Chinese Journal of Animal Nutrition >

2023 , Vol. 35 >Issue 7: 4632 - 4642

DOI: https://doi.org/10.12418/CJAN2023.430

RESEARCH PAPER

Glycyrrhizin Alleviates Lipopolysaccharide-Induced Inflammatory Response in IPEC-J2 Cells via c-Jun N-Terminal Protein Kinase Pathway

  • ZHAO Xiaohan , 1, 2 ,
  • SI Wei 2 ,
  • ZHAO Qingyu 2 ,
  • ZHANG Junmin 2 ,
  • ZHAO Jinshan , 1, * ,
  • QIN Yuchang , 2, *
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  • 1 College of Animal Science and Technology, Qingdao Agricultural University, Qingdao 266109, China
  • 2 State Key Laboratory of Animal Nutrition, Institute of Animal Sciences of Chinese Academy of Agricultural Sciences, Beijing 100193, China
* ZHAO Jinshan, professor, ;
QIN Yuchang, professor,

Received date: 2022-12-31

  Online published: 2023-07-11

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Abstract

This study was to elucidate the molecular mechanisms of glycyrrhizin (GL) alleviating lipopolysaccharide (LPS)-induced inflammatory response in IPEC-J2 cells. The IPEC-J2 cells were selected as the study object. The viability of IPEC-J2 cells after incubation with GL of different concentrations (0, 10, 50, 100, 200 and 500 μmol/L) for different time (12, 24 and 48 h) was measured by CCK-8 assay to determine the appropriate treatment condition of GL. There were four groups in this study, namely control group [IPEC-J2 cells were grown for 24 h and then treated with dimethyl sulfoxide (DMSO) for 12 h followed by Dulbecco’s phosphate buffered saline (DPBS) co-treatment for 12 h], LPS group (IPEC-J2 cells were grown for 24 h and then treated with DMSO for 12 h followed by LPS co-treatment with 10 μg/mL for 12 h), GL group (IPEC-J2 cells were grown for 24 h and then treated with 50 μmol/L GL for 12 h followed by DPBS co-treatment for 12 h) and GL+LPS group ((IPEC-J2 cells were grown for 24 h and then treated with 50 μmol/L GL for 12 h followed by 10 μg/mL LPS co-treatment for 12 h). Enzyme linked immunosorbent assay (ELISA) method was used to detect the levels of interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and high mobility group protein 1 (HMGB1) in the supernatant of IPEC-J2 cells treated with LPS and GL. The mRNA expression levels of IL-1β, IL-6 and TNF-α in cells were detected by real-time fluorescent quantitative PCR (RT-qPCR). Expression levels of proteins related with Toll-like receptor 4 (TLR4) signal pathway were detected by Western blotting method and immunofluorescence staining method. The results showed as follows: 1) compared with no GL added, which was treated with 50 μmol/L GL for 24 h had no significant effect on the viability of IPEC-J2 cells (P>0.05), so the appropriate concentration and time for GL were 50 μmol/L and 24 h, respectively. 2) Compared with the control group, the level and mRNA expression level of IL-6 in the LPS group were significantly increased (P<0.05); compared with the LPS group, the level and mRNA expression level of IL-6 in the GL+LPS group were significantly decreased (P<0.05). 3) Compared with the control group, LPS stimulation significantly increased the protein expression levels of phosphorylated c-Jun N-terminal protein kinase 1/3 (p-JNK1/3) and c-Jun in cells (P<0.05); compared with the LPS group, pretreatment with GL could down-regulate the protein expression levels of p-JNK1/3 and c-Jun in cells (P<0.05), but had no significant effects on the protein expression levels of p38 mitogen-activated protein kinase (p38 MAPK) and nuclear factor kappa-B p65 (NF-κB p65) in cells (P>0.05). 4) The fluorescence intensity of phosphorylated c-Jun (p-c-Jun) in cell nucleus in the LPS group was significantly decreased compared with the control group (P<0.05), while pretreatment with GL could significantly down-regulate the fluorescence intensity of p-c-Jun in cell nucleus (P<0.05). In conclusion, GL alleviates LPS-induced inflammatory response in IPEC-J2 cells by down-regulating the gene expression of IL-6 and related protein expression of c-Jun N-terminal protein kinase (JNK) signaling pathway.

Key words： glycyrrhizin; IPEC-J2 cells; JNK signaling pathway; anti-inflammatory function

Cite this article

ZHAO Xiaohan , SI Wei , ZHAO Qingyu , ZHANG Junmin , ZHAO Jinshan , QIN Yuchang . Glycyrrhizin Alleviates Lipopolysaccharide-Induced Inflammatory Response in IPEC-J2 Cells via c-Jun N-Terminal Protein Kinase Pathway[J]. Chinese Journal of Animal Nutrition, 2023 , 35(7) : 4632 -4642 . DOI: 10.12418/CJAN2023.430

