Role of Prostasome-derived miRNA-146a in Regulating TLR-4/NF-κB Pathway and Mechanism of Action of Anemone Tomentosa in Treating Chronic Inflammation in a Rat Model of Experimental Autoimmune Prostatitis

doi:10.12114/j.issn.1007-9572.2022.0731

Chinese General Practice ›› 2023, Vol. 26 ›› Issue (20): 2518-2524.DOI: 10.12114/j.issn.1007-9572.2022.0731

• Original Research • Previous Articles Next Articles

Role of Prostasome-derived miRNA-146a in Regulating TLR-4/NF-κB Pathway and Mechanism of Action of Anemone Tomentosa in Treating Chronic Inflammation in a Rat Model of Experimental Autoimmune Prostatitis

1. Department of Andrology, Ren'ai Branch of the First Affiliated Hospital of Guangxi University of Chinese Medicine, Nanning 530001, China
2. Guangxi Key Laboratory of Molecular Biology of Preventive Medicine of Traditional Chinese Medicine, First School of Clinical Medicine of Guangxi University of Chinese Medicine, Nanning 530023, China
3. Graduate School, Guangxi University of Chinese Medicine, Nanning 530001, China

Received:2022-10-11 Revised:2022-12-02 Published:2023-07-15 Online:2023-02-01
Contact: WU Jinyu

前列腺小体miRNA-146a/TLR-4/NF-κB通路在EAP大鼠慢性炎症中的作用及大火草干预的研究

1．530001　广西壮族自治区南宁市，广西中医药大学第一附属医院仁爱分院男科
2．530023　广西壮族自治区南宁市，广西中医药大学第一临床医学院广西中医药防治医学分子生物重点实验室
3．530001　广西壮族自治区南宁市，广西中医药大学研究生院

通讯作者: 吴金玉
作者简介:
作者贡献：陆良喜、吴金玉、黄志敏提出研究的构思与设计，负责研究的实施推进，并进行结果的分析与解释；陆良喜负责论文起草；史宏负责研究设计的可行性分析和实验过程指导；陆良喜、黄志敏负责动物模型的制备、实验指标的检测；王文杰负责统计分析和绘制图表；邹涵、张知英负责数据收集和整理；陆良喜、吴金玉负责最终版本修订，对论文负责。所有作者确认论文终稿。
基金资助:
广西自然科学基金资助项目(2020GXNSFBA297101)

Abstract

Abstract:

Background

It is urgently necessary to find new drugs for chronic prostatitis (CP) as desired treatment effect is still yet to be achieved. Our previous clinical study has found that Anemone tomentosa (Maxim.) Péi has a good effect on CP, but the specific mechanism of action remains to be further studied.

Objective

To investigate the role of prostasome-derived miRNA-146a in regulating toll-like receptor 4/nuclear factor-κB (TLR-4/NF-κB) pathway and the mechanism of action of Anemone tomentosa in treating chronic inflammation in experimental autoimmune prostatitis (EAP) rats.

Methods

This study was conducted between December 2021 and June 2022. Forty-two Wistar rats were equally divided into seven groups using a random number table, including normal group, model group, low-, medium- and high-dose Anemone tomentosa groups, miRNA-146a mimics and inhibitor groups. After drug intervention, miRNA-146a-5p mRNA in prostasome was detected by real-time polymerase chain reaction, and TLR-4, inhibitor of NF-κB (IκB), phosphorylation of IκB (p-IκB) and tumor necrosis factor receptor-associated factor 6 (TRAF6) protein were detected by western blot, and inflammatory cytokines were detected by enzyme-linked immunosorbent assay.

Results

Compared with normal group, the expression level of miRNA-146a-5p mRNA in prostasome was down-regulated in each of the other six groups (P<0.01). Compared with model group, the expression level of miRNA-146a-5p mRNA in prostasome was up-regulated in medium- and high-dose Anemone tomentosa groups, and miRNA-146a mimics group (P<0.01), but was down-regulated in miRNA-146a inhibitor group (P<0.01). In Anemone tomentosa and miRNA-146a mimics groups, the expression levels of TLR-4, p-IκBα and TRAF6 proteins in prostate tissues were significantly down-regulated (P<0.01), the expression levels of IκBα proteins in prostate tissues were significantly up-regulated (P<0.01), and serum levels of TNF-α, interleukin (IL) -6, IL-8 and interferon-γ were significantly down-regulated (P<0.05), indicating that Anemone tomentosa and miRNA-146a mimics significantly reduced the inflammatory cell infiltration and improved the pathological injury in prostate tissue.

