Toll样受体4在对乙酰氨基酚致小鼠肝损伤过程中对肝脏再生的影响

陈明月; 郑秀良; 乔亚琴; 沈海涛; 路燕

doi:10.3969/j.issn.1001-5256.2023.05.017

Toll样受体4在对乙酰氨基酚致小鼠肝损伤过程中对肝脏再生的影响

DOI: 10.3969/j.issn.1001-5256.2023.05.017

安徽医科大学第二附属医院消化内科，合肥 230601

基金项目:

国家自然科学基金青年科学基金项目 (81800524)

伦理学声明：本研究方案于2018年3月经由安徽医科大学实验动物伦理委员会审批，批号：LLSC20180080，符合实验室动物管理与使用准则。

利益冲突声明：本文不存在任何利益冲突。

作者贡献声明：陈明月负责分析数据，撰写论文和参与课题设计；郑秀良、乔亚琴、沈海涛参与实验操作和修改论文；路燕负责课题设计和修改论文。

详细信息

通信作者:
路燕，luyancathrine@163.com (ORCID: 0000-0001-5116-5145)

计量
- 文章访问数: 499
- HTML全文浏览量: 71
- PDF下载量: 69
- 被引次数: 0
出版历程
- 收稿日期: 2022-09-30
- 录用日期: 2022-11-15
- 出版日期: 2023-05-20

Effect of Toll-like receptor 4 on liver regeneration during acetaminophen-induced liver injury in mice

Department of Gastroenterology, The Second Affiliated Hospital of Anhui Medical University, Hefei 230601, China

Research funding:

National Natural Science Foundation of China Youth Science Fund Project (81800524)

More Information

Corresponding author: LU Yan, luyancathrine@163.com (ORCID: 0000-0001-5116-5145)

