唐 莼, 李 昀, 林小軍, 葉靜華, 李偉念, 何志翔, 李芳菲, 蔡小燕△
(1廣州市第一人民醫(yī)院風濕免疫科,廣東 廣州 510180; 2中山大學附屬第三醫(yī)院心胸外科,廣東 廣州 510630)
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泌乳素促進類風濕關節(jié)炎患者外周血單個核細胞分泌白細胞介素-6*
唐 莼1, 李 昀2, 林小軍1, 葉靜華1, 李偉念1, 何志翔1, 李芳菲1, 蔡小燕1△
(1廣州市第一人民醫(yī)院風濕免疫科,廣東 廣州 510180;2中山大學附屬第三醫(yī)院心胸外科,廣東 廣州 510630)
目的: 探討類風濕關節(jié)炎(RA)患者血清泌乳素(PRL)水平與疾病活動程度的關系,以及PRL促進外周血單個核細胞(PBMCs)分泌白細胞介素-6(IL-6)的機制。方法:收集我院2015年3月至9月40例初治RA患者臨床及實驗室資料。采用化學發(fā)光免疫分析法(CLIA)檢測血清PRL水平,ELISA檢測IL-6水平,RT-qPCR 檢測泌乳素受體(PRLR) mRNA的表達,Western blot法檢測MAPK通路相關蛋白p-p38的蛋白水平。結果:RA患者血清PRL水平明顯升高(P<0.01),活動期RA患者PRL水平明顯高于非活動期RA患者(P<0.01)。PRL水平與DAS28評分、ESR和CRP呈正相關(P<0.01)。RA患者PBMCs中PRLR水平明顯升高(P<0.01)。PRL可誘導PBMCs分泌IL-6,siRNA沉默PRLR或采用MAPK通路抑制劑可抑制IL-6的產生。結論:RA患者血清PRL升高與DAS28評分、ESR和CRP呈正相關,PRL可作為預測RA嚴重程度的指標。PRL通過與PRLR相互作用,激活p38 MAPK通路,從而促進IL-6分泌。
類風濕關節(jié)炎; 泌乳素; 白細胞介素-6; p38
類風濕性關節(jié)炎(rheumatoid arthritis,RA)是一種慢性、彌漫性的自身免疫性疾病,其主要侵犯外周關節(jié)引起滑膜炎癥,進而導致不可逆的關節(jié)破壞,同時關節(jié)外的多處器官包括心、肺、雙眼及皮膚均可受累[1]。目前認為RA以關節(jié)炎癥、滑膜增生和血管翳形成為主要特征,但迄今為止,其發(fā)病機制仍未完全闡明。研究表明,類風濕關節(jié)炎的疾病進展與異常的免疫系統(tǒng)釋放前炎癥細胞因子密切相關,從而導致持續(xù)性滑膜炎癥和關節(jié)軟骨或骨的破壞。其中白細胞介素-6(interleukin-6,IL-6)是一種多效性前炎癥細胞因子,也是RA患者血清和滑液中含量最豐富的細胞因子。IL-6是RA的致病源頭因素之一,IL-6參與RA致病過程中早期B和T細胞的活化,并參與全程致病過程[2]。泌乳素(prolactin,PRL)主要由垂體前葉產生,在細胞免疫和體液免疫過程發(fā)揮重要作用[3]。絲裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)通路是調節(jié)細胞生長、分化、對環(huán)境應激適應、炎癥反應等多種病理生理過程的途徑。RA患者中PRL是否通過MAPK通路促進IL-6釋放尚無文獻報告。本研究通過檢測RA患者PRL、PRLR、IL-6水平及MAPK通路 p-p38蛋白的表達,探討PRL通過MAPK通路促進PBMCs分泌IL-6的作用機制。
1 研究對象
收集2015年3~9月廣州市第一人民醫(yī)院風濕科收治的40例初治RA患者臨床資料,所有患者均符合2009年美國風濕病協(xié)會的RA 分類診斷標準[4]。排除常見引起繼發(fā)性高泌乳素血癥的原因,如泌乳素瘤、下丘腦或垂體病變、甲狀腺功能減退、慢性腎功能不全、嚴重肝病、藥物(口服避孕藥、雌激素、抗精神病藥、抗抑郁藥、西米替丁等)、妊娠及哺乳等。其中男9例,女31例,年齡(30.5±16.3)歲(16~61歲)。根據(jù) RA疾病活動度積分(disease activity score 28,DAS28)進行活動評分,DAS28≥2.6為活動期,DAS28<2.6為穩(wěn)定期。RA活動患者26例,穩(wěn)定患者14例。同時采用20例健康體檢成人標本為對照組。
