昆母汤对RA滑膜成纤维细胞增殖及细胞因子表达的影响
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摘要
目的：
类风湿关节炎(rheumatoid arthritis, RA)是一种以关节滑膜炎症为主要表现的自身免疫性疾病。成纤维样滑膜细胞(fibroblast-like synoviocyte, FLS)是滑膜组织的重要组成部分,其分泌高水平促炎性细胞因子、化学趋化因子、基质蛋白降解酶等,持续刺激滑膜细胞,作用于滑膜信号转导途径的不同部位,引起细胞内蛋白激酶的持续激活,从而导致滑膜信号转导异常以及滑膜细胞的增殖与凋亡失衡,引起关节的炎症和破坏。TNF-α具有多种生物活性,是一种重要的生理炎性介质,主要由单核巨噬细胞产生,通过作用于靶细胞上的独特受体结合而发生作用。TNF-α是RA发病机理中居于重要地位的促炎症细胞因子,参与RA的发生发展过程,能够刺激滑膜成纤维细胞增生及分泌IL-6、粒细胞-巨噬细胞集落刺激因子(GM-CSF)、趋化因子IL-8以及基质金属蛋白酶(MMPs)和前列腺素(PGE2)等效应分子。在不同的组织中,TNF-α的生物学作用不同,TNF-α主要通过和不同细胞表面受体结合发挥其生物学作用。TNF-α两个受体为TNFRI和TNFRII,为肿瘤坏死因子受体家族,与TNF-α都有高度的亲和力,TNFRI胞内具有死亡结构域(death domain, DD),而TNFRII却不含死亡结构域。TNFRII缺乏DD序列,以抗细胞凋亡作用为主,它主要通过募集TRAFS信号分子激活多种信号传导通路,从而表达炎性细胞因子。临床研究证实,昆母汤具有抗炎、免疫调节作用,但对于RA滑膜细胞的影响,至今尚未见报道。为了进一步研究昆母汤对治疗RA的潜在应用价值,我们观察了不同浓度的昆母汤对TNF-α诱导的RA-FLS的影响.通过体外培养类风湿关节患者滑膜成纤维细胞,观察昆母汤对RA-FLS异常增殖的影响,以及TNF-α受体TNFRI和TNFRII的表达,相关炎性细胞因子IL-1β、IL-6、IL-8、 MMP-3以及NO, iNOS, COX-2, PGE2的表达的影响,从体外培养滑膜细胞增殖和细胞因子表达的角度探讨昆母汤调控细胞因子的靶点和抗类风湿关节炎的作用机制,为昆母汤治疗RA的中医理论提供实验依据和开拓新途径。
方法：
1.类风湿关节炎成纤维样滑膜细胞的原代培养和传代鉴定：在无菌条件下取类风湿关节炎的滑膜组织运用两步消化酶法分离纯化类风湿关节炎滑膜细胞,传代后采用倒置显微镜和流式细胞技术表面抗体标记的鉴定,处于对数生长期的3-5代RA-FLS用于实验。
2.四甲基偶氮唑蓝法(MTT)检测细胞生长的抑制率：体外培养RA-FLS细胞生长抑制率检测：取RA-FLS,按以下方法分成5组：空白组、甲氨蝶呤(MTX)阳性MTX组(含1μg/ml的MTX)和各剂量昆母汤组(分别含100,200,400μg/ml的昆母汤醇提物),干预后用MTT检测24h、48h、72h细胞增殖抑制情况。
3.采用荧光定量RT-PCR法,检测各组细胞相关炎性因子IL-1β、IL-6、IL-8、 MMP-3、iNOS、COX-2、TNF受体TNFRI,TNFRII的mRNA表达：分为6组：空白组、TNF-α模型组、甲氨蝶呤(MTX)阳性MTX组(含1μ g/ml的MTX)和各浓度昆母汤组(分别含100,200,400μ g/ml的昆母汤醇提物)。除空白组,其余各组均加入TNF-α lOng/L,细胞培养24h后运用RT-PCR法检测mRNA表达情况。
4.运用ELISA法检测上述各组处理后细胞培养液上清中的IL-1β、IL-6、IL-8、 MMP-3、PGE2的蛋白表达情况。
5. Griess测NO法检测各组MTX组和昆母汤高中低剂量组上清液中NO的分泌水平。
结果
1.细胞培养实验,在倒置显微镜下可观察原代滑膜细胞生长情况,以成纤维细胞样细胞为主,细胞呈树杈型或多角形或梭形。滑膜组织分离纯化出的成纤维细胞进行流式细胞术表面标记的鉴定。结果显示CD14、CD3阳性率均小于2%、而CD90阳性率达93%以上。成纤维细胞纯度较高。
2.MTT实验结果显示：MTT检测结果表明,随着干预时间的延长,各组RA-FLS的OD值增高而抑制率随着时间和剂量增多而逐步升高,呈现时间和剂量依赖性。加药干预24h时,各组RA-FLS和空白组对照,差异无统计学意义(P>0.05),增殖无明显抑制作用,当药物干预48h后,和空白组比较,低剂量昆母汤组无明显抑制作用差异无统计学意义(P>0.05), MTX、昆母汤中、高剂量组均可抑制RA-FLS的增殖,与TNF-α组相比,差异有显著统计学意义(P＜0.01),高剂量组对RA-FLS的抑制率差异有显著性(P＜0.01),抑制程度较MTX组高；72h后,各加药组均出现抑制作用,昆母汤不同剂量组随着药物浓度增加,抑制作用更为显著,呈剂量依赖性。低剂量组出现抑制作用,差异有统计学意义(P＜0.05),而MTX组、昆母汤醇提液中、高剂量组对RA-FLS的抑制程度明显,差异显著(P＜0.01夕。与低剂量组比较,中、高剂量组差异有统计学意义。各组细胞增殖率随着时间的延长,增殖率增高,抑制率也逐渐升高,在72h时,细胞增值率最高,而抑制率也是最高。在相同时间点,药物浓度越高,抑制率越高。MTX组抑制率介于中高剂量组之间。
3.昆母汤对RA-FLS TNFRI和’TNFRII表达的影响
半定量荧光PCR结果显示,TNF-α诱导后对各组TNFRII的mRNA表达均有促进作用,表达量增多,和空白组相比,差异有统计学意义(P<0.05)。而TNFRI的mRNA表达均出现下降。药物干预24h后,低剂量组TNFRI和TNFRII的mRNA表达差异均无统计学意义(P＞0.05),而MTX、中、高剂量组TNFRII明显降低,TNFRI则略有升高,差异有统计学意义(P＜0.05)。而MTX、高剂量组差异显著(P<0.01)。
4.昆母汤对RA滑膜成纤维细胞相关细胞因子IL-1β、IL-6、IL-8表达的影响
RT-PCR结果显示：加入TNF-α诱导后各组IL-1β、IL-6、IL-8的mRNA表达量明显升高,和空白组比较,mRNA表达量其差异有统计学意义(P＜0.05),提示TNF-α均可诱导IL-1β、IL-6、IL-8mRNA的表达,在加药干预24h后,相对于TNF-α组,低剂量组IL-6、IL-8的RNA表达量差异没有统计学意义(P＞0.05),而对MTX组和中、高剂量组TNF-α组细胞IL-6、IL-8的RNA表达量均低于TNF-α组,说明均可抑制RNA表达量(P<0.05),差异有统计学意义,MTX、高剂量组差异显著(P<0.01)。对IL-6的作用,MTX介于中、高剂量组之间,而对IL-8的作用,高剂量组和MTX组相当。低、中剂量组对IL-1β的抑制作用不明显,差异无统计学意义(P>0.05),而MTX组、高剂量组均有显著差异(P<0.01)。ELISA检测结果显示,TNF-α诱导后对各炎性因子IL-1β、IL-6、IL-8的蛋白表达均有促进作用,蛋白表达量增多,和空白组相比,差异有统计学意义(P＜0.05)。加入药物干预24h后,和TNF-α诱导组对比,低剂量组细胞培养上清IL-6、IL-8含量差异无统计学意义,中、高剂量差异有统计学意义(P<0.05), MTX、高剂量昆母汤不同剂量组明显降低(P＜0.01)。低、中剂量组对IL-1β蛋白表达影响和TNF-α组差异无统计学意义(P>0.05),而MTX组和高剂量组对IL-1β蛋白表达的抑制作用有差异显著性(P<0.05)。
5.昆母汤对RA-FLS细胞因子MMP3表达的影响
RT-PCR结果显示：TNF-α均可诱导MMP-3的mRNA的表达加入TNF-α诱导后各组的RNA表达量明显升高,各组对MMP-3的RNA抑制作用和TNF-α组对比,除低剂量组,其余各组具有显著差异,MTX中、高剂量组均有显著差异(P<0.01), MTX组与中剂量组作用相当,差异无统计学意义(P>0.05)。ELISA检测结果显示,TNF-α诱导后对MMP-3蛋白表达均有促进作用,蛋白表达量增多,和空白组相比,差异有统计学意义(P<0.05)。药物干预24h后,各组对MMP-3蛋白的抑制作用和TNF-α组对比,除低剂量组,其余各组具有显著差异,而中、高剂量组均有显著差异(P<0.01),MTX组与高剂量组作用相当,差异无统计学意义(P>0.05)。
6.昆母汤对RA-FLS细胞因子NO和iNOS表达的影响
各组NO检测结果显示：各组加入TNF-α诱导后各组的RNA表达量明显升高,和空白组比较,其差异有统计学意义(P<0.01)。在加药干预24h后,相对于TNF-α组,低剂量组NO量差异没有统计学意义(P>0.05),而对MTX组和中、高剂量组均低于TNF-α组,均可抑制NO,差异有统计学意义,且差异显著(P＜0.01)。荧光PCR检测结果显示：TNF-α均可诱导iNOS的分泌,表达加入TNF-α诱导后各组的RNA表达量明显升高和空白组比较其差异有统计学意义(P<0.05)。在加药干预24h后,相对于TNF-α组,低剂量组的RNA表达量差异没有统计学意义(P>0.05),而对MTX组和中、高剂量组TNF-α组细胞RNA表达量均低于TNF-α组,均可抑制RNA表达量,差异有统计学意义,且差异显著(P＜0.01)。
