Emodin Ameliorates High Glucose-Induced Podocyte Apoptosis via Regulating AMPK/mTOR-Mediated Autophagy Signaling Pathway / 中国结合医学杂志

OBJECTIVE@#To investigate the effect of emodin on high glucose (HG)-induced podocyte apoptosis and whether the potential anti-apoptotic mechanism of emodin is related to induction of adenosine-monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR)-mediated autophagy in podocytes (MPC5 cells) in vitro.@*METHODS@#MPC5 cells were treated with different concentrations of HG (2.5, 5, 10, 20, 40, 80 and 160 mmol/L), emodin (2, 4, 8 µ mol/L), or HG (40 mmol/L) and emodin (4 µ mol/L) with or without rapamycin (Rap, 100 nmol/L) and compound C (10 µ mol/L). The viability and apoptosis of MPC5 cells were detected using cell counting kit-8 (CCK-8) assay and flow cytometry analysis, respectively. The expression levels of cleaved caspase-3, autophagy marker light chain 3 (LC3) I/II, and AMPK/mTOR signaling pathway-related proteins were determined by Western blot. The changes of morphology and RFP-LC3 fluorescence were observed under microscopy.@*RESULTS@#HG at 20, 40, 80 and 160 mmol/L dose-dependently induced cell apoptosis in MPC5 cells, whereas emodin (4 µ mol/L) significantly ameliorated HG-induced cell apoptosis and caspase-3 cleavage (P<0.01). Emodin (4 µ mol/L) significantly increased LC3-II protein expression levels and induced RFP-LC3-containing punctate structures in MPC5 cells (P<0.01). Furthermore, the protective effects of emodin were mimicked by rapamycin (100 nmol/L). Moreover, emodin increased the phosphorylation of AMPK and suppressed the phosphorylation of mTOR. The AMPK inhibitor compound C (10 µ mol/L) reversed emodin-induced autophagy activation.@*CONCLUSION@#Emodin ameliorated HG-induced apoptosis of MPC5 cells in vitro that involved induction of autophagy through the AMPK/mTOR signaling pathway, which might provide a potential therapeutic option for diabetic nephropathy.

Therapeutic mechanism of emodin for treatment of rheumatoid arthritis: a network pharmacology-based analysis / 南方医科大学学报

OBJECTIVE@#To investigate the therapeutic mechanism of emodin in the treatment of rheumatoid arthritis (RA) using a network pharmacology-based method and validate this mechanism in a fibroblast-like synovial cell line.@*METHODS@#The PubChem, Targetnet, SwissTargetPrediction, Genecards, OMIM, and DisGeNET databases were searched to obtain emodin targets and RA-related genes. A protein-protein interaction (PPI) network was constructed, and GO and KEGG pathway enrichment analyses were carried out to analyze the intersection genes. AutoDock4.2.6 software was used to simulate molecular docking between emodin and its candidate targets. In a cultured fibroblast-like synovial cell line (MH7A), the effects of different concentrations of emodin on proliferation of tumor necrosis factor-α (TNF-α)-induced cells were investigated using CCK-8 assay, cell scratch experiment and flow cytometry; the changes in the expressions of nuclear factor-κB (NF-κB) pathway proteins were detected using Western blotting, and the mRNA expressions of the hub genes were examined with RT-qPCR.@*RESULTS@#We identified 32 intersection genes of emodin and RA, and the key targets including CAPS3, ESR1, and MAPK14 involved mainly the NF-κB signaling pathway. Cell scratch experiment and flow cytometry demonstrated a strong inhibitory effect of emodin on MH7A cell proliferation. Treatment with TNF-α significantly increased the cellular expressions of the NF-κB pathway proteins, which were obviously lowered by treatment with 80 μmol/L emodin. The results of RT-qPCR showed that TNF-α treatment obviously up-regulated the expressions of the hub genes COX2 and P38MAPK, and emodin treatment significantly down-regulated the expressions of MAPK and PTGS2 and up-regulated the expression of CASP3.@*CONCLUSION@#The therapeutic effect of emodin on RA is mediated mainly through regulation of cell proliferation, apoptosis, and the NF-κB pathway.

Mechanism of emodin in relieving neuropathic pain by regulating serum metabolism / 中国中药杂志

The present study investigated the effect of emodin on the serum metabolite profiles in the chronic constriction injury(CCI) model by non-target metabolomics and explored its analgesic mechanism. Twenty-four Sprague Dawley(SD) rats were randomly divided into a sham group(S), a CCI group(C), and an emodin group(E). The rats in the emodin group were taken emodin via gavage once a day for fifteen days(50 mg·kg~(-1)) on the first day after the CCI surgery. Mechanical withdrawal threshold(MWT) and thermal withdrawal threshold(TWL) in each group were performed before the CCI surgery and 3,7, 11, and 15 days after surgery. After 15 days, blood samples were collected from the abdominal aorta. The differential metabolites were screened out by non-target metabolomics and analyzed with Kyoto Encyclopedia of Genes and Genomes(KEGG) and ingenuity pathway analysis(IPA). From the third day after CCI surgery, the MWT and TWL values were reduced significantly in both CCI group and emodin group, compared with the sham group(P<0.01). At 15 days post-surgery, the MWT and TWL values in emodin group increased significantly compared with the CCI group(P<0.05). As revealed by non-target metabolomics, 72 differential serum metabolites were screened out from the C-S comparison, including 41 up-regulated and 31 down-regulated ones, while 26 differential serum metabolites from E-C comparison, including 10 up-regulated and 16 down-regulated ones. KEGG analysis showed that the differential metabolites in E-C comparison were enriched in the signaling pathways, such as sphingolipid metabolism, arginine biosynthesis, glycerophospholipid metabolism, and tryptophan metabolism. IPA showed that the differential metabolites were mainly involved in the lipid metabolism-molecular transport-small molecule biochemistry network. In conclusion, emodin can exert an analgesic role via regulating sphingolipid metabolism and arginine biosynthesis.

