Sheng Mai Yin shows anti-fatigue, anti-hypoxia and cardioprotective potential in an experimental joint model of fatigue and acute myocardial infarction.

ETHNOPHARMACOLOGICAL RELEVANCE: Cardiovascular disease (CVD) and fatigue are two common diseases endangering human life and health that may interact and reinforce one another. Myocardial infarction survivors frequently experience fatigue, and acute myocardial infarction (AMI) is one of the most common cardiovascular diseases that cause fatigue-induced sudden death. Sheng Mai Yin (SMY), a Chinese medicine prescription, is traditionally used for the treatment of diabetes and cardiovascular disease, and has been demonstrated to reduce fatigue and safeguard cardiac function. AIM OF THE STUDY: This study aims to investigate the effects and underlying mechanisms of SMY in treating fatigue and AMI. MATERIALS AND METHODS: The pharmacological mechanisms of SMY in treating fatigue and AMI were predicted by bioinformatics and network pharmacology methods. After administering SMY at high, medium and low doses, the swimming time to exhaustion, hemoglobin level, serological parameters and hypoxia tolerance time were detected in C57BL/6N mice, and the left ventricular ejection fractions (LVEF), left ventricular fractional shortening (LVFS), grasp strength, cardiac histopathology, serological parameters and the expression of PINK1 and Parkin proteins were examined in Wistar rats. RESULTS: 371 core targets for SMY and 282 disease targets for fatigue and AMI were obtained using bioinformatics and network pharmacology methods. Enrichment analysis of target genes revealed that SMY might interfere with fatigue and AMI through biological processes such as mitochondrial autophagy, apoptosis, and oxidative stress. For in vivo experiments, SMY showed significant anti-fatigue and hypoxia tolerance effects in mice; It also improved the cardiac function and grasp strength, decreased their cardiac index, myocardial injury and fibrosis degree, and induced serological parameters levels and the expression of PTEN-induced putative kinase 1 (PINK1) and Parkin proteins in myocardium, suggesting that SMY may exert cardioprotective effects in a joint rat model of fatigue and AMI by inhibiting excessive mitochondrial autophagy. CONCLUSION: This study revealed the anti-fatigue, anti-hypoxia and cardioprotective effects of SMY in a joint model of fatigue-AMI, and the pharmacological mechanism may be related to the inhibition of mitochondrial autophagy in cardiomyocytes through the PINK1/Parkin pathway. The discoveries may provide new ideas for the mechanism study of traditional Chinese medicine, especially complex prescriptions, in treating fatigue and AMI.

Acute, subchronic toxicity and genotoxicity studies of JointAlive, a traditional Chinese medicine formulation for knee osteoarthritis.

AIM: In vivo and in vitro toxicity tests of JointAlive® were studied in animal models to support the safe use of JointAlive® as a drug for knee osteoarthritis treatment. METHODS: The acute toxicity study in Sprague Dawley (SD) rats was conducted at a 20 g/kg bw/day dose of JointAlive®. For 13-week subchronic toxicity tests, SD rats were orally dosed daily with 0.5, 1.5 and 5 g/kg bw/day of JointAlive®. To assess the potential genotoxicity, Ames test, cellular chromosome aberration and mouse micronucleus test in vivo were carried out. RESULTS: Based on a lack of notable findings other than histopathology finding of co-incidental prostate inflammation at the high dose, the "No Observed Adverse Effect Level (NOAEL)" of JointAlive® was concluded as 5 g/kg bw/day in males and females. Results also indicated that JointAlive® has no risk of genotoxicity. CONCLUSIONS: General toxicity and genotoxicity studies empirically demonstrated that JointAlive® poses a low risk of potential health risks, providing safety supports for the application of JointAlive® as a potential drug candidate to treat knee osteoarthritis.

Quantitative proteomics reveals the therapeutic effects of RFAP against depression via pathway regulation of long-term depression and potentiation.

