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. 2023 Apr 20;43(4):552–559. [Article in Chinese] doi: 10.12122/j.issn.1673-4254.2023.04.07

小檗碱抑制类风湿关节炎患者的成纤维样滑膜细胞的自噬并促进其凋亡: 基于下调ROS/mTOR信号通路

Berberine inhibits autophagy and promotes apoptosis of fibroblast-like synovial cells from rheumatoid arthritis patients through the ROS/mTOR signaling pathway

Shiye ZONG 1, Jing ZHOU 2, Weiwei CAI 1, Yun YU 1, Ying WANG 1, Yining SONG 1, Jingwen CHENG 1, Yuhui LI 1, Yi GAO 1, Baihai WU 1, Hao XIAN 1, Fang WEI 1,*
PMCID: PMC10202790  PMID: 37202190

Abstract

Objective

To evaluate the regulatory effect of berberine on autophagy and apoptosis balance of fibroblast-like synoviocytes (FLSs) from patients with in rheumatoid arthritis (RA) and explore the mechanism.

Methods

The inhibitory effect of 10, 20, 30, 40, 50, 60, 70, and 80 μmol/L berberine on RA-FLS proliferation was assessed using CCK-8 method. Annexin V/PI and JC-1 immunofluorescence staining was used to analyze the effect of berberine (30 μmol/L) on apoptosis of 25 ng/mL TNF-α- induced RA-FLSs, and Western blotting was performed to detect the changes in the expression levels of autophagy- and apoptosis-related proteins. The cells were further treated with the autophagy inducer RAPA and the autophagy inhibitor chloroquine to observe the changes in autophagic flow by laser confocal detection of mCherry-EGFP-LC3B. RA-FLSs were treated with the reactive oxygen species (ROS) mimic H2O2 or the ROS inhibitor NAC, and the effects of berberine on ROS, mTOR and p-mTOR levels were observed.

Results

The results of CCK-8 assay showed that berberine significantly inhibited the proliferation of RA-FLSs in a time- and concentration-dependent manner. Flow cytometry and JC-1 staining showed that berberine (30 μmol/L) significantly increased apoptosis rate (P < 0.01) and reduced the mitochondrial membrane potential of RA-FLSs (P < 0.05). Berberine treatment obviously decreased the ratios of Bcl-2/Bax (P < 0.05) and LC3B-II/I (P < 0.01) and increased the expression of p62 protein in the cells (P < 0.05). Detection of mCherry-EGFP-LC3B autophagy flow revealed obvious autophagy flow block in berberine-treated RA-FLSs. Berberine significantly reduced the level of ROS in TNF-α-induced RA-FLSs and upregulated the expression level of autophagy-related protein p-mTOR (P < 0.01); this effect was regulated by ROS level, and the combined use of RAPA significantly reduced the pro-apoptotic effect of berberine in RA-FLSs (P < 0.01).

Conclusion

Berberine can inhibit autophagy and promote apoptosis of RA-FLSs by regulating the ROS-mTOR pathway.

Keywords: rheumatoid arthritis, berberine, fibroblast-like synoviocytes, autophagy, apoptosis, reactive oxygen species, mTOR

类风湿关节炎(RA)是一种全身性自身免疫性疾病，主要临床表现为关节滑膜炎症、滑膜异常增生、血管翳形成以及骨和软骨破坏[1]。成纤维样滑膜细胞(FLSs)[2]约占关节滑膜细胞总量的70%[3]，是RA主要的效应细胞[4]，在疾病进展过程中呈现“类肿瘤样增殖”，同时分泌多种基质金属蛋白酶(MMPs)，包括MMP2、MMP9等，以及大量促炎症细胞因子包括肿瘤坏死因子-α(TNF-α)、白细胞介素-1β、白细胞介素-6等，进而导致软骨侵蚀和骨质破坏[4]。

细胞通过自噬可在一定程度上清除外来微生物[5]、促进氧化还原平衡[6]、维持基因组稳态[7]减少伤害性刺激促进的细胞凋亡，保证细胞继续存活[8]；但是，过度自噬也可会导致细胞发生Ⅱ型程序性死亡。值得注意的是，RA临床研究显示，RA患者的滑膜组织中FLSs的自噬水平明显高于骨性关节炎(OA)患者，且其自噬水平与RA患者红细胞沉降率，C反应蛋白，类风湿因子水平呈正相关，提示RA的自噬水平与疾病的活动度和严重程度明显相关[9-11]。因此抑制RA-FLSs自噬，促进其凋亡可能是RA临床治疗的有效策略。