7.昆母汤对RA-FLS细胞因子COX-2和PGE2表达的影响
半定量荧光PCR结果显示,TNF-α诱导后对各组COX-2的mRNA表达均有促进作用,表达量增多,和空白组相比,差异有统计学意义(P＜0.05)。药物干预24h后,和TNF-α组对比,低剂量组COX-2的mRNA表达差异有统计学意义(P<0.05),而MTX、中、高剂量组明显降低(P＜0.01)。ELISA检测结果显示,TNF-α诱导后对各组的PGE2蛋白表达均有促进作用,和空白组相比,差异有统计学意义(P＜0.05)。药物干预24h后,和TNF-α组对比,低剂量组细胞PGE2含量差异有统计学意义,而MTX、中、高剂量差异有统计学意义(P＜0.05)。
结论
昆母汤可抑制RA-FLS细胞的增殖和TNF-α诱导的细胞炎性因子IL-1β、IL-6, IL-8、MMP-3以及NO, iNOS, COX-2, PGE2的表达,抑制TNF-α和TNFRII结合,推测昆母汤可能通过抑制TNF-α和TNFRII结合,抑制细胞因子的产生,从而抑制阻断TNF-α介导的细胞增殖,滑膜炎症的增生和骨破坏。这为昆母汤治疗RA提供了实验基础和理论依据。
Objectives
Rheumatoid arthritis (RA) is the autoimmune diseases with the main performance of joint synovial inflammation. Fibroblast-like synoviocytes (FLS) is an important component of the synovial tissue, secreting cytokines high level to promote the inflammatory chemokines, matrix mental protein degradation enzymes, etc., which stimulates synovial cells through acting on different parts of the synovial signal transduction pathways, it can cause the activation of intracellular protein kinase, resulting in synovial abnormal signal transduction and synovial cell proliferation and apoptosis imbalance, and inflammation and damage of the joints. TNF alpha has a variety of biological activities, it is a kind of important physiological inflammatory mediators, mainly produced by mononuclear macrophages, TNF alpha in the pathogenesis of RA is occupies the important position of proinflammatory cytokines through the role of their unique receptors on target cell, and it participates in the development process of RA, stimulates the synovial fibroblasts proliferation and secretion of chemokines such as IL-6, granulocyte macrophage colony stimulating factor (GM-CSF), IL-8and the matrix metalloproteinases (MMPs) and prostaglandin (PGE2), and other cytokines. In different tissues TNF alpha play a role of the biology of various cell surface receptors mainly through the TNF alpha. Two receptors for TNF alpha are TNFRI and TNFRII, they belong to tumor necrosis factor receptor family, and have high affinity with TNF alpha, TNFRI intracellular has death domain,(DD), whereas TNFRII excluding death domain structure. TNFRII lack of DD sequence is given priority to resistant to apoptosis role, it mainly raises TRAFS signal molecular activation of multiple signaling pathways, and expression of inflammatory cytokines. Clinical studies had confirmed that Kunmu Dedoction has anti-inflammatory, immune regulating function, but in RA synovial cell, its influence of has not been reported. In order to further research the potential application value of KUNMU Dedoction in treating RA, we observed the different concentrations of KUNMU Dedoction of TNF alpha induced RA FLS influence.-by in vitro culture rheumatoid joint synovial fibroblasts from patients, to observe KUNMU Dedoction the influence of abnormal proliferation and TNF alpha receptor TNFRI and TNFRII expression, relating to the inflammatory cytokines IL-1beta, IL-6, IL-8, MMP-3, and NO, iNOS, COX-2, PGE2expression, and to observe the effect of the in vitro proliferation and cytokine expression culturing synovial cells, and discusses KUNMU Dedoction regulation of cytokines targets and action mechanism of RA, to provide experimental basis and new ways of development KUNMU Dedoction treatment theory of traditional Chinese medicine.
Methods
1.RA fibroblast-like synoviocytes of primitive culture and extend the identification:We use two-step enzymatic digestion of RA synovial cell separation and purification under aseptic conditions in RA synovial tissue, and represented by inverted microscope and flow cytometry technology after surface antibody marker identification. Three to five generation of fibroblast-like synoviocyte in the logarithmic growth period are used in experiments.
