Therapeutic Mechanism of Baicalin in Experimental Colitis Analyzed Using Network Pharmacology and Metabolomics.

Background: Baicalin is an important active flavonoid isolated from the roots of Scutellaria baicalensis (S. baicalensis), a well-known traditional Chinese herb used in treating inflammatory bowel disease (IBD). The objectives of this study were to assess the potential benefit of baicalin in experimental colitis, as well as to investigate metabolic biomarkers of experimental colitis in conjunction with network pharmacology. Methods: Using a widely utilized network pharmacology technique, baicalin's targets and pathways were predicted. Simultaneously, experimental colitis was induced by intrarectal administration of TNBS. Histopathology examinations were performed to confirm pathological changes. Plasma samples were examined by using an untargeted metabolomics technique based on ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) to screen differential metabolites and associated metabolic pathways. Additionally, network pharmacology and integrated analysis of metabolomics were used to identify the primary targets. Results: Through network pharmacology research, tumor necrosis factor (TNF), interleukin 6 (IL6), serine/threonine-protein kinase (AKT1), and other 7 proteins were found to be the main targets of baicalin against IBD. The untargeted metabolomics results showed that 47 metabolites in glycerophospholipids and sphingolipid metabolism were involved as key pathways in the experimental colitis model group. 19 metabolites, including Sphingomyelin (SM d42:2, SM d42:1, SM d34:1), Lysophosphatidic acids (LPA 18:4), 1-Palmitoylglycerophosphocholine, and 17(18)-EpETE were demonstrated as key metabolites for baicalin to exert effects. Moreover, udp-glucose ceramide glucosyltransferase (UGCG), sphingomyelin synthase 1 (SGMS1), and sphingosine kinase (SPHK1) were predicted as sphingolipids-linked targets of baicalin against experimental colitis by integrative analysis. Conclusion: Based on these results, it implies that sphingolipid metabolism and sphingolipid signaling pathway might be acted as therapeutic mechanism for baicalin against experimental colitis.

Geranylgeranyl pyrophosphate depletion by statins compromises skeletal muscle insulin sensitivity.

BACKGROUND: Statins are widely prescribed cholesterol-lowering drugs but have been shown to increase the risk of type 2 diabetes mellitus. However, the molecular mechanisms underlying the diabetogenic effect of statins are still not fully understood. METHODS: The effects of geranylgeranyl transferase I and II (GGTase I and II) inhibition on insulin-stimulated glucose uptake and GLUT4 translocation, and the dependence of these effects on insulin signalling were investigated in skeletal muscle cells. The protective effects of geranylgeranyl pyrophosphate (GGPP) and its precursor geranylgeraniol (GGOH) on simvastatin-induced insulin resistance were evaluated in vitro and in vivo. The effect of GGTase II inhibition in skeletal muscle on insulin sensitivity in vivo was confirmed by adeno-associated virus serotype 9 (AAV9)-mediated knockdown of the specific subunit of GGTase II, RABGGTA. The regulatory mechanisms of GGTase I on insulin signalling and GGTase II on insulin-stimulated GLUT4 translocation were investigated by knockdown of RhoA, TAZ, IRS1, geranylgeranylation site mutation of RhoA, RAB8A, and RAB13. RESULTS: Both inhibition of GGTase I and II mimicked simvastatin-induced insulin resistance in skeletal muscle cells. GGPP and GGOH were able to prevent simvastatin-induced skeletal muscle insulin resistance in vitro and in vivo. GGTase I inhibition suppressed the phosphorylation of AKT (Ser473) (-51.3%, P < 0.01), while GGTase II inhibition had no effect on it. AAV9-mediated knockdown of RABGGTA in skeletal muscle impaired glucose disposal without disrupting insulin signalling in vivo (-46.2% for gastrocnemius glucose uptake, P < 0.001; -52.5% for tibialis anterior glucose uptake, P < 0.001; -17.8% for soleus glucose uptake, P < 0.05; -31.4% for extensor digitorum longus glucose uptake, P < 0.01). Inhibition of RhoA, TAZ, IRS1, or geranylgeranylation deficiency of RhoA attenuated the beneficial effect of GGPP on insulin signalling in skeletal muscle cells. Geranylgeranylation deficiency of RAB8A inhibited insulin-stimulated GLUT4 translocation and concomitant glucose uptake in skeletal muscle cells (-42.8% for GLUT4 translocation, P < 0.01; -50.6% for glucose uptake, P < 0.001). CONCLUSIONS: Geranylgeranyl pyrophosphate regulates glucose uptake via GGTase I-mediated insulin signalling-dependent way and GGTase II-mediated insulin signalling-independent way in skeletal muscle. Supplementation of GGPP/GGOH could be a potential therapeutic strategy for statin-induced insulin resistance.