《成方便读》中的四妙丸和《丹溪心法》中的二妙散皆是治疗RA的经典中药方剂，其中黄柏是两味方剂中的君药。现代药理学研究证实，小檗碱(BBR)是黄柏中的主要活性成分，具有抗炎抗菌等多种药理活性，并对实验性关节炎具有良好的治疗作用。我们课题组前期在佐剂性关节炎(AA)大鼠模型中也证实BBR可调控巨噬细胞极化发挥抗关节炎作用，并显著抑制AA大鼠的滑膜的异常增生，改善AA大鼠的骨破坏[12]。同时多项研究报道，BBR可通过多种途径调控肝癌细胞、脂肪细胞自噬[13-15]。但是，BBR对于RA-FLSs的自噬凋亡调节作用及其机制尚未见报道。因此，本研究以RA患者FLSs为研究对象，初步探讨BBR对RA-FLSs凋亡/自噬调控作用及其作用机制。

1. 材料和方法

1.1. 实验材料

1.1.1. 实验细胞和药物

蚌埠医学院伦理委员会批准实验方案(伦科批字[2020]第018号)，蚌埠医学院第一附属医院RA患者知情同意，并签署知情同意书，进行全膝关节/髋关节膝关节置换术后收集滑膜组织。将滑膜组织切成2 mm×2 mm的小块，双抗浸泡后贴于培养瓶中，细胞培养箱中培养爬取FLSs。实验中使用3-8代；BBR(CAS Number: 633-65-8， > 98% purity，大连美仑生物科技有限公司)，TNF-α(PROTEINTECH)，雷帕霉素(RAPA)、氯喹(CQ)、N-乙酰半胱氨酸(NAC，上海麦克林公司)。

1.1.2. 主要试剂和仪器

DMEM高糖培养基(GIBCO)，胎牛血清(FBS)、0.25% 乙二胺四乙酸(EDTA)胰酶、磷酸缓冲盐溶液(PBS)，Cell Counting Kit-8(CCK-8)细胞计数试剂(APE×BIO)、2, 2'-二辛可宁酸(BCA)蛋白浓度测定试剂盒及二甲基亚砜(DMSO；北京索莱宝)，B淋巴细胞瘤-2基因(Bcl2)Polyclonal Antibody，Rabbit-anti-LC3、B淋巴细胞瘤-2 Associated X(Bax)Rabbit、LC3B、SQSTM1/p62、Beclin-1、β-actin、mTOR、Phospho-mTOR(Ser2448)(CST)，CO2细胞培养箱(上海Heal Force)，酶标仪(BioTek)，FACSVerse流式细胞仪(BD)，倒置显微镜、双光子激光共聚焦显微镜(Olympus)，Fusion FX7 Spectra多功能成像仪(Vilber)。

1.2. 实验方法

1.2.1. 细胞培养

RA-FLSs细胞培养于含有20% FBS的DMEM培养基中，置于37 ℃、5% CO2培养箱。当细胞密度达到70%~80%时，加0.25% 的胰酶消化，对细胞进行1∶2~1∶3传代培养。

1.2.2. CCK-8法检测细胞增殖抑制

用含血清的培养液制备细胞悬液(5×104 cell/mL)，取90 μL加至96孔培养板，TNF-α浓度为25 ng/mL，BBR组浓度分别有8个终浓度：10，20，30，40，50，60，70，80 μmol/L，10 μL，每组设6个复孔，培养24、48、72 h，终止培养前，每孔加入CCK-8 10 μL，继续培养2~4 h后，测吸光度A490 nm。细胞存活率(%)=[(处理组A-空白对照组A)(/阴性对照组A-空白对照组A)]×100%，实验重复3次。

1.2.3. 流式细胞仪检测细胞凋亡

将细胞接种于6孔板，待细胞完全贴壁后，加入BBR(30 μmol/L)继续培养24 h后，用冷PBS冲洗2遍，收集冲洗液至相应离心管中做好标记，每孔加入胰酶消化，收集消化后的细胞，1000 r/min，4 ℃离心5 min收集处理后的各组细胞，用PBS洗涤细胞2次1000 r/min，4℃离心5 min，加入100 μL的Binding Buffer重悬细胞；每管加入5 μL Annexin V-FITC、5 μL Propidium Iodide，轻轻混匀；避光、室温反应10 min；加入400 μL的Binding Buffer在1 h内，用流式细胞仪检测。

