Reagents
Mouse insulin (EMD Millipore), TNF-α and IL-6 (Abcam) ELISA kits, anti-DNA (cat. no. AC30-10) monoclonal antibody (Novus Biologicals, Ltd.), D-Luciferin, sodium salt D-Fluorescein sodium salt (cat. no. 40901ES01; Shanghai Yeasen), LUC-labeled lentiviral particles (cat. No. GM-0220PC, Shanghai; Genomeditech), RIPA lysis buffer, nitrocellulose membranes and an SABC (rabbit IgG)-POD kit were obtained from Beijing Solarbio Science & Technology Co., Ltd. A blood glucose meter and test strips were acquired from ACCU-CHEK (Accu-ChekSoftclix;). TRIzol®was purchased from Invitrogen (Thermo Fisher Scientific, Inc.). Antibodies against PI3K, P85, phospho-PI3K P85 (cat. no.Y607), insulin receptor β (IR), p-IR (cat. no.Y1185); PTEN, p-PTEN (cat. no.S380+T382+T383), glucose transporter 4 (cat. no.GLUT4)and IGF-1R were obtained from Abcam. Antibodies against Akt, p-Akt (ser 473), ERK andp-ERK (1/2) (Thr202/Tyr204) were obtained from Cell Signaling Technology, Inc. Anti-GAPDH, anti-mouse IgG, and anti-rabbit IgG were obtained from Cell Signaling Technology, Inc. Other reagents used were of analytical grade.
Animals and experimental design
Six-week old female db/db mice (18-20g) were purchased from Model Animal Research Center of Nanjing University. All mice were housed individually with a 12 h light/dark cycle at 23±2℃ and a humidity of 55±10%, with free access to water and food. The mice were allowed to adapt to these conditions for one week prior to beginning the experimental procedures. The animal procedures were approved by the Animal Care and Use Committee on the Ethics of Animal Experiments of Taizhou University of Science and Technology. All related facilities and experimental procedures were performed according to the guidelines described by the Technical Standards for the Testing & Assessment of Health Food (2003).
Cell culture
HUC-MSCs were provided by Jilin TuohuaBioengineering Co. Ltd. (Siping). HUC-MSCs were immediately obtained from healthy mothers during routine term elective caesarean section births. Fully informed consent was obtained several weeks prior to delivery. Ethics approval was obtained from the Ethics Committee of the Siping Center Hospital (approval no. SC-2017-010). HUC-MSCs were isolated and propagated, as previously described [18].
HUC-MSCs treatment study
The db/db mice were divided into four groups: db/m (n=10), db/m+HUC-MSCs (intraperitoneal injection, IP; n=10), db/m+HUC-MSCs (tail vain injection, IV; n=10), db/m+HUC-MSCs (skeletal muscle injection, IM; n=10), db/db (n=15), db/db+HUC-MSCs (intraperitoneal injection, IP; n=15), db/db+HUC-MSCs (tail vain injection) anddb/db+HUC-MSCs (skeletal muscle injection). A total of 1x107 HUC-MSCs (passage 4) were resuspended in 0.7 ml saline and administered by IP/IV; or 1x108 (passage 4) in 2ml saline were administered by local injection IM for the db/m+HUC-MSCs and db/db+HUC-MSCs groups. The other groups were given an equivalent volume of saline as the db/m and db/db groups, and both groups were fed a normal chow diet. To determine the effect of HUC-MSCs, survival rate, weight, blood glucose, ALT, AST, Cre, BUN, γ-GT and the levels of serum insulin, were measured at the mentioned times.
Bioluminescence imaging
HUC-MSCs cells were infected with luciferase-lentivirus (CMV-Luciferase-PGK-Puro, cat. No. GM-0220PC, Shanghai Genomeditech Co., Ltd., Shanghai, China) to establish HUC-MSCs-luc cell stably expressing luciferase. The HUC-MSCs-luc skeletal muscle transplantation db/db mice were anesthetized using isoflurane, and injected intraperitoneally with 100 μl of 15 mg/ml D-luciferin in Sodium salt D solution (Shanghai Yeasen Biotechnology Co., Ltd., Shanghai, China). Subsequently, the mice were imaged using an in vivo imaging system (FOBI; Berthold Technologies).In vivo imaging was performed prior to sacrifice.
Calculation of IR index
IR calculated as follows: HOMA IR = serum insulin mmol/l x blood glucose mmol/l/22.5 [19].
Histological examination of the skeletal muscle
A portion of the extracted skeletal muscle was immediately fixed in PBS mixed with 4% paraformaldehyde and embedded in paraffin. The sections (4 µm in thickness) were stained with hematoxylin and eosin (H&E). Histological analysis was performed using a light microscope (DM4000B photomicroscope; Leica Microsystems, Inc.).
