Celastrol (98.0%) was purchased from Sichuan Weikeqi Biotech Co., Ltd. FITC and Cy5.5-NHS were purchased from MedChemExpress. Pluronic F-127 was purchased from Sigma-Aldrich. All the chemicals used in this study were of analytical grade.
Preparation of K3-HBc/CLT NCs
The amino acid sequence of HBc-183 is the same as the previously reported . K3 peptide accompanied with two glycine-rich linkers was incorporated to the surface-exposed major immunodominant loop region of HBc-183 (between the residues 78 and 81) to yield K3-HBc NCs. The objective plasmid carrying the K3-HBc gene was purchased from Shanghai Generay Biotech Co., Ltd. The corresponding vectors were transformed into E. coli strain BL21 (DE3). The expression and purification of K3-HBc protein were performed as the previous description .
Ultra-small CLT nanodots were fabricated by droplet-confined crystallization during the freeze-drying of frozen emulsion . CLT-containing dichloromethane was mixed with an aqueous solution containing F127 (1.0 wt%), and then converted into an O/W emulsion by sonification. The emulsion was immediately immersed in liquid nitrogen and rapidly frozen, followed by freeze-drying. The resulted CLT nanodots were harvested and washed by centrifugation-redispersion cycles to remove free F127.
The bioengineered K3-HBc NCs were reversibly disassembled with 8 M urea at 25 °C for 3 h. After the dissociation, ultra-small CLT nanodots were added and the solution was gently stirred for 2 h. Then, the reassembly of K3-HBc NCs was conducted by dialysis in an assembling buffer at 4 °C and free CLT nanodots was removed by SuperdexTM-75 column. To calculate the loading capacity, the concentration of CLT was determined by UV–vis spectrophotometer.
The morphology of ultra-small CLT nanodots, K3-HBc NCs and K3-HBc/CLT NCs were observed using a transmission electron microscope (TEM, JEM-1400, JEOL). The hydrodynamic diameter sizes of NCs and zeta potential of CLT nanodots were respectively determined by Zeta Sizer (Nano Series, Malvern). Besides, the diameter distributions of CLT nanodots and NCs were obtained through counting at least 100 particles in TEM images.
The FITC-labeled HBc-183 or K3-HBc NCs were performed as the previous description . Briefly, FITC was dissolved in DMSO, and then were mixed with HBc-183 or K3-HBc NCs and gently vortexed for 2 h in the dark at 25 °C. The mixtures were dialyzed in PBS buffer at 4 °C for 48 h to remove unreacted FITC molecules. Cy5.5-NHS was labeled to HBc-183 or K3-HBc NCs in the same methods with FITC labeling.
In vitro drug release
Dialysis bags (MWCO = 3000 Da) containing 2 mL of K3-HBc/CLT and CLT solution (CLT, 2 mg) were separately submerged in release medium(50 mL) at 37 °C while stirring at 100 r/min. At predetermined time points (0.25, 0.5, 1, 2, 4, 8, 12, 24, 36 and 48 h), 1 mL aliquots of the solution were withdrawn to determinate the released drug. Then an equal volume of fresh buffer was added to keep the volume constant and to ensure sink conditions throughout the whole process. These samples were determined by high performance liquid chromatography (HPLC) to calculate the cumulative amount of released drug and plot the percentage of drug released.
Animals model and therapeutic experiments
The male Sprague Dawley rats (6–8 weeks) and male BALB/C mice (6–8 weeks) were purchased from animal center in Third Military Medicine University (Chongqing, China). All animal care and experiments were conducted in compliance with the requirements of the National Act on the use of experimental animals (China) and were approved by the Institutional Animal Care and Ethic Committee of Third Military Medicine University.
UUO was a well-established experimental model of renal fibrosis . Animals were anesthetized with 1% pentobarbital (10 μL g−1), the left ureter was exposed by a tilted incision which near the lower edge of the left rib 1 cm, and then it was obstructed by two-point ligations with 4–0 silk sutures. The abdominal incision was sealed with the same silk suture, and animals were returned to the cages. Sham-operated mouse underwent the same procedure except ligation.
The rats were randomized into three groups: the sham group treated with PBS, the UUO group treated with 0.9% NaCl, and UUO + CLT group received free CLT (1 mg kg−1). PBS, 0.9% NaCl or free CLT was injected intraperitoneally every two days. The body weights were recorded every two days. All animals were sacrificed on day 14. Kidney index from each group were measured (obstructed kidney weight/body weight × 100% or non-obstructed kidney/body weight × 100%). The heart, liver, spleen, lung kidney and brain were harvested immediately. Tissues were washed, fixed and stained with H&E. Histological lesions were observed under the microscope (Olympus BX51, Japan).
