HMGB1在肺纤维化中作用的研究
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摘要
研究背景与目的:
特发性肺纤维化(IPF)是特发性间质性肺疾病最常见的类型,其预后差,生存中位时间仅为3至4年。虽然目前对IPF的发病机制有一定的认识,但对其可能的病因及确切的细胞分子机制还未明了,迄今为止尚无有效的治疗手段。IPF最显著的病理特征是成纤维细胞灶的存在。成纤维细胞灶数目的增多与IPF病程的进展和预后不良相关。成纤维细胞灶的关键效应细胞是肌纤维母细胞。表达α平滑肌肌动蛋白(α-SMA)是肌纤维母细胞区别于成纤维细胞的一个标志性特征。它能促进纤维形成,是生成细胞外基质和促纤维化相关细胞因子的重要来源。因此,阐明炎症损伤与异常修复过程中α-SMA基因的转录调控机制,对于揭示特发性纤维化发病的细胞分子机制具有重要的意义。在前期工作中,我们应用α-SMA启动子为钓饵,在博莱霉素(BLM)诱导肺纤维化的小鼠肺组织中筛选到一个高迁移率组蛋白B1家族(HMGB1)成员,分子量约20KDa,较之经典的HMGB1缺失C端的30个酸性氨基酸残基序列(GenBank登陆号:BAC34367, HMGB34367),是HMGB1的截断分子形式。HMGB1作为核蛋白存在于几乎所有的真核细胞中,它能够稳定核小体形状,作为转录调控因子具有调节某些基因转录活化的作用。最近的研究表明HMGB1是一种晚期炎症因子,能被免疫细胞主动分泌或被损伤和坏死的细胞释放到细胞外环境,触发炎症反应。基于这些已有的发现,我们推测HMGB1可能是联系IPF病理过程中肺损伤和异常修复的关键信号分子。为了证实这一推测,在本研究中,我们探讨了HMGB1以及它的截断分子HMGB34367,在博来霉素诱导的肺纤维化病理过程中的作用,并着重探讨了HMGB34367在IPF的上皮-间充质转分化发生发展中的地位。
研究内容和方法:
第一部分:HMGB1在BLM诱导的肺纤维化小鼠模型肺组织和IPF患者肺组织中的表达。
对BLM诱导的肺纤维化小鼠模型肺组织和IPF患者肺组织予以常规病理学检测,观察病理学改变。应用免疫组化、免疫荧光染色和western blot检测肺组织HMGB1和SMA的蛋白表达情况,检测HMGB1在肺纤维化病理过程中的作用。
第二部分:HMGB34367 RNAi对小鼠肺纤维化病理过程的影响
给予HMGB34367 siRNA干预其在BLM诱导肺纤维化模型小鼠肺组织中的表达,探讨HMGB1的截断分子形式HMGB34367蛋白是否参与了诱导肌纤维母细胞活化过程。首先经气管灌注BLM构建肺纤维化小鼠模型,然后于实验第2、5、8天经鼻滴入HMGB34367 siRNA或siRNA阴性对照。实验第10天处死小鼠留取肺组织进行常规病理学检测,观察病理学改变。应用免疫组化、免疫荧光染色和western blot检测肺组织HMGB1和SMA的蛋白表达变化,探讨HMGB1的高表达对肌纤维母细胞活化的影响。
第三部分:HMGB1对上皮细胞转分化的诱导作用
将HMGB34367真核表达载体pcDNA3.0-HMGB34367转染到上皮细胞16HBE中,提取细胞核蛋白,用EMSA和超迁移(Super shift)实验检测HMGB34367蛋白与α-SMA启动子CArGB基序特异性的结合。将α-SMA启动子红色荧光报告质粒和绿色荧光标记的HMGB34367真核表达载体质粒( pDsRed-SMA +pEGFP-N2-HMGB34367)共转染到上皮细胞中,通过流式细胞仪检测细胞表达红色荧光和绿色荧光强度,探讨过量表达HMGB34367对α-SMA启动子活化的影响。培养人肺泡上皮细胞株A549,予细胞因子混合刺激剂Mix(TGF-β1+IL-1β+IFN-γ)孵育,转染HMGB34367 siRNA干扰其在细胞中的表达,RT-PCR和免疫荧光检测α-SMA和HMGB1基因和蛋白水平的表达。阐明炎症微环境诱导内源性HMGB1对肺纤维化病理过程的影响。
结果:
第一部分:HMGB1在BLM诱导的肺纤维化小鼠模型肺组织和IPF患者肺组织中的的表达。
病理学观察可见:BLM诱导了小鼠肺组织局灶性纤维化病变,在胸膜下区域可见肺泡间隔增厚,肺泡腔破坏,上皮脱落,肺间质大量炎症细胞浸润。Masson胶原染色显示BLM组小鼠肺组织中胶原沉积明显增多,免疫组化检测可见肺组织HMGB1和α-SMA表达增强。IPF患者肺组织中也观察到同样的结果。IPF患者肺组织双重免疫荧光染色揭示肺细支气管的一些上皮细胞同时表达α-SMA和HMGB1,两者可共定位在细胞内同一区域。
第二部分:HMGB34367 RNAi对小鼠肺纤维化病理过程的影响
组织病理学观察表明,与BLM组和siRNA阴性对照组相比, HMGB34367 siRNA显著减轻了BLM诱导的小鼠肺纤维化损害;相应的免疫组化结果证实肺组织中HMGB1的表达明显下调;与此同时HMGB34367 siRNA组小鼠损伤的上皮细胞和上皮下细胞的α-SMA表达也明显减少。Western-Blot的结果和免疫组化类似。
第三部分:HMGB1对气道上皮细胞转分化的诱导作用
EMSA和超迁移(super shif)结果显示HMGB34367蛋白可与α-SMA启动子CArGA基序特异性结合。流式细胞仪检测结果显示共转染质粒pDsRed-SMA+pEGFP-N2-HMGB34367组红/绿色荧光相对值较对照组明显升高。在TGF-β1刺激的条件下,前者红/绿色荧光相对值较对照组增强更为明显。混合刺激剂诱导A549细胞表型向间充质样细胞转化,细胞内HMGB1的表达量增多。RT-PCR结果表明,加入混合刺激剂Mix作用后刺激了A549细胞α-SMA和HMGB34367基因的表达。免疫荧光结果揭示混合刺激剂处理48小时后,一些上皮细胞出现α-SMA的阳性表达。HMGB34367RNAi能有效降低HMGB1的表达,与此同时可见α-SMA的基因和蛋白水平的表达也相应降低。HMGB34367 RNAi具有特异性地阻断α-SMA的诱导表达能力。
结论:
1、HMGB1的诱导表达在肺纤维化发病过程中发挥了重要的作用。
