Mechanism of Astragalus mongholicus Bunge ameliorating cerebral ischemia-reperfusion injury: Based on network pharmacology analysis and experimental verification.

ETHNOPHARMACOLOGICAL RELEVANCE: Astragalus mongholicus Bunge (AMB) is a herb with wide application in traditional Chinese medicine, exerting a wealth of pharmacological effects. AMB has been proven to have an evident therapeutic effect on ischemic cerebrovascular diseases, including cerebral ischemia-reperfusion injury (CIRI). However, the specific mechanism underlying AMB in CIRI remains unclear. AIM OF THE STUDY: This study aimed to investigate the potential role of AMB in CIRI through a comprehensive approach of network pharmacology and in vivo experimental research. METHODS: The intersection genes of drugs and diseases were obtained through analysis of the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database and Gene Expression Omnibus (GEO) database. The protein-protein interaction (PPI) network was created through the string website. Meanwhile, the gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was carried out using R studio, and thereafter the key genes were screened. Then, the molecular docking prediction was made between the main active ingredients and target genes, and hub genes with high binding energy were obtained. In addition, molecular dynamic (MD) simulation was used to validate the result of molecular docking. Based on the results of network pharmacology, we used animal experiments to verify the predicted hub genes. First, the rat middle cerebral artery occlusion and reperfusion (MACO/R) model was established and the effective dose of AMB in CIRI was determined by behavioral detection and 2,3,5-Triphenyltetrazolium chloride (TTC) staining. Then the target proteins corresponding to the hub genes were measured by Western blot. Moreover, the level of neuronal death was measured using hematoxylin and eosin (HE) and Nissl staining. RESULTS: Based on the analysis of the TCMSP database and GEO database, a total of 62 intersection target genes of diseases and drugs were obtained. The KEGG enrichment analysis showed that the therapeutic effect of AMB on CIRI might be realized through the advanced glycation endproduct-the receptor of advanced glycation endproduct (AGE-RAGE) signaling pathway in diabetic complications, nuclear factor kappa-B (NF-κB) signaling pathway and other pathways. Molecular docking results showed that the active ingredients of AMB had good binding potential with hub genes that included Prkcb, Ikbkb, Gsk3b, Fos and Rela. Animal experiments showed that AWE (60 g/kg) could alleviate CIRI by regulating the phosphorylation of PKCß, IKKß, GSK3ß, c-Fos and NF-κB p65 proteins. CONCLUSION: AMB exerts multi-target and multi-pathway effects against CIRI, and the underlying mechanism may be related to anti-apoptosis, anti-inflammation, anti-oxidative stress and inhibiting calcium overload.

Cinobufacini injection delays hepatocellular carcinoma progression by regulating lipid metabolism via SREBP1 signaling pathway and affecting macrophage polarization.

ETHNOPHARMACOLOGICAL RELEVANCE: Cinobufacini injection, an aqueous extract of the toad, is a commonly used anti-tumor animal herbal medicine in clinical practice. It has the effects of detoxifying, reducing swelling, and relieving pain. AIMS OF THE STUDY: To investigate the effects of Cinobufacini injection on hepatocellular carcinoma progression by regulating lipid metabolism and macrophage polarization in the tumor microenvironment and to identify the potential molecular mechanisms. MATERIALS AND METHODS: To establish the axillary transplantation tumor model of hepatocellular carcinoma Hepa1-6 in C57BL/6 mice, and to evaluate the inhibitory effect of Cinobufacini injection on hepatocellular carcinoma in vivo as well as drug delivery security. Combined metabolomics and transcriptomics analysis of the effect of Cinobufagin Injection on tumor microenvironment. An in vitro mouse co-culture model of peritoneal macrophages and Hepa1-6 cells was established to research the effects of Cinobufacini injection on macrophage polarization, hepatocellular carcinoma cell growth, migration, and changes in lipid metabolism. Cinobufacini injection inhibition of the AMPK/SREBP1/FASN signaling pathway regulating cholesterol metabolism and affecting macrophage polarization was examined using qRT-PCR, lentiviral transfection, immunofluorescence, and Western blot. RESULT: In vivo experiments demonstrated that Cinobufacini injection treatment significantly inhibited the growth of Hepa1-6 hepatomas, along with a reduction in cholesterol content and a decrease in the percentage of M2 macrophages in tumor tissue. In vitro, we found that Cinobufacini injection inhibits IL-4-induced M2 macrophage polarization, reduces the cholesterol content of Hepa1-6 cells in a co-culture system, and inhibits the promotion of hepatocellular carcinoma cells by M2 macrophages. In addition, successful overexpression of SREBP1 in Hepa1-6 cells showed more pronounced cellular activity whereas Cinobufacini injection inhibited this change and reduced intracellular lipid levels. CONCLUSION: Cinobufacini injection inhibits cholesterol synthesis within the tumor microenvironment via the AMPK/SERBP1/FASN signaling pathway, which in turn blocks the M2 polarization of macrophages, leading to the weakening of hepatocellular carcinoma growth and migration, and the promotion of its apoptosis. Our findings provide an important Introduction to understanding the molecular mechanism of Cinobufacini injection's anticancer activity and provide reliable theoretical and experimental support for its clinical application.

