浏览全部资源
扫码关注微信
Phillygenin Mitigates LPS/ATP-induced L02 Cell Inflammation by Regulating P2X7R/NF-
κ B/NLRP3 Inflammasome Signaling Pathway- Chinese Journal of Experimental Traditional Medical Formulae Vol. 28, Issue 14, Pages: 61-69(2022)
- 作者机构：
成都中医药大学 药学院 西南中药资源国家重点实验室，成都 611137
- 作者简介：
- 基金信息：
DOI：10.13422/j.cnki.syfjx.20220808
CLC： R2-0;R22;R285.5;R289;R33Published：20 July 2022，
Published Online：28 February 2022，
Received：11 November 2021，
- 稿件说明：
扫 描 看 全 文
邓英,赵兴桃,周梦婷等.连翘脂素通过调控P2X7R/NF-κB/NLRP3炎性小体信号通路缓解LPS/ATP诱导的L02细胞炎症[J].中国实验方剂学杂志,2022,28(14):61-69.
DENG Ying,ZHAO Xingtao,ZHOU Mengting,et al.Phillygenin Mitigates LPS/ATP-induced L02 Cell Inflammation by Regulating P2X7R/NF-κB/NLRP3 Inflammasome Signaling Pathway[J].Chinese Journal of Experimental Traditional Medical Formulae,2022,28(14):61-69.
邓英,赵兴桃,周梦婷等.连翘脂素通过调控P2X7R/NF-κB/NLRP3炎性小体信号通路缓解LPS/ATP诱导的L02细胞炎症[J].中国实验方剂学杂志,2022,28(14):61-69. DOI： 10.13422/j.cnki.syfjx.20220808.
DENG Ying,ZHAO Xingtao,ZHOU Mengting,et al.Phillygenin Mitigates LPS/ATP-induced L02 Cell Inflammation by Regulating P2X7R/NF-κB/NLRP3 Inflammasome Signaling Pathway[J].Chinese Journal of Experimental Traditional Medical Formulae,2022,28(14):61-69. DOI： 10.13422/j.cnki.syfjx.20220808.
摘要
目的
2
为研究连翘脂素（Phillygenin，PHI）对脂多糖（LPS）和腺嘌呤核苷三磷酸（ATP）联合诱导L02细胞中的抗炎药效和对P2X7受体（P2X7R），NOD样蛋白结构域受体3（NLRP3）和核转录因子-
κ
B（NF-
κ
B）表达的影响。
方法
2
本研究采用先给予100 μg
·
L
-1
LPS处理24 h后，再使用5 mmoL·L
-1
ATP 5 h建立L02细胞炎症模型。给药组在给予LPS处理的同时给予不同浓度PHI（100、50、25 mg·L
-1
）培养6 h，随后换液，继续用LPS处理18 h，ATP处理5 h后，采用实时荧光定量聚合酶链式反应（Real-time PCR）和蛋白免疫印迹法（Western blot）检测L02细胞中白细胞介素-1
β
（IL-1
β
）、白细胞介素-18（IL-18）、P2X7R、NLRP3、胱天蛋白酶-1前体（pro-Caspase-1）、裂解胱天蛋白酶-1（cleaved Caspase-1）、NF-
κ
B、NF-
κ
B抑制蛋白
α
（I
κ
B
α
） mRNA和蛋白表达。通过分子对接实验预测P2X7R与PHI能否结合，以及DCFH-DA活性氧（ROS）荧光探针检测细胞中ROS的累积。采用小干扰核糖核酸（siRNA）沉默P2X7R，并随后通过Real-time PCR检测IL-1
β
、IL-18、P2X7R、NLRP3、Caspase-1、NF-
κ
B、I
κ
B
α
mRNA表达情况。
结果
2
Real-time PCR和Western blot分析显示，与空白组比较，模型组IL-1
β
、IL-18的表达均有所升高（
P
<
0.05）；与模型组比较，给药组明显下调了IL-1
β
、IL-18 mRNA和蛋白的表达（
P
<
0.05）。分子对接结果显示PHI与P2X7R有较好的结合作用。Real-time PCR和Western blot分析显示，与空白组比较，模型组P2X7R的表达明显上调（
P
<
0.05）；与模型组比较，给药组下调了P2X7R mRNA和蛋白表达（
P
<
0.05）。ROS荧光探针分析显示，与空白组比较，ROS的含量明显升高（
P
<
0.05）；与模型组比较，给药组明显降低了ROS的积累（
P
<
0.05）。Real-time PCR和Western blot分析显示，与空白组比较，模型组NLRP3炎性小体，NF-
κ
B的表达明显升高（
P
<
0.05）；与模型组比较，给药组明显降低NLRP3、cleaved-Caspase-1和上调NF-
κ
B、I
κ
B
α
mRNA及蛋白表达（
P
<
0.05）。Real-time PCR分析显示，与模型组比较，siRNA沉默P2X7R后，IL-1
β
、IL-18、P2X7R、NLRP3、Caspase-1、NF-
κ
B、I
κ
B
α
mRNA表达明显降低（
P
<
0.05），PHI具有同样的作用，二者合用后，基因表达进一步下降。
结论
2
P2X7R为NF-
κ
B/NLRP3信号通路的上游，PHI可能是通过下调P2X7R从而抑制NF-
κ
B/NLRP3信号通路的表达从而缓解LPS/ATP诱导的L02细胞炎症，提示P2X7R可能是PHI发挥抗炎作用的靶标。
Abstract
Objective
2
