Effect of B28Bn(6–14) treatment on prostate tumor growth <i>in vivo</i>. A

<p>. Effect of intratumoral injection<b>.</b> BALB/c nude mice bearing DU145 tumors were administered the peptide (5 mg/kg) or PBS daily on days 9-14 (arrows-indicated) post inoculation. ***, <i>P</i><0.001 versus PBS from day 23 to the end, or B28 from day 25 to the end. <b>B</b>. B28Bn(6–14)-induced tumor tissue disruption. Mice bearing DU145 tumor grafts (200–300 mm³) were intratumorally administered a single, 50 µl dose of 50 µg B28Bn(6–14) or PBS. At 24 h post injection, the animals were sacrificed and the tumor tissues were routinely stained with H&E. <b>C</b>. Effect of intraperitoneal injection<b>.</b> Mice bearing tumors received the peptide (15 mg/kg) or PBS on days 7-13 (arrows-indicated) post inoculation. *, <i>P</i><0.05 versus PBS or B28 from day 13 to the end. <b>D</b>. <i>In vivo</i> cytotoxicity of the liver and kidney was determined by histological examination. At the end of i.p. therapy with B28Bn(6–14) or PBS, liver and kidney from the sacrificed animals were routinely stained with H&E. The histopathologic architecture was analyzed by light microscopy. The original magnification was indicated. The tumor volume was calculated as length×width²×0.5. Differences in tumor growth were analyzed by one-way ANOVA test.</p

Targeted cytotoxicity of B28Bn(6–14) on tumor cells is directed by the Bn(6–14) motif. A

<p>. Comparison of the cytotoxicity of Bn(6–14), B28, the conjugate B28Bn(6–14) and B28Bn(2–7). DU145 and PC-3 cells were treated with increasing concentrations of the peptide for 1 h and cell viability was measured. <b>B</b>. Comparison of the cytotoxicity of B28Bn(6–14) in tumor and non-malignant cells (<b>left panel</b>). The IC₅₀ values were calculated from the respective cell viability curves (<b>right panel</b>). <b>C</b>. Live/Dead assay for DU145 tumor cells and HSF normal cells. Cells were dual stained with SYTO 9 and PI after treatment with 5 µM B28Bn(6–14) for 45 min. The living cells fluoresced green, while the dead cells fluoresced red. Scale bar is 50 µm. <b>D</b>. Cellular uptake of FITC-labeled B28Bn(2–7), Bn(6–14), and B28Bn(6–14) in DU145 and HSF cells were tested with FITC-labeled IgG1 as control. Cells were incubated with 5 µM FITC-labeled peptides (red line) or IgG1 (blue line) for 30 min, washed with PBS, and analyzed by FACS. The percentage of positive cells was indicated.</p

Peptide secondary structure prediction and measurement.

<p><b>A</b>. A peptide secondary structure consensus prediction was performed based on the amino acid sequence using a set of online methods, including MLRC, PHD, and SOPM. Each random coil (c), extended strand (e), α helix (h), and ambiguous state (?) corresponded to an amino acid in the peptide sequence. <b>B</b>. The actual secondary structure contents of the peptides were measured by far-ultraviolet (UV) circular dichroism (CD) spectrum. The CD spectra of B28 (dashed line), Bn(6–14) (dot-dashed line) and B28Bn(6–14) (solid line) were recorded in 2 mM sodium phosphate buffer (pH 7.4) at 25°C.</p

Effect of B28Bn(6–14)-induced apoptosis and necrosis in tumor cells. A

<p>. Detection of B28Bn(6–14)-induced apoptotic cells under a microscope. Cells treated with B28Bn(6–14) for 30 min were subjected to dual staining with FITC-Annexin V (green) and PI (red). Cells showing Annexin V−/PI-, Annexin V+/PI-, Annexin V+/PI+ were considered living, early apoptotic, and necrotic, respectively, in the merged image (PCM, phase contrast microscope). <b>B</b>. FACS analysis of B28Bn(6–14)-induced apoptosis. DU145 cells were either treated with 5 µM B28Bn(6–14) for different time, or treated with B28Bn(6–14) at different concentrations for 10 min. Cells were stained and analyzed by FACS. Cells in early apoptotic and necrotic stages are indicated as the percentage of total cells counted. <b>C</b>. Inhibition of B28Bn(6–14)-induced apoptosis with the Pan-caspase inhibitor, Z-VAD-FMK. Cells were pre-incubated with Z-VAD-FMK, treated with B28Bn(6–14), and cell viability was determined. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001, Student’s <i>t</i> test. <b>D</b>. B28Bn(6–14)-induced caspase-3 activation and DNA fragmentation. Cells were treated with B28Bn(6–14) and stained either with the cleaved caspase-3 antibody or the TUNEL kit. Compared with untreated cells (CK merge), the positive cells presented green fluorescence with DAPI-stained nuclei. <b>E</b>. Caspase-3 Colorimetric Assay. Caspase-3 activity was presented as the fold of untreated cells. 2 µg/ml TRAIL was used as the positive control. <b>F</b>. Evaluation of B28Bn(6–14)-induced necrosis. After treatment with 5 µM B28Bn(6–14), the release of lactate dehydrogenase (LDH) into the culture supernatant was detected. LDH levels were presented as the percentage of the max release from the cells treated with 0.9% Triton X-100. <b>G</b>. Assessment of B28Bn(6–14)-induced hemolysis. Human erythrocytes in PBS were treated with the peptide for 16 h. Hemolysis was presented as the percentage of the absorbance at 540 nm from erythrocytes treated with 0.1% Triton X-100. Scale bar is 10 µM for all images.</p