2. Tet RAmethyl azo thiazole blue method (MTT) detection of cell growth inhibition rate:FLS was cultured in vitro-cell growth inhibition test: take an fibroblast-like synoviocyte, according to the following method is divided into5groups:blank group, the methotrexate (MTX) positive MTX group (including1ug/ml of MTX and each dose Kunmu Dedoction group (respectively, including Kunmu Dedoction alcohol extract from the g/ml), intervents with determined by MTT test after24h,48h,72h cell proliferation inhibition.
3. Using the fluorescent quantitative RT-PCR method, testing each cell related inflammatory cytokines IL-1beta, IL-6, IL-8and MMP-3, iNOS, COX-2and TNF receptor TNFRI, TNFRII mRNA expression:which are divided into 6groups:blank group, the TNF alpha model, methotrexate (MTX) positive MTX group (including1mu g/ml of MTX) and the concentration of kunduz mother decoction group (respectively, including Kunmu dedoction alcohol extract from the g/ml). Except the blank group, the rest of the groups are added the TNF alpha10ng/L,24h after cell culture using RT-PCR method to detect the mRNA expression.
4. Using the ELISA method to detect the groups after treatment cell culture supernatant of IL-1beta, IL-6, IL-8and MMP-3, PGE2protein expression.
5. Griess NO measurement method is used todetect each group including dose MTX group and kun soup group NO secretion level in the supernatant.
Results
1. Cell culture experiments, and observed under inverted microscope primary synovial cell growth situation, mainly on fibroblast cells, cell a fork shaped or polygon or fusiform. Synovial tissue separation and purification of fibroblasts in flow cytometry surface marker identification. Results show that the CD14, CD3positive RAte less than2%, and CD90positive RAte of more than93%. Fibroblasts purity is higher.
2. Determined by MTT experiment results show that the determined by MTT test results show that with the extension of intervention time, the groups of fibroblast-like synoviocyte-inhibition RAte0D value increases with time and dose increased g RAdually rise, rendering time and dose dependent.24h dosing intervention, groups of fibroblast-like synoviocyte and blank control group, there was no statistically significant difference (P>0.05), no obvious inhibitory effect on proliferation, while48h after drug intervention, compared to blank group, low dose he mother soup group without obvious inhibition there was no statistically significant difference (P>0.05), MTX, mother kun soup, high dose group are inhibits the proliferation of RA-FLS, compared with TNF alpha group, with significant difference statistically significant (P<0.01), high dose group of fibroblast-like synoviocyte-inhibition RAte have significant difference (P<0.01), the inhibition degree higher than MTX group; After72h, the dosing inhibition was observed in all groups, mother kun soup different dose groups with the increase of drug concentration, more significant inhibitory effect, are dose dependent. Inhibitory effect, low dose group the difference was statistically significant (P<0.05), while MTX group of elder brother, mother soup alcohol ext RAction liquid medium and high dose groups significantly, the rest RAint of the fibroblast-like synoviocyte-significant difference (P<0.01). Compared with low dose group, middle, high dose group difference was statistically significant. Each cell proliferation RAte over time, increased proliferation RAte, the inhibition RAte also increased, at the time of72h, cells increment RAte is highest, and the inhibition RAte was the highest. At the same time, the higher the drug concentration, the inhibition RAte is higher. MTX group inhibition RAte between middle and high dose group.
