Role of NLRC5 in the Activation of Hepatic Stellate Cells Induced by TGF-β1 and Reversal of Hepatic Fibrosis

doi:10.12114/j.issn.1007-9572.2020.00.351

Abstract

Abstract: Background　Liver fibrosis is a pathological change common to various chronic liver diseases.During this process，transforming growth factor-β1（TGF-β1） is significantly increased，which induces the activation of hepatic stellate cells（HSC），promoting process of liver fibrosis.NLRC5，the largest member of NLR family，is an important negative regulator of inflammatory pathway.There is evidence that NLRC5 plays a key role in the development and reversal of liver fibrosis by regulating inflammatory signals and liver fibrosis response.Objective　To investigate the role of NLRC5 in the activation of HSCs induced by TGF-β1 and the reversal of liver fibrosis.Methods　The experiment was implemented from March to July 2018.A total of 24 8-week-old male C57BL/6 mice（body weight 15-20 g） were selected and equally divided into experimental group，normal control group and reversal group using a table of random numbers.After the liver fibrosis model was established in experimental and reversal groups，the mice in all groups were sacrificed，and liver tissues were taken out for measuring liver histological changes with HE staining，Masson trichrome staining and α-smooth muscle actin（α-SMA） immunohistochemistry，respectively.NLRC5 and its mRNA，phosphorylated inhibitor protein αIκBα（P-IκBα），I κ B α，cytoplasmic p65 and nuclear p65 were detected.LX-2 cells were cultured in vitro and were divided into control group，TGF-β1 group and TGF-β1+MDI group.The levels of α-SMA，Col1α1 and Col1α1 mRNA，and NLRC5 and NLRC5 mRNA in LX-2 cells were detected.After being treated with siRNA，activated LX-2 cells were divided into NLRC5-siRNA group，control-siRNA group and normal cell group.NLRC5 and its mRNA，α-SMA，Col1α1，P-IκBα，IκBα，cytoplasmic and nuclear p65 were detected.Results　The detection using HE staining showed that in the normal control group，hepatic cell cords were neat and orderly，without necrotic degeneration and inflammatory cell infiltration，but in the experimental group，potty necrosis，regeneration，steatosis and ballooning degeneration occurred.And in the reversal group，the liver restored ruddy and the structure tended to be normal.The detection with Masson trichrome staining demonstrated that compared with the normal control group，significantly increased blue collagen fibers were observed in the experimental group under the light microscopy，and the increase of blue collagen fibers was also seen in the reversal group but to a lesser degree.The proportion of α-SMA positive cells in the experimental group was higher than that in the normal control group and the reversal group（P<0.05）.The mean levels of NLRC5 and NLRC5 mRNA in liver tissue of experimental group were higher than those of normal control group and reversal group（P<0.05）.The experimental group showed higher mean levels of P-IκBα and nuclear p65 and lower mean level of cytoplasmic p65 in the liver tissue compared with normal control and reversal groups（P<0.05）.The mean levels of α-SMA，Col1α1 and Col1α1 mRNA of LX-2 cells in TGF-β1 group were higher than those in control group and TGF-β1+MDI group（P<0.05）.The mean levels of NLRC5 and NLRC5 mRNA in LX-2 cells of TGF-β1 group were higher than those of control group and TGF-β1+MDI group（P<0.05）.The mean NLRC5 level in the NLRC5-siRNA group was lower than that in the Control-siRNA group〔（0.679±0.045）vs（1.260±0.071）〕（P<0.001）.The mean levels of α-SMA，Col1α1 and Col1α1 mRNA in the NLRC5-siRNA group were lower than those in the Control-siRNA group，but were higher than those in the normal cell group（P<0.05）.Compared to either normal cell group or Control-siRNA group，NLRC5-siRNA group showed higher mean levels of P-IκBα and nuclear p65，and lower mean level of cytoplasmic p65（P<0.05）.Conclusion　NLRC5 can regulate and interfere with key processes related to liver fibrosis by regulating TGF-β1-induced HSC activation and ECM synthesis.It may play a key role in regulating the progression and reversal of liver fibrosis through the NF-κB signaling pathway