1.2.4. Western blot法检测

LC3B，p62，Beclin-1，Bcl-2，Bax，mTOR，p-mTOR等蛋白表达收集各组处理后细胞，用4 ℃预冷的PBS洗涤3次，加入80 μL细胞裂解液(含相应蛋白酶、磷酸酶抑制剂)，冰上裂解30 min，提取细胞总蛋白，BCA法测蛋白浓度。10% SDS-PAGE聚丙烯酰胺凝胶电泳分离蛋白，置于冰上220mA恒流转移100 min转至PVDF上，用5%脱脂牛奶的封闭液4 ℃封闭过夜。洗膜后，加入相应一抗抗体(1∶1000稀释)过夜，辣根过氧化物酶标记的二抗(1∶2000稀释)反应2 h。按照ECL显影试剂盒说明，将化学发光增强液A和B两种试剂1∶1混匀涂抹于PVDF膜上。用Vilber凝胶成像系统检测和成像，实验重复3次。

1.2.5. 质粒转染

将5 μL Lipofectamine 2000和5 μL质粒溶液分别与250 μL Opti-MEM混合避光室温放置。5 min后将两种溶液充分混合(混合液体的总体积为510 μL)，室温放置20 min。弃去培养皿中原来的培养基，2 mL PBS清洗2次，20min后将混合液加入培养皿中，DMEM补足至培养皿内液体总体积为2 mL，随后将细胞放回培养箱，6 h后更换含血清的新鲜培养基继续培养，48 h后按具体的实验设计方案进行相关药物处理。

1.2.6. 活性氧检测

将细胞以2×105/mL的密度接种于六孔板，待细胞长至60%左右，药物处理，药物分组为：Control组、TNF-α(25 ng/mL)组、TNF-α + BBR (30 μmol/L)组、H2O2(1 mmol/L)组、TNF-α + NAC(50 μmol/L)组；加入浓度为5 μmol/L的DCFH-DA探针避光染色。在37 ℃细胞培养箱中孵育30 min后，荧光显微镜激发光为488 nm检测氧化态细胞，收集数据并保存文件，使用image J进行数据分析。

1.2.7. 统计学分析

采用SPSS 19.0统计软件进行分析处理，实验数据以均数±标准差表示。组间差异比较采用单因素方差分析和Dunnett's-t检验，P < 0.05表示两组间差异有统计学意义。

2. 结果

2.1. BBR对RA-FLSs增殖的影响

CCK结果显示(图 1)，与空白对照组相比，TNF-α刺激后，RA-FLSs增殖率显著增加(P < 0.01)，BBR对RA-FLSs的增殖抑制作用呈时间和浓度依赖性，表明BBR对TNF-α诱导的RA-FLSs的异常增殖具有显著抑制作用。根据BBR对RA-FLSs的增殖抑制率，我们选择BBR(30 μmol/L)作用24 h为后续实验条件。

图 1.

图 1

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BBR对RA-FLSs增殖抑制作用

Effect of Berberine (BBR) on proliferation of cultured RA-FLSs. n=3, *P < 0.05, **P < 0.01 vs control group.

2.2. BBR对RA-FLSs凋亡的影响

通过流式细胞术检测BBR对RA-FLSs凋亡率，结果显示(图 2A)，TNF-α刺激后RA-FLSs凋亡率无显著变化，但BBR(30 μmol/L)给药24h后，RA-FLSs凋亡率显著增加(P < 0.01，图 2C)。Western blot实验检测凋亡相关蛋白的表达情况(图 2B、C)显示TNF-α处理后抑凋亡蛋白Bcl-2表达增加，促凋亡蛋白Bax表达降低，Bcl-2/Bax比值显著增加(P < 0.01)；而BBR处理后，相较于TNF-α诱导组下调Bcl-2表达，上调Bax蛋白表达，显著降低Bcl-2/Bax的比值(P < 0.01)。BBR对RA-FLSs线粒体膜电位的影响结果显示(图 3)，TNF-α刺激后，JC-1通过线粒体膜进入线粒体内明显增多，红色荧光的多聚体显著增加，而BBR处理后，线粒体膜电位去极化，JC-1从线粒体内释放，绿色荧光的单体形式显著增加(P < 0.01)。

图 2.

图 2

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BBR对RA-FLSs凋亡的影响

Effect of BBR on apoptotic rate and apoptotic proteins in RA-FLSs. A: Apoptosis of RA-FLSs treated with BBR for 24 h detected by flow cytometry. B: Expression of Bcl-2 and Bax in BBR-treated RA-FLSs (n=3). C: Quantitative analysis of apoptosis rate and Bcl-2/Bax ratio (n=3). **P < 0.01 vs control group, ##P < 0.01 vs TNF-α group.

图 3.