3. The mother kun soup for fibroblast-like synoviocyte-TNFRI and TNFRII expression effect
Semi quantitative fluorescence PCR results showed that TNF alpha after induction, has promoting effect to each TNFRII mRNA expression, expression increased, compared with the blank group, the difference was statistically significant (P<0.05). And mRNA expression of TNFRI all fell.24h after drug Intervention, low dose group TNFRI and TNFRII mRNA expression differences had no statistical significance (P>0.05), while MTX TNFRII, medium and high dose group was obviously reduced, TNFRI is slightly higher, the difference was statistically significant (P<0.05). While MTX, significant difference was found in high dose group (P<0.01).
4. Kunmu dedoction for RA synovial fibroblasts related cytokines IL-1beta, IL-6, IL8expression
Rt-pcr results showed that after joining TNF alpha induced IL-1beta, constituting the mRNA expression of IL-6, IL-8volume increased significantly, compared with blank group, the amount of mRNA expression difference was statistically significant (P<0.05), suggesting the TNF alpha beta can be induced IL-1, IL-6, IL-8mRNA expression, in24h after dosing intervention, relative to the TNF alpha group, low dose group of IL-6, IL-8RNA expression quantity differences had statistics significance (P>0.05), and the MTX group and middle, high dose group of TNF alpha cells of IL-6, IL-8RNA expression quantity are lower than TNF alpha group, all can inhibit RNA expression quantity (P<0.05), the difference was statistically significant, MTX, significant difference was found in high dose group (P<0.01). On the role of il-6, MTX between medium and high dose group, and to the role of IL-8, high dose group and MTX group. Low, medium dose group have no obvious inhibition of IL-1beta, there was no statistically significant difference (P>0.05), while MTX group, high dose group were significantly different (P<0.01). ELISA test results showed that TNF alpha beta after induction of inflammatory factor, IL-1, IL-6, IL-8has the promoting effect of protein expression, protein expression increased, compared with the blank group, the difference was statistically significant (P<0.05). In24h after drug intervention, and TNF alpha cont RAst induced group, low dose group of cell culture supernatant IL-6, IL-8content is no statistically significant differences, middle, high dose difference was statistically significant (P<0.05), MTX, high dose mother kun soup different dose groups decreased significantly (P<0.01). Low, medium dose group of IL-1beta protein expression effect and TNF alpha group has no statistically significant difference (P>0.05), while MTX group and high dose group of beta protein expression of IL-1inhibition has a significant difference (P<0.05).
5. Kunmu dedoction for fibroblast-like synoviocyte-MMP3expression of cytokines
Rt-pcr results showed that TNF alpha can induce the mRNA expression of MMP-3to join the TNF alpha group after induction of RNA expression quantity increased significantly, groups of RNA inhibition of MMP-3, and TNF alpha group compared, in addition to the low dose group, the rest of the group has significant difference, MTX medium and high dose groups were significantly different (P<0.01), MTX group effect and the middle dose group, there was no statistically significant difference (P>0.05). ELISA test results showed that TNF alpha induced the expression of MMP-3protein after all have promoting effect, protein expression increased, compared with the blank group, the difference was statistically significant (P<0.05).24h after drug intervention, the inhibition of MMP-3protein and TNF alpha group compared, in addition to the low dose group, the rest of the group has significant difference and the medium and high dose groups were significantly different (P<0.01), MTX group and high dose group, there was no statistically significant difference (P>0.05).
6. Kunmu dedoction for fibroblast-like synoviocyte cytokines NO and iNOS expression
Groups NO test results showed that each group to join the TNF alpha group after induction of RNA expression quantity increased significantly, compared with blank group, the difference was statistically significant (P<0.01). In24h after dosing intervention, compared with the TNF alpha group, low dose group NO difference NO statistical significance (P>0.05), and the MTX group and middle, high dose group were lower than TNF alpha group, all can inhibit the NO, the difference was statistically significant, and significant difference (P<0.01). Fluorescent PCR detection results showed that TNF alpha can be induced iNOS secretion, express the TNF alpha group after induction of RNA expression quantity increased significantly compared with blank group the difference was statistically significant (P<0.05). In24h after dosing intervention, compared with the TNF alpha group, low dose group of the RNA expression difference was not statistically significance (P>0.05), and the MTX group and middle, high dose group of cell RNA expression quantity of TNF alpha group were lower than TNF alpha group, all can inhibit RNA expression quantity, the difference was statistically significant, and significant difference (P<0.01).
7. Kunmu dedoction for fibroblast-like synoviocyte-cytokine effects of cox-2and PGE2expression
Semi quantitative fluorescence PCR results showed that TNF alpha after induction of each mRNA expression of cox-2has promoting effect, expressing quantity increased, compared with the blank group, the difference was statistically significant (P<0.05).24h after drug intervention, cont RAst and TNF alpha group, low dose group of cox-2mRNA expression difference was statistically significant (P<0.05), while MTX, medium and high dose group decreased obviously (P<0.01). ELISA test results showed that TNF alpha for each group after induction of PGE2protein expression had promoting effect, compared with the blank group, the difference was statistically significant (P<0.05).24h after drug intervention, and comparison of TNF alpha group, low dose group PGE2content difference was statistically significant, and MTX, medium and high dose difference was statistically significant (P<0.05).
Conclusion
Kunmu dedoction inhibits the proliferation of fibroblast-like synoviocyte cells, and suppress the expression of TNF alpha induced inflammatory cytokines of IL-1beta, IL-6, IL-8, MMP-3, and NO, iNOS, COX-2, PGE2, it can also suppress TNF alpha and TNFRII combination of speculation and inhibit the TNF alpha female soup combined with TNFRII, inhibition of cytokine production, thus inhibits the block TNF alpha mediating cell proliferation, synovial inflammation, hyperplasia of the cartilage and bone destruction. This reseach of Kunmu Dedoction provides the experimental basis and way for RA.