摘要

摘要: 目的观察抑制Toll样受体4(TLR4)是否影响对乙酰氨基酚(APAP)致小鼠肝损伤过程中的肝脏再生，初步探讨TLR4参与肝脏再生的机制。方法将78只雄性CD-1小鼠采用随机数字表法分为9组，其中对照组(正常对照组、溶剂对照组、抑制剂对照组)每组6只，实验组(APAP 24 h组、TAK-242+APAP 24 h组、APAP 48 h组、TAK-242+APAP 48 h组、APAP 72 h组、TAK-242+APAP 72 h组)每组10只。实验组小鼠给予单剂量腹腔注射APAP(300 mg/kg)，TAK-242在APAP注射前3 h以3 mg/kg剂量腹腔注射。在不同时间点收集各组小鼠血清和肝脏组织。采用生化方法检测小鼠血清ALT水平，HE染色检测肝脏组织病理改变。RT-PCR、Western blot、免疫组化方法检测Cyclin D1、PCNA、Ki-67、STAT3、p-STAT3的表达。正态分布的计量资料两组间比较采用成组t检验；多组间比较采用单因素方差分析，进一步两两比较采用LSD-t检验。非正态分布的计量资料两组间比较采用Mann-Whitney U检验；多组间比较及进一步两两比较均采用Kruskal-Wallis H检验。结果与正常对照组相比，APAP 24 h组和APAP 48 h组的血清ALT水平均明显较高(P值均＜0.05)；TAK-242+APAP 24 h和48 h组的血清ALT水平均明显高于同时间点APAP组(P值均＜0.05)。HE染色结果显示，APAP处理的小鼠肝脏可见典型的小叶中心性坏死，TAK-242+APAP 24 h和48 h组小鼠肝脏的坏死面积均显著大于同时间点APAP组(P值均＜0.05)。RT-PCR、Western blot、免疫组化结果显示，TAK-242+APAP 24 h、48 h和72 h组Cyclin D1 mRNA和蛋白表达水平均明显低于同时间点的APAP组(P值均＜0.05)；TAK-242+APAP 24 h、48 h和72 h组PCNA mRNA表达水平均明显低于同时间点的APAP组(P值均＜0.05)，TAK-242+APAP 24 h和48 h组PCNA蛋白表达水平均明显低于同时间点的APAP组(P值均＜0.05)；TAK-242+APAP 24 h和72 h组Ki-67 mRNA表达水平均明显低于同时间点的APAP组(P值均＜0.05)，TAK-242+APAP 24 h、48 h和72 h组Ki-67蛋白表达水平均明显低于同时间点的APAP组(P值均＜0.05)。此外，TAK-242+APAP 24 h和48 h组STAT3磷酸化水平均显著低于同时点的APAP组(P值均＜0.05)。结论 TLR4可能通过提高STAT3磷酸化水平促进APAP诱导的小鼠肝损伤过程中的肝脏再生。
- 化学性与药物性肝损伤 /
- 醋氨酚 /
- Toll样受体4 /
- 肝再生 /
- 小鼠, 近交ICR
Abstract: Objective To investigate whether Toll-like receptor 4 (TLR4) inhibition affects liver regeneration during acetaminophen (APAP)-induced liver injury in mice, as well as the mechanism of TLR4 involved in liver regeneration. Methods A total of 78 male CD-1 mice were divided into nine groups using a random number table, i.e., three control groups (normal control group, solvent control group, inhibitor control group) with 6 mice in each group and six experimental groups (APAP 24-hour group, TAK-242+APAP 24-hour group, APAP 48-hour group, TAK-242+APAP 48-hour group, APAP 72-hour group, TAK-242+APAP 72-hour group) with 10 mice in each group. The mice in the experimental groups were given a single dose of intraperitoneally injected APAP (300 mg/kg), and TAK-242 was intraperitoneally injected at a dose of 3 mg/kg at 3 hours before APAP administration. Serum and liver tissue samples were collected at different time points. The biochemical method was used to measure the serum level of alanine aminotransferase (ALT); HE staining was used to observe liver pathological changes; RT-PCR, Western blot, and immunohistochemistry were used to measure the expression levels of Cyclin D1, PCNA, Ki-67, STAT3, and p-STAT3. The t-test was used for comparison of normally distributed continuous data between two groups; a one-way analysis of variance was used for comparison between multiple groups, and the least significant difference t-test was used for further comparison between two groups. The Mann-Whitney U test was used for comparison of non-normally distributed continuous data between two groups, and the Kruskal-Wallis H test was used for comparison between multiple groups and further comparison between two groups. Results Compared with the normal control group, the APAP 24-hour and 48-hour groups had a significantly higher serum level of ALT (both P < 0.05), and the TAK-242+APAP 24-hour and 48-hour groups had a significantly higher serum level of ALT than the APAP group at the same time point (both P < 0.05). HE staining showed typical central lobular necrosis in the liver of APAP-treated mice, and the TAK-242+APAP 24-hour and 48-hour groups had a significantly larger necrotic area than the APAP group at the same time point (both P < 0.05). RT-PCR, Western blot, and immunohistochemistry showed that the TAK-242+APAP 24-hour, 48-hour, and 72-hour groups had significantly lower mRNA and protein expression levels of Cyclin D1 than the APAP group at the same time point (all P < 0.05); the TAK-242+APAP 24-hour, 48-hour, and 72-hour groups had a significantly lower mRNA expression level of PCNA than the APAP group at the same time point (all P < 0.05), and the TAK-242+APAP 24-hour and 48-hour groups had a significantly lower protein expression level of PCNA than the APAP group at the same time point (all P < 0.05); the TAK-242+APAP 24-hour and 72-hour groups had a significantly lower mRNA expression level of Ki-67 than the APAP group at the same time point (all P < 0.05), and the TAK-242+APAP 24-hour, 48-hour, and 72-hour groups had a significantly lower protein expression level of Ki-67 than the APAP group at the same time point (all P < 0.05). In addition, the TAK-242+APAP 24-hour and 48-hour groups had a significantly lower phosphorylation level of STAT3 than the APAP group at the same time point (both P < 0.05). Conclusion TLR4 may promote liver regeneration by increasing the phosphorylation level of STAT3 during APAP-induced liver injury in mice.
- Chemical and Drug Induced Liver Injury /
- Acetaminophen /
- Toll-Like Receptor 4 /
- Liver Regeneration /
- Mice, Inbred ICR

HTML全文

图 1 小鼠血清ALT水平比较

Figure 1. Comparison of serum ALT levels in mice

下载: 全尺寸图片幻灯片

图 2 小鼠肝脏HE染色结果 (×100)

注：a，正常对照组；b，溶剂对照组；c，抑制剂对照组。

Figure 2. HE staining results of mouse liver (×100)

下载: 全尺寸图片幻灯片

图 3 抑制TLR4对肝损伤恢复的影响(HE染色，×100)

注：a，正常对照组；b，APAP 24 h组；c，TAK-242+APAP 24 h组；d，APAP 48 h组；e，TAK-242+APAP 48 h组；f，APAP 72 h组；g，TAK-242+APAP 72 h组。

Figure 3. Effect of inhibition of TLR4 on recovery from liver injury (HE staining, ×100)