图 3

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BBR对RA-FLSs线粒体膜电位的影响

JC-1 staining for detecting mitochondrial membrane potential in RA-FLSs treated with TNF-α alone or in combination with BBR. A: Immunofluorescence assay for detecting JC-1 expression levels (Original magnification: ×200). B: Quantitative analysis of JC-1 staining intensity in each group. *P < 0.05 vs control group, ##P < 0.01 vs TNF-α group.

2.3. BBR对RA-FLSs自噬的影响

Western blot结果显示(图 4A、B)，BBR处理后显著下调RA-FLSs中LC3II/I蛋白(P < 0.01)和Beclin-1的表达(P < 0.05)，上调p62表达水平(P < 0.01)。激光共聚焦显微镜进行串联荧光探针及荧光共定位检测自噬流检测结果显示(图 4C)，TNF-α和RAPA(自噬诱导剂)处理后，细胞内红色和黄色荧光亮点增加，表明自噬流和自噬小体数目增加，自噬增强。而BBR处理后红色亮点和绿色亮点均明显减少，自噬抑制剂CQ作用后，由于CQ是抑制溶酶体导致自噬溶酶体中的pH值较高，EGFP荧光信号在进入溶酶体后没有发生淬灭，因此可见细胞内绿色亮点明显增加，红色荧光亮点和黄色荧光亮点数量均减少。

图 4.

图 4

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BBR对RA-FLSs自噬水平的影响

Effect of Berberine (BBR) on autophagy of TNF-α-induced RA-FLSs. A: Western blotting for detecting expressions of p62 and LC3B in RA-FLSs. B: Quantitative analysis of p62 and LC3B expression levels with Image J (n=3). C: Confocal fluorescent microscopy of LC3 puncta in cells transfected with a mCherry-GFP-LC3B plasmid for 48 h, followed by control solvent (PBS) or BBR treatment for 24 h (×600). The experiments were performed in triplicate. *P < 0.05, **P < 0.01 vs control group, #P < 0.05, ##P < 0.01 vs TNF-α group.

2.4. BBR对RA-FLSs中ROS，mTOR表达水平的影响

TNF-α处理后，ROS表达水平显著升高(P < 0.01)，在使用BBR干预后，显著降低了活化RA-FLSs内的ROS水平(图 5A)。Western blot检测自噬负调控蛋白p-mTOR的表达水平，结果显示TNF-α处理会显著降低p-mTOR的表达水平，BBR干预后可以显著上调p-mTOR的表达水平(图 5 B、C，P < 0.01)。

图 5.

图 5

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BBR对RA-FLSs中ROS和mTOR表达水平的影响

Regulatory effect of BBR on ROS and mTOR in RA-FLSs. A: Immunofluorescence assay for detecting ROS expression level (×200). B, C: Western blotting for detecting mTOR and p-mTOR protein levels in RA-FLSs. **P < 0.01 vs control group, ##P < 0.01 vs TNF-α group, $$P < 0.01 vs H2O2 group.

2.5. RAPA对BBR促RA-FLSs凋亡作用的影响

将自噬诱导剂RAPA(也是mTOR的靶向抑制剂)与BBR联合使用，检测对RA-FLSs凋亡率的影响，结果显示BBR合用RAPA后，RA-FLSs凋亡率相较于BBR组显著降低(图 6，P < 0.01)。

图 6.

图 6

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RAPA对BBR促RA-FLSs凋亡的影响

Effect of modulating mTOR level on apoptosis of RA-FLSs. A: Apoptosis of RA-FLSs detected by flow cytometry after treatment with BBR and RAPA for 24 h. B: Quantitative analysis of apoptosis rate in each group (n=3). ##P < 0.01 vs TNF-α group, & & P < 0.01 vs TNF-α+BBR group.

3. 讨论

RA是我国最常见的系统性风湿病，病情多反复且逐渐加重，最终导致关节结构破坏造成残疾[16]。异常活化的RA-FLSs具有明显的凋亡抗性，引起关节滑膜过度增生并分泌大量的促炎症细胞因子、趋化因子和MMPs，促进慢性滑膜炎症和关节骨质破坏[3]。近年来对于RA-FLSs的凋亡自噬研究逐渐成为热点，但其具体机制尚不明确。本研究聚焦BBR通过ROS/mTOR抑制RA-FLSs自噬促进凋亡的作用及机制，研究结果表明，BBR可以显著抑制TNF-α刺激的RA-FLSs过度增殖，下调RA-FLSs自噬，促进凋亡，其机制与下调ROS/mTOR信号通路有关。