2 主要試劑
抗泌乳素受體(prolactin receptor, PRLR)抗體、p38抗體、p-p38抗體和PRLRsiRNA購自Santa Cruz;泌乳素化學發(fā)光免疫分析(chemiluminescence immunoassay,CLIA)試劑盒購自Siemens Healthcare Diagnostics;IL-6 ELISA檢測試劑盒和泌乳素蛋白檢測試劑盒購自R&D Systems;轉染、RT-qPCR和Western blot實驗所需試劑均購自Invitrogen;DNA純化盒購自Promega;淋巴細胞分離液購自Nycomed;MAPK抑制劑PD98059購自Sigma-Aldrich。
3 主要方法
3.1 外周血單個核細胞(peripheral blood mononuclear cells, PBMCs)分離 用10 mL肝素抗凝管采集患者及對照者肘靜脈血5 mL,根據(jù)淋巴細胞分離的要求進行密度梯度離心,獲得的PBMCs用RPMI-1640培養(yǎng)以進行進一步實驗。
3.2 實時熒光定量PCR(RT-qPCR)實驗 TRIzol法提取PBMCs總RNA并定量。按試劑盒說明進行逆轉錄合成cDNA。采用MiniOpticon PCR儀(Bio-Rad)進行RT-qPCR檢測,GAPDH作為內參照。PRLR的上游引物為5’-GCAAGCAGTACACCTCCATG-3’,下游引物為 5’-GAGCGTGAACCAACCAGTTT-3’;IL-6的上游引物為5’-CAAATTCGGTACATCCTC-3’,下游引物為 5’-CTGGCTTGTTCCTCACTA-3’;GAPDH的上游引物為5’-ACCACAGTCCATGCCATCAC-3’,下游引物 為5’-TCCACCACCCTGTTGCTGTA-3’。反應體系為20 μL,反應條件為95 ℃ 3 min;95 ℃ 10 s,55 ℃ 30 s,39個循環(huán);在每個循環(huán)延伸末端點收集熒光信號,繪制擴增曲線?;虻谋磉_量用2-ΔΔCt表示。
3.3 Western blot實驗 預冷PBS洗滌細胞3次,加入全蛋白裂解液后冰上裂解10 min收集蛋白。將抽提蛋白用BCA法定量后,取50 μg上樣,于100 V進行SDS-PAGE;電泳,結束后以90 V、90 min將蛋白轉移至PVDF膜; 5% TBS牛奶室溫封閉30 min, I 抗(0.5~1 mg/L)室溫孵育1 h,HRP結合的 II 抗室溫孵育1 h。每次抗體孵育后用TBST洗膜15 min 3次。ECL試劑盒進行發(fā)光反應,壓片、顯影、定影,觀察蛋白條帶并進行ImageJ圖像分析。
3.4 酶聯(lián)免疫吸附實驗(ELISA) 將PBMCs細胞1 500 r/min水平離心10 min,取上清按IL-6 ELISA檢測試劑盒的要求檢測IL-6水平。
3.5 化學發(fā)光免疫分析 血清PRL檢測采用CLIA法,根據(jù)Siemens PRL試劑盒要求檢測。根據(jù)試劑盒要求,定義PRL濃度>17.7 μg/L(男性)或29.2 μg/L(女性)為高泌乳素血癥。
3.6 PRL刺激PBMCs 將分離得到的RA患者及健康成人的PBMCs和培養(yǎng)液鋪于6孔板內,使得每孔細胞數(shù)量為1×109/L,然后加入濃度為200 μg/L的PRL。處理后的細胞放入37 ℃、5% CO2的培養(yǎng)箱內培養(yǎng)48 h,再采用ELISA法檢測IL-6水平。
