摘要
背景：骨关节炎（Osteoarthritis，OA）是由一组异质性较大的导致各种关节病症的综合征组成，它的特征是关节软骨和一般骨结构的退行性变。现有的治疗方法已经能有效的缓解OA的一些症状，如疼痛、关节僵硬、关节绞索等，但是在逆转关节不断进行的退行性变上还是无能为力的。所以弄清关节退化的机制对于控制OA病程，保护关节结构非常重要。颞下颌关节（temporomandibularjoint，TMJ）在颅面部的生长发育以及各种基本功能中起到重要作用，OA在TMJ中有较高的发生率。许多研究显示，OA的发生与关节内明显升高的炎性因子密切相关，如一氧化氮（nitricoxide，，NO）、肿瘤坏死因子（tumornecrosisfactor-alpha，TNF）、白介素1（interleukin-1beta，IL-1β)等。在对OA进行治疗的过程中，对这些炎性因子进行干预会起到软骨保护作用。很多研究表明颞下颌关节紊乱综合征（temporomandibulardisorder,TMD）在女性中的发病率和严重程度都明显高于男性，特别是在雌激素缺乏的女性中，这提示雌激素有一定的关节保护作用。事实上，大量的研究已经证明雌激素能通过它对炎性因子的抑制作用而起到软骨保护作用。但是关于雌激素软骨保护作用的具体机制目前仍不清楚。
瞬时感受器电位离子通道家族(transientreceptorpotential,TRP)香草素受体亚家族（transientreceptorpotentialvanilloid4，TRPV4）是TRP家族的一员，它是一种非选择性阳离子通道，对钙离子有中等通透性。TRPV4广泛分布于耳蜗毛细胞、默克尔细胞、感觉神经节、游离神经末梢和皮肤A及C纤维终端等处。现有文献显示TRPV4与疼痛相关，可能有软骨保护的功效。虽然雌激素和TRPV4都被证实有一定的软骨保护作用，并且雌激素和钙离子流之间有密切的联系，但是关于TRPV4和雌激素之间的关系的研究还尚未见报道。在本研究中，我们发现在炎性条件下雌激素能在大鼠下颌髁突软骨细胞（mandibularcondylarchondrocytes,MCCs）上以转录后调控的方式使miR-203（MicroRNA-203）表达下调，继而增强TRPV4的表达，随之导致NO产物的下降。本研究论证了在髁突软骨细胞上TRPV4和雌激素可能的相互作用机制，为解释雌激素的软骨保护作用提供了依据。
方法：在本研究中通过检测TRPV4和miR-203在SD大鼠MCCs的表达，研究了二者在雌激素对NO的抑制过程中的作用。首先取材SD大鼠髁突软骨，进行MCCs原代培养，接着观察细胞在接受了不同的刺激后【不同浓度的雌二醇（17β-estradiol，E2）（10-9-10-6M）加Lipopolysa-ccharide（LPS）,TRPV4的激动剂（4α-phorbol12,13-didecanoate,4α－PDD）TRPV4的抑制剂（RutheniumRed）,miR－203】细胞上清中NO、TNF-α、IL-1β的含量的改变。采用Westernblot和RT-PCR分别检测TRPV4蛋白和mRNA水平的变化。并采用real-timePCR测定MCCs在接受不同浓度的E2刺激后，细胞内miR-203的变化情况。同时采用虫荧光素酶报告实验验证TRPV4是否是miR-203的靶基因。
结果：无论是基因水平还是蛋白水平TRPV4都在MCCs丰富表达。在LPS刺激的情况下，E2能抑制NO的浓度，这种抑制作用可被4α-PDD增强，且被RutheniumRed所逆转。并且，E2能通过抑制miR-203的表达，进而增强TRPV4在MCCs上的表达水平。虫荧光素酶报告基因实验证实TRPV4是miR-203的靶基因。转染miR-203模拟物可下调TRPV4蛋白表达水平，并降低E2对NO的抑制作用，与RutheniumRed的作用效果一致。
结论：雌激素E2可以抑制NO的表达，miR-203及其靶蛋白TRPV4可能参与了这一过程。
第一部分
TRPV4在SD大鼠髁突软骨细胞表达的鉴定
许多研究表明TRPV4在炎性痛和神经痛方面起到重要作用。MimiN.Phan（2009）等人报道TRPV4已经在猪的软骨细胞上得到鉴定。但是目前尚未见到TRPV4在MCCs上表达的报导。所以本部分研究的主要目的是证实TRPV4在SD大鼠MCCs上表达。在这部分研究中，首先原代培养了MCCs，然后分别用免疫组化和Westernblot两种方法检测了TRPV4在蛋白水平的表达，用RT-PCR方法检测了TRPV4在基因水平的表达。结果显示TRPV4无论是在基因水平还是蛋白水平都在SD大鼠上MCCs上丰富表达。
第二部分
雌激素通过TRPV4调控炎性因子的研究
发生在TMJ的OA多青睐于女性。事实上大部分有着严重的关节吸收的女性都是雌激素缺乏的患者。但是雌激素在关节软骨究竟起到什么样的作用还存在着很多争议。近年来，越来越多的证据表明雌激素可以通过它的抗炎性因子的作用发挥软骨保护功效。另据报道TRPV4可通过细胞间的信号传递而被性激素调控，但是关于TRPV4与性激素之间的真正作用机制还研究得很少。本部分的研究证实E2能在雌鼠的MCCs上调TRPV4的表达。在这部分研究中，使用酶联免疫吸附测定法（ELISA）和激光共聚焦显微镜检测了在E2、E2+4α-PDD和E2+RutheniumRed的刺激下MCCs内NO、TNF-α、IL-1β和钙离子浓度的变化。结果我们发现在LPS(20ng/ml)刺激的情况下，E2能明显抑制NO、TNF-αandIL-1β的产生。并且，由LPS刺激引起的NO的升高会被TRPV4的抑制剂RutheniumRed加强，相反会被TRPV4的激动剂4α-PDD减弱，这意味着E2对NO的抑制作用是TRPV4依赖性的。本部分研究中还发现E2对TNF-α和IL-1β的抑制作用与TRPV4没有明显的联系。本部分研究的结果为TRPV4在MCCs上的抗炎作用提供了依据，并证实TRPV4参与了E2对NO的抑制作用。