Effects of Emodin Derivative on Cell Cycle, Apoptosis and NF-κB Pathway in Burkitt Lymphoma Cells / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect and mechanism of a novel emodin derivative YX-18 on Burkitt lymphoma (BL) cells.@*METHODS@#MTT assay was used to detect the effect of YX-18 on the proliferation of BL cell lines CA46 and Raji. Annexin V-PE/7-AAD double staining assay was used for detecting the effect of YX-18 on the apoptosis of CA46 and Raji cells. PI/RNase staining was used to test the effect of YX-18 on CA46 and Raji cell cycle. JC-1 method was used to measure the changes of mitochondrial membrane potential after YX-18 treatment, and DAPI staining was used to detect the morphology of apoptotic cells. Western blot was used to analyze the distribution changes of NF-κB pathway protein (P65, P-P65, IκB, P-IκB) in the cytoplasm and cell nucleus, and also the expression changes of cyclin-related protein P21, CDK2, P-CDK2, Cycling D1, Cycling E1, and the apoptosis-related protein Caspase-3, Caspase-8, Caspase-9 and the proliferation-related protein C-MYC, BCL-2 by YX-18. Real-time fluorescence-quantitative PCR was used to evaluate the effects of YX-18 on mRNA levels of C-MYC and Ki-67 genes in CA46 and Raji cells, and EBNA-1 and EBER genes of EBV in Raji (EBV@*RESULTS@#Novel Emodin derivative YX-18 could effectively inhibit the proliferation of BL cell lines CA46 and Raji, showing a time-dependent effect (24, 48 and 72 h: r@*CONCLUSION@#The novel emodin derivative YX-18 can significantly inhibit the proliferation of Burkitt lymphoma cells, and induce the cell apoptosis and cycle arrest. The inhibitory effect of YX-18 on the proliferation of Burkitt lymphoma cells may be related with the effect of Caspase apoptosis pathway, the proliferation and apoptosis-related molecules, such as C-MYC and Ki-67, and also to the inhibition of NF-κB pathway.

Emodin alleviates hypertrophic scar formation by suppressing macrophage polarization and inhibiting the Notch and TGF-β pathways in macrophages

Hypertrophic scar (HS) formation is a common complication that develops after skin injury; however, there are few effective and specific therapeutic approaches for HS. Emodin has previously been reported to inhibit mechanical stress-induced HS inflammation. Here, we investigated the molecular mechanisms underlying the inhibitory effects of emodin on HS formation. First, we conducted in vitro assays that revealed that emodin inhibited M1 and M2 polarization in rat macrophages. We subsequently established a combined rat model of tail HS and dorsal subcutaneous polyvinyl alcohol (PVA) sponge-induced wounds. Rats were treated with emodin or vehicle (DMEM). Tail scar specimens were harvested at 14, 28, and 42 days post-incision and subjected to H&E staining and Masson's trichrome staining. Histopathological analyses confirmed that emodin attenuated HS formation and fibrosis. Macrophages were separated from wound cells collected from the PVA sponge at 3 and 7 days after implantation. Flow cytometry analysis demonstrated that emodin suppressed in vivo macrophage recruitment and polarization at the wound site. Finally, we explored the molecular mechanisms of emodin in modulating macrophage polarization by evaluating the expression levels of selected effectors of the Notch and TGF-β pathways in macrophages isolated from PVA sponges. Western blot and qPCR assays showed that Notch1, Notch4, Hes1, TGF-β, and Smad3 were downregulated in response to emodin treatment. Taken together, our findings suggested that emodin attenuated HS formation and fibrosis by suppressing macrophage polarization, which is associated with the inhibition of the Notch and TGF-β pathways in macrophages.

Emodin ameliorates high-glucose induced mesangial p38 over-activation and hypocontractility via activation of PPARgamma

Early stage diabetic nephropathy is characterized by elevated glomerular filtration. Recent studies have identified high-glucose induced p38 MAPK (p38) over-activation in mesangial cells. Mesangial hypocontractility is the major underlying mechanism, however, no ameliorating agents are currently available. We investigated the protective effects of emodin on high-glucose induced mesangial cell hypocontractility. Mesangial cells were cultured under normal (5.6 mM) and high glucose (30 mM) conditions. Emodin was administrated at doses of 50 mg/l and 100 mg/l. Angiotension II stimulated cell surface reductions were measured to evaluate cell contractility. p38 activity was detected using Western blotting. To further explore the possible mechanism of emodin, expression of the peroxisome proliferator-activated receptor gamma (PPARgamma) was measured and its specific inhibitor, gw9662, was administrated. Our results showed: (1) high-glucose resulted in a 280% increase in p38 activity associated with significant impairment of mesangial contractility; (2) emodin treatment dose-dependently inhibited high-glucose induced p38 over-activation (a 40% decrease for 50 mg/l emodin and a 73% decrease for 100 mg/l emodin), and mesangial hypocontractility was ameriolated by emodin; (3) both the PPARgamma mRNA and protein levels were elevated after emodin treatment; (4) inhibition of PPARgamma using gw9662 effectively blocked the ameliorating effects of emodin on high-glucose induced p38 over-activation and mesangial hypocontractility. Emodin effectively ameliorated p38 over-activation and hypocontractility in high-glucose induced mesangial cells, possibly via activation of PPARgamma.