To explore the effects of phillygenin （PHI） on the inflammation in L02 cells induced by lipopolysaccharide （LPS） and adenosine triphosphate （ATP） and the expression of purinergic 2X7 receptor （P2X7R）， NOD-like receptor family pyrin domain containing 3 （NLRP3）， and nuclear factor kappa B （NF-
κ
B） expression.
Method
2
In this study， the inflammation model was induced in L02 cells by 100 μg·L
-1
LPS treatment for 24 h and 5 mmoL·L
-1
ATP treatment for 5 h. The cells in the PHI groups were cultured with PHI （100， 50， 25 mg·L
-1
） for 6 h in the LPS treatment period， followed by LPS treatment for another 18 h. After ATP treatment for 5 h， the mRNA and protein expression of interleukin-1
β
（IL-1
β
）， interleukin-18 （IL-18）， P2X7R， NLRP3， Caspase-1 precursor （pro-Caspase-1）， cleaved Caspase-1， NF-
κ
B， and NF-
κ
B inhibitor protein
α
（I
κ
B
α
） in L02 cells was detected by real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） and Western blot， respectively. Molecular docking was used to predict whether P2X7R could bind to PHI， and DCFH-DA was employed to detect the accumulation of reactive oxygen species （ROS） in cells. P2X7R was silenced by small interfering ribonucleic acid （siRNA）， and then the mRNA expression of IL-1
β
， IL-18， P2X7R， NLRP3， Caspase-1， NF-
κ
B， and I
κ
B
α
was detected by Real-time PCR.
Result
2
Real-time PCR and Western blot showed that compared with the normal group， the model group showed increased expression of IL-1
β
and IL-18 （
P
<
0.05）， and compared with the model group， the PHI groups showed down-regulated IL-1
β
， IL-18 mRNA and protein expression （
P
<
0.05）. Molecular docking suggested a good binding effect of PHI to P2X7R. Real-time PCR and Western blot analysis showed that the expression of P2X7R in the model group was significantly up-regulated compared with that in the normal group （
P
<
0.05）， and compared with the model group， the PHI groups showed down-regulated mRNA and protein expression of P2X7R （
P
<
0.05）. DCFH-DA results showed that compared with the normal group， the model group showed increased content of ROS （
P
<
0.05）， and compared with the model group， the PHI groups decreased the accumulation of ROS （
P
<
0.05）. As demonstrated by Real-time PCR and Western blot， compared with the normal group， the model group showed increased expression of NLRP3 inflammasome and NF-
κ
B （
P
<
0.05）， and compared with the model group， the PHI groups significantly decreased the mRNA and protein expression of NLRP3 and cleaved Caspase-1， and up-regulated the mRNA and protein expression of NF-
κ
B and I
κ
B
α
（
P
<
0.05）. Real-time PCR analysis showed that compared with the results in the model group， after silencing P2X7R by siRNA， the mRNA expression of IL-1
β
， IL-18， P2X7R， NLRP3， Caspase-1， NF-
κ
B， and I
κ
B
α
was decreased （
P
<
0.05）. PHI exerted the same effect， and the mRNA expression was further reduced after the combination of them.