Ethnopharmacological relevance: RFAP is a compound extraction complex of four Traditional Chinese Medicine (TCM), including the dry bark of Paeonia lactiflora Pall. (Radix Paeoniae Alba), Gardenia jasminoides J. Ellis (Fructus Gardeniae), Albizia julibrissin Durazz. (Albizia julibrissin Durazz), and Paeonia × suffruticosa Andrews (Peony bark). Not only RFAP but also the individual ingredients have been commonly used for the treatment of depression in the clinic. However, the underlying mechanism of pharmacology is difficult to interpret since its holistic and multidrug nature. Aim of the study: This study aimed to elucidate the potential antidepressant mechanism of RFAP in the treatment of chronic unpredictable mild stress (CUMS) rats' model via the quantitative proteomics approach. Materials and methods: We established the CUMS rats' model and evaluated the efficacy of RFAP using multiple behavior assays, including the sugar preference test, open field test, and forced swimming test. Then label-free quantitative proteomics analyses were performed to evaluate the integrated changes of proteome profiling in control, CUMS, RFAP low dose, and RFAP high dose groups. Finally, we validated the critical changed proteins in the pathways of long-term depression and potentiation via RT-PCR and Western blotting assays. Results: We successfully established the CUMS rats' model. The behavior assays indicated that the rats demonstrated a tendency to behavioral despair after four weeks. Label-free quantitative proteomics showed that 107 proteins were significantly upregulated and 163 proteins were downregulated in the CUMS group compared to the control group. These differentially expressed proteins were involved in long-term potentiation, long-term depression, nervous system development, neuronal synaptic structural constituent of ribosome, ATP metabolic process, learning or memory, and cellular lipid metabolic process. RFAP treatment partially restored the differentially expressed protein profile. The protective effect of RFAP on behavioral assessment were consistent with the results of proteomics. Conclusions: The results indicated that RFAP exerted a synergistic effect on CUMS by regulating long-term inhibition and potentiation-related proteins.

Investigation Driven by Network Pharmacology on Potential Components and Mechanism of DGS, a Natural Vasoprotective Combination, for the Phytotherapy of Coronary Artery Disease.

Phytotherapy offers obvious advantages in the intervention of Coronary Artery Disease (CAD), but it is difficult to clarify the working mechanisms of the medicinal materials it uses. DGS is a natural vasoprotective combination that was screened out in our previous research, yet its potential components and mechanisms are unknown. Therefore, in this study, HPLC-MS and network pharmacology were employed to identify the active components and key signaling pathways of DGS. Transgenic zebrafish and HUVECs cell assays were used to evaluate the effectiveness of DGS. A total of 37 potentially active compounds were identified that interacted with 112 potential targets of CAD. Furthermore, PI3K-Akt, MAPK, relaxin, VEGF, and other signal pathways were determined to be the most promising DGS-mediated pathways. NO kit, ELISA, and Western blot results showed that DGS significantly promoted NO and VEGFA secretion via the upregulation of VEGFR2 expression and the phosphorylation of Akt, Erk1/2, and eNOS to cause angiogenesis and vasodilation. The result of dynamics molecular docking indicated that Salvianolic acid C may be a key active component of DGS in the treatment of CAD. In conclusion, this study has shed light on the network molecular mechanism of DGS for the intervention of CAD using a network pharmacology-driven strategy for the first time to aid in the intervention of CAD.

Chinese Herbal Extracts Exert Neuroprotective Effect in Alzheimer's Disease Mouse Through the Dopaminergic Synapse/Apoptosis Signaling Pathway.

