MMP/TIMP失衡在肺部疾病中的研究进展

基质金属蛋白酶(matrix metalloproteinases,MMPs)是一类锌依赖性内肽酶家族,可以特异性降解细胞外基质(extracellular matrix,ECM)。基质金属蛋白酶组织抑制因子(tissue inhibitor of metalloproteinases,TIMPs)是MMPs的内源性抑制剂,可抑制MMPs、整合素-金属蛋白酶以及聚蛋白多糖酶。TIMPs/MMPs之间的平衡可以调节组织重塑、修复和再吸收。二者在人体中保持平衡状态,近年来发现二者失衡可导致多种疾病的发生,本文主要对MMPs/TIMPs失衡及作用机制在肺部疾病中的研究进展进行简单综述。     

腹腔一次注射法构建百草枯致小鼠肺间质纤维化模型

目的建立一种简便易行的百草枯(PQ)致肺间质纤维化动物模型。方法 33只C57BL/6J小鼠随机分为实验组(30只)和对照组(3只)。实验组腹腔一次性注射PQ 10 mg/kg,并于染毒后第2、5、7、14、28天处死小鼠,对照组腹腔注射生理盐水并于第28天处死。观察两组小鼠的一般情况、肺组织大体结构和肺组织病理学改变。结果实验组小鼠在染毒后2 h即出现中毒性改变,肺组织在第28天出现了明显的纤维化改变。结论腹腔一次性注射PQ能简便、可靠的构建出肺纤维化动物模型,可用于进一步研究PQ中毒所致的肺间质纤维化发病机制和治疗方法。     

基质金属蛋白酶及其组织抑制剂研究进展

基质金属蛋白酶家族是细胞外基质降解过程中的重要酶类,组织金属蛋白酶抑制剂是基质金属蛋白酶的天然抑制物。研究证实,细胞外基质中基质金属蛋白酶及其组织抑制剂的失衡与多种病理机制有关,尤其与肿瘤的侵袭和转移密切相关。本就基质金属蛋白酶及其组织抑制剂的性质、结构以及功能进行了综述。     

血红素氧合酶-1 与肺纤维化

血红素氧合酶-1(Hene Oxygenase-1)是一种氧化应激反应蛋白,广泛存在全身各组织器官。HO-1及催化产物组成了重要的内源性保护系统,具有调控炎症、抗氧化损伤及抗细胞凋亡等作用,对于组织器官具有保护作用。肺纤维化发病机制复杂,氧化应激是肺纤维化的致病机制之一。HO-1是一种重要的抗氧化剂,其通过多种途径参与致病,在肺纤维化致病过程中发挥重要作用。     

己酮可可碱对肺纤维化大鼠MMP-2和TIMP-1表达的影响

目的观察己酮可可碱(pentoxifylline, PTX)对博来霉素致肺纤维化大鼠肺组织基质金属蛋白酶2(matrix metalloproteinase-2)和基质金属蛋白酶组织抑制剂1(tissue inhibitor of matrix metalloproteinase-1)表达的影响,初步探讨其抗肺纤维化的作用机制.方法 SD大鼠36只,随机分为模型组、治疗组和对照组.模型组和治疗组气管内注射博来霉素诱导肺纤维化,对照组在相同条件下给予生理盐水.第二天起治疗组大鼠腹腔给予己酮可可碱6mg/kg.d,其余两组相同条件下给予生理盐水.治疗的第7d和28d,处死动物取出肺组织,用RT-PCR和免疫组化ABC法观察各组鼠肺组织MMP-2和TIMP-1表达的变化. 结果与模型组比较,治疗组经PTX作用的第7d和28d肺组织中MMP-2和TIMP-1 mRNA的基因转录均有减少,MMP-2 mRNA表达分别降低33.4%和35.5%(P＜0.001),TIMP-1 mRNA表达分别降低25.3%和33.0%(P＜0.05).免疫组化结果则显示,PTX作用的第7d和28dMMP-2分别较模型组降低30.7%和41.7%(P＜0.05),TIMP-1分别降低13.1%和19.8%(P＜0.05).结论 PTX对肺纤维化不同时期肺组织中MMP-2和TIMP-1的表达均有一定程度的降低作用,其可能通过调整MMP-2和TIMP-1比值使其趋于平衡,从而延缓甚至抑制纤维化的进程.     