Mevalonate pathway orchestrates insulin signaling via RAB14 geranylgeranylation-mediated phosphorylation of AKT to regulate hepatic glucose metabolism.

Statin use accompanies with increased risk of new onset of type 2 diabetes, however, the underlying mechanisms remain not be fully understood and effective prevention strategies are still lacking. Herein, we find that both pharmacological and genetic inhibition of GGTase II mimic the disruption of simvastatin on hepatic insulin signaling and glucose metabolism in vitro. AAV8-mediated knockdown of liver RABGGTA, the specific subunit of GGTase II, triggers systemic glucose metabolism disorders in vivo. By adopting a small-scale siRNA screening, we identify RAB14 as a regulator of hepatic insulin signaling and glucose metabolism. Geranylgeranylation deficiency of RAB14 inhibits the phosphorylation of AKT (Ser473) and disrupts hepatic insulin signaling and glucose metabolism possibly via impeding mTORC2 complex assembly. Finally, geranylgeranyl pyrophosphate (GGPP) supplementation is sufficient to prevent simvastatin-caused disruption of hepatic insulin signaling and glucose metabolism in vitro. Geranylgeraniol (GGOH), a precursor of GGPP, is able to ameliorate simvastatin-induced systemic glucose metabolism disorders in vivo. In conclusion, our data indicate that statins-targeted mevalonate pathway regulates hepatic insulin signaling and glucose metabolism via geranylgeranylation of RAB14. GGPP/GGOH supplementation might be an effective strategy for the prevention of the diabetic effects of statins.

Statins induce skeletal muscle atrophy via GGPP depletion-dependent myostatin overexpression in skeletal muscle and brown adipose tissue.

Myopathy is the major adverse effect of statins. However, the underlying mechanism of statin-induced skeletal muscle atrophy, one of statin-induced myopathy, remains to be elucidated. Myostatin is a negative regulator of skeletal muscle mass and functions. Whether myostatin is involved in statin-induced skeletal muscle atrophy remains unknown. In this study, we uncovered that simvastatin administration increased serum myostatin levels in mice. Inhibition of myostatin with follistatin, an antagonist of myostatin, improved simvastatin-induced skeletal muscle atrophy. Simvastatin induced myostatin expression not only in skeletal muscle but also in brown adipose tissue (BAT). Mechanistically, simvastatin inhibited the phosphorylation of forkhead box protein O1 (FOXO1) in C2C12 myotubes, promoting the nuclear translocation of FOXO1 and thereby stimulating the transcription of myostatin. In differentiated brown adipocytes, simvastatin promoted myostatin expression mainly by inhibiting the expression of interferon regulatory factor 4 (IRF4). Moreover, the stimulative effect of simvastatin on myostatin expression was blunted by geranylgeranyl diphosphate (GGPP) supplementation in both myotubes and brown adipocytes, suggesting that GGPP depletion was attributed to simvastatin-induced myostatin expression. Besides, the capacities of statins on stimulating myostatin expression were positively correlated with the lipophilicity of statins. Our findings provide new insights into statin-induced skeletal muscle atrophy. Graphical headlights 1. Simvastatin induces skeletal muscle atrophy via increasing serum myostatin levels in mice; 2. Simvastatin promotes myostatin expression in both skeletal muscle and brown adipose tissue through inhibiting GGPP production; 3. The stimulating effect of statins on myostatin expression is positively correlated with the lipophilicity of statins.

A "protective umbrella" nanoplatform for loading ICG and multi-modal imaging-guided phototherapy.

