Salvianolic Acid B Significantly Suppresses the Migration of Melanoma Cells via Direct Interaction with ß-Actin.

Melanoma is the most aggressive and difficult to treat of all skin cancers. Despite advances in the treatment of melanoma, the prognosis for melanoma patients remains poor, and the recurrence rate remains high. There is substantial evidence that Chinese herbals effectively prevent and treat melanoma. The bioactive ingredient Salvianolic acid B (SAB) found in Salvia miltiorrhiza, a well-known Chinese herbal with various biological functions, exhibits inhibitory activity against various cancers. A375 and mouse B16 cell lines were used to evaluate the main targets and mechanisms of SAB in inhibiting melanoma migration. Online bioinformatics analysis, Western blotting, immunofluorescence, molecular fishing, dot blot, and molecular docking assays were carried out to clarify the potential molecular mechanism. We found that SAB prevents the migration and invasion of melanoma cells by inhibiting the epithelial-mesenchymal transition (EMT) process of melanoma cells. As well as interacting directly with the N-terminal domain of ß-actin, SAB enhanced its compactness and stability, thereby inhibiting the migration of cells. Taken together, SAB could significantly suppress the migration of melanoma cells via direct binding with ß-actin, suggesting that SAB could be a helpful supplement that may enhance chemotherapeutic outcomes and benefit melanoma patients.

Ponicidin-induced conformational changes of HSP90 regulates the MAPK pathway to relieve ulcerative colitis.

ETHNOPHARMACOLOGICAL RELEVANCE: Ulcerative colitis (UC) is a recurring chronic intestinal disease that can be debilitating and in severe cases, may further lead to cancer. However, all these treatment techniques still suffer from drug dependence, adverse effects and poor patient compliance. Therefore, there is an urgent need to seek new therapeutic strategies. In traditional Chinese medicine, Rabdosia rubescens (Hemsl.) H.Hara has the effects of clearing heat-toxin and promoting blood circulation to relieve pain, it is wildly used for treating inflammatory diseases such as sore throats and tonsillitis. Ponicidin is an important molecule for the anti-inflammatory effects of Rabdosia rubescens, but it has not been studied in the treatment of colitis. HSP90 is the most critical regulator in the development and progression of inflammatory diseases such as UC. AIM OF THE STUDY: The aim of this study was to explore the anti-inflammatory activity of ponicidin and its mechanism of effect in vitro and in vivo, as well as to identify the target proteins on which ponicidin may interact. MATERIAL AND METHODS: 2.5% (w/v) dextran sulfate sodium (DSS) was used to induce C57BL/6 mice to form an ulcerative colitis model, and then 5 mg/kg and 10 mg/kg ponicidin was given for treatment, while the Rabdosia rubescens extract group and Rabdosia rubescens diterpene extract group were set up for comparison of the efficacy of ponicidin. At the end of modeling and drug administration, mouse colon tissues were taken, and the length of colon was counted, inflammatory factors and inflammatory signaling pathways were detected. RAW264.7 cells were induced to form cell inflammation model with 1 µg/mL Lipopolysaccharide (LPS) for 24 h. 1 µM, 2 µM and 4 µM ponicidin were given at the same time, and after the end of the modeling and administration of the drug, the inflammatory factors and inflammatory signaling pathways were detected by qRT-PCR, western blotting, immunofluorescence and other methods. In vitro, target angling and combined with mass spectrometry were used to search for relevant targets of ponicidin, while isothermal titration calorimetry (ITC), protease degradation experiments and molecular dynamics simulations were used for further confirmation of the mode of action and site of action between ponicidin and target proteins. RESULTS: Ponicidin can alleviate DSS and LPS-induced inflammation by inhibiting the MAPK signaling pathway at the cellular and animal levels. In vitro, we confirmed that ponicidin can interact with the middle domain of HSP90 and induce the conformational changes in the N-terminal domain. CONCLUSION: These innovative efforts identified the molecular target of ponicidin in the treatment of UC and revealed the molecular mechanism of its interaction with HSP90.

Insight into the mechanism of Xiao-Chai-Hu-Tang alleviates irinotecan-induced diarrhea based on regulating the gut microbiota and inhibiting Gut ß-GUS.

