异钩藤碱上调miR-192-5p对TNF-α诱导的人支气管上皮细胞凋亡和炎症因子释放的作用及其机制

doi:10.13481/j.1671-587X.20210308

Abstract

Abstract: Objective

To observe the effect of isorhynchophylline （IRN） on TNF-α-induced the apoptosis and the release of inflammatory factors in the human bronchial epithelial （16HBE） cells， and to explore its possible mechanism.

Methods

After treated with different concentrations［0（control group），2.5， 5.0， 10.0， 20.0， 30.0 and 40.0 μmol·L^－1］ of IRN for 24 h， the 16HBE cells were cultured with 20 mg·L^－1 TNF-α for 18 h. The 16HBE cells were divided into control group， TNF-α group （20 mg·L^－1）， IRN group （20 μmol·L^－1）， TNF-α+IRN group （20 mg·L^－1 TNF-α and 20 μmol·L^－1 IRN）， TNF-α+IRN+miR-192-5p inhibitor group （20 mg·L^－1 TNF-α， 20 μmol·L^－1 IRN and 50 nmol·L^－1 miR-192- 5p inhibitor） and TNF-α+IRN+pcDNA3.1 CXCR5 group ［20 mg·L^－1 TNF-α， 20 μmol·L^－1 IRN and 2 μmol·L^－1 pcDNA3.1-C-X-C chomokine receptor type 5（CXCR5）］. CCK-8 method was used to detect the viabilities of 16HBE cells in various groups； Real-time fluorescence quantitative PCR（RT-qPCR） method was used to detect the expression levels of miR-192-5p and CXCR5 mRNA in the 16HBE cells in various groups；Western blotting method was used to detect the expression levels of interleukins-1β （IL-1β）and monocyte chemoattactant protein-1 （MCP-1），B cell lymphoma-2（Bcl-2）， Cleaved-Caspase 3， CXCR5， and phosphorylated p38（p-P38）， c-Jun N-terminal kinase （JNK）， c-Jun， and nuclear factor-κB （NF-κB） p65 proteins in the 16HBE cells in various groups； ELISA method was used to detect the levels of IL-1β and MCP-1 in supernatant of the 16HBE cells in various groups. TargetScan7.1 website was used to predict the target genes and the targeted binding association between miR-192-5p and CXCR5 was verified by dual-luciferase reporter gene assay.

Results

Compared with control group， the viability of the 16HBE cells in TNF-α group was significantly decreased （P<0.05）；compared with control group， the viabilities of 16HBE cells in 5.0，10.0，20.0， and 30.0 μmol·L^-1 IRN groups were increased （P<0.05）. Compared with control group， the expression levels of miR-192-5p and Bcl-2 proteins in the 16HBE cells in TNF-α group were significantly decreased （P<0.05）， the expression levels of IL-1β， MCP-1，Cleaved-Caspase-3， p-p38， p-JNK， p-c-Jun and p-NF-κB p65 proteins， the apoptotic rate and the levels of IL-1β and MCP-1 in supernatant of the 16HBE cells were significantly increased （P<0.05）； compared with TNF-α group， the expression levels of miR-192-5p and Bcl-2 protein in the 16HBE cells in TNF-α+IRN group were significantly increased （P<0.05）， the the expression levels of IL-1β， MCP-1，Cleaved-Caspase-3， p-p38， p-JNK， p-c-Jun and p-NF-κB p65 proteins， the apoptotic rate and the levels of IL-1β and MCP-1 in supernatant of the 16HBE cells were significantly decreased （P<0.05）； compared with TNF-α+IRN+NC inhibitor group， the expression levels of miR-192-5p and Bcl-2 protein in the 16HBE cells in TNF-α+IRN+ miR-192-5p inhibitor group were significantly decreased （P<0.05），the expression levels of IL-1β， MCP-1，Cleaved-Caspase-3， p-p38， p-JNK， p-c-Jun and p-NF-κB p65 proteins，and the apoptotic rate and the levels of IL-1β and MCP-1 in supernatant of the 16HBE cells were significantly increased （P<0.05）. Compared with NC mimic and NC inhibitor groups， the expression levels of CXCR5 mRNA and protein in the 16HBE cells in miR-192-5p mimic group were significantly decreased （P<0.05）， and the expression levels of CXCR5 mRNA and protein in the 16HBE cells in miR-192-5p inhibitor group were significantly increased （P<0.05）. Compared with control group， the expression level of CXCR5 protein in the 16HBE cells in TNF-α group was significantly increased （P<0.05）， the cell viability was decreased （P<0.05）， and the apoptotic rate and the levels of IL-1β and MCP-1 in supernatant of the 16HBE cells were increased （P<0.05）；compared with TNF-α group， the expression level of CXCR5 protein in the 16HBE cells in TNF-α+IRN group was significantly decreased （P<0.05），the cell viability was increased （P<0.05），and the apoptotic rate and the levels of IL-1β and MCP-1 were significantly decreased （P<0.05）； compared with TNF-α+IRN+ pcDNA3.1 group， the expression level of CXCR5 protein in the 16HBE cells in TNF-α+IRN+ pcDNA3.1 CXCR5 group was significantly increased （P<0.05），the cell viability was decreased （P<0.05），and the apoptotic rate and the levels of IL-1β and MCP-1 in supernatant of the 16HBE cells were increased （P<0.05）.