类风湿关节炎(rheumatoid arthritis, RA)是一种以关节滑膜炎症为主要表现的自身免疫性疾病。成纤维样滑膜细胞(fibroblast-like synoviocyte, FLS)是滑膜组织的重要组成部分,其分泌高水平促炎性细胞因子、化学趋化因子、基质蛋白降解酶等,持续刺激滑膜细胞,作用于滑膜信号转导途径的不同部位,引起细胞内蛋白激酶的持续激活,从而导致滑膜信号转导异常以及滑膜细胞的增殖与凋亡失衡,引起关节的炎症和破坏。TNF-α具有多种生物活性,是一种重要的生理炎性介质,主要由单核巨噬细胞产生,通过作用于靶细胞上的独特受体结合而发生作用。TNF-α是RA发病机理中居于重要地位的促炎症细胞因子,参与RA的发生发展过程,能够刺激滑膜成纤维细胞增生及分泌IL-6、粒细胞-巨噬细胞集落刺激因子(GM-CSF)、趋化因子IL-8以及基质金属蛋白酶(MMPs)和前列腺素(PGE2)等效应分子。在不同的组织中,TNF-α的生物学作用不同,TNF-α主要通过和不同细胞表面受体结合发挥其生物学作用。TNF-α两个受体为TNFRI和TNFRII,为肿瘤坏死因子受体家族,与TNF-α都有高度的亲和力,TNFRI胞内具有死亡结构域(death domain, DD),而TNFRII却不含死亡结构域。TNFRII缺乏DD序列,以抗细胞凋亡作用为主,它主要通过募集TRAFS信号分子激活多种信号传导通路,从而表达炎性细胞因子。临床研究证实,昆母汤具有抗炎、免疫调节作用,但对于RA滑膜细胞的影响,至今尚未见报道。为了进一步研究昆母汤对治疗RA的潜在应用价值,我们观察了不同浓度的昆母汤对TNF-α诱导的RA-FLS的影响.通过体外培养类风湿关节患者滑膜成纤维细胞,观察昆母汤对RA-FLS异常增殖的影响,以及TNF-α受体TNFRI和TNFRII的表达,相关炎性细胞因子IL-1β、IL-6、IL-8、 MMP-3以及NO, iNOS, COX-2, PGE2的表达的影响,从体外培养滑膜细胞增殖和细胞因子表达的角度探讨昆母汤调控细胞因子的靶点和抗类风湿关节炎的作用机制,为昆母汤治疗RA的中医理论提供实验依据和开拓新途径。
方法：
1.类风湿关节炎成纤维样滑膜细胞的原代培养和传代鉴定：在无菌条件下取类风湿关节炎的滑膜组织运用两步消化酶法分离纯化类风湿关节炎滑膜细胞,传代后采用倒置显微镜和流式细胞技术表面抗体标记的鉴定,处于对数生长期的3-5代RA-FLS用于实验。
2.四甲基偶氮唑蓝法(MTT)检测细胞生长的抑制率：体外培养RA-FLS细胞生长抑制率检测：取RA-FLS,按以下方法分成5组：空白组、甲氨蝶呤(MTX)阳性MTX组(含1μg/ml的MTX)和各剂量昆母汤组(分别含100,200,400μg/ml的昆母汤醇提物),干预后用MTT检测24h、48h、72h细胞增殖抑制情况。
3.采用荧光定量RT-PCR法,检测各组细胞相关炎性因子IL-1β、IL-6、IL-8、 MMP-3、iNOS、COX-2、TNF受体TNFRI,TNFRII的mRNA表达：分为6组：空白组、TNF-α模型组、甲氨蝶呤(MTX)阳性MTX组(含1μ g/ml的MTX)和各浓度昆母汤组(分别含100,200,400μ g/ml的昆母汤醇提物)。除空白组,其余各组均加入TNF-α lOng/L,细胞培养24h后运用RT-PCR法检测mRNA表达情况。
4.运用ELISA法检测上述各组处理后细胞培养液上清中的IL-1β、IL-6、IL-8、 MMP-3、PGE2的蛋白表达情况。
5. Griess测NO法检测各组MTX组和昆母汤高中低剂量组上清液中NO的分泌水平。
结果
1.细胞培养实验,在倒置显微镜下可观察原代滑膜细胞生长情况,以成纤维细胞样细胞为主,细胞呈树杈型或多角形或梭形。滑膜组织分离纯化出的成纤维细胞进行流式细胞术表面标记的鉴定。结果显示CD14、CD3阳性率均小于2%、而CD90阳性率达93%以上。成纤维细胞纯度较高。
2.MTT实验结果显示：MTT检测结果表明,随着干预时间的延长,各组RA-FLS的OD值增高而抑制率随着时间和剂量增多而逐步升高,呈现时间和剂量依赖性。加药干预24h时,各组RA-FLS和空白组对照,差异无统计学意义(P>0.05),增殖无明显抑制作用,当药物干预48h后,和空白组比较,低剂量昆母汤组无明显抑制作用差异无统计学意义(P>0.05), MTX、昆母汤中、高剂量组均可抑制RA-FLS的增殖,与TNF-α组相比,差异有显著统计学意义(P＜0.01),高剂量组对RA-FLS的抑制率差异有显著性(P＜0.01),抑制程度较MTX组高；72h后,各加药组均出现抑制作用,昆母汤不同剂量组随着药物浓度增加,抑制作用更为显著,呈剂量依赖性。低剂量组出现抑制作用,差异有统计学意义(P＜0.05),而MTX组、昆母汤醇提液中、高剂量组对RA-FLS的抑制程度明显,差异显著(P＜0.01夕。与低剂量组比较,中、高剂量组差异有统计学意义。各组细胞增殖率随着时间的延长,增殖率增高,抑制率也逐渐升高,在72h时,细胞增值率最高,而抑制率也是最高。在相同时间点,药物浓度越高,抑制率越高。MTX组抑制率介于中高剂量组之间。
3.昆母汤对RA-FLS TNFRI和’TNFRII表达的影响
半定量荧光PCR结果显示,TNF-α诱导后对各组TNFRII的mRNA表达均有促进作用,表达量增多,和空白组相比,差异有统计学意义(P<0.05)。而TNFRI的mRNA表达均出现下降。药物干预24h后,低剂量组TNFRI和TNFRII的mRNA表达差异均无统计学意义(P＞0.05),而MTX、中、高剂量组TNFRII明显降低,TNFRI则略有升高,差异有统计学意义(P＜0.05)。而MTX、高剂量组差异显著(P<0.01)。
4.昆母汤对RA滑膜成纤维细胞相关细胞因子IL-1β、IL-6、IL-8表达的影响
RT-PCR结果显示：加入TNF-α诱导后各组IL-1β、IL-6、IL-8的mRNA表达量明显升高,和空白组比较,mRNA表达量其差异有统计学意义(P＜0.05),提示TNF-α均可诱导IL-1β、IL-6、IL-8mRNA的表达,在加药干预24h后,相对于TNF-α组,低剂量组IL-6、IL-8的RNA表达量差异没有统计学意义(P＞0.05),而对MTX组和中、高剂量组TNF-α组细胞IL-6、IL-8的RNA表达量均低于TNF-α组,说明均可抑制RNA表达量(P<0.05),差异有统计学意义,MTX、高剂量组差异显著(P<0.01)。对IL-6的作用,MTX介于中、高剂量组之间,而对IL-8的作用,高剂量组和MTX组相当。低、中剂量组对IL-1β的抑制作用不明显,差异无统计学意义(P>0.05),而MTX组、高剂量组均有显著差异(P<0.01)。ELISA检测结果显示,TNF-α诱导后对各炎性因子IL-1β、IL-6、IL-8的蛋白表达均有促进作用,蛋白表达量增多,和空白组相比,差异有统计学意义(P＜0.05)。加入药物干预24h后,和TNF-α诱导组对比,低剂量组细胞培养上清IL-6、IL-8含量差异无统计学意义,中、高剂量差异有统计学意义(P<0.05), MTX、高剂量昆母汤不同剂量组明显降低(P＜0.01)。低、中剂量组对IL-1β蛋白表达影响和TNF-α组差异无统计学意义(P>0.05),而MTX组和高剂量组对IL-1β蛋白表达的抑制作用有差异显著性(P<0.05)。
5.昆母汤对RA-FLS细胞因子MMP3表达的影响
RT-PCR结果显示：TNF-α均可诱导MMP-3的mRNA的表达加入TNF-α诱导后各组的RNA表达量明显升高,各组对MMP-3的RNA抑制作用和TNF-α组对比,除低剂量组,其余各组具有显著差异,MTX中、高剂量组均有显著差异(P<0.01), MTX组与中剂量组作用相当,差异无统计学意义(P>0.05)。ELISA检测结果显示,TNF-α诱导后对MMP-3蛋白表达均有促进作用,蛋白表达量增多,和空白组相比,差异有统计学意义(P<0.05)。药物干预24h后,各组对MMP-3蛋白的抑制作用和TNF-α组对比,除低剂量组,其余各组具有显著差异,而中、高剂量组均有显著差异(P<0.01),MTX组与高剂量组作用相当,差异无统计学意义(P>0.05)。
6.昆母汤对RA-FLS细胞因子NO和iNOS表达的影响
各组NO检测结果显示：各组加入TNF-α诱导后各组的RNA表达量明显升高,和空白组比较,其差异有统计学意义(P<0.01)。在加药干预24h后,相对于TNF-α组,低剂量组NO量差异没有统计学意义(P>0.05),而对MTX组和中、高剂量组均低于TNF-α组,均可抑制NO,差异有统计学意义,且差异显著(P＜0.01)。荧光PCR检测结果显示：TNF-α均可诱导iNOS的分泌,表达加入TNF-α诱导后各组的RNA表达量明显升高和空白组比较其差异有统计学意义(P<0.05)。在加药干预24h后,相对于TNF-α组,低剂量组的RNA表达量差异没有统计学意义(P>0.05),而对MTX组和中、高剂量组TNF-α组细胞RNA表达量均低于TNF-α组,均可抑制RNA表达量,差异有统计学意义,且差异显著(P＜0.01)。