Background: Alzheimer's disease (AD) as an age-related, irreversible neurodegenerative disease, characterized by cognitive dysfunction, has become progressively serious with a global rise in life expectancy. As the failure of drug elaboration, considerable research effort has been devoted to developing therapeutic strategies for treating AD. TCM is gaining attention as a potential treatment for AD. Gastrodia elata Blume, Polygala tenuifolia Willd., Cistanche deserticola Ma, Rehmannia lutinosa (Gaertn.)DC., Acorus gramineus Aiton, and Curcuma longa L. (GPCRAC) are all well-known Chinese herbs with neuroprotective benefits and are widely used in traditional Chinese decoction for AD therapy. However, the efficacy and further mechanisms of GPCRAC extracts in AD experimental models are still unclear. The purpose of this study was to investigate the synergistic protective efficacy of GPCRAC extracts (composed of extracts from these six Chinese medicines), and the protein targets mediated by GPCRAC extracts in treating AD. Methods: Scopolamine-induced cognitive impairment mouse model was established to determine the neuroprotective effects of GPCRAC extracts in vivo, as shown by behavioral tests and cerebral cholinergic function assays. To identify the potential molecular mechanism of GPCRAC extracts against AD, label-free quantitative proteomics coupled with tandem mass spectrometry (LC-MS/MS) were performed. The integrated bioinformatics analysis was applied to screen the core differentially expressed proteins in vital canonical pathways. Critical altered proteins were validated by qPCR and Western blotting. Results: Administration of GPCRAC extracts significantly recovered scopolamine-induced cognitive impairment, as evidenced by the improved learning and memory ability, increased Ach content and ChAT activity, as well as decreased AchE activity in the hippocampus of mice. In total, 390 proteins with fold-change>1.2 or <0.83 and p < 0.05 were identified as significant differentially expressed proteins, of which 110 were significantly up-regulated and 25 were significantly down-regulated between control and model group. By mapping the significantly regulated proteins, we identified five hub proteins: PPP2CA, Gsk3ß, PP3CC, PRKACA, and BCL-2 that were associated with dopaminergic synapse and apoptosis signaling pathway, respectively. Western blotting and QPCR demonstrate that the expression levels of these core proteins could be significantly improved by the administration of GPCRAC extracts. These pathways and some of the identified proteins are implicated in AD pathogenesis. Conclusion: Administration of GPCRAC extracts was effective on alleviating scopolamine-induced cognitive impairment, which might be through modulation of dopaminergic synapse and apoptosis signaling pathway. Consequently, our quantitative proteome data obtained from scopolamine-treated model mice successfully characterized AD-related biological alterations and proposed novel protein biomarkers for AD.

Quantitative proteomics reveal the protective effects of EDS against osteoarthritis via attenuating inflammation and modulating immune response.

ETHNOPHARMACOLOGICAL RELEVANCE: Epimedium brevicornu Maxim, Dioscorea nipponica Makino, and Salvia miltiorrhiza Bunge formula (EDS) are three traditional Chinese medicines commonly combined and used to treat osteoarthritis (OA). However, the mechanism of its therapeutic effect on OA is still unclear. AIM OF THE STUDY: The aim of this study was to investigate the potential anti osteoarthritis mechanism of EDS in the treatment of OA rats' model by quantitative proteomics. MATERIALS AND METHODS: A papain-induced rat OA model was established, and then EDS was intragastrically administered for 28 days. A label-free quantification proteomics was performed to evaluate the holistic efficacy of EDS against OA and identify the possible protein profiles mechanisms. The expression levels of critical changed proteins were validated by RT-qPCR and Western blotting. The effects of EDS were then assessed by evaluating pathologic changes in the affected knee joint and measuring pressure pain threshold, acoustic reflex threshold, angle of joint curvature. RESULTS: Proteomics analysis showed that 62 proteins were significantly upregulated and 208 proteins were downregulated in OA group compared to control group. The changed proteins were involved in activation of humoral immunity response, complement cascade activation, leukocyte mediated immunity, acute inflammatory response, endocytosis regulation, and proteolysis regulation. The EDS treatment partially restored the protein profile changes. The protective effects of EDS on pathologic changes in OA rats' knee joint and pain threshold assessment were consisted with the proteomics results. CONCLUSIONS: The results suggest that EDS exerted synergistic therapeutic efficacies to against OA through suppressing inflammation, modulating the immune system, relieving joint pain, and attenuating cartilage degradation.