养猪业作为我国当前养殖业中的重点产业,对于我国农业发展有着相当重要的作用。对于动物来说,肠道不仅是营养物质消化吸收的主要场所,也是机体最大的免疫和内分泌器官,因此充分了解并维护猪的肠道健康是猪养殖的核心[1]。肠道上皮是由单层上皮细胞构成的绒毛样组织,肠上皮细胞作为肠道的物理屏障,将肠腔内容物与内部环境分隔,降低侵入性外来物造成感染的风险,故上皮细胞的完整性对于维持外部和内部环境的稳态至关重要[2-3]。当肠道在生理或病理情况下发生炎症时,肠道上皮细胞都会分泌信号分子,如黏蛋白、细胞因子和趋化因子,以防止肠道中有害微生物的入侵[4-5]。脂多糖(lipopolysaccharide,LPS)是革兰氏阴性菌细胞壁中的一种关键脂质成分,可触发机体及细胞的免疫反应。LPS活化Toll样受体4(Toll-like receptor 4,TLR4)后,将信号呈递给下游反应因子髓样分化因子88(myeloid differentiation factor 88,MyD88),从而激活核因子-κB(nuclear factor-κB,NF-κB)信号通路,入核刺激炎症细胞因子白细胞介素-1β(interleukin-1β,IL-1β)、白细胞介素-6(interleukin-6,IL-6)和肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)等的产生[6-7]。除此之外,丝裂原活化蛋白激酶(mitogen-activated protein kinases,MAPK)是一类丝氨酸-苏氨酸蛋白激酶,其介导的信号转导通路在机体的炎性反应中发挥重大作用[8]。猪发生肠道炎症会引起腹泻、采食量下降以及体重下降,严重时甚至会引起猪的死亡,影响生产效率,制约养猪业的发展[9]。因此,寻找缓解猪肠道上皮细胞炎症的药物,减轻肠道炎症对养猪业的影响,是目前养猪业中亟需解决的问题。
甘草为豆科甘草属植物,是一种古老且常用的药食同源中草药[10],甘草中的主要功能组分有皂苷类化合物、黄酮类化合物和甘草多糖[11]。甘草酸(glycyrrhizin,GL)是甘草皂苷类化合物中的一种,具有广泛的药理作用,如抗炎、抗氧化、抗病毒等[12]。有研究表明,GL可以显著降低放射性肠炎中的肠道损伤以及炎症因子TNF-α、IL-6、IL-1β的分泌,并且GL作为高迁移率族蛋白B1(high mobility group box protein 1,HMGB1)的特异性抑制剂,可以通过抑制HMGB1/TLR4信号通路,减轻小鼠放射性肠炎的发生[13]。同时,GL能通过下调LPS诱导的小鼠子宫内膜上皮细胞TLR4相关蛋白表达和NF-κB活化来抑制炎症相关因子的释放[14]。除此之外,GL还可以通过抑制鸡的MAPK信号通路中的c-Jun氨基末端蛋白激酶(c-Jun N-terminal protein kinase,JNK)和p38丝裂原活化蛋白激酶(p38 mitogen-activated protein kinase,p38 MAPK)的表达来抵抗支原体引发的炎症和细胞凋亡[15]。但目前有关GL在猪肠道炎症中作用的研究还较少,本试验通过探究GL对LPS诱导的IPEC-J2细胞炎症的影响来阐明GL的抗炎作用机制,以期为猪肠道炎症研究提供参考。

1 材料与方法

1.1 试验试剂

胎牛血清(fetal bovine serum,FBS)、DMEM/F12细胞培养基、青霉素-链霉素、0.25%胰蛋白酶、免疫印迹超敏发光液和BCA Protein Assay Kit购自Thermo Fisher Scientific公司;LPS购自Sigma Aldrich公司;RIPA裂解液(中)、蛋白酶抑制剂、蛋白磷酸酶抑制剂、TBS溶液和TBST溶液购自北京华兴博创基因技术有限公司;GL(分析标准品,HPLC≥98%)、封闭用山羊血清和抗荧光淬灭剂购自于上海源叶生物科技有限公司;二甲基亚砜(dimethyl sulfoxide,DMSO)和杜氏磷酸缓冲液(Dulbecco’s phosphate buffered saline,DPBS)购自于Gibco公司;Cell Counting Kit-8(CCK-8)细胞活力检测试剂盒和蛋白上样缓冲液购自北京索莱宝科技有限公司;兔抗磷酸化c-Jun氨基末端蛋白激酶1/3(phosphorylated c-Jun N-terminal protein kinase 1/3,p-JNK1/3)、c-Jun、磷酸化c-Jun(phosphorylated c-Jun,p-c-Jun)、p38 MAPK、核因子-κB p65(nuclear factor kappa-B p65,NF-κB p65)、磷酸化核因子-κB p65(phosphorylated nuclear factor kappa-B p65,p-NF-κB p65)、甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase,GAPDH)、Anti-rabbit IgG、Anti-rabbit IgG (H+L)、Anti-mouse IgG、Anti-mouse IgG (H+L)抗体购自Cell Signaling Technology公司;猪IL-1β、IL-6、TNF-α和HMGB1的酶联免疫吸附测定(enzyme linked immunosorbent assay,ELISA)试剂盒购自江苏雨桐科技有限公司;细胞总RNA提取试剂盒购自天根生化科技有限公司;反转录试剂盒和荧光定量试剂盒购自宝日医生物技术有限公司。