2、HMGB34367蛋白作为HMGB1蛋白的截断分子形式,能够与α-SMA启动子特异性结合,作为α-SMA基因启动子的特异性转录激活子,诱导α-SMA基因的转录活化,在上皮-间质转分化过程中发挥作用。
3、HMGB1可能是联系IPF病理过程中肺损伤和异常修复的关键信号分子
Rationale:
Idiopathic pulmonary fibrosis (IPF), which is the most common idiopathic interstitial lung disease, has the worse prognosis, with a median survival of only 3 to 4 years. Although much has been learned recently about the pathogenesis of IPF, the etiology and precise cellular and molecular mechanisms involved are not established. So far, no specific therapy has been proven unequivocally effective in IPF. The most notable characteristic feature of IPF is presence of fibroblastic foci. Increased numbers of fibroblastic foci are associated with disease progression and a worse prognosis in IPF. The key effector cell in fibroblastic foci is the myofibroblast. Myofibroblasts, with their characteristicα-smooth muscle actin (α-SMA) expression, arise de novo in fibrosis, and they are thought to be the primary source of heightened matrix and profibrogenic cytokine expression. Hence, understanding the mechanism responsible for abnormal activation and transcriptional regulation ofα-SMA gene is important to uncover the fibrotic pathogenesis. We have previously identified a 20Kda nuclear protein (GenBank number:BAC34367) that is a C-terminal truncated form of high mobility group B-1(HMGB1) in the lung of bleomycin-treated mice, with an increased binding toα-SMA promoter. HMGB1 is a nuclear protein that is present in almost all eukaryotic cells, and it functions to stabilize nucleosome formation and acts as a transcription-factor like protein that regulates the expression of several genes. Recent studies identify HMGB1 as a delayed mediator of inflammation, damaged or necrotic cells can release HMGB1 into the extracellular milieu, where it triggers inflammatory responses. Based on these findings described as above, we hypothesize that HMGB1 may function as a key signal molecular that links lung injury to abnormal repair in the development of IPF. To validate the contribution of HMGB1 to the pathogenesis of the disease, we evaluated the role of HMGB34367, a C-terminal truncated form of HMGB1, in the initiation and progression of lung fibrosis induced by BLM, especially clarified its role in epithelial-myofibroblast transdifferentiation underlying the pathogenesis of IPF.
Methods
The first part:
Investigation of the expression of HMGB1 on the lung tissues of mice with pulmonary fibrosis induced by BLM and IPF patients.
The lung tissues sections were prepared from BLM-treated mice and IPF patients according to routine protocols for pathological examination, where immunostainings toα-SMA and HMGB1 were performed on determination of the role of HMGB1 in the lung fibrotic process.