7.昆母汤对RA-FLS细胞因子COX-2和PGE2表达的影响
半定量荧光PCR结果显示,TNF-α诱导后对各组COX-2的mRNA表达均有促进作用,表达量增多,和空白组相比,差异有统计学意义(P＜0.05)。药物干预24h后,和TNF-α组对比,低剂量组COX-2的mRNA表达差异有统计学意义(P<0.05),而MTX、中、高剂量组明显降低(P＜0.01)。ELISA检测结果显示,TNF-α诱导后对各组的PGE2蛋白表达均有促进作用,和空白组相比,差异有统计学意义(P＜0.05)。药物干预24h后,和TNF-α组对比,低剂量组细胞PGE2含量差异有统计学意义,而MTX、中、高剂量差异有统计学意义(P＜0.05)。
结论
昆母汤可抑制RA-FLS细胞的增殖和TNF-α诱导的细胞炎性因子IL-1β、IL-6, IL-8、MMP-3以及NO, iNOS, COX-2, PGE2的表达,抑制TNF-α和TNFRII结合,推测昆母汤可能通过抑制TNF-α和TNFRII结合,抑制细胞因子的产生,从而抑制阻断TNF-α介导的细胞增殖,滑膜炎症的增生和骨破坏。这为昆母汤治疗RA提供了实验基础和理论依据。
Objectives
Rheumatoid arthritis (RA) is the autoimmune diseases with the main performance of joint synovial inflammation. Fibroblast-like synoviocytes (FLS) is an important component of the synovial tissue, secreting cytokines high level to promote the inflammatory chemokines, matrix mental protein degradation enzymes, etc., which stimulates synovial cells through acting on different parts of the synovial signal transduction pathways, it can cause the activation of intracellular protein kinase, resulting in synovial abnormal signal transduction and synovial cell proliferation and apoptosis imbalance, and inflammation and damage of the joints. TNF alpha has a variety of biological activities, it is a kind of important physiological inflammatory mediators, mainly produced by mononuclear macrophages, TNF alpha in the pathogenesis of RA is occupies the important position of proinflammatory cytokines through the role of their unique receptors on target cell, and it participates in the development process of RA, stimulates the synovial fibroblasts proliferation and secretion of chemokines such as IL-6, granulocyte macrophage colony stimulating factor (GM-CSF), IL-8and the matrix metalloproteinases (MMPs) and prostaglandin (PGE2), and other cytokines. In different tissues TNF alpha play a role of the biology of various cell surface receptors mainly through the TNF alpha. Two receptors for TNF alpha are TNFRI and TNFRII, they belong to tumor necrosis factor receptor family, and have high affinity with TNF alpha, TNFRI intracellular has death domain,(DD), whereas TNFRII excluding death domain structure. TNFRII lack of DD sequence is given priority to resistant to apoptosis role, it mainly raises TRAFS signal molecular activation of multiple signaling pathways, and expression of inflammatory cytokines. Clinical studies had confirmed that Kunmu Dedoction has anti-inflammatory, immune regulating function, but in RA synovial cell, its influence of has not been reported. In order to further research the potential application value of KUNMU Dedoction in treating RA, we observed the different concentrations of KUNMU Dedoction of TNF alpha induced RA FLS influence.-by in vitro culture rheumatoid joint synovial fibroblasts from patients, to observe KUNMU Dedoction the influence of abnormal proliferation and TNF alpha receptor TNFRI and TNFRII expression, relating to the inflammatory cytokines IL-1beta, IL-6, IL-8, MMP-3, and NO, iNOS, COX-2, PGE2expression, and to observe the effect of the in vitro proliferation and cytokine expression culturing synovial cells, and discusses KUNMU Dedoction regulation of cytokines targets and action mechanism of RA, to provide experimental basis and new ways of development KUNMU Dedoction treatment theory of traditional Chinese medicine.
Methods
1.RA fibroblast-like synoviocytes of primitive culture and extend the identification:We use two-step enzymatic digestion of RA synovial cell separation and purification under aseptic conditions in RA synovial tissue, and represented by inverted microscope and flow cytometry technology after surface antibody marker identification. Three to five generation of fibroblast-like synoviocyte in the logarithmic growth period are used in experiments.