1.2 细胞培养

使用含有10% FBS的DMEM/F12细胞培养基培养IPEC-J2细胞,培养条件为37 ℃、5%CO2的细胞培养箱,当细胞融合度达到80%左右时,用胰蛋白酶进行消化传代。

1.3 细胞活力检测

将IPEC-J2细胞按照1×104个/孔接种到96孔细胞培养板内,分别在培养0、24、36 h后加入1 μL含有不同浓度GL的DMSO溶液,使培养基中GL终浓度分别达到0、10、50、100、200、500 μmol/L,再分别培养48、24、12 h后,使用CCK-8试剂盒检测细胞活力,操作步骤按照说明书进行。每个GL处理的浓度、时间设置6个重复。

1.4 试验分组

根据1.3所得试验结果确定GL处理的浓度和时间。试验共设4个组,即对照组(IPEC-J2细胞生长24 h后,经DMSO处理12 h再经DPBS共同处理12 h)、LPS组(IPEC-J2细胞生长24 h后,经DMSO处理12 h再经10 μg/mL的LPS共同处理12 h)、GL组(IPEC-J2细胞生长24 h后,经50 μmol/L的GL处理12 h再经DPBS共同处理12 h)和GL+LPS组(IPEC-J2细胞生长24 h后,经50 μmol/L的GL处理12 h再经10 μg/mL的LPS共同处理12 h)。

1.5 炎症细胞因子分泌及基因表达检测

将IPEC-J2细胞以6×105个/孔的细胞数接种于六孔板。按照1.4中方法分组处理细胞后收集细胞培养基,以1 000 r/min离心5 min去除颗粒物质,取上清液按照ELISA检测试剂盒说明书检测IL-1β、IL-6、TNF-α和HMGB1水平。收取细胞,提取细胞总RNA并反转录合成cDNA。NCBI数据库查询得到猪IL-1β、IL-6、TNF-α和GAPDH基因序列,使用Primer Premier 6.0设计引物(表1),引物由北京新时代众和科技有限公司合成。以GAPDH为内参基因,进行实时荧光定量PCR检测。反应体系为10 μL,其中上、下游引物各0.2 μL,cDNA 2 μL,TB Green 5 μL,ROXⅡ 0.2 μL,ddH2O 2.4 μL。使用两步法PCR反应程序进行实时荧光定量PCR。第1步为95 ℃预变性30 s;第2步为95 ℃变性5 s,60 ℃延伸34 s,40个循环;第3步为95 ℃ 15 s,65 ℃ 1 min,95 ℃ 15 s的熔解曲线分析。采用2-ΔΔCt法计算目的基因mRNA表达水平。
表1 实时荧光定量PCR引物

Table 1 Primers for RT-qPCR

基因
Genes		 引物序列
Primer sequence (5'—3')		 GenBank登录号
GenBank accession No.		 产物长度
Product length/bp
白细胞介素-1β
IL-1β		 F:CCTGAACCTGCCAAGGAA
R:TTGCCACAATCACAGACACC		 NM_214055.1 150
白细胞介素-6
IL-6		 F:GCTGCAGTCACAGAACGAGT
R:CAGGTGCCCCAGCTACATTA		 NM_214399.1 118
肿瘤坏死因子-α
TNF-α		 F:CTGTCCCTCGGCTTTGACAT
R:GCTTGTCACTCGAATTTTGAGA		 NM_214022.1 82
甘油醛-3-磷酸脱氢酶
GAPDH		 F:TGTCCACCTTCCAGCAGATGT
R:AGCTCAGTAACAGTCCGCCTAGA		 NM_001206359.1 132

1.6 蛋白质免疫印迹(Western blotting)检测

将IPEC-J2细胞以3×106个/孔的细胞数接种于10 cm细胞培养皿中,按照1.4中的方法分组处理并收取细胞后,使用含有蛋白酶抑制剂和蛋白磷酸酶抑制剂的RIPA裂解液(中)于冰上提取蛋白。使用BCA试剂盒检测样品蛋白浓度。按照20 μg每孔的蛋白上样量使用10%分离胶进行电泳。观察到溴酚蓝染料电泳至离胶底部约5 mm左右处后开始电转。根据蛋白大小将膜裁下进行封闭。使用含有5%脱脂奶粉的TBST于室温下封闭1 h。使用含有5%脱脂奶粉的TBST按照抗体使用说明书推荐比例稀释TLR4、p-JNK1/3、c-Jun、p38 MAPK、NF-κB p65和NF-κB p-p65抗体,4 ℃孵育过夜。经TBST洗涤3次后,使用二抗(1∶5 000)室温孵育1 h,并再次使用TBST洗涤3次,使用超敏发光液于Tanon 5200凝胶成像系统发光拍摄。结果使用ImageJ 1.52软件进行统计分析。