The second part:
Effects of HMGB34367 RNAi on the pathological process of pulmonary fibrosis To validate if the truncated form of HMGB1 is involved in activation of myofibroblasts, the BLM-treated mice were administered intranasally with either HMGB34367 siRNA or control siRNA (20μl /per time )on day 2, 5, 8 after BLM addition. The lung tissues were isolated for pathological examination. Immunostaining toα-SMA and HMGB1 and western blot were performed for determination of effect of the increased level of HMGB1 on activation of myofibroblasts.
The third part:
The role of HMGB1 in the transdifferentiation of pulmonary epithelial cells into myofibroblasts
The 16HBE cells transfected with pcDNA3.0-HMGB34367 were subjected to performance of Electrophoretic Mobility Shift Assay(EMSA) and Super shift for detection of the binding activity of HMGB34367 withα-SMA promoter CarGB motif. The flowcytometry assay for measurement of the ratio of red fluorescence (RFP protein expressed under guidance ofα-SMA promoter) to green fluorescence of GPF-HMGB34367 fusion protein .was performed on qualification ofα-SMA promoter’s activation in response to over-expression of HMGB34367 in the cells after co-transfected with pGPF-HMGB34367 and pDsRed-SMA . To elucidate endogenous induction of HMGB1 by inflammatory micro-environment in lung fibrotic process,the A549 cells were cultured with cytokines-mix (TGF-β1+TNF-α+ IFN-γ) in absence or presence of HMGB34367-RNAi followed by RT-PCR and immunofluorescent staining to test expression ofα-SMA and HMGB1,
Result
The first part:
Investigation of the expression of HMGB1 on the lung tissues of BLM-induced pulmonary fibrosis mice and IPF.
Pathological examination demonstrated that BLM administration induced focal fibrotic lesions in mice lungs, primarily in the subpleural regions with thickened or thickening interalveolar septa with obvious alveolar destruction, mesenchyma infiltration with inflammatory cells and epithelial shedding. By Masson's trichrome staining, it was demonstrated an increased level of collagen synthesis. .Immunostaining displayed expression of both HMGB1 andα-SMA with a significant elevated level on the lung tissues of BLM-treated mice. In the pulmonary tissue of patients with IPF, we observed the same as above. Using double immunofluorescent staining, with antibodies againstα-SMA and HMGB1, we further observed certain cells located at bronchiolar epithelium of the pulmonary tissue of IPF with double staining ofα-SMA and HMGB1.
