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BACKGROUND: Cyclic adenosine monophosphate (cAMP) levels are directly activated by adenylate cyclase (AC) and play an anti-inflammatory role in chronic obstructive pulmonary disease (COPD). Previously, we have shown that isoforskolin (ISOF) can effectively activate AC1 and AC2 in vitro, improve pulmonary ventilation and reduce the inflammatory response in COPD model rats, supporting that ISOF may be a potential drug for the prevention and treatment of COPD, but the mechanism has not been explored in detail. METHODS: The potential pharmacological mechanisms of ISOF against COPD were analyzed by network pharmacology and multi-omics based on pharmacodynamic study. To use specific agonists, inhibitors and/or SiRNA for gene regulation function studies, combined qPCR, WB were applied to detect changes in mRNA and protein expression of important targets PIK3C3, AKT, mTOR, SPP1 and AQP4 which related to ISOF effect on COPD. And the key inflammatory factors detected by ELISA. RESULTS: Bioinformatics suggested that the anti-COPD pharmacological mechanism of ISOF was related to PI3K-AKT signaling pathway, and suggested target protein like PIK3C3, AQP4, SPP1, AKT, mTOR. Using the AQP4 inhibitor,or inhibiting SPP1 expression by siRNA-SPP1 could block the PIK3C3-AKT-mTOR pathway and ameliorate chronic inflammation. ISOF showed cAMP-promoting effect then suppressed AQP4 expression, together with decreased level of IL-1ß, IL-6, and IL-8. CONCLUSIONS: These findings demonstrate ISOF controlled the cAMP-regulated PIK3C3-AKT-mTOR pathway, thereby alleviating inflammatory development in COPD. The cAMP/AQP4/PIK3C3 axis also modulate Th17/Treg differentiation, revealed potential therapeutic targets for this disease.
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OBJECTIVE: To reveal the molecular mechanism of the antagonistic effect of traditional Chinese medicine Tianma formula (TF) on dementia including vascular dementia (VaD) and Alzheimer's disease (AD) and to provide a scientific basis for the study of traditional Chinese medicine for prevention and treatment of dementia. METHOD: The TF was derived from the concerted application of traditional Chinese medicine. We detected the pharmacological effect of TF in VaD rats. The molecular mechanism of TF was examined by APP/PS1 mice in vivo, Caenorhabditis elegans (C. elegans) in vitro, ELISA, pathological assay via HE staining, and transcriptome. Based on RNA-seq analysis in VaD rats, the differentially expressed genes (DEGs) were identified and then verified by quantitative PCR (qPCR) and ELISA. The molecular mechanisms of TF on dementia were further confirmed by network pharmacology and molecular docking finally. RESULTS: The Morris water maze showed that TF could improve the cognitive memory function of the VaD rats. The ELISA and histological analysis suggested that TF could protect the hippocampus via reducing tau and IL-6 levels and increasing SYN expression. Meanwhile, it could protect the neurological function by alleviating Aß deposition in APP/PS1 mice and C. elegans. In the RNA-seq analysis, 3 sphingolipid metabolism pathway-related genes, ADORA3, FCER1G, and ACER2, and another 5 nerve-related genes in 45 key DEGs were identified, so it indicated that the protection mechanism of TF was mainly associated with the sphingolipid metabolism pathway. In the qPCR assay, compared with the model group, the mRNA expressions of the 8 genes mentioned above were upregulated, and these results were consistent with RNA-seq. The protein and mRNA levels of ACER2 were also upregulated. Also, the results of network pharmacology analysis and molecular docking were consistent with those of RNA-seq analysis. CONCLUSION: TF alleviates dementia by reducing Aß deposition via the ACER2-mediated sphingolipid signaling pathway.
RESUMO
BACKGROUND: Chronic obstructive pulmonary disease (COPD) is characterized by limited airflow due to pulmonary and alveolar abnormalities from exposure to cigarette smoke (CS). Current therapeutic drugs are limited and the development of novel treatments to prevent disease progression is challenging. Isoforskolin (ISOF) from the plant Coleus forskohlii is an effective activator of adenylyl cyclase (AC) isoforms. Previously we found ISOF could attenuate acute lung injury in animal models, while the effect of ISOF on COPD has not been elucidated. PURPOSE: In this study, we aimed to evaluate the efficacy of ISOF on COPD and reveal its potential mechanisms. METHODS: A rat model of COPD was established by long-term exposure to CS, then the rats were orally administered with ISOF (0.5, 1 and 2 mg/kg). The pulmonary function, lung morphology, inflammatory cells and cytokines in serum or bronchoalveolar lavage fluid (BALF) were evaluated. Transcriptomics, proteomics and network pharmacology analysis were utilized to identify potential mechanisms of ISOF. Droplet digital PCR was used to detect the mRNA expression of AC1-10 in donor lung tissues. AC activation was determined in recombinant human embryonic kidney 293 (HEK293) cells stably expressing human AC isoforms. In addition, ISOF caused trachea relaxation ex vivo were assessed in isolated trachea rings from guinea pigs. RESULTS: ISOF significantly ameliorated pathological damage of lung tissue and improved pulmonary function in COPD rats. ISOF treatment decreased the number of inflammatory cells in peripheral blood, and also the levels of pro-inflammatory cytokines in serum and BALF. Consistent with omics-based analyses, ISOF markedly downregulated the mTOR level in lung tissue. Flow cytometry analysis revealed that ISOF treatment reduced the ratio of Th17/Treg cells in peripheral blood. Furthermore, the expression levels of AC1 and AC2 are relatively higher than other AC isoforms in normal lung tissues, and ISOF could potently activate AC1 and AC2 in vitro and significantly relax isolated guinea pig trachea. CONCLUSION: Collectively, our studies suggest that ISOF exerts its anti-COPD effect by improving lung function, anti-inflammation and trachea relaxation, which may be related to AC activation, mTOR signaling and Th17/Treg balance.