Conclusion

Prostasome-derived miRNA-146a can participate in local pathological changes of prostate via regulating the activation of TLR-4/NF-κB pathway in EAP rats. The mechanism of action of Anemone tomentosa inhibiting chronic inflammation in EAP rats may be related to its regulation of prostasome-derived miRNA-146a modulating the TLR-4/NF-κB pathway.

Key words: Chronic prostatitis, Prostasome, miRNA-146a, TLR-4, NF-κB, Rats, Znflammation

摘要：

背景

目前对慢性前列腺炎（CP）的治疗未达到预期的疗效，亟需寻找新的治疗药物，本课题组前期研究发现大火草〔Anemone tomentosa （Maxim.）Péi〕对CP有较好的疗效，但具体作用机制还有待进一步研究。

目的

探讨前列腺小体源性微小RNA-146a（miRNA-146a）调控Toll样受体4（TLR-4）/核转录因子κB（NF-κB）通路在自身免疫性前列腺炎（EAP）大鼠慢性炎症中的作用及大火草干预的可能机制。

方法

2021年12月—2022年6月，将42只Wistar大鼠采用随机数字表法分正常组、模型组、大火草低剂量组、大火草中剂量组、大火草高剂量组、miRNA-146a激动剂（mimics）组、miRNA-146a抑制剂（inhibitor）组，每组6只。药物干预后，采用实时荧光定量聚合酶链式反应（RT-PCR）检测前列腺小体miRNA-146a-5p mRNA，采用蛋白质印迹法检测TLR-4、细胞内抑制性蛋白κB（IκB）、磷酸化细胞内抑制性蛋白κB（p-IκB）、肿瘤坏死因子受体相关因子6（TRAF6），酶联免疫吸附剂测定法（ELISA）检测炎症因子。

结果

与正常组比较，各组前列腺小体miRNA-146a-5p mRNA表达均下调（P<0.01）。与模型组比较，大火草中、高剂量组，miRNA-146a mimics组前列腺小体miRNA-146a-5p mRNA表达均上调（P<0.01）；miRNA-146a inhibitor组前列腺小体miRNA-146a-5p mRNA表达下调（P<0.01）。大火草和miRNA-146a mimics可以显著降低前列腺组织TLR-4、p-IκBα、TRAF6蛋白表达（P<0.01）；促进前列腺组织IκBα蛋白表达上调（P<0.01）；降低大鼠血清中肿瘤坏死因子α（TNF-α）、白介素6（IL-6）、白介素8（IL-8）、干扰素γ（IFN-γ）水平（P<0.05）。大火草和miRNA-146a mimics可以减轻前列腺组织炎症细胞浸润，改善前列腺组织病理损伤。

结论

前列腺小体源性miRNA-146a可以调节TLR-4/NF-κB通路参与EAP大鼠前列腺局部病理变化。大火草抗EAP大鼠慢性炎症的作用机制可能与其调控前列腺小体源性miRNA-146a/TLR-4/NF-κB通路有关。

关键词: 慢性前列腺炎, 前列腺小体, 微小RNA-146a, Toll样受体4, 核转录因子κB, 大鼠, 炎症

LU Liangxi,SHI Hong,HUANG Zhimin, et al. Role of Prostasome-derived miRNA-146a in Regulating TLR-4/NF-κB Pathway and Mechanism of Action of Anemone Tomentosa in Treating Chronic Inflammation in a Rat Model of Experimental Autoimmune Prostatitis[J]. Chinese General Practice, 2023, 26(20): 2518-2524. DOI: 10.12114/j.issn.1007-9572.2022.0731.
陆良喜,史宏,黄志敏等. 前列腺小体miRNA-146a/TLR-4/NF-κB通路在EAP大鼠慢性炎症中的作用及大火草干预的研究[J]. 中国全科医学, 2023, 26(20): 2518-2524. DOI: 10.12114/j.issn.1007-9572.2022.0731.