The second part:
Effects of HMGB34367 RNAi on the pathological process of pulmonary fibrosis
Treatment of HMGB34367 siRNA significantly attenuated the lung fibrotic lesion in the mice induced by BLM. Correspondingly, immunohistochemistry examination demonstrated that compare with the mice with challenge by BLM alone or plus with siRNA control, the expression of HMGB1 were obviously decreased in the BLM-induced mice with the treatment of HMGB34367 siRNA .,α-SMA expression in the injured epithelial cells and subepithelial cells was correspondingly down-regulated .The same results were also detected by Western-Blot.
The third part:
The role of HMGB1 in the transdifferentiation of airway epithelial cells into myofibroblasts
EMSA and super shif revealed that the truncated form of HMGB1 can specifically bind to CArG B motif. By Flow cytometry, without and with TGFβ1, RFP/GFP signal ratio was detected with a higher level in the cells co-transfected with pDsRed-SMA+pEGFP-N2-HMGB34367 plasmids than those co-transfected with control plasmids. Following with treatment of cytokines-mix, the A549 cells appeared myofibroblastic morphology concomitant with highly expressed intracellular HMGB1. As shown by the results of RT-PCR and immunofluorescent staining, transcriptional induction ofα-SMA and HMGB34367 genes were detected in the A549 cells after treatment of the Mix .α-SMA positive staining were observed in some of the treated A549 cells. It was shown that HMGB34367RNAi has an ability to specifically block the induction ofα-SMA at protein or gene level.
Conclusion
1、Induction of HMGB1 plays an important role in the process of lung fibrosis
2、The truncated form of HMGB1, HMGB34367, can act as a transcriptional factor for up-regulation of expression ofα-SMA.gene, contributing to epithelial cell to myofibroblast transdifferentiation.
3. HMGB1 may function as a key signal molecular that links lung injury to abnormal repair in the development of IPF.
特发性肺纤维化(IPF)是特发性间质性肺疾病最常见的类型,其预后差,生存中位时间仅为3至4年。虽然目前对IPF的发病机制有一定的认识,但对其可能的病因及确切的细胞分子机制还未明了,迄今为止尚无有效的治疗手段。IPF最显著的病理特征是成纤维细胞灶的存在。成纤维细胞灶数目的增多与IPF病程的进展和预后不良相关。成纤维细胞灶的关键效应细胞是肌纤维母细胞。表达α平滑肌肌动蛋白(α-SMA)是肌纤维母细胞区别于成纤维细胞的一个标志性特征。它能促进纤维形成,是生成细胞外基质和促纤维化相关细胞因子的重要来源。因此,阐明炎症损伤与异常修复过程中α-SMA基因的转录调控机制,对于揭示特发性纤维化发病的细胞分子机制具有重要的意义。在前期工作中,我们应用α-SMA启动子为钓饵,在博莱霉素(BLM)诱导肺纤维化的小鼠肺组织中筛选到一个高迁移率组蛋白B1家族(HMGB1)成员,分子量约20KDa,较之经典的HMGB1缺失C端的30个酸性氨基酸残基序列(GenBank登陆号:BAC34367, HMGB34367),是HMGB1的截断分子形式。HMGB1作为核蛋白存在于几乎所有的真核细胞中,它能够稳定核小体形状,作为转录调控因子具有调节某些基因转录活化的作用。最近的研究表明HMGB1是一种晚期炎症因子,能被免疫细胞主动分泌或被损伤和坏死的细胞释放到细胞外环境,触发炎症反应。基于这些已有的发现,我们推测HMGB1可能是联系IPF病理过程中肺损伤和异常修复的关键信号分子。为了证实这一推测,在本研究中,我们探讨了HMGB1以及它的截断分子HMGB34367,在博来霉素诱导的肺纤维化病理过程中的作用,并着重探讨了HMGB34367在IPF的上皮-间充质转分化发生发展中的地位。