Immunofluorescence (IF) studies
Skeletal muscle sections were subjected to signal-direct IF staining of human DNA (1:10; ANA), followed by incubation with Alexa Flour 488-conjugated secondary antibodies (OriGene Technologies, Inc.). Nuclei were counterstained with Hoechst (Invitrogen; Thermo Fisher Scientific, Inc.). All sections were scanned, and images were acquired using a laser scanning confocal microscope (FV1000; Olympus Corporation).
Immunohistochemical (IHC) staining
After de-paraffinization, the sections were incubated with a 3% H2O2 solution to block endogenous peroxidases. Antigen retrieval was performed using 0.1 M sodium citrate (pH 6.0) for 60 min. Sections were incubated with anti-PI3K, anti-GLUT4, anti-p-IR, anti-p-PTEN (1:100; Abcam), or anti-p-ERK (1:100; CST) antibodies overnight at 4℃, and a horseradish peroxidase-conjugated secondary antibody and diaminobenzidine substrate were added sequentially. Following hematoxylin counterstaining and dehydration, the sections were mounted and observed using a Leica M4000B photomicroscope (Leica Microsystems, Inc.).
ELISA
The concentrations of TNF-α, IL-6 (all from Abcam) and serum insulin (EMD Millipore) were determined using specific ELISA kits.
Reverse transcription-quantitative (RT-q)PCR
Gene expression of c-fos, c-myc, JNK, GSK-3β, FOXO1, GLUT4, PTEN and IRS-1in skeletal muscle section was analyzed using RT-qPCR. TRIzol®reagent was used to extract RNA, and cDNA was synthesized using a reverse transcription kit (Takara Bio, Inc.). qPCR containing was performed using a SYBR Premix EX TaqTMcDNA with specific gene primers, and genes were amplified using a 7300PULAS system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The relative expression of each gene was determined and normalized to the expression of 18S and calculated using the 2-ΔΔCq method. The sequences of the primers used are: 18S-forward 5’-ACTCAACACGGGAAACCTCAC-3’ and reverse, 5’-TCGCTCCACCAACTAAGAACG-3’; c-fos-forward, 5’-GGAATTAACCTGGTGCTGGATTG-3’ and reverse, 5’-GAACATTGACGCTGAAGGACTAC-3’;c-myc-forward, 5’-CTATCACCAGCAACAGCAGAG-3’ and reverse, 5’-ACATAGGATGGAGAGCAGAGC-3’; JNK-forward, 5’-ACATAGGATGGAGAGCAGAGC-3’ and reverse, 5’-CATTGACAGACGGCGAAGAC-3’; GSK-3β-forward, 5’-CACCGCTCCTTCCTTCCTTC-3’and reverse, 5’-GACTCCTCTTCCTAACCACCTG-3’;FoxO1-forward, 5’-CAGCCTTGAGCAGCCTAATG-3’and reverse, 5’-AGACTGGGAAACACCGATGG-3’; GLUT4-forward, 5’-ACGGATAGGGAGCAGAAACC-3’ and reverse, 5’-CAGCACAGGACACTCATCTTC-3’; PTEN-forward, 5’-AGAGATTGGCTGCTGTCCTG-3’ and reverse, 5’-TGGTTAAGTCATTGCTGCTGTG-3’; IRS-1-forward, 5’-AGCAGCAGTAGCAGCATCAG-3’and reverse,5’-TTACCGCCACCACTCTCAAC-3’; IGF-1-forward, 5’-TATGGAGATGGGAGGGTTTCAG-3’ and reverse, 5’-GTAGGCACAGCATTCGTTAGG-3’; mTOR-forward, 5’-GCAGCAACAGTGAGAGTGAAG-3’ and reverse, 5’-CAAGGAGATAGAACGGAAGAAGC-3’.
Western blot analysis
Protein samples from skeletal muscle section were resolved using 8 or 10% SDS-PAGE and transferred to PVDF membranes (EMD Millipore). Membranes were blocked in 5% nonfat dry milk, and subsequently incubated with primary antibodies againstp-PI3K/PI3K, p-AKT/Akt, p-IR/IR, p-PTEN/PTEN and IGF-1R (1:500; Abcam), p-ERK/ERK1/2and GAPDH (1:500; CST Biological Reagents Co., Ltd.) overnight. Horseradish peroxidase-conjugated anti-rabbit IgG or anti-mouse IgG (1:7,000; CST Biological Reagents Co., Ltd.) were used as the secondary antibodies. PVDF membranes were developed using an Image-ProPlus system.
Statistical analysis
Data are represented as the mean ± standard deviation and analyzed using a one-way ANOVA or a two-tailed unpaired Student’s t-test. P-values were adjusted for multiple comparisons using Bonferroni correction. Analyses were performed using GraphPad Prism version 7 (GraphPad Software, Inc.). P<0.05 was considered to indicate a statistically significant difference.