1.7 免疫荧光检测

将IPEC-J2细胞以5×104个/孔的细胞数接种于放有爬片的24孔板中,按照1.4中的方法分组处理细胞。除去培养基,DPBS洗涤3次后,4%多聚甲醛37 ℃固定1 h,再洗涤后用0.5% Triton X-100室温下通透30 min,洗涤后用含有10%山羊血清的DPBS在37 ℃下封闭1 h,用5%山羊血清配制的p-c-Jun一抗工作液(1∶100)4 ℃过夜,山羊抗兔二抗工作液(1∶200)37 ℃避光孵育1 h,4'6-二脒基-2-苯基吲哚(4'6-diamidino-2-phenylindole,DAPI)避光孵育5 min,防荧光淬灭剂封片,激光共聚焦显微镜观察并拍照。结果使用ImageJ 1.52软件进行统计分析。

1.8 数据统计与分析

使用SPSS 23.0统计软件分析数据。采用单因素方差分析(one-way ANOVA)程序分析细胞活力、炎症细胞因子分泌和基因mRNA表达水平以及相关信号通路蛋白表达水平的统计学差异,差异显著时采用Duncan氏法进行多重比较。P<0.05表示差异显著,P<0.01表示差异极显著。

2 结果与分析

2.1 GL对IPEC-J2细胞活力的影响

为确定GL的处理条件,用不同浓度(0、10、50、100、200、500 μmol/L)GL分别处理IPEC-J2细胞12、24和48 h。如图1所示,处理12 h时,不同浓度GL对细胞活力无显著影响(P>0.05);处理24 h时,GL浓度为50 μmol/L时细胞活力高于未添加GL时,而GL浓度为10、100、200、500 μmol/L时细胞活力均低于未添加GL时,但不同甘草酸浓度间差异不显著(P>0.05);处理48 h时,随着GL浓度的升高,细胞活性呈先升后降的趋势,当浓度为500 μmol/L时,细胞活力急剧下降,显著低于其他浓度时(P<0.05)。因此,本试验选择50 μmol/L的GL处理24 h作为后续处理条件。
图1 GL对IPEC-J2细胞活力的影响

同一处理时间时,数据点标注不同小写字母表示不同甘草酸浓度间差异显著(P<0.05),相同字母表示不同甘草酸浓度间差异不显著(P>0.05)。

Fig.1 Effects of GL on viability of IPEC-J2 cells

On the same treatment time, data points with different small letters indicated significant difference between different GL concentrations (P<0.05), while with the same letter indicated no significant difference between different GL concentrations (P>0.05).

2.2 GL对LPS刺激后IPEC-J2细胞中炎症细胞因子分泌的影响

为探究GL在IPEC-J2炎症反应中的作用,用LPS刺激IPEC-J2产生炎症,LPS作用TLR4后,激活相关信号通路引起炎症细胞因子IL-1β、IL-6及TNF-α等的表达。本试验用50 μmol/L的GL预处理IPEC-J2细胞12 h,再用10 μg/mL的LPS共同处理12 h,采用ELISA法检测细胞上清液中IL-6、IL-1β、TNF-α的水平。如图2-A、图2-B和图2-C所示,与对照组相比,LPS刺激细胞后,细胞上清液中IL-1β、IL-6、TNF-α的水平显著升高(P<0.05),GL预处理可显著降低LPS刺激后细胞上清液中IL-6的水平(P<0.05),但对IL-1β、TNF-α的水平无显著影响(P<0.05)。LPS刺激细胞后,细胞内HMGB1被动释放,诱导炎症反应。GL作为HMGB1的抑制剂,可能通过抑制HMGB1的活性来缓解炎症,因此本试验采用ELISA法检测了细胞上清液中HMGB1的水平,如图2-D所示,细胞上清液中HMGB1的水平各组间无显著差异(P>0.05)。
图2 GL对LPS刺激后IPEC-J2细胞上清液中炎症细胞因子水平的影响

数据柱标注不同小写字母表示组间差异显著(P<0.05),无字母或相同字母表示组间差异不显著(P>0.05)。下图同。

Fig.2 Effects of GL on levels of inflammatory cytokines in supernatant of IPEC-J2 cells stimulated with LPS

Data columns with different small letters indicated significant difference between groups (P<0.05), while without letter or with the same letter indicated no significant difference between groups (P>0.05). The same as below.

2.3 GL对LPS刺激后IPEC-J2细胞中炎症细胞因子mRNA表达水平的影响

本试验进一步检测了细胞中IL-6、TNF-α、IL-1β的mRNA表达水平变化。如图3-A所示,相对于对照组,LPS刺激显著增加了细胞中IL-6的mRNA表达水平(P<0.05);相对于LPS组,GL预处理显著降低了LPS刺激后细胞中IL-6的mRNA表达水平(P<0.05)。如图3-B和图3-C所示,细胞中TNF-α、IL-1β的mRNA表达水平各组间无显著差异(P>0.05)。
图3 GL对LPS刺激后IPEC-J2细胞中炎症细胞因子mRNA表达水平的影响

Fig.3 Effects of GL on mRNA expression levels of inflammatory cytokines in IPEC-J2 cells stimulated with LPS