Key words: Liver cirrhosis, Transforming growth factor beta1, Hepatic stellate cells, NLRC5, Mice

摘要： 背景　肝纤维化是多种慢性肝病的共同病理改变。在肝纤维化过程中，转化生长因子-β1（TGF-β1）高度表达，诱导肝星状细胞（HSC）活化并促进纤维化的进展。NLRC5作为最主要的核苷酸结合寡聚化结构域NOD样受体（NLRs）家族成员，是炎症途径中的重要负性调节因子。有证据表明，NLRC5是肝纤维化及其逆转的关键调节因子，可以通过调节炎症信号和肝纤维化反应参与肝纤维化的发展及逆转。目的　探讨NLRC5在TGF-β1诱导的HSC活化及逆转中的作用，并分析其与肝纤维化的关系。方法　2018年3—7月选取24只8周龄雄性C57BL/6小鼠（体质量15~20 g），根据随机数字表法分为实验组、正常对照组和逆转组，每组8只。实验组和逆转组建立肝纤维化模型，然后处死小鼠，进行HE、Masson染色和α平滑肌肌动蛋白（α-SMA）免疫组化，观察各组肝纤维化程度；检测小鼠肝组织NLRC5及其mRNA、磷酸化抑制蛋白α IκBα（P-IκBα）、IκBα、胞质p65、核p65水平。体外培养LX-2细胞，将培养的细胞分为对照组、TGF-β1组及TGF-β1+MDI组；检测各组LX-2细胞中α平滑肌肌动蛋白（α-SMA）、Ⅰ型胶原α1（Col1α1）及其mRNA水平，NLRC5及其mRNA水平。siRNA干扰NLRC5后，将激活的LX-2细胞分为NLRC5-siRNA组、Control-siRNA组及正常细胞组，检测NLRC5及其mRNA、α-SMA、Col1α1、P-IκBα、IκBα、胞质p65、核p65。结果　HE染色结果显示正常对照组肝细胞索排列整齐，无坏死变性、炎性细胞浸润现象；实验组小鼠肝组织存在点状坏死和再生现象，可见脂肪变性、气球样变性；逆转组可见肝脏恢复红润，结构趋于正常。Masson染色结果显示与正常对照组相比，实验组小鼠镜下可见大量蓝色胶原纤维；而逆转组胶原纤维增生相比实验组有一定程度的改善。实验组小鼠肝组织中α-SMA阳性细胞比例高于正常对照组和逆转组（P<0.05）。实验组小鼠肝组织中NLRC5、NLRC5 mRNA水平高于正常对照组、逆转组（P<0.05）。实验组小鼠肝组织P-IκBα、核p65水平高于正常对照组及逆转组，胞质p65水平低于正常对照组及逆转组（P<0.05）。TGF-β1组LX-2细胞的α-SMA、Col1α1及其mRNA水平均高于对照组、TGF-β1+MDI组（P<0.05）。TGF-β1组LX-2细胞中NLRC5及其mRNA水平高于对照组、TGF-β1+MDI组（P<0.05）。NLRC5-siRNA组NLRC5水平（0.679±0.045）低于Control-siRNA组（1.260±0.071）（P<0.001）。NLRC5-siRNA组α-SMA、Col1α1及其mRNA水平低于Control-siRNA组，高于正常细胞组（P<0.05）。NLRC5-siRNA组P-IκBα和核p65水平高于正常细胞组和Control-siRNA组，胞质p65水平低于正常细胞组和Control-siRNA组（P<0.05）。结论　NLRC5可调节TGF-β1诱导的HSC活化和细胞外基质（ECM）合成，并可以调节和干扰与肝纤维化相关的关键过程，其可能通过核因子（NF）-κB信号通路在调节肝纤维化进展及逆转中起关键作用。

关键词: 肝硬化, 转化生长因子&beta, 1, 肝星状细胞, NLRC5, 小鼠

ZHANG Yan*,FAN Xiaoxiang,ZHANG Meiwu, et al. Role of NLRC5 in the Activation of Hepatic Stellate Cells Induced by TGF-β1 and Reversal of Hepatic Fibrosis[J]. Chinese General Practice, 2020, 23(24): 3051-3059. DOI: 10.12114/j.issn.1007-9572.2020.00.351.
张燕*,范晓翔,章美武等. LRC5调节转化生长因子-β1诱导的肝星状细胞活化及逆转对肝纤维化的影响[J]. 中国全科医学, 2020, 23(24): 3051-3059. DOI: 10.12114/j.issn.1007-9572.2020.00.351.

