Sanzi Yangqin Decoction alleviated epithelial barrier damage in acute lung injury by mediating CD40 in alveolar macrophages to inhibit necroptosis of pulmonary epithelial cells.

Abstract

BACKGROUND: Acute lung injury (ALI) is a severe pulmonary condition characterized by inflammation and damage to the epithelial barrier of the lungs. Sanzi Yangqin Decoction (SZYQ) is a traditional therapeutic formula for lung diseases and has been shown to play a role in the regulation of ALI; however, the specific mechanisms by which it modulates the epithelial barrier remain unclear. PURPOSE: This study aims to elucidate the specific mechanisms by which SZYQ regulates epithelial barrier injury in ALI. METHODS: We first identified the constituents for SZYQ in plasma through UHPLC-QTOF-MS. Subsequently, we utilized the GEO database in conjunction with network pharmacology to predict the mechanism by which SZYQ regulates epithelial barrier damage in ALI. An in vitro ALI epithelial barrier injury model was established using LPS-induced MLE-12 cells to evaluate the efficacy and mechanism of SZYQ. Additionally, an in vivo ALI model was created through intranasal LPS administration to validate the in vitro findings. To explore the key target, PCR array analysis was conducted on lung tissues, revealing that SZYQ functions by regulating CD40 in alveolar macrophages (AMs). Following this, clodronate liposomes were employed for AMs depletion, and CD40 ligand (CD40L) stimulation of MH-S and MLE-12 cells co-culture was utilized to verify the pharmacological effects of SZYQ both in vivo and in vitro. RESULTS: This study identified a total of 18 constituents for SZYQ in plasma. GEO database analysis results suggest that SZYQ may alleviate epithelial barrier damage in ALI by exerting anti-necroptotic effects. Both in vitro and in vivo results indicated that SZYQ inhibits the expression of key necroptosis proteins RIP1, RIP3, and MLKL, alleviates DRP1-mediated mitochondrial fission, regulates ROS and Carelease, promotes the proliferation and differentiation of type II alveolar epithelial cells (AT2), and thereby mitigates epithelial barrier damage in ALI. Following the depletion of AMs in vivo, the necroptosis of AT2, mitochondrial fission, and lung epithelial barrier injury in ALI mice were exacerbated; while the regulatory capacity of SZYQ on these factors was diminished. The co-culture experiments involving MH-S cells and MLE-12 cells demonstrated that SZYQ exerts an anti-necroptotic effect on AT2 by inhibiting the expression of CD40 on AMs. Additionally, SZYQ improved epithelial barrier function by promoting the proliferation and differentiation of AT2. CONCLUSIONS: SZYQ improved epithelial barrier function in ALI by mediating the CD40 of AMs. Its mechanism of action involved inhibiting necroptosis in AT2, alleviating mitochondrial fission, and promoting the proliferation and differentiation of AT2.