下载: 全尺寸图片幻灯片

图 4 肝组织cyclin D1、PCNA的蛋白表达

注：a，Cyclin D1蛋白表达水平；b，PCNA蛋白表达水平。

Figure 4. Cyclin D1, PCNA protein expressions in liver tissue

下载: 全尺寸图片幻灯片

图 5 免疫组化染色检测肝组织Ki-67蛋白表达(×100)

注：a，正常对照组；b，APAP 24 h组；c，TAK-242+APAP 24 h组；d，APAP 48 h组；e，TAK-242+APAP 48 h组；f，APAP 72 h组；g，TAK-242+APAP 72 h组。

Figure 5. Ki-67 protein expression in liver tissue detected by Immunohistochemical staining (×100)

下载: 全尺寸图片幻灯片

图 6 各组Ki-67阳性细胞累积光密度值比较

Figure 6. Comparison of integrated option density of Ki-67 positive cells

下载: 全尺寸图片幻灯片

图 7 肝组织STAT3磷酸化蛋白的表达

Figure 7. Expression of phosphorylated STAT3 protein in liver tissue

下载: 全尺寸图片幻灯片

表 1 RT-PCR引物序列

Table 1. RT-PCR primer sequences

基因	上游(5′-3′)	下游(5′-3′)
Cyclin D1	AGGCGGATGAGAACAAGCAG	CCTTGTTTAGCCAGAGGCCG
Ki-67	CCATCATTGACCGCTCCTT	CTGCCAGTGTGCTGTTCTAC
PCNA	GGGTTGGTAGTTGTCGCTGT	CCAAGGAGACGTGAGACGAG
GAPDH	GACATGCCGCCTGGAGAAAC	AGCCCAGGATGCCCTTTAGT

下载: 导出CSV

表 2 APAP对小鼠血清ALT及肝脏组织的影响

Table 2. Effects of APAP on serum ALT and liver tissue in mice

组别	动物数(只)	ALT(U/L)	肝脏坏死面积(%)
正常对照组	6	12.339±4.245	0
APAP 24 h组	10	1 449.848±209.491¹⁾	41.600(38.617~46.502)¹⁾
APAP 48 h组	10	281.702±140.592¹⁾	36.050(25.127~45.718)¹⁾
APAP 72 h组	10	45.251±4.298	0 (0~1.903)
H值		17.857	16.035
P值		＜0.001	0.001
注：与正常对照组比较，1)P＜0.05。

下载: 导出CSV

表 3 抑制TLR4在各时间点对APAP肝损伤小鼠血清ALT及肝脏组织的影响

Table 3. Effects of TLR4 inhibition on serum ALT and liver tissue of mice with APAP-induced liver injury at different time point

组别	动物数(只)	24 h	48 h	72 h
ALT(U/L)
APAP组	10	1 449.848±209.491	281.702±140.592	45.251±4.298
TAK-242+APAP组	10	3 484.212±960.336	1 151.505±656.722	56.995±24.763
t值		-4.628	-2.896	-1.045
P值		0.002	0.040	0.352
肝脏坏死面积(%)
APAP组	10	41.600(38.617~46.502)	36.050(25.127~45.718)	0(0~1.903)
TAK-242+APAP组	10	58.554(55.889~73.409)	59.558(54.856~60.591)	2.713(0~8.289)
Z值		-2.610	-2.611	-1.294
P值		0.008	0.008	0.310

下载: 导出CSV

表 4 APAP对小鼠肝组织Ki-67 mRNA、Cyclin D1 mRNA、PCNA mRNA表达的影响

Table 4. Effects of APAP on the expression of Ki-67 mRNA, Cyclin D1 mRNA and PCNA mRNA in mouse liver

组别	动物数(只)	Ki-67 mRNA相对表达量	Cyclin D1 mRNA相对表达量	PCNA mRNA相对表达量
正常对照组	6	1.002±0.072	1.034±0.325	1.009±0.162
APAP 24 h组	10	1.027±0.184	1.061±0.102	1.347±0.115¹⁾
APAP 48 h组	10	83.566±23.362¹⁾	3.707±0.255¹⁾	5.327±0.162¹⁾
APAP 72 h组	10	39.316±10.916¹⁾	2.417±0.336¹⁾	1.739±0.086¹⁾
F值		27.853	88.659	657.231
P值		＜0.001	＜0.001	＜0.001
注：与正常对照组比较，1)P＜0.05。

下载: 导出CSV

表 5 抑制TLR4对小鼠肝组织Ki-67 mRNA、Cyclin D1 mRNA、PCNA mRNA表达的影响

Table 5. Effects of TLR4 inhibition on the expression of Ki-67 mRNA, Cyclin D1 mRNA and PCNA mRNA in mouse liver