2. Tet RAmethyl azo thiazole blue method (MTT) detection of cell growth inhibition rate:FLS was cultured in vitro-cell growth inhibition test: take an fibroblast-like synoviocyte, according to the following method is divided into5groups:blank group, the methotrexate (MTX) positive MTX group (including1ug/ml of MTX and each dose Kunmu Dedoction group (respectively, including Kunmu Dedoction alcohol extract from the g/ml), intervents with determined by MTT test after24h,48h,72h cell proliferation inhibition.
3. Using the fluorescent quantitative RT-PCR method, testing each cell related inflammatory cytokines IL-1beta, IL-6, IL-8and MMP-3, iNOS, COX-2and TNF receptor TNFRI, TNFRII mRNA expression:which are divided into 6groups:blank group, the TNF alpha model, methotrexate (MTX) positive MTX group (including1mu g/ml of MTX) and the concentration of kunduz mother decoction group (respectively, including Kunmu dedoction alcohol extract from the g/ml). Except the blank group, the rest of the groups are added the TNF alpha10ng/L,24h after cell culture using RT-PCR method to detect the mRNA expression.
4. Using the ELISA method to detect the groups after treatment cell culture supernatant of IL-1beta, IL-6, IL-8and MMP-3, PGE2protein expression.
5. Griess NO measurement method is used todetect each group including dose MTX group and kun soup group NO secretion level in the supernatant.
Results
1. Cell culture experiments, and observed under inverted microscope primary synovial cell growth situation, mainly on fibroblast cells, cell a fork shaped or polygon or fusiform. Synovial tissue separation and purification of fibroblasts in flow cytometry surface marker identification. Results show that the CD14, CD3positive RAte less than2%, and CD90positive RAte of more than93%. Fibroblasts purity is higher.
2. Determined by MTT experiment results show that the determined by MTT test results show that with the extension of intervention time, the groups of fibroblast-like synoviocyte-inhibition RAte0D value increases with time and dose increased g RAdually rise, rendering time and dose dependent.24h dosing intervention, groups of fibroblast-like synoviocyte and blank control group, there was no statistically significant difference (P>0.05), no obvious inhibitory effect on proliferation, while48h after drug intervention, compared to blank group, low dose he mother soup group without obvious inhibition there was no statistically significant difference (P>0.05), MTX, mother kun soup, high dose group are inhibits the proliferation of RA-FLS, compared with TNF alpha group, with significant difference statistically significant (P<0.01), high dose group of fibroblast-like synoviocyte-inhibition RAte have significant difference (P<0.01), the inhibition degree higher than MTX group; After72h, the dosing inhibition was observed in all groups, mother kun soup different dose groups with the increase of drug concentration, more significant inhibitory effect, are dose dependent. Inhibitory effect, low dose group the difference was statistically significant (P<0.05), while MTX group of elder brother, mother soup alcohol ext RAction liquid medium and high dose groups significantly, the rest RAint of the fibroblast-like synoviocyte-significant difference (P<0.01). Compared with low dose group, middle, high dose group difference was statistically significant. Each cell proliferation RAte over time, increased proliferation RAte, the inhibition RAte also increased, at the time of72h, cells increment RAte is highest, and the inhibition RAte was the highest. At the same time, the higher the drug concentration, the inhibition RAte is higher. MTX group inhibition RAte between middle and high dose group.