第三部分
miRNA-203通过对其靶基因TRPV4的作用参与了雌激素对NO的下调
在过去的几年中，许多的研究表明miR-203参与了不同的恶性肿瘤的病理过程，同时也有证据显示它在炎症疾病中起到了重要作用。相对于健康人，miR-203在多种炎性疾病病人的细胞中表达明显增高，这意味着miR-203可能是一种促炎性的因子。在本部分研究中，MCCs首先暴露于不同浓度（10-9-10-6M）的E2+LPS,rutheniumred,4α－PDD,以及过表达的miR-203，然后细胞上清被提取，用Greiss法、RT-PCRandWesternblot分别对NO、TRPV4的基因和蛋白水平进行检测。在E2刺激过后，miR-203的基因水平用Real-timePCR进行了检测。同时虫荧光素酶报告基因实验还验证了miR-203的靶基因是否为TRPV4。
RT-PCR结果显示在接受过E2（10-9-10-6M）刺激72小时后，TRPV4mRNA水平明显下降，同时Westernblot显示TRPV4蛋白水平明显上调。这两个相反的结果表明E2可能转录后调控TRPV4的基因水平。Real-timePCR结果显示：相对于空白对照,E2能使miR-203的表达明显下调。在虫荧光素酶报告基因实验中，首先构建了包含TRPV4-3’UTR区的虫荧光素酶报告基因载体，然后将miR-203模拟物及其对照分别和荧光素酶报告载体和一起转染进了293T细胞，结果发现相对于miR-203模拟物对照，用TRPV4-3’-UTR和miR-203模拟物转染的细胞内相对荧光素梅活性明显下降。这表明TRPV4确实是miR-203的靶基因。既然E2抑制NO是TRPV4依赖性的，并且E2能下调靶基因是TRPV4的miR-203，就可以假设E2下调NO部分是因为通过解除了miR-203对TRPV4的抑制而形成的。实际上，在接受了E2刺激的MCCs上，Westernblot已经证实转染了miR-203模拟物后，TRPV4蛋白水平相对于转染miR-203模拟物对照的明显下降，这个结果表明miR-203能在细胞暴露于E2后调控TRPV4的表达。根据第二部分的实验结果已经知道E2能在LPS刺激的情况下下降N0的水平，并且这个效果能被TRPV4的抑制剂RutheniumRed所逆转。在转染了miR-203模拟物的细胞内，有着和RutheniumRed同样的效果，即转染了miR-203模拟物后的MCCs，E2对NO的抑制作用消失，NO含量明显升高。
本部分试验结果证实了E2对NO的下调作用部分是因为通过抑制miR-203，继而作用于TRPV4,使其表达上调形成的。
Osteoarthritis (OA) is a heterogeneous group of conditions thatcauses joint symptoms and signs, which are associated with defectiveintegrity of articular cartilage in addition to related changes in theunderlying bone at the joint margins. The hallmark of OA is aprogressive degradation of articular cartilage and the general jointstructure. Current available pharma cological interventions mayeffectively handle the joint sy mptoms such as joint pain, tenderness,stiffness, locking, but they have limitations in terms of re versing theaccelerated cartilage degradation. Studies have showed that secretedpro-inflammatory molecules are among the critical mediators of thedisturbed processes implicated in OA pathophysiology. Interventionsmodulating pro-inflamma tory molecules might havechondroprotective role in OA treatment. The temporomandibular joint(TMJ) plays an i mportant role in craniofacial growth and function,and shows a high incidence of OA. M oreover, many studies showedthat fema le had higher incidence and more severe symptoms oftemporomandibular joint disorders（TMD） than male, especially inthose who had signs of (17β-estradiol)E2deficiency, implying thechondroprotective effect of E2. Exactly, there was a growing body ofevidence suggesting that E2had chondroprotective role via itsinhibitory effect on pro-inflammatory cytokines. However, theprecise mechanism of E2chondroprotective effe ct was still wanted.