To study the therapeutic effect of K3-HBc/CLT in the UUO model, 24 mice were randomly divided into four groups (n = 6): sham, UUO + 0.9% NaCl, UUO + CLT and UUO + K3-HBc/CLT groups. The mice were treated with CLT (1 mg kg−1) by intraperitoneal injection or K3-HBc/CLT (CLT, 1 mg kg−1) by tail vein injection. Both CLT and K3-HBc/CLT were administered every two days. The mice were treated with an equal volume of 0.9% NaCl in UUO + 0.9% NaCl group. All mice were sacrificed on day 14. The organs were harvested for H&E staining. Before animals were sacrificed, blood was collected and centrifuged at 2500 rpm for 15 min. The serum was measured on a chambray 240 automatic biochemical analyzer (Radyto, China) for the following analytes: BUN,CREA, AST, ALT, TBIL and LDH-L, which indicates renal, liver and heart functions, respectively.
Cell culture and TGF-β-induced EMT processes in vitro
HK-2cells were purchased fromStem Cell Bank, Chinese Academy of Sciences. The HK-2 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/F12 medium (Hyclone, SH30023.01, Thermo Scientific, USA) supplemented with 10% fetal bovine serum (ScienCell, Carlsbad, CA, USA), 100 U mL−1 penicillin/streptomycin (Beyotime, Shanghai, China). Cells were cultured in a humidified atmosphere containing 5% CO2 at 37 °C.
Serum-starved HK-2 cells were pre-incubated with or without the CLT (500 nM) or K3-HBc/CLT (CLT, 500 nM) for 1 h for the in vitro experiments. Then the cells were stimulated with TGF-β1 (5 ng mL−1) at the indicated time points. HK-2 cell morphology was observed by phasecontrast microscopy and the cell lysate was harvested for further study.
Cellular uptake of K3-HBc NCs in vitro
HK-2 cells were seeded onto sterilized microscope coverslips placed in 6-well plates at a density of 2 × 105 cells/well and incubated in DMEM medium supplemented with 10% fetal bovine serum for 24 h at 37 °C with 5% CO2. Afterwards, the cells were treated with Cy5.5-labeled K3-HBc/CLT NCs or HBc-183/CLT NCs under the equivalent NCs concentration (20 μg mL−1) and co-incubated for 0.5 and 1 h. Subsequently, the cells were washed 5 min with PBS for three times, fixed with cold 4% paraformaldehyde for 20 min and washed three times with PBS. DAPI was used to stain the nuclei of the cells. Finally, the cells were observed with a confocal laser scanning microscope (Zeiss LSM 700, Zeiss, Thornwood, Germany).
The cellular uptake of FITC-labeled-K3-HBc/CLT NCs or HBc-183/CLT NCs in HK-2 cells was quantified by flow cytometry. Briefly, HK-2 cells (1 × 105 cells/well) were seeded in 12-well plates and incubated for 24 h. Then, cells were incubated with FITC-labeled K3-HBc/CLT NCs or HBc-183/CLT NCs under the equivalent concentration (20 μg mL−1). At predetermined times (0.5, 1, 2 and 4 h), cells were washed with PBS and detached using trypsin. Finally, cells were suspended in cold PBS, which was then immediately analyzed by BD FACS Verse flow cytometer (BD Biosciences, San Jose, CA, USA).
The siRNA transfection experiments were performed as reported previously . The reagents for silencing of gene expression were obtained from Sangon Biotechnology. We employed siRNAs specifically targeting mRNA for humanmegalin and a nontargeting scramble-sequence siRNA (siSCR) as a negative control. HK-2 cells were transfected with siSCR or siRNA targeting LRP2 for 72 h. Then cells were incubated with FITC-labeled K3-HBc/CLT NCs for 1 h, the cellular uptake efficiency was analyzed by flow cytometry.
Distribution of K3-HBc/CLT NCs in vivo
Mice on day 1 or day 7 after the UUO received Cy5.5-labeled HBc-183/CLT or K3-HBc/CLT NCs via the tail vein. Four hours post injection, the mice were sacrificed and the major organs including heart, liver, spleen, lung and kidney were excised and imaged using an imaging system (IVIS Spectrum, Caliper LifeSciences, USA).
The ability of K3-HBc/CLT to inhibited cell senescence in UUO mice kidney was measured by SA-β-Gal staining. Frozen mouse kidney sections (4 μm thickness) were used for detection of SA-β-Gal. The activity of SA-β-Gal was analyzed with SA-β-Gal staining kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions.
Western blot analysis
Total proteins were isolated from kidney and HK-2 cells and quantified as described previously . Protein samples (30 μg/lane) were resolved using sodium dodecyl sulfate–polyacrylamide electrophoresis and then transferred onto polyvinylidene difluoride membranes (PVDF, Millipore, USA). The membranes were incubated at 4 °C overnight with α-SMA (1:300, Abcam, Cambridge, MA, USA), fibronectin (1:300, Santa Cruz, CA, USA), vimentin (1:500, Santa Cruz, CA, USA), E-cadherin (1:1000, Proteintech, Wuhan, China), Megalin (1:100, Santa Cruz, CA, USA) and HBc (1:1000, Abcam, Cambridge, MA, USA), followed by incubation with horseradish peroxidase-conjugated goat anti-mouse (1:10,000, Santa Cruz, CA, USA) or goat anti-rabbit IgG secondary antibodies (1:10,000, Santa Cruz, CA, USA) at room temperature for 1 h. The immunoblots were imaged by the chemiluminescence western blot detection system (Bio-Rad ChemiDoc MP, California, USA) with GAPDH (1:5000, ZENBIO, Chengdu, China) as the loading control.