3.7 細胞轉染 選擇對數(shù)生長期的PBMCs進行轉染,轉染前1 d將生長良好的細胞接種到6孔板中,每孔中接種約5×104個細胞,加入0.5 mL含10% PBS的RPMI-1640培養(yǎng)液,在37 ℃、5% CO2培養(yǎng)箱中培養(yǎng)24 h,當細胞密度達到50%~80%時,根據(jù)Invitrogen 公司的轉染試劑說明書,將PRLRsiRNA轉染至PBMCs以沉默PRLR。然后在37 ℃、5% CO2條件下培養(yǎng)4~6 h,更換正常培養(yǎng)液以終止轉染。轉染后,于24 h內收集細胞,檢測瞬時表達情況及IL-6水平。
3.8 RA相關血液學指標的檢測 采集患者及對照組血液,由我院檢驗科按照要求檢測RA相關血液學指標,包括:(1) 血沉(erythrocytes edimentation rate,ESR)采用魏氏法,其正常值分別為男性≤15 mm/h,女性≤20 mm/h; (2) C反應蛋白(C-reactive protein, CRP)采用散射比濁法,其正常值為<6 mg/L;(3) 類風濕因子(rheumatoid factor,RF)采用散射比濁法,其正常值為≤15 IU/mL。
4 統(tǒng)計學處理
應用統(tǒng)計學軟件SPSS 19.0進行統(tǒng)計學處理。計量數(shù)據(jù)用均數(shù)±標準差(mean±SD)表示,兩組比較采用t檢驗,多組比較用單因素方差分析檢驗。計數(shù)資料組間比較采用2檢驗。PRL與臨床參數(shù)的相關性分析運用Pearson相關分析方法。以P<0.05為差異有統(tǒng)計學顯著性。
1 RA患者血清PRL水平明顯升高
RA患者PRL水平明顯高于健康組(P<0.01);而活動期RA患者PRL水平明顯高于非活動期RA患者(P<0.01),見圖1。根據(jù)血清PRL水平,將RA患者分為高PRL組和正常組,結果提示,高PRL組的活動期RA患者明顯多于非活動期RA患者(P<0.01),見表1。
Figure 1.PRL was increased in the RA patients. A: serum PRL levels of RA patients and healthy controls were detected by CLIA; B: serum PRL levels of active or inactive RA patients were detected by CLIA. Mean±SD.**P<0.01vscontrol;##P<0.01vsinactive RA.
圖1 RA患者血清PRL明顯升高
表1 血清PRL水平與RA患者疾病活動的相關性
Table 1. The correlation between serum PRL and disease activity of RA patients
GroupRAActive(n=26)Inactive(n=14)2PNormalPRL(n=19)71212.2980.001HighPRL(n=21)192
2 RA患者血清PRL與臨床參數(shù)的關系
RA患者血清PRL水平與DAS28評分呈正相關(P<0.01)。同時,PRL水平與ESR和CRP呈正相關(P<0.01),而與RF水平無明顯相關性,見圖2。
3 RA患者PBMCs中PRLR高表達
RT-qPCR檢測PBMCs中PRLR的表達,結果發(fā)現(xiàn)RA患者PBMCs的PRLR水平明顯高于對照組(P<0.01),見圖3。
Figure 2.The correlations between serum PRL and clinical parameters of the RA patients. PRL levels were positively correlated with DAS28 score (A), ESR level (B) and CRP level (C) of the RA patients.
圖2 RA患者血清PRL與臨床參數(shù)的關系
Figure 3. The mRNA expression of PRLR in the PBMCs of the RA patients was enhanced (detected by RT-qPCR).**P<0.01vscontrol.