Eucommia, Cuscuta, and Drynaria Extracts Ameliorate Glucocorticoid-Induced Osteoporosis by Inhibiting Osteoclastogenesis Through PI3K/Akt Pathway.

Osteoporosis is one of the most common diseases in the world which resulted in heavy socioeconomic burden and a public health threat. Glucocorticoid-induced osteoporosis (GIO) is the most common secondary reason of osteoporosis. Therapeutic strategies using traditional Chinese medicine are under investigation for osteoporosis, with efforts to improve efficacy and clarify the mechanism. The combination of Eucommia, Cuscuta, and Drynaria is widely used in traditional Chinese decoction for osteoporosis treatment, but the experimental efficacy and mechanism are still unclear. Administration of E.C.D. extracts (Eucommia, Cuscuta, and Drynaria) in experimental GIO rats resulted in decreased urinal calcium, phosphorus loss, and decreased expression of RANKL, CTX in serum, increased serum calcium, phosphorus, and OPG level. E.C.D. extracts also improved bone density, structural integrity, and biomechanical function in experimental GIO rats. These finding were associated with E.C.D. extracts' treatment efficacy to GIO in vivo. The balance between osteoclast and osteoblast activity is essential for bone remodeling and bone related disease. The E.C.D. extracts inhibited Raw 264.7 cell differentiation to osteoclast in vitro. On the other hand, it promoted OPG expression of bone marrow mesenchymal stromal cells (MSCs) which can suppress the osteoclast genesis. E.C.D. extracts also increased the Wnt1 and Runx2 expression which are related to osteoblast formation. It also regulated the paracrine effect of MSC to inhibit osteoclast differentiation. The analysis of HPLC and comprehensive pharmacology identified the constituents of E.C.D. extracts and the potential osteoporosis-related targets mediated by E.C.D. extracts. The KEGG enrichment analysis suggested that PI3K/Akt pathway may be involved in the regulation osteoclast genesis by E.C.D. extracts and the result of Western blot of vitro assays proved it. Collectively, these data demonstrate E.C.D. extracts can inhibit osteoclast differentiation to foster experimental osteoporosis both in vivo and in vitro and it may exert the function of inhibiting osteoclast differentiation through PI3K/Akt pathway.

Effect of grape seed proanthocyanidins on tumor vasculogenic mimicry in human triple-negative breast cancer cells.

Vasculogenic mimicry (VM) refers to the unique ability of highly aggressive tumor cells to mimic the pattern of embryonic vasculogenesis, which was associated with invasion and metastasis. The grape seed proanthocyanidins (GSPs) had attracted much attention as a potential bioactive anti-carcinogenic agent. However, GSPs regulation of VM and its possible mechanisms in a triple-negative breast cancer cells (TNBCs) remain not clear. Therefore, we examined the effect of GSPs on VM information in HCC1937 cell model. In this study, we identified the VM structure via the three-dimensional (3D) matrix in vitro. Cell viability was measured using the CCK8 assay. The effects of GSPs on human triple-negative breast cancer cells (TNBCs) HCC1937 in terms of related proteins of VM information were determined using western blot analysis. In vitro, the tubular networks were found in highly invasive HCC1937 cells but not in the non-invasive MCF-7 cells when plated on matrigel. The number of vascular channels was significantly reduced when cells were exposed in GSPs (100 µg/ml) and GSPs (200 µg/ml) groups (all p<0.001). Furthermore, we found that treatment with GSPs promoted transition of the mesenchymal state to the epithelial state in HCC1937 cells as well as reducing the expression of Twist1 protein, a master EMT regulator.GSPs has the ability to inhibit VM information by the suppression of Twist1 protein that could be related to the reversal of epithelial-to-mesenchymal (EMT) process. It is firstly concluded that GSPs may be an potential anti-VM botanical agent for human TNBCs.