Total RNA was extracted from the obstructed kidney mice treated with 0.9%NaCl, CLT, or K3-HBc/CLT for 14 days using Trizol reagent (TaKaRa, Japan) according to the manufacturer’s recommendations. Subsequently, 1 μg RNA was used to reverse transcribed cDNA by PrimeScript RT reagent Kit with gDNA Eraser (TaKaRa, Japan). cDNA abundance was de-termined by qPCR with SYBRPRIME qPCR Kit (Bioground, China). The following primers were used: Cdknla forward, 5′-tgatctgctgctcttttcc-3′, and reverse, 5′-tacattcccttccagtcca-3′; Gadd45 forward, 5′-agaagaccgaaaggatgga-3′, and reverse, 5′-cgtaatggtgcgctgac-3′; Sfn forward, 5′-cgacagtgctggggaag-3′, and reverse, 5′-ccaaggtgtggctgaaca-3′; Rprm forward, 5′-acgcaaacctgtcggagt-3′, and reverse, 5′-tgccacctgctgctgtat-3′; Cdk4 forward, 5′-gctgaaattggtgtcggt-3′, and reverse, 5′-cctccagaatccttaaca-3′; GAPDH forward, 5′-ggttgtctcctgccgacttca-3′, and reverse, 5′-tggtccagggtttcttactcc-3′. GAPDH serves as an endogenous control.
Histology and immunohistochemical staining
Kidney tissues harvested from animals on day 14 were fixed in 4% paraformaldehyde, embedded in paraffin and cut into 4 μm thick per section. The sections were stained with H&E and Masson’s trichrome staining. The collagen deposition in the obstructed kidney tissue was assessed by Masson’s trichrome staining. At least 10 randomly selected cortical fields were observed under the microscope (Olympus BX51, Japan) and the renal fibrotic area was semi-quantitated using image-pro plus 6.0 software.
The immunohistochemistry experiment was performed as previously described . Briefly, sections mounted on slides were blocked with 5% BSA for 1 h and incubated over night at 4 °C with primary antibodies against α-SMA (1:200), collagen I (1:500, Santa Cruz, CA, USA), TGF-β (1:200), p16 (1:500) and p21 (1:500). The slides were then stained with a goat anti-rabbit IgG secondary antibody (1:50, Beyotime, jiangsu, China). The results were analyzed using a 3,3′-diaminobenzidine (DAB) assay kit (Servicebio, Wuhan, China). The slides were visualized by a microscope (Olympus BX51, Japan) and were semi-quantitated based on the intensity and spread of positive staining in terms of IOD using image-pro plus 6.0 software.
To examine the localization of K3-HBc NCs in the kidney, immunohistochemical studies were performed in BALB/c mice. The FITC-labeled K3-HBc NCs or HBc-183 NCs was administered to mice via tail vein injection. At 1 h post injection, mice were sacrificed and harvested kidneys were fixed in 4% paraformaldehyde for 24 h. The primary antibody was rabbit anti-FITC antibody (1:500, Sangon Biotech, Shanghai, China). The immunohistochemical staining and analysis was performed as described above.
RNA-seq and bioinformatic analysis
RNA-seq experiment was performed as previous descriped . Briefly, RNA was isolated from kidney tissues in sham, UUO, UUO + CLT and UUO + K3-HBc/CLT groups using the Trizol kit according to the manufacturer ‘s instructions, respectively. Sample integrity, quality and purity were determined accordingly. cDNA synthesis was performed from DNase1 treated RNA samples using ImProm-II Reverse Transcriptase (Promega Corp., Madison, WI). Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations. The sequencing service is performed by Novogene Corporation (Beijing, China). Raw reads were generated using an Illumina Novaseq 6000 platform by paired-end sequencing. After quality filtering, the clean reads were mapped onto the mouse reference genome and the read count for each gene was derived from the mapping results obtained by Feature Counts. All read counts were normalized to fragments per kilo bases per million mapped reads (FPKM). DEseq2 was used to determine differential expressions. Transcripts with an adjusted p value, 0.05 were accepted as being differential. Differentially expressed genes from the different comparisons and the subjected biological process and molecular functional pathways were analyzed using the Gene Ontology, Kyoto Encyclopedia of Genes and Genomes (KEGG) and Reactome biochemical pathway databases.
Quantitative results were expressed as mean ± SEM. Statistical differences among groups were checked by One-way ANOVA followed by Newman-Keuls multiple comparisons test or Student’s unpaired two-tailed t test from GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA). p < 0.05 was considered statistically significant.