研究内容和方法:
第一部分:HMGB1在BLM诱导的肺纤维化小鼠模型肺组织和IPF患者肺组织中的表达。
对BLM诱导的肺纤维化小鼠模型肺组织和IPF患者肺组织予以常规病理学检测,观察病理学改变。应用免疫组化、免疫荧光染色和western blot检测肺组织HMGB1和SMA的蛋白表达情况,检测HMGB1在肺纤维化病理过程中的作用。
第二部分:HMGB34367 RNAi对小鼠肺纤维化病理过程的影响
给予HMGB34367 siRNA干预其在BLM诱导肺纤维化模型小鼠肺组织中的表达,探讨HMGB1的截断分子形式HMGB34367蛋白是否参与了诱导肌纤维母细胞活化过程。首先经气管灌注BLM构建肺纤维化小鼠模型,然后于实验第2、5、8天经鼻滴入HMGB34367 siRNA或siRNA阴性对照。实验第10天处死小鼠留取肺组织进行常规病理学检测,观察病理学改变。应用免疫组化、免疫荧光染色和western blot检测肺组织HMGB1和SMA的蛋白表达变化,探讨HMGB1的高表达对肌纤维母细胞活化的影响。
第三部分:HMGB1对上皮细胞转分化的诱导作用
将HMGB34367真核表达载体pcDNA3.0-HMGB34367转染到上皮细胞16HBE中,提取细胞核蛋白,用EMSA和超迁移(Super shift)实验检测HMGB34367蛋白与α-SMA启动子CArGB基序特异性的结合。将α-SMA启动子红色荧光报告质粒和绿色荧光标记的HMGB34367真核表达载体质粒( pDsRed-SMA +pEGFP-N2-HMGB34367)共转染到上皮细胞中,通过流式细胞仪检测细胞表达红色荧光和绿色荧光强度,探讨过量表达HMGB34367对α-SMA启动子活化的影响。培养人肺泡上皮细胞株A549,予细胞因子混合刺激剂Mix(TGF-β1+IL-1β+IFN-γ)孵育,转染HMGB34367 siRNA干扰其在细胞中的表达,RT-PCR和免疫荧光检测α-SMA和HMGB1基因和蛋白水平的表达。阐明炎症微环境诱导内源性HMGB1对肺纤维化病理过程的影响。
结果:
第一部分:HMGB1在BLM诱导的肺纤维化小鼠模型肺组织和IPF患者肺组织中的的表达。
病理学观察可见:BLM诱导了小鼠肺组织局灶性纤维化病变,在胸膜下区域可见肺泡间隔增厚,肺泡腔破坏,上皮脱落,肺间质大量炎症细胞浸润。Masson胶原染色显示BLM组小鼠肺组织中胶原沉积明显增多,免疫组化检测可见肺组织HMGB1和α-SMA表达增强。IPF患者肺组织中也观察到同样的结果。IPF患者肺组织双重免疫荧光染色揭示肺细支气管的一些上皮细胞同时表达α-SMA和HMGB1,两者可共定位在细胞内同一区域。
第二部分:HMGB34367 RNAi对小鼠肺纤维化病理过程的影响
组织病理学观察表明,与BLM组和siRNA阴性对照组相比, HMGB34367 siRNA显著减轻了BLM诱导的小鼠肺纤维化损害;相应的免疫组化结果证实肺组织中HMGB1的表达明显下调;与此同时HMGB34367 siRNA组小鼠损伤的上皮细胞和上皮下细胞的α-SMA表达也明显减少。Western-Blot的结果和免疫组化类似。
第三部分:HMGB1对气道上皮细胞转分化的诱导作用
EMSA和超迁移(super shif)结果显示HMGB34367蛋白可与α-SMA启动子CArGA基序特异性结合。流式细胞仪检测结果显示共转染质粒pDsRed-SMA+pEGFP-N2-HMGB34367组红/绿色荧光相对值较对照组明显升高。在TGF-β1刺激的条件下,前者红/绿色荧光相对值较对照组增强更为明显。混合刺激剂诱导A549细胞表型向间充质样细胞转化,细胞内HMGB1的表达量增多。RT-PCR结果表明,加入混合刺激剂Mix作用后刺激了A549细胞α-SMA和HMGB34367基因的表达。免疫荧光结果揭示混合刺激剂处理48小时后,一些上皮细胞出现α-SMA的阳性表达。HMGB34367RNAi能有效降低HMGB1的表达,与此同时可见α-SMA的基因和蛋白水平的表达也相应降低。HMGB34367 RNAi具有特异性地阻断α-SMA的诱导表达能力。
结论:
1、HMGB1的诱导表达在肺纤维化发病过程中发挥了重要的作用。
2、HMGB34367蛋白作为HMGB1蛋白的截断分子形式,能够与α-SMA启动子特异性结合,作为α-SMA基因启动子的特异性转录激活子,诱导α-SMA基因的转录活化,在上皮-间质转分化过程中发挥作用。
3、HMGB1可能是联系IPF病理过程中肺损伤和异常修复的关键信号分子
Rationale:
Idiopathic pulmonary fibrosis (IPF), which is the most common idiopathic interstitial lung disease, has the worse prognosis, with a median survival of only 3 to 4 years. Although much has been learned recently about the pathogenesis of IPF, the etiology and precise cellular and molecular mechanisms involved are not established. So far, no specific therapy has been proven unequivocally effective in IPF. The most notable characteristic feature of IPF is presence of fibroblastic foci. Increased numbers of fibroblastic foci are associated with disease