Elucidating the role of Rhodiola rosea L. in sepsis-induced acute lung injury via network pharmacology: emphasis on inflammatory response, oxidative stress, and the PI3K-AKT pathway.

CONTEXT: Sepsis-induced acute lung injury (ALI) is associated with high morbidity and mortality. Rhodiola rosea L. (Crassulaceae) (RR) and its extracts have shown anti-inflammatory, antioxidant, immunomodulatory, and lung-protective effects. OBJECTIVE: This study elucidates the molecular mechanisms of RR against sepsis-induced ALI. MATERIALS AND METHODS: The pivotal targets of RR against sepsis-induced ALI and underlying mechanisms were revealed by network pharmacology and molecular docking. Human umbilical vein endothelial cells (HUVECs) were stimulated by 1 µg/mL lipopolysaccharide for 0.5 h and treated with 6.3, 12.5, 25, 50, 100, and 200 µg/mL RR for 24 h. Then, the lipopolysaccharide-stimulated HUVECs were subjected to cell counting kit-8 (CCK-8), enzyme-linked immunosorbent, apoptosis, and Western blot analyses. C57BL/6 mice were divided into sham, model, low-dose (40 mg/kg), mid-dose (80 mg/kg), and high-dose (160 mg/kg) RR groups. The mouse model was constructed through caecal ligation and puncture, and histological, apoptosis, and Western blot analyses were performed for further validation. RESULTS: We identified six hub targets (MPO, HRAS, PPARG, FGF2, JUN, and IL6), and the PI3K-AKT pathway was the core pathway. CCK-8 assays showed that RR promoted the viability of the lipopolysaccharide-stimulated HUVECs [median effective dose (ED50) = 18.98 µg/mL]. Furthermore, RR inhibited inflammation, oxidative stress, cell apoptosis, and PI3K-AKT activation in lipopolysaccharide-stimulated HUVECs and ALI mice, which was consistent with the network pharmacology results. DISCUSSION AND CONCLUSION: This study provides foundational knowledge of the effective components, potential targets, and molecular mechanisms of RR against ALI, which could be critical for developing targeted therapeutic strategies for sepsis-induced ALI.

Salidroside attenuates sepsis-associated acute lung injury through PPP1R15A mediated endoplasmic reticulum stress inhibition.

BACKGROUND: This study explored the role and mechanism of salidroside in sepsis-associated acute lung injury (ALI). METHOD: Sepsis-associated ALI in rats was induced by cecal ligation and perforation method, while lipopolysaccharide (LPS) was used to stimulate cell model. After the treatment of salidroside and dexamethasone (DEX), hematoxylin and eosin staining was applied to evaluate the rat lung injury. Next, the number of total cells and neutrophils in rat bronchoalveolar lavage fluid (BALF) was counted, and the lung wet/dry (W/D) ratio and water content were measured. The neutrophil elastase activity and inflammatory factor levels in BALF were detected by Elastase Assay kit and ELISA. The expressions of PPP1R15A and endoplasmic reticulum (ER) stress-related genes were detected by quantitative reverse transcription polymerase chain reaction and western blot. And the effects of silenced PPP1R15A and tauroursodeoxycholic acid (TUDCA) on cell viability, apoptosis and endoplasmic reticulum (ER) stress were determined by cell counting kit-8 assay, flow cytometry and western blot. RESULT: The lung injury, inflammation and edema in sepsis model rats were alleviated by salidroside and DEX, meanwhile salidroside increased the viability and inhibited apoptosis in LPS-treated cells. The expression of PPP1R15A was decreased in sepsis models and increased by salidroside, and salidroside down-regulated the ER stress-related protein expressions in vitro and in vivo. Silenced PPP1R15A reversed the effect of salidroside on cell viability, apoptosis and ER stress, whereas TUDCA could counteract the above effect of silenced PPP1R15A. CONCLUSION: Salidroside targeted PPP1R15A to ameliorate lung injury in sepsis through inhibiting ER stress.

Potential biomarkers for inflammatory response in acute lung injury.

Acute lung injury (ALI) is a severe respiratory disorder occurring in critical care medicine, with high rates of mortality and morbidity. This study aims to screen the potential biomarkers for ALI. Microarray data of lung tissues from lung-specific geranylgeranyl pyrophosphate synthase large subunit 1 knockout and wild-type mice treated with lipopolysaccharide were downloaded. Differentially expressed genes (DEGs) between ALI and wild-type mice were screened. Functional analysis and the protein-protein interaction (PPI) modules were analyzed. Finally, a miRNA-transcription factor (TF)-target regulation network was constructed. Totally, 421 DEGs between ALI and wild-type mice were identified. The upregulated DEGs were mainly enriched in the peroxisome proliferator-activated receptor signaling pathway, and fatty acid metabolic process, while downregulated DEGs were related to cytokine-cytokine receptor interaction and regulation of cytokine production. Cxcl5, Cxcl9, Ccr5, and Cxcr4 were key nodes in the PPI network. In addition, three miRNAs (miR505, miR23A, and miR23B) and three TFs (PU1, CEBPA, and CEBPB) were key molecules in the miRNA-TF-target network. Nine genes including ADRA2A, P2RY12, ADORA1, CXCR1, and CXCR4 were predicted as potential druggable genes. As a conclusion, ADRA2A, P2RY12, ADORA1, CXCL5, CXCL9, CXCR1, and CXCR4 might be novel markers and potential druggable genes in ALI by regulating inflammatory response.