圖3 RA患者PBMCs的PRLR mRNA表達增高
4 PRL誘導PBMCs分泌IL-6
PRL誘導的RA患者PBMCs分泌IL-6的水平明顯升高(P<0.01)。用siRNA沉默PRLR后,IL-6的表達水平明顯下調(P<0.01),見圖4。
5 PRL通過MAPK通路誘導PBMCs分泌IL-6
采用MAPK抑制劑PD98059作用于PBMCs后,PRL誘導的分泌IL-6水平明顯下降(P<0.01)。Western blot法檢測p-p38的蛋白水平,結果發(fā)現(xiàn)PRL可誘導PBMCs上調p-p38的水平,而用siRNA沉默PRLR后,p-p38的蛋白水平明顯下降,見圖5。
IL-6是具有多種生物活性的多效性促炎癥細胞因子,近年來的研究表明,IL-6/可溶性IL-6受體(soluble interleukin-6 receptor,sIL-6R)與RA的發(fā)病機制和疾病進展密切相關。IL-6/sIL-6R可以打破Thl7/Treg的平衡[5],促進B細胞發(fā)生、分化,并產生大量自身抗體[6]。類風濕關節(jié)炎患者血清及關節(jié)滑液中IL-6/sIL-6R的濃度較高,其水平與受累關節(jié)數(shù)量、疾病活動、晨僵持續(xù)時間、X線改變等密切相關。目前,抗sIL-6R的人源化抗體Tocilizumab已上市,并在RA的治療中取得顯著療效。PRL主要由垂體前葉產生,影響乳腺的生長和分化,促進乳汁分泌。研究表明,PRL參與細胞免疫和體液免疫過程[3, 7],影響人類免疫細胞的增殖和分化[8],近期研究表明, PRL與RA的發(fā)病機制相關,RA患者血清及滑膜液中的PRL 水平明顯升高,其水平與類風濕關節(jié)炎疾病活動度及影像學Larsen 評分相關[9]。
Figure 4.IL-6 release from the PBMCs was induced by PRL. A: PBMCs were stimulated by PRL. The IL-6 release was tested by ELISA. Mean±SD.n=12.**P<0.01vsvehicle. B:PRLRwas knockdown by transfection withPRLRsiRNA (si-PRLR) in the PBMCs of RA patients. The knockdown effect was certificated by Western blot. Mean±SD.n=6.**P<0.01vscontrol. C: the IL-6 release was detected by ELISA. Mean±SD.n=12.*P<0.05,**P<0.01vsPRL+si-PRLR;##P<0.01vssi-PRLR.
圖4 PRL誘導RA 病人的PBMCs分泌IL-6
Figure 5.IL-6 release induced by PRL was interrupted by MAPK inhibitor. A: the IL-6 release was detected by ELISA. Mean±SD.n=12.*P<0.05,**P<0.01vsPRL+PD98059;##P<0.01vsPD98059. B: the protein level of phosphorylated p38 was detected by Western blot. Mean±SD.n=12.*P<0.05,**P<0.01vsPRL+si-PRLR;##P<0.01vssi-PRLR.