Conclusion

IRN can reduce the TNF-α-induced apoptosis and release of inflammatory factors in the human bronchial epithelial cells， and its mechanism may be related to the miR-192-5p/MAPKs/NF-κB signal pathway.

Key words: isorhynchophylline, chronic obstructive pulmonary disease, human bronchial epithelial cells, miR-192-5p, C-X-C chemokine receptor type 5

CLC Number:

Congling HOU, Xiaofan LU, Xiaoting LEI, Bin LI, Runyang ZHAO. Effect of isorhynchophylline on apoptosis and release of inflammatory factors in human bronchial epithelial cells induced by TNF-α via up-regulating miR-192-5p and its mechanism[J].Journal of Jilin University(Medicine Edition), 2021, 47(3): 595-607.

Figures/Tables 23

Fig.1

Fig. 2

Fig. 3

Fig. 4

Fig. 5

Fig. 6

Tab.1

Fig. 7

Fig. 8

Fig. 9

Tab.2

Fig. 10

Fig. 11

Fig. 12

Fig. 13

Fig. 14

Fig. 15

Fig. 16

Fig.17

Fig. 18

Fig. 19

Fig. 20

Tab.3

References

1	LI C N， CHEN J X， YUAN W W， et al. Preventive effect of ursolic acid derivative on particulate matter 2.5-induced chronic obstructive pulmonary disease involves suppression of lung inflammation［J］. IUBMB Life， 2020， 72（4）： 632-640.
2	MA X H， JIAO X G， WU J X， et al. The function of Ophiocordyceps sinensis in airway epithelial cell senescence in a rat COPD model［J］. Can Respir J， 2018， 2018： 6080348.
3	LI S， JIN Z， LU X. MicroRNA-192 suppresses cell proliferation and induces apoptosis in human rheumatoid arthritis fibroblast-like synoviocytes by downregulating caveolin 1［J］. Mol Cell Biochem， 2017， 432（1/2）： 123-130.
4	ZHENG J， ZHU L J， IOK IN I， et al. Bone marrow-derived mesenchymal stem cells-secreted exosomal microRNA-192-5p delays inflammatory response in rheumatoid arthritis［J］. Int Immunopharmacol， 2020， 78： 105985.
5	WANG Y， YU C D， ZHANG H Y. Lipopolysaccharides-mediated injury to chondrogenic ATDC5 cells can be relieved by Sinomenine via downregulating microRNA-192［J］. Phytother Res， 2019， 33（7）： 1827-1836.
6	ZHANG F， SUN A S， YU L M， et al. Effects of isorhynchophylline on angiotensin Ⅱ-induced proliferation in rat vascular smooth muscle cells［J］. J Pharm Pharmacol， 2008， 60（12）： 1673-1678.
7	YUAN D， MA B， YANG J Y， et al. Anti-inflammatory effects of rhynchophylline and isorhynchophylline in mouse N9 microglial cells and the molecular mechanism［J］. Int Immunopharmacol， 2009， 9（13/14）： 1549-1554.
8	CHEN F F， QI W， SUN J H， et al. Urinary metabolites of isorhynchophylline in rats and their neuroprotective activities in the HT22 cell assay［J］. Fitoterapia， 2014， 97： 156-163.
9	NDAGIJIMANA A， WANG X M， PAN G X， et al. A review on indole alkaloids isolated from Uncaria rhynchophylla and their pharmacological studies［J］. Fitoterapia， 2013， 86： 35-47.
10	ZHU J Y， WANG W Q， WU X. Isorhynchophylline exerts anti-asthma effects in mice by inhibiting the proliferation of airway smooth muscle cells： The involvement of miR-200a-mediated FOXC1/NF-κB pathway［J］. Biochem Biophys Res Commun， 2020， 521（4）： 1055-1060.