Conclusion
2
PHI is presumed to suppress the expression of the NLRP3/NF-
κ
B signaling pathway by down-regulating upstream P2X7R to alleviate the LPS/ATP-induced inflammation in L02 cells， suggesting that P2X7R may be the target of PHI against inflammation.
关键词
连翘脂素P2X7RL02细胞NOD样蛋白结构域受体3（NLRP3）炎性小体核转录因子-κB（NF-κB）
Keywords
phillygeninpurinergic 2X7 receptor （P2X7R）L02 cellNOD-like receptor family pyrin domain containing 3 （NLRP3） inflammasomenuclear factor kappa B （NF-κB）
references
YANGI B，CENGIZ USTUNER M，DINCER M，et al.Propolis protects endotoxin induced acute lung and liver inflammation through attenuating inflammatory responses and oxidative stress［J］.J Med Food，2018，21（11）：1096-1105.
曾祥伟，冯倩，张莹莹，等.葛根素对炎症相关疾病研究进展［J］.中国药理学通报，2018，34（1）：8-11.
SUN M J，CAO Z Q，LENG P.The roles of galectins in hepatic diseases［J］.J Mol Histol，2020，51（5）：473-484.
LEE H N，NA H K，SURH Y J.Resolution of inflammation as a novel chemopreventive strategy［J］.Semin Immunopathol，2013，35（2）：151-161.
冯瑞雪，张再康.论连翘疏肝通经、发汗止呕之厥功［J］.河北中医学院学报，1994（4）：29.
HU N，WANG C，DAI X，et al.Phillygenin inhibits LPS-induced activation and inflammation of LX2 cells by TLR4/MyD88/NF-κB signaling pathway［J］.J Ethnopharmacol，2020，248：112361.
龚莉虹，余琳媛，胡乃华，等.连翘抗炎药效物质基础及其作用机理研究进展［J］.中药与临床，2019，10（1）：43-49.
全云云，袁岸，龚小红，等.连翘抗炎药效物质基础筛选研究［J］.天然产物研究与开发，2017，29（3）：435-438，471.
DENG H，ZHANG Y，LI G G，et al.P2X7 receptor activation aggravates NADPH oxidase 2-induced oxidative stress after intracerebral hemorrhage［J］.Neural Regen Res，2021，16（8）：1582-1591.
LI Z，HUANG Z，ZHANG H，et al.P2X7 receptor induces pyroptotic inflammation and cartilage degradation in osteoarthritis via NF-κB/NLRP3 crosstalk［J］.Oxid Med Cell Longev，2021，2021：8868361.
LEESON H C，KASHERMAN M A，CHAN-LING T，et al.P2X7 receptors regulate phagocytosis and proliferation in adult hippocampal and SVZ neural progenitor cells： Implications for inflammation in neurogenesis［J］.Stem Cells，2018，36（11）：1764-1777.
LI R，WANG J，LI R，et al.ATP/P2X7-NLRP3 axis of dendritic cells participates in the regulation of airway inflammation and hyper-responsiveness in asthma by mediating HMGB1 expression and secretion［J］.Exp Cell Res，2018，366（1）：1-15.
曹淑花，袁绍鹏，侯琦.P2X7与呼吸系统炎症性疾病的研究进展［J］.药学学报，2013，48（8）：1183-1188.
ZHANG Q，HU F，GUO F，et al.Emodin attenuates adenosine triphosphate‑induced pancreatic ductal cell injury in vitro via the inhibition of the P2X7/NLRP3 signaling pathway［J］.Oncol Rep，2019，42（4）：1589-1597.
XU H，XIONG C，HE L，et al.Trans-resveratrol attenuates high fatty acid-induced P2X7 receptor expression and IL-6 release in PC12 cells： Possible role of p38 MAPK pathway［J］.Inflammation，2015，38（1）：327-337.
AMOROSO F，CAPECE M，ROTONDO A，et al.The P2X7 receptor is a key modulator of the PI3K/GSK3β/VEGF signaling network： Evidence in experimental neuroblastoma［J］.Oncogene，2015，34（41）：5240-5251.
HUANG C，YU W，CUI H，et al.P2X7 blockade attenuates mouse liver fibrosis［J］.Mol Med Rep，2014，9（1）：57-62.
LARROUYET-SARTO M L，TAMURA A S，ALVES V S，et al.P2X7 receptor deletion attenuates oxidative stress and liver damage in sepsis［J］.Purinergic Signal，2020，16（4）：561-572.