JAK抑制剂托法替尼可阻断多种炎性细胞因子的信号转导，在临床上目前主要用于对甲氨蝶呤疗效不足或无法耐受的中重度活动性RA成人患者的治疗。最新的研究表明，托法替尼对RA-FLSs的自噬抑制作用，有助于其在RA患者治疗中疗效的发挥，而对RA-FLSs的凋亡并无明显影响[17]。事实上，自噬在不同细胞环境中的影响不甚相同，其与凋亡之间的关系较为复杂[10]。有报道在RA-FLSs中，TNF-α、IL-17、IL-38等促炎症细胞因子引起线粒体功能失调，促进自噬和增加凋亡抵抗[18]，Dinesh等[19]报道的BBR抑制IL-21诱导的AAFLSs过度增殖，促进凋亡，同时抑制自噬。我们在实验中也观察到TNF-α刺激后RA-FLSs自噬显著增强，凋亡显著减少。BBR可以显著下调RA-FLSs中自噬标志蛋白LC3BⅡ/Ⅰ的比例和自噬蛋白Beclin-1的表达，上调p62的表达，同时自噬流结果也证实，BBR可以减少RA-FLSs自噬通量。这些结果均表明BBR能抑制RAFLSs的过度自噬，发挥促RA-FLSs凋亡的作用。值得注意的是，自噬诱导剂RAPA可促进RA-FLSs氨基甲酰化，而在RA患者中也发现了抗氨基甲酰化蛋白的特异性IgG抗体[20]。RA队列研究的结果表明，抗瓜氨酸化波形蛋白抗体与抗氨基甲酰化蛋白抗体密切相关[21]。此外，在缺氧条件下，RA-FLSs的自噬水平和IV型肽基精氨酸脱氨酶(PADI4)表达显著上调，表现出过度增殖和凋亡抵抗[22]。PADI4被认为是介导和促进组蛋白瓜氨酸化的关键酶，与RA自身抗体的产生和调节密切相关。因此，RA-FLSs的凋亡减少、自噬增加，可能与促进RA患者体内抗瓜氨酸抗体的形成有关，抑制RA-FLSs的过度自噬促进其凋亡，不仅能够减轻关节滑膜的过度增生，还将有助于抑制RA病人体内瓜氨酸化抗原的呈递和炎症免疫反应的持续活化[23, 24]。

RA过度增殖的关节滑膜中往往伴随着氧化应激失衡，导致ROS大量积聚，增加的ROS参与介导RA-FLSs促炎症细胞因子的分泌和其迁移功能的增强[25, 26]。文献报道，ROS可以通过多种途径参与细胞自噬的调控，包括促进自噬通路相关蛋白Beclin-1 Ser90/Ser93磷酸化，阻碍Beclin-1和Bcl-2结合，诱导自噬发生[18]，以及通过TLR4诱导RA-FLSs自噬[27]。同时，抑制ROS介导的氧化应激反应也被证实在RA-FLSs中具有良好的促凋亡效果[28]。我们的结果发现，ROS能促进RA-FLSs中自噬关键调节分子mTOR的活化，BBR显著下调TNF-α刺激后RA-FLSs中的ROS水平，上调p-mTOR的表达水平。为探究BBR发挥促RA-FLSs凋亡作用是否通过抑制自噬，我们进一步加入mTOR抑制剂RAPA，结果显示，RAPA可以显著降低BBR促RA-FLSs凋亡的作用。随着对自噬与凋亡机制研究的深入，ROS在细胞内的水平也被证实在二者交互作用间扮演重要角色，特别是在PI3K/Akt信号通路[29]，这也更好地解释了为何BBR也可以通过PI3K/Akt通路抑制IL-21/IL-21R介导的AA-FLSs自噬，促进其凋亡[19]。

综上所述，RA-FLSs的凋亡/自噬平衡的改变可能主要是受到ROS通路的调控。本研究表明，BBR可以下调RA-FLSs异常升高的氧化应激水平，下调ROS/mTOR通路，抑制RA-FLSs过度自噬，促进其凋亡，发挥对RA的治疗作用，其具体机制值得进一步研究和阐明。

Biography

宗世烨，在读硕士研究生，E-mail: 2514083935@qq.com

Funding Statement

国家自然科学基金(81703529)；安徽省教育厅自然科学重大项目(KJ2021ZD0086)；蚌埠医学院512人才项目(by51202203)；国家大学生创新创业训练计划项目(201910367036，202010367037)；蚌埠医学院创新药物创新团队(BYKC201904)

Supported by National Natural Science Foundation of China (81703529)

Contributor Information

宗 世烨 (Shiye ZONG), Email: 2514083935@qq.com.

魏 芳 (Fang WEI), Email: weifangmailbox@126.com.

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