2.4 GL对LPS刺激后IPEC-J2细胞中TLR4信号通路相关蛋白表达水平的影响

为了探究GL缓解LPS刺激下IPEC-J2炎症发生的相关机制,本试验采用Western blotting和免疫荧光染色法检测了细胞TLR4信号通路中NF-κB和MAPK信号通路相关蛋白表达水平的变化。采用Western blotting法检测细胞中NF-κB p65表达水平,由图4-B和图4-C可知,细胞中NF-κB p65及p-NF-κB p65的蛋白表达水平各组间均无显著差异(P>0.05)。LPS作用TLR4后还可以通过活化MAPK信号通路,介导胞外信号进入细胞调节炎症反应,因此本试验采用Western blotting法检测了细胞中p38 MAPK、p-JNK1/3以及c-Jun的蛋白表达水平。由图4-D可知,p38 MAPK的蛋白表达水平不同组间无显著差异(P>0.05)。由图4-E和图4-F可知,相对于对照组,LPS组细胞中p-JNK1/3和c-Jun的蛋白表达水平显著升高(P<0.05);相对于LPS组,GL预处理后显著降低了LPS刺激后细胞中p-JNK1/3和c-Jun的蛋白表达水平(P<0.05)。采用免疫荧光法检测了细胞核中p-c-Jun的蛋白表达水平变化,由图5-A、图5-B可知,LPS组细胞核中p-c-Jun的荧光强度显著高于对照组(P<0.05);相对于LPS组,GL预处理可以显著减弱细胞核中p-c-Jun的荧光强度(P<0.05)。以上结果说明,相对于对照组,LPS刺激显著增加了p-JNK1/3的水平,并显著增加了p-c-Jun和c-Jun的水平,且相对于LPS组,GL预处理可以显著降低p-JNK1/3的水平,并进一步显著降低p-c-Jun与c-Jun的水平。
图4 GL对LPS刺激后IPEC-J2细胞中TLR4信号通路相关蛋白表达水平的影响

图A为Western blotting法检测细胞中TLR4信号通路相关蛋白的表达情况;图B~图E分别为细胞中NF-κB p65、p-NF-κB p65、p38 MAPK、p-JNK1/3和c-Jun的蛋白表达水平。

Fig.4 Effects of GL on expression levels of proteins related with TLR4 signal pathway in IPEC-J2 cells stimulated with LPS

Figure A showed the expression of proteins related with TLR4 signal pathway in cells detected by Western blotting; figures B to E showed the protein expression levels of NF-κB p65, p-NF-κB p65, p38 MAPK, p-JNK1/3 and c-Jun in cells, respectively.

图5 GL对LPS刺激后IPEC-J2细胞核中JNK信号通路相关蛋白荧光强度的影响

图A为激光共聚焦显微镜观察到的细胞核中p-c-Jun的荧光图像;B为细胞核中p-c-Jun的荧光强度。

Fig.5 Effects of GL on fluorescence intensity of protein associated with JNK signal pathway in IPEC-J2 cell nucleus stimulated with LPS

Figure A showed the fluorescence images of p-c-Jun in cell nucleus visualized by confocal microscopy; figure B showed the fluorescence intensity of p-c-Jun in cell nucleus.