TRPV4is a me mb er of TRP super family of Ca2+-permeablenon-selective cation channels. TRPV4is widely expressed in cochlear hair cells, vibrissal Merkel cells, se nsory ganglia as well as in freenerve endings and cutaneous A and C-fibers terminals, etc. Recentresearch showed that TRPV4might have chondroprotective role inknee joints of the mouse.
Although E2and TRPV4have each been reported to bechondroprotective and the correlation of E2and Ca2+influx have alsobeen demonstrated in a wi de variety of cell types, the molecularmechanism between E2and TRPV4was still unknown. In our study,we found that E2could down-regulate NO expr ession by stimulatingTRPV4expression in a post-transcriptional regulating way viainhibiting miR-203, which directly targets TRPV4, in fe male ratmandibular condylar chondrocytes (MCCs). Our findingsdemonstrated the possible molecular mechanism between E2andTRPV4i n MCCs, which was helpful for the identification of thechondroprotective effect of E2.
The present study was undertaken to examine alterations inexpression of miR-203and transient receptor potential vanilloid4(TRPV4) in fe male Sprague–Dawley rats MCCs, and analyze theirroles in the down-regulating process of NO by E2. First, we culturedSD rats MCCs. The MCCs were exposure to E2（10-9-10-6M） andLipopolysaccharide (LPS) plus ruthenium red,4α-phorbol12,13-didecanoate(4α-PDD) as well as over-expressed miR-203, thecellular supernatants were analyzed for TNF-α、 IL-1βand NOconcentrations, TRPV4gene and protein level was me asured byRT-PCR and Western blot analysis, respectively.mi R-203gene levelwas measured by quantitative PCR after the treatment with E2（10-9-10-6M）. Dual luciferase activity assay was perfor med toidentify the target gene of miR-203.
The results showed that TRPV4was expressed abundantly at theRNA and protein levels. The inhibition effect on NO production under LPS-stimulation by E2（10-9-10-6M）was blocked by RutheniumRed and enhanced by4α-PDD, respectively. Furthermore, E2coulddown-regulate miR-203expression followed with increasing TRPV4expression level in MCCs. Dual luciferase activity assay suggestedthat TRPV4was the direct target gene of miR-203. Enforc ed miR-203reduced TRPV4protein level and blocked the inhibition effect on NOproduction under LPS-stimulation by E2as Ruthenium Re d.
Our foundings first suggested that E2might down-regulate NOproduction, at least in part, by inhibiting miR-203via targetingTRPV4.
Part Ⅰ
amounting evidence suggested that TRPV4channel might play animportant role in inflammatory and neuropathic pain. Mimi N. Phanet al. reported that TRPV4was also present in porcine articularchondrocytes.However, there was no research about the location ofTRPV4in MCCs. The aim of the present study was to demo nstrate thepresence of TRPV4in MCCs. In this part, Primary SD rats MCCswere cultured.TRPV4RNA and protein expression was measuredbyRT-PCR、 i mmunolabeling and Western blotting. The resultsshowed that TRPV4was expressed abundantly at the RNA and proteinlevel. Our findings indicate that TRPV4is present in MCCs.