维生素B_3与帕金森病的防治

帕金森病(PD)是以运动功能失调为主要表现的神经退行性疾病,其病理特征是黑质致密部多巴胺能神经元的丢失和路易氏小体的形成。线粒体能量供应障碍和氧化应激在PD发生与发展过程中起重要作用,抗氧化和改善线粒体功能的物质可延缓PD的发展。维生素B_3是体内氧化还原体系的重要组分,参与抗氧化、抗炎、促进自噬以及维护神经元正常结构和功能,提示补充维生素B_3可能是延缓PD的方法之一。     

百草枯通过SOCE途径促进肺纤维化的发生

摘要 目的：探讨SOCE对百草枯（PQ）致肺纤维化过程的具体调控作用。方法：体外培养肺泡上皮A549细胞,设置分组为对照组、PQ组和PQ+SKF96365组。对照组不作任何处理;PQ组给予800 μM的PQ溶液处理细胞24 h;PQ+SKF96365组先加入10 μM的SKF96365预处理细胞2 h,然后再加800 μM PQ处理24 h。免疫荧光检测PQ染毒后SOCE相关蛋白的活化和胞内分布、NFATc1的表达和入核情况。清洁级小鼠40只,随机分为空白对照组、PQ组、SKF96365组和PQ+SKF96365组。PQ组及PQ+SKF96365组给予百草枯（20 mg/kg）一次性腹腔注射,对照组给予等量生理盐水;SKFS96365组及PQ+SKF96365组每天腹腔注射一次10 mg/kg的SKF96365溶液,持续3天,行HE染色、Masson染色观察肺组织病理状态改变以及胶原纤维的变化情况。结果：与正常组相比,PQ染毒组STIM1蛋白活化出现寡聚化现象并且ORAI1、TRPC1膜定位显著;PQ引起细胞质内NFATc1信号向细胞核内转移,密度差异具有统计学意义（P<0.01）,使用SOCE抑制剂后NFATc1信号减弱向细胞质内转移（P<0.01）。与对照组相比,PQ中毒的小鼠肺泡结构被破坏,肺间质大量胶原纤维沉积;与PQ组相比,PQ+SKF96365组肺泡结构相对完整并且间质胶原纤维沉积减少。结论：PQ中毒可活化SOCE并通过增加NFAT入核激活下游转录信号,促进肺纤维化的发生。     

PARs在IPF中的作用及其靶向药物研究进展

特发性肺纤维化(idiopathic pulmonary fibrosis, IPF)是一种病因不明的慢性、进行性、纤维化性间质性肺炎,治愈率低而死亡率很高,目前有效的治疗药物较为匮乏。蛋白酶激活受体(protease activated receptors, PARs)的不可逆地激活对维持肺功能具有重要作用,PARs的异常表达与活化可促进炎症和纤维化过程。因此,在特发性肺纤维化的治疗中,靶向PARs可能成为一种新的治疗手段。该文介绍了PARs在促进特发性肺纤维化进程中的功能及作用机制,并且总结了靶向PARs的抗肺纤维化药物的研究进展。     

博莱霉素致肺间质成纤维细胞MMP-2/TIMP-1表达失衡

观察博莱霉素对肺间质成纤维细胞中基质金属蛋白酶-2（MMP-2）及组织金属蛋白酶抑制剂-1（TIMP-1）表达的影响,探讨博莱霉素引起肺纤维化的机制。体外培养肺间质成纤维细胞,并向培养基中加入博莱霉素,在作用不同时间后收集样本,采用酶谱图测定细胞培养上清液中MMP-2酶活性、ELISA测定TIMP-1量,免疫组织化学法检测细胞中MMP-2、TIMP-1的原位表达,RT-PCR法检测MMP-2和TIMP-1的mRNA水平。结果发现,博莱霉素在2h、12h促进MMP-2的分泌,24h后无促分泌作用;而2-48h,MMP-2的原位表达及mRNA均不受博莱霉素的影响;博莱霉素从12h开始促进TIMP-1及mRNA的表达,并持续至48h。结果表明博莱霉素可引起肺间质戍纤维细胞MMP-2／TIMP-1表达失衡,并可能参与肺纤维化的发生。     