3 讨论

LPS也称为内毒素,是革兰氏阴性菌外膜的主要生物活性成分[16]。LPS会导致猪肠道炎症,损害肠道健康,故目前LPS刺激IPEC-J2细胞产生炎症是目前较为理想的研究模型[17]。甘草是目前最常用的中草药之一,南朝医学家陶弘景将甘草尊为“国老”。GL作为甘草中主要的活性功能成分,在2003年被联合国粮农组织和世卫组织食品添加剂联合专家委员会列入了使用名单,已经被证实在体内体外都具有重要的抗炎、抗氧化、抗菌等作用[18]。有研究表明,GL可缓解沙门氏菌诱导的小鼠空肠炎症反应以及肠道菌群失调[19]。除此之外,GL还可以提高肉鸡的生长性能与免疫状态[20]。但当前GL在缓解猪肠道炎症中发挥的作用的相关研究还比较少见,本试验通过研究GL对LPS诱导的IPEC-J2细胞炎症反应的影响,证明了GL对于LPS诱导IPEC-J2细胞炎症的抑制作用可能与JNK信号通路有关。
在炎症发生过程中,细胞因子水平变化起着至关重要的作用,IL-6是一种促炎细胞因子,发挥传递病原体入侵信号或传递组织损伤信号[21],从而刺激急性炎症反应发生的作用。在生理条件下,IL-6几乎无法检测到[22],但IL-6分泌过度则会导致机体炎症相关疾病的产生[23]。有研究发现,在大鼠溃疡性结肠炎模型中,IL-6的水平显著升高,茜草可以通过下调IL-6的水平改善结肠组织病理学变化以及炎症反应[24]。Wu等[25]发现,通过下调大鼠神经炎症中IL-6的水平,可以缓解大鼠的神经炎症,减轻神经元损失和认知缺陷的症状。在本试验中,LPS刺激IPEC-J2细胞产生炎症,细胞中IL-6的基因表达及分泌水平显著升高,但GL预处理可以显著降低LPS刺激后IL-6水平的升高,改善细胞炎症,这表明GL可以有效缓解LPS诱导的IPEC-J2细胞中IL-6的产生,从而调节炎症的发生。
LPS作用TLR4后,可能会激活NF-κB和MAPK信号通路,调节机体炎症反应[26]。NF-κB是几乎存在于所有细胞中的一种转录因子,在细胞炎症反应中发挥重要作用,可诱导TNF-α、IL-1β以及IL-6等促炎细胞因子的产生[27]。有研究表明,GL能抑制LPS活化的TLR4/NF-κB信号通路,减轻小鼠IEC-6细胞的炎性损伤[28]。在本研究中,GL可显著降低LPS刺激下IPEC-J2细胞中TNF-α、IL-1β的分泌,但LPS刺激下TNF-α、IL-1β的转录水平虽高于对照组但无显著差异,与前人结果不一致。在LPS刺激单核细胞的相关研究中,研究人员发现LPS刺激下的单核细胞合成分泌TNF-α及IL-1β存在剂量以及时间依赖性[29]。本试验中,10 μg/mL的LPS刺激IPEC-J2细胞12 h后,TNF-α、IL-1β可能已过合成分泌峰值,导致其基因表达水平相对于对照组无显著差异,但具体机制还有待进一步研究。除此之外,GL以及LPS对NF-κB p65蛋白表达水平无显著影响,故本试验中GL可能是通过其他炎症相关通路来缓解LPS引起的细胞炎症。HMGB1是一种核蛋白,广泛存在于哺乳动物细胞中。细胞受到外界刺激后可被动释放HMGB1参与机体炎症的发生[30]。有研究表明,GL作为HMGB1的抑制剂,可以通过抑制TLR4和NF-κB信号通路抑制NOD样受体热蛋白结构域相关蛋白3(NOD-like receptor thermal protein domain associated protein 3, NLRP3)的产生来改善大鼠的坏死性肠道炎症[31]。但在本研究中,LPS刺激后IPEC-J2细胞培养液中HMGB1水平无显著变化。张芸等[32]在关于LPS诱导RAW264.7细胞内HMGB1转位及释放的研究中也有类似结果,该研究发现RAW264.7细胞接受LPS刺激后活化和释放HMGB1存在剂量依赖关系,较小剂量的LPS不会显著增加细胞内HMGB1的释放,且与其他炎症因子相比,HMGB1的释放时间较为延迟,可能是由于本试验中LPS刺激时间不足以引起IPEC-J2细胞中HMGB1的释放。MAPK信号通路是真核生物信号传递网络中的重要途径之一,是细胞增殖、分化、凋亡以及炎症发生中关键的信号通路[33]。MAPK分为4个亚族,其中JNK信号通路可被外界刺激激活后作用下游多种转录因子[34],其中包括c-Jun、ETS样蛋白1(ETS-like protein 1,ELK1)、ETS2等,产生生长、分化、炎症等生理效应。Ye等[35]研究发现,姜黄素类似物C66可以通过抑制JNK磷酸化来防止心肌细胞的炎症和凋亡,保护心脏免受肥胖引起的心肌损伤。也有研究表明,反式-4-甲氧基肉桂醛抑制了巨噬细胞中由LPS诱导的JNK磷酸化以及p-c-Jun的表达,从而降低诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)和环氧化酶-2(cyclooxygenase-2,COX-2)的水平[36]。综上可知,JNK和c-Jun在机体炎症进程中发挥着重要的作用,故可通过抑制JNK和c-Jun的磷酸化来调节机体炎症的发生。本试验发现,LPS刺激IPEC-J2细胞后,促炎细胞因子IL-6的水平升高,通过检测上游信号通路相关的蛋白的表达水平发现,LPS刺激下细胞中p-JNK1/3、p-c-Jun及c-Jun的蛋白表达水平显著升高,而GL可以通过调节JNK信号通路相关蛋白的表达缓解炎症发生,说明GL可以通过下调JNK信号通路来缓解LPS引起的IPEC-J2细胞的炎症反应,该结果可为GL作为饲料添加剂的使用提供理论依据。

4 结论

综上所述,GL可以通过减弱由LPS刺激引起的JNK磷酸化,进而减少c-Jun和p-c-Jun的水平,降低下游IL-6的基因表达和分泌,来减轻IPEC-J2细胞的炎症反应。

References

Publishing order | Descend order by publishing year | Descend order by cited within
[1]
江慧青, 李宁. 植物提取物对猪肠道健康的影响[J]. 养猪, 2022(1):15-18.

JIANG H Q, LI N. The effects of the plant extracts on intestinal health of pigs[J]. Swine Production, 2022(1):15-18. (in Chinese)

[2]
OKUMURA R, TAKEDA K. Roles of intestinal epithelial cells in the maintenance of gut homeostasis[J]. Experimental and Molecular Medicine, 2017, 49(5):e338.

[3]
GOTO Y, IVANOV I I. Intestinal epithelial cells as mediators of the commensal-host immune crosstalk[J]. Immunology and Cell Biology, 2013, 91(3):204-214.