11	XI Z N， QIAO Y H， WANG J F， et al. Gastrodin relieves inflammation injury induced by lipopolysaccharides in MRC-5 cells by up-regulation of miR-103［J］. J Cell Mol Med， 2020， 24（2）： 1451-1459.
12	ZHU J Y， WANG W Q， WU X. Isorhynchophylline exerts anti-asthma effects in mice by inhibiting the proliferation of airway smooth muscle cells： The involvement of miR-200a-mediated FOXC1/NF-κB pathway［J］. Biochem Biophys Res Commun， 2020， 521（4）： 1055-1060.
13	WANG M， LI H， ZHAO Y X， et al. Rhynchophylline attenuates allergic bronchial asthma by inhibiting transforming growth factor-β1-mediated Smad and mitogen-activated protein kinase signaling transductions in vivo and in vitro［J］. Exp Ther Med， 2019， 17（1）： 251-259.
14	ZHOU Z， SU Y H， FA X N. Isorhynchophylline exerts anti-inflammatory and anti-oxidative activities in LPS-stimulated murine alveolar macrophages［J］. Life Sci， 2019， 223： 137-145.
15	XIAN Y F， LIN Z X， MAO Q Q， et al. Protective effect of isorhynchophylline against β-amyloid-induced neurotoxicity in PC12 cells［J］. Cell Mol Neurobiol， 2012， 32（3）： 353-360.
16	YAMAMOTO M， SINGH A， RUAN J， et al. Decreased miR-192 expression in peripheral blood of asthmatic individuals undergoing an allergen inhalation challenge［J］. BMC Genomics， 2012， 13： 655.
17	LOU L L， TIAN M Y， CHANG J X， et al. MiRNA-192-5p attenuates airway remodeling and autophagy in asthma by targeting MMP-16 and ATG7［J］. Biomedecine Pharmacother， 2020， 122： 109692.
18	LI H C， LIN Y B， LI C， et al. Expression of miRNAs in serum exosomes versus hippocampus in methamphetamine-induced rats and intervention of rhynchophylline［J］. Evid Based Complement Alternat Med， 2018， 2018： 8025062.
19	ZHANG K， LIANG Y X， FENG Y C， et al. Decreased epithelial and sputum miR-221-3p associates with airway eosinophilic inflammation and CXCL17 expression in asthma［J］. Am J Physiol Lung Cell Mol Physiol， 2018， 315（2）： L253-L264.
20	HAN Z， ZHU Y， CUI Z， et al. MicroRNA Let-7f-1-3p attenuates smoke-induced apoptosis in bronchial and alveolar epithelial cells in vitro by targeting FOXO1［J］. Eur J Pharmacol， 2019， 862： 172531.
21	ZHANG D F， WU Y B， SUN G Y. miR-192 suppresses T follicular helper cell differentiation by targeting CXCR5 in childhood asthma［J］. Scand J Clin Lab Invest， 2018， 78（3）： 236-242.
22	SONG Y， QU R， ZHU S H， et al. Rhynchophylline attenuates LPS-induced pro-inflammatory responses through down-regulation of MAPK/NF-κB signaling pathways in primary microglia［J］. Phytother Res， 2012， 26（10）： 1528-1533.
23	LIU J F， LEE C W， LIN C Y， et al. CXCL13/CXCR5 interaction facilitates VCAM-1-dependent migration in human osteosarcoma［J］. Int J Mol Sci， 2020， 21（17）： E6095.
24	WU X B， CAO D L， ZHANG X， et al. CXCL13/CXCR5 enhances sodium channel Nav1.8 current density via p38 MAP kinase in primary sensory neurons following inflammatory pain［J］. Sci Rep， 2016， 6： 34836.

