Exploring the pharmacological mechanisms and key active ingredients of total flavonoids from Lamiophlomis rotata (Benth.) Kudo against rheumatoid arthritis based on multi-technology integrated network pharmacology.

ETHNOPHARMACOLOGICAL RELEVANCE: Lamiophlomis rotata (Benth.) Kudo (LR, Lamiaceae) is a traditional Tibetan medicinal material in China. Tibetan medicine classic and research report suggested that LR could be used to cure rheumatoid arthritis (RA). However, the anti-RA active ingredients and pharmacological mechanisms of LR have not been elucidated. AIM OF THE STUDY: To explore the mechanisms and key active ingredients of total flavonoids from LR (TFLR) against RA. MATERIALS AND METHODS: First, the mechanisms of TFLR against RA were investigated on collagen-induced arthritis (CIA) rat model by analyzing paw appearance, paw swelling, arthritis score, spleen index, thymus index, inflammatory cytokine (TNF-α, IL-1ß, IL-6 and IL-17) levels in serum, histopathology of ankle joint and synovium from knee joint (hematoxylin-eosin, safranin O-fast green and DAB-TUNEL staining), and apoptosis-related protein (PI3K, Akt1, p-Akt, Bad, p-Bad, Bcl-xL and Bcl-2) levels in the synovium of ankle joints (Western blot). Then, the crucially active ingredients of TFLR against RA were explored by network pharmacology, ingredient analysis, in vitro metabolism and TNF-α-induced human RA synovial fibroblast MH7A proliferation assays. Network pharmacology was applied to predict the key active ingredients of TFLR against RA. The ingredient analysis and in vitro metabolism of TFLR were performed on HPLC, and MH7A proliferation assay were applied to evaluate the predicted results of network pharmacology. RESULTS: TFLR shown excellently anti-RA effect by reducing paw swelling, arthritis score, spleen index, thymus index and inflammatory cytokine (IL-1ß, IL-6 and IL-17) levels, and improving the histopathological changes of ankle joint and synovium from knee joint in CIA rats. Results of Western blot indicated that TFLR reversed the changes of PI3K, p-Akt, p-Bad, Bcl-xL and Bcl-2 levels in the ankle joint synovium of CIA rats. Results of network pharmacology exhibited that luteolin was identified as the pivotal active ingredient of TFLR against RA. The ingredient analysis of TFLR indicated that the main ingredient in TFLR was luteoloside. The in vitro metabolism study of TFLR suggested that luteoloside could be converted to luteolin in artificial gastric juice and intestinal juice. Results of MH7A proliferation assay showed that there was no significant difference between TFLR and equal luteoloside on the viability of MH7A cells, indicating that luteoloside was the key active ingredient of TFLR against RA. Additionally, the luteolin (same mol as luteoloside) showed better inhibitory effect on the viability of MH7A cells than luteoloside. CONCLUSION: TFLR showed anti-RA effect, and the mechanism was related to promoting synovial cell apoptosis mediated by PI3K/Akt/Bad pathway. Meanwhile, this work indicated that luteoloside was the key active ingredient of TFLR against RA. This work lays a foundation for providing TFLR product with clear mechanism and stable quality to treat RA.

Integrated strategy of network pharmacology, molecular docking, HPLC-DAD and mice model for exploring active ingredients and pharmacological mechanisms of Penthorum chinense Pursh against alcoholic liver injury.

ETHNOPHARMACOLOGICAL RELEVANCE: Penthorum chinense Pursh (PCP, Saxifragaceae) is an edible plant and frequently-used Chinese herbal medicine, and is commonly used as Miao medicine in China. It showed well effect on alcoholic liver injury (ALI), but studies on its active ingredients and mechanisms against ALI remain at the starting stage. AIM OF THE STUDY: This work aims to explore the active ingredients and pharmacological mechanisms of PCP against ALI. MATERIALS AND METHODS: First, network pharmacology was applied to decipher the potential active ingredients and pharmacological mechanisms of PCP against ALI by ingredient identification, ADMET evaluation, target identification, network construction and analysis, protein-protein interaction (PPI) analysis, and gene enrichment analysis. Second, molecular docking was used to explore the interaction between key active ingredient and hub protein of PCP against ALI. Then, the ingredient analysis of PCP aqueous extract and semiquantitative analysis of key active ingredient were carried out on HPLC-DAD. Subsequently, mice with ALI were used to investigate the therapeutic effect or verify the predicted mechanisms of PCP or key active ingredient against ALI by analyzing body weight, liver index, ALT and AST activities in serum and liver tissues, oxidation related indices (SOD activity, GSH level and MDA level) in liver tissues, histopathology of liver tissues (oil red O, hematoxylin-eosin and DAB-TUNEL staining), and changes of related proteins (PI3K, Akt, p-Akt, Bax and Bcl-2) in liver tissues with the aid of Western blot. RESULTS: Network pharmacology showed that the active ingredients and related genes of PCP against ALI comprised 10 ingredients and 52 genes. Based on the result of ingredient analysis of PCP aqueous extract, quercitrin was identified as the key active ingredient of PCP against ALI. PPI analysis indicated that AKT1 was the hub gene of PCP against ALI, and molecular docking suggested that there were good interaction between quercetin and Akt1 protein. Gene enrichment analysis showed that the pivotal molecular mechanism of PCP against ALI might be to inhibit hepatocyte apoptosis via activation of PI3K-Akt signaling pathway. PCP and quercitrin showed anti-ALI effect by offsetting weight loss and increase of liver index, and reversing the imbalance of oxidative stress and histopathological changes of liver tissues (abnormal fatty acid metabolism, hepatic cord swelling and inflammatory cell infiltration) in mice with ALI. PCP caused the decrease of DAB-TUNEL-positive cells, upregulated the anti-apoptotic proteins (PI3K, Akt and p-Akt) levels and the ratio of p-Akt/Akt, and downregulated pro-apoptotic protein (Bax) level and the ratio of Bax/Bcl-2 in liver tissues of mice with ALI, indicating that the mechanism of PCP against ALI involved in inhibiting hepatocyte apoptosis via activation of PI3K-Akt signaling pathway. CONCLUSION: PCP and quercitrin showed well anti-ALI effect. The key active ingredient of PCP against ALI was identified as quercitrin. The underlying pharmacological mechanisms of PCP against ALI may be related to PI3K-Akt signaling pathway-mediated inhibition of hepatocyte apoptosis. This work provided new evidence to support the application of PCP in treatment of ALI, and a research basis for the research and development of functional foods or drugs against ALI from PCP.