大黄治疗急性百草枯中毒的作用研究

目的:通过观察PQ中毒患者SOD、TNF-α、IL-6、PaO2、PaCO2、BUN和Cr水平,及血浆PQ浓度的变化,旨在探讨大黄在治疗PQ中毒中的作用。方法:36例急性百草枯中毒患者随机分为对照组(n=15)和治疗组(n=21),所有患者均给予常规治疗,对照组应用甘露醇导泻,治疗组应用生大黄混悬液导泻,采用酶联免疫吸附法(enzyme linked immunosorbent assay,ELISA)检测所有患者血清SOD、TNF-α、IL-6水平,高效液相色谱法(High Performance Liquid Chromatography,HPLC)测定患者血浆PQ浓度,并测定PaO2、PaCO2、BUN和Cr。结果:治疗组患者第1、3、7天PaO2、SOD水平显著高于对照组,PaCO2、BUN、Cr、TNF-α、IL-6水平和PQ浓度亦较对照组均显著降低(P0.05)。结论:大黄可降低PQ中毒患者血中PQ浓度,抑制TNF-α、IL-6的表达,明显升高SOD的水平,改善PQ中毒患者肺、肾功能。     

HIF‐1α regulates EMT via the Snail and β‐catenin pathways in paraquat poisoning‐induced early pulmonary fibrosis

Paraquat (PQ) poisoning‐induced pulmonary fibrosis is one of the primary causes of death in patients with PQ poisoning. Hypoxia‐inducible factor‐1α (HIF‐1α) and epithelial‐mesenchymal transition (EMT) are involved in the progression of pulmonary fibrosis. Snail and β‐catenin are two other factors involved in promoting EMT. However, the relationship among HIF‐1α, Snail and β‐catenin in PQ poisoning‐induced pulmonary fibrosis is not clear. Our research aimed to determine whether the regulation of HIF‐1α in EMT occurs via the Snail and β‐catenin pathways in PQ poisoning‐induced pulmonary fibrosis. Sixty‐six Sprague–Dawley rats were randomly and evenly divided into a control group and a PQ group. The PQ group was treated with an intragastric infusion of a 20% PQ solution (50 mg/kg) for 2, 6, 12, 24, 48 and 72 hrs. A549 and RLE‐6TN cell lines were transfected with HIF‐1α siRNA for 48 hrs before being exposed to PQ. Western blotting, real‐time quantitative PCR, immunofluorescence, immunohistochemistry and other assays were used in our research. In vivo, the protein levels of HIF‐1α and α‐SMA were increased at 2 hrs and the level of ZO‐1 (Zonula Occluden‐1) was reduced at 12 hrs. In vitro, the transient transfection of HIF‐1α siRNA resulted in a decrease in the degree of EMT. The expression levels of Snail and β‐catenin were significantly reduced when HIF‐α was silenced. These data demonstrate that EMT may be involved in PQ poisoning‐induced pulmonary fibrosis and regulated by HIF‐1α via the Snail and β‐catenin pathways. Hypoxia‐inducible factor‐1α may be a therapeutic target for the treatment of PQ poisoning‐induced pulmonary fibrosis.     

Rapamycin attenuates the paraquat-induced pulmonary fibrosis through activating Nrf2 pathway

Oxidative stress is a key regulator of idiopathic pulmonary fibrosis. Paraquat (PQ)-induced pulmonary fibrosis seriously endangers people's health. Rapamycin has been reported to alleviate PQ-induced pulmonary fibrosis, but its underlying mechanism is unclear. The nuclear factor E2-related factor 2 (Nrf2) plays an important regulatory role in the antioxidant therapy of PQ-induced pulmonary fibrosis. In this study, we tried to confirm that rapamycin attenuates PQ-induced pulmonary fibrosis by regulating Nrf2 pathway. In vivo, we proved that rapamycin could inhibit the degree of PQ-induced oxidant stress as well as enhanced the expression of Nrf2. In vitro, rapamycin decreased the upregulated effects of cell death and apoptosis, fibrosis-related factors expression and fibroblast-to-myofibroblast transformation by PQ treatment. In vivo, rapamycin treatment reduced fibrosis degree and the expression of fibrosis-related factors in lung tissues of rat treated PQ. Furthermore, we also found that Nrf2 knockdown reduced the inhibitory effect of rapamycin on PQ-induced pulmonary fibrosis, as well as decreased Nrf2 transfer from the cytoplasm into the nucleus. Our findings demonstrated that the protective effect of rapamycin is associated with the activation of the Nrf2 pathway in pulmonary fibrosis induced by PQ poisoning.     

