Alkaloid extract of Chuanxiong Rhizoma alleviates liver fibrosis by regulating calcium ion communication between endoplasmic reticulum and mitochondria

To decipher the anti-fibrosis effects and mechanisms of alkaloid extract of Chuanxiong Rhizoma (CX AL ) by modulating endoplasmic reticulum (ER) and mitochondrial disorders through combination of network pharmacological methods with molecular biology. After identifying the components of CX AL through UHPLC-HRMS, a bile duct ligation (BDL)-induced liver fibrosis mouse model was established and CX AL was administered in a dose gradient as 5, 10 and 20 mg/kg. Then, liver serum level, expression analysis of liver fibrosis-related genes, pathological staining and immunofluorescence (IF) staining were performed to detect the efficacy of CX AL . Based on target sets from open online databases, network pharmacology and cell type enrichment analysis were performed to locate the pathways and the cell type targeted by CX AL . Furthermore, qPCR, immunofluorescence, ROS-related assays, Mito-SOX, mitochondrial-associated ER membrane (MAM) identification, and calcium ion (Ca 2+ ) flux detection techniques were applied in vitro to evaluate the efficacy of CX AL on the ER, mitochondria, and MAMs in AML12 cells following DCA treatment. Serological and hepatic histopathological findings indicated the administration of CX AL significantly reduced BDL-increased plasma hyaluronic acid (HA), laminin (LN), total bile acids (TBA) and alkaline phosphatase (ALP) levels, pro-fibrotic markers and liver histopathological changes during liver fibrosis. A total of 57 common targets between the 206 targets of represented CX AL components from TCMSP Database/DrugBank Database and 500 targets associated wtih liver fibrosis from the Gene Cards database were enriched in ER/mitochondrial-related pathways and were specifically localized to hepatocytes in liver. Further in vivo and in vitro investigations revealed that CX AL ameliorated ER stress, mitochondrial dysfunction, MAMs structural damage and aberrant Ca 2+ flux. Mechanistically, as MAMs serve as a bridge for Ca 2+ communication between the ER and the mitochondria, it found that CX AL might inhibit Ca 2+ flux from the ER to the mitochondria through the MAM-associated component, inositol 1,4,5-trisphosphate receptor type 1 (ITPR1). Meanwhile, CX AL upregulated mitofusin 2 (MFN2) to promote the retrieval of Ca 2+ from the mitochondria into the ER, thus reducing the accumulation of Ca 2+ in the mitochondria. The administration of CX AL exhibits notable anti-fibrotic properties in the BDL-induced mouse model. The protective effects of CX AL on the ER stress and mitochondrial dysfunction can be attributed to the restoration of Ca 2+ influx between the ER and the mitochondrial, mediated by MAM proteins ITPR1 and MFN2.