References

［1］邱冰峰，徐骐，徐汤舟，等．拉米夫定对乙型肝炎肝硬化患者肝纤维化状态与血小板参数的影响研究［J］．中华医院感染学杂志，2016，26（8）:1700-1702．DOI:10.11816/cn.ni.2016-153154.
QIU B F，XU Q，XU T Z，et al．Influence of lamivudine for fibrosis status and platelet parameters of patients with hepatitis B cirrhosis［J］．Chin J Nosocomiol，2016，26（8） :1700-1702．DOI:10.11816/cn.ni.2016-153154.
［2］刘怀鄂，游晶，洪敏，等．辅助性T淋巴细胞17/调节性 T 淋巴细胞平衡在乙型肝炎病毒感染相关肝纤维化进展过程中的作用研究［J］．中国全科医学，2016，19（18）：2126-2129．DOI:10.3969/j.issn.1007-9572.2016.18.004.
LIU H E，YOU J，HONG M，et al．Study on the role of the balance Th17/treg in the development of hepatitis B virus - related liver fibrosis［J］．Chinese General Practice，2016，19（18）：2126-2129．DOI:10.3969/j.issn.1007-9572.2016.18.004.
［3］劳远翔，贺福初，姜颖.肝星状细胞的去活化机制与抗肝纤维化治疗［J］．中华肝脏病杂志，2015，23（1）:77-80．DOI:10.3760/cma.j.issn.1007-3418.2015.01.019.
LAO Y X，HE F C，JIANG Y.Deactivation mechanisms of hepatic stellate cells and their implications for anti-hepatic fibrosis therapy［J］．Chin J Hepatol，2015，23（1）:77-80．DOI:10.3760/cma.j.issn.1007-3418.2015.01.019.
［4］夏家露，严醒，刘亚如，等．SOCS3对四氯化碳诱导的小鼠肝纤维化进展和逆转的影响及机制研究［J］．中国药理学通报，2017，33（9）：1215-1221．DOI:10.3969/j.issn.1001-1978.2017.09.008.
XIA J L，YAN X，LIU Y R，et al．Effect of SOCS3 on progress of liver fibrosis and reversein mice induced by carbon tetrachloride and its mechanism［J］．Chinese Pharmacological Bulletin，2017，33（9）：1215-1221．DOI:10.3969/j.issn.1001-1978.2017.09.008.
［5］DOLASIA K，BISHT M K，PRADHAN G，et al．TLRs/NLRs：Shaping the landscape of host immunity［J］．Int Rev Immunol，2018，37（1）：3-19．DOI:10.1080/08830185.2017.1397656.
［6］CHANG G B，LIU X P，MA T，et al．A mutation in the NLRC5 promoter limits NF-κB signaling after Salmonella Enteritidis infection in the spleen of young chickens［J］．Gene，2015，568（2）：117-123．DOI:10.1016/j.gene.2015.05.023.
［7］LI L，XU T，HUANG C，et al．NLRC5 mediates cytokine secretion in RAW264.7 macrophages and modulated by the JAK2/STAT3 pathway［J］．Inflammation，2014，37（3）：835-847．DOI:10.1007/s10753-013-9804-y.
［8］CHEN Z，DING T，MA C G.Dexmedetomidine（DEX） protects against hepatic ischemia/reperfusion（I/R） injury by suppressing inflammation and oxidative stress in NLRC5 deficient mice［J］．Biochem Biophys Res Commun，2017，493（2）：1143-1150．DOI:10.1016/j.bbrc.2017.08.017.