Ameliorative role of aspirin in paraquat‐induced lung toxicity via mitochondrial mechanisms

Paraquat (PQ) has accounted for numerous suicide attempts in developing countries. Aspirin (ASA) as an adjuvant treatment in PQ poisoning has an ameliorative role. And, it's uncoupling of mitochondrial oxidative phosphorylation role has been well established. The current study aimed at examining the aspirin mechanism on lung mitochondria of rats exposed to PQ. Male rats were randomly allocated in five groups: Control group, PQ group (50 mg/kg; orally, only on the first day), and PQ + ASA (100, 200, and 400 mg/kg; i.p.) groups for 3 weeks. Mitochondrial indices and respiratory chain‐complex activities were determined. PQ induced lung interstitial fibrosis; however, ASA (400 mg/kg) led to decrease in this abnormal alteration. In comparison with PQ group, complex II and IV activity, and adenosine triphosphate content in ASA groups had significantly increased; however, reactive oxygen species production, mitochondrial membrane permeabilization, and mitochondrial swelling were significantly reduced. In conclusion, aspirin can alleviate lung injury induced by PQ poisoning by improving mitochondrial dynamics.     

甘草酸二铵对百草枯中毒致急性肺损伤大鼠TLR-4表达的影响

目的探讨甘草酸二铵(DG)对百草枯(PQ)中毒致急性肺损伤(ALI)大鼠的保护作用及其机制。方法选择健康SD大鼠50只,随机分为百草枯组(PQ组)、甘草酸二铵组(DG组)和正常对照组(NS组),PQ组和DG组予百草枯100mg/kg灌胃1次,DG组于灌胃后立即腹腔注射DG 50mg/kg,每日1次,对照组与PQ组注射等剂量的生理盐水。观察至48h处死大鼠,取肺组织检测肺湿干重比;肺组织HE染色评价肺组织损伤情况;采用RT-PCR法检测肺组织TLR-4mRNA和NF-κB mRNA的表达情况。另选择健康SD大鼠50只,分组及各组处置方法同上,观察其7天内死亡率。结果 PQ组与DG组肺湿干比、肺组织TLR-4mRNA和NF-κB mRNA表达较NS组明显升高(P0.01);DG组各指标明显低于PQ组(P0.01)。HE染色结果,NS组肺部结构正常,PQ组、DG组可见肺组织水肿、出血及炎性细胞浸润等肺损伤表现,DG组病变轻于PQ组。群体死亡率比较,PQ组7天死亡率为90%,DG组为40%,NS组无死亡。结论甘草酸二铵可减轻百草枯中毒致急性肺损伤大鼠肺部炎症,其机制可能与降低TLR-4、NF-κB的表达有关。     

Therapeutical Effects and Mechanism of Salubrinal Combined with Ulinastatin on Treating Paraquat Poisoning

To explore therapeutic effects and underlying mechanism of Salubrinal combined with Ulinastatin (UTI) on acute Paraquat (PQ) poisoning. Four hundred rats were randomly allocated into UTI group, SAL group, SAL + UTI and control group according to random number table with 100 rats in each group. Acute PQ poisoning models were established, and all rats received UTI, Salubrinal, SAL + UTI and normal saline injection, respectively. Afterward, we analyzed the change of lung tissue and explored the mechanism. Acute PQ poisoning caused significantly damage in rat lung tissue structure, and UTI could effectively repair lung tissue damage. Salubrinal suppressed hemorrhage and fibrosis, but promoted inflammatory infiltration. In contrast, UTI + Salubrinal suppressed hemorrhage, fibrosis and inflammatory infiltration, but could not improve lung tissue damage. Expression of LC3 and Bcl-2 showed statistically significant difference among different groups (p < 0.05). LC3 and Bcl-2 levels in UTI group were much higher than in the other groups, and LC3 and Bcl-2 levels in UTI + SAL group was second higher. LC expression in SAL group was lower than in UTI group and UTI + SAL group with Bcl-2 in control group significantly lower than in the other groups (p < 0.05). Expression of Caspase-3 and Bcl-2/Bax in lung tissue in different groups had statistically significant difference (p < 0.05). Caspase-3 in UTI group was lower than in the other groups; however, Bcl-2/Bax in UTI group was higher than in the other groups (p < 0.05). Acute PQ poisoning can cause endoplasmic reticulum stress–autophagy in rat, and UTI can increase Bcl-2 expression, decrease Caspase-3, which can inhibit progress of lung injury by suppressing apoptosis and exert good therapeutic effects. Although salubrinal has marked effects on protecting lung tissue, it can increase Bcl-2 expression, which is not beneficial to lung tissue protection. The underlying mechanism still needs further exploration.     