3. The mother kun soup for fibroblast-like synoviocyte-TNFRI and TNFRII expression effect
Semi quantitative fluorescence PCR results showed that TNF alpha after induction, has promoting effect to each TNFRII mRNA expression, expression increased, compared with the blank group, the difference was statistically significant (P<0.05). And mRNA expression of TNFRI all fell.24h after drug Intervention, low dose group TNFRI and TNFRII mRNA expression differences had no statistical significance (P>0.05), while MTX TNFRII, medium and high dose group was obviously reduced, TNFRI is slightly higher, the difference was statistically significant (P<0.05). While MTX, significant difference was found in high dose group (P<0.01).
4. Kunmu dedoction for RA synovial fibroblasts related cytokines IL-1beta, IL-6, IL8expression
Rt-pcr results showed that after joining TNF alpha induced IL-1beta, constituting the mRNA expression of IL-6, IL-8volume increased significantly, compared with blank group, the amount of mRNA expression difference was statistically significant (P<0.05), suggesting the TNF alpha beta can be induced IL-1, IL-6, IL-8mRNA expression, in24h after dosing intervention, relative to the TNF alpha group, low dose group of IL-6, IL-8RNA expression quantity differences had statistics significance (P>0.05), and the MTX group and middle, high dose group of TNF alpha cells of IL-6, IL-8RNA expression quantity are lower than TNF alpha group, all can inhibit RNA expression quantity (P<0.05), the difference was statistically significant, MTX, significant difference was found in high dose group (P<0.01). On the role of il-6, MTX between medium and high dose group, and to the role of IL-8, high dose group and MTX group. Low, medium dose group have no obvious inhibition of IL-1beta, there was no statistically significant difference (P>0.05), while MTX group, high dose group were significantly different (P<0.01). ELISA test results showed that TNF alpha beta after induction of inflammatory factor, IL-1, IL-6, IL-8has the promoting effect of protein expression, protein expression increased, compared with the blank group, the difference was statistically significant (P<0.05). In24h after drug intervention, and TNF alpha cont RAst induced group, low dose group of cell culture supernatant IL-6, IL-8content is no statistically significant differences, middle, high dose difference was statistically significant (P<0.05), MTX, high dose mother kun soup different dose groups decreased significantly (P<0.01). Low, medium dose group of IL-1beta protein expression effect and TNF alpha group has no statistically significant difference (P>0.05), while MTX group and high dose group of beta protein expression of IL-1inhibition has a significant difference (P<0.05).
5. Kunmu dedoction for fibroblast-like synoviocyte-MMP3expression of cytokines
Rt-pcr results showed that TNF alpha can induce the mRNA expression of MMP-3to join the TNF alpha group after induction of RNA expression quantity increased significantly, groups of RNA inhibition of MMP-3, and TNF alpha group compared, in addition to the low dose group, the rest of the group has significant difference, MTX medium and high dose groups were significantly different (P<0.01), MTX group effect and the middle dose group, there was no statistically significant difference (P>0.05). ELISA test results showed that TNF alpha induced the expression of MMP-3protein after all have promoting effect, protein expression increased, compared with the blank group, the difference was statistically significant (P<0.05).24h after drug intervention, the inhibition of MMP-3protein and TNF alpha group compared, in addition to the low dose group, the rest of the group has significant difference and the medium and high dose groups were significantly different (P<0.01), MTX group and high dose group, there was no statistically significant difference (P>0.05).
6. Kunmu dedoction for fibroblast-like synoviocyte cytokines NO and iNOS expression
Groups NO test results showed that each group to join the TNF alpha group after induction of RNA expression quantity increased significantly, compared with blank group, the difference was statistically significant (P<0.01). In24h after dosing intervention, compared with the TNF alpha group, low dose group NO difference NO statistical significance (P>0.05), and the MTX group and middle, high dose group were lower than TNF alpha group, all can inhibit the NO, the difference was statistically significant, and significant difference (P<0.01). Fluorescent PCR detection results showed that TNF alpha can be induced iNOS secretion, express the TNF alpha group after induction of RNA expression quantity increased significantly compared with blank group the difference was statistically significant (P<0.05). In24h after dosing intervention, compared with the TNF alpha group, low dose group of the RNA expression difference was not statistically significance (P>0.05), and the MTX group and middle, high dose group of cell RNA expression quantity of TNF alpha group were lower than TNF alpha group, all can inhibit RNA expression quantity, the difference was statistically significant, and significant difference (P<0.01).
7. Kunmu dedoction for fibroblast-like synoviocyte-cytokine effects of cox-2and PGE2expression
Semi quantitative fluorescence PCR results showed that TNF alpha after induction of each mRNA expression of cox-2has promoting effect, expressing quantity increased, compared with the blank group, the difference was statistically significant (P<0.05).24h after drug intervention, cont RAst and TNF alpha group, low dose group of cox-2mRNA expression difference was statistically significant (P<0.05), while MTX, medium and high dose group decreased obviously (P<0.01). ELISA test results showed that TNF alpha for each group after induction of PGE2protein expression had promoting effect, compared with the blank group, the difference was statistically significant (P<0.05).24h after drug intervention, and comparison of TNF alpha group, low dose group PGE2content difference was statistically significant, and MTX, medium and high dose difference was statistically significant (P<0.05).
Conclusion
Kunmu dedoction inhibits the proliferation of fibroblast-like synoviocyte cells, and suppress the expression of TNF alpha induced inflammatory cytokines of IL-1beta, IL-6, IL-8, MMP-3, and NO, iNOS, COX-2, PGE2, it can also suppress TNF alpha and TNFRII combination of speculation and inhibit the TNF alpha female soup combined with TNFRII, inhibition of cytokine production, thus inhibits the block TNF alpha mediating cell proliferation, synovial inflammation, hyperplasia of the cartilage and bone destruction. This reseach of Kunmu Dedoction provides the experimental basis and way for RA.
引文
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