Qingluoyin granules protect against adjuvant-induced arthritis in rats via downregulating the CXCL12/CXCR4-NF-κB signalling pathway.

CONTEXT: Qingluoyin (QLY) is a traditional Chinese medicine (TCM) formula which has been used in treating human rheumatoid arthritis (RA) for years in China. OBJECTIVE: This study investigates the effect of QLY granules on adjuvant arthritis (AA) and the possible mechanism. MATERIALS AND METHODS: Sprague-Dawley (SD) rats were injected with Complete Freund's adjuvant (CFA) to induce the AA model. After the onset of arthritis, rats received intragastric administrations of the QLY granules (1.35, 2.70, and 5.40 g/kg) or Tripterygium glycosides (TG) tablets (positive drug, 10 mg/kg) for 14 d. After 28 d immunization, the symptoms, inflammatory parameters and molecular mechanisms were investigated. RESULTS: In the QLY granule (1.35, 2.70, and 5.40 g/kg) therapy groups, the arthritis index decreased to 6.30 ± 2.06, 5.80 ± 1.55, 5.30 ± 1.16 compared with the model (9.00 ± 3.01), paw swelling decreased to 1.56 ± 0.40, 1.28 ± 0.38, 1.12 ± 0.41 mL compared with the model (2.22 ± 0.73 mL). QLY granules (1.35, 2.70 and 5.40 g/kg) significantly reduced the thymus and the spleen indexes, inhibited the production of pro-inflammatory cytokines, and alleviated the pathological changes of joints compared with the model group. Furthermore, the treatment of QLY granules (2.70 and 5.40 g/kg) markedly inhibited CXCL12, CXCR4 (in spleen and synovium) and p-NF-κB p65 (in synovium) protein expression of AA rats. CONCLUSIONS: QLY granules have obvious therapeutic effects on AA rats, which may be associated with downregulating the CXCL12/CXCR4-NF-κB signalling pathway. QLY granules can be used as a candidate for the treatment of RA, which deserves further study.

[Efficacy of Kangshuailing Gao on benign prostatic hyperplasia in rats].

OBJECTIVE: To investigate the effect of Kangshuailing Gao (KG) on benign prostatic hyperplasia (BPH) in rats and its action mechanisms. METHODS: Fifty BPH model rats were randomized into five groups of equal number, BPH model control, finasteride control, and high-, medium- and low-dose KG, to be treated intragastrically with distilled water, finasteride solution at 0.52 mg/kg, and KG solution at 4.16, 2.08 and 1.04 g/kg respectively once a day for 30 days consecutively. Another 10 normal healthy rats were taken as blank controls. The rats were weighed once a week during the treatment. The wet weight and index of the prostate were obtained after treatment, followed by measurement of the contents of serum estradiol (E2) and dihydrotestosterone (DHT), testosterone (T) and hypoxia-inducible factor-1α (HIF-1α) in the prostatic tissue, and observation of histomorphological changes in the prostate under the light microscope. RESULTS: Compared with the BPH model control group, high- and medium-dose KG significantly reduced the prostate wet weight (ï¼»0.84 ± 0.08ï¼½ vs ï¼»0.69 ± 0.04ï¼½ and ï¼»0.71 ± 0.07ï¼½ g, P < 0.01), the prostatic index (ï¼»0.28 ± 0.03ï¼½% vs ï¼»0.20 ± 0.02ï¼½% and ï¼»0.22 ± 0.03ï¼½%, P < 0.01), and the levels of T (ï¼»4.63 ± 1.25ï¼½ vs ï¼»2.44 ± 0.47ï¼½ and ï¼»2.91 ± 0.69ï¼½ ng/L, P < 0.01) and DHT (ï¼»154.44 ± 20.25ï¼½ vs ï¼»88.23 ± 13.63ï¼½ and ï¼»90.52 ± 16.44ï¼½ nmol/L, P < 0.01), but increased the level of E2 (ï¼»0.95 ± 0.24ï¼½ vs ï¼»1.19 ± 0.14ï¼½ and ï¼»1.20 ± 0.22ï¼½ nmol/L, P < 0.01) in the serum. High-dose KG remarkably reduced the overexpression of HIF-1α in the prostate tissue of the BPH model rats (P < 0.01) and alleviated such BPH-related symptoms as epithelium thinning, intraglandular secretion reduction, and interstitial substance decrease. CONCLUSIONS: Kangshuailing Gao acted effectively on BPH in the model rats by reducing the androgen level, balancing the estrogen/androgen ratio, and downregulating the expression of HIF-1α in the prostate tissue.