Paraquat Induces Epithelial-Mesenchymal Transition-Like Cellular Response Resulting in Fibrogenesis and the Prevention of Apoptosis in Human Pulmonary Epithelial Cells

The aim of this study is to investigate the molecular mechanisms underlying delayed progressive pulmonary fibrosis, a characteristic of subacute paraquat (PQ) poisoning. Epithelial-mesenchymal transition (EMT) has been proposed as a cause of organ fibrosis, and transforming growth factor-β (TGF-β) is suggested to be a powerful mediator of EMT. We thus examined the possibility that EMT is involved in pulmonary fibrosis during PQ poisoning using A549 human alveolar epithelial cells in vitro. The cells were treated with various concentrations of PQ (0–500 μM) for 2–12 days. Short-term (2 days) high-dose (>100 μM) treatments with PQ induced cell death accompanied by the activation of caspase9 as well as a decrease in E-cadherin (an epithelial cell marker), suggesting apoptotic cell death with the features of anoikis (cell death due to the loss of cell-cell adhesion). In contrast, long-term (6–12 days) low-dose (30 μM) treatments with PQ resulted in a transformation into spindle-shaped mesenchymal-like cells with a decrease of E-cadherin as well as an increase of α-smooth muscle actin (α-SMA). The mesenchymal-like cells also secreted the extracellular matrix (ECM) protein fibronectin into the culture medium. The administration of a TGF-β1 receptor antagonist, SB431542, almost completely attenuated the mesenchymal transformation as well as fibronectin secretion, suggesting a crucial role of TGF-β1 in EMT-like cellular response and subsequent fibrogenesis. It is noteworthy that despite the suppression of EMT-fibrogenesis, apoptotic death was observed in cells treated with PQ+SB431542. EMT-like cellular response and subsequent fibrogenesis were also observed in normal human bronchial epithelial (NHBE) cells exposed to PQ in a TGF-β1-dependent manner. Taken together, our experimental model reflects well the etiology of PQ poisoning in human and shows the involvement of EMT-like cellular response in both fibrogenesis and resistance to cell death during subacute PQ poisoning of pulmonary epithelial cells.     

Polydatin alleviates bleomycin-induced pulmonary fibrosis and alters the gut microbiota in a mouse model

To investigate the effect and mechanism of polydatin on bleomycin (BLM)-induced pulmonary fibrosis in a mouse model. The lung fibrosis model was induced by BLM. The contents of TNF-α, LPS, IL-6 and IL-1β in lung tissue, intestine and serum were detected by ELISA. Gut microbiota diversity was detected by 16S rDNA sequencing; R language was used to analyse species composition, α-diversity, β-diversity, species differences and marker species. Mice were fed drinking water mixed with four antibiotics (ampicillin, neomycin, metronidazole, vancomycin; antibiotics, ABx) to build a mouse model of ABx-induced bacterial depletion; and faecal microbiota from different groups were transplanted into BLM-treated or untreated ABx mice. The histopathological changes and collagen I and α-SMA expression were determined. Polydatin effectively reduced the degree of fibrosis in a BLM-induced pulmonary fibrosis mouse model; BLM and/or polydatin affected the abundance of the dominant gut microbiota in mice. Moreover, faecal microbiota transplantation (FMT) from polydatin-treated BLM mice effectively alleviated lung fibrosis in BLM-treated ABx mice compared with FMT from BLM mice. Polydatin can reduce fibrosis and inflammation in a BLM-induced mouse pulmonary fibrosis model. The alteration of gut microbiota by polydatin may be involved in the therapeutic effect.     