［9］MARRONE G，SHAH V H，GRACIA-SANCHO J.Sinusoidal communication in liver fibrosis and regeneration［J］．J Hepatol，2016，65（3）：608-617．DOI:10.1016/j.jhep.2016.04.018.
［10］BALA，CSAK T，SAHA B，et al．The pro-inflammatory effects of miR-155 promote liver fibrosis and alcohol-induced steatohepatitis［J］．J Hepatol，2016，64（6）：1378-1387．DOI:10.1016/j.jhep.2016.01.035.
［11］NIELSEN S R，QUARANTA V，LINFORD A，et al．Macrophage-secreted granulin supports pancreatic cancer metastasis by inducing liver fibrosis［J］．Nat Cell Biol，2016，18（5）：549-560．DOI:10.1038/ncb3340.
［12］JIANG S，ZHANG Y，ZHENG J H，et al．Potentiation of hepatic stellate cell activation by extracellular ATP is dependent on P2X7R-mediated NLRP3 inflammasome activation［J］．Pharmacol Res，2017，117：82-93．DOI:10.1016/j.phrs.2016.11.040.
［13］徐涛，彭云云，李琳，等．pEGFP-C2-NLRC5重组质粒的构建及其表达［J］．安徽医科大学学报，2014，49（1）：5-8．DOI:10.19405/j.cnki.issn1000-1492.2014.01.002.
XU T，PENG Y Y，LI L，et al．Construction of recombinant plasmid pEGFP-C2-NLRC5 and its expression［J］．Acta Universitatis Medicinalis Anhui，2014，49（1）：5-8．DOI:10.19405/j.cnki.issn1000-1492.2014.01.002.
［14］李顶春，李武.TGF-β/Smad、MAPK/ERK、NF-κB信号通路对肝纤维化的影响［J］．现代免疫学，2017，37（5）：427-432.
［15］张望，朱萱.NOX介导的氧化应激对肝纤维化相关信号通路调控的研究进展［J］．山东医药，2017，57（9）：100-103．DOI:10.3969/j.issn.1002-266X.2017.09.035.
［16］LIU C，YUAN X，TAO L，et al．Xia-Yu-Xue decoction（XYXD） reduces carbon tetrachloride（CCl4）-induced liver fibrosis through inhibition hepatic stellate cell activation by targeting NF-κB and TGF-β1 signaling pathways［J］．BMC Complement Altern Med，2015，15：201．DOI:10.1186/s12906-015-0733-1.
［17］CHEN X Q，LIU X，WANG Q X，et al．Pioglitazone inhibits angiotensin II-induced atrial fibroblasts proliferation via NF-κB/TGF-β1/TRIF/TRAF6 pathway［J］．Exp Cell Res，2015，330（1）：43-55．DOI:10.1016/j.yexcr.2014.08.021.
［18］HAN S，DING S，MIAO X，et al．TGF-β1 expression in regulatory NK1.1-CD4+ NKG2D+ T cells dependents on the PI3K-p85α/JNK，NF-κB and STAT3 pathways［J］．Am J Cancer Res，2018，8（3）:489-501.
［19］DAWOODY NEJAD L，BIGLARI A，ANNESE T，et al．Recombinant fibromodulin and decorin effects on NF-κB and TGFβ1 in the 4T1 breast cancer cell line［J］．Oncol Lett，2017，13（6）:4475-4480．DOI:10.3892/ol.2017.5960.

