Chemicals and reagents
The reagents purchased from Cell Signaling Technology (Danvers, MA, USA) included primary antibodies against MLC (8505), phospho-MLC (3674), AMPK (2983), phospho-AMPK (2971), GAPDH (2118), and α-tubulin (3873); and Assay Kit for detecting the Activation of RhoA (8789), Compound C and FITC-Phalloidin (P5282) were obtained from Sigma Aldrich (Saint Louis, MO, USA). Linagliptin and Y-27632 (S1049) were provided by Med Chem Express (Newark, NJ, USA).
Culture of HRGECs and glucose management
We obtained HRGECs (PS-4000) from ScienCell Research Laboratories (Kirkland, WA, USA) and nurtured them in a medium that contains 10% serum and 1% growth supplement for endothelial cells (ScienCell Research Laboratories). After pretreating with or without linagliptin for 2 h, HRGECs were activated by normal glucose (NG;5.5 mM ) or high glucose (HG;30 mM ) (R&D Systems). 24 h later, they were examined using Western blotting or immuno- fluorescence assay.
As we described previously, HRGECs were treated with lysis buffer that contained phosphatase inhibitors and proteinase (Roche, USA). We determined protein concentration using the BCA assay (Beyotime, Beijing, China). After isolation using SDS-PAGE, the same amount of protein was conveyed to PVDF membranes. Following blockade with 5% non-fat milk, the membranes were incubated with a primary antibody overnight at 4 °C. Further, the membranes were reacted with a secondary antibody conjugated with horseradish peroxidase (HRP) for 2 h at 24℃. After being spotted by the ECL Super Signal reagent (Pierce, 34078), bands of protein were envisioned using an analysis system of digital gel images (Bio-Rad, Hercules, CA, USA).
F-actin labeling assay
We grew confluent HRGEC monolayers on glass coverslips that were precoated with 0.1% gelatin for an immunofluorescence assay. After being treated with 4% paraformaldehyde for 10 min, the HRGECs were blocked with PBS, including 1% bovine serum albumin. Afterward, the HRGECs were hatched with FITC-Phalloidin for 1 h and then labeled by DAPI. After acquiring cellular images with a laser-scanning confocal microscope (FV1000-IX81, Olympus), confocal images were analyzed using FV10-ASW Viewer software (Ver 4.1, Olympus Life Science, Japan).
Assessment of transendothelial electrical resistance (TEER)
We seeded HRGECs on transwell inserts (0.4 µm pore, Millipore, USA) and cultivated them to confluence. The HRGEC monolayer’s TEER was assessed using a Millicell-ERS voltohmmeter (Millipore, Burlington, MA, USA). The value of resistance was indicated as the unit of Ω·cm2. The TEER of the transwell was documented and normalized by deducting its basal value in each group.
The examination of transendothelial albumin leakage
HRGECs were developed to confluence on a pore transwell insert (0.4 μm; 3413, Coring). Following the treatment with NG or HG for 24 h in the presence or absence of inhibitors, medium including albumin labeled with horseradish peroxidase (HRP) (50 μg/mL; Solarbio, Beijing, China) was appended into the top chamber. The concentration of the albumin in the chamber was determined using the TMB Soluble Substrate kit (Solarbio). The value of absorbance was quantified using a microplate reader (Elx 800, BioTek). Afterward, the permeability coefficint of albumin (Pa) was computed using the formula Pa =[A]/t ×1/A ×V/[L]. In the equation, [A] and [L] symbolize albumin’s concentration in the bottom and top chambers , correspondingly ; t represents time (s), A signifies the region of the membrane (cm2), and V denotes the bottom chamber’s volume (uL).
Assessing Rho activity
We employed a detection kit of the Active Rho to examine the Rho activity of HRGECs. In brief, HRGECs bred in Petri dishes (100 mm) were remedied with glucose (5.5 or 30 mM) following pretreatment with or without linagliptin (100 nM) for 1 h. After lysing with a buffer of cell lysis, HRGECs were reared with rhotekin Rho-binding peptide restrained on agarose (GST-Rhotekin-RBD), which facilitated the descent of GTP-bound Rho. Western blotting was used to examine activated GTP-Rho and total RhoA.
The measurement of DPP-4 activity
DPP-4 activity was measured by an Activity Assay Kit (Biovision Milpitas, CA). In brief, cultured HRGECs were homogenized in an assay buffer of DPP-4 and then centrifuged to eliminate insoluble components. The samples were fine-tuned to a final volume of 50 µl and loaded to a 96-well plate for the examination. After mixing with the substrate (2 µl) of Gly-Pro-7-Amino-4-Methylcoumarin (AMC), the samples were incubated for 30 min at 37°C. AMC releasing from the substrate was detected using a fluorescence spectrophotometer at an excitation of 360 nm and an emission of 460 nm.
Data are expressed as the mean ± standard error. Differences among multiple groups were analyzed using one-way ANOVA followed post hoc by the Newman–Keuls test. Comparisons between two groups were performed by Student’s t-test. Values of P ≤ 0.05 were considered to be significant. Statistical analyses were conducted using GraphPad Prism 7.0 software (La Jolla, CA, USA).