Salidroside regulates inflammatory pathway of alveolar macrophages by influencing the secretion of miRNA-146a exosomes by lung epithelial cells.

The purpose of this study was to explore the investigative mechanism of salidroside (SAL) on LPS-induced acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). The exosomes from RLE-6TN are extracted and identified by transmission electron microscopy, particle size analysis and protein marker detection, and co-cultured with NR8383 cells. The ALI/ARDS model of SD rats was established by LPS (10 mg/kg) intratracheal instillation. Following a four-hour intratracheal instillation of LPS, 50 µl of RLE-6TN exosomes were injected through the tail vein. After that, SAL and miR-146a antagomir were injected into the tail vein for 72 h, respectively. As the changes of HE stain, body weight and ALI score are observed. The expression of miR-146a, TLR4, NF-kB, IRAK1, TRAF6 and their related proteins were detected by RT-PCR and Western blot, respectively. TNF-α, IL-6, IL-8 and IL-1 ß inflammatory factors were detected by ELISA. The expression of miR-146a, NF-kB, IRAK, TRAF6 and related inflammatory factors in LPS-induced NR8383 was significantly higher than that in the control group, while SAL has greatly reduced the expression of TLR4 mediated NF-kB inflammatory pathway and related inflammatory factors. SAL can significantly improve the LPS-induced lung morphological abnormalities, slowed down the rate of weight loss in rats, and reducing the ALI score. The expression trend of NF-kB, IRAK, TRAF6 and related inflammatory factors in rats' lung tissues was consistent with that in NR8383 cells. SAL has a protective effect on ALI/ARDS caused by sepsis, which is likely to be developed to a potential treatment for the disease. To sum up, this study provides a new theoretical basis for the treatment of ALI/ARDS with SAL.

Thymosin­ß 4 induces angiogenesis in critical limb ischemia mice via regulating Notch/NF­κB pathway.

Thymosin­ß 4 (Tß4) has been reported to exert a pro­angogenic effect on endothelial cells. However, little is known on the role and underlying mechanisms of Tß4 on critical limb ischemia (CLI). The present study aimed therefore to investigate the mechanisms and pro­angiogenic effects of Tß4 in CLI mice. Tß4 overexpression lentiviral vector was first transfected into HUVEC and CLI mice model, and inhibitors of Notch pathway (DAPT) and NF­κB pathway (BMS) were also applied to HUVEC and CLI mice. Subsequently, MTT, tube formation and wound healing assays were used to determine the cell viability, angiogenesis and migratory ablity of HUVEC, respectively. Western blotting, reverse transcription, quantitative PCR, immunofluorescence and immunohistochemistry were used to detect the expression of the angiogenesis­related factors angiopoietin­2 (Ang2), TEK receptor tyrosine kinase 2 (tie2), vascular endothelial growth factor A (VEGFA), CD31 and α­smooth muscle actin (α­SMA) and the Notch/NF­κB pathways­related factors NOTCH1 intracellular domain (N1ICD), Notch receptor 3 (Notch3), NF­κB and p65 in HUVEC or CLI mice muscle tissues. The results demonstrated that Tß4 not only enhanced the cell viability, angiogenesis and migratory ability of HUVEC but also promoted the expression of Ang2, tie2, VEGFA, N1ICD, Notch3, NF­κB, and phosphorylated (p)­p65 in HUVEC. In addition, Tß4 promoted the expression of CD31, α­SMA Ang2, tie2, VEGFA, N1ICD and p­p65 in CLI mice muscle tissues. Treatment with DAPT and BMS had opposite effects of Tß4, whereas Tß4 reversed the effect of DAPT and BMS. The findings from the present study suggested that Tß4 may promote angiogenesis in CLI mice via regulation of Notch/NF­κB pathways.

Salidroside alleviates paraquat-induced rat acute lung injury by repressing TGF-ß1 expression.

OBJECTIVE: This study was designed to investigate the protective effects of salidroside (SDS) via suppressing the expression of transforming growth factor-ß1 (TGF-ß1) in rat acute lung injury (ALI) induced by paraquat (PQ) and to explore the potential molecular mechanisms. METHODS: A total of 90 male rats (190-210 g) were randomly and evenly divided into 9 groups: control group, PQ groups (4 groups), and PQ + SDS groups (4 groups). The rats in control group were treated with equal volume of saline intraperitoneally. The rats in PQ groups were exposed to PQ solution (20 mg/kg) by gastric gavage for 1, 6, 24, and 72 hours, respectively. The rats in PQ + SDS groups were intraperitoneally injected once with SDS (10 mg/kg) every 12 hours after PQ perfusion. Pulmonary pathological changes were observed by hematoxylin and eosin (HE) staining. The expression of TGF-ß1 and the mRNA were evaluated by immunohistochemical (IHC) scoring and real time quantitative reverse transcription polymerase chain reaction (real-time qRT-PCR), respectively. RESULTS: SDS alleviated the symptoms of PQ induced ALI. Moreover, SDS reduced the expression of the inflammatory cytokine TGF-ß1 including TGF-ß1 IHC scores (at each time point from 6 to 72 hours after PQ perfusion) and mRNA level (at each time point from 1 to 72 hours after PQ perfusion) compared with PQ groups (P < 0.05). CONCLUSION: SDS alleviated the pulmonary symptoms of PQ-induced ALI, at least partially, by repressing inflammatory cell infiltration and the expression of TGF-ß1 resulting in delayed lung fibrosis.