Part Ⅱ
OA in TMJ fav ors wo me n over men. It was the fact that mostfe male patients with severe condylar resorption had signs andsymptoms of E2deficiency. However, there was still an unresolved paradox with respect to the exactly role of estrogens in articularcartilage. Currently, increasing evidence indicated that estrogensmight ha ve chondroprotective effect through its anti-inflammationeffect. It was reported that ion channel activity could be regulated bysex hormones, either directly or indirectly via the generation ofintracellular signals. However,the molecular mechanism betweensex hormo nes and TRPV4was still unknown.Our results demonstratedthat E2could up-regulate TRPV4in fe male MCCs. In this part, wemeasured the production of NO、TNF-α、 IL-1βand changes in Ca2+signaling using ELISA and laser cofous microscopy respectively atthe stimulation of E2and TRPV4agonist/antagonists (4α-PDD,Ruthenium Red) in MCCs. I n a situation of LPS(20ng/ml)stimulation,we found that E2（10-9-10-6M）could significantly inhibit NO、T N F-αand IL-1β production. In addition, NO production induced by LPScould significantly increased by TRPV4inhibitor Ruthenium Red,while4αPDD, a TRPV4specific activator could significantlydownregulate NO production. Further more, The inhibition effect onNO production by E2（10-9-10-6M）was blocked by Ruthenium Red andenhanced by4α-PDD, respectively, which suggested that E2mightinhibite NO production in a TRPV4-dependent manner. However, wealso found that The inhibition effect on TNF-α and IL-1β productionby E2（10-9-10-6M）were no relation with TRPV4. Our results providedthe proof of the favorable effect of TRPV4in anti-inflammation onMCCs and suggested that TRPV4was a necessary part in NOinhibition by E2.
Part Ⅲ
In the last few years it had become clear that miR-203wereinvolved in the pathogenesis of various malignant tumors. At the same ti me, evidence for the involvement of miR-203in inflammatorydisorders appeared. Elevated levels of miR-203lead to increasedsecretion of MMP-1and IL-6, indicating that mi R-203might be aproinflammatory and joint-destructive factor. Exactly, in our study,we also found that over-expression of mi R-203resulted in increasedsecretion of NO which was one of the major proinflammat ory factorin OA via targeting TRPV4in MCCs. In this part, Primary SD ratsMCCs were exposure to E2（10-9-10-6M）and LPS plus ruthenium red,4α－PDD as well as over-expressed miR-203, the cellularsupernatants were analyzed for NO concentrations, TRPV4gene andprotein level was measured by RT-PCR and Western blot analysis,respectively.miR-203gene level was measured by quantitative PCRafter the treatment with E2（10-9-10-6M）. Dual luciferase activityassay was performe d to identify the target gene of miR-203.
RT-PCR ana lysis showed that TRPV4mR NA level wassignificantly down-regulated after treatment with E2（10-8-10-6M）for72hr, while the protein level was significantly up-regulated afterexposure to E2(10-9-10-6M) for the same time period. Thisinconsistent result suggested that E2might post-transcriptionallyupregulate TRPV4protein level.
we hypothesized that E2could inhibit miRNAs which targetedTRPV4.， so we detected miR-203expression level changes in MCCsafter the treatment of E2. Real-time quantification of mi R-203bystem-loop RT-PCR exactly showed that miR-203was significantlydown-regulated in MCCs aft er incubating with E2（10-9-10-6M） for72hr compared with control.
TargetScan5.1predicted that TRPV4was the target gene of themi R-203conservely between different species. To explore whetherTRPV4was the target gene of miR-203, we constructed a luciferase reporter vector with the putative TRPV4-3’ UTR target site for themi R-203downstream of the luciferase gene (TRPV4-3’-UTR).Luciferase reporter vector together with the miR-203mi mic or themi RNA mimic control was transfected into293T cells, a significantdecrease in relative luciferase activity was observed whenTRPV4-3’-UTR was co-transfected with the miR-203mimic but n otwith the mi RNA mimic control, suggesting that TRPV4is the directtarget gene of the miR-203.
Since E2could inhibit NO pr oduction in a TRPV4-dependentmanner and TRPV4targeted mi R-203was also downregulated by E2,we hypothesized that E2could down-regulate NO production at leastin part by inhibiting miR-203via targeting TRPV4. Exactly, in MCCs,Western blot assay revealed that those transfected with miR-203mi mic exhibited greatly decreased TRPV4protein level comparedwith the mi RNA mimic control transfected cells after the treatmentwith different concentrations of E2. This result proved that miR-203could mo dulate TRPV4expression at the exposure of E2. Asmentioned above, E2could inhibit NO production in LPS-activatedMCCs. The effect could be blocked by TRPV4inhibitor RutheniumRed. In miR-203mi mic transfected cells, we also found this effect.Those miR-203mi mic transfected cells showed significantlyincreased NO production after treatment with E2. Taken all together,our foundings suggested that E2might down-regulate NO production,at least in part, by inhibiting mi R-203via targeting TRPV4.
引文
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