Figures/Tables 7

References 20

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基因	引物
miRNA-146a-5p	5'-TGAGAACTGAATTCCATGGGTT-3'
U6	5'-CGCAAGGATGACACGCAAAT-3'

基因	引物
miRNA-146a-5p	5'-TGAGAACTGAATTCCATGGGTT-3'
U6	5'-CGCAAGGATGACACGCAAAT-3'

组别	只数	miRNA-146a-5p
正常组	6	1.123±0.140
模型组	6	0.439±0.116^a
miRNA-146a mimics组	6	0.848±0.093^ab
miRNA-146a inhibitor组	6	0.212±0.028^ab
大火草低剂量组	6	0.535±0.118^a
大火草中剂量组	6	0.724±0.105^ab
大火草高剂量组	6	0.747±0.116^ab
F值		45.455
P值		<0.001

组别	只数	miRNA-146a-5p
正常组	6	1.123±0.140
模型组	6	0.439±0.116^a
miRNA-146a mimics组	6	0.848±0.093^ab
miRNA-146a inhibitor组	6	0.212±0.028^ab
大火草低剂量组	6	0.535±0.118^a
大火草中剂量组	6	0.724±0.105^ab
大火草高剂量组	6	0.747±0.116^ab
F值		45.455
P值		<0.001

组别	只数	TLR-4〔M（QR）〕	TRAF6〔M（QR）〕	IκBα〔M（QR）〕	p-IκBα（±s）
正常组	6	0.212（0.050）	0.500（0.052）	0.600（0.019）	0.342±0.043
模型组	6	0.763（0.154）^a	1.160（0.085）^a	0.368（0.098）^a	1.032±0.079^a
miRNA-146a mimics组	6	0.350（0.108）^ab	0.881（0.137）^ab	0.521（0.135）^b	0.816±0.042^ab
miRNA-146a inhibitor组	6	0.935（0.189）^ab	1.198（0.238）^a	0.413（0.123）^a	1.179±0.079^ab
大火草低剂量组	6	0.577（0.091）^ab	0.832（0.160）^ab	0.432（0.079）^a	0.777±0.051^ab
大火草中剂量组	6	0.376（0.055）^ab	0.755（0.252）^ab	0.462（0.144）^a	0.576±0.033^ab
大火草高剂量组	6	0.286（0.041）^ab	0.660（0.140）^ab	0.534（0.063）^ab	0.456±0.034^ab
H（F）值		38.389	35.632	26.330	183.603^c
P值		<0.001	<0.001	<0.001	<0.001

Role of Prostasome-derived miRNA-146a in Regulating TLR-4/NF-κB Pathway and Mechanism of Action of Anemone Tomentosa in Treating Chronic Inflammation in a Rat Model of Experimental Autoimmune Prostatitis

前列腺小体miRNA-146a/TLR-4/NF-κB通路在EAP大鼠慢性炎症中的作用及大火草干预的研究

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组别	只数	TNFα	IL-6	IL-8	IFN-γ
正常组	6	19.589±2.334	78.776±6.796	24.696±1.682	34.953±4.613
模型组	6	53.760±8.883^a	165.526±19.027^a	54.831±8.738^a	77.453±14.843^a
miRNA-146a mimics组	6	31.071±5.242^ab	104.187±13.447^ab	34.788±4.647^ab	54.570±6.281^ab
miRNA-146a inhibitor组	6	61.889±6.150^ab	181.079±18.012^a	62.099±6.097^ab	94.530±10.562^ab
大火草低剂量组	6	47.137±4.768^ab	145.426±15.726^ab	46.512±5.846^ab	73.483±6.794^a
大火草中剂量组	6	38.292±3.514^ab	117.838±13.913^ab	37.103±4.286^ab	59.738±7.556^ab
大火草高剂量组	6	27.717±5.198^ab	86.934±9.526^b	30.117±3.337^b	47.082±7.514^ab
F值		45.267	44.909	38.307	30.854
P值		<0.001	<0.001	<0.001	<0.001

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