Group	IL-1β	MCP-1
Control	48.21±3.24	65.34±5.11
TNF-α	510.40±19.32^*	482.80±24.87^*
TNF-α+IRN	173.20±13.90^△	125.60±15.32^△
TNF-α+IRN+NC inhibitor	171.91±10.81	127.35±12.20
TNF-α+IRN+ miR-192-5p inhibitor	365.62±18.53^#	299.58±24.21^#

Group	p-p38/p38	p-JNK/JNK	p-c-Jun/c-Jun	p?NF?κB p65/ NF?κB p65
Control	1.00±0.09	1.00±0.05	1.00±0.06	1.00±0.06
TNF-α	3.51±0.15^*	2.64±0.10^*	2.23±0.08^*	2.41±0.18^*
TNF-α+IRN	2.38±0.12^△	1.59±0.16^△	1.24±0.03^△	1.54±0.14^△
TNF-α+IRN+NC inhibitor	2.35±0.07	1.65±0.11	1.21±0.06	1.49±0.09
TNF-α+IRN+ miR-192-5p inhibitor	3.14±0.07^#	2.29±0.06^#	1.56±0.09^#	1.97±0.02^#

Group	IL-1β	MCP-1
Control	52.17±3.53	70.68±5.08
TNF-α	497.15±14.89^*	511.57±27.24^*
TNF-α+IRN	154.32±13.24^△	182.11±15.68^△
TNF-α+IRN+pcDNA3.1	150.62±11.35	179.00±12.28
TNF-α+IRN+ pcDNA3.1 CXCR5	267.35±16.55^#	290.85±24.49^#