组别	动物数(只)	24 h	48 h	72 h
Ki-67 mRNA相对表达量
APAP组	10	1.027±0.184	83.566±23.362	39.316±10.916
TAK-242+APAP组	10	0.366±0.129	70.145±31.141	5.950±0.678
t值		5.107	0.597	5.284
P值		0.007	0.583	0.006
Cyclin D1 mRNA相对表达量
APAP组	10	1.061±0.102	3.707±0.255	2.417±0.336
TAK-242+APAP组	10	0.678±0.073	2.918±0.487	1.837±0.202
t值		6.116	2.871	2.957
P值		0.001	0.028	0.025
PCNA mRNA相对表达量
APAP组	10	1.347±0.115	5.327±0.162	1.739±0.086
TAK-242+APAP组	10	0.830±0.075	2.937±0.144	0.620±0.035
t值		6.529	19.074	20.909
P值		0.003	＜0.001	＜0.001

下载: 导出CSV

参考文献(27)

[1]	LOUVET A, NTANDJA WANDJI LC, LEMAÎTRE E, et al. Acute liver injury with therapeutic doses of acetaminophen: A prospective study[J]. Hepatology, 2021, 73(5): 1945-1955. DOI: 10.1002/hep.31678.
[2]	WANG YW, LIANG YR. Research progress on liver transplantation for drug-induced liver injury[J]. Ogran Transplant, 2022, 13(3): 338-343. DOI: 10.3969/j.issn.1674-7445.2022.03.009. 王砚伟, 梁雨荣. 药物性肝损伤肝移植治疗进展[J]. 器官移植, 2022, 13(3): 338-343. DOI: 10.3969/j.issn.1674-7445.2022.03.009.
[3]	ASRANI SK, DEVARBHAVI H, EATON J, et al. Burden of liver diseases in the world[J]. J Hepatol, 2019, 70(1): 151-171. DOI: 10.1016/j.jhep.2018.09.014.
[4]	NASH E, SABIH AH, CHETWOOD J, et al. Drug-induced liver injury in Australia, 2009-2020: the increasing proportion of non-paracetamol cases linked with herbal and dietary supplements[J]. Med J Aust, 2021, 215(6): 261-268. DOI: 10.5694/mja2.51173.
[5]	STRAVITZ RT, LEE WM. Acute liver failure[J]. Lancet, 2019, 394(10201): 869-881. DOI: 10.1016/S0140-6736(19)31894-X.
[6]	BHUSHAN B, APTE U. Liver regeneration after acetaminophen hepatotoxicity: Mechanisms and therapeutic opportunities[J]. Am J Pathol, 2019, 189(4): 719-729. DOI: 10.1016/j.ajpath.2018.12.006.
[7]	BHUSHAN B, GUNEWARDENA S, EDWARDS G, et al. Comparison of liver regeneration after partial hepatectomy and acetaminophen-induced acute liver failure: A global picture based on transcriptome analysis[J]. Food Chem Toxicol, 2020, 139: 111186. DOI: 10.1016/j.fct.2020.111186.
[8]	ROCHA DM, CALDAS AP, OLIVEIRA LL, et al. Saturated fatty acids trigger TLR4-mediated inflammatory response[J]. Atherosclerosis, 2016, 244: 211-215. DOI: 10.1016/j.atherosclerosis.2015.11.015.
[9]	MARLINI M, MABUCHI A, MALLARD BL, et al. Delayed liver regeneration in C3H/HeJ mice: possible involvement of haemodynamic and structural changes in the hepatic microcirculation[J]. Exp Physiol, 2016, 101(12): 1492-1505. DOI: 10.1113/EP085727.
[10]	HOSHINO K, TAKEUCHI O, KAWAI T, et al. Cutting edge: Toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene product[J]. J Immunol, 1999, 162(7): 3749-3752.
[11]	MATSUNAGA N, TSUCHIMORI N, MATSUMOTO T, et al. TAK-242 (resatorvid), a small-molecule inhibitor of Toll-like receptor (TLR) 4 signaling, binds selectively to TLR4 and interferes with interactions between TLR4 and its adaptor molecules[J]. Mol Pharmacol, 2011, 79(1): 34-41. DOI: 10.1124/mol.110.068064.
[12]	ONO Y, MAEJIMA Y, SAITO M, et al. TAK-242, a specific inhibitor of Toll-like receptor 4 signalling, prevents endotoxemia-induced skeletal muscle wasting in mice[J]. Sci Rep, 2020, 10(1): 694. DOI: 10.1038/s41598-020-57714-3.