HOQUE R，SOHAIL M A，SALHANICK S，et al.P2X7 receptor-mediated purinergic signaling promotes liver injury in acetaminophen hepatotoxicity in mice［J］.Am J Physiol Gastrointest Liver Physiol，2012，302（10）：G1171-G1179.
LE DARÉ B，FERRON P J，GICQUEL T.The purinergic P2X7 receptor-NLRP3 inflammasome pathway： A new target in alcoholic liver disease？［J］.Int J Mol Sci，2021，22（4）：2139.
FIGLIUOLO V R，SAVIO L，SAFYA H，et al.P2X7 receptor promotes intestinal inflammation in chemically induced colitis and triggers death of mucosal regulatory T cells［J］.Biochim Biophys Acta Mol Basis Dis，2017，1863（6）：1183-1194.
KAWANO A，TSUKIMOTO M，MORI D，et al.Regulation of P2X7-dependent inflammatory functions by P2X4 receptor in mouse macrophages［J］.Biochem Biophys Res Commun，2012，420（1）：102-107.
ZEMPO H，SUGITA Y，OGAWA M，et al.A P2X7 receptor antagonist attenuates experimental autoimmune myocarditis via suppressed myocardial CD4+ T and macrophage infiltration and NADPH oxidase 2/4 expression in mice［J］.Heart Vessels，2015，30（4）：527-533.
张海丽，游雷鸣，刘慧，等.汉黄芩素对LPS和ATP联合诱导的巨噬细胞炎症反应的抑制作用［J］.中国免疫学杂志，2019，35（9）：1059-1063.
刘富群，高崎，王丹丹，等.银杏酮酯抑制LPS/ATP诱导原代小胶质细胞NLRP3炎症小体的激活机制研究［J］.中国中药杂志，2018，43（16）：3346-3352.
崔东娟，刘涛，赵艳红.香叶木素降低NLRP3炎性小体活性缓解CCl4诱导的肝损伤大鼠肝纤维化［J］.中国免疫学杂志，2019，35（5）：555-559.
BOARU S G，BORKHAM-KAMPHORST E，VAN DE LEUR E ，et al.NLRP3 inflammasome expression is driven by NF-κB in cultured hepatocytes［J］.Biochem Biophys Res Commun，2015，458（3）：700-706.
MURAKAMI T，RUENGSINPINYA L，NAKAMURA E，et al.Cutting edge： G protein subunit β1 negatively regulates NLRP3 inflammasome activation［J］.J Immunol，2019，202（7）：1942-1947.
俞建顺，陈芝芸，吴黎艳，等.胡柚皮黄酮对非酒精性脂肪性肝炎小鼠肝脏NLRP3炎症小体的影响［J］.中国药学杂志，2019，54（24）：2076-2081.
陈洪涛，陈月，吴诗品.NLRP3、AIM2、IFI16炎症小体在慢性乙型病毒性肝炎患者PBMC中的活化水平和与HBV感染的相关性分析［J］.暨南大学学报：自然科学与医学版，2017，38（4）：322-329.
刘雯，郭文洁，徐强，等.NLRP3炎症小体调控机制研究进展［J］.药学学报，2016，51（10）：1505-1512.
YOSHIDA K，OKAMURA H，HIROSHIMA Y，et al.PKR induces the expression of NLRP3 by regulating the NF-κB pathway in Porphyromonas gingivalis-infected osteoblasts［J］.Exp Cell Res，2017，354（1）：57-64.
宋蔚.桂花源连翘脂素的护肝作用及其机制研究［D］.武汉：华中科技大学，2015.
0
Views
8
下载量
3
CSCD
- H-PDFDownload
- L-PDFDownload
- BibTeXDownload
- EndnoteDownload
- RefMan Download
- NoteExpressDownload
- NoteFirstDownload
Publicity Resources
Related Articles
Related Author
Related Institution
- Postal code：100700
- Tel：010-84076882 Email：syfjx_2010@188.com
- Technical support is provided by Beijing Founder electronics co., LTD 京ICP备11006657号-10
京公网安备11010802024621 - It is recommended to read the content of this site in Chrome&IE9+. Please switch to extreme mode in browser 360.
- Cookies We use cookies to help provide and enhance our service and tailor content. By continuing, you agree to the use of cookies.