progression and a worse prognosis in IPF. The key effector cell in fibroblastic foci is the myofibroblast. Myofibroblasts, with their characteristicα-smooth muscle actin (α-SMA) expression, arise de novo in fibrosis, and they are thought to be the primary source of heightened matrix and profibrogenic cytokine expression. Hence, understanding the mechanism responsible for abnormal activation and transcriptional regulation ofα-SMA gene is important to uncover the fibrotic pathogenesis. We have previously identified a 20Kda nuclear protein (GenBank number:BAC34367) that is a C-terminal truncated form of high mobility group B-1(HMGB1) in the lung of bleomycin-treated mice, with an increased binding toα-SMA promoter. HMGB1 is a nuclear protein that is present in almost all eukaryotic cells, and it functions to stabilize nucleosome formation and acts as a transcription-factor like protein that regulates the expression of several genes. Recent studies identify HMGB1 as a delayed mediator of inflammation, damaged or necrotic cells can release HMGB1 into the extracellular milieu, where it triggers inflammatory responses. Based on these findings described as above, we hypothesize that HMGB1 may function as a key signal molecular that links lung injury to abnormal repair in the development of IPF. To validate the contribution of HMGB1 to the pathogenesis of the disease, we evaluated the role of HMGB34367, a C-terminal truncated form of HMGB1, in the initiation and progression of lung fibrosis induced by BLM, especially clarified its role in epithelial-myofibroblast transdifferentiation underlying the pathogenesis of IPF.
Methods
The first part:
Investigation of the expression of HMGB1 on the lung tissues of mice with pulmonary fibrosis induced by BLM and IPF patients.
The lung tissues sections were prepared from BLM-treated mice and IPF patients according to routine protocols for pathological examination, where immunostainings toα-SMA and HMGB1 were performed on determination of the role of HMGB1 in the lung fibrotic process.
The second part:
Effects of HMGB34367 RNAi on the pathological process of pulmonary fibrosis To validate if the truncated form of HMGB1 is involved in activation of myofibroblasts, the BLM-treated mice were administered intranasally with either HMGB34367 siRNA or control siRNA (20μl /per time )on day 2, 5, 8 after BLM addition. The lung tissues were isolated for pathological examination. Immunostaining toα-SMA and HMGB1 and western blot were performed for determination of effect of the increased level of HMGB1 on activation of myofibroblasts.