DOI PMID

[4]
OSWALD I P. Role of intestinal epithelial cells in the innate immune defence of the pig intestine[J]. Veterinary Research, 2006, 37(3):359-368.

PMID

[5]
ARCE C, RAMÍREZ-BOO M, LUCENA C, et al. Innate immune activation of swine intestinal epithelial cell lines (IPEC-J2 and IPI-2I) in response to LPS from Salmonella typhimurium[J]. Comparative Immunology,Microbiology and Infectious Diseases, 2010, 33(2):161-174.

DOI

[6]
XIE K H, SU G Q, CHEN D W, et al. The immunomodulatory function of the porcine β-defensin 129:alleviate inflammatory response induced by LPS in IPEC-J2 cells[J]. International Journal of Biological Macromolecules, 2021, 188:473-481.

DOI

[7]
王斌. L-色氨酸缓解动物肠道炎症的作用机制研究[D]. 博士学位论文. 北京: 中国农业大学, 2018.

WANG B. Study of the mechanism for the regulatory role of L-tryptophan in alleviating intestinal inflammation in animals[D]. Ph.D.Thesis. Beijing: China Agricultural University, 2018. (in Chinese)

[8]
XU M J, WANG J Y, ZHANG X Y, et al. Polysaccharide from Schisandra chinensis acts via LRP-1 to reverse microglia activation through suppression of the NF-κB and MAPK signaling[J]. Journal of Ethnopharmacology, 2020, 256:112798.

DOI

[9]
由大鹏, 张絮颖, 刘丽娜, 等. 替代抗生素饲料添加剂研究进展[J]. 畜牧与饲料科学, 2021, 42(3):39-42,52.

YOU D P, ZHANG X Y, LIU L N, et al. Research advances on substitutes for antibiotics additives[J]. Animal Husbandry and Feed Science, 2021, 42(3):39-42,52. (in Chinese)

[10]
刘一. 甘草提取物及有效成分的抗菌活性研究[D]. 硕士学位论文. 延吉: 延边大学, 2013.

LIU Y. Study on antibacterial activity of extractive and active constituent of glycyrrhizae[D]. Master’s Thesis. Yanji: Yanbian University, 2013. (in Chinese)

[11]
ZHU Z H, TAO W W, LI J P, et al. Rapid determination of flavonoids in licorice and comparison of three licorice species[J]. Journal of Separation Science, 2016, 39(3):473-482.

DOI PMID

[12]
WANG G F, HIRAMOTO K, MA N, et al. Glycyrrhizin attenuates carcinogenesis by inhibiting the inflammatory response in a murine model of colorectal cancer[J]. International Journal of Molecular Sciences, 2021, 22(5):2609.

DOI

[13]
ZHANG X M, HU X, OU J Y, et al. Glycyrrhizin ameliorates radiation enteritis in mice accompanied by the regulation of the HMGB1/TLR4 pathway[J]. Evidence-based Complementary and Alternative Medicine, 2020, 2020:8653783.

[14]
WANG X R, HAO H G, CHU L. Glycyrrhizin inhibits LPS-induced inflammatory mediator production in endometrial epithelial cells[J]. Microbial Pathogenesis, 2017, 109:110-113.

DOI

[15]
WANG Y J, WANG L L, LUO R L, et al. Glycyrrhizic acid against Mycoplasma gallisepticum-induced inflammation and apoptosis through suppressing the MAPK pathway in chickens[J]. Journal of Agricultural and Food Chemistry, 2022, 70(6):1996-2009.

DOI

[16]
SHI L, FANG B, YONG Y H, et al. Chitosan oligosaccharide-mediated attenuation of LPS-induced inflammation in IPEC-J2 cells is related to the TLR4/NF-κB signaling pathway[J]. Carbohydrate Polymers, 2019, 219:269-279.

DOI PMID

[17]
LI H J, HAN R, YONG F, et al. The protective effect of Eleutheroside E against the mechanical barrier dysfunction triggered by lipopolysaccharide in IPEC-J2 cells[J]. Research in Veterinary Science, 2023, 154:1-7.

DOI

[18]
李林霏, 毛福英, 李斯琦, 等. 甘草甜素的药理活性、作用机制及其应用进展[J]. 中华中医药学刊, 2022, 40(1):242-247.

LI L F, MAO F Y, LI S Q, et al. Pharmacological activity,mechanism of action and application progress of glycyrrhizin[J]. Chinese Archives of Traditional Chinese Medicine, 2022, 40(1):242-247.

[19]
WANG B K, YE X L, ZHOU Y H, et al. Glycyrrhizin attenuates Salmonella typhimurium-induced tissue injury,inflammatory response,and intestinal dysbiosis in C57BL/6 mice[J]. Frontiers in Veterinary Science, 2021, 8:648698.

DOI

[20]
高嘉豪, 乔彦杰, 连科迅, 等. 甘草酸对脂多糖应激黄羽肉仔鸡免疫性能的影响[J]. 中国畜牧兽医, 2022, 49(9):3419-3427.