Control release of mitochondria-targeted antioxidant by injectable self-assembling peptide hydrogel ameliorated persistent mitochondrial dysfunction and inflammation after acute kidney injury

<p>Persistent mitochondrial injury occurs after acute kidney injury (AKI) and mitochondria-targeted antioxidant Mito-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) (MT) has shown benefits for AKI, but its efficiency is limited by short half-life and side effect <i>in vivo</i>. Self-assembling peptide (SAP) hydrogel is a robust platform for drug delivery. This study aims to develop an SAP-based carrier to slow release MT for enhancing its long-term therapeutic potency on AKI. The KLD with aspartic acid (KLDD) was designed. The microstructure and <i>in vitro</i> release of MT was assayed. The protective role of MT-loaded SAP (SAP-MT) hydrogel on renal mitochondrial injury, tubular apoptosis, and inflammation was evaluated in mice at five days after ischemia-reperfusion injury (IRI). Our results showed that KLDD could self-assemble into cross-linked nanofiber hydrogel and it had lower release rate than free MT and KLD hydrogel. Compared to IRI and free MT mice, SAP-MT mice exerted reduced renal mitochondria-produced ROS (mtROS) and improved mitochondrial biogenesis and architecture. Consequently, SAP-MT mice showed less renal tubular cell apoptosis, kidney injury marker kidney injury molecule-1 (Kim-1) expression, lower level of pro-inflammatory factors expression, and macrophages infiltration than those of IRI and free MT mice. This study suggested that SAP-MT ameliorated IRI due to its extended mitochondrial protection role than free MT and thus improved the long-term outcomes of AKI.</p

GLP-1 receptor agonist ameliorates obesity-induced chronic kidney injury via restoring renal metabolism homeostasis - Fig 1

<p>(A)Representative HE, PAS staining (scale bar = 50 μm) and IHC staining for α-SMA (scale bar = 100 μm) of kidney from control, HFD and HFD + Lira group. (B) IHC staining for IL-6 and MCP-1 in kidney of different groups (scale bar = 100 μm). (C)Quantification of IL-6 and MCP-1 protein detected by IHC staining. (D-E) Morphological analysis of glomerular size, glomerulosclerosis and interstitial fibrosis in renal sections. (F)Quantification of α-SMA detected by IHC staining(** p<0.01, compared with control;^# p<0.05,^{# #} p<0.01, compared with HFD).</p

GLP-1 receptor agonist ameliorates obesity-induced chronic kidney injury via restoring renal metabolism homeostasis - Fig 4

<p>(A)Oil Red O (ORO) staining of kidney from control, HFD and HFD + Lira group (scale bar = 100 μm).(B) Quantification of kidney MDA level. (C) Real-time PCR analysis for CD36, L-FABP, SREBP-1c, FAS, PPAR-α and CPT1 mRNA. (D) IHC staining for renal PPAR-α, FABP1 and CPT1(scale bar = 100 μm). (E) Quantification of PPAR-α, FABP1 and CPT1proteindetected by IHC staining (* p<0.05, ** p<0.01, compared with control;^# p<0.05, ^{# #} p<0.01, compared with HFD).</p

GLP-1 receptor agonist ameliorates obesity-induced chronic kidney injury via restoring renal metabolism homeostasis - Fig 6

<p>(A) IHC staining for PGC1α, p-AMPK and Sirt1of kidney from control, HFD and HFD + Lira group (scale bar = 100μm). (B) Real-time PCR analysis for Sirt1 and PGC1α mRNA. (C) Quantification of PGC1α, p-AMPK and Sirt1 protein detected by IHC staining. (D) Determination of renal NAD⁺/NADH ratio by commercial kit (** p<0.01 compared with control; ^{# #} p<0.01 compared with HFD).</p

GLP-1 receptor agonist ameliorates obesity-induced chronic kidney injury via restoring renal metabolism homeostasis - Fig 2

<p>OPLS-DA loading plots derived from renal lipid extracts of ¹H NMR spectra in (A) HFD group <i>vs</i> control group and (B) HFD+ Lira group <i>vs</i> HFD group. Note: 1. Total Cholesterol (C₁₈H₃), 2. Fatty acid residues (ω-CH₃), 3. Total Cholesterol (C₂₆H₃, C₂₇H₃), 4. Free Cholesterol (C₁₉H₃), 5. Fatty acid residues ((CH₂-)_n), 6. Fatty acid residues (COCH₂-CH₂), 7. Fatty acid residues (-CH₂ of ARA+EPA), 8. Fatty acid residues (CH₂-CH = 9. Fatty acid residues (-CO-CH₂), 10. Fatty acid residues (-CH = CH-CH₂-CH = CH-), 11. Phosphatidylethanolamine (-CH₂-NH₂), 12. Sphingomyelin (-CH₂-N-(CH₃)₃), 13, Phosphatidylcholine (-CH₂-N-(CH₃)₃), 14. Total phospholipids (Glycerol (C₃H₂)), 15. Triglycerides (C₁H and C₃H of glycerol), 16. Triglycerides(C₂H of glycerol), 17. Fatty acid residues (-CH = CH-).</p

General and biochemical parameters of rats in different groups.

<p>General and biochemical parameters of rats in different groups.</p