The third part:
The role of HMGB1 in the transdifferentiation of pulmonary epithelial cells into myofibroblasts
The 16HBE cells transfected with pcDNA3.0-HMGB34367 were subjected to performance of Electrophoretic Mobility Shift Assay(EMSA) and Super shift for detection of the binding activity of HMGB34367 withα-SMA promoter CarGB motif. The flowcytometry assay for measurement of the ratio of red fluorescence (RFP protein expressed under guidance ofα-SMA promoter) to green fluorescence of GPF-HMGB34367 fusion protein .was performed on qualification ofα-SMA promoter’s activation in response to over-expression of HMGB34367 in the cells after co-transfected with pGPF-HMGB34367 and pDsRed-SMA . To elucidate endogenous induction of HMGB1 by inflammatory micro-environment in lung fibrotic process,the A549 cells were cultured with cytokines-mix (TGF-β1+TNF-α+ IFN-γ) in absence or presence of HMGB34367-RNAi followed by RT-PCR and immunofluorescent staining to test expression ofα-SMA and HMGB1,
Result
The first part:
Investigation of the expression of HMGB1 on the lung tissues of BLM-induced pulmonary fibrosis mice and IPF.
Pathological examination demonstrated that BLM administration induced focal fibrotic lesions in mice lungs, primarily in the subpleural regions with thickened or thickening interalveolar septa with obvious alveolar destruction, mesenchyma infiltration with inflammatory cells and epithelial shedding. By Masson's trichrome staining, it was demonstrated an increased level of collagen synthesis. .Immunostaining displayed expression of both HMGB1 andα-SMA with a significant elevated level on the lung tissues of BLM-treated mice. In the pulmonary tissue of patients with IPF, we observed the same as above. Using double immunofluorescent staining, with antibodies againstα-SMA and HMGB1, we further observed certain cells located at bronchiolar epithelium of the pulmonary tissue of IPF with double staining ofα-SMA and HMGB1.
The second part:
Effects of HMGB34367 RNAi on the pathological process of pulmonary fibrosis
Treatment of HMGB34367 siRNA significantly attenuated the lung fibrotic lesion in the mice induced by BLM. Correspondingly, immunohistochemistry examination demonstrated that compare with the mice with challenge by BLM alone or plus with siRNA control, the expression of HMGB1 were obviously decreased in the BLM-induced mice with the treatment of HMGB34367 siRNA .,α-SMA expression in the injured epithelial cells and subepithelial cells was correspondingly down-regulated .The same results were also detected by Western-Blot.
The third part:
The role of HMGB1 in the transdifferentiation of airway epithelial cells into myofibroblasts
EMSA and super shif revealed that the truncated form of HMGB1 can specifically bind to CArG B motif. By Flow cytometry, without and with TGFβ1, RFP/GFP signal ratio was detected with a higher level in the cells co-transfected with pDsRed-SMA+pEGFP-N2-HMGB34367 plasmids than those co-transfected with control plasmids. Following with treatment of cytokines-mix, the A549 cells appeared myofibroblastic morphology concomitant with highly expressed intracellular HMGB1. As shown by the results of RT-PCR and immunofluorescent staining, transcriptional induction ofα-SMA and HMGB34367 genes were detected in the A549 cells after treatment of the Mix .α-SMA positive staining were observed in some of the treated A549 cells. It was shown that HMGB34367RNAi has an ability to specifically block the induction ofα-SMA at protein or gene level.
Conclusion
1、Induction of HMGB1 plays an important role in the process of lung fibrosis
2、The truncated form of HMGB1, HMGB34367, can act as a transcriptional factor for up-regulation of expression ofα-SMA.gene, contributing to epithelial cell to myofibroblast transdifferentiation.
3. HMGB1 may function as a key signal molecular that links lung injury to abnormal repair in the development of IPF.
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