[1]	Jing HUANG,Ming DING,Xiaoli ZHU,Pingsheng CHEN,Shuhua HAN. Pulmonary epithelioid hemangioendothelioma complicated with chronic obstructive pulmonary disease:A case report and literature review [J]. Journal of Jilin University(Medicine Edition), 2021, 47(1): 196-202.
[2]	LIANG Xiaobo, WU Dehong, WANG Xing, ZHANG Yun, WANG Xiaoyun, LI Guoping. Evaluation on mouse models of chronic obstructive pulmonary disease induced by intranasal instillation of cigarette smoke extract and lipopolysaccharide [J]. Journal of Jilin University(Medicine Edition), 2019, 45(06): 1454-1458.
[3]	REN Fangping, JIANG Yanwen. Application of N-acetylcysteine inhalation in treatment of elderly patients with acute exacerbation of chronic obstructive pulmonary disease [J]. Journal of Jilin University(Medicine Edition), 2019, 45(05): 1141-1145.
[4]	HOU Hailong, TANG Ying, QU Xinglong, HUA Shucheng. Clinical effect of dexmedetomidine combined with non-invasive positive pressure ventilation in treatment of patients with AECOPD complicated with pulmonary encephalopathy and evaluation on its safety [J]. Journal of Jilin University(Medicine Edition), 2018, 44(05): 1014-1019.
[5]	WANG Xiaoyun, LAN Nan, TANG Hongmei, YUAN Xiefang, WANG Xing, LI Guoping. Effect of Lyn on expression of MUC5AC in human bronchial epithelial cells induced by house dust mite and its mechanism [J]. Journal of Jilin University Medicine Edition, 2018, 44(02): 211-215.
[6]	ZHANG Xueyang, MA Ji, MENG Guangping, WANG Qi, LI Wei, XU Yanling, ZHANG Jie, GAO Peng. Analysis on prevalence of chronic obstructive pulmonary disease based on pulmonary function screening in Changchun urban area of Jilin province [J]. Journal of Jilin University Medicine Edition, 2017, 43(05): 1047-1052.
[7]	QI Huiqin, ZHANG Lianlian, XING Liping, SUN Luyao, LIU Ying, YU Zhenxiang. Changes of serum TGF-β and MMP-9 levels in patients with chronic obstructive pulmonary disease after treated with ipratropium bromide combined with N-acety-L-cysteine and their clinical significances [J]. Journal of Jilin University Medicine Edition, 2016, 42(04): 777-782.
[8]	LI Na, HE Ye, LI Minchao. Analysis on correlation between expression level of pressure-sensitive channel protein and airway remodeling in COPD patients [J]. Journal of Jilin University Medicine Edition, 2016, 42(02): 277-282.
[9]	WANG Guoqiang, MU Jia, LI Yuan, YUAN Yuze, WANG Yubo, SONG Chenxue, XIE Jingshu, ZHENG Jingtong, WANG Fang. Influence of NLRP3,AIM2 and CASP1 genes in acute exacerbations of chronic obstructive pulmonary diseases [J]. Journal of Jilin University Medicine Edition, 2016, 42(01): 130-133.
[10]	TANG Wenfang, LIU Rihui, YU Yaqin, LIU Jin, GAO Peng, WANG Ke. Prevalence of COPD among Chinese people aged 40 years and over from 2000 to 2014:A Meta-analysis [J]. Journal of Jilin University Medicine Edition, 2015, 41(05): 961-968.
[11]	WU Zhonglian, HUANG Xuekuan, LUO Yan, ZHANG Chaonan, JIANG Juan. Effects of BeiMuGuaLou powder on expression level of angiotensin Ⅱ in lung tissue of rats with chronic obstructive pulmonary disease [J]. Journal of Jilin University Medicine Edition, 2015, 41(03): 527-531.
[12]	CHEN Xin, LIU Te, WANG Shuyue, ZHANG Wenjing, XIE Jingfang, QIN Yuanming, QIAN Donghua, YE Lin. Effect of daily mean temperature on hospital admissions of patients with acute exacerbation of chronic obstructive pulmonary disease [J]. Journal of Jilin University Medicine Edition, 2015, 41(02): 389-392.
[13]	LI Fang, GUAN Wenxia, REN Fei, TONG Xuexia, SUN Yuning. Expressions of Sirt-1 and Hif-1α in peripheral blood mononuclear cells of patients with chronic obstructive pulmonary disease and their significances [J]. Journal of Jilin University Medicine Edition, 2015, 41(02): 356-361.
[14]	ZHANG Wenjing, JING Hongyu, XU Hongbang, QIAN Donghua. Analysis on association between polymorphisms of IL-4-33C/T and IL-6-572C/G and susceptibility of chronic obstructive pulmonary disease [J]. Journal of Jilin University Medicine Edition, 2015, 41(01): 145-149.
[15]	XU Xiao-guang，JIANG Zhen-yu，DU Min-juan，YANG Ya-qin，JIANG Ying-chao. Evaluation on effectiveness of salmeterol/fluticasonepropionate combined with N-acetylcysteine in treatment of chronic obstructive pulmonary disease [J]. Journal of Jilin University Medicine Edition, 2014, 40(04): 870-874.

Effect of isorhynchophylline on apoptosis and release of inflammatory factors in human bronchial epithelial cells induced by TNF-α via up-regulating miR-192-5p and its mechanism

RichHTML

PDF (PC)

Like

Knowledge

Abstract

Cite this article

share this article

Figures/Tables 23

References

Related Articles 15

Metrics

Comments

Recommended 0