In order to prevent the aggregation of ICG and enhance its stability, a novel nanoplatform (TiO2:Yb,Ho,F-ß-CD@ICG/HA) was designed for NIR-induced phototherapy along with multi-mode imaging(UCL/MRI/Flu). In this nanosysytem: TiO2:Yb,Ho,F was used as upconversion materials and applied in vivo for the first time; ß-CD acted as a "protective umbrella" to load separated ICG and avoid the low phototherapy efficiency because of its aggregation; HA was the capping agent of ß-CD to prevent ICG unexpected leaking and a target to recognize CD44 receptor. The nanosystem exhibited excellent size (~200 nm) and photo- and thermal-stability, preferable reactive oxygen yield and temperature response (50.4 °C) under 808 nm laser. It could efficiently target and suppress tumor growth. The imaging ability (UCL/MRI) of TiO2:Yb,Ho,F could facilitate diagnosis of the tumor, especially for deep tissues. Altogether, our work successfully improved the phototherapy efficacy through incorporating the ICG into the cavity of ß-CD and applied TiO2:Yb,Ho,F for upconversion imaging in vivo.

A Hollow NaGdF4 /AFn Nanosystem Based on "Relay Race" Release for Therapy.

To develop a multifunctional nanomaterial for dual-mode imaging and synergetic chemotherapy, curcumin (CUR) was physically entrapped into hollow upconversion NaGdF4 nanomaterial, then apoferritin (AFn) loaded with doxorubicin (DOX) was attached to the NaGdF4 surface. Subsequent modification with the targeting reagent folic acid (FA) led to generation of the CUR/NaGdF4 -DOX/AFn-FA conjugate for cancer treatment. X-ray diffraction, scanning (SEM) and transmission electron microscopy (TEM), and Fourier transform infrared (FTIR) spectroscopy demonstrated the successful preparation of hexagonal-phase NaGdF4 and NaGdF4 -AFn-FA. Moreover, no toxicity was observed for NaGdF4 -AFn-FA. In vitro and in vivo experiments demonstrated that the two drugs are sequentially released from the nanocomposites. This two-drug system showed strong growth inhibitory effects on MCF-7 cells. Upconversion luminescence imaging and magnetic resonance (MR) imaging of NaGdF4 -AFn-FA were carried out. The results of this study show that NaGdF4 -AFn-FA can be used for targeted anticancer drug delivery as well as imaging, a novel multi-pronged theranostic system for tumor treatment.

A "win-win" nanoplatform: TiO2:Yb,Ho,F for NIR light-induced synergistic therapy and imaging.

To avoid the defect of low energy transfer efficiency in core-shell UCNP-TiO2 NPs, doping rare earth into TiO2 and improving the photocatalytic activity of TiO2 itself under Vis-NIR light might be a more direct and efficient strategy for high 1O2 production. Here, we designed a TiO2:Yb,Ho,F-ß-CD@DTX/HA nanoplatform using TiO2:Yb,Ho,F as the core, ß-CD as the drug carrier, hyaluronic acid (HA) as the capping agent and target, and then applied it for 808 nm induced photodynamic-chemotherapy and 980 nm upconversion fluorescence/MR imaging. The results were as follows: (i) for TiO2 as a photosensitizer, after doping Yb, Ho, F into TiO2, it could directly generate reactive oxygen species under an 808 nm laser; the dopants enhanced the absorption under the UV-Vis-NIR region and increased the electron-hole pair separation. (ii) For TiO2 as the upconversion host, F and Ho also endowed TiO2:Yb,Ho,F with enhanced upconversion fluorescence under a 980 nm laser and T2-MRI contrast performance (r2 = 30.71 mM-1 s-1), respectively, thus, facilitating imaging for deep tissues. (iii) The HA shell outside of ß-CD prevented the unexpected leaking of DTX, which improved the target abilities and achieved the enzyme-responsive drug release. The in vitro and in vivo studies also demonstrated the nanosystem could efficiently suppress tumor growth by combination therapy and had excellent imaging (UCL/MR) ability. Particularly, our work was the first example that utilized TiO2 simultaneously as a photosensitizer and upconversion host, which simplified the core-shell UCNP-TiO2 nanocomposites and reached a "win-win" cooperation in NIR-induced photodynamic therapy and UCL imaging.