The Protective Effect of Total Flavones from Rhododendron simsii Planch. on Myocardial Ischemia/Reperfusion Injury and Its Underlying Mechanism.

OBJECTIVES: Total flavones from Rhododendron simsii Planch. (TFR) are the effective part extracted from the flowers of Rhododendron simsii Planch. and have obvious protective effects against cerebral ischemic or myocardial injuries in rabbits and rats. However, their mechanism of cardioprotection is still unrevealed. Therefore, the present study was designed to investigate the effect of TFR on myocardial I/R injury and the underlying mechanism. METHODS: TFR groups were treated by gavage once a day for 3 days at a dose of 20, 40, and 80 mg/kg, respectively, and then the model of myocardial I/R injury was established. Myocardial infarction, ST-segment elevation, and the expression of UTR, ROCK1, ROCK2, and p-MLC protein in rat myocardium were determined at 90 min after reperfusion. UTR siRNA in vivo transfection and competition binding assay method were used to study the relationship between the protective effect of TFR and UTR. RESULTS: The expression of UTR protein markedly decreased in myocardium of UTR siRNA transfection group rats. TFR could significantly reduce the infarct size and inhibit the increase of RhoA activity and ROCK1, ROCK2, and p-MLC protein expressions both in WT and UTR knockdown rats. The reducing rate of TFR in myocardial infarction area, RhoA activity, and ROCK1, ROCK2, and p-MLC protein expressions in UTR knockdown rats decreased markedly compared with that in WT rats. In addition, TFR had no obvious effect on the increase of ΣST in UTR knockdown rats in comparison with that in model group. In particular, TFR could significantly inhibit the combination of [125I]-hu-II and UTR, and IC50 was 0.854 mg/l. CONCLUSIONS: The results indicate that the protective effect of TFR on I/R injury may be correlated with its blocking UTR and the subsequent inhibition of RhoA/ROCK signaling pathway.

The effect of ethyl acetate extract from persimmon leaves on Alzheimer's disease and its underlying mechanism.

BACKGROUND: Alzheimer's disease (AD) is one of the most prevalent neurodegenerative disorders characterized by neuronal loss in the brain and cognitive impairment. AD is now considered to be the third major cause of death in developed countries, after cardiovascular disease and cancer. Persimmon leaves are used as a popular folk medicine to treat hypertension, angina and internal haemorrhage in Cyangbhina, and it has been reported that ethyl acetate extract of persimmon leaves (EAPL) displays a potential therapeutic effect on neurodegenerative diseases. HYPOTHESIS/PURPOSE: This study was designed to investigate the effects of EAPL on AD, to clarify the possible mechanism by which EAPL exerts its beneficial effects and prevents AD, and to determine the major constituents involved. STUDY DESIGN: AD model was established by bilateral injection of Aß1-42 into the hippocampus of rats. The cognitive performance was determined by the Morris water maze and step-down tests. Superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), malondialdehyde (MDA), apoptosis, total and phosphorylated c-Jun NH2-terminal kinase (JNK/p-JNK), caspase-3, Bax and Bcl-2 were determined. In addition, a sensitive and reliable LC-QTOF-MS method was applied to identify the major compounds present in EAPL. RESULTS: EAPL at doses of 200mg/kg, 400mg/kg could markedly reduce the latency, significantly increase the time in the first quadrant and number of the target crossing times in Morris water maze test, markedly increase the latency and reduce the number of errors in the step-down test, significantly inhibit the reductions in SOD and GSH-Px activities, and increase the level of MDA. In addition, EAPL treatment attenuated neuronal apoptosis in the hippocampus, reduced the expression of p-JNK, caspase-3, and the relative ratio of Bax/Bcl-2. Meanwhile, 32 constituents were identified by LC-QTOF-MS/MS assays. CONCLUSION: The results indicate that EAPL has a potent protective effect on cognitive deficits induced by Aß in rats and this effect appears to be associated with the regulation of the antioxidative defense system and the mechanism of mitochondrial-mediated apoptosis. Furthermore, analysis of the LC-MS data suggests that flavonoids and triterpenoids may be responsible for the potential biological effects of EAPL.