圖5 MAPK抑制劑可阻斷PRL誘導PBMCs分泌IL-6
本研究發(fā)現(xiàn),RA患者PBMCs中PRLR表達水平明顯高于對照組,提示PBMCs中PRL可能與PRLR相互作用而產生病理效應。大量證據(jù)表明,PRL/PRLR在糖代謝、脂代謝及細胞、體液免疫調節(jié)及腫瘤發(fā)生發(fā)展中具有重要作用[10],可促進細胞增殖、分化及細胞因子的產生[11]。本研究發(fā)現(xiàn)RA患者PBMCs經(jīng)過PRL處理后,分泌IL-6明顯升高,而siRNA沉默PRLR后,PRL誘導的IL-6分泌作用明顯受到抑制,提示PRL/PRLR相互作用在PBMCs分泌IL-6的機制中具有關鍵作用。
MAPK是一組能被不同的細胞外刺激,如細胞因子、神經(jīng)遞質、激素、細胞應激及細胞黏附等激活的絲氨酸-蘇氨酸蛋白激酶,MAPK通路調節(jié)細胞生長、分化、對環(huán)境的應激適應、炎癥反應等多種重要的細胞病理生理過程[12]。RA患者中PRL是否通過MAPK通路促進IL-6釋放目前暫無相關文獻報告。本研究發(fā)現(xiàn),PRL可誘導MAPK通路蛋白p-p38表達明顯升高,而siRNA沉默PRLR后,p-p38表達水平明顯下降,同時IL-6分泌明顯減少。以上結果提示RA患者PBMCs中PRL通過與PRLR相互作用激活MAPK通路,從而促進IL-6分泌。
綜上所述,RA患者血清PRL水平明顯升高,且與DAS28評分、ESR和CRP呈正相關,提示PRL可作為預測RA嚴重程度的指標。PBMCs表達高水平PRLR,PRL通過激活MAPK通路促進IL-6分泌,提示PRL/PRLR-MAPK p38-IL-6通路在RA發(fā)病中具有一定作用,對其進一步研究將有利于對RA發(fā)病機制的深入理解。
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(責任編輯: 林白霜, 羅 森)
Prolactin induces interleukin-6 expression in PBMCs of rheumatoid arthritis patients
TANG Chun1, LI Yun2, LIN Xiao-jun1, YE Jing-hua1, LI Wei-nian1, HE Zhi-xiang1, LI Fang-fei1, CAI Xiao-yan1
(1DepartmentofRheumatology,GuangzhouFirstPeople’sHospital,Guangzhou510180,China;2DepartmentofCardiothoracicSurgery,TheThirdAffiliatedHospitalofSunYat-senUniversity,Guangzhou510630,China.E-mail:xycai@medmail.com.cn)
AIM: To investigate the correlation between serum prolactin (PRL) levels and disease activity in rheumatoid arthritis (RA) patients, and the regulatory role of PRL in interleukin-6 (IL-6) release from peripheral blood mononuclear cells (PBMCs), and to explore the MAPK-related mechanism of IL-6 release in PBMCs. METHODS: The clinicopathologic and hematologic parameters of 40 new-onset RA patients in the Department of Rheumatology of our hospital between March and September 2015 were collected. Chemilumineseent immunoassay (CLIA) was used to detect the serum PRL levels in the 40 RA patients and 20 healthy controls. The levels of IL-6 secretion by the PBMCs were evaluated using ELISA. Quantitative real-time PCR was employed to examine the mRNA expression of prolactin receptor (PRLR). MAPK pathway protein p-p38 levels were evaluated by Western blot. RESULTS: Serum PRL level in the RA patients was significantly higher than that in the healthy controls (P<0.01). Serum PRL level in active RA patients was significantly higher than that in inactive RA patients (P<0.01). Serum PRL level was positively correlated with DAS28, ESR and CRP (P<0.01). The expression of PRLR in the PBMCs was markedly increased in the RA patients than that in the healthy samples (P<0.01). Exposure of the PBMCs to PRL in the culture increased the release of IL-6, which was abolished byPRLRgene silencing or blocking the MAPK pathway. CONCLUSION: Serum PRL level is related to DAS28, ESR and CRP of RA patients and could be used as a predictor of disease activity. PRL/PRLR-p38 MAPK-IL-6 pathway may play a central role in the pathogenesis of RA.
Rheumatoid arthritis; Prolactin; Interleukin-6; p38
1000- 4718(2016)11- 2062- 05
2016- 05- 19
2016- 09- 18
廣東省科技計劃項目(No.2012B031800274)
R363; R593.2
A
10.3969/j.issn.1000- 4718.2016.11.024
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△ 通訊作者 Tel: 020-81048664; E-mail: xycai@medmail.com.cn