肺痹汤2号调控AT2细胞内质网应激抵抗凋亡促进肺泡修复改善小鼠肺纤维化的机制OA

Objective:To investigate the intervention mechanism of the traditional Chinese medicine Number 2 Feibi recipe(N2FBR)in idiopathic pulmonary fibrosis(IPF),focusing on its effects on endoplasmic reticulum(ER)stress,apoptosis,stemness maintenance,and regenerative capacity of alveolar type Ⅱ epithelial cells(AT2 cells),and to validate the modern translational pathway of the theory of"deficiency of Zong Qi leading to pulmonary atelectasis and atrophy".Methods:A mouse model of pulmonary fibrosis was induced by bleomycin(BLM).Mice were randomly divided into blank control,model,low-,and high-dose N2FBR intervention groups(9.1,18.2 g·kg-1),and prednisolone intervention group(6.5 mg·kg-1).Pulmonary histopathological changes and collagen deposition were evaluated using hematoxylin-eosin(HE)and Masson's trichrome staining.Hydroxyproline(HYP)content was measured by the alkaline hydrolysis method.Lung coefficient and pulmonary function parameters were evaluated.The mRNA expression levels of fibrosis-related factors,including collagen type Ⅰ alpha 1 chain(ColⅠa1),alpha-smooth muscle actin(α-SMA),and tissue inhibitor of metalloproteinase 1(Timpl),were detected by real-time polymerase chain reaction(Real-time PCR).Cell apoptosis was assessed using the terminal deoxynucleotidyl transferase dUTP nick-end labeling(TUNEL)assay.Apoptosis of AT2 cells was further evaluated by double immunofluorescence staining for surfactant protein C(SPC)and cysteine-aspartic protease-3(Caspase-3).Endoplasmic reticulum(ER)stress in AT2 cells was examined by double staining for SPC and protein kinase R-like endoplasmic reticulum kinase(PERK).Ultrastructural changes of ER and lamellar bodies in AT2 cells were observed by transmission electron microscopy(TEM).The expression levels of key proteins involved in ER stress and apoptosis pathways,including PERK,activating transcription factor 4(ATF4),and Caspase-3,were detected by Western blot.Double immunofluorescence staining of SPC and Ki-67 antigen(Ki-67)was performed to evaluate the proliferative capacity of AT2 cells.Lineage tracing technology(labeling AT2 cells with GFP)combined with Krt8 labeling was used to evaluate intermediate differentiation states,and morphological transformation of AT2 cells into alveolar type Ⅰ epithelial cells(AT1)was observed.Results:BLM-induced mice exhibited significant structural disruption of lung tissue,increased collagen deposition,elevated lung coefficient,decreased pulmonary function,and upregulation of fibrosis-related factors(P＜0.01).High-dose N2FBR treatment significantly ameliorated lung tissue damage and dysfunction,significantly reduced HYP content(P＜0.01),and significantly downregulated ColⅠa1,α-SMA,and Timp1 expression(P＜0.01).Apoptosis analysis showed increased TUNEL-positive and Caspase-3-positive AT2 cells in the model group,which was significantly reduced by high-dose N2FBR treatment.TEM revealed swollen ER structures in AT2 cells of the model group,which tended to return to normal following treatment.PERK protein staining analysis showed evident ER stress in AT2 cells of the model group,which were markedly alleviated in the treatment group.The expression levels of ER stress-related proteins PERK and ATF4,as well as the apoptosis-related protein Caspase-3,were elevated in the model group and significantly reduced after treatment.TEM also revealed disrupted lamellar body structures in the model group,which tended to recover in the treatment group.Regarding the proliferative capacity of AT2 cells,the proportion of Ki-67+SPC+AT2 cells significantly increased in the treatment group(P＜0.01).Lineage tracing showed that the proportion of keratin 8-positive green fluorescent protein-positive(Krt8+GFP+)cells increased in the model group,indicating differentiation arrest.This proportion was significantly reduced in the treatment group,and the morphology of GFP+cells exhibited a flattened,extended shape,suggesting restored differentiation toward AT1 cells.Conclusion:N2FBR alleviates ER stress in AT2 cells,reduces AT2 cell apoptosis,restores lamellar body structure and function,enhances proliferation activity,and alleviates differentiation arrest to promote differentiation into AT1 cells,thereby repairing the alveolar epithelium and effectively blocking the progression of pulmonary fibrosis.Its traditional Chinese medicine mechanism of"replenishing Zong Qi,harmonizing Qi and blood,and unblocking pulmonary meridians"closely aligns with the modern regulatory pathway of AT2 stem cells,providing a novel theoretical basis and experimental evidence for the intervention of IPF with traditional Chinese medicine.