Polydatin suppresses the development of lung inflammation and fibrosis by inhibiting activation of the NACHT domain-, leucine-rich repeat-, and pyd-containing protein 3 inflammasome and the nuclear factor-κB pathway after Mycoplasma pneumoniae infection

Mycoplasma pneumoniae (MP) can infect both the upper and lower respiratory tracts. Polydatin (PD), a traditional Chinese medicine, is known to have anti-inflammation and antifibrosis properties. However, the protective effects of PD against MP pneumonia (MPP) remain unclear. So, the aim of this study was to describe the therapeutic effects and underlying mechanisms of PD against MPP. BALB/c mice were assigned to three groups: a normal control group, MP infection group, or PD-treated MP infection group. BEAS-2B cells transfected with or without NACHT domain-, leucine-rich repeat-, and pyd-containing protein 3 (NLRP3) were used to confirm the protective mechanisms of PD. Immunohistochemical analysis, Western blot analysis, enzyme-linked immunosorbent assay, and flow cytometry were used in this study. The results showed that PD treatment suppressed MP-induced lung injury in mice by suppressing the expression of inflammatory factors and inhibiting the development of pulmonary fibrosis. Meanwhile, PD treatment inhibited activation of the NLRP3 inflammasome and nuclear factor κB (NF-κB) pathway. Overexpression of NLRP3 reversed the protective effect of PD against MP-induced injury of BEAS-2B cells. Taken together, these results indicate that PD treatment suppressed the inflammatory response and the development of pulmonary fibrosis by inhibiting the NLRP3 inflammasome and NF-κB pathway after MP infection.     

Adiponectin Attenuates Lung Fibroblasts Activation and Pulmonary Fibrosis Induced by Paraquat

Pulmonary fibrosis is one of the most common complications of paraquat (PQ) poisoning, which demands for more effective therapies. Accumulating evidence suggests adiponectin (APN) may be a promising therapy against fibrotic diseases. In the current study, we determine whether the exogenous globular APN isoform protects against pulmonary fibrosis in PQ-treated mice and human lung fibroblasts, and dissect the responsible underlying mechanisms. BALB/C mice were divided into control group, PQ group, PQ + low-dose APN group, and PQ + high-dose APN group. Mice were sacrificed 3, 7, 14, and 21 days after PQ treatment. We compared pulmonary histopathological changes among different groups on the basis of fibrosis scores, TGF-β₁, CTGF and α-SMA pulmonary content via Western blot and real-time quantitative fluorescence-PCR (RT-PCR). Blood levels of MMP-9 and TIMP-1 were determined by ELISA. Human lung fibroblasts WI-38 were divided into control group, PQ group, APN group, and APN receptor (AdipoR) 1 small-interfering RNA (siRNA) group. Fibroblasts were collected 24, 48, and 72 hours after PQ exposure for assay. Cell viability and apoptosis were determined via Kit-8 (CCK-8) and fluorescein Annexin V-FITC/PI double labeling. The protein and mRNA expression level of collagen type III, AdipoR1, and AdipoR2 were measured by Western blot and RT-PCR. APN treatment significantly decreased the lung fibrosis scores, protein and mRNA expression of pulmonary TGF-β₁, CTGF and α-SMA content, and blood MMP-9 and TIMP-1 in a dose-dependent manner (p<0.05). Pretreatment with APN significantly attenuated the reduced cell viability and up-regulated collagen type III expression induced by PQ in lung fibroblasts, (p<0.05). APN pretreatment up-regulated AdipoR1, but not AdipoR2, expression in WI-38 fibroblasts. AdipoR1 siRNA abrogated APN-mediated protective effects in PQ-exposed fibroblasts. Taken together, our data suggests APN protects against PQ-induced pulmonary fibrosis in a dose-dependent manner, via suppression of lung fibroblast activation. Functional AdipoR1 are expressed by human WI-38 lung fibroblasts, suggesting potential future clinical applicability of APN against pulmonary fibrosis.