BACKGROUND: Irinotecan (CPT-11, Camptosar@) is a first-line drug for metastatic colorectal cancer. CPT-11-induced diarrhea, which is closely related to the concentrations of ß-glucuronidase (ß-GUS) and SN-38 in the gut, largely limits its clinical application. PURPOSE: Herein, Xiao-Chai-Hu-Tang (XCHT), a traditional Chinese formula, was applied to mitigate CPT-11-induced toxicity. This study initially explored the mechanism by which XCHT alleviated diarrhea, especially for ß-GUS from the gut microbiota. METHODS: First, we examined the levels of the proinflammatory cytokines and the anti-inflammatory cytokines in the intestine. Furthermore, we researched the community abundances of the gut microbiota in the CPT-11 and XCHT-treated mice based on 16S rRNA high-throughput sequencing technology. Meanwhile, the level of SN-38 and the concentrations of ß-GUS in intestine were examined. We also resolved the 3D structure of ß-GUS from gut microbiota by X-ray crystallography technology. Moreover, we used virtual screening, SPR analysis, and enzyme activity assays to confirm whether the main active ingredients from XCHT could selectively inhibit ß-GUS. RESULTS: In XCHT-treated mice, the levels of the proinflammatory cytokines decreased, the anti-inflammatory cytokines increased, and the community abundances of beneficial Firmicutes and Bacteroidota improved in the gut microbiota. We also found that the concentrations of ß-GUS and the level of SN-38, the major ingredient that induces diarrhea in the gut, significantly decreased after coadministration of XCHT with CPT-11 in the intestine. Additionally, we revealed the structural differences of ß-GUS from different gut microbiota. Finally, we found that EcGUS had good affinity with baicalein and meanwhile could be selectively inhibited by baicalein from XCHT. CONCLUSIONS: Overall, XCHT could relieve the delayed diarrhea induced by CPT-11 through improving the abundance of beneficial gut microbiota and reduced inflammation. Furthermore, based on the three-dimensional structure, baicalein, especially, could be used as a candidate EcGUS inhibitor to alleviate CPT-11-induced diarrhea.

Zn2+ regulates human oxalate metabolism by manipulating oxalate decarboxylase to treat calcium oxalate stones.

A high concentration of oxalate is associated with an increased risk of kidney calcium oxalate (CaOx) stones, and the degradation of exogenous oxalate mostly depends on oxalate-degrading enzymes from the intestinal microbiome. We found that zinc gluconate supplement to patients with CaOx kidney stones could significantly improve the abundance of oxalate metabolizing bacteria in humans through clinical experiments on patients also subjected to antibiotic treatment. The analysis of clinical samples revealed that an imbalance of Lactobacillus and oxalate decarboxylase (OxDC) was involved in the formation of CaOx kidney stones. Then, we identified that Zn2+ could be used as an external factor to improve the activity of OxDC and promote Lactobacillus in the intestinal flora, and this treatment achieved a therapeutic effect on rats with stones aggravated by antibiotics. Finally, by analyzing the three-dimensional structure of OxDC and completing in vitro experiments, we propose a model of the Zn2+-induced reduction of CaOx kidney stone symptoms in rats by increasing the metabolism of oxalate through the positive effects of Zn2+ on Lactobacillus and OxDC.

Salvianolic acid B suppresses hepatic fibrosis by inhibiting ceramide glucosyltransferase in hepatic stellate cells.

UDP-glucose ceramide glucosyltransferase (UGCG) is the first key enzyme in glycosphingolipid (GSL) metabolism that produces glucosylceramide (GlcCer). Increased UGCG synthesis is associated with cell proliferation, invasion and multidrug resistance in human cancers. In this study we investigated the role of UGCG in the pathogenesis of hepatic fibrosis. We first found that UGCG was over-expressed in fibrotic livers and activated hepatic stellate cells (HSCs). In human HSC-LX2 cells, inhibition of UGCG with PDMP or knockdown of UGCG suppressed the expression of the biomarkers of HSC activation (α-SMA and collagen I). Furthermore, pretreatment with PDMP (40 µM) impaired lysosomal homeostasis and blocked the process of autophagy, leading to activation of retinoic acid signaling pathway and accumulation of lipid droplets. After exploring the structure and key catalytic residues of UGCG in the activation of HSCs, we conducted virtual screening, molecular interaction and molecular docking experiments, and demonstrated salvianolic acid B (SAB) from the traditional Chinese medicine Salvia miltiorrhiza as an UGCG inhibitor with an IC50 value of 159 µM. In CCl4-induced mouse liver fibrosis, intraperitoneal administration of SAB (30 mg · kg-1 · d-1, for 4 weeks) significantly alleviated hepatic fibrogenesis by inhibiting the activation of HSCs and collagen deposition. In addition, SAB displayed better anti-inflammatory effects in CCl4-induced liver fibrosis. These results suggest that UGCG may represent a therapeutic target for liver fibrosis; SAB could act as an inhibitor of UGCG, which is expected to be a candidate drug for the treatment of liver fibrosis.

