较，aP<0.01；与单纯衣霉素组比较，bP<0.01
表1 实时荧光定量反转录PCR法检测大鼠肝组织中内质网应激与细胞焦亡相关标志物mRNA的引物序列及产物大小
基因名称 引物序列（5'→3'） 产物大小（bp） GRP78 上游：GATGTAGGCACGGTGGTTGG下游：GTGAAGGCCACATACGACGG 131 CHOP 上游：AAGCCCTCGCTCTCCAGATT下游：TCCTTCATGCGCTGTTTCCC 132 NLRP3 上游：CAGCGATCAACAGGCGAGAC下游：AGAGATATCCCAGCAAACCTATCCA 141 caspase-1 上游：GACAAGATCCTGAGGGCAAA下游：GGTCTCGTGCCTTTTCCATA 163 消皮素D 上游：GGCAGCATCCTTGAGTGTCT下游：ATGGGGTGCTCTGTTCCAAG 199 IL-1β 上游：AGGCTTCCTTGTGCAAGTGT下游：TGAGTGACACTGCCTTCCTG 230 GAPDH 上游：AGACAGCCGCATCTTCTTGT下游：CTTGCCGTGGGTAGAGTCAT 207 注：GRP78为葡萄糖调节蛋白78，CHOP为CCAAT/增强子结合蛋白同源蛋白，NLRP3为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，caspase-1为胱天蛋白酶1，IL-1β为白细胞介素1β，GAPDH为3-磷酸甘油醛脱氢酶 
下载: 导出CSV
表2 假伤组与2个严重烫伤组大鼠伤后72 h血清中AST和ALT含量比较（U/L，
组别 鼠数（只） AST ALT 假伤组 5 106±13 42±6 单纯烫伤组 5 640±22 157±8 烫伤+肉碱组 5 519±50 121±10 F值 372.98 252.14 P值 <0.001 <0.001 t1值 -46.78 -25.98 P1值 <0.001 <0.001 t2值 4.93 6.06 P2值 0.001 <0.001 注：AST为天冬氨酸转氨酶，ALT为丙氨酸转氨酶；F值、P值为3组间各指标总体比较所得；t1值、P1值为单纯烫伤组与假伤组各指标比较所得；t2值、P2值为烫伤+肉碱组与单纯烫伤组各指标比较所得
下载: 导出CSV
-
图(7) / 表(2)
计量
- 文章访问数: 221
- HTML全文浏览量: 95
- PDF下载量: 25
- 被引次数: 0