[13]	WANG H, LI X, DONG G, et al. Toll-like receptor 4 inhibitor TAK-242 improves fulminant hepatitis by regulating accumulation of myeloid-derived suppressor cell[J]. Inflammation, 2021, 44(2): 671-681. DOI: 10.1007/s10753-020-01366-y.
[14]	YU W, ZHANG LJ, LU Y, et al. Role of STAT3 in hepatocyte regeneration after acetaminophen-induced hepatocellular injury in mice[J]. J Clin Hepatol, 2021, 37(4): 857-862. DOI: 10.3969/j.issn.1001-5256.2021.04.026. 余旺, 章礼久, 路燕, 等. STAT3在对乙酰氨基酚所致小鼠肝损伤后肝细胞再生中的作用[J]. 临床肝胆病杂志, 2021, 37(4): 857-862. DOI: 10.3969/j.issn.1001-5256.2021.04.026.
[15]	YAGI S, HIRATA M, MIYACHI Y, et al. Liver regeneration after hepatectomy and partial liver transplantation[J]. Int J Mol Sci, 2020, 21(21): 8414. DOI: 10.3390/ijms21218414.
[16]	FAUSTO N, CAMPBELL JS, RIEHLE KJ. Liver regeneration[J]. Hepatology, 2006, 43(2 Suppl 1): S45-S53. DOI: 10.1002/hep.20969.
[17]	YANG R, MIKI K, HE X, et al. Prolonged treatment with N-acetylcystine delays liver recovery from acetaminophen hepatotoxicity[J]. Crit Care, 2009, 13(2): R55. DOI: 10.1186/cc7782.
[18]	SHAO S, ZHANG Y, LI G, et al. The dynamics of cell death patterns and regeneration during acute liver injury in mice[J]. FEBS Open Bio, 2022, 12(5): 1061-1074. DOI: 10.1002/2211-5463.13383.
[19]	BHUSHAN B, EDWARDS G, DESAI A, et al. Liver-specific deletion of integrin-linked kinase in mice attenuates hepatotoxicity and improves liver regeneration after acetaminophen overdose[J]. Gene Expr, 2016, 17(1): 35-45. DOI: 10.3727/105221616X691578.
[20]	FAN X, CHEN P, TAN H, et al. Dynamic and coordinated regulation of KEAP1-NRF2-ARE and p53/p21 signaling pathways is associated with acetaminophen injury responsive liver regeneration[J]. Drug Metab Dispos, 2014, 42(9): 1532-1539. DOI: 10.1124/dmd.114.059394.
[21]	YU L, WANG L, CHEN S. Endogenous toll-like receptor ligands and their biological significance[J]. J Cell Mol Med, 2010, 14(11): 2592-2603. DOI: 10.1111/j.1582-4934.2010.01127.x.
[22]	KULKARNI OP, HARTTER I, MULAY SR, et al. Toll-like receptor 4-induced IL-22 accelerates kidney regeneration[J]. J Am Soc Nephrol, 2014, 25(5): 978-989. DOI: 10.1681/ASN.2013050528.
[23]	LIANG J, ZHANG Y, XIE T, et al. Hyaluronan and TLR4 promote surfactant-protein-C-positive alveolar progenitor cell renewal and prevent severe pulmonary fibrosis in mice[J]. Nat Med, 2016, 22(11): 1285-1293. DOI: 10.1038/nm.4192.
[24]	SHAO C, JING Y, ZHAO S, et al. LPS/Bcl3/YAP1 signaling promotes Sox9+HNF4α+ hepatocyte-mediated liver regeneration after hepatectomy[J]. Cell Death Dis, 2022, 13(3): 277. DOI: 10.1038/s41419-022-04715-x.
[25]	YIN S, WANG H, PARK O, et al. Enhanced liver regeneration in IL-10-deficient mice after partial hepatectomy via stimulating inflammatory response and activating hepatocyte STAT3[J]. Am J Pathol, 2011, 178(4): 1614-1621. DOI: 10.1016/j.ajpath.2011.01.001.
[26]	FU H, DONG R, ZHANG Y, et al. Tmub1 negatively regulates liver regeneration via inhibiting STAT3 phosphorylation[J]. Cell Signal, 2019, 55: 65-72. DOI: 10.1016/j.cellsig.2018.12.013.
[27]	HU K, XU J, FAN K, et al. Nuclear accumulation of pyruvate kinase M2 promotes liver regeneration via activation of signal transducer and activator of transcription 3[J]. Life Sci, 2020, 250: 117561. DOI: 10.1016/j.lfs.2020.117561.