GAO J H, QIAO Y J, LIAN K X, et al. Effects of glycyrrhizic acid on immune performance of lipopolysaccharide-stressed yellow feather broilers[J]. China Animal Husbandry & Veterinary Medicine, 2022, 49(9):3419-3427. (in Chinese)

[21]
TANAKA T, NARAZAKI M, KISHIMOTO T. IL-6 in inflammation,immunity,and disease[J]. Cold Spring Harbor Perspectives in Biology, 2014, 6(10):a016295.

[22]
HIRANO T. IL-6 in inflammation,autoimmunity and cancer[J]. International Immunology, 2021, 33(3):127-148.

DOI

[23]
GHASEMI H. Roles of IL-6 in ocular inflammation:a review[J]. Ocular Immunology and Inflammation, 2018, 26(1):37-50.

DOI

[24]
QIN W, LUO H, YANG L, et al. Rubia cordifolia L. ameliorates DSS-induced ulcerative colitis in mice through dual inhibition of NLRP3 inflammasome and IL-6/JAK2/STAT3 pathways[J]. Heliyon, 2022, 8(8):e10314.

DOI

[25]
WU Y Y, SUN Z P, WANG T T, et al. Maresin 1 alleviates sevoflurane-induced neuroinflammation in neonatal rats via JAK2/STAT3/IL-6 pathways[J]. International Immunopharmacology, 2022, 108:108912.

DOI

[26]
LI Z G, QI X L, ZHANG X, et al. TRDMT1 exhibited protective effects against LPS-induced inflammation in rats through TLR4-NF-κB/MAPK-TNF-α pathway[J]. Animal Models and Experimental Medicine, 2022, 5(2):172-182.

DOI PMID

[27]
郇长超. HMGB1在猪流行性腹泻病毒感染中作用机制的研究[D]. 博士学位论文. 南京: 南京农业大学, 2016.

XUN C C. The mechanism of HMGB1 in porcine epidemic diarrhea virus infection[D]. Ph.D.Thesis. Nanjing: Nanjing Agricultural University, 2016 (in Chinese)

[28]
TANG Y H, YUE Z S, LI G S, et al. Effect of β-ecdysterone on glucocorticoid-induced apoptosis and autophagy in osteoblasts[J]. Molecular Medicine Reports, 2018, 17(1):158-164.

[29]
马铮铮. 脆弱类杆菌LPS对人外周血单核细胞分泌TNF-α和IL-1β的影响[D]. 硕士学位论文. 长沙: 中南大学, 2006.

MA Z Z. Effects of LPS on secretion of TNF-α and IL-1β by human peripheral blood mononuclear cells[D]. Master’s Thesis. Changsha: Central South University, 2006. (in Chinese)

[30]
罗敏, 肖婷婷, 曾星, 等. 甘草酸对LPS诱导的IEC-6细胞NF-κB通路及炎症因子表达的影响[J]. 中国免疫学杂志, 2019, 35(10):1160-1163,1168.

LUO M, XIAO T T, ZENG X, et al. Effects of glycyrrhizic acid on LPS-induced activation of IEC-6 cells NF-κB pathway and expression of inflammatory factors[J]. Chinese Journal of Immunology, 2019, 35(10):1160-1163,1168. (in Chinese)

[31]
YU R, JIANG S, TAO Y, et al. Inhibition of HMGB1 improves necrotizing enterocolitis by inhibiting NLRP3 via TLR4 and NF-κB signaling pathways[J]. Journal of Cellular Physiology, 2019, 234(8):13431-13438.

DOI PMID

[32]
张芸, 赵中夫. LPS诱导RAW264.7细胞内HMGB1转位及释放实验研究[J]. 长治医学院学报, 2008, 22(4):245-247.

ZHANG Y, ZHAO Z F. HMGB1 translocates from the nucleus to the cytoplasm and releases in RAW264.7 cell activated by LPS[J]. Journal of Changzhi Medical College, 2008, 22(4):245-247. (in Chinese)

[33]
HUANG G H, SHI L Z, CHI H B. Regulation of JNK and p38 MAPK in the immune system:signal integration,propagation and termination[J]. Cytokine, 2009, 48(3):161-169.

DOI

[34]
WANG H, LI X, XIA B, et al. Amelioration of chronic prostatitis by fractions of Mongolian medicine Hosta plantaginea flowers via inhibition of NF-κB,MAPKs,JAK-STAT,and PI3K-Akt signaling pathways in rats[J]. Journal of Ethnopharmacology, 2023, 307:116245.

DOI

[35]
YE L, CHEN X J, WANG M X, et al. Curcumin analogue C66 attenuates obesity-induced myocardial injury by inhibiting JNK-mediated inflammation[J]. Biomedicine & Pharmacotherapy, 2021, 143:112121.

DOI

[36]
SRISOOK K, MANKHONG S, CHIRANTHANUT N, et al. Anti-inflammatory effect of trans-4-methoxycinnamaldehyde from Etlingera pavieana in LPS-stimulated macrophages mediated through inactivation of NF-κB and JNK/c-Jun signaling pathways and in rat models of acute inflammation[J]. Toxicology and Applied Pharmacology, 2019, 371:3-11.

DOI

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