[1]	YU Xueke, LI Mengling, PENG Siyuan, SHEN Yueming, LIANG Lunxi, ZENG Ya. Influence of Gastroesophageal Variceal Bleeding on One-year Mortality and Associated Factors in Patients with Liver Cirrhosis [J]. Chinese General Practice, 2023, 26(14): 1745-1752.
[2]	WANG Xia, YANG Jinhui, ZHENG Mengyao, JIANG Ting, XU Zhiyuan, MA Honglin. Advances in Diagnostic Criteria for Acute-on-chronic Liver Failure [J]. Chinese General Practice, 2023, 26(07): 886-892.
[3]	SHI Xuexiu, YUN Wenjing, WANG Haixu, ZHAO Yingying, YANG Yanan, SUN Tongwen. Clinical Features of Hepatic Cirrhosis in Hypopituitarism [J]. Chinese General Practice, 2023, 26(06): 699-703.
[4]	CHEN Ran, YANG Haoran, SHI Huilian, LIU Qiong, TANG Ting. Hotspots and Trends in Research on Nutrition in Cirrhosis from 1991 to 2021: a Visualized Review [J]. Chinese General Practice, 2022, 25(32): 4091-4098.
[5]	Keng CHEN, Yiyao DENG, Shunlai SHANG, Qinggang LI, Xiangmei CHEN. Chinese Herbal Formula Shenyi Improves Kidney Injury and Inhibits the Activation of the Alternative Complement Pathway in a Mouse Model of Lupus Nephritis [J]. Chinese General Practice, 2022, 25(26): 3298-3307.
[6]	Jingwei CHENG, Yangqing LIU, Yanfang WANG. Effect of Myostatin Gene Knockout on Browning of White Fat and Related Gene Expression Levels in a Mouse Model of Type 2 Diabetes Mellitus [J]. Chinese General Practice, 2022, 25(21): 2610-2617.
[7]	Ming KONG, Manman XU, Yu CHEN. Study on the Relationship between the Levels of Trace Elements and Vitamins in Peripheral Blood of Patients with End-stage Liver Disease and the Etiology and Severity of Disease [J]. Chinese General Practice, 2022, 25(12): 1455-1459.
[8]	ZHANG Yushuang, YU Fuyang, WU Zhongbing, WANG Yiran, LI Jing. Analysis of Gut Flora in a Mouse Model of Esophageal Squamous Cell Carcinoma in Situ [J]. Chinese General Practice, 2022, 25(08): 945-951.
[9]	TIAN Caiyun，HU Han，ZHANG Guoyuan，LIN Shide. Recent Developments in the Diagnosis of Hepatogenous Diabetes　 [J]. Chinese General Practice, 2021, 24(9): 1158-1164.
[10]	ZHANG Jingyi，TANG Yingmei. Novel Advances in the Relationship between Chronic Liver Disease and Thyroid Disorder　 [J]. Chinese General Practice, 2021, 24(33): 4281-4286.
[11]	GUO Maodong，HU Liang，CHEN Yanping，SHI Xin，YE Xiaohua，WANG Qunying，ZHANG Yunyun，DING Jin. A Prediction Nomogram for High-risk Esophageal Varices in Cirrhosis Patients　 [J]. Chinese General Practice, 2021, 24(30): 3848-3855.
[12]	LYU Yanhang，WU Shanshan，WANG Zhenchang，YE Xuejin，FU Yanqing，DUAN Guijiao，SU Xiaowen，NONG Xiaoxin. Clinical，Serum Inflammatory Cytokine，Immune，and Oxidative Stress Responses to Rougan Huaxian Granules with Transplantation of Bone Mesenchymal Stem Cells in Decompensated Cirrhosis Patients　 [J]. Chinese General Practice, 2021, 24(3): 355-362.
[13]	CHEN Shaohua，WANG Chinyun，WU Changgui，ZHAO Xiao，XU Hao，SHI Qi，LIANG Qianqian. Efficacy and Mechanism of Action of Danggui Niantong Decoction against Acute Gouty Arthritis in a Mouse Model：an Experimental Study　 [J]. Chinese General Practice, 2021, 24(24): 3116-3121.
[14]	ZHANG Guoyuan，TIAN Caiyun，HU Han，LIN Shide. Recent Progress in Immune Dysfunction of Neutrophils in Patients with Cirrhosis　 [J]. Chinese General Practice, 2021, 24(21): 2734-2743.
[15]	PAN Yuli，ZENG Xuan，LIN Xiujin，YU Miuyu，ZHANG Shengjie，CHEN Fenglan，WU Junduan. Relationship of the Risk of Adulthood Depression with Serum CRP，IP-10，TGF-β1 and Childhood Trauma：a Case-control Study　 [J]. Chinese General Practice, 2021, 24(11): 1349-1353.