参龙煎剂调控细胞凋亡铁死亡抑制NFIL3防治特发性肺纤维化的机制

Mechanism of Shenlong Decoction inhibiting NFIL3 via apoptosis and ferroptosis in preventing and treating idiopathic pulmonary fibrosis

目的:探讨参龙煎剂对特发性肺纤维化(IPF)细胞铁死亡及凋亡与炎症的关系,以及抗IPF机制。方法:将60只Wistar大鼠随机分为空白组、假手术组、模型组、参龙煎剂组、吡非尼酮组。博来霉素(BLM)5 mg/kg诱导建立IPF模型,28 d取材。HE染色法观察肺组织的病理变化,Masson染色观察肺纤维化状态,免疫组化明确肺泡上皮细胞α-SMA、E-cadherin表达水平。免疫荧光染色对肺组织凋亡和铁死亡水平进行检测,及其与NFIL3共定位,明确纤维组织中凋亡和铁死亡水平的差异,细胞铁死亡及凋亡与炎症的关系。结果:体内实验结果显示,参龙煎剂组可改善IPF大鼠一般情况,缓解呼吸急促,改善心率、精神状态等;改善肺组织病变程度;抑制肺组织胶原蛋白与α-SMA表达,提升E-cadherin表达水平;测序结果显示参龙煎剂明显减少NFIL3的表达水平;与模型组比较,参龙煎剂组Tunel与α-SMA共定位显著增多(P<0.01),FSP1与α-SMA共定位显著减少(P<0.01)。体外实验结果显示,与模型组比较,含药血清组凋亡水平显著升高(P<0.01);与模型组比较,含药血清组细胞迁移水平下降,铁死亡抑制剂组迁移能力提高,铁死亡诱导剂组细胞迁移能力下降;与模型组比较,参龙煎剂组FSP1与NFIL3共定位显著减少(P<0.05);与模型组比较,参龙煎剂组Caspase12、Bax表达水平显著升高(P<0.01),Bcl-2、FSP1、NFIL3、TNF-α表达水平显著降低(P<0.01,P<0.05)。结论:参龙煎剂可能通过凋亡和铁死亡途径抑制炎症反应,进而抑制肌成纤维细胞转化防治IPF。

Objective:To investigate the relationship between Shenlong Decoction(SLJJ)on apoptosis,ferroptosis and inflammation of idiopathic phlmonary fibrosis(IPF)cells,and its anti-IPF mechanism.Methods:Sixty Wistar rats were divided into blank group,sham operation group,model group,SLJJ and pirfenidone group.IPF model was established after induction of Recombinant Bleomycin(BLM)5 mg/kg,and materials were collected after 28 d.Pathological changes of lung tissues were observed by HE staining,and pulmonary fibrosis was observed by Masson staining.The expression levels ofα-SMA and E-cadherin in alveolar epithelial cells were determined by immunohistochemistry.The levels of apoptosis and ferroptosis in lung tissues were detected by immunofluorescence staining,and co-located them with NFIL3 to clarify the differences of apoptosis and ferroptosis levels in fibrous tissues,and the relationship between cell ferroptosis and apoptosis and inflammation.Results:In vivo experiment results showed that SLJJ group improved the general condition of IPF rats,alleviated shortness of breath,improved heart rate,mental state,etc.,as well as improved the pathological degree of lung tissue,inhibited the expression of collagen in lung tissue andα-SMA,increased the expression of E-cadherin.Sequencing results showed that SLJJ group significantly reduced the expression level of NFIL3.Compared with the model group,the co-localization of Tunel andα-SMA was significantly increased(P<0.01),the co-localization of FSP1 andα-SMA was significantly decreased in the SLJJ group(P<0.01).In vitro experiment results showed that,compared with the model group,the apoptosis level of drugcontaining serum group was significantly increased(P<0.01).Compared with the model group,the level of cell migration in the drug-containing serum group decreased,the iron death inhibitor group increased,and the iron death inducer group decreased.Compared with model group,the co-localization of FSP1 and NFIL3 in SLJJ group was significantly decreased(P<0.05).Compared with the model group,the expression levels of Caspase12 and Bax in SLJJ group were significantly increased(P<0.01),the expression levels of Bcl-2,FSP1,NFIL3 and TNF-αwere significantly decreased(P<0.01,P<0.05).Conclusion:SLJJ may inhibit myofibroblast transformation through ferroptosis and apoptosis pathway,and then inhibit inflammatory response to prevent and treat IPF.