Shikonin ameliorated mice colitis by inhibiting dimerization and tetramerization of PKM2 in macrophages.

Dysregulated immune response plays a pivotal role in Ulcerative colitis. In lamina propria of inflammatory colonic mucosa, macrophages tend to polarize into M1 type and metabolically reprogram to aerobic glycolysis. PKM2 orchestrates glucose metabolic switch in macrophages, which tetramer has high pyruvate kinase activity, while which dimer mainly works as a protein kinase to stabilize HIF-1α and mediate anabolism. Shikonin is a potent PKM2 inhibitor derived from traditional Chinese medicine Arnebiae Radix with anti-inflammatory and anticarcinogen activities. However, it is unclear which conformation of PKM2 is inhibited by Shikonin, and whether this inhibition mediates pharmacological effect of Shikonin. In this study, we examined the efficacy of Shikonin on dextran sulfate sodium-induced mice colitis and determined the states of PKM2 aggregation after Shikonin treatment. Results showed that Shikonin dose-dependently alleviated mice colitis, down-regulated expression of F4/80, iNOS and CD86, decreased IFN-γ, IL-1ß, IL-6 and TNF-α, while increased IL-10 in mice colon. Furthermore, Shikonin suppressed the pyruvate, lactate production and glucose consumption, inhibited the pyruvate kinase activity and nuclear translocation of PKM2, and decreased both dimerization and tetramerization of PKM2 in macrophages. In vitro assay revealed that Shikonin bounded to PKM2 protein, inhibited the formation of both dimer and tetramer, while promoted aggregation of PKM2 macromolecular polymer. TEPP-46, an activator of PKM2 tetramerization, attenuated the ameliorative effect of Shikonin on disuccinimidyl suberate mice. In summary, Shikonin improved mice colitis, which mechanism may be mediated by inhibiting dimerization and tetramerization of PKM2, suppressing aerobic glycolysis reprogram, improving mitochondrial dynamic, and therefore alleviating inflammatory response of macrophages.

UGT1A1 dysfunction increases liver burden and aggravates hepatocyte damage caused by long-term bilirubin metabolism disorder.

UGT1A1 is the only enzyme that can metabolize bilirubin, and its encoding gene is frequently mutated. UGT1A1*6 (G71R) is a common mutant in Asia which leads to the decrease of UGT1A1 activity and abnormal bilirubin metabolism. However, it is not clear whether low UGT1A1 activity-induced bilirubin metabolism disorder increases hepatocyte fragility. ugt1a+/- mice were used to simulate the UGT1A1*6 (G71R) population. Under the same CCl4 induction condition, ugt1a+/- mice showed severer liver damage and fibrosis, indicating that ugt1a1 dysfunction increased liver burden and aggravated hepatocyte damage. In the animal experiment with a continuous intraperitoneal injection of bilirubin, the ugt1a+/- mice livers had more serious unconjugated bilirubin accumulation. The accumulated bilirubin leads to hyperphosphorylation of IκB-α, Ikk-ß, and p65 and a significant increase of inflammatory factor. The α-SMA and Collagen I proteins markedly up-regulated in the ugt1a+/- mice livers. Immunofluorescence and confocal microscopy showed that hepatic stellate cells and Kupffer cells were activated in ugt1a+/- mice. Comprehensive results show that there was a crosstalk relationship between low UGT1A1 activity-bilirubin-liver damage. Furthermore, cell experiments confirmed that unconjugated bilirubin activated the NF-κB pathway and induced DNA damage in hepatocytes, leading to the significant increase of inflammatory factors. UGT1A1 knockdown in hepatocytes aggravated the toxicity of unconjugated bilirubin. Conversely, overexpression of UGT1A1 had a protective effect on hepatocytes. Finally, Schisandrin B, an active ingredient with hepatoprotective effects, extracted from a traditional Chinese medicinal herb, which could protect the liver from bilirubin metabolism disorders caused by ugt1a1 deficiency by downregulating p65 phosphorylation, inhibiting Kupffer cells, reducing inflammation levels. Our data clarified the mechanism of liver vulnerability caused by cross-talk between low UGT1A1 activity bilirubin, and provided a reference for